Trends Sci. 2025; 22(10): 10615

Exploring Antibacterial Potential of Anhuienoside E from Nigella sativa Linn:

A Promising Candidate Against Dental Caries In Vitro and In Silico Studies

Rizal Padilah¹, Dikdik Kurnia^1,*, Tri Mayanti¹ and Denny Nurdin²

¹Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,

West Java 45363, Indonesia

²Department of Conservative Dentistry, Faculty of Dentistry, Universitas Padjadjaran, Jawa Barat 40132, Indonesia

(^*Corresponding author’s e-mail: [email protected])

Received: 9 May 2025, Revised: 4 June 2025, Accepted: 15 June 2025, Published: 15 July 2025

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 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].

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

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% - 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 11 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 F_254S 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 F_254S 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. Huang [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].

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) Evangelina et al. [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].

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% - 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 h 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 h 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 h 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.

In silico molecular docking

Preparation of docking

The enzymes used in molecular docking were MurA and Gtfs which have PDB ID: 8FKL Schormann et al. [25] and 1UAE Kurnia et al. [26] whose 3D structures were downloaded on the website www.rcsb.org. Conformation validation of MurA and Gtf enzymes using Ramachandran Plot [27]. The 2 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.

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. 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

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 [30-32].

Results and discussion

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 3 bacteria (test results are in Table 1) which indicated the compound components potentially as antibacterial agent [33]. The methanol extract was further fractionated into 11 fractions and tested for antibacterial activity. Based on the results of antibacterial testing of the 11 fractions, it showed that fraction 7 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].

Table 1 The results of the inhibition test of N. sativa seed methanol extract against 3 oral pathogenic bacteria

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 3 oral pathogenic bacteria.

Abbreviation: nd (Note Determine), mm (millimeters), - (no inhibition), M (mean), SD (Standard Deviation)

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].

Chemical shift of carbon compound (1) (brown solids, 100 mg) showed ¹³C-NMR (CD₃OD, 700 MHz) 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) 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 Tables 3 and 4.

Based on the ¹³C-NMR data, compound 1 contained 60 types of carbon. DEPT, ¹H-NMR spectrum and HSQC correlation showed compound 1 contained 8 quaternary carbons, 5 methine carbons, 24 oxidized methine carbons, one sp² methine, 9 methyl, and thirteen methylene. HMBC spectrum shows proton to-carbon correlation as far as 2 to 5 bonds and the COSY spectrum shows proton to proton correlation as far as 3 bonds are suspected of compound 1 being a burnt triterpenoid group [38]. Carbon 60 and carbon 52 experienced the addition of sugar substituents as many as 5 pieces shown by the presence of carbon and anomeric protons as many as 5. Three sugars namely glucose’- glucose”-rhamnose’ were bound to carbon 60 and 2 glc-rha sugars at carbon 52. The placement of the number and type of sugar was based on the HMBC correlation of the anomeric proton to carbon 60 and 52 proton to anomeric carbon. Shi et al. [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 12. 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 Figures S(1) - S(7)) [43].

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

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

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 and MBC of 1000 [44]. Anhuienoside E (1) showed that did not give activity for killing E. faecalis but only gave inhibition activity. Anhuienoside E had 5 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.

Table 5 MIC and MBC test results of Anhuienoside E (1) against 3 oral pathogenic bacteria.

Abbreviation: MIC (minimum inhibition concentration), MBC (minimum bactericidal concentration), - (inactive) M (mean), SD (Standard Deviation).

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).

Figure 3 Chemical structure of Anhuienoside D (derivate 2), derivate 8, Anhuienoside F (derivate 4), and Oleanolic acid (derivate 9).

Figure 4 Chemical structure of Anhuienoside E (1), Flaccisdoside II (derivate 6), Flaccisdoside III (derivate 7), chlorhexidine and Apigenin (positive control).

Molecular docking

Molecular docking was performed on Anhuienoside E (1) and derivates on 2 key enzymes from caries-causing bacteria (the structures are shown in Figures 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 2 coordinate torsions ϕ and ψ (ψ 180, ϕ > 0). Based on the Karplus equation, the Ramachandran plot was divided into 4 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.

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: