TRENDS IN SCIENCES 202x; xx(xx): xxxxx RESEARCH ARTICLE

https://doi.org/

Exploring Antibacterial Potential of Anhuienoside E from Nigella sativa

Linn: A Promising Candidate Against Dental Caries In Vitro And In Silico
Studies

Highlights
1. Anhuienoside E was successfully isolated from the methanol extract of N. sativa seeds.
2. Anhuienoside E has antibacterial activity against S. mutans, S. sanguinis, E. faecalis based on MIC and MBC
tests.
3. Anhuienoside E has the potential to inhibit Gtf and MurA enzymes based on In silico molecular docking
study.

Graphical abstract
Trends Sci. 202x; xx(xx): xxxxx 2 of 22

Abstract
Dental caries is a chronic disease suffered by almost the entire population in the world. The main bacterium
that causes dental caries is Streptococcus mutans, which has the enzyme glucosyltransferase as a virulence factor.
Other fatogenic bacteria that play a role in exacerbating the biofilm form of dental caries such as Streptococccus
sanguinis and Enterococcus faecalis have cell wall defenses catalyzed by the MurA enzyme. Chlorhexidine has
been reported as a treatment for dental caries but has developed resistance over time. Nigella sativa L. seeds have
been widely recognized to have many health benefits such as antibacterial, antioxidant and antifungal. The caries-
causing antibacterial activity of N. sativa seeds has not been widely reported. The aim of this study was to isolate
antibacterial compounds from N. sativa against S. mutans, S. sanguinis, E. faecalis, predict the mechanism of
compounds and their derivatives by in silico molecular docking and pharmacokinetic analysis by ADMET and
drug-likeness methods. Isolation of compounds was carried out by column chromatography with bioassay
guidance, inhibitory mechanism and sugar substituent effects were predicted by in silico molecular docking,
pharmacokinetics and drug-likeness analysis were predicted by ADMET and Lipinski rules. Anhuienoside E was
successfully isolated from N. sativa extract and demonstrated antibacterial activity, which had moderate MIC and
MBC values of 625 μg/mL and 1000 μg/mL against S. mutans, S. sanguinis, E. faecalis. Anhuienoside E had
moderate binding affinity value compared to its derivatives with ∆G values of −6.84 and −8.67 Kcal/mol against
MurA and Gtf. ADMET pharmacokinetic analysis and drug-likeness evaluation suggest that Anhuienoside E and
its derivatives may serve as non-toxic, non-oral drug candidates. In conclusion, Anhuienoside E has potential
antibacterial activity to be developed as an alternative to chlorhexidine. The amount and type of sugar substituents
greatly affect the antibacterial activity of Anhuienoside E.

Keywords: Nigella sativa L., dental caries, antibacterial, anhuienoside E

Introduction
Dental caries is a chronic disease that affects nearly the entire global population. According to Indonesia's
2018 Basic Health Research (Riskesdas) data, 95% of children aged 5 to 6 years suffer from dental caries [1]. The
main bacteria that causes dental caries is Streptococcus mutans. S. mutans secretes extracellular polysaccharides
by glucosyltransferase enzymes and gives the signals to other bacteria to form colonies on the biofilm layer [2–
3]. Streptococcus sanguinis and Enterococcus faecalis receiving signals from S. mutans work together to make a
stronger biofilm layer into caries [4–5]. Both of these bacteria have cell walls composed of peptidoglycans, which
are synthesized by the MurA enzyme. Various treatments of dental caries have been reported in previous studies.
Chlorhexidine is one of antiseptics that has been used commercially. Chlorhexidine targets the destruction of
bacterial cell membranes. The ability of bacteria to regulate the DltA operon causes the increasing of
hydrophobicity on the surface of cell membranes so chlorhexidine is becoming resistance [6]. Treatment of dental
caries with more specific targets needs to be explored, one of which is by targeting the glucosyltransferase (Gtf)
and Muramidase A (MurA) enzymes.
Gtf is an enzyme that catalyzes the synthesis of extracellular polysaccharides on the tooth surface. It is
secreted by Streptococcus mutans as a virulence factor and breaks the glycosidic bond in sucrose, producing
glucose and fructose. Glucose monomers are polymerized and act as a signal for other bacteria to form colonies
on the teeth surface (biofilm) which then form caries [7].
MurA is the first enzyme that catalysed the formation of peptidoglycans to build bacterial cell wall. Uridine
diphosphate N-acetylglucosamine is converted into UDP-MurNac by MurA enzyme. In the next stage, it will be
attached to sugar molecules to form peptidoglycans. Therefore, inhibition of this enzyme becomes very potent so
bacteria will grow abnormalities or die [8–9]. Exploration of natural compounds that have potential as caries
antibacterials by in silico molecular docking of inhibitory mechanisms against MurA and Gtf enzymes needs to
be done. One of the natural sources which has antibacterial agent is the seeds of Black Cumin (Nigella sativa
Linn.).
N. sativa Linn. is an herbal plant and has been known for a long time among people in all parts of the world.
The high benefits of this plant, especially in the seeds, make it often used as an alternative to conventional
medicine. N. sativa seeds are widely used as an herbal medicine in Indonesia called “Jamu”. The method of use
is pounded then boiled and filtered water. The efficacy of this “Jamu” is believed to treat various diseases such as
inflammation, diabetes and cold medicine. Several researchers revealed the benefits of extracts from N. sativa
seeds as antibacterial, antioxidant, immunomodulatory and antiviral [10–11].
The content of secondary metabolite compounds found in N. sativa seeds such as flavonoids, triterpenoids,
saponins, alkaloids and essential oils provide diverse bioactivities [12]. Natural product compounds have diverse
functional groups such as hydroxy, carbonyl, amine and ester. The role of these functional groups involves redox
reactions in prokaryotic cells. So that the presence of functional groups from compounds in N. sativa will increase
antibacterial activity [13–16]. The main compound isolated from the essential oil of N.sativa seeds is
thymoquinone, which has potential therapeutic effects against atherosclerosis, cancer, and diabetes [17–19].
Trends Sci. 202x; xx(xx): xxxxx 3 of 22

The treatment of dental caries with N. sativa seed compounds has not been widely reported. The study of
the mechanism of inhibition of Gtf and MurA enzymes by compounds from N. sativa through molecular docking
approach is very useful to know the treatment action of compounds. Therefore, compounds from N. sativa have
the potential to be explored as natural caries drug candidates with a review of mechanism of action studies. This
study aimed to identify antibacterial compounds from N. sativa seed extract effective against S. mutans, S.
sanguinis, and E. faecalis. to determine their mechanisms of action targeting MurA and Gtf enzymes, and to
predict their pharmacokinetic properties through ADMET and drug-likeness analyse.

Materials and methods

1. Isolation of Anhuienoside E (1)

N. sativa seeds were obtained from Indonesian local market at Jl. Pasar Barat No.44, Kebun Jeruk, Andir,
Bandung in July 2023. Dried N. sativa seeds were ground then macerated, extracted and purified using n-hexane,
ethyl acetate, methanol and water solvents from Sigma Aldrich Co. Ltd. (St. Louis, MO, USA) and Merck Co.
Ltd. A total of 2 kg of dried N. sativa seeds were ground and macerated using 4 liters of methanol for 3 days, then
filtered and concentrated using a rotatory evaporator. The methanol extract was tested for antibacterial activity
against S. mutans ATCC 25175, S. sanguinis ATCC 10556 and E. faecalis ATCC 29212 at concentrations of 1,
2, 3, 4 and 5%. The extract that was known to have oral antibacterial activity was partitioned as much as 30 g
using a normal phase chromatography column Silica G 60 F254 as much as 300 g. The extract was eluted by n-
hexane, ethyl acetate, methanol and water of 300 mL for 3 times, respectively until get eleven fractions. The
fraction that had good antibacterial activity with the large amount of mass was fraction 7 (F-7) (methanol fraction,
14.52 g) which is continued to the purification stage. Analysis of spot patterns in F-7 used TLC (Thin Layer
Chromatography) ODS (Octadecylsilane) RP-18 F254S and Silica G 60 F254 (Merck, Darmstadt, Germany). It was
observed under UV light at wavelengths of 254 and 365 nm and sprayed with 10% H2SO4 solution in ethanol
(v/v). 1 g of F-7 was performed column chromatography using ODS RP-18 F254S and elution was performed using
30 mL of a methanol–water mixture (9:1, v/v) with a 2% v/v gradient of increasing polarity, resulting in 32
subfractions.[20] Column results were analyzed using TLC and observed the stain pattern under UV light 254 and
365 nm, then sprayed with H2SO4 10% stain reagent in ethanol (v/v) [21].

2. Structure Elucidation of Compound 1

Structure elucidation of Compound 1 was conducted by spectroscopic instruments. Identification of

functional groups of compound 1 using Infrared spectrophotometer (FTIR Shimadzu 8400)[22], identification of
proton and carbon chemical environment of compounds with 1D and 2D spectra (1H-NMR, 13C-NMR, DEPT
135°, HMQC, 1H-1H COSY, HMBC) using BRUKER NMR spectrometer AVANCE NEO 700 MHz series.
Molecular mass measurement of compounds using Mass Spectrometer (MS Aquity TQD, Waters Corporation,
MA, USA) [23].

3. Antibacterial Testing

The methanol extract of N. sativa seeds was tested for antibacterial activity against S. mutans ATCC 25175,
S. sanguinis ATCC 10556 and E. faecalis ATCC 29212 at concentrations of 1, 2, 3, 4 and 5% by disc diffusion
method. Positive control (chlorhexidine 2%), negative control methanol and extract as much as 20 μL were placed
on paper disks and then placed on Muller Hinton agar media (purchased from Merck Co. Ltd. and Sigma Aldrich)
containing 100 μL bacteria ½ McFarland. Agar media was incubated for 24 hours at 37 ℃. Determination of the
active fraction used the same procedure as the N. sativa seed methanol extract assay.
MIC testing was carried out on compound 1 by microdilution method at a concentration of 2% using
microplate-96 wells. All wells of microplate-96 wells were filled with Muller Hinton liquid media, then 100 μL
of methanol solvent was added as control. Compound 1 was added to the wells without methanol as much as 100
μL. Dilutions were performed by the microdilution method on Microplate-96 wells. Bacteria with a turbidity level
of 0.5 McFarland as much as 5 μL were added to the wells. The microplate-96 wells were incubated for 24 hours
at 37°C. After incubation, the absorbance was observed on the Biochrom EZ Read 400 ELISA tool with a
wavelength of 620 nm and the MIC value was obtained. MBC testing is done by spreading clear media microplate-
96 wells containing compound 1 and bacteria on Muller Hinton agar media then incubated at 37°C for 24 hours
so that the MBC value can be determined [24]. Antibacterial testing of zone of inhibition was performed in
triplicate, MIC and MBC were performed in duplicate.
Trends Sci. 202x; xx(xx): xxxxx 4 of 22

4. In silico Molecular Docking

4.1 Preparation of Docking

The enzymes used in molecular docking were MurA and Gtfs which have PDB ID: 8FKL[25] and 1UAE
[26] whose 3D structures were downloaded on the website www.rcsb.org. Conformation validation of MurA and
Gtf enzymes using Ramachandran Plot [27]. The two Ramachandran-conforming enzyme PDB codes 8FKL and
1UAE were separated with bile and ligand natives using the Biovia discovery studio. Coordinates of the enzyme
active site were analyzed on the web server http://sts.bioe.uic.edu/castp/index [28]. Enzyme preparation using
Biovia Discovery Studio 2021 software. The structures of compound 1 and its derivatives were drawn in
ChemDraw 15.0 and energy minimization was performed in Chem3D Ultra 8.0. Chlorohexidine and apigenin as
positive controls were downloaded on the page https://pubchem.ncbi.nlm.nih.gov with CID: 9552079 and
5280443 codes.

4.2 Molecular docking

Molecular docking was run using AutoDockTools-1.5.7 software.[29] Receptors (Gtf and MurA) were
pretreated by adding Kollman charge while ligands compound 1, derivates and positive control were added to
Compute Gasteiger. Docking was performed on receptor active site regions obtained from calculations on the
CASTp webserver [28]. Docking parameters using Genetic algorithms with 100 runs. Visualization of docking
results (type of interaction and number of interactions) was observed in Biovia discovery studio. Adding grid box
area is better

5. ADMET and Drug-likeness Analysis

ADMET analysis of compound 1 and derivatives using web server https://biosig.lab.uq.edu.au/pkcsm. The
chemical structure of compound 1 and its derivatives is made in smile format which is drawn on the web
http://www.swissadme.ch. Determination of drug-likeness using web server https://tox.charite.de/protox3 [30–
31][32].

Results and discussion

1. Isolation of Compounds from N. sativa Seeds

Maceration of 2 kg of N. sativa seeds using 4L methanol was concentrated to obtain 46.5 g of extract. The
methanol extract of N. sativa seeds was tested for antibacterial activity against S. mutans, S. sanguinis and E.
faecalis through inhibition zone test. The test results of the extract showed active as antibacterial against the three
bacteria (test results are in Table 1) which indicated the compound components potentially as antibacterial agent
[33]. The methanol extract was further fractionated into eleven fractions and tested for antibacterial activity. Based
on the results of antibacterial testing of the eleven fractions, it showed that fraction seven had good oral
antibacterial activity, so it was continued to the purification stage (test results are in Table 2). The isolation of
compounds from the active fraction aims to obtain the most active isolate. Some isolates showed more active
antibacterial activity than the fraction and others were less active depending on their nature in the antagonistic or
synergistic fraction [34].
Fraction F-7 from the initial partition was purified using ODS RP-18 F254S chromatography column with
2% gradient H2O-methanol eluent starting from 9:1 solvent ratio until 32 fractions were obtained. The purification
results were analyzed using TLC. Based on the KLT analysis on the ODS RP-18 plate, F-7-30 (100 mg) has a
single stain that does not fluoresce under UV lights 254 and 365 nm and is brown after being sprayed with H2SO4.
This fraction is thought to be a pure isolate which is called compound 1 [35–36].

2. Structure Elucidation of Compound (1)

The results of measurements using IR spectrophotometer indicated that compound 1 has an OH group at
maks 3367 cm-1, C-H sp3 stretch at maks 2940 cm-1, C=O stretch vibrations at maks 1634 cm-1, aliphatic C=C
stretch vibrations at maks 1455 cm-1, gem dimethyl C(CH3)2 stretch vibrations at maks 1385 cm-1, C-O stretch
vibrations at maks 1058 cm-1, and alkene C-H bending at maks 635 cm-1 [37].
Trends Sci. 202x; xx(xx): xxxxx 5 of 22

Chemical shift of carbon compound (1) (brown solids, 100 mg) showed 13C-NMR (CD3OD, 700 MHz) δ
13.8 ppm (C-1), 16.6 ppm (C-2), 17.8 ppm (C-3), 17,9 ppm (C-4), 18.0 ppm (C-5), 18.8 ppm (C-6), 23.9 ppm (C-
7), 24.2 ppm (C-8), 24.6 ppm (C-9), 26,4 ppm (C-10), 26.6 ppm (C-11), 28.9 ppm (C-12), 31.6 ppm (C-13), 33.3
ppm (C-14), 33.3 ppm (C-15), 33.5 ppm (C-16), 34.8 ppm (C-17), 37.6 ppm (C-18), 39.7 ppm (C-19), 40.6 ppm
(C-20), 42.5 ppm (C-21), 43.9 ppm (C-22), 44.0 ppm (C-23), 47.1 ppm (C-24), 48.0 ppm (C-25), 48.1 ppm (C-
26), 49.9 ppm (C-27 Rha), 61.8-82.4 ppm (C-(28-52 sugar), 95.7, 101.3, 104.2, 104.6 and 106.5 ppm (C-(53-
57anomeric), 123.7 ppm (C-58), 144.0 ppm (C-59), 178.0 ppm (C-60); 1H-NMR (CD3OD, 700 MHz) δ 0.72 ppm
(3H, s; H-1), 0.82 ppm (3H, s; H-4), 0.94 ppm (3H, s; H-16), 0.97 ppm (3H, s; H-8), 1.00 ppm (3H, s; H-2), 1.19
ppm (3H, s; H-10), 1.25 ppm (2H, t J = 6, H-14), 1.28 ppm (3H, s; H-3), 1.29 ppm (3H, s; H-5), 1.39 ppm (2H, t;
J = 12, H-17), 1.51 ppm (2H, t; J = 12, H-6), 1.63 ppm (2H, t; J = 6, H-7), 1.64 ppm (3H, s; H-28), 1.67 ppm (4H,
t; J = 6, H-9&15), 1.71 ppm (4H, t; J = 6, H-11&12), 1.78 ppm (2H, d; J = 6, H-24), 1.79 ppm (2H, q; J = 6, H-
24), 1.92 ppm (1H, t; J = 6, H-27), 1.93 ppm (1H, t; J = 6, H-25), 2.89 ppm (1H, q; J = 6, H-21), 3.87 ppm (1H,
t; J= 6; H-52), 3.89 ppm (1H, t; J= 6; H-58), 3.90-4.1 ppm (21H; H-sugar), 4.42 ppm (1H, d; J= 7.8 H-55
anomeric), 4.51 ppm (1H, d; J = 7,5 H-57 anomeric), 4.55 ppm (1H, d; J = 6.2 H-56 anomeric), 5.26 ppm (1H, d;
J = 16.3 H-54 anomeric), 5.36 ppm (1H, d; J = 8.1 H-53 anomeric). 1H, 13C- and 2D-NMR chemical shift data of
compound 1 are shown in Table 3-4.
Based on the 13C-NMR data, compound 1 contained sixty types of carbon. DEPT, 1H-NMR spectrum and
HSQC correlation showed compound 1 contained eight quaternary carbons, five methine carbons, twenty-four
oxidized methine carbons, one sp2 methine, nine methyl, and thirteen methylene. HMBC spectrum shows proton
to-carbon correlation as far as two to five bonds and the COSY spectrum shows proton to proton correlation as
far as three bonds are suspected of compound 1 being a burnt triterpenoid group [38]. Carbon sixty and carbon
fifty-two experienced the addition of sugar substituents as many as five pieces shown by the presence of carbon
and anomeric protons as many as five. Three sugars namely glucose’- glucose”-rhamnose’ were bound to carbon
sixty and two glc-rha sugars at carbon fifty-two. The placement of the number and type of sugar was based on the
HMBC correlation of the anomeric proton to carbon sixty and fifty-two proton to anomeric carbon.[39]
Configuration of sugar group in compound 1 was β which was indicated by the shear of the anomeric carbon ≥99
and α at ≤95 [40–41]. NMR spectrum showed that compound 1 had the molecular formula C60H98O6 with a degree
of unsaturation of twelve. Measurement of the molecular mass of compound 1 using a Water Acquit UPLC type
triquadrupole [M+Na]+ at m/z 1257 confirmed the alleged structure of compound 1 (Shown in Figure 1) [42].
Based on the literature, compound 1 has proton and carbon chemical shifts similar to Anhuienoside E isolated
from N. sativa seeds. In addition, based on MS measurements, it has a similar molecular weight, thus confirming
that compound 1 is Anhuienoside E (all spectra in Figure S1-S7) [12–43].

3. Antibacterial Testing of Anhuienoside E (1)

Antibacterial testing of Anhuienoside E (1) against S. mutans, S. sanguinis and E. faecalis was carried out
by microdilution method. The test results are in Table 5 which showed moderate activity with MIC values of 625
μg⁄mL and MBC of 1000 μg⁄mL [44]. Anhuienoside E (1) showed that did not give activity for killing E. faecalis
but only gave inhibition activity. Anhuienoside E had five sugar groups and other functional groups such as
alkenes and carbonyls that could inhibit bacteria through the destruction of cell membranes and inhibit enzymes
that have important catalytic activities in bacteria [45]. The moderate MIC values were very relevant to MBC
values that were in the inactive range. The study of the antibacterial mechanism of Anhuienoside E (1) and the
effect of sugar groups on antibacterial activity was predicted by molecular docking against key pathogenic
enzymes of oral bacteria.
Trends Sci. 202x; xx(xx): xxxxx 6 of 22

Figure 1: Structure of Anhuienoside E (1) with thick line COSY spectrum correlation and red line HMBC correlation.

Figure 2: Chemical structure of Anhuienoside C (derivate 3) and Cussonoside B (derivate 5)

Trends Sci. 202x; xx(xx): xxxxx 7 of 22

Figure 3: Chemical structure of Anhuienoside D (derivate 2), derivate 8, Anhuienoside F (derivate 4), and Oleanolic acid (derivate 9)
Trends Sci. 202x; xx(xx): xxxxx 8 of 22

Figure 4: Chemical structure of Anhuienoside E (1), Flaccisdoside II (derivate 6), Flaccisdoside III
(derivate 7), chlorhexidine and Apigenin (positive control)
Trends Sci. 202x; xx(xx): xxxxx 9 of 22

4. Molecular docking

Molecular docking was performed on Anhuienoside E (1) and derivates on two key enzymes from caries-
causing bacteria (the structures are shown in Figure 2-4). MurA enzyme was an enzyme that catalysed
peptidoglycan at the initiation stage representing E. faecalis and S. sanguinis bacteria. Gtf enzyme with PDBID
8FKL isolated from S. mutans catalysed the formation of extracellular polysaccharides starting from the cleavage
of sucrose glycosides [46]. Both enzymes were analysed for conformational similarity of amino acid bile before
characterization and after visualization in the protein data bank using the Ramachandran plot. Ramachandaran
plot describes the conformation of α-helical and β-sheet bonds in protein peptides with two coordinate torsions ϕ
and ψ (ψ 180, ϕ>0). Based on the Karplus equation, the Ramachandran plot was divided into four regions to
determine whether the conformation of amino acids was still stable during X-ray and does not damage the α-
helical and β-sheet bonds. One [A, B, L] region was highly favoured, additional allowed regions [a, b, l, p],
generously allowed regions [~a, ~b, ~l, ~p] and disallowed regions as shown in Figure 5. Amino acid residues
from proteins that will be used for molecular docking should not occupy disallowed regions of more than 2% to
ensure conformational conformity with the actual protein structure [47–49]. Both MurA and Gtf have a disallowed
region value of 0%, indicating that their conformations are consistent with the original structures and can be used
for in silico molecular docking.

(1) (2)
Figure 5: Ramachandran plot of MurA enzyme (1) and Gtf enzyme (2)

Anhuienoside E (1), Anhuienoside C (3), Anhuienoside D (2), Anhuienoside F (4), Cussonoside B (5),
Flaccidoside II (6), and Flaccidoside III (7), Derivate (8), Oleanolic acid (9), chlorhexidine, apigenin were docked
with MurA and Gtf enzymes. Compounds 1–9 were subjected to molecular docking to compare their inhibitory
activity against MurA and Gtf enzymes, with a focus on variations in the number and type of sugar substituents.
Sugar groups had many hydroxyl functional groups that can interact by hydrogen bond with enzymes. Molecular
docking results demonstrate that sugar groups significantly influence the strength of interactions between ligands
(compounds) and receptors (enzymes), as characterized by varying delta G (∆G) values, as shown in Figures 5
and 6. ∆G is a parameter representing the strength of ligand-receptor binding, measured in kcal/mol, and is derived
from the following equation:

∆G= -RT ln K
The ∆G value decreases as the K value (the equilibrium constant between protein and ligand) increases [50]. The
greater the value of K the reaction goes towards the product or the stronger the protein-ligand association. Docking
is performed at the receptor (enzyme) active site, meaning the ligand (compound) acts as a competitive inhibitor
against the native ligand or cofactor. Ligand binding in the active site region can alter enzyme stability or inhibit
its activity [51].
Anhuienoside E (1) had a relatively moderate ∆G value compared to other compounds and positive controls
against the MurA enzyme. The number of glucose units significantly affects ligand–protein binding affinity, as
evidenced by Anhuienoside F (4), which exhibited the lowest binding affinity (the highest ∆G value) toward
Trends Sci. 202x; xx(xx): xxxxx 10 of 22

MurA among the tested compounds, due to the presence of six sugar groups (Figure 6). The results of docking
visualization in Figure 8 showed that almost all compounds interact with ligands through sugar groups excepted
compounds four and seven with carbonyl esters. Sugar groups covered other functional groups in terpenoid
structure because the interactions formed (hydrogen bonds) were stronger. MurA enzyme structure was also
relatively small, so bulk compounds such as Anhuienoside E were difficult to interact with the active site area.
MurA facilitates glycosidic bond formation in peptidoglycan biosynthesis, and the presence of sugar groups may
disrupt this activity, preventing bacterial cell wall formation [12]. The type of sugar affected the value of ∆G.
Almost all compounds (1-8) interacted by hydrogen bond with the enzyme through the sugar rhamnose. The
hydroxy position at number four on rhamnose tended to be the most electronegative because there was an EDG
(electron donating group) group, namely either of C-anomeric and hydroxy group at carbon five which pushed
electrons through the induction effect [52]. Compound 9 did not show the lowest ∆G value, likely due to the
formation of a single hydrogen bond, highlighting the influence of the type and number of sugar groups on MurA
enzyme inhibition. In addition, the positive control, chlorhexidine, did not show a higher ∆G value than some of
the tested compounds (1-9), suggesting that Anhuienoside E, with proper derivatization, has potential as an
alternative to chlorhexidine.
The docking results of compounds 1-9 with Gtf enzyme showed various ∆G values (shown in Figure 7).
Flaccisdoside III (7) exhibited the lowest ∆G value through its interaction with the Gtf enzyme, specifically at the
Glc'-Glc'' glycosidic bond (Figure 9). The Gtf enzyme catalyzes the synthesis of extracellular polysaccharides,
initiating the process by cleaving the glycosidic bond of sucrose (Glc–fructose) [7]. The presence of Flaccisdoside
III (7) in the active site region decreased the catalytic activity of the enzyme through hydrogen bonds formed from
aspartic amino acids with Glc'-acid protons and Glc'-oxygen. In addition, in other compounds 1-8 there were
hydrogen bonds (Shown in Figure 9) with Gtf which were very dominant from Glc to Gtf amino acid residues in
the active site region. Anhuienoside E had inhibitory activity similar to the positive control Apigenin as shown
by the ∆G values in Figure 7. This finding is supported by the number of hydrogen bond interactions formed by
the glucose moieties of Anhuienoside E. All sugar-containing derivatives demonstrated stronger inhibitory
activity against the Gtf enzyme compared to the positive control.

5. ADMET and Drug-likeness Analysis

ADMET analysis results of compounds 1-9 are show in Table 6. Analysis of Absorption (A), Distribution
(D), Metabolism (M), Excretion (E) and Toxicity (T) was conducted to investigate the feasibility of these as
antibacterial drugs. Absorption parameters (Intestinal Absorption and Water Solubility) indicated the drug's
ability to be absorbed by the body based on its water solubility. The range of water solubility was log 95% of
drugs: -6.0 to 0.5 [53]. Only compounds five, eight and nine were absorbed by the body and the rest were carried
by the blood to all body tissues. This data showed that Anhuienoside E, can reach the target of pathogenic bacteria.
Distribution parameters (VDss, CNS and BBB): all compounds are poor distributed in the blood or in the
bloodstream. A good VDss range was 0.5 to 3 log L/Kg. The drug candidate should not affect the central nervous
system (CNS) and BBB (Blood-Brain Barrier). The drug concentration should be focused on the disease target
[54–55]. BBB and CNS >2.0 indicated strong absorption, whereas 0.1-2.0 indicated moderate absorption and <0.1
indicated low absorption. Analysis of the effect of compounds 1-9 on central nervous system metabolism of
CYP1A2, CYP2D6, CYP2C9, and CYP2C19 showed that all compounds did not inhibit the four enzymes. These
enzymes were CYP isoenzymes that catalysed the metabolism of drugs so they could be digested or absorbed by
the body [56]. Good drugs did not inhibit the activity of these enzymes. Compounds 1-9 exhibited substantial total
clearance values, indicating how easily these compounds, particularly thymoquinone, can be secreted and their
overall benefit to the body. Acute oral toxicity analysis with the LD50 parameter in compounds 1-9 showed the
possibility of being harmful if ingested. This parameter was used to measure how toxic or non-toxic the oral drug
is when tested on rats. LD50 values in the range of 50 mg/kg ≤ LD50 >5 mg/kg indicated the drug fatal if swallowed,
toxic in the range of 300 mg/kg ≤ LD50 >50 mg/kg, oral drugs could be harmful if swallowed in the range of 2000
mg/Kg-5000 mg/Kg, and non-toxic in the range of LD50 >5000 mg/kg [56].
The potential of a compound as an oral drug candidate can also be assessed through its physicochemical
properties, particularly its compliance with Lipinski's Rule of Five (RO5) [57]. RO5 rules must have a specific
gravity of less than 500 Daltons, the number of H bond acceptors was less than 10 bonds, H bond donors were
less than 5, lipophilicity was less than 5 bonds and molar refractivity was 40-130. A compound was allowed to
be oral drug-like if there had only a maximum one deviation from the RO5 rule. RO5 analysis of compounds 1-9
was contained in Table 7. The compound that complied with RO5 parameters was only compound 9. Compounds
1-8 do not qualify as oral drugs, as they have a Violation score of 4. However, they have the potential to be used
as mouthwash as an alternative to chlorhexidine [58].
Trends Sci. 202x; xx(xx): xxxxx 11 of 22

Docking results of Compounds with MurA enzyme

0
delta G (Kcal/mol)

-5
-5.38 -4.75
-10 -6.86 -6.45 -7.31 -8.13
-9.18 -9.39 -8.94
-11.66 -10.96
-15
1 2 3 4 5 6 7 8 9 A B

Figure 6: Graph of binding affinity (∆G) values between compounds (1-9), apigenin (A), chlorhexidine (B) with
MurA enzyme

Docking results of Compounds with Gtf enzyme

0
delta G (Kcal/mol)

-5

-10 -8.67 -8.35 -7.97

-9.66 -8.85 -10.4
-10.85-10.41-11.76
-15 -11.86-11.78
1 2 3 4 5 6 7 8 9 A B

Figure 7: Graph of binding affinity (∆G) values between compounds (1-9), apigenin (A), chlorhexidine (B) with
Gtf enzyme
Trends Sci. 202x; xx(xx): xxxxx 12 of 22

1. 2. 5. VAL 4. ARG
A:163 A:120 PRO
A:121
VAL
THR GLU
A:122
A:326 A:329
THR
A:326 ARG
A:120 PRO
A:298
VAL
VAL LYS
A:327
VAL VAL A:161 A:160
LYS A:161
A:327
A:160 THR
ALA VAL
PRO A119 A:362
A:327
A:122

3.
A. PHE
ASN
B. ARG SER
PRO A:332 THR
A:303 A:67 A:120 A:162
VAL A:326 LEU
PRO A:161 A:124
VAL A:298 GLU
VAK A:326 LYS
A:163
A:327 A:160
ARG
A:227 TRP ARG
GLU HIS
A:298 A:91
A:329 ASN A:125
A:253

6. VAL
7. 9. 8.
A:161

THR
A:326

LYS
A:160 ARG
ALA A:91
VAL A:119 VAL
VAL PRO
A:122 PRO A:327
A:161 A:121
A:298
ARG ALA LYS
ASN ASP GLU
A:120 A:119 A:160
A:184 A:159 A:329

C.

Interactions:
Hydrogen bond
π -Alkyl
π -sigma
π -donor Hydrogen bond

Figure 8: 2D visualization of docking results of Anhuienoside E (1), Anhuienoside D (2), Anhuienoside C (3), Anhuienoside F (4), Cussonoside B (5), Flaccidoside II (6),
Flaccidoside III (7), their derivatives (9), Oleanolic acid (9), Chlorhexidine (A), Apigenin (B) docked with MurA and 3D visualization (C)
Trends Sci. 202x; xx(xx): xxxxx 13 of 22

1. ASP 2. 5. TYR 4. TYR

THR SER A:688 A:700 A:657
A:326 A:690 LYS TYR
A:657 ASN
A:172
GLY A:714
A:689 ASP
PRO
A:688 PRO
ASP A:173
ILE A:713
A:688 ASP
A:401
A:688
LYS
TYR TYR
A:715 SER
A:657 PHE A:700
A:691 A:690
LYS LYS
A:653 PHE
A:650 A:712

3.
A. PRO
PHE
ASP
B. ARG SER
A:332
A:303 THR A:688 A:120 A:162
VAL A:326 LEU
A:162 GLU
VAL PRO A:124
A:325 SER LYS
A:163 A:298 VAL A:160
GLY A:690
A:327
A:689 PHE
GLU A:396 TRP HIS ARG
A:329 A:95 A:125 A:91
LYS PHE TYR
A:712 A:619 A:394

6. 7. 9. 8.

GLY PHE
ASP A:689 A:650
LYS A:688
A:712
LYS
ASN A:664 ASP
A:655 ALA
A:688
A:649
LYS GLN
A:457 SER A:648
LYS
A:395 PRO GLY
A:712
A:713 A:451

C.

Interactions:
Hydrogen bond
π -Alkyl
π -sigma
π -donor Hydrogen bond

Figure 9: 2D visualization of docking results of Anhuienoside E (1), Anhuienoside D (2), Anhuienoside C (3), Anhuienoside F (4), Cussonoside B (5), Flaccidoside II (6),
Flaccidoside III (7), their derivatives (8), Oleanolic acid (9), Chlorhexidine (A), Apigenin (B) docked with Gtf and 3D visualization (C).
Trends Sci. 202x; xx(xx): xxxxx 14 of 22

Conclusions
The pure compound Anhuienoside E (1) was successfully isolated from the methanol extract of N. sativa seeds and
demonstrated moderate antibacterial activity against S. mutans, S. sanguinis, and E. faecalis. In silico molecular docking
analysis indicated that derivatization of Anhuienoside E (1) is necessary to improve its antibacterial potency by targeting
the inhibition of MurA and Gtf enzymes. ADMET and drug-likeness analyses revealed that Anhuienoside E (1) is not
suitable for oral administration. However, it shows potential as a mouthwash agent and could serve as a promising
alternative to chlorhexidine, which has shown increasing resistance in the treatment of dental caries. In the present study,
derivatization of Anhuienoside E (1) was not performed. Further research is required to enhance its antibacterial efficacy,
particularly through sugar moiety modifications aimed at MurA and Gtf enzyme inhibition.

Acknowledgements
The authors are grateful to Academic Leadership Grant (ALG) Prof. Dikdik Kurnia, M.Sc., Ph.D. Universitas
Padjadjaran Indonesia for all research facilities

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

No generative AI or AI-assisted technologies were used in the writing of this manuscript.

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Names of 5-7 referees

We encourage authors to submit the names of 5-7 referees (not from your own country) suitable to review the work
to aid in the peer review process.
1. EL-ASHRY, El Sayed H. , University of Alexandria, Egypt
2. HEMMATEENEJAD, Bahram, Shiraz University, Iran
3. SINGH, Ajaya Kumar, Govt. V. Y. T. PG. Autonomous College, India
4. KANNAN, Narayanan, Asian Institute of Medicine, Science & Technology, Bedong, Malaysia
5. ISMAIL, Ahmad, Universiti Putra Malaysia, Malaysia
Trends Sci. 202x; xx(xx): xxxxx 18 of 22
Trends Sci. 202x; xx(xx): xxxxx 19 of 22
Table 1. The results of the inhibition test of N. sativa seed methanol extract against three oral pathogenic bacteria
Chlorhexidine
1% 2% 3% 4% 5%
No. Bacteria (2%)
M±SD M±SD M±SD M±SD M±SD M±SD
1 S. mutans ATCC 25175 nd nd nd 6.9±0.15 10.5±0.1 17.8±0.1
2 S. sanguinis ATCC 10556 nd nd nd 8.7±0.05 8.9±0.05 21.9±0.05
3 E. faecalis ATCC 29212 11.2±0.1 12.2±0.05 13.2±0.05 14.5±0.05 15.9±0.05 17.3±0.05
Abbreviation: nd (Note Determine), mm (millimeters), M (mean), SD (Standard Deviation)

Table 2. Inhibition zone test results of fractions 1-11 of N. sativa seed methanol extract against three oral pathogenic bacteria
Inhibition Zone (mm) at 5% Concentration
Chlorhexidine
No. Bacteria 1 2 3 4 5 6 7 8 9 10 11
(2%)
M±SD M±SD M±SD M±SD M±SD M±SD M±SD M±SD M±SD M±SD M±SD
M±SD
S. mutans
1 nd 7.5±0.1 8.9±0.1 8.3±0.05 7.3±0.1 7.7±0.1 7.2±0.1 - - - - 17,8±0.0
ATCC 25175
S. sanguinis
2 nd 9.5±0.1 9.0±0.15 9.0±0.1 8.9±0.05 9.6±0.1 9.7±0.1 8.6±0.15 8.4±0.05 8.7±0.1 8.8±0.15 21,9±0.0
ATCC 10556
E. faecalis
3 nd nd 13.4± 14.4±0.1 nd nd 8.6±0.1 nd nd nd 10.9±0.15 17,25±0.0
ATCC 29212
Abbreviation: nd (Note Determine), mm (millimeters), - (no inhibition), M (mean), SD (Standard Deviation)
Trends Sci. 202x; xx(xx): xxxxx 20 of 22

Table 3 Chemical shift data of 1H, 13C- and 2D-NMR of compound 1

c HMBC
No.C H (ppm) COSY
(ppm) (Σ𝐇; multiplicity; J (Hz))
2
J 3
J
1 13.8 0.72 (3H; s) - - -
2 16.6 1.00 (3H; s) - - -
3 17.8 0.82 (3H; s) - - -
4 17.9 1.29 (3H; s) - H-1 -
5 18.0 1.29 (3H; s) - - -
6 18.8 1.29 (2H; d; 6) - - -
7 23.9 1.67 (1H; d;6) - - -
8 24.1 0.82 (3H; s) - - -
9 24.5 1.78 (2H; q; 12) - - -
10 26.4 0.94 (3H; s) - - -
11 26.6 3.65 (2H; d; 12) - - -
12 28.9 1.92 (2H; t; 12) - - -
13 31.6 - H-8, H-13 - -
14 33.3 1.71 (2H; t; 12) - - -
15 33.3 1.28 (1H; d; 12) - - -
16 33.5 1.6 (1H; d; 12) - - -
17 34.8 1.19 (3H; s) - - -
18 37.6 - H-2 - -
19 39.7 3.27 (2H; t; 6) - - -
20 40.57 - H-3, H-5 - -
21 42.47 3.35 (2H; t; 6) - - -
22 43.9 - - - -
23 43.9 - - - -
24 47.2 3.27 (2H; t; 6) - - H-25
25 48.0 1.63 (2H; d; 6) - H-2 H-24
26 48.1 - - - -
27 48.2 2.89 (1H; d; 6) - H2 -
28 49.8 3.93 (1H; d; 6) - - -
29 61.8 3.86 (2H; d; 6) - - -
30 64.6 3.79 (2H; d; 6) - - -
31 69.8 3.55 (2H; d; 6) - - -
32 66.9 3.72 (1H; s) - - -
33 70.6 3.42 (1H; s) - - -
34 70.8 3.93 (1H; d; 6) - - -
35 70.9 3.99 (1H; d; 6) - - -
36 71.0 3.48 (1H; d; 6) - - -
37 72.1 3.44 (1H; d; 6) - - H-57
38 72.3 3.59 (1H; d; 12) - - -
39 72.7 3.65 (1H; d; 6) - - -
40 737 3.68 (1H; d; 6) - - H-53
41 73.7 3.09 (1H; d; 6) - - -
42 74.1 4.10 (1H; d; 12) - - -
43 75.2 3.59 (1H; d; 12) - - -
44 76.1 3.59 (1H; d; 12) - - -
45 76.6 3.59 (1H; d; 12) - - -
46 76.7 3.59 (1H; d; 12) - - -
47 77.5 3.53 (1H; d; 12) - - -
48 77.9 3.68 (1H; d; 6) - - -
Trends Sci. 202x; xx(xx): xxxxx 21 of 22

Table 4 Chemical shift data of 1H, 13C- and 2D-NMR of compound 1

c H (ppm) HMBC
No.C 2 3 COSY
(ppm) (Σ𝐇; multiplicity; J (Hz)) J J
48 77.9 3.68 (1H; d; 6) - - -
49 78.1 3.59 (1H; d; 12) - - -
50 79.5 3.53 (1H; d; 12) - - -
51 82.0 3.68 (1H; d; 6) - - -
52 82.3 3.59 (1H; d; 12) - - -
53 95.7 5.35 (1H; d; 6) - H-30 H-40
54 101.3 5.27 (1H; d; 12) - H-28 -
55 102.7 4.42 (1H; d; 6) - H-52 -
56 104.0 4.53 (1H; d; 12) - H-10 -
57 104.6 4.50 (1H; d; 6) - H-30, H-29 H-37
58 123.7 3.90 (1H; m; 6) - - -
59 144.0 - - - -
60 178.0 - - H-53 -
Trends Sci. 202x; xx(xx): xxxxx 22 of 22

Table 5. MIC and MBC test results of Anhuienoside E (1) against three oral pathogenic bacteria
MIC (μg⁄mL) MBC( μg⁄mL)
No. Bacteria
M±SD M
1 S. mutans ATCC 25175 625±0.05 1000
2 S. sanguinis ATCC 10556 625±0.05 1000
3 E. faecalis ATCC 29212 625±0.1 -
Abbreviation: MIC (minimum inhibition concentration), MBC (minimum bactericidal concentration), - (inactive) M (mean), SD (Standard Deviation)

Table 6. ADMET analysis results of compounds 1-9

Properties Parameters 1 2 3 4 5 6 7 8 9
Intestinal Absorption (% Absorbed) 0 0 0 0 19.964 0 0 10.885 100
Absorption
Water Solubility (log mol/L) -2.886 -2.78 -2.859 -2.892 -2.795 -2.891 -2.892 -2.884 -3.006
BBB Permeability (log BB) -2.567 -2.339 -2.038 -2.631 -1.542 -2.292 -2.441 -1.695 -0.152
Distribution CNS Permeability (log PS) -5.931 -5.048 -4.792 -5.935 -4.257 -5.05 -5.379 -3.829 -1.084
Volume Distribution (VDss) (log L/Kg) -0.161 -0.282 -0.217 -0.002 -0.261 -0.093 -0.06 -0.387 -1.182
CYP1A2 No No No No No No No No No
CYP2D6 No No No No No No No No No
Metabolism Inhibitor of CYP2C9 No No No No No No No No No
CYP2C19 No No No No No No No No No
CYP3A4 No No No No No No No No No
Excretion Total Clearance log mL/Min/Kg -0.023 -0.016 -0.057 -0.055 0.008 -0.091 -0.016 -0.1 -0.081
Acute Oral Toxicity Lethal Dose 50% mg/Kg 2.482 2.483 2.488 2.508 2.588 2.482 2.484 2.497 2.861

Table 7. Drug-Likeness Analysis of Lipinski's Rule of Five (RO5) compounds 1-9

Rule Parameter 1 2 3 4 5 6 7 8 9
Molecular mass (Less than 500 Dalton) (g/mol) 1234 1058 1058 1366 927 1189 1205 881 456
Hydrogen bond acceptors (Less than 10) 25 21 20 29 17 24 25 15 3
Hydrogen bond donor (Less than 5) 14 12 12 17 10 14 15 9 2
Lipinski’s
Molar Refractivity (40–130) 294.68 258.65 257.56 321.17 232.23 288.78 289.95 225.59 136.65
Rule of Five
LogP (Less than 5) -1.13 -0.37 -0.1 -3.42 1.17 -1.24 -2.27 2.65 7.23
Violation 4 4 4 4 4 4 4 4 2
Drug-likeness No No No No No No No No Yes