Secondary Metabolites from Piper crocatum as Natural Inhibitors of Oral Pathogens: In Vitro and In Silico Insights into Streptococcus mutans and Candida albicans

doi:10.21203/rs.3.rs-7679103/v1

Background

Dental caries is a widespread infectious disease caused by complex interactions between microorganisms in oral biofilms. Streptococcus mutans plays a central role through acid production and glucan-mediated biofilm formation, while Candida albicans enhances biofilm stability and tissue damage. Their virulence factors, glucosyltransferase B (GtfB) and secreted aspartyl proteinase 5 (Sap5), are key molecular targets for therapeutic intervention. Natural compounds offer safer alternatives to synthetic antimicrobials in caries prevention.

Methods

Two bioactive compounds isolated from Piper crocatum were evaluated against S. mutans and C. albicans. In vitro assays included antibacterial, antifungal, and antibiofilm testing. In silico molecular docking and dynamics simulations were performed to examine compound interactions with GtfB and Sap5 enzymes.

Results

Both compounds inhibited microbial growth and reduced biofilm formation in vitro. Computational analysis revealed strong binding affinities and stable interactions with GtfB, supporting their potential to interfere with glucan-mediated adhesion and proteolytic activity.

Conclusion

The tested compounds demonstrated promising antibacterial, antifungal, and antibiofilm effects against S. mutans and C. albicans. Their dual targeting of GtfB and Sap5 highlights their potential as natural agents for dental caries prevention. Further in vivo studies are required to validate these findings.

Dental caries

Streptococcus mutans

Candida albicans

in vitro

in silico

Dental caries is one of the most prevalent chronic infectious diseases worldwide and remains a major public health concern, particularly in developing countries [1, 2]. This condition is characterized by the demineralization of dental hard tissues caused by the activity of plaque biofilm microorganisms that ferment carbohydrates into acids [3]. For decades, Streptococcus mutans has been recognized as the primary etiological agent of dental caries due to its acidogenicity, aciduricity, and ability to form biofilms through glucan production from sucrose [4].

However, recent evidence has highlighted that caries pathogenesis is polymicrobial and not solely attributed to bacteria [5, 6]. Candida albicans, an opportunistic fungus commonly present in the oral cavity, has also been implicated in the initiation and progression of caries, particularly in children and immunocompromised patients [7]. The coexistence of C. albicans and S. mutans within dental plaque enhances biofilm structural integrity, increases tolerance to acidic environments, and exacerbates tooth tissue destruction [8]. This synergistic interaction is mediated by glucans produced by the glucosyltransferase B (GtfB) enzyme of S. mutans, which facilitate the adhesion and colonization of C. albicans through surface recognition and the formation of a complex biofilm matrix [9, 10].

In addition, C. albicans possesses specific virulence factors such as secreted aspartyl proteinase 5 (Sap5), a proteolytic enzyme involved in tissue invasion, host protein degradation, and biofilm development [11, 12]. Thus, inhibiting the activity of these virulence enzymes represents a promising strategy for caries prevention and control.

Synthetic antimicrobial agents are commonly used to inhibit microbial growth and biofilm formation [13, 14]. However, their application is often associated with side effects such as antimicrobial resistance, mucosal irritation, and disruption of the oral microbiota balance [15, 16]. Consequently, natural bioactive compounds have attracted increasing interest as safer and more environmentally friendly therapeutic alternatives [17, 18]. In this regard, mouthwash formulations are considered advantageous because topical application enables direct interaction with biofilms at the infection site, ensuring effective concentrations while minimizing systemic side effects compared to oral administration [19–21].

In this study, two compounds isolated from Piper crocatum were evaluated for their antimicrobial and antibiofilm activities against S. mutans and C. albicans through both in vitro and in silico approaches. The in vitro assays encompassed antibacterial and antifungal testing, while the in silico analysis focused on assessing the binding affinity of these compounds toward GtfB and Sap5, the key virulence enzymes involved in caries pathogenesis. This study aims to explore the potential of these compounds as natural antibiofilm agents for the prevention and management of dental caries.

General experimental procedures

NMR spectra were recorded using a Bruker 700 MHz instrument. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained with a Xevo G2-XS QTOF mass spectrometer (Waters Corp.), integrated with a Waters ACQUITY UPLC system (Waters Corp., Milford, MA). Column chromatography (CC) was performed using RP-18 gel (63–212 µm, Fujifilm Wako, Osaka, Japan) and Silica Gel (0.063–0.2 mm, Merck, Darmstadt, Germany). For thin layer chromatography (TLC), precoated silica gel plates RP-18 F254S (0.25 mm, Merck) and 60 F254 (0.25 mm, Merck) were employed. Detection of sample spots was carried out by spraying with 10% aqueous sulfuric acid followed by heating [22].

Plant sample and identification

Fresh leaves of Piper crocatum (Ruiz and Pav.) were collected in September 2022 from a private property in Cikarang Barat, Bekasi, West Java, Indonesia, with permission from the landowner. The plant material was taxonomically identified by Dr. Budi Irawan, M.Si, at the Biosystematics and Molecular Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang, Indonesia (identification number: 93/LBM/IT/9/2022). A voucher specimen (#113) has been deposited at the Herbarium Jatinangor, Plant Taxonomy Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jatinangor, Indonesia. The plant samples were sectioned into smaller pieces and initially air-dried in a shaded area [23].

Extraction and isolation

To obtain the pure compound, 15 kg of dried, chopped P. crocatum leaves were macerated in 150 L of 70% methanol at room temperature (25°C) for 72 h. The resulting methanolic extract was filtered and concentrated under reduced pressure using a Buchi Rotavapor R-100 (Switzerland), yielding 25 g of crude extract. This concentrated extract was then fractionated by column chromatography on Silica G 60 (particle size 0.063–0.200 mm), using a solvent system of n-hexane and ethyl acetate with a stepwise 10% gradient, starting from 100% n-hexane to 100% ethyl acetate, resulting in eleven fractions (Fr. 1–11). Guided by bioactivity, each fraction was tested for antimicrobial activity against S. mutans, and C. albicans using the disc diffusion method [24, 25].

Fractions 5 and 7 exhibited strong activity against both tested microorganisms. Fr. 5 (2305.7 mg) was further separated by column chromatography on Silica G 60, using a 2.5% gradient elution of n-hexane–ethyl acetate from 100% n-hexane to a 6:4 ratio, resulting in 17 subfractions (Fr.5.1 – Fr.5.17). Compound 1 (26 mg) was isolated from subfraction F5.10 and purified by recrystallized from n-hexane. Meanwhile, Fraction 7 (1933 mg) was separated using column chromatography on ODS RP-18, eluted with methanol – water at a 2.5% gradient from 100% methanol to 100% water, yielding 41 subfractions (Fr. 7.1–Fr. 7.41). Subfraction Fr. 7.3 was further purified by column chromatography on ODS RP-18 using a 1% gradient of methanol–water (from 10:0 to 8:2), from which Compound 2 (20 mg) was obtained from subfraction F7.3.16.

In vitro antimicrobial activities

Microbial strains and media

S. mutans ATCC 25175 and C. albicans ATCC 10231 from the American Type Culture Collection (ATCC) were used in this study. For bacterial cultivation and assays, Mueller-Hinton agar (MHA) and brain heart infusion (BHI) media were utilized. C. albicans was cultured using potato dextrose broth and potato dextrose agar (PDA), both obtained from Sigma-Aldrich (USA). Prior to testing, bacterial stock cultures were incubated in their respective media at 37°C for 24 hours. The bacterial suspension turbidity was then standardized to 0.5 McFarland using a Biochrom microplate reader at a wavelength of 620 nm, resulting in a final concentration of 10⁷ CFU/mL [26, 27].

Disk diffusion test

Antibacterial activity of the fractions (Fr. 1–11) and the isolated compound were evaluated using the disk diffusion method [28]. The sample was prepared at a concentration of 2%, with chlorhexidine (Sigma-Aldrich, USA) as the positive control. A 5 mL aliquot of brain heart infusion (BHI) medium (Sigma-Aldrich, USA) was inoculated with 100 µL of bacterial suspension and incubated at 37°C for 48 hours. After incubation, the bacterial turbidity was standardized to the 0.5 McFarland standard (approximately 10⁷ CFU/mL) using spectrophotometric measurement at 620 nm. Subsequently, 100 µL of the standardized bacterial suspension was evenly spread on the surface of the agar medium (Sigma-Aldrich, USA). Paper disks (6 mm diameter, Grainger Approved, Origin, USA) were loaded with 20 µL of each test fraction or chlorhexidine and placed onto the inoculated agar plates. The plates were then incubated at 37°C for 24 hours to observe zones of inhibition [29].

MIC and MBC determination

The broth microdilution method was employed to determine the MIC values for compounds 1 and 2. For the MIC assay, 100 µL of broth medium was added to each well of a 96-well microplate (NEST Biotechnology, Wuxi, China). Wells in columns A-1, B-1, E-1, and F-1 added 100 µL of solvent, while wells in columns C-1, D-1, G-1, and H-1 were added with 100 µL of compound 1 at an initial concentration of 2500 µg/mL. Serial dilutions were conducted across columns 1 to 12. Subsequently, 5 µL of the bacterial and fungal suspension was added to wells in columns E through H. Following a 48-hour incubation at 37°C, optical density was measured at 620 nm using a microplate reader (Biochrom Ltd., Cambridge, UK). To determine the minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC), aliquots from wells containing the test compound, media, and bacterial suspension (columns G and H) were plated onto agar medium and incubated for an additional 48 hours at 37°C [30, 31].

Molecular docking of compounds with target proteins

Molecular docking was initiated with the preparation of the target receptors, namely glucosyltransferase B (PDB ID: 8FG8), serine-rich protein A (PDB ID: 5EQ3), and secreted aspartyl protease 5 (Sap5, PDB ID: 2QZX), which were retrieved from the RCSB Protein Data Bank in .pdb format. Receptors were separated from their native ligands using Discovery Studio, followed by the removal of water molecules, non-essential metal ions, and cofactors. The cleaned receptor structures were then saved in .pdb format, and subsequently processed in AutoDock by adding Kollman charges and polar hydrogens before being converted into .pdbqt format. Ligand preparation involved isolating the native co-crystallized ligands for redocking as well as constructing the isolated compounds using ChemDraw 3D, followed by energy minimization with the MM2 force field to obtain the most stable conformation, and then saved as .pdb files. Docking validation was performed by redocking the native ligands into the active sites of the receptors, with grid box dimensions and coordinates set according to each enzyme (GtfB: x = − 9.725, y = 18.09, z = 6.804, 40 × 40 × 40 Å; Sap5: x = 34.912, y = 7.487, z = 61.269, 44 × 44 × 44 Å). The docking protocol was considered valid when the RMSD value was less than 2.0 Å. Docking simulations were then carried out using the Genetic Algorithm with 100 runs, and the results were generated in dock.dlg files after autogrid and autodock execution [32].

Molecular dynamics simulation

Partial atomic charges of compounds 1–3 were calculated using the semi-empirical Austin Model 1-Bond Charge Correction (AM1-BCC) quantum mechanics method, implemented in the AmberTools21 antechamber module. Topologies for the ligands were generated using Generalized Amber Force Fields 2 (GAFF2), and the complexes were dissolved in a 10 Å grid with TIP3P water molecules. Charge neutralizing ions (Na⁺ and Cl-) were added to achieve a salt concentration of 0.15 M using the tleap utility in AmberTools21 [33]. Molecular dynamics simulations for each protein-ligand complex were performed using GPU-accelerated Particle-Mesh Ewald Molecular Dynamics (PMEMD) with periodic boundary conditions on Amber2027 software. The procedure starts with two sequential energy minimization steps. At first, a positional restraint of 25 kcal mol-¹ Å-² is applied to the protein-ligand complex, followed by a reduction of the restraint to 5 kcal mol-¹ Å-² in the second step. The system temperature was then raised to 300 K under the NVT (Number-Volume-Temperature) ensemble for 50 ps. Next, the simulation switches to the NPT (Number-Pressure-Temperature) ensemble to equilibrate the system density to 1 g cm-³ for 50 ps. During the subsequent NVT equilibration, the restraint on the solute is gradually reduced by 1 kcal mol-¹ Å-² every 50 ps until it is completely removed. Production molecular dynamics simulations were performed for 100 ns at 300 K under NPT conditions to generate trajectories for each system. Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) analyses were performed using the R programming language to evaluate complex stability and potential inhibition during simulation [34].

Identification of isolated compound

Compound 1 is a yellow crystal, showing fluorescence at UV λ254 and 365 nm, and appears as a yellow stain with 10% sulfuric acid spray and heating, shown in S-1 (Supplementary Data) UV (MeOH) λmax: 358 and 208.9 nm. IR (KBr pellet) νmax: 3788, 3249, 2946, 1664, and 1593 cm^-1. TQD-MS with negative ionization mode (ESI-) showed m/z 343.93. The ¹H-NMR (CDCl₃, 700 MHz) showed signals at δ_H 12.64 ppm (1H, s, 5-OH), 7.71 ppm (1H, dd, J = 1.98 Hz, H-2′), 7.68 ppm (1H, dd, J = 2.02, 8.4 Hz, H-6′), 6.46 ppm (1H, d, J = 2.2 Hz, H-8), 6.37 ppm (1H, d, J = 2.5 Hz, H-6), 3.98 ppm (3H, s, 3′-OMe), 3.88 ppm (3H, s, 7-OMe), and 3.86 ppm (3H, s, 3-OMe). The spectrum of ¹³C-NMR (CDCl₃, 175 MHz) displayed 18 chemical shifts at δ_C 178.9 (C-4), 165.6 (C-7), 162.2 (C-5), 156.9 (C-2), 156.1 (C-9), 148.5 (C-4′), 146.5 (C-3′), 139.0 (C-3), 122.8 (C-6′), 122.6 (C-1′), 114.7 (C-5′), 111.0 (C-2′), 106.2 (C-10), 98.0 (C-6), 92.3 (C-8), 60.3 (3-OMe), 56.3 (3′-OMe), and 56.0 (7-OMe).

The ¹³C-NMR and DEPT 135° spectra of compound 1 displayed 18 carbon signals, including one carbonyl (δ_C 178.9 ppm), seven oxygenated aromatic quaternary carbons (δ_C 162.2-178.9 ppm), one non-oxygenated sp² quaternary carbon (δ_C 122.6 ppm), five sp² methine carbons (δ_C 92.3-122.8 ppm), and three oxygenated methyl carbons (δ_C 55.8-60.2 ppm). The ¹H-NMR spectrum showed a singlet at δ_H12.64 ppm, typical of a chelated hydroxyl group, along with aromatic proton signals consistent with ortho- and meta-substituted protons, and three methoxy singlets at δ_H 3.98, 3.88, and 3.86 ppm. These features are characteristic of a flavonoid with a flavone backbone, further supported by 2D NMR data. The 1D and 2D NMR spectra were consistent with the data reported in the literature [35, 36], thereby confirming the identity of the compound as pachypodol. The chemical structures of pachypodol (1) are shown in Fig. 1.

Identification of Compound 2

Compound 2 is a colorless oil, showing fluorescence at UV light λ254 nm, and appears as a yellow stain with 10% sulfuric acid spray and heating (See Supplementary Data S-2) UV (MeOH) λ_max: 267.9 and 208.9 nm. IR (KBr pellet) ν_max: 3436, 3076, 2938, 1686, 1589, 1509, 1288, and 1125 cm^-1. TQD-MS with negative ionization mode (ESI-) showed m/z 417.28. The NMR spectrum of compound 2 (CDCl₃; ¹H-NMR 700 MHz; ¹³C-NMR 175 MHz) showed ¹H signals at δ_H 6.28 (1H, s, H-2), 6.26 (1H, s, H-6), 3.33 (1H, d, J = 6.5 Hz, H-7), 1.30 (3H, d, J = 7.1 Hz, H-9), 4.01 (1H, s, H-4′), 6.14 (1H, s, H-6′), 2.77 (2H, d, J = 7.0 Hz, H-7′), 5.90 (1H, ddt, J = 10.2, 17.0 Hz, H-8′), and 5.21–5.30 (2H, m, H-9′). The methoxy protons resonated at δ 3.68 (3H, s, 3-OMe), 3.78 (3H, s, 5-OMe), 3.77 (3H, s, 4-OMe), 3.78 (3H, s, 3′-OMe), and 3.43 (3H, s, 5′-OMe). The ¹³C-NMR spectrum displayed signals at δC 192.4 (C-2′), 153.1 (C-3, C-5), 137.2 (C-1′, C-4), 134.6 (C-8′), 133.8 (C-1), 125.2 (C-6′), 119.0 (C-9′), 106.2 (C-2, C-6), 95.9 (C-3′, C-5′), 79.1 (C-4′), 59.6 (C-7), 49.3 (C-8), 35.0 (C-7′), and 17.7 (C-9), together with methoxy carbons at δC 61.0 (4-OMe), 56.1 (5-OMe, 3′-OMe), 55.6 (3-OMe), and 54.8 (5′-OMe).

The ¹³C-NMR and DEPT 135° spectra of compound 2 revealed 23 carbons, including eight quaternary carbons (two sp³ at δ_C 49.2 and 95.9 ppm, and six sp² at δ_C 133.8, 137.2, 152.7, 153.1 (2C), and 192.4 ppm), two methylenes at δ_C 35.1 and 119.2 ppm, seven methines (δ_C49.3, 59.6, 79.1, 106.2 (2C), 125.5, and 134.6 ppm), and six methyl groups comprising one sp³ (δ_C 17.7 ppm) and five methoxy carbons (δ_C 54.8, 55.6, 56.1 (2C), and 61.0 ppm). The ¹H-NMR spectrum showed resonances corresponding to five methoxy groups (δ_H 3.43, 3.68, 3.77, 3.78 (2×) ppm), aromatic/olefinic protons (δ_H 6.14, 6.26, 6.28 ppm), and a methyl doublet at δ_H 1.30 ppm (J = 7.1 Hz), along with methine and allylic signals at δ_H 3.33, 4.01, 5.21, 5.30, and 5.90 ppm. Further NOESY analysis revealed stereochemical correlations among protons at C-4, C-16, C-11, and C-9. The 1D and 2D NMR spectra were consistent with the data reported in the literature [37], thereby confirming the identity of the compound as nectamazin B. Its chemical structure is shown in Fig. 1.

Antimicrobial activity assay of compounds

Investigation of the antimicrobial effects of pachypodol (1) and nectamazin B (2) against S. mutans and C. albicans was carried out through Kirby-Bauer agar diffusion, MIC and MBC assay. The MIC value is referred to as strong category if MIC 101-500, moderate if 501-1000, and weak if MIC > 1000 μg/mL [38, 39].

The findings, as displayed in Table 1, indicates that compounds 1 and 2 have strong inhibitory activity against the two microbes. The MBC values exceed the MIC, which clearly indicates that this compound possesses bacteriostatic properties rather than being bactericidal. This suggests that both pachypodol and nectamazin B is capable of inhibiting S. mutans and C. albicans growth without eliminating the bacteria, thereby exhibiting a growth-inhibitory effect.

Table 1 In vitro antibacterial and antifungal activity of pachypodol and nectamazin B from P. crocatum against S. mutans and C. albicans

Compound	Microorganism	Inhibition zone (mm)	MIC (µg/mL)	MBC/MFC (µg/mL)
1	S.mutans ATCC 25175	10.2	156.25	1250
1	C. albicans ATCC 10231	9.0	156.25	625
2	S.mutans ATCC 25175	10.67	156.25	625
2	C. albicans ATCC 10231	9.61	156.25	625

Molecular docking study

The docking results of pachypodol (1) and nectamazin B (2) against C. albicans virulence proteins (Gtf B and Sap 5) are presented in Table 2. Pachypodol exhibited the strongest binding affinity toward Gtf B with a ΔG of −8.05 kcal/mol and an inhibition constant (Ki) of 1.26 μM, which is superior compared to the native ligand (ΔG −6.85 kcal/mol, Ki 9.51 μM). In contrast, its interaction with Sap 5 was relatively weaker (ΔG −6.67 kcal/mol, Ki 12.89 μM).

Nectamazin B demonstrated moderate affinity toward Gtf B (ΔG −6.70 kcal/mol, Ki 12.2 μM), which is comparable to the native ligand. Interestingly, nectamazin B showed a stronger affinity toward Sap 5 (ΔG −7.56 kcal/mol, Ki 2.88 μM), nearly equivalent to that of the native ligand (ΔG −7.61 kcal/mol, Ki 2.63 μM). These findings suggest that pachypodol may serve as a potent inhibitor of Gtf B, while nectamazin B displays preferential activity toward Sap 5, indicating their potential as selective antifungal agents targeting distinct virulence factors of C. albicans.

Table 2 Binding affinity data and inhibition constants of compounds 1 and 2 against Gtf B and Sap 5 enzymes.

Compound	Protein Target	ΔG (kcal/mol)	Ki (µM)
Pachypodol (1)	Gtf B	-8.05	1.26
Pachypodol (1)	Sap 5	-6.67	12.89
Nectamazin B (2)	Gtf B	-6.70	12.2
Nectamazin B (2)	Sap 5	-7.56	2.88
Ligan Native	Gtf B	-6.85	9.51
Ligan Native	Sap 5	-7.61	2.63

The molecular docking interaction profiles of pachypodol (1) and nectamazin B (2) with enzymes Gtf B and Sap 5 are summarized in Fig. 2 and Fig. 3. For the Gtf B enzyme (Fig. 2), pachypodol formed multiple stabilizing interactions, including conventional hydrogen bonds with ASP A:454, TYR A:404, and HIS A:561, as well as a covalent interaction with ASP A:883, TYR A:890, and GLU A:489. Additionally, several hydrophobic contacts were observed (Pi–alkyl with LEU A:407 and ALA A:452; Pi–Pi T-shaped with TRP A:491; and alkyl with PHE A:881, VAL A:453, ALA A:490). These diverse interactions may explain the strong binding affinity of pachypodol to Gtf B (ΔG −8.05 kcal/mol, Ki 1.26 μM). In comparison, nectamazin B interacted with fewer residues. It mainly formed hydrogen bonds with ASP A:552 and TRP A:491, along with a covalent bond involving ASP A:451 and GLU A:489. The reduced number of hydrophobic and electrostatic contacts relative to pachypodol may underlie its weaker affinity toward Gtf B.

In Sap 5 enzyme (shown in Fig. 3), both compounds exhibited multiple binding interactions. Pachypodol formed conventional hydrogen bonds with TYR B:225 and ARG B:120, as well as covalent interactions with residues such as TYR B:221, ASP B:86, and SER B:118. Furthermore, hydrophobic stabilization was mediated by Pi–alkyl interactions with ILE B:123, ILE B:30, and ALA B:119. Interestingly, nectamazin B displayed a richer binding profile with Sap 5. It established three hydrogen bonds (TYR B:225, ASP B:86, and TYR B:225), as well as multiple covalent and hydrophobic interactions (e.g., LYS B:50, TRP B:51, ARG B:86). Importantly, nectamazin B also engaged in Pi–anion and Pi–hydrogen donor interactions, which may contribute to its strong binding affinity (ΔG −7.56 kcal/mol, Ki 2.88 μM), comparable to the native ligand. Overall, these findings suggest that pachypodol exhibits a higher binding strength toward Gtf B, supported by multiple hydrogen bonds and hydrophobic interactions, whereas nectamazin B preferentially binds Sap 5 through diverse hydrogen bonding, hydrophobic, and Pi interactions. This differential binding behavior highlights their potential as selective inhibitors against distinct virulence proteins of C. albicans.

Molecular dynamics

The stability and binding affinity of the protein–ligand complexes were assessed using molecular dynamics simulations combined with MM/GBSA calculations. The RMSD trajectories (Fig. 4a) indicated that the GtfB–native ligand and GtfB–compound 1 (Pachypodol) complexes remained relatively stable throughout the 100 ns simulation, with mean RMSD values of 2.096 ± 0.325 Å and 1.823 ± 0.312 Å, respectively. In contrast, the GtfB–compound 2 (Nectamazin B) complex exhibited higher structural deviations (2.983 ± 0.410 Å), reflecting reduced stability.

Residue-level flexibility, as shown in the RMSF profiles (Fig. 4b), revealed that both the native ligand and compound 2 complexes followed similar fluctuation patterns across most residues, while compound 1 induced localized flexibility in loop regions. These shifts may reflect conformational adjustments that facilitate stronger binding interactions.

The energetic stability of the complexes was further evaluated using MM/GBSA analysis (Table 5). Compound 1 displayed the most favorable binding free energy (−28.223 ± 5.394 kcal/mol), significantly stronger than both the native ligand (−7.092 ± 4.877 kcal/mol) and compound 2 (−15.042 ± 3.020 kcal/mol). To explore binding stability over time, MM/GBSA binding free energy values were computed using a 10-ns sliding window approach (Fig. 5). Compound 1 complex consistently exhibited the lowest ΔG values, remaining between −30 and −20 kcal/mol for most of the trajectory, highlighting its robust binding affinity. In contrast, compound 2 showed moderate stability with binding energies fluctuating around −15 to −20 kcal/mol, while the native ligand displayed weaker and more variable interactions, with values often closer to −10 kcal/mol.

Taken together, the RMSD, RMSF, and MM/GBSA results consistently demonstrate that compound 1 (Pachypodol) forms the most stable and energetically favorable complex with GtfB, outperforming both the native ligand and compound 2 (Nectamazin B).

Table 5 RMSD and MMGBSA value of complex protein and ligand after 100 ns molecular dynamics simulation

Complex (Protein-Ligand)	RMSD (Å)		MMGBSA (kcal/mol)
Complex (Protein-Ligand)	Mean SD	Median MAD	Mean SD	Median MAD
Gtf B - Native	2.096 0.325	2.078 0.314	-7.092 4.877	-5.832 5.313
Gtf B - Compound 1	1.823 0.312	1.841 0.305	-28.223 5.394	-29.516 3.372
Gtf B - Compound 2	2.983 0.410	2.994 0.361	-15.042 3.020	-13.983 2.373

The hydrogen bonding profiles of the complexes were also analyzed to evaluate the stability of ligand–protein interactions over the simulation period (Fig. 6). Compound 1 maintained between one and three hydrogen bonds consistently throughout the trajectory, with intermittent formation of up to four bonds, indicating stable and persistent interactions within the binding pocket. In contrast, compound 2 formed a higher number of hydrogen bonds overall, but these interactions were more fluctuating and less stable, suggesting weaker binding persistence despite frequent contacts. The native ligand showed fewer hydrogen bonds, typically fluctuating between zero and two, with intermittent peaks up to three, reflecting less stable hydrogen-bonding interactions compared to compound 1 and compound 2.

These findings highlight that although compound 2 exhibited a greater number of hydrogen bonds, their dynamic instability reduces their overall contribution to binding affinity. Conversely, compound 1 formed fewer but more stable hydrogen bonds, complementing its favorable MM/GBSA binding energy and lower RMSD, thereby reinforcing its potential as the most stable binder among the tested ligands.

ADMET and drug-likeness

The ADMET predictions of the lead compounds provided further insights into their potential suitability as oral care agents (Table 6). Both compound 1 (Pachypodol) and compound 2 (Nectamazin B) showed good intestinal absorption as indicated by favorable Caco-2 permeability values, suggesting that they could efficiently permeate epithelial membranes if administered systemically. However, for a mouthwash formulation, local retention and safety are more critical than systemic absorption.

In terms of distribution, neither compound was predicted to cross the blood–brain barrier (BBB−), which minimizes potential central nervous system side effects. Both exhibited high plasma protein binding (>85%), although this parameter is less relevant for topical use in oral formulations. With respect to metabolism, the two compounds showed distinct profiles. Pachypodol was predicted to be a substrate and potential inhibitor of CYP1A2, while Nectamazin B was associated with CYP3A4 interaction. Although such interactions are important in systemic drug design, they are of lower concern for a mouthwash, as the formulation is not intended for extensive systemic circulation.

Excretion analysis indicated low clearance values and moderate half-lives for both compounds, suggesting reasonable metabolic stability. Toxicity assessment revealed low LD50 values for both ligands, classifying them as compounds with relatively low acute toxicity risk. Neither compound was predicted to be hepatotoxic or mutagenic in the Ames test. Importantly, Pachypodol was classified as non-sensitizing and non-immunotoxic, making it safer for repeated oral exposure. In contrast, Nectamazin B showed a potential for skin sensitization and immunotoxicity, raising safety concerns for topical use.

Taken together, the ADMET evaluation suggests that Pachypodol exhibits a more favorable pharmacological and safety profile for development as an oral rinse agent compared to Nectamazin B, aligning well with its stronger binding stability and favorable interaction patterns observed in molecular dynamics simulations.

Table 6 Predicted ADMET properties of Pachypodol (1) and Nectamazin B (2) based on in silico analysis, including absorption, distribution, metabolism, excretion, and toxicity parameters

Properties		Pachypodol (1)		Nectamazin B (2)
Properties		Value	Interpretation	Value	Interpretation
Absorption	Caco-2	-5,118	Good permeability	-4,993	Good permeability
Distribution	BBB	0,001	BBB-	0,685	BBB-
Distribution	PPB	96,172	High plasma protein binding	88,605	High plasma protein binding
Metabolism	CYP1A2	0,999	Substrate and potential inhibitor	0,003	Not a substrate or inhibitor
	CYP2D6	0,054	Not a substrate or inhibitor	0,011	Not a substrate or inhibitor
	CYP3A4	0,008	Not a substrate or inhibitor	0,901	Substrate and potential inhibitor
	CYP2B6	0,026	Not a substrate or inhibitor	0,0	Not a substrate or inhibitor
Excretion	Clearance	4,708	Low clearance	7,197	Low clearance
Excretion	T1/2	1,308	Moderate half-life	1,428	Moderate half-life
Toxicity	LD50	0,427	Low toxicity	0,633	Low toxicity
	Hepatotoxicity	0,404	Non-hepatotoxic	0,423	Non-hepatotoxic
	Mutagenicity (Ames)	0,588	Non-mutagenic (Ames test)	0,314	Non-mutagenic (Ames test)
	Skin sensitization	0,54	Non-sensitizer	0,93	Potential sensitizer
	Immunotoxicity	0,088	Non-immunotoxic	0,206	Potential immunotoxicity

The emergence of antimicrobial resistance has prompted the exploration of strategies beyond systemic antibiotic therapy. For oral infections, a mouthwash formulation offers distinct advantages, because it delivers the active compound directly to the site of infection, minimizes systemic exposure, and reduces the risk of resistance development [40]. Unlike conventional antibiotics that exert broad-spectrum activity and disrupt the commensal microbiota, compounds targeting specific virulence factors provide a more selective approach. In this study, attention was focused on glucosyltransferase B (GtfB) of Streptococcus mutans and secreted aspartyl protease 5 (Sap5) of Candida albicans, enzymes that are critical for biofilm formation and pathogenicity but not essential for microbial survival [41–43]. Targeting these enzymes therefore represents a promising strategy to impair virulence while mitigating selective pressure for resistance.

Two flavonoid derivatives, Pachypodol (compound 1) and Nectamazin B (compound 2), were successfully isolated from P. crocatum and evaluated for their antimicrobial potential. The in vitro assays confirmed inhibitory activity against both S. mutans and C. albicans (Table 1). Pachypodol and Nectamazin B produced comparable inhibition zones (9.0–10.67 mm) and identical MIC values (156.25 µg/mL), though their MBC/MFC values differed slightly. Notably, Nectamazin B demonstrated a lower MBC against S. mutans, suggesting stronger bactericidal activity, while both compounds were active against C. albicans. These results support their potential as antimicrobial agents for oral care applications.

The molecular docking studies provided mechanistic insights into these activities. Pachypodol exhibited the strongest affinity for GtfB (ΔG = − 8.05 kcal/mol, Ki = 1.26 µM), outperforming both the native ligand and Nectamazin B (Table 2). This suggests its superior ability to interfere with glucan synthesis and biofilm formation by S. mutans. In contrast, Nectamazin B showed higher affinity for Sap5 (ΔG = − 7.56 kcal/mol, Ki = 2.88 µM), comparable to the native ligand, indicating antifungal potential through inhibition of C. albicans protease activity. Thus, the docking results highlight a complementary profile, with Pachypodol primarily acting as an anti-caries agent and Nectamazin B showing stronger antifungal interactions.

To further validate docking outcomes, molecular dynamics simulations were performed. RMSD and RMSF analyses revealed that the GtfB–Pachypodol complex was more stable than those of the native ligand or Nectamazin B, showing minimal fluctuations during the 100 ns trajectory. MM/GBSA calculations confirmed that Pachypodol formed the most energetically favorable complex (− 28.22 kcal/mol), substantially stronger than Nectamazin B (− 15.04 kcal/mol) or the native ligand (− 7.09 kcal/mol). Hydrogen bond analysis further supported this observation, as Pachypodol maintained fewer but more stable hydrogen bonds, whereas Nectamazin B formed a larger number of less persistent interactions. This consistent stability strengthens the case for Pachypodol as the most promising inhibitor of GtfB.

The pharmacokinetic and safety aspects were assessed through ADMET profiling. Both compounds demonstrated good predicted absorption and were classified as BBB−, an advantageous property for topical oral application since systemic CNS effects are undesirable. Neither compound was hepatotoxic or mutagenic. However, Nectamazin B was predicted to have skin sensitization and immunotoxicity potential, raising safety concerns for use in long-term oral care products. Pachypodol, in contrast, was classified as non-sensitizing and non-immunotoxic. Radar chart analysis further emphasized this distinction, with Pachypodol’s physicochemical properties largely within the optimal drug-likeness range, while Nectamazin B showed multiple deviations that may limit its pharmaceutical applicability.

Taken together, the integration of in vitro assays, molecular docking, molecular dynamics simulations, and ADMET profiling provides strong evidence that Pachypodol is a superior Candidate for development as a mouthwash active ingredient. It combines moderate antimicrobial activity with robust binding to GtfB, stable and energetically favorable protein–ligand interactions, and a safe pharmacological profile. While nectamazin B also exhibits antimicrobial activity and higher affinity toward Sap5, its reduced stability and less favorable ADMET properties limit its potential for oral rinse formulations. Overall, Pachypodol emerges as a promising natural compound to impair S. mutans biofilm formation and support oral health, with reduced risk of resistance compared to conventional antibiotics.

Two secondary metabolites isolated via bioactivity guided from P. crocatum, pachypodol (1) and nectamazin B (2), exhibited antimicrobial activity against S. mutans and C. albicans. Integrated in vitro and in silico analyses revealed that Pachypodol displayed the strongest inhibitory potential against GtfB, supported by favorable docking scores, stable interactions in molecular dynamics simulations, and the most advantageous binding free energy profile. ADMET predictions further indicated that pachypodol is safe, non-immunotoxic, and possesses optimal physicochemical properties for formulation. In contrast, nectamazin B showed higher affinity toward Sap5 and comparable antifungal activity, but its reduced stability and predicted sensitization risk may limit its applicability. Collectively, the findings highlight pachypodol (1) as the most promising Candidate for development as a mouthwash agent, providing targeted inhibition of virulence enzymes with lower risk of resistance compared to systemic antibiotics.

ADMET : Absorption, distribution, metabolism, excretion, and toxicity

BBB : Blood–brain barrier

CYP : Cytochrome P450

DEPT : Distortionless enhancement by polarization transfer

GTF B : Glucosyltransferase B

HR-ESI-MS : High-resolution electrospray ionization mass spectrometry

LD50 : Lethal dose 50%

MBC : Minimum bactericidal concentration

MFC : Minimum fungicidal concentration

MHA : Mueller-Hinton agar

MIC : Minimum inhibitory concentration

MMGBSA : Molecular mechanics generalized born surface area

NMR : Nuclear magnetic resonance

NOESY : Nuclear overhauser effect spectroscopy

PDA : Potato dextrose agar

PPB : Plasma protein binding

RMSD : Root-mean-square deviation

RMSF : Root-mean-square fluctuation

SAP : Secreted aspartyl protease

T1/2 : Half life

Ethics approval and consent to participate

Permission to collect plant samples was obtained from the landowner of the private property. No further ethical approval was required for this study.

Consent for publication

Not applicable.

Clinical Trial Number

Clinical trial number: Not applicable

Competing interests

The authors declare no competing interests.

Funding

This research was funded by Penelitian Fundamental Reguler (PFR) Grant (1549/UN6.3.1/PT.00/2025) and Academic Leadership Grant (ALG) Prof. Dikdik Kurnia, M.Sc, Ph.D, Indonesia (504/UN6.WR3/TU.00/2024, March 13, 2024).

Author Contribution

DK conceived and supervised the study. HDAD and DN performed the experimental work and data acquisition. NAT contributed to data analysis, interpretation, and manuscript preparation. EA assisted in methodology and validation. SZKA contributed to data curation. All authors discussed the results and contributed to the final manuscript.

Acknowledgement

The authors are grateful to Universitas Padjadjaran for supporting all research facilities.

Data Availability

All data generated or analyzed during this study are included in this published article and its supplementary materials.

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No competing interests reported.

SupplementaryDatapaperBMCrev1.docx

Secondary Metabolites from Piper crocatum as Natural Inhibitors of Oral Pathogens: In Vitro and In Silico Insights into Streptococcus mutans and Candida albicans

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

General experimental procedures

Plant sample and identification

Extraction and isolation

Microbial strains and media

Disk diffusion test

MIC and MBC determination

Molecular docking of compounds with target proteins

Molecular dynamics simulation

Results

Discussion

Conclusions

Abbreviations

Declarations

Clinical Trial Number

Competing interests

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1