Molecular docking studies, in vitro-brine shrimp lethal assay and antibacterial assessment of embelin, vilangin and phenyl vilangin against endodontic pathogen, Enterococcus faecalis

Article abstract of DOI:10.1007/s11164-021-04559-8

Complete debridement and disinfection of the root canal system are the primary requirement for successful endodontic treatment. Enterococcus faecalis is the most commonly found organism in a high percentage of root canal failures as a single organism or as a major component of the flora. Primary goal of this study is to assess the antibacterial effect of phytochemical agents namely embelin, vilangin and phenyl vilangin, respectively, against E. faecalis. And this is the first time that these materials are reported for antibacterial activity against the pathogen, E. faecalis. Our second goal is to predict the interaction between the active structure and the protein in the binding mode, and the third goal is to screen for cytotoxicity by brine shrimp lethality bioassay. All the compounds showed low to elevated level of antibacterial activity. Particularly, compound embelin, as well as its combination with NaOCl in the ratio 1:1, exhibits zone of inhibition value of 29 ± 0.89 and 32 ± 0.98?mm at a concentration of 80?μL and the MIC value of embelin was found to be 78.10?μg/mL. All the compounds exhibit low toxicity to high toxicity. The LC₅₀ value of tested compounds was in the range of 17.5–80.0?μL with respect to positive control (vincristine sulphate). The result of lethality reveals the phenyl vilangin exhibits very minimal toxicity even under higher concentration, and in future, it may be investigated further for biocompatibility studies. The results of molecular docking reveal that embelin showed good interaction against 2JER protein with a higher binding energy value of ? 7.2. Hence, these finding reveals that embelin and phenyl vilangin may be used as root canal irrigants in future. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-021-04559-8

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-021-04559-8
Molecular docking studies, in vitro‑brine shrimp lethal
assay and antibacterial assessment of embelin, vilangin
and phenyl vilangin against endodontic pathogen,
Enterococcus faecalis
S. Jayalakshmi1 · K. Narendran2 · E. Sukumar3 · J. Irshad Ahamed4 ·
M. S. Nivedhitha5 · S. Rajesh Kumar6
Received: 22 March 2021 / Accepted: 28 July 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021
Abstract
Complete debridement and disinfection of the root canal system are the primary
requirement for successful endodontic treatment. Enterococcus faecalis is the most
commonly found organism in a high percentage of root canal failures as a single
organism or as a major component of the fora. Primary goal of this study is to
assess the antibacterial efect of phytochemical agents namely embelin, vilangin and
phenyl vilangin, respectively, against E. faecalis. And this is the frst time that these
materials are reported for antibacterial activity against the pathogen, E. faecalis. Our
second goal is to predict the interaction between the active structure and the protein
in the binding mode, and the third goal is to screen for cytotoxicity by brine shrimp
lethality bioassay. All the compounds showed low to elevated level of antibacterial
activity. Particularly, compound embelin, as well as its combination with NaOCl in
the ratio 1:1, exhibits zone of inhibition value of 29 0.89 and 32 0.98 mm at a
concentration of 80 µL and the MIC value of embelin was found to be 78.10 µg/mL.
All the compounds exhibit low toxicity to high toxicity. The LC50 value of tested
*
S. Jayalakshmi
jayalakshmisomasundaram@saveetha.com
1
White Lab, Material Research Centre, Saveetha Dental College, Saveetha Institute of Medical
and Technical Sciences (Deemed University), Chennai 600 077, India
2
3
4
Department of Chemistry, Saveetha Engineering College, Thandalam, Chennai 602 105, India
Research and Development, VPro Biotech, Puducherry, Tamilnadu 605 110, India
Department of Chemistry, LIFE (Loyola Institute of Frontier Energy), Loyola College,
University of Madras, Nungambakkam, Chennai, Tamil Nadu 600034, India
5
6
Department of Conservative Dentistry and Endodontics, Saveetha Dental College and Hospitals,
Saveetha Institute of Medical and Technical Sciences (Deemed University), Chennai 600 077,
India
Department of Pharmacology, Saveetha Dental College and Hospitals, Saveetha Institute
of Medical and Technical Sciences (Deemed University), Chennai 600 077, India
Vol.:(0123456789)
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S. Jayalakshmi et al.
compounds was in the range of 17.5–80.0 µL with respect to positive control (vin-
cristine sulphate). The result of lethality reveals the phenyl vilangin exhibits very
minimal toxicity even under higher concentration, and in future, it may be investi-
gated further for biocompatibility studies. The results of molecular docking reveal
that embelin showed good interaction against 2JER protein with a higher binding
energy value of−7.2. Hence, these fnding reveals that embelin and phenyl vilangin
may be used as root canal irrigants in future.
Graphic abstract
Keywords Embelin · Vilangin · phenyl vilangin · Lethal assay · Cytotoxic · Docking
and disc difusion
Introduction
Enterococci are gram-positive pathogen, possessing the ability to grow in the pres-
ence or absence of oxygen. E. faecalis is found in 4–40% of primary endodontic
infections. In fact, failed root canal treatment cases are nine times more likely to
contain E. faecalis than primary endodontic infections. Enterococcus faecalis
is a microorganism commonly detected in asymptomatic, persistent endodontic
infections. Its prevalence in such infections ranges from 24 to 77%. Unlike pri-
mary endodontic infections, which are polymicrobial in nature and dominated by
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Molecular docking studies, in vitro-brine shrimp lethal assay…
gram-negative anaerobic rods, the microorganisms involved in secondary infections
are composed of one or a few bacterial species. Among that, E. faecalis is the major
and most commonly found organism in a high percentage of root canal failures and
it is able to survive in the root canal as a single organism or as a major component of
the fora [1]. Enterococci survive very harsh environments including extreme alka-
line pH (9.6) and salt concentrations.
Sodium hypochlorite is an efective irrigant for all presentations of E. faecalis
including its existence as a bioflm [2]. The major drawback of sodium hypochlo-
rite is that at high concentration of chloride ions, it possesses excellent antibacterial
efect but at the same time, more cytotoxic towards normal tissues [3]. In order to
resolve this issue, we are in constant search of an alternative irrigant over hypochlo-
rite. By considering the biological structure and protein structure of E. faecalis, we
preferred to try embelin, vilangin and phenyl vilangin as alternate phytochemical
irrigants for endodontic treatment.
The aim of the present investigation is to assess the antibacterial activity of embe-
lin, vilangin and phenyl vilangin against E. faecalis by disc difusion method, and
also, the MIC value was assessed for antibacterial investigation [4, 5]. Brine shrimp
lethality assay was performed to study about the cytotoxicity of phytochemical irri-
gants based on the mortality percentage of nauplii and LC50 value. Finally, molecu-
lar docking method which is a recent technique to get insight about the exact bind-
ing location of the ligand and protein was also done. Embelin, vilangin, and phenyl
vilangin ligands are selected to dock at the active areas of 2JER of E. faecalis pro-
tein, and the binding energy value was calculated based on the docking efciency.
Materials and methods
Chemicals and reagents
Embelin, formaldehyde, benzaldehyde, sodium hypochlorite, DMSO and Vincris-
tine sulphate were sourced from Sigma Aldrich. Enterococcus Faecalis, Mueller-
Hinton agar (MHA) and Sabouraud Dextrose agar were sourced from Sisco research
laboratory chemicals, Mumbai, The optimized ligand molecules were docked into
refned Enterococcus Faecalis protein (PDB ID: 2JER) protein using “LigandFit” in
the AutoDock 4.2.6
Synthesis of vilangin and phenyl vilangin
Compound embelin was isolated from the berries of Embelia ribes Burm.f. (Fam-
ily: Primulaceae). Vilangin and phenyl vilangin were synthesised by simple conden-
sation of embelin with formaldehyde and benzaldehyde in acetic acid medium as
reported by Bheema sankara Rao, and the synthetic scheme is shown in Fig. 1 [6].
The complete synthetic procedure and characterisation of phenyl vilangin involving
Mass and NMR were reported in our previous work [7]
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S. Jayalakshmi et al.
Fig. 1 Synthesis of vilangin (2) and phenyl vilangin (3) from embelin (1) by reacting with formaldehyde
(a) and benzaldehyde (b)
Antimicrobial activity
Disc difusion method
The antimicrobial activity of embelin and its derivatives were investigated
through agar disc difusion method. Mueller-Hinton agar (MHA) medium and
Sabouraud Dextrose agar (SDA) medium are employed to study the antibacte-
rial activity of samples against E. faecalis. Nutrient agar slant loaded with stock
cultures was maintained at 4 °C. Active cultures for the study were prepared by
transferring a loop full of culture from the stock cultures into the test tubes con-
taining nutrient broth that were incubated at 37 °C for 24 h. Sabouraud Dextrose
agar and Mueller-Hinton agar media were poured and uniformly dispersed into
the petri plates. Once the medium solidifed, the inoculums were spread on the
solid plates with sterile swab moistened with the bacterial suspension. 5 mm
diameter of sterile discs was charged with various concentration (5 µL, 10 µL,
20 µL, 40 µL and 80 µL) of DMSO solution of sample under investigation and
placed on the agar plates. The plates were incubated for 24 h, at 37 °C. All deter-
minations were studied in triplicates. The diameters of zone of inhibition were
correlated with standard hypochlorite. Determination of zone of inhibition by disc
difusion method was performed in two categories as shown in Table 1 (Determi-
nation of zone of inhibition of sample against standard hypochlorite) and Table 2
(Determination of zone of inhibition of sample having 1:1 ratio of hypochlorite
and embelin derivatives.
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Molecular docking studies, in vitro-brine shrimp lethal assay…
Table 1 Zone of inhibition (mm) of samples and standard hypochlorite against E. Faecalis pathogen
Sample
Zone of inhibition (mm) at various concentration
5 µL
10 µL
20 µL
40 µL
80 µL
Embelin
Vilangin
Phenyl vilangin
Sodium hypochlorite
10 1.01
9 0.95
10 0.82
20 1.1
14 0.65
12 0.98
13 1.01
25 0.82
20 0.55
14 0.50
15 0.85
29 46
23 0.65
16 0.87
19 0.54
32 0.78
29 0.89
17 0.75
23 0.88
40 0.65
Table 2 Zone of inhibition (mm) of sample (Compound:HOCl, 1:1 ratio) and minimum inhibitory con-
centration of compound and standard hypochlorite against E. Faecalis pathogen
Sample
Zone of inhibition (mm) at various concentration
MICa (µg/mL)
5 µL
10 µL
20 µL
40 µL
80 µL
Embelin:HOCl
Vilangin:HOCl
13 0.5
21 0.97 24 0.89 29 0.23 32 0.98
13 0.78 15 0.56 18 0.65 19 0.56 312.39
78.10
9
0.87
Phenyl vilangin:HOCl 12 0.23 15 0.97 17 0.21 21 0.98 26 0.75 156.20
Sodium hypochlorite 39.05
–
–
–
–
–
aMinimum Inhibitory Concentration values are highlighted for embelin, vilangin and phenyl vilangin and
sodium hypochlorite
Minimal inhibitory concentration (MIC)
Serial dilution of twofold was preferred to attain the concentration range of
1250–9.762 µg/mL. All the compounds including standard hypochlorite of difer-
ent concentration range as mentioned above were prepared in sterile microwells to
determine minimum inhibitory concentration. According to Sarker et al. [8], mini-
mal inhibitory concentration was determined in nutrient broth accompanied with
resazurin dye. All the samples and standard hypochlorite were dissolved in 1%
DMSO and added to nutrient broth to a maximum dose of 1250 µg/mL. The micro-
titre plates were enclosed and the substances were agitated by plate shaker at the
incubation temperature of 38 °C for 24 h. The microbial growth was observed by
colour change in the wells.
Brine shrimp lethality assay
Hatching of the brine shrimp
In the present investigation, we employed brine shrimp lethality to determine the
cytotoxicity of embelin, vilangin and phenyl vilangin [9, 10]. The brine shrimp eggs
were procured from Philadelphia (USA). A rectangular jar containing 3 L of water
was mixed with 28g ms of NaCl, and the jar was provided with proper aeration
using air pump. About 15 gms of brine shrimp was sprinkled into the jar and the
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S. Jayalakshmi et al.
compartment was illuminated by using 60–100 Watt bulb, placed few distance away
from the jar. After incubation for 24 h, the hatched nauplii were separated from the
empty egg and utilised for the experiment.
Bioassay
The suspension of brine shrimp nauplii (10 nos) was charged into test tube contain-
ing diferent concentrations (5 µL, 10 µL, 20 µL, 40 µL, 80 µL) of embelin, vilangin,
phenyl vilangin and Vincristine sulphate (Control). And the tubes were left at room
temperature for 24 h. After 24 h, the number of non-motile nauplii in each tube was
counted. Again after 48 h, the number of survivors was observed and the mortality
was recorded. The mortality endpoint of this bioassay is absence of controlled for-
ward motion of nauplii for 30 s of observation. The mortality percentage of nauplii
in each test tube was calculated using the formula given as follows:
[
]
% Mortality = Number of dead nauplii∕Total number of nauplii under investigation ∗ 100
Molecular docking
Protein preparation for docking
The 3D structure of Agmatine Catabolism of Enterococcus Faecalis (PDB ID:
2JER) was downloaded from Protein Data Bank (PDB), and before initiating the
docking simulations, all non-protein molecules were removed from 2JER; for any
alternative atom locations, only the frst location was retained. All the docking cal-
culations were performed by using AutoDock 4.2.6. E. faecalis protein was modifed
by adding polar hydrogens and then kept rigid in the docking process, whereas all
the torsional bonds of ligands were set free by Ligand module in AutoDock Tools.
Ligand preparation for docking
The phenyl vilangin ligand was built using Chemsketch and optimized using “Pre-
pare Ligands” in the AutoDock 4.2.6 for docking studies. The optimized ligand mol-
ecules were docked into refned E. faecalis protein (PDB ID: 2JER) using “Ligand
Fit” in the AutoDock 4.2.6.
Computational details
All theoretical computations (quantum chemical calculations) were performed
in Becke3-Lee-Yang-parr (B3LYP) with 6–311 + + G (d,p) functional by Gauss-
ian 09 W program package on a computing system [11]. The electronic prop-
erties like HOMO and LUMO energies were confrmed using the DFT method.
For achieving chemical reactivity of the molecule, the molecular electrostatic
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Molecular docking studies, in vitro-brine shrimp lethal assay…
potential (MEP) surface and the frontier molecular orbitals (FMOs) were visu-
alized using the Gauss View 05 software. In order to understand the electronic
properties, the theoretical UV–Visible spectra have been studied using TD-DFT
method with 6e311 + + G (d,p) basis set for the gas phase.
Results and discussion
Chemistry
The chemistry of these compounds (1–3) has undergone a surge of interest
because of the active pharmacore unit, 2,5-dihydroxy-[1, 4]-benzoquinone [12,
13] and the hydrophobic nature of alkyl chain from embelin, vilangin and ben-
zene from phenyl vilangin. In our present investigation, carbon labelled C5 and
C6 of benzoquinone derivative plays a vital role in reactivity (condensation) with
formaldehyde and benzaldehyde because of the presence of hydroxyl unit (–OH)
at C5 and active methylene unit at C6. The resonance stabilised dianion structure
of embelin and phenyl vilangin shown in Fig. 2 exhibits more electron density
at C6 and C3 position, which is also refected from the molecular electrostatic
potential diagram of phenyl vilangin. From the colour scheme of MEPs, red rep-
resents electron rich, that is the region where the hydroxyl hydrogen involve in
resonance with quinonoid carbonyl oxygen and the partially positive charge rep-
resented by blue colour and that is due to the + I efect of alkyl chain at C3 posi-
tion. The reactivity of embelin with formaldehyde and benzaldehyde was also
refected from the yield of the vilangin and phenyl vilangin. The reactivity of
anion at C6 of embelin towards the carbonyl carbon of formaldehyde and benza-
ldehyde difers due to steric reason, because benzaldehyde is more sterically hin-
dered by benzene ring than formaldehyde. Also from the MEPs, the green colour
cloud is a neutral region, which comprises of hydrophobic alkyl as well phenyl
unit. Hence, this green region only is responsible for the efcient interaction of
bacterial cell wall, since it is made up of hydrophobic lipoproteins.
Fig. 2 Structure of embelin (1), phenyl vilangin (3) and its resonance stabilized dianion (1a and 1b)
1 3

S. Jayalakshmi et al.
Investigation of antibacterial activity
In the present study, benzoquinone derivatives were tested for their antibacterial
activity against E. Faecalis bacteria, (gram-positive). The antibacterial activities
of benzoquinone derivatives are being increasingly documented. Depending upon
the nature of bacterial structure, some chemical structural modifcation is required
to enhance the antibacterial activity. In the present work, the benzoquinone deriva-
tives viz., embelin, vilangin and phenyl vilangin and the standard hypochlorite were
tested for their antibacterial activity against E. Faecalis pathogen. The diameter of
zone of inhibition and the MIC values are highlighted in Tables 1 and 2. It is explicit
from the result that embelin and its combination with NaOCl (1:1) exhibit very good
antibacterial activity. Particularly 1:1 ratio of embelin and NaOCl exhibits zone of
inhibition value of 32 0.98 mm at a concentration of 80 µL. The trend of zone of
inhibition is found to be embelin>phenyl vilangin>vilangin. In addition to antibac-
terial assay minimum inhibitory concentration against E. faecalis was also investi-
gated at diferent concentrations. From Table 2, it is clear that E. Faecalis is sensi-
tive towards embelin next to standard NaOCl at a concentration of 78.10 µg/mL.
Again the trend of MIC is similar to zone of inhibition. According to Overtone’s
concept of cell permeability, the lipid membrane surrounded by the cell favours the
entry of only lipid soluble materials due to which liposolubility is an vital criterion
controlling the antibacterial activity.
The hydrophobic/hydrophilic balance of chemical structure is usually regarded
as the key factor to achieve pronounced activity against microbes and low toxicity
to mammalian cells. In general, the gram-positive bacterial cell walls are usually
thicker than gram-negative bacteria and the wall is usually made up of peptidogly-
can layers and lipopolysaccharide. In our present study, the hydroxyl group in all
the compounds exhibits hydrophilic interactions as electrostatic and hydrogen bond-
ing with that of peptidoglycan layers and the same hydrogen bonding interaction of
embelin with that of Enterococcus Faecalis protein is also refected on docking stud-
ies shown in Figs. 5, 6, 7. On the other view, the undecyl unit (C11H23) at the third
carbon of embelin is hydrophobic and is responsible for the adsorption and penetra-
tion of lipopolysaccharides. These two types of interaction can signifcantly impact
the antimicrobial activity of surfaces. Based on the above discussion, the trend of
hydrophobic character is phenyl vilangin>vilangin>embelin. Thus, the presence of
low hydrophobic and the presence of carbonyl carbon and hydroxyl group have a
signifcant infuence on antibacterial activity.
Chemistry in antimicrobial activity
E. faecalis is the major bacteria which is responsible for endodontic infections
and commonly found in high contents in root canal failure. Enterococcus and few
Streptococcus species are the limited gram-positive bacteria shows signifcant level
of glutathione (GSH). One of the signifcant functions of glutathione is to protect
the cells against toxic substances by regulation of intracellular level of K+, whose
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Molecular docking studies, in vitro-brine shrimp lethal assay…
transport and accumulation plays a vital role in maintenance of cell turgor and
homeostasis of intracellular pH. Presence of glutathione in bacterial cell was found
to intensify their resistance towards selective antibiotics. A probe through the litera-
ture reveals [14–18] that exposure of bacterial cell towards foreign bodies leads to
decrease in the level of thiol content, thus the potential of bacterial cells to maintain
a high level of glutathione largely determines their resistance to toxic substances. A
survey through the literature reveals Streptococcus agalactiae, Streptococcus ther-
mophilus, and Enterococcus faecalis grown in Todd-Hewitt medium must have the
ability to synthesis GSH. The synthesis of GSH by these bacteria is the frst reported
case of GSH production by prokaryotes. Hence, it is more signifcant to focus on
the mechanism behind chemical interaction with bacterial cell. In our present anti-
microbial investigation, the trend in antimicrobial activity follows the order embe-
lin>phenyl vilangin>vilangin. The possible mechanism of embelin with bacterial
cell is shown in Fig. 3. E. faecalis being a gram-positive bacterium is usually made
up of thicker cell wall than gram-negative bacteria, and the chemical constituent of
cell wall consists of usually peptidoglycan layers and lipopolysaccharide. Adsorp-
tion and penetration are the two important factors which decide the frst step of
interaction between embelin and bacterial cell. The presence of hydrophobic unde-
cyl unit (C11H23) at third carbon of embelin may be responsible for the adsorption
and penetration of hydrophobic lipopolysaccharides. Complete exploration through
the literature reveals, α, β-unsaturated carbonyl compounds viz., conjugated diones
enable Michael addition of endogenous peptide glutathione to the olefnic bond. In
Fig. 3 Plausible mechanism of embelin with bacterial cell through Michael addition of endogenous pep-
tide glutathione to the olefnic bond
1 3

S. Jayalakshmi et al.
our present investigation, there exists same Michael addition at carbon C6 of embe-
lin with nucleophilic thiol unit of glutathione that leads to the formation of oxo
anion at C4 carbon as shown in Figs. 3 and 4. The next step involves intra-molecular
rearrangement with simultaneous elimination of hydroxyl group in the form of H2O
at C5 carbon. The possible outcome of this mechanism is found to be in good agree-
ment with the work reported by Hubert et al. [19]. The net result from the mech-
anism indicates the loss of cells natural identity by decrease in the concentration
of glutathione content. Thus, the efective reaction of embelin with glutathione is
responsible for the antimicrobial activity. In general, most of the antimicrobial stud-
ies are concluded based on the zone of inhibition, culture and colony count. When
we go for imaging tools, confocal microscopy can be used to study the volume of
live and dead bacteria. Even though all these tools are valuable, they are technique
sensitive, are prone for contamination during processing leading to altered results.
But all these studies usually do not discuss the chemistry or mechanism behind the
antimicrobial activity. This makes our study very unique where we have discussed in
detail the mechanism behind the antimicrobial activity of embelin concluding that it
could be a better alternative as an irrigant in the disciplines of endodontic.
Brine shrimp cytotoxicity studies
The brine shrimp lethality assay represents a rapid, inexpensive and simple it also
proved to be a convenient method for monitoring biological activities of synthe-
sised compound under investigation. The present study shows that hatched nauplii
are unique for the preliminary cytotoxicity study. The mortality percentage and
LC50 values of the brine shrimp lethality bioassay obtained for the synthesised com-
pound extracts and that of the positive control, Vincristine sulphate, are presented in
Fig. 4 Suggested pathway for the nucleophilic substitution at embelin
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Molecular docking studies, in vitro-brine shrimp lethal assay…
1 3

S. Jayalakshmi et al.
Table 3, respectively. The present study determined that the extent of lethality was
directly proportional to the concentration of the sample under study.
There was no efect of lethality on any of the concentrations of the extract for
24 h. The brine shrimp were still actively moving in the test materials. All the syn-
thesised compounds were non-toxic towards brine shrimp for 24 h of exposure but
there is increase in toxicity at 48 h. Of the three synthesised benzoquinone deriva-
tives (1–3), the mortality rate of embelin and vilangin after 48 h was increasing with
increase in concentration from 5 to 80 µL with LC50 value of 17.5 and 40 µL. In
particular, phenyl vilangin does not exhibit any mortality up to 4 µL and exhibited
50% mortality at concentration of 80 µL and it exhibited LC50 value under the same
concentration. Overall, the maximum mortality took place at a concentration range
of 40–80 µL. The result of lethality reveals that phenyl vilangin exhibits very mini-
mal toxicity even under higher concentration, and in future, it may be investigated
further for biocompatibility studies. The overall trend in lethality follows the order
1>2>3. This may be attributed to the increase in hydrophobicity from embelin to
phenyl vilangin. The present fndings suggest that these compounds when used as
root canal irrigants will be more compatible with the harmony of periapical tissues.
Molecular docking studies
Molecular docking studies were performed on embelin, vilangin and phenyl vilangin
with Agmatine Catabolism of Enterococcus Faecalis (PDB ID: 2JER) target pro-
tein. Enterococcus faecalis makes ATP from Agmatine in three steps catalysed by
Agmatine deiminase (AgDI), Putrescine transcarbamylase (PTC), and Carbamate
kinase (CK). An antiporter exchanges Putrescine for Agmatine. We have cloned
the E. faecalis ef0732 and ef0734 genes of the reported gene cluster for Agmatine
catabolism, over expressed them in Escherichia coli, purifed the products, charac-
terized them functionally as PTC and AgDI, and crystallized and X-ray difracted
them. The 1.65-Angstroms-resolution structure of AgDI forming a covalent adducts
with an Agmatine-derived amidine reaction intermediate is described. We provide
defnitive identifcation of the gene cluster for Agmatine catabolism and confrm that
Ornithine is a genuine but poor PTC substrate, suggesting that PTC (found here to
be trimeric) evolved from Ornithine transcarbamylase. N-(Phosphonoacetyl)-putres-
cine was prepared and shown to strongly (K (i)=10 nM) and selectively inhibit PTC
and to improve PTC crystallization. We fnd that E. faecalis AgDI, which is com-
mitted to ATP generation, closely resembles the AgDIs involved in making polyam-
ines, suggesting the recruitment of a polyamine-synthesizing AgDI into the AgDI
pathway. The arginine deiminase (ADI) pathway of arginine catabolism probably
supplied the genes for PTC and CK but not those for the Agmatine/Putrescine anti-
porter, and thus the AgDI and ADI pathways are not related by a single “en bloc”
duplication event. The AgDI crystal structure reveals a tetramer with a fve-blade
propeller subunit fold, proves that AgDI closely resembles ADI despite a lack of
sequence identity, and explains substrate afnity, selectivity, and Cys357-medi-
ated-covalent catalysis. A three-tongued agmatine-triggered gating opens or blocks
access to the active centre [20].
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Molecular docking studies, in vitro-brine shrimp lethal assay…
In drug discovery to reduce the time and cost and increase the efciency of
the medicine, molecular docking method has been used. It is a recent technique
to get insight study of the exact binding location of the ligand and protein [21].
In this study, we can understand the transport of small molecule called ligand in
biological systems. Auto Dock suite 4.2.6 is an excellent tool to get insights into
the molecular mechanism of ligand–protein interactions bind to a receptor of the
well-known three-dimensional structure. Embelin, vilangin, and phenyl vilangin
ligands optimized structures obtained from Gaussian 09 were docked using Auto
Dock Tools. These ligands were selected to be docked at the active areas of three
receptors 2JER of E. faecalis protein which were downloaded from the protein
data bank (RCSB). In this protein, Chain-A was selected for docking studies.
Ligand structures were made for docking by incorporating non-polar hydrogen
(H) atoms, adding Gasteiger partial charges and defning rotatable bonds. A max-
imum grid box size of 112 × 126 × 126 Å was created using Auto Grid. The grid
box was assigned at the centre of the protein using x, y, and z coordinates of
21.936, − 0.33, and 62.649 were created with a grid spacing of 0.492 Å at the
binding site for 2JER, respectively. Hundred docking runs were used to obtain
the best-ft proteins and it had very high binding energy. Docked structures were
visualized and a 2D (dimensional) graphic representation of protein–ligand inter-
action was created using Discovery studio visualize 4.0.
The ligand- receptor residue interaction, hydrogen bonds, distance of hydro-
gen bonds and molecular docking binding energies (kcal/mol) were obtained and
shown in Table 4. Among these three compounds, embelin exhibited the high-
est binding energy value of − 7.2 against 2JER protein which may be the reason
for its high biological activity. The diferent hydrogen bond interaction, hydrogen
bond receptor-side surface interaction, and 2D-hydrogen bonds interaction repre-
sentations are shown in Figs. 5, 6, 7.
Table 4 The ligand- receptor residue interaction between embelin, vilangin and phenyl vilangin with
2JER protein
S.No Pub chem. Id Compound name Binding
Residue interac-
Type of bond Distance(Å)
energy (kcal/ tion
mol)
1
2
3
2JER
2JER
2JER
Embelin
Vilangin
− 7.2
[ALA265]NH–O Hydrogen
H–OH[GLY209] Hydrogen
[ASN117]NH–O Hydrogen
OH–O[GLY180] Hydrogen
2.29
2.38
2.11
3.02
3.39
3.08
3.30
2.45
− 5.8
Phenyl Vilangin − 6.9
C–O[GLU295]
C–O[GLU213]
C–O[GLU213]
[ILE299]N–O
Hydrogen
Hydrogen
Hydrogen
Hydrogen
Bold indicates a pub chem id of protein code for E. faecalis bacteria subjected for docking studies
1 3

S. Jayalakshmi et al.
Fig. 5 a Hydrogen bond interaction of embelin with 2JER protein. b Hydrogen bond receptor-side sur-
face interaction of embelin with 2JER protein. c 2D-Hydrogen bond interaction representation of embelin
with 2JER protein
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Molecular docking studies, in vitro-brine shrimp lethal assay…
Fig. 5 (continued)
HOMO–LUMO analysis (Phenyl Vilangin)
In the present work, phenyl vilangin is subject for Gaussian studies, since the mol-
ecule exhibits appreciable results on antibacterial and brine shrimp lethal assay. The
highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular
orbital (LUMO) are known as frontier molecular orbitals (FMOs). Chemical stabil-
ity is mostly infuenced using the frontier orbitals (HOMO and LUMO). The FMOs
plays a signifcant role in the optical and electrical properties and quantum chemis-
try [22]. The HOMO represents electron-donating ability, while the LUMO repre-
sents electron accepting ability [23]. The HOMO and LUMO energies are very help-
ful for physicists and chemists and are very important terms in quantum chemistry.
The graphic representation of energies of molecular orbitals with molecular formula
of phenyl vilangin (C41H56O8) is shown in Fig. 8 (positive phase is represented by
the red colour and negative phase represented in green colour). The HOMO lying
at−6.2929 eV and spreads around over the entire molecule whereas the LUMO
located at−4.0835 eV with large anti-bonding character, which reveals that the
eventual charge transfer occurs within the molecule and the frontier orbital energy
gap is 2.2095 eV.
In general both the HOMO and LUMO orbitals are the main orbital that helps to
explain the chemical reactivity and kinetic stability of molecule. Dipole moments
(Debye) of the title compound phenyl vilangin are shown in Table 5, representing that
the dipole moment of this title compound can support more interaction with high dipole
1 3

S. Jayalakshmi et al.
Fig. 6 a Hydrogen bond interaction of vilangin with 2JER protein. b Hydrogen bond receptor-side
surface interaction of vilangin with 2JER protein. c 2D-Hydrogen bond interaction representation of
vilangin with 2JER protein
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Molecular docking studies, in vitro-brine shrimp lethal assay…
Fig. 6 (continued)
moment species especially in the biological systems. From Table 5, it is observed that
title compound of phenyl vilangin has been exhibited a low value of chemical hardness
η (eV) value of 1.1047 and high value of global softness S (eV) value of 0.2601; there-
fore, this compound has chemically reactive. Furthermore, the lowering in the HOMO
and LUMO energy gap describes the ultimate charge transfer interactions that happen
within the molecule and also possessed very good chemical and biological activities.
The lower value of electron afnity A=4.0835 (eV) for the phenyl vilangin mol-
ecule has accepted electrons for this reaction. As a result of having LUMO and HOMO
energy gap (ΔE), the ionization potential (I), the electron afnity (A), global hardness
(ɳ) chemical potential (μ), global electrophilicity (ω) values can be estimated by the
following formula.
ꢀ = I + A∕2(Electronegativity)
(1)
ꢀ = −(I + A) ∕2(Chemical potential)
(2)
ꢀ = I − A∕2(Chemical hardness)
(3)
S = 1∕2ꢀ (chemical softness)
(4)
ꢀ = ꢁ2∕2ꢂ (Electrophilicity index)
(5)
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S. Jayalakshmi et al.
Fig. 7 a Hydrogen bond interaction of phenyl vilangin with 2JER protein. b Hydrogen bond Receptor-
side surface interaction of phenyl vilangin with 2JER protein. c 2D-Hydrogen bond interaction represen-
tation of phenyl vilangin with 2JER protein
Molecular electrostatic potential
Molecular electrostatic potential is a signifcant tool to better understand the
relative polarity and reactivity of a molecule. Also, MEP is a valuable tool for
expecting and examining the molecular interaction like hydrogen bonding, drug-
receptor and enzyme–substrate interactions. The optimized structures at DFT/
B3LYP/6–311+ +G (d, p) level of theory are used to generate MEPs of the isolated
compound and phenyl vilangin is displayed in Fig. 9a and b. The MEP is a 3D-plot of
1 3

Molecular docking studies, in vitro-brine shrimp lethal assay…
Fig. 7 (continued)
Fig. 8 Molecular orbital energy level diagram with HOMO and LUMO orbital of phenyl vilangin
1 3

S. Jayalakshmi et al.
Table 5 Calculated quantum
chemical parameters HOMO–
LUMO results phenyl vilangin
Quantum Parameters (eV)
Phenyl vilangin
(HOMO–
LUMO)
SCF energy (a.u)
Dipole moment (Debye)
EHOMO
ELUMO
∆Egap
−2197.221595
7.8207
−6.2929
−4.0835
2.2095
I= − EHOMO
A=−ELUMO
ꢀ =
6.2929
4.0835
5.1882
I+A
2
μ=−(I+A)/2
η=(I−A)/2
−5.1882
1.1047
0.2601
1
S=
2ꢀ
ꢀ2
ω=
2ꢁ
12.1832
electrostatic potential mapped onto electron density surface which depicts the size,
shape, charge density and site of chemical reactivity of the molecules. In the colour
scheme of MEPs, red represents electron rich, partially negative charge which is the
favoured site for electrophilic attack, blue corresponds to electron defcient, partially
positive charge which is the favoured site for nucleophilic attack, yellow coloured as
slightly electron rich region; green coloured as neutral, respectively. Molecular elec-
trostatic potential mapping is extremely helpful in the study of the molecular struc-
ture with its physiochemical property relationships and also biological studies [24].
The colour code of this map is in the range between−8.799×10−2(deepest red) to
8.799×10−2 (deepest blue). It can be seen that the negative regions are mainly over
the oxygen atom and the positive potential sites are around the hydrogen atoms.
UV–vis spectral analysis
The frontier molecular orbital acting a main role in discovering bioactive, electrical,
in addition, optical properties. The test UV–Visible spectrum of phenyl vilangin title
molecule has been recorded with methanol as a solvent and compared with a simu-
lated spectrum with the 6–311+ +G(d,p) basis set in Fig. 10a, calculated using the
TDDFT/B3LYP method, Fig. 10b.
Absorption spectroscopy of most of the organic molecules is based on transi-
tions π–π*and σ–σ* in the UV–Vis region [25]. The UV–Vis absorption spec-
trum of phenyl vilangin is shown in Fig. 10 a and b and its exhibits a spectral
range 300–800 nm. The theoretical UV–Visible spectra image was obtained from
Gauss-Sum 3.0 program [10]. The signifcant absorption peaks observed are at
788, 664, and 617 nm. The most intense UV bands are observed due to absorption
from highest occupied molecular orbital and lowest unoccupied molecular orbital
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Molecular docking studies, in vitro-brine shrimp lethal assay…
Fig. 9 a Molecular electrostatic potential (Overall view) of the compound phenyl vilangin. b Molecular
electrostatic potential (Inner view) of phenyl vilangin
(HOMO to LUMO). In order to understand electronic transitions of compound,
TD-DFT calculations on electronic absorption spectra were performed. Experi-
mentally observed peaks are in good deal with the theoretical values calculated
by TD-DFT method. The values of wavelength, electronic excitation energies and
oscillator strength calculated by TD-DFT method are tabulated in Table 6.
1 3

S. Jayalakshmi et al.
Fig. 10 a and b UV–Visible Spectrum of phenyl vilangin a Experimental b Theoretical
Table 6 The UV–visible wavelength (λ), band gap energy (ΔE) and oscillator strength (f) for phenyl
vilangin calculated by TD-DFT/B3LYP method
Experimental
Calculated by TD-DFT/B3LYP/6–311++G(d,p)
λmax (nm)
Log (ε)
λmax (nm)
E(eV)
(f)
788
664
617
1.0
1.5
1.3
788 HOMO → LUMO (95%)
664 HOMO-1 → LUMO (61%)
617 HOMO-3 → LUMO (55%)
1.5716
1.8659
2.0092
0.0094
0.0233
0.0109
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Molecular docking studies, in vitro-brine shrimp lethal assay…
Conclusion
Thus, the major conclusion drawn from this work is that the selection of suitable
chemical structure with balanced hydrophobic and hydrophilic ratio decides its
use as a root canal irrigant in the discipline of endodontics.
The prime outcome of this work reveals embelin as a very good antimicrobial
agent and phenyl vilangin as a good cytocompatible agent. The resonance sta-
bilised dianion structure of embelin and phenyl vilangin exhibits more electron
density at carbon position C6 and C3, which is also refected from the molecu-
lar electrostatic potential diagram of phenyl vilangin. The plausible mechanism
proposed for antibacterial activity was found to be in good agreement with MIC
value of embelin. It is certain from this work that by fne tuning the balance
between hydrophobic and hydrophilic ratio, both antimicrobial property and bio-
compatible property can be achieved. This work may be extended further by syn-
thesizing few more derivatives of embelin by incorporating various hydrophilic
groups, and their impact on antimicrobial and biocompatibility properties can be
investigated.
Declarations
Confict of interest The authors have no confict of interest.
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