Design, synthesis and drug resistance reversal potential of novel curcumin mimics Van D: Synergy potential of curcumin mimics

Article abstract of DOI:10.1016/j.bioorg.2020.104454

Being crucial part of plant-based novel discovery of drug from natural resources, a study was done to explore the antibacterial potential of curcumin mimics in combination with antibiotics against multidrug resistant isolates of Pseudomonas aeruginosa. Th

Full text of DOI:10.1016/j.bioorg.2020.104454

Journal Pre-proofs
Design, synthesis and drug resistance reversal potential of novel curcumin
mimics Van D
Synergy potential of curcumin mimics
Gaurav Raj Dwivedi, Sadiya Khwaja, Arvind Singh Negi, Swati S. Panda, A.
Swaroop Sanket, Sanghamitra Pati, Amit Chand Gupta, D.U. Bawankule,
Debabrata Chanda, Rajni Kant, Mahendra P. Darokar
PII:
S0045-2068(20)31752-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.104454
YBIOO 104454
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
26 June 2020
3 October 2020
1 November 2020
Please cite this article as: G. Raj Dwivedi, S. Khwaja, A. Singh Negi, S.S. Panda, A. Swaroop Sanket, S. Pati, A.
Chand Gupta, D.U. Bawankule, D. Chanda, R. Kant, M.P. Darokar, Design, synthesis and drug resistance
reversal potential of novel curcumin mimics Van D, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/
j.bioorg.2020.104454
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Design, synthesis and drug resistance reversal potential of novel
curcumin mimics Van D
Synergy potential of curcumin mimics
Gaurav Raj Dwivedi1#*, Sadiya Khwaja# 2,4, Arvind Singh Negi2,4*, Swati S Panda5, A.
Swaroop Sanket5, Sanghamitra Pati5, Amit Chand Gupta3, D.U. Bawankule3,4, Debabrata
Chanda3,4, Rajni Kant1 and Mahendra P. Darokar3,4
1Microbiology Department, ICMR-Regional Medical Research Centre, Gorakhpur 273013, Uttar Pradesh, India;
2Chemical Sciences Division; 3Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants,
Near Kukrail Picnic Spot, P.O. CIMAP, Lucknow-226015 India
4Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India.
5ICMR-Regional Medical Research Centre, Bhubaneshwar 751023, Odisha, India;
#Both the authors contributed equally
*Corresponding authors:
1. Dr. Arvind Singh Negi
CSIR-Central Institute of Medicinal and Aromatic Plants, Near Kukrail Picnic Spot, P.O. CIMAP,
Lucknow-226015 India,+91-5222718583; Fax: +91-522-2342666; Email: as.negi@cimap.res.in
2. Dr. Gaurav Raj Dwivedi,
Microbiology Department, ICMR-Regional Medical Research Centre, Gorakhpur 273013, Uttar
Pradesh, India
Email: grd.rmrcb@gov.in; grdnikhil@gmail.com
1

Abstract
Being the crucial part of plant-based novel discovery of drug from natural resources, a study was
done to explore the antibacterial potential of curcumin mimics in combination with antibiotics against
multidrug resistant isolates of Pseudomonas aeruginosa. The best candidate Van D, a curcumin
mimics reduced the MIC of tetracycline (TET) up to 16 folds against multidrug resistant clinical
isolates. VanD further inhibited the efflux pumps as evident by ethidium bromide efflux and by in-
silico docking studies. In another experiment, it was also found that Van D inhibits biofilm synthesis.
This derivative kills the KG-P2, an isolate of P. aeruginosa in a time dependent manner, the post-
antibiotic effect (PAE) of tetracycline was extended as well as mutant prevention concentration (MPC)
of TET was also decreased. In Swiss albino mice, Van D reduced the proinflammatory cytokines
concentration. In acute oral toxicity study, this derivative was well tolerated and found to be safe up
to 1000 mg/kg dose. To the best of our knowledge, this is the first report on curcumin mimics as
synergistic agent via inhibition of efflux pump.
Keywords: Drug resistance reversal, curcumin based derivative, efflux pump, acute oral toxicity.
2

1. Introduction
In the developing and under-developed world, communicable diseases are the key cause of illness
and death [1]. Among the wide range of infectious diseases, bacterial diseases are concerned due to
prevalence of the multidrug resistance (MDR) [2–4]. Over-expression of the efflux transporters,
down-expression of porin forming genes, biofilm synthesis and production of different types of beta
lactamase are the key mechanisms for MDR and among these, efflux pumps distinguish and
proficiently expel a wide series of distinct compounds through the cell [3,5–7]. Due to limited
treatment options these MDR bacteria put an extra burden, cost and boost infections severity many
times [8–10]. According to the latest report of WHO priority list, P. aeruginosa is placed in the
category of ‘critical’ which needs more attention for discovery and development of novel drugs. P.
aeruginosa, is a principal causative representative of cystic fibrosis complications with substantial
illness and deaths [11–13].
Drug resistant P. aeruginosa became a global issue. Efflux pumps being utilized by P. aeruginosa
contribute resistance of highest level in harmonization with biofilms and barriers of outer membrane
[11,14–16]. Efflux pumps are the main cause of MDR which narrow down the treatment [3]. These
pumps make the antibiotics inefficient and on the other hand drug transporters narrow down treatment
options against GNB [10]. For MDR P. aeruginosa, carbapenem group of antibiotics were regarded
as the best treatment option. But, with the emergence of carbapenemases, drug discovery programs
against these superbugs have become more challenging [13]. In the recent past, P. aeruginosa became
one of the main intimidations for drug discovery and development [13].
Antimicrobial with broad spectrum activity, competent cost as well as quite lower toxicity make
tetracycline group of antibiotics as choice drugs for the treatment of many life threatening infections
[17–19]. These are effective against prokaryotic organisms and also against some eukaryotic parasites
[18]. From the last four decades, drug tansporters are one of the key causes of MDR in bacteria [3,20].
For the development of effective combination therapy, these transporters are regarded as the “key”
drug targets and efflux pump inhibitors as popular choice to inverse the mechanism of resistance [21–
24]; [21,23,25]
Continuance of previous endeavors on combinatorial potential of natural compounds and their
derivatives [14,26–33], present study depicts the combination potential of curcumin mimics with
tetracycline and other antibiotics. Inspired from the previous findings, it was intended to synthesize
derivatives possessing strong antibacterial activity and the structural learning of curcumin was
utilized in the designing of this series. The curcumin mimics were assessed for their antibacterial
efficacy individually and in combination with TET and other antibiotics.
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The best derivative Van D, was further appraised for mode of action study which was done by efflux
pump inhibition assay, biofilm inhibitory action, quantitative expression analysis, molecular docking
and drug likeliness analysis. Acute oral toxicity of this molecule was also studied in Swiss albino
mice. The derivative was also able to minimize the pro-inflammatory cytokines in LPS induced
septicemia.
2. Results
2.1. Chemical synthesis
Curcumin was isolated from the rhizomes of Curcuma longa as per our reported method [34].
Eighteen diverse curcumin mimics were synthesized through two series of starting substrates via
Claisen Schmidt reaction (Fig.1.). For compounds CD1 to CD6 and CD8, terephthaldehyde was taken
as a common dialdehyde and diverse acetophenones were condensed using 7% alkali in methanol at
room temperature to get compounds CD1 to CD6 and CD8. Few compounds were prepared by
condensing 1,3-diacetylbenzene as a common substrate with diverse aldehydes in the presence of 7%
alkali in methanol at room temperature to get CD7 and CD9. While the rest of the products were
synthesized also by condensing 1,3-diacetylbenzene as a common substrate with different aldehydes
but in presence of 1,4-dioxane-conc. HCl under reflux at about 110 ⁰C to get CD11, CD13, CD15,
and Van M (CD17) as monomer units while CD 10, CD12, CD14, CD16, and Van D (CD18) as the
dimer units. All the products were confirmed by spectroscopy.
Fig.1.
2.2. Sensitivity profiling and procurement of most resistant clinical isolate
As per initial and present study of antibiotic sensitivity profiling, KGP2 was the most resistant clinical
isolate among the used 10 clinical isolates (Table S1)[14]. Among all KGP2 and U-1926 isolates of
P. aeruginosa were also found to be MBL producers (Table S2) while remaining were negative to
MBL production. KGP2 was also the best biofilm producer (Fig. 2). Based on all the results, it can
be concluded that KGP2 isolate was highly resistant to different group of antibiotics due to MBL
production, biofilm and efflux pumps. KGP2 an isolate of P. aeruginosa was used for the further drug
resistance reversal and mode of action study.
Fig. 2.
2.3. Antibacterial and combination studies
Antibacterial activity of curcumin mimics (CD1 to CD18) was done against the highest resistant
clinical isolate KG-P2 of P. aeruginosa, the MIC of these derivatives varied from 200-800 mg/L. In
combination, these compounds were able to decrease the MIC of clinically used antibiotics (Amikacin,
norfloxacin, ofloxacin, tobramycin, ampicillin, cefoxitin, cefotaxime, oxy-tetracycline and
4

tetracycline) by 2 to 16 folds (Table S3), Among all the used antibiotics, Van D showed the highest
reduction in the MIC of tetracycline upto 16 folds (Table 1). However the results are given against
the most resistant isolate KG-P2 and the best combination antibiotic tetracycline (Table 1). In this
study, compound Van D, the best representative was found even better than other compounds and
equivalent to PAβN in terms of fold reduction of MIC of TET (Table 1). The fold reduction of MIC
of TET varied from 2fold to 16 folds. On the basis of fractional inhibitory concentration index (FICI),
CUR the strating molecule showed the synergistic interaction along with the it’s derivatives CD-2,
CD-6, CD-7, CD-11,CD-12, CD-14 and VanD while other derivatives exhibited no interaction with
TET (Table 1). Hence, combination of Van D with TET was selected for further investigations.
Table1
2.4. Time kill kinetic study
Treatment of TET at MIC, 2xMIC and 4xMIC, cell viability of KG-P2 was reduced by ten folds as
compared to untreated control (Fig. 3A). However, the bactericidal active ty of TET was achieved at
low concentration when used in combination with Van D (12.5 mg/L) (Fig. 3B).
Fig. 3.
2.5. Mutation prevention concentration
A drug sensitive strain of P. aeruginosa MTCC-741 was used for mutant selection study. At 800 mg/L
(8×MIC) of TET, no mutant was selected and hence defined as the MPC. The MPC of TET was
reduced to 400 mg/L in combination withVan D (12.5 mg/L) (Table 2).
Table 2
2.6. Post antibiotic effect (PAE)
The PAE of antibiotic TET alone and in combination with Van D (12.5 mg/L) was determined against
KG-P2, the alone antibiotic TET exhibited a PAE of 1.83, 2.55 and 3.75 h at 0.25× MIC, 0.5×MIC
and MIC, respectively. However, PAE of TET was lengthened to 2.12, 2.79 and 4.37 h respectively
at the same concentrations in combination with Van D (12.5 mg/L) (Table 3). In the combination of
CUR (50 mg/L) the PAE of TET was lengthened to 2.05, 2.48 and 3.95 h respectively at 0.25× MIC,
0.5×MIC and MIC, respectively.
The combinatorial potential as well as promising results of in-vitro drugaability of Van D encouraged
us to go forward for the mode of action studies.
Table 3
2.7. MBL inhibitory study
5

As KGP2 clinical isolate was producing the MBL which is responsible for the resistance towards
carbapenems (the last resort against Gram negatives). In this study KGP2 clinical isolate was showing
no zone of inhibition either imipenem alone or in presence of Van D, however, a clear zone of
inhibition was observed imipenem and EDTA (Fig. 4) In the same way curcumin was also showing
no clear zone of inhibition with the imipenem (Figure is not given).
Fig. 4.
2.8. Biofilm Inhibitory potential of Van D
In order to understand the mechanism of actionVan D was also evaluated for biofilm inhibitory study
in which Van D and CUR was able to inhibit the biofilm synthesis (Fig. 5).
Fig. 5.
2.9. Ethidium bromide efflux pump studies
In order to understand the mechanism of action, efflux pump inhibitory potential of Van D was done
using ethidium bromide efflux assay in multidrug resistant clinical isolate KG-P2. As evident in Fig.
6, relative fluorescence was significantly decreased in non-treated cells., However relative
fluorescence decrease was restricted in the presence of Van D and CUR.
Fig. 6.
2.10. Expression profile of efflux pump genes
In qPCR study, expression profile of the efflux pump genes such as mexA, mexB and oprM was done
in the presence of TET, Van D, CUR and PaβN alone and in combination. The Van D affected the
expression pattern of efflux pump genes, especially RND tripartite complex (mexA, mexB, and
oprM). As per Figure 7, an up-regulation of these three efflux pump genes was detected when the
cells were treated with tetracycline alone at 400 mg/L concentration. However, Van D, CUR and
PaβN were found to down-regulate the expression of efflux pump genes alone and in combination
with TET (Fig. 7).
Fig. 7.
2.11. Effect of Van D on pro-inflammatory cytokine production in LPS-induced endotoxemia in
mice
Serum level of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) was significantly increased in
LPS challenged vehicle treated mice when compared to normal mice. Oral administration of Van D
6

before LPS challenge significantly (p<0.05) inhibited TNF-α, IL-6 and IL-1β production in dose
dependant manner at the dose rate of 3, 10, 30 mg/kg body weight (Fig. 8, Table S4).
Fig.8.
2.12. In-silico molecular docking study
In the in-silico molecular docking study, Van D was reported to dock best with MexA (PDB ID:
1VF7) followed by MexB (PDB ID: 3W9I) and OprM (PDB ID: 3D5K). The compound Van D
interacted with MexA protein (docking score of -8.29 kcal/mol) at several sites like glutamic acid and
arginine by forming hydrogen bonds (one each) & proline, valine, leucine, arginine, and alanine
interacted by forming alkyl bonds [Supplementary information, Table S7]. The ligand curcumin when
interacted with MexA (docking score of -7.61 kcal/mol) was observed to interacting with two
hydrogen bonds at different amino acid residue positions (glutamine at 46 and leucine at 138)
however leucine 138 also formed a sigma bond; two alkyl bonds were formed by arginine at 133 and
44.
Van D when interacted with MexB (docking score of -8.3 kcal/mol) formed two hydrogen bonds
with alanine and leucine whereas proline was observed to form alkyl bonds; glutamic acid formed an
anionic interaction and alanine formed a sigma bond with the compound. In comparison to Van D,
when curcumin interacted with the protein MexB (docking score of -7.92 kcal/mol), arginine 716,
serine 721, and asparagine 676 formed hydrogen bonds whereas phenylalanine 617 (formed amide
pi-stacked bond), and alanine 618, proline 717 formed alkyl bonds. The amino acid residues aspartic
acid 681 and glutamic acid 825 form the non-conventional hydrogen bonds.
When the interaction of Van D with OprM (docking score of -6.18 kcal/mol) was studied, only one
hydrogen bond was observed to be formed by Glutamic acid. However, proline and isoleucine formed
sigma and alkyl bonds respectively. For the same protein, when the interaction of curcumin was
studied (docking score of -5.72 kcal/mol), phenylalanine 85, glutamic acid 125, alanine 123 formed
three hydrogen bonds whereas threonine 122 and isoleucine 88 formed non-conventional hydrogen
bonds. The residue isoleucine 88 was also observed to be forming one alkyl bond with curcumin.
Supplementary file table S5, S6 and S7 describe insilico druggability and interacting residues with
VanD, CUR and positive control.
Table 4
Fig.9.
2.13. Acute oral toxicity of Van D
Nowadays, safety has become the most important concern and a key criterion for successful new
drugs due to regulatory, societal and legal concerns. Therefore, Van D was evaluated for safety
7

aspects in rodent model (Table 5 and Fig.10). Four different oral doses (5 mg/kg, 50 mg/kg, 300
mg/kg, and 1000 mg/kg) of Van D were given to Swiss-albino mice as a single dose. Throughout the
experiment, there was no morbidity or mortality in experimental animals treated with Van D up to
the dose level 1000 mg/kg in the acute oral toxicity test. Most biochemical parameters showed non-
significant changes. However, ALP and cholesterol levels showed an increase which were statistically
insignificant in a group of animals treated with the compound at 1000 mg/kg as a single acute oral
dose. Overall, Van D was quite safe and well tolerated by experimental animals in acute oral toxicity.
However, experiments with prolonged exposure of drug candidate will be required during future
development through sub-acute, chronic and sub-chronic toxicity experiments.
Table 5
Fig. 10.
Discussion
After the discovery and development of antibiotics penicillin and streptomycin (which were supposed
to be the ground-breaking innovations of Fleming, Domagk, Dubos, Waksman and their many co-
workers) chemotherapy was rejuvenated and found a momentum. These antibiotics were a miracle
which saved millions of lives from many diseases including deadly pneumonia and tuberculosis.
Antibiotics transformed the chemotherapy industry also. Infectious diseases were regarded as
diseases of the past in 1980s with comprehensive life expectancy [13,19]. After the 1980s the era of
negligence started towards the available treatment systems which gave rise to the era of drug
resistance. The uncontrolled use of antibiotics have promoted several human pathogenic bacteria such
as P. aeruginosa to perceive resistance to multiple drugs [33,35,36]. As a result of multidrug
resistance (MDR) phenomenon, the evolution of “superbugs” with serious threat to available sets of
antibiotics has taken a great attention in the scientific community [6,37,38]. Since the discovery of
the novel antibiotic ‘Teixobactin’, a MDR Gram positive bacteria inhibitor, there has not been a
greater progress to find new antibiotics against MDR-Gram negative bacteria [39,40]. However
initiatives by FDA, the Infectious Diseases Society of America (IDSA), and the European Medicines
Agency have shown great promising approaches to identify new antibiotics in the coming future [41].
Chemically, curcumin is a diferuloylmethane possessing a keto-enol tautomerism. It is a highly
conjugated system having dienone arrangement which exhibits potential antioxidant activity. Present
study aimed at similar conjugated enone type core with minor modification where centralized
methane group was r eplaced with a phenyl ring to have greater conjugation for better efficacy. Thus,
similar bis-enone system was as diferuloylbenzene core in our planned curcumin mimics. There was
no definite trend in structure activity relationship. However, diferuloylbenzene type curcumin mimics
8

(CD-7, CD-9 to CD-16, Van M and Van D) were much more effective as compared to bis
cinnamoylbenzene type system (CD-1 to CD-6 and CD-8). Among the diferuloylbenzene type
curcumin mimics, Van M and Van D possessed the similar substitutions in the terminal phenyl rings
as in curcumin. But Van D has much more higher conjugation as compared to Van M due to dimer
type compound. Overall, Van D possessed very close structural similarity with curcumin among all
these synthesized curcumin mimics thatswhy it might exhibited the best bioactivity.
13
Structures of Van M and Van D were established by spectroscopy. C NMR and High resolution
1
mass value of Van M at 296 amu suggested its molecular formulae as C18H16O4. In H NMR, it
showed one methyl as singlet at δ2.88 and one methoxy as singlet at δ3.97. The deshielded chemical
shift of methyl indicated it as acetyl methyl (CH3CO). It showed nine methines (CH) in the aromatic
region. Two of the methines were at δ7.15-7.22 and δ7.77-7.82 as doublet with coupling constants of
15.6 Hz indicating two olefinic CH in trans fashion. Deshielded chemical shift of one of the CH
designated it as α/β-unsaturated ketone system. The presence of ketonic group was also confirmed in
13C NMR at δ190.13. Presence of four aromatic quaternaty carbons deshielded beyond δ135ppm
(δ137.79, 139.34, 146.56, & 149.05) indicated them as oxygenated aromatic syetem. Thus, the based
on all these spectral data its chemical structure was established as (E)-1-(3-acetylphenyl)-3-(4-
hydroxy-3-methoxyphenyl)prop-2-en-1-one (Van M).
1H NMR of Van D exhibited signal at δ4.03 for two methoxy groups and fourteen proton signals in
aromatic region. Ten of the proton signals in aromatic region were aromatic protons while a doublet
signals for two protons at δ 7.47-7.51 and another at δ 7.84-7.89 with coupling constant of 15.3 Hz
clearly indicated two trans double bonds. Downfield chemical shift of second trans double bond at δ
7.84-7.89 indicated presence bis Cinnamoyl type arrangements. It was further confirmed by 13C NMR
where a ketone group was present at at δ190.73. On considering 13C NMR signals and High
resolurion-mass its molecular weight was determined as 430amu with chemical formulae of C26H22O6.
13
In C NMR it showed fourteen signals for carbon. Further, DEPT-135 and DEPT-90 experiments
clearly indicated presence of two methyls as methoxy groups, fourteen methines (CH). Presence of
deshielded quaternary carbons at δ139.30, 146.51, 147.32, 149.06 indicated them as oxygenated
aromatic carbons. Considering all these spectral assignments, the chemical structure of Van D was
established as 1,3-Bis-(E/E)-[3-(3-Methoxy,4-hydroxyphenyl)-1-oxo-2-en-yl]-benzene.
Curcumin, a natural phenolic compound possesses a wide spectrum of pharmacological activities [42].
Previously, curcumin has shown antibacterial activity in combination with β-lactam and quinolones.
In another study, it was reported to reverse methicillin resistance in S. aureus via affecting penicillin
binding protein 2a (PBP2a), cell wall and cell membrane damage [43,44] In our study, we planned to
9

have curcumin mimics with additional conjugated system to better activity. In our studies, the
curcumin mimics, Van D has exhibited drug resistance reversal activity better than curcumin.
Curcumin alone exhibited MIC at 200 mg/L, on the other hand MIC of others derivatives varied from
200 mg/L to 800 mg/L . Individually, neither CUR (MIC 200 mg/L) nor VanD(MIC 200 mg/L) and
other derivatives should be consider as good antibacterial. Further ,in combinatorial study, MIC of
TET was reduced by eight-fold for curcumin and four to sixteen-fold reductions in MIC was for Van
D. Both curcumin and Van D showed synergism with TET. However, Van D was better than
curcumin as it reduced the MIC of different antibiotics efficiently. Comparable observations have
also been reported earlier [26,45–47].
Individual bacterial effect of TET was attained at higher concentration (2xMIC, 4xMIC) and the same
effect of TET was observed at low concentration when studied in combination with Van D. Similar
study by many groups reported that bactericidal effect was attained at at quite low concentration when
tested in combinations with natural/semisynthetic compounds [48–50]. Van D was able to lenthen
the post antibiotic effect of TET and was also able to reduce the mutation prevention concentration
of TET which enhances the suitability of Van D to be used as therapeutic agent.
In efflux inhibition assay, Van D was able to inhibit the efflux of ethidium bromide as compared to
PAβN and curcumin. Accumulation and efflux of ethidium bromide indicate involvement of efflux
pumps in the resistance mechanism, particularly in gram negative bacteria such as P. aeruginosa
[11,51–53]. In Gram-negative bacteria, porin proteins allow a wide variety of compounds to pass
through and reach the inner part of the membrane [15,54]. Porin proteins are down regulated
whenever a cell senses the presence of noxious agents [55]. The role of efflux pumps have been well
correlated and proved to exhibit multidrug resistant (MDR) phenotype [8,51,55,56].
In the present study, we observed over-expression of mexA, mexB and oprM genes upon treating
tetracycline the clinical isolate KG-P2 of P. aeruginosa which was also observed in our previous
studies [14,55,56]. Upon the treatment of Van D alone and in combination, down-regulation of the
genes was observed implicating the role and involvement of these genes in tetracycline resistance
exhibited by the isolate.
Endotoxemia is the dysregulation of systemic immunity followed by failure of multiple organs [57].
Lipopolysaccharide (LPS or endotoxin) is a major factor triggering pathogenesis in endotoxemia [58].
Certain features like production of inflammatory cytokines, modulation of adhesion molecules,
activation of leukocytes and disbalance of vascular integrity take place during this infection [59]. The
monocytes and macrophages also come into action during this period [60].
10

Intraperitoneal administration of lipopolysaccharide (LPS), has been shown to cause sepsis thus
resulting in death of the cell. [57,61]. Changes include the release of inflammatory cytokines which
are inducted by recognition of lipid A region by TLR-4 [62]. The presence of endotoxins in the blood
is called endotoxemia. Lipopolysaccharide (LPS), an endotoxin is the major mediator responsible for
triggering the cellular and humoral responses which leads to septic shock. This finding is correlated
to the previous finding that natural molecules and their analogues are able to inhibit the production
of pro-inflammatory cytokines in LPS-induced endotoxemia in mice [59,63,64].
Our observations from the acute oral toxicity study show that the compound Van D has no adverse
side effects when parenterally administered. Thus, we conclude the efficacy of the compound for
consideration in combination drug discovery studies. However, we propose that the utility of this
molecule should further be validated by studying sub-acute, chronic or sub-chronic experiments for
any adverse effect on prolonged exposure.
Corroborating our results with previous studies, we propose the acceptance of Van D as it is able to
interact with cell components by showing synergism with the antibiotics [65].
3. Conclusion
The curcumin mimics Van D was able to reduce the MIC of TET through multiple mechanisms like
efflux pump inhibition, biofilm inhibition and modulation of efflux pump genes. Van D may be
helpful in the management of MDR P. aeruginosa by (i) lowering the dose of antibiotics; (ii) reducing
the drug resistance development frequency; and (iii) increasing the efficacy of antibiotics. These
results may give an insight to design novel antibacterial agents in economic and natural resources.
4. Material and methods
4.1. Standard therapeutics
Ampicillin (AMP), streptomycin, ofloxacin (OFL), nalidixic acid, tetracycline and chloramphenicol
were procured from Sigma-Aldrich, USA.
4.2. Chemistry
The synthesis and spectral data of compounds (CD1 to CD16) have also been given in our reported
work [34].
General procedure for the synthesis of Van D, Van M and CD10 to CD16
Synthesis of 1,3-Bis-(E/E)-[3-(3-Methoxy,4-hydroxyphenyl)-1-oxo-2-en-yl]-benzene (Van D, CD18):
To a stirred solution of 1,3-diacetylbenzene (810 mg, 5 mmol) in 1,4-dioxane (10 mL), conc.
hydrochloric acid (0.5 mL) was added. To this vanillin (1.82 g, 12 mmol) was added in small portions
and the reaction mixture was refluxed for 4 h. After every one-hour interval conc. HCl was added
11

(0.5 mLx2) and refluxed. On completion, reaction mixture was poured into crushed ice, extracted
with ethyl acetate (3x20 mL), organic layer was washed with water (2x30 mL) and dried over
anhydrous sodium sulphate and evaporated in vacuo. The residue thus obtained was purified through
column chromatography over silica gel (60-120 mesh) and eluted with the increasing polarity of ethyl
acetate in hexane to get a minor product (monomer, Van M) followed by the desired dimer (major
product) Van D as amorphous solid.
Van M (CD17): Yield= 211 mg (39.3%); m.p.=155-158oC; 1H NMR (CDCl3, 300MHz): δ 2.66 (s,
3H, CH3CO), 3.977 (s, 3H, OCH3), 6.95-6.97 (d, 1H, 12-CH, J=8.1 Hz ),7.15-7.22 ( d, 1H, 11-CH,
J= 16.5 Hz ), 7.37 (s,1H, 15-CH), 7.42-7.45 (d, 1H, 8-CH, J= 9.9 Hz), 7.61-7.64 (m, 1H, 3-CH),7.77-
7.82 ( d, 1H, 9-CH, J=15.6 Hz ), 8.14-8.22 (m, 2H, 2 & 4-CH), 8.57 (s, 1H, 6-CH), 9.82 (s, 1H, OH);
13C NMR (CDCl3, 75MHz): δ27.19, 30.08, 56.50, 110.49, 115.34, 119.50, 124.23, 127.65, 128.47,
129.41, 132.45, 133.16, 137.79, 139.34, 146.56, 149.05, 190.13; ESI-MS for C26H22O6 (MeOH) =
297 [M+H]+, 319 [M+Na]+; Negative mode = 295 [M-H]- ; ESI-TOF-HRMS (MeOH) for C18H16O4
calculated 297.1126 for [M+H]+, found = 297.1120.
Van D (CD18): Yield=1184 mg (53%); m.p.= 82-86oC; 1H NMR (CDCl3, 300MHz): δ 4.036 (s, 6H,
2xOCH3), 7.04 (d, 2H, CH aromatic, J=8.1Hz), 7.33 (bs, 4H, 4xCH aromatic), 7.47-7.51(d, 2H,
2x=CH, J=15.3Hz ), 7.68-7.73(bm, 1H,CH aromatic, J=7.5Hz), 7.84-7.89 (d, 2H, 2xCH=, J=15.3 Hz
), 8.26-8.28 (d, 2H, 2xCH aromatic, J=7.5Hz ), 8.68 (s, 1H, CH aromatic); 13C NMR (CDCl3, 75MHz):
δ 56.50, 110.49, 115.36, 119.69, 124.26, 127.69, 128.57, 129.34, 132.59, 139.30, 146.51, 147.32,
149.06, 190.37; ESI-MS for C26H22O6 (MeOH) = 453[M+Na]+, 469[M+K]+; Negative mode =
429[M-H]-; ESI-TOF-HRMS (MeOH) for C26H22O6 calculated 431.1494 for [M+H]+, found,
431.1487.
1',3'-Bis-(E/E)-[3-(3",4"-dimethoxyphenyl)-1-oxo-2-enyl]-benzene (CD10)
Yield=72%, yellow oil; 1H NMR (300MHz, CDCl3): δ3.93 (s, 6H, 2×OCH3), 3.96 (s, 6H, 2×OCH3),
6.91 (bd, 2H, CH, aromatic), 7.08-7.34 (d, 4H, CH, aromatic), 7.41-7.46 (d, 2H, =CH, J=15.6Hz),
7.61 (t, 1H, CH, aromatic), 7.78-7.84 (d, 2H, CH=, J=15.6Hz), 8.19 (bd, 2H, CH, aromatic), 8.62 (s,
13
1H, CH, aromatic); C NMR (75MHz, CDCl3): δ56.4, 110.6, 111.6, 119.9, 123.9, 128.1, 129.3,
132.6, 139.3, 146.3, 149.7, 152.1, 190.3; ESI-MS for C28H26O6 (MeOH): 459 [M+H]+, 481 [M+Na]+,
497[M+K]+; ESI-TOF-HRMS (MeOH) for C28H26O6calculated, 459.1807 for [M+H]+;found,
459.1802.
(E)-1-(3'-acetylphenyl)-3-(3",4"-dimethoxyphenyl)prop-2-en-1-one (CD11)
12

Yield= 26.5%, yellow solid; m. p. = 145-150 ⁰ C; 1H NMR (300 MHz, CDCl3): δ2.60 (s, 3H, CH3),
3.96 (s, 6H, 2×OCH3), 6.89-6.92 (d, 1H, 5”-CH, J=8.1Hz), 7.38-7.44 (d,1H, 2-CH, J=15.6Hz),7.06
(s, 1H, CH, aromatic), 7.17(s, 1H, 2”-CH), 7.59-7.64 (m, 1H, 5’-CH), 7.78-7.83 (d,1H, 3-CH, J= 15.6
Hz), 8.19-8.22 (d, 2H, 4’&6’-CH, J=7.8 Hz), 8.57 (s, 1H, 2’-CH);
ESI-MS for C19H18O4 (MeOH): 311[M+H]+, 333[M+Na]+, 349[M+K]+; ESI-TOF-HRMS (MeOH)
for C19H18O4 calculated, 311.1283 for [M+H]+;found, 311.1279.
1',3'-Bis-(E/E)-[3-(3",4",5"-trimethoxyphenyl)-1-oxo-2-enyl]-benzene (CD12)
1
Yield=96%, yellow oil; H NMR (300MHz, CDCl3): δ4.02 (s, 18H, 6×OCH3), 6.98 (s, 4H, CH,
aromatic), 7.36-7.41 (d, 2H, CH=,J=15Hz), 7.68 (bs, 1H, CH, aromatic), 7.76-7.81 (d, 2H, =CH,
13
J=15.6Hz), 8.22 (bs, 2H, CH, aromatic), 8.64 (s, 1H, CH, aromatic); C NMR (75MHz, CDCl3):
δ56.3, 61.0, 105.9, 120.9, 128.3, 129.1, 130.1, 132.4, 138.8, 146.0, 153.5, 189.9; ESI-MS for
C30H30O8 (MeOH): 519 [M+H]+, 541 [M+Na]+, 557[M+K]+; ESI-TOF-HRMS (MeOH) for C30H30O8
calculated, 519.2018 for [M+H]+; found, 519.1963.
(E)-1-(3'-acetylphenyl)-3-(4"-hydroxyphenyl) prop-2-en-1-one (CD13)
Yield= 34.5%, yellow solid; m.p.= 203-206⁰ C; 1H NMR (300MHz, CDCl3 , DMSOd6 few drops):
δ2.62 (s,3H, CH3-C=O), 6.88-6.91 (d, 2H, 2”&6”-CH, J=8.1 Hz), 7.40-7.45 (d, 1H, 2-CH, J= 15.6Hz),
7.54-7.65 (m, 3H, 3”&5”-CH & 5’-CH), 7.74-7.80 (d, 1H, 3-CH, J=15.6 Hz), 8.16-8.19 (bm, 2H,
13
4’&6’-CH), 8.54 (s, 1H, 2’-CH), 9.71 (s, 1H, exchangeable, OH) ; C NMR (75 MHz, CDCl3,
DMSOd6 few drops): δ31.92, 121.38, 123.26, 131.04, 133.09, 134.13, 135.81, 137.00, 137.77,
142.44, 144.09, 151.27, 165.66, 194.79, 202.71; ESI-MS for C17H14O3 (MeOH): Positive mode:
267[M+H]+, 289[M+Na]+, 305[M+K]+; Negative mode: 265[M-H]-; ESI-TOF-HRMS (MeOH) for
C17H14O3 calculated, 267.1021 for [M+H]+; found, 267.1005.
1',3'-Bis-(E/E)-[3-(4"-hydroxyphenyl)-1-oxo-2-enyl]-benzene (CD14)
1
Yield=69%, yellow solid; m.p.=235-238⁰C; H NMR (300MHz, CDCl3): δ6.92-7.08 (d, 4H, CH,
aromatic), 7.69-7.76 (bd, 8H, CH, aromatic & 2×=CH), 8.20 (bd, 2H, CH, 2×CH=), 8.62 (bs, 1H,
CH, aromatic); 13C NMR (75MHz, CDCl3): δ116.3, 119.1, 127.0, 128.2, 129.4, 131.2, 132.4, 139.4,
145.3, 189.1; ESI-MS for C24H18O4 (MeOH): 371 [M+H]+, 393 [M+Na]+, 409 [M+K]+; ESI-TOF-
HRMS (MeOH) for C24H18O4 calculated, 371.1283 for [M+H]+; found, 371.1277.
(E)-1-(3'-acetylphenyl)-3-(3"-hydroxy-4"-methoxyphenyl)prop-2-en-1-one (CD15)
Yield=17.1 %, brown solid; m.p.= 149-153 ⁰C; 1H NMR (300MHz, Pyridine-d5):δ2.67(s, 3H, CH3-
C=O), 3.88 (s, 3H, OCH3), 6.83-6.86(d, 1H, CH, aromatic, J= 8.1 Hz), 7.15 (bs, 1H, CH, aromatic),
7.43-7.46 (d, 1H, CH, aromatic, J=7.5 Hz), 7.60 (s, 1H, CH, aromatic), 7.65-7.70 (d, 1H, 2-CH,
J=15.6Hz), 7.98-8.03 (d, 1H, 3-CH, J=15.9Hz), 8.05-8.08 (m, 1H, CH, aromatic), 8.21-8.24 (d, 1H,
13

CH, aromatic, J=6.9 Hz), 8.43 (s, 1H, 2’-CH); 13C NMR (75MHz, Pyridine-d5): δ27.96, 31.27, 57.14,
113.48, 117.07, 121.10, 130.06, 130.64, 133.55, 134.23, 139.20, 140.64, 147.36, 149.85, 151.09,
151.45, 152.78, 190.52, 198.65; ESI-MS for C18H16O4 (MeOH): Positive mode: 297[M+H]+,
319[M+Na]+; Negative mode: 295 [M-H]-; ESI-TOF-HRMS (MeOH) for C18H16O4 calculated,
297.1126 for [M+H]+; found, 297.1120.
1',3'-Bis-(E/E)-[3-(3"-hydroxy-4"-methoxyphenyl)-1-oxo-2-enyl]-benzene (CD16)
1
Yield=79%, dark yellow solid; m.p.=147-150⁰C; H NMR (300MHz, CDCl3): δ3.91 (s, 6H,
2×OCH3), 7.84-7.86 (bd, 2H, CH, aromatic, J=8.1Hz), 7.13-7.16 (bd, 2H, CH, aromatic), 7.33-7.39
(d, 2H, =CH, J=16.8Hz), 7.58 (bs, 1H, CH, aromatic), 7.73-7.78 (d, 2H, CH=, J=15.6Hz), 8.16-8.19
(bs, 2H, CH, aromatic), 8.60 (s, 1H, CH, aromatic); 13C NMR (75MHz, CDCl3): δ56.4, 111.1, 113.8,
120.3, 123.3, 128.7, 129.4, 132.6, 139.2, 146.1, 146.4, 149.5, 190.2; ESI-MS for C26H22O6 (MeOH):
431 [M+H]+, 453 [M+Na]+; ESI-TOF-HRMS (MeOH) for C26H22O6 calculated, 431.1494 for
[M+H]+; found, 431.1485.
General procedure for the synthesis of CD1 to CD6 and CD8):
To a solution of 7% KOH in methanol (10 mL), 1, 4- terephthaldehyde (300 mg, 1.12 mmol) was
added and stirred at room temperature. To this acetophenone derivative (2.4 mmol) was added and
the reaction mixture was stirred for 7-8 hours. After completion of reaction, methanol was distilled
off, residue was diluted with water (10 mL), acidified with 5% HCl (10 mL) and extracted with ethyl
acetate (3x10 mL). After this the ethyl acetate layer was washed with water and dried over
anhydrous sodium sulphate and finally dried in-vacuo. Then the crude residue was further purified
through column chromatography using solvents ethyl acetate-hexane to isolate the pure compound.
1',4'-Bis-(E/E)-[1-(3",4"-dimethoxylphenyl)-prop-1-oxo-2-enyl]benzene (CD1)
Yield=61%, yellow solid.; m.p.=218-220⁰C; 1H NMR (300MHz, CDCl3): δ4.02 (s, 12H, 4×OCH3),
6.93-6.96 (d, 2H, CH, aromatic, J=8.7Hz), 7.47-7.52 (d, 2H, =CH, J=15Hz), 7.73-7.78 (d, 2H, CH=,
J=15Hz), 7.52-8.11 (m, 8H, CH, aromatic); ESI-MS for C28H26O6 (MeOH): 481[M+Na]+; ESI-TOF-
HRMS (MeOH) for C28H26O6 calculated, 459.1807 for [M+H]+; found, 459.1801.
1',4'-Bis-(E/E)-[1-(4"-methylphenyl)-prop-1-oxo-2-enyl]benzene (CD2)
Yield=64%, yellow solid; m.p.=234-235 ⁰C; 1H NMR (300MHz, CDCl3): δ2.43 (s, 6H, 2×CH3), 7.24-
7.31 (m, 6H, CH, aromatic), 7.53-7.58 (d, 2H, =CH, J=15.9Hz), 7.66 (s, 2H, CH, aromatic), 7.75-
7.781 (d, 2H, CH=, J=15.9Hz), 7.94 (m, 4H, CH, aromatic); ESI-MS for C26H22O2 (MeOH):367
[M+H]+, 405[M+K]+; ESI-TOF-HRMS (MeOH) for C26H22O2 calculated, 367.1698 for
[M+H]+;found, 367.1696.
1',4'-Bis-(E/E)-[1-(3",4"-methylenedioxyphenyl)-prop-1-oxo-2-enyl]benzene (CD3)
14

Yield=53%, yellow solid; m.p.=242-245⁰C; 1H NMR (300MHz, CDCl3): δ6.10 (s, 4H, 2×O-CH2-O),
6.92-6.95 (d, 2H, CH, aromatic, J=8.1Hz), 7.53-7.58 (d, 2H, =CH, J=15.6Hz), 7.57 (d, 4H, CH,
aromatic) 7.68-7.70 (d, 4H, CH, aromatic, J=6.9Hz), 7.79-7.84 (d, 2H, CH=, J=15.6Hz); ESI-MS for
C26H18O6 (MeOH): 465[M+K]+; ESI-TOF-HRMS (MeOH) for C26H18O6 calculated, 427.1181 for
[M+H]+; found, 427.1179.
1',4'-Bis-(E/E)-[1-(4"-fluorophenyl)-prop-1-oxo-2-enyl]benzene (CD4)
1
Yield=94%, yellow solid; m.p.=242-245⁰C; H NMR (300MHz, CDCl3): δ7.27-7.33 (m, 6H, CH,
aromatic), 7.60-7.65 (d, 2H, =CH, J=15.6Hz), 7.77 (bs, 4H, CH, aromatic), 7.86-7.92 (d, 2H, CH=,
J=15.6Hz), 8.12-8.15 (m, 2H, CH, aromatic); ESI-MS for C24H16F2O2 (MeOH): Positive mode: 397
[M+Na]+; Negative mode: 373 [M-H]-; ESI-TOF-HRMS (MeOH) for C24H16O2 calculated, 375.1196
for [M+H]+; found, 399.1194.
1',4'-Bis-(E/E)-[1-(4"-bromophenyl)-prop-1-oxo-2-enyl]benzene (CD5)
Yield=63%, yellow solid; m.p.=262-264⁰C; 1H NMR (300MHz, CDCl3): δ7.48--7.53 (d, 2H, =CH,
J=15.6Hz), 7.63-7.68 (m, 6H, CH, aromatic), 7.71-7.82 (m, 4H, CH, 2XCH= & 2xaromatic), 7.94
(m, 4H, CH, aromatic); ESI-MS for C24H16Br2O2 (MeOH): Positive mode: 535[M+K]+; Negative
mode: 495 [M-H]-; ESI-TOF-HRMS (CH3CN) for C24H16Br2O2 calculated, 494.9595 for [M+H]+;
found,494.9549.
1',4'-Bis-(E/E)-[1-(4"-methoxylphenyl)-prop-1-oxo-2-enyl]benzene (CD6)
1
Yield=92%, yellow solid; m.p.=239-242⁰C; H NMR (300MHz, CDCl3): δ3.90 (s, 6H, 2×OCH3),
7.01 (d, 4H, CH, aromatic), 7.51-7.56 (d, 2H, =CH, J=14.7Hz), 7.61 (s, 4H, CH, aromatic), 7.77-7.83
(d, 2H, CH=, J=15.9Hz), 8.10 (d, 4H, CH, aromatic); ESI-MS for C26H22O4 (MeOH): 399 [M+H]+;
ESI-TOF-HRMS (CH3CN) for C26H22O4 calculated, 399.1596 for [M+H]+; found, 399.1594.
1',4'-Bis-(E/E)-[1-(3",4",5"-trimethoxylphenyl)-prop-1-oxo-2-enyl]benzene (CD8)
1
Yield=96%, yellow solid; m.p.=216-218⁰C; H NMR (300MHz, CDCl3): δ3.93 (s, 18H, 6×CH3),
7.27 (s, 4H, CH, aromatic), 7.48-7.53 (d, 2H, =CH, J=15.6Hz), 7.76 (s, 4H, CH, aromatic), 7.76-7.81
(d, 2H, CH=, J=15.6Hz); 13C NMR (75MHz, CDCl3): δ56.8, 61.3, 106.6, 123.1, 129.3, 133.7, 137.3,
143.2, 143.8, 153.6, 189.2; ESI-MS for C30H30O8 (MeOH): 519 [M+H]+, 541[M+Na]+; ESI-TOF-
HRMS (CH3CN) for C30H30O8 calculated, 519.2018 for [M+H]+; found, 519.2016.
Synthesis of CD7 and CD9
To a 7% KOH solution (10 mL) in methanol, 400mg 1,3-diacetyl benzene (2.47 mmol) was added
and stirred at room temperature. To this stirred reaction mixture, benzaldehyde derivative (5 mmol)
was added and further stirred at room temperature for 7-8 h. The work up and purification process to
get the pure compound was same as described earlier.
15

1',3'-Bis-(E/E)-[3-(3",4"-methylenedioxyphenyl)-1-oxo-2-enyl]-benzene (CD7)
Yield=60%, yellow solid; m.p.=205-208⁰C; 1H NMR (300MHz, CDCl3): δ6.16 (s, 4H, 2×O-CH2-O),
6.83 (m, 2H, CH, aromatic), 7.12 (s, 2H, CH, aromatic, J=8.1Hz), 7.43 (d, 2H, CH, aromatic), 7.76-
7.81 (d, 2H, =CH, J=16.5Hz), 7.90-7.95 (d, 2H, CH=, J=15.5Hz), 8.26 (d, 2H, CH, aromatic), 8.46
(d, 2H, CH, aromatic); 13C NMR (75MHz, CDCl3): δ101.5, 102.5, 107.9, 108.1, 108.6, 209.4, 120.4,
127.1, 128.7, 129.9, 130.1, 133.4, 137.4, 139.2, 145.6, 148.9, 150.6, 189.6;
ESI-MS for C26H18O6 (MeOH):449 [M+Na]+, 465[M+K]+; ESI-TOF-HRMS (MeOH) for C26H18O6
calculated, 427.1181 for [M+H]+; found, 427.1175.
1',3'-Bis-(E/E)-[3-(4"-N,N-dimethylphenyl)-1-oxo-2-enyl]-benzene (CD9)
Yield=71%, orange solid; m.p.=149-152⁰C; 1H NMR (300MHz, CDCl3): δ3.04 (s, 12H, 4×N-CH3),
6.68 (d, 4H, CH, aromatic, J=8.7Hz), 7.35-7.40 (d, 2H, =CH, J=15.6Hz), 7.56-7.61 (d, 5H, CH,
aromatic), 7.80--7.85 (d, 2H, CH=, J=15.6Hz), 8.15-8.18 (d, 2H, CH, aromatic), 8.60 (s, 1H, CH,
aromatic); 13C NMR (75MHz, CDCl3): δ40.4, 111.1, 112.2, 113.3, 116.8, 122.88, 128.3, 129.1, 131.1,
132.1, 139.7, 146.9, 152.6, 190.3; ESI-MS for C28H28N2O2 (MeOH):425 [M+H]+, 447[M+Na]+,
463[M+K]+; ESI-TOF-HRMS (MeOH) for C28H28N2O2 calculated, 425.2229 for [M+H]+; found,
425.2127.
4.3. Bacterial strains
Clinical isolates of P. aeruginosa coded as U-1926, PS-1, R-574, R-581, R-583, 1922, 1925, P-1923,
P-1816 and KG-P2 were procured from Microbiology Department, King George Medical University
Lucknow, India. These isolates were profiled for antibiotic susceptibility test and were resistant to
ampicillin, nalidixic acid, ofloxacin, chloramphenicol etc. For bacterial culture, standard Mueller
Hinton Agar (MHA ) and broth (Hi-Media, Mumbai, India) were the standard media used for
screening.
4.4. Antibacterial potential of antibiotics and curcumin mimics
MIC was determined by a 2-fold serial microbroth dilution method in 96-well microtiter plates as per
Clinical and Laboratory Standards Institute guidelines [66]. The concentrations of test compounds
were reduced upto appropriate dilutions (1600 to 12.5 mg/L) and were assayed against P. aeruginosa
isolates. Preliminary inoculum (5× 105 cfu/mL) was used and the plate was incubated at 37°C for 24
h, and results were noted as per CLSI guidelines.
4.5. Metallo β-Lactamase production/inhibition test
16

Isolates were phenotypically characterized for MBL production (Imipenem with or without EDTA)
using Ezy MIC™ Strips (Himedia laboratories, Mumbai, India). Briefly, lawn of overnight grown
culture of clinical isolates of P. aeruginosa (turbidity of 0.5 McFarland) was prepared and Ezy MIC™
strips were gently placed on the centre. Results were observed and described in terms of ratio of
values of Imipenem + EDTA (IPM+EDTA). A ratio of >8 was considered to be positive else negative.
MBL inhibitory potential of Van D was also evaluated with imipenem.
4.6. In vitro combination studies
Broth checkerboard assay was conducted for this study [67]. 150 µL of pH adjusted Mueller-Hinton
broth was pipetted and the dilution was adjusted such that each well had different concentrations of
antibiotics and curcumin mimics. The concentration of tetracycline was adjusted in between 12.5 -
1600 mg/L and for PAβN whereas 3.25 to 200 mg/L for Van D. Results were documented in different
terms of type of interaction, fold reduction and fractional inhibitory concentration index (FICI). The
FICI index was measured in terms of synergism (<0.5), no interaction (0.5-4.0) and antagonism(>4.0)
[68].
4.7. Time kill studies
Time kill kinetics was done using as previously described method [69,70]. It performed tetracycline
alone and in combination with Van D at MIC, 2xMIC and 4xMIC concentrations. Replicative studies
were done along with appropriate control and the results were plotted for log10 cfu/mL v/s time (in
hours). Similar design was also adopted for combinations of antibiotics and test compounds at the
reduced concentrations.
4.8. Postantibiotic effect
Curcumin mimics Van D along with curcumin were also evaluated for post antibiotic effect by using
the earlier described method by Gudmundsson et al [70,71]. 0.5 mL of isolate KGP-2 (0.5 McFarland
standard) was added to 5 mL fresh broth containing MIC (1600 mg/L), 0.5× MIC (800 mg/L), and
0.25× MIC (400 mg/L) concentrations of tetracycline alone and in combination with Van D (12.5
mg/L). A dilution ratio of 1:1000 was achieved for each tube and incubated at 37°C with shaking for
2 h. Sampling was done from every tube for cell viability assessment at regular intervals. The
postantibiotic effect (PAE) measured as the difference in the time for growth in the exposed culture
(T), and the corresponding unexposed control (C) was measured as per formula PAE=T–C.
4.9. Mutant prevention concentration (MPC)
17

The MPC of tetracycline was determined using P. aeruginosa MTCC 741 as per protocol of Heisig
and Tschorny [72]. TET alone or in the presence of Van D was used to assay the MPC at different
concentrations (2× MIC, 4× MIC, 8× MIC, and 16× MIC). Results were obtained by dividing the
total number of colonies after 48 h of incubation at 37 °C on the antibiotic-containing plate by the
total number of colony forming units plated.
4.10.
Ethidium bromide efflux studies
The efflux of ethidium bromide (EtBr) was measured by flurometric method [73]. Log phase cells of
KGP2 were grown up in 10 mL MHB (pH 7.3±0.2) with 0.6 at 600 nm optical density (OD). The
cells were harvested by centrifuging at 16,060×g for 3 min. The harvested cells were then washed
with phosphate buffer saline. Now suspended cells were treated with 50 mg/L of ethidium bromide
and incubated for 60 min at 25°C in the absence/presence of 12.5 mg/L of Van D. The EtBr treated
bacterial suspension was again settled down by centrifuging at 16,060 × g for 3 min, the pellet was
resuspended in cold 1xPBS. Aliquots of the suspended cells were distributed in 96 well plates. Loss
of fluorescence was recorded for 30 min at a regular interval of 1 min at the excitation and emission
wavelength of 530 nm and 585 nm respectively using spectrofluorometer (FLUOstarOomega-BMG
Labtech, Ortenberg, Germany).
4.11.
Biofilm inhibition studies
Biofilm inhibition assay was done as per standard protocol which include biofilm formation and
impact of inhibitors on the biofilm synthesis [74,75]. The 24 h culture was diluted upto 1/100. In 96
well plate the diluted bacterial culture was added except negative control (Only put 100 µL broth).
Different inhibitor (Van D alone and combination with antibiotic) was added at subinhibitory
concentration. After adding the inhibitors, the plate was incubated for 48 h. The plate was washed
by simply inverting the plate. After the plate was washed by 0.7% saline (NaCl) water twice and
washed it by inverting the plate. Then the plate was incubated for 3 h. Crystal violet was added and
incubated it for 20 mins. After that the plate was washed by normal saline twice and acetic acid was
added. Next, it was transferred into a new 96 well plate and scanned it under ELISA reader.
4.12.
qRT-PCR analysis
Transcriptional profiling of efflux pump genes present in KGP2 isolate of P. aeruginosa, belonging
to resistance nodulation division family (mexA, mexB and oprM) was dones in presence of Van D.
Briefly, cells were harvested at mid-log phase in presence of sub-inhibitory concentrations of TET
and Van D (alone and in combination). SYBR GreenER qPCR super-mix (Invitrogen, Carlsbad, CA,
USA) was used for quantification of the template using 7900HT fast real-time PCR system
18

(Invitrogen, Carlsbad, CA, USA). Results were presented and recorded in RQ after normalization of
D-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) an indigenous control.
4.13. In-vivo study
As a standard protocol approved by the Institutional Animal Ethics Committee (IAEC), in-vivo
study was done following the Government of India’s Committee for the Purpose of Control and
Supervision of Experimental Animals (CPCSEA).
4.13. 1. LPS-induced endotoxemia in mice
Protocol and guidelines of the experiment were approved by Institutional Animal Ethical Committee
(IAEC) vide ref. No. CIMAP/IAEC2016-19/05. Six to eight weeks of age BALB/c mice (male) each
of 18–22 gm of weight were obtained from Institutional animal house (CSIR-CIMAP, Lucknow).
Mice were acclimatized for a week before the initiation of actual experiment to the animal room.
Animals were given standard mice feed and ad libitum drinking water under standard room conditions
of 25 ± 3°C and alternate dark-to-light cycle of 12 h each. After acclimatization, the mice were
separated randomly into 6 experimental groups of 6 mice (n=6) each. LPS (5ꢀmg/kg) was injected
intraperitoneally in mice to induce endotoxemia. A uniform suspension of test compound Van D was
prepared in 0.7% CMC (carboxymethyl cellulose) and orally administered 2 h before LPS injection.
Group 1: Normal Control administered CMC
Group 2: Vehicle Control administered CMC + LPS
Group 3: Van D (3 mg/kg) + LPS
Group 4: Van D (10 mg/kg) + LPS
Group 5: Van D (30 mg/kg) + LPS
Blood was collected 2 h after LPS administration from the orbital plexus. Serum was separated and
stored at -80°C until analysis. Pro-inflammatory Cytokines (IL-1β, IL-6 and TNF-α) were determined
in serum using commercially available mouse specific enzyme immunoassay kits (BD Biosciences,
USA) by following the manufactures protocol.
4.13.2. Determination of Pro-inflammatory Cytokines by enzyme-linked immunosorbent assay
Enzyme Immunoassay (EIA) Kits (BD Biosciences, USA) were used to measure concentrations of
IL-1β, IL-6 and TNF-α in mice serum as per manufacturer’s protocol. The values of IL-1β, IL-6 and
TNF-α were expressed as pg/mL. %Inhibition of pro-inflammatory cytokine production was
calculated as follows:
19

% Inhibition =100 x (concentration of vehicle control- concentration of test treatment)/ O.D. of
concentration of vehicle control.
Where vehicle control indicates cells treated with vehicles in LPS-induced inflammation.
4. 13.3. Statistical analysis
Results are presented as the means ± SE. Differences with a p value <0.05 were considered significant.
4.14. In-silico study
Molecular docking studies were carried out using Autodock v 4.2.6 to understand the ligand and
receptor interaction. 3D structures of mexA, mexB, and mexC were retrieved from Protein Databank.
The ligands except VanD were retrieved from the PubChem database. The structures (both ligand and
protein) were refined by adding polar hydrogens and applying the appropriate Gasteiger charge.
The center search space was 0.375 Å and grid dimensions were maintained at x: -11.568; y: 9.824;
z:-29.989. Following docking, visualization of interacting amino acid residues with the ligand was
done using Discovery Studio v 19.1
4.15 Safety studies of Van D
Acute oral toxicity studies were carried out in Swiss albino mice for effectiveness of Van D.
Protocols and guidelines were designed by taking into consideration in accordance with those set by
Organization for Economic Co-operation and Development (OECD) test guideline No 423 (1987)
and after approval of protocols by Institutional Animal Ethical Committee (IAEC) vide ref. No.
CIMAP/IAEC/2016-19/01, dated 09/2/2017.
Briefly, five groups each comprising 3 male and 3 female mice (n=6) weighing between 20-25 g were
taken and divided as one control and four treatment groups. Mice were acclimatized in the
experimental environment for 7 days prior to the actual experimentation. The animals were
maintained at 22±50C with humidity control and also on an automatic dark and light cycle of 12 h
each. The animals were given standard mice feed and ad libitum drinking water. Mice of Group-I
were kept as control group and animals of Groups-II, III, IV and V were kept as treatment groups.
The test compound Van D was solubilized in minimum quantity of DMSO and then re-hydrated
in 0.7% CMC (carboxymethylcellulose) in double distilled water and given to animals of Groups II,
III, IV and V once orally at four different doses i.e. 5, 50, 300 and 1000 mg/kg body weight
respectively. Control animals were given the same quantity of the vehicle. The animals were observed
for any sign of ill health and mortality at an hourly interval on the day of administration of test
compound Van D. Thereafter, a daily clinical examination was carried out including changes in skin,
eyes, mucous membrane, occurrence of secretion and excretion and also responses like respiratory
patterns, lachrymation, pilo-erection, changes in gait, posture and response to handling were recorded
20

([76]. Further, on 7th day of the experiment body weights were recorded and blood and serum samples
were collected from all the animals and analysed for hematological parameters (total WBC, RBC,
differential leucocytes count, haemoglobin percentage) and liver function test (biochemical
parameters like ALP, SGPT, SGOT, bilirubin, serum protein and tissue protein activity, lipid profile
(triglycerides, total cholesterol), and also kidney function test (creatinine level). The animals were
then sacrificed and were necropsied for any gross pathological changes. Weights of vital organs like
heart, liver, kidney etc. were also recorded [77].
Abbreviations
TET, Tetracycline; PAE, Post-Antibiotic Effect; MPC, Mutant Prevention Concentration; MDR,
Multidrug resistance; GNB, Gram Negative Bacteria; MBL, Metallo-β-lactamase; MIC, Minimum
Inhibitory Concentration; PAβN, Phenylalanine-arginine-β-naphthylamide; FICI, Fractional
Inhibitory Concentration Index; MTCC, Microbial Type of Culture Collection; EDTA,
Ethylenediamine Tetra Acetic Acid; RND, Root–Nodulation–cell Division; CUR, Curcumin; TNF-
α, Tumour Necrosis Factor-alpha; MexA/MexB and OprM, Types of Efflux pump Proteins; LPS,
Lipopolysaccharide; TLR-4, Toll-Like Receptor-4; AMP, Ampicillin; OFL, Ofloxacin; CDCl3,
Deuterated chloroform; ESI-TOF-HRMS, Electrospray Ionisation-Time of flight-High Resolution
Mass Spectrometry; MHA, Mueller Hinton Agar; qRT-PCR, Quantitative Real Time Polymersase
Chain Reaction; GAPDH, D-Glyceraldehyde-3-Phosphate-Dehydrogenase; IAEC, Institutional
Animal Ethics Committee; CPCSEA, Committee for the Purpose of Control and Supervision of
Experimental Animals; OECD, Organization for Economic Co-operation and Development; SGPT,
Serum Glutamic Pyruvic Transaminase; SGOT, Serum Glutamic-Oxaloacetic Transaminase.
Declarations of Competing Interest
The authors declare to have no competing interest.
Acknowledgements
This work was financed by ‘Science and Engineering Research Board (SERB) with project file NO.
SB/YS/LS-77/2014. Thanks are also due to Prof. DP singh of BBAU and Director of CSIR-CIMAP
for providing facilities for this study. Miss Sadiya Khwaja acknowledges to Council of Scientific and
Industrial Research for her Senior Research Fellowship.
21

Appendix A. Supplementary material
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