Essential Oils Enriched Dant-Kanti-Gandush (Oil-pulling) Inhibits Inter-kingdom Biofilm Formation on Orthodontic Fixtures and Ameliorates Cariogenic Virulence Factors of Oral Pathogens

Essential Oils Enriched Dant-Kanti-Gandush (Oil-pulling) Inhibits Inter-kingdom Biofilm Formation on Orthodontic Fixtures and Ameliorates Cariogenic Virulence Factors of Oral Pathogens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Essential Oils Enriched Dant-Kanti-Gandush (Oil-pulling) Inhibits Inter-kingdom Biofilm Formation on Orthodontic Fixtures and Ameliorates Cariogenic Virulence Factors of Oral Pathogens Acharya Balkrishna, Harshita Jonwal, Nem Kumar Ngpoore, Yash Varshney, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6928179/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Orthodontic fixtures provide a conducive niche for microbial colonization and inter-kingdom biofilm formation, exacerbating oral hygiene challenges. Conventional mouthwashes, though effective, are associated with adverse effects and potential antimicrobial resistance. Oil pulling is an Indian traditional method of oral detoxification. This study evaluates a blend of six essential oils (referred to as DKG) from Syzygium aromaticum, Mentha piperita, Eucalyptus globulus, Zanthoxylum armatum , and Ocimum sanctum , mixed with coconut and sesame carrier oils, as a potential oil-pulling formulation. Gas chromatography–mass spectrometry confirms the phytochemical composition of DKG. Antimicrobial assays demonstrate MIC₅₀ values of DKG ranging from 0.10% (v/v) to 0.45% (v/v) against Streptococcus pyogenes, Streptococcus mutans, Proteus mirabilis and Candida albicans , respectively. DKG exposure delays the exponential phase and perturbs the growth of these pathogens. The cariogenic traits of S. mutans are impaired at ≥ 1.0× MIC₅₀ DKG, showing reduced biofilm formation, decreased acid production, and lower survival under acidic stress. DKG inhibits C. albicans biofilms at ≥ 1.0× MIC₅₀, prevents yeast-to-hyphae transition, and disrupts cell wall integrity by reducing ergosterol. SEM analysis shows reduced microbial density, fragmented hyphae, and disrupted bacterial aggregation. These findings highlight plant-based DKG, an anticariogenic alternative for maintaining oral health in individuals with orthodontic fixtures. Oil pulling Essential oil Dant-Kanti-Gandush orthodontic fixtures S. mutans-C. albicans cross-kingdom biofilms cariogenic oral health Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Dental caries, commonly known as tooth decay or cavity formation, is one of the most prevalent global health issues, affecting individuals of all ages 1 . According to the WHO Global Oral Health Status Report (2022), nearly 3.5 billion people are affected by oral diseases, with 3 out of 4 people living in middle-income countries. Specifically, about 2 billion people suffer from caries of permanent teeth, and 514 million children are affected by caries of primary teeth. Interestingly, oral cavity harbors a complex microbiome, consisting of approximately 700 bacterial species that form biofilms on surfaces like teeth, gingiva, and prosthetic devices 2 , 3 . Streptococcus mutans and Candida albicans , are key contributors to dental caries and contribute to biofilm formation and acid production, which leads to enamel demineralization and development of cavities 4 Therefore, maintaining a balanced oral microbiota and practicing good oral hygiene are critical in preventing the formation of dental caries and related infections. Biofilm formation is a critical factor in the development and progression of dental caries, as it enables pathogenic bacteria and fungi to thrive in the oral cavity. The primary cariogenic bacterium, S. mutans , initiates biofilm development by adhering to salivary pellicles on tooth surfaces, aided by extracellular polymeric substances (EPS), mainly glucans produced by glucosyltransferases 5 . These glucans facilitate bacterial aggregation and biofilm maturation, making the biofilm highly resilient and structured. Additionally, Candida albicans , another key pathogen involved in biofilm formation, produces organic acids such as pyruvate and formate, which lower the pH of the surrounding environment, further promoting enamel demineralization 6 . This acidic environment created by S. mutans and C. albicans enhances bacterial survival and accelerates tooth decay 7 , 8 . Streptococcus pyogenes has been associated with dental plaque while, Proteus mirabilis , is commonly linked to urinary tract infections, has also been isolated from the oral cavity 9 , 10 . Orthodontic surfaces provide niche for bacterial adhesion and biofilm development, they contribute to increased bacterial load, enamel demineralization, and inflammation of the gingiva 11 . Therefore, preventing biofilm formation is essential in preventing dental caries and preserving oral health, particularly in patients with orthodontic fixtures, where biofilm control is challenging yet crucial for long-term oral health maintenance. Oil pulling, also known as Gandusha or Kavala Graha in Ayurvedic medicine, is a traditional Indian remedy practiced for centuries to maintain oral hygiene and overall health. The technique involves swishing edible oils such as coconut, sesame, or sunflower oil in the mouth for several minutes to facilitate oral detoxification and enhance oral hygiene. It is believed that oil pulling works by emulsifying and trapping bacteria within the oil, effectively reducing microbial load, dissolving plaque biofilms, and promoting gum health. Unlike commercial mouthwashes, oil pulling is safe, accessible, and free from side effects such as staining, unpleasant aftertaste, or allergic reactions, making it a favorable adjunct to conventional oral hygiene practices 12 , 13 . Coconut oil contains lauric acid, which has shown antimicrobial properties, significantly reducing the levels of Streptococcus mutans , a key bacterium responsible for dental caries. Studies have demonstrated that oil pulling with antimicrobial oils not only reduces plaque accumulation but also promotes gum health by combating harmful bacteria and supporting oral hygiene. In orthodontic settings, essential oils such as eucalyptol have been shown to reduce biofilm thickness and bacterial colonization on orthodontic fixtures, including brackets and arch wires 14 . Other essential oil components like limonene and linalool disrupt bacterial cell membranes, inhibiting the growth of S. mutans , further highlighting their role in managing biofilm-related complications 15 , 16 The current study demonstrates the antimicrobial activity of essential oils of Dant-Kanti-Gandush (DKG) against oral pathogens, including S. mutans , P. mirabilis , S. pyogenes , and C. albicans. DKG is a combination of essential oil extracted from clove ( Syzygium aromaticum ), peppermint ( Mentha piperita ), eucalyptus ( Eucalyptus globulus ), prickly ash ( Zanthoxylum armatum ), and basil ( Ocimum sanctum ). The oral pathogens showed reduced growth dynamics in a dose-dependent manner upon exposure to DKG. Notably, DKG disrupts the cross-kingdom colonization and biofilm formation of S. mutans and C. albicans on orthodontic braces. These findings suggest that DKG may serve as a safe, natural adjunct for managing dental caries and biofilm-associated oral infections, reinforcing the role of Ayurveda as a complementary and alternative medicine. MATERIAL AND METHODS Procurement of test article, microbial strains and growth conditions The test article, a blend of essential oil present in Dant-Kanti-Gandush (Batch# PRF/CHI/0424/0288) was supplied by Herbal Chemistry Department, Patanjali Research Foundation, Haridwar, India. The study employed specific microbial strains, which were sourced from Microbial Type Culture Collection (MTCC), CSIR-Institute of Microbial Technology (Chandigarh, India). The bacterial strains used included Streptococcus mutans (MTCC 497), Streptococcus pyogenes (MTCC 442), and Proteus mirabilis (MTCC 1429), as well as the fungal strain Candida albicans (MTCC 183). Bacterial species were cultured in Brain Heart Infusion (BHI) broth (HiMedia, India), while the yeast was cultured in Yeast Extract Peptone Dextrose (YPD) broth (HiMedia, India). Single isolated colonies of each microorganism were inoculated in their respective media and incubated at 35–37°C for 24–48 hours. For routine maintenance, these strains were sub-cultured onto Brain Heart Infusion agar and Yeast Extract Peptone Dextrose agar and preserved as glycerol stocks at − 80°C for long-term storage. Gas chromatography-mass spectrometry (GC-MS/MS) Analysis of Essential Oil GC-MS/MS (7000D GC/MS triple quad with 7890B GC system, Agilent-USA) was performed using mass hunter software to identify, quantify, and characterize chemical compounds present in DKG. Separation was carried out using Agilent HP-5MS capillary column (30 m x 0.25 mm, 0.25 µm) Helium was used as carrier gas at a flow rate of 1 mL/min. The temperature of the split injector was maintained at 280°C and the split ratio was 20:1. The column temperature was initially set at 60°C without hold, then ramped at 5°C/min to 100°C (held for 1 min), followed by an increase at 3°C/min to 160°C (held for 3 minutes), and finally ramped at 10°C/min to 280°C, where it was held for 1 minute. The GC-MS temperature was 230°C and ionization potential was 70 eV. Antimicrobial effects of Dant-Kanti-Gandush Essential Oil The antibacterial activity of DKG was evaluated using the disk diffusion method. The optical density (OD) of an overnight-grown culture was adjusted to 0.06 at 600 nm, and the bacterial culture was swabbed onto Muller Hinton Agar, while Candida albicans was swabbed on Yeast Peptone Dextrose agar. Sterile disks carrying varying concentrations of DKG (5% (v/v), 25% (v/v), 50% (v/v), and 100% (v/v)) were placed to the agar plates. A sterile disk impregnated with sterile water was included as a negative control and placed at the center of the plate. The plates were incubated at 37°C for 24 hours. Microbroth- dilution assay The antibacterial activity of DKG was assessed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI, 2015) guidelines. The Minimum Inhibitory Concentration (MIC) test was conducted using 96-well tissue culture microplates, each containing 100 µL of Brain Heart Infusion (BHI) medium (Himedia, India). The stock solution of DKG was initially diluted to 10% v/v by adding sterile autoclaved water, and this was transferred to the first well. Two-fold serial dilutions were subsequently performed, resulting in concentrations ranging from 0.02% (v/v) to 5.0% (v/v). The bacterial inoculum (OD 0.06–0.08 at 600 nm) was added to each well, except for the blank wells, which contained only the medium contain respective DKG concentration. The plates were then incubated at 37°C for 24 h. The experiments were repeated in three biological replicates. Growth kinetic assessment in oral pathogens The microbial cultures were adjusted to an optical density (OD) 0.06–0.08 and treated with different concentrations of DKG (0.25× MIC 50 , 0.50× MIC 50 , 1.0× MIC 50 , 2.0× MIC 50 and 4.0× MIC 50 ) in 96-well microtiter plate. Growth kinetics of the bacterial and yeast cultures were recorded every two hours while their incubation at 37°C for 24 hours in Infinite 2000 Pro microplate reader (Tecan Group Ltd., Switzerland). All experiments were performed in independent triplicate to ensure consistency and reproducibility. The recorded absorbance, after blank correction, was plotted using a non-linear regression curve fit function. Assessment of Biofilm formation Biofilm biomass assessment was performed by crystal violet staining method and scanning electron microscopy. Actively growing S. mutans culture adjusted at optical density of 0.1 at 600nm in BHI media (supplemented with 1% sucrose) was used to establish biofilm on the sterile cover slips in the 6-well plate. Biofilm in C. albicans was developed on the sterile cover slips in the 6-well plate in YPD (supplemented with 50 mM glucose). For dual-species co-culturing, equal volumes (1:1) of the above cultures in their respective media were added to sterile coverslips in a 6-well plate. Subsequently, DKG at different concentrations (0.5× MIC 50 : 0.25% (v/v), 1× MIC 50 : 0.50% (v/v), and 2× MIC 50 : 1.0% (v/v)) was added to the S. mutans culture and allowed to develop biofilm under aerobic incubation in an incubator with 5% CO 2 at 37°C 17 . The supernatant was aspirated after 72 h of incubation and the tightly adhered cells forming biofilms were washed twice with PBS to remove planktonic bacterial cells. Biofilm was fixed with 2.0% formaldehyde for 20 minutes, followed by crystal violet (0.1%) staining for 30 minutes. Once dried, the stained biofilms were photographed under an TS2 inverted brightfield microscope (Nikon, Japan). The surface area covered by the biofilm was evaluated using ImageJ software (US National Institutes of Health, Bethesda, Maryland, USA). The biofilms formed by co-cultured inter-kingdom species, S. mutans and C. albicans were examined using a scanning electron microscope (FlexSEM1000, Hitachi, Japan). Glycolytic pH drop assay Using a pH-drop experiment, the impact of DKG on S. mutans acidogenicity was assessed. The actively growing S. mutans culture was harvested, washed and resuspended in the buffer containing 50 mM potassium chloride (KCl) and 1 mM magnesium chloride (MgCl₂) in the presence of DKG at different concentrations (0.25× MIC 50 , 0.50× MIC 50 , 1.0× MIC 50 , 2.0× MIC 50 and 4.0× MIC 50 ). Glucose was added to a final concentration of 55.6 mM, and the initial pH of the mixtures was adjusted to 7.2–7.4 using 1.0 M potassium hydroxide. Change in the pH was recorded (Laboholic Microprocessor pH meter, India) at 20-minute intervals over a total duration of 100 minutes 18 , 19 . The experiment was conducted in triplicate to ensure reproducibility. Acid tolerance assay Acid tolerance is defined as the ability of bacteria to survive in acidic environments, a critical characteristic of S. mutans , which employ both constitutive and acid-inducible mechanisms (Matsui et al., 2010). The effect of DKG on the acidurity of S. mutans was assessed by exposing bacteria to an acidic pH of 5.0. Actively growing S. mutans bacterial cells were harvested to resuspend in TYEG (Tryptone Yeast Extract Glucose) broth (pH 5.0) in the presence of DKG at sub-inhibitory and inhibitory concentrations of DKG (0.5%, 1%, and 2% (v/v)) at 37°C for 6 and 24 hours 19 . The untreated control group contained no DKG. After incubation, viability of S. mutans was determined from all treatment conditions by plating the bacterial cells on Brain heart infusion agar plates. The experiment was independently repeated three times to ensure reproducibility (15). Hyphal growth assessment in C. albicans Candida albicans cultured in Yeast Peptone Dextrose (YPD) medium supplemented with 10% Fetal Bovine Serum to induce hyphal formation 20 . Yeast culture adjusted to an optical density of 0.1 at 600 nm was inoculated in hyphal-inducing media (YPD medium containing 10% Fetal Bovine Serum) in the presence of DKG (0.5× MIC 50 : 0.05% (v/v), 1× MIC 50 : 0.10% (v/v), and 2× MIC 50 : 2.0% (v/v)) at 37°C with shaking for 6 h and 24 h. After incubation cell morphology was observed and photographed using a Zeiss Observer.Z1 microscope (Carl Zeiss, Jena, Germany) (16). UHPLC-DAD based ergosterol detection From an overnight-grown culture, a 100 mg pellet of untreated yeast cells and cells treated with DKG (at 0.5x, 1.0x and 2.0x MIC 50 ) was dissolved in 0.25 mL of methanol and homogenized by adding sterile stainless-steel balls for 10 minutes. This solution was then centrifuged for 5 min at 10000 rpm and 100 µl of supernatant was transferred in injecting vials and injected into the system. Ergosterol standard (of 1 mg/mL) in methanol was used to prepare 1000 ppm standard solution. 0.1 ml of this standard solution was diluted to 10 ml to prepare 10 µg/mL working stock. Ergosterol content of the treated and untreated C. albicans was analyzed on Prominence-XR UHPLC system (Shimadzu, Japan) equipped with Quaternary pump (Nexera XR LC-20AD XR), DAD detector (SPD-M20 A), Auto-sampler (Nexera XR SIL-20 AC XR), Degassing unit (DGU-20A 5R) and Column oven (CTO-10 AS VP). Separation was achieved using a Shodex C18-4E (5 µm, 4.6*250 mm) column subjected to isocratic elution with a flow rate of 1.0 mL/min. The mobile phase was used for the analysis consisted of the ratio of methanol: acetonitrile (80:20). 50 µL of standard and test solutions were injected and wavelength was set at 280 nm. Scanning electron microscopy on orthodontic brackets The dual-species biofilms were formed using the same method as described in the above section, with the only difference being that clean orthodontic brackets and rings (Koden orthodontic brackets, provided by Dental Clinic and Research Centre, Patanjali Ayurved Hospital, Haridwar, India) replaced the coverslips in the six-well plate. Subsequently, DKG treatment (1.0% (v/v)) was added to the co-cultures and plates were aerobically incubated at 37°C for 72 hours in 5% CO₂ (17). Orthodontic brackets and rings were thoroughly washed to remove planktonic cells, fixed in formaldehyde and air dried before imaging under SEM scanning electron microscope (FlexSEM1000, Hitachi, Japan) to observe the effect of DKG on dual-species biofilms. RESULTS Gas Chromatography-tandem mass spectrometry (GC-MS/MS) analysis of Dant-Kanti-Gandush Essential Oils (DKG) The oil-pulling formulation, DKG consists of 90% (v/v) Sesamum indicum (sesame) oil and 8% Cocos nucifera (coconut) oil. The oil base, constituting 98% of the formulation, is supplemented with a 1.5% essential oil blend from Syzygium aromaticum , Mentha piperita , Eucalyptus globules , Zanthoxylum armatum and Ocimum sanctum , in a ratio of 2:3:8:1:1, respectively (Table 1 ). The essential oil blend of Dant-Kanti-Gandush (hereafter, called as DKG) was subjected to detailed microbiological testing, and GC-MS/MS analysis for phytochemical profiling. The chromatograph generated demonstrated prominent peaks, which were subsequently identified by matching mass fragmentation data with the National Institute of Standards and Technology (NIST, USA) library (MS Search 2.2). Notably, the identified phytometabolites, D-limonene, eucalyptol, linalool, menthol and eugenol were further validated and quantified against their respective reference standard (Fig. 1 A). The quantified content percentage (w/w) for D-limonene, eucalyptol, linalool, menthol and eugenol were 5.64%, 21.79%, 3.34%, 5.55%, and 3.32%, respectively (Fig. 1 B) Table 1 Composition of Dant-Kanti-Gandush. S. No. English name Scientific name Plant Part Form Qty. (g) 1 Essential oils Clove Syzygium aromaticum Buds Oil 0.20 2 Peppermint Mentha piperita Leaves Oil 0.30 3 Eucalyptus Eucalyptus globules Leaves Oil 0.80 4 Prickly ash Zanthoxylum armatum Seeds Oil 0.10 5 Basil Ocimum sanctum Leaves Oil 0.10 6 Carrier oils Coconut Cocos nucifera Endosperm Oil 8.00 7 Sesame Sesamum indicum Seeds Oil 90.00 8 Flavour - - - 0.50 Dant-Kanti-Gandush demonstrated remarkable antimicrobial activity against oral pathogens The study tested the anti-microbial activity of DKG, blend of essential oils, against few common oral pathogens associated with dental caries and periodontal infections, such as Streptococcus mutans , Proteus mirabilis , Streptococcus pyogenes , and Candida albicans . Initial screening by disc diffusion demonstrated the potential antimicrobial activity of DKG, tested at 50% (v/v) and 100% (v/v) against these oral pathogens. DKG against Streptococcus pyogenes showed a zone of inhibition of 10 mm ± 0.40 at 50% (v/v) and 11.5 mm ± 0.54 at 100% (v/v). The bacterial lawn of Proteus mirabilis exhibited < 10 mm and 12.1 mm ± 0.75 zone at 50% (v/v) and 100% (v/v), respectively. DKG against Streptococcus mutans showed < 10 mm and 10.8 mm ± 1.16 of clear zone at 50% (v/v) and 100% (v/v), respectively. Most prominent clearance with zone diameters of 11.8 mm ± 1.83, 19 mm ± 2.96 and 25 mm ± 2.28 at 25% (v/v), 50% (v/v) of DKG, respectively was observed against Candida albicans (Fig. 2 A). Carrier oils of Dant Kanti Gandush, sesame oil and coconut oil, were also tested in these experiments with minimal effects (Data not shown). Minimum inhibitory concentrations (MIC) corresponding to MIC 50 and MIC 90 were also determined using broth microdilution method to evaluate the DKG concentrations that inhibited 50% and 90% of visible microbial growth, respectively. For Streptococcus pyogenes , DKG exhibited MIC 50 and MIC 90 at 0.10% (v/v) and 0.17% (v/v), respectively (Fig. 2 B). MIC 50 and MIC 90 for DKG in Proteus mirabilis were evaluated to be 0.29% (v/v) and 1.59% (v/v), respectively (Fig. 2 C). MIC 50 and MIC 90 in Streptococcus mutans were evaluated as 0.45% (v/v) and 1.70% (v/v), respectively (Fig. 2 D). DKG demonstrated MIC 50 and MIC 90 in Candida albicans at 0.11% (v/v) and 0.64% (v/v), respectively (Fig. 2 D). Dant-Kanti-Gandush decelerated the exponential phase of oral pathogens The time-kill assay in Fig. 3 (A-D), demonstrated a time-dependent anti-microbial activity of DKG over a 22 h incubation period. The untreated controls from all four pathogens tested exhibited continuous logarithmic growth up to 22 h, whereas DKG exposure retarted the growth progression profiles, significantly. In Streptococcus pyogenes , a delay in the onset of the exponential phase was evident at 1.0× and 2.0× of MIC 50 compared to the untreated control (Fig. 3 A). Similarly, in Proteus mirabilis , DKG not only delayed the exponential phase by 2–4 h, but the pathogen also achieved the stationary phase at half the growth density (0.5 and 0.2 optical density at 1.0× and 2.0× MIC 50 , respectively) compared to the untreated (Fig. 3 B). The most pronounced inhibition in growth dynamics was observed in Streptococcus mutans , where exponential growth phase was observed after 12 h at 1.0× MIC 50 , compared to 2 h in the untreated. Interestingly, at the highest dose of 2.0× MIC 50 , no growth progression was observed (Fig. 3 C). However, in Candida albicans , the exponential phase initiated after 12–14 h of growth at 2.0× MIC 50 compared to 4–6 h in the untreated (Fig. 3 D). Collectively, DKG limited the microbial proliferation rate and even inhibited the pathogens to achieve usual population density and biomass required for achieving stationary phase. Dant-Kanti-Gandush suppresses the cariogenic properties in S. mutans S. mutans contribute substantially to oral biofilm formation, enamel demineralization posing risks to oral and overall health. DKG was evaluated for its impact on the biofilm forming potential of S. mutans . Untreated samples exhibited dense biofilm formation, whereas DKG treatment at 0.5× MIC 50 and above showed a dose-dependent significant disruption in the biofilms formed (Fig. 4 A). ImageJ analysis revealed a significant reduction in the surface area covered by biofilm in the presence of DKG. The surface area covered reduced by ~ 30%, ~ 50% and ~ 70% at 0.5×, 1.0× and 2.0× MIC 50 of DKG (Fig. 4 B). The effect of DKG on S. mutans acidogenicity was determined by monitoring glycolytic pH drop (Fig. 4 C). In untreated cultures, pH decreased from 7.01 ± 0.03 to 3.89 ± 0.12 within 100 minutes, indicating acid production capabilities of S. mutans . However, DKG treatment significantly delayed the pH drop, with terminal pH values of 5.30 ± 0.61, 5.95 ± 0.61, and 6.95 ± 0.00 at 0.5×, 1.0×, and 2.0× MIC 50 , respectively (Fig. 4 C). Higher concentrations of DKG notably impeded acid production. An exponential decay curve was generated on the obtained pH values to calculate Tau (τ) constant. The decay rate (τ −1 ) evaluated by fit function for untreated (41.14), 0.5×MIC 50 (83.30), 1.0×MIC 50 (74.36) and ambiguous results for 2.0×MIC 50 indicated inhibited acidogenicity potential in S. mutans in the presence of DKG targeted. In addition, the acid tolerance capability of S. mutans , when evaluated in the presence of DGK, on the contrary showed notable reduction in the number of viable S. mutans colonies at 6 h and 24 h under acid stress. At 6 h, viable counts calculated as CFU/mL reduced from 725 × 10⁶ (Untreated) to 329 × 10⁶ (0.5×MIC 50 ) 118 × 10⁶ (1.0×MIC 50 ) and 17 × 10⁶ (2.0×MIC 50 ) (Fig. 4 D). Whereas, by 24 h, further reductions were observed from 517 × 10⁶ (Untreated) to 189 × 10⁶ (0.5×MIC 50 ) 4 × 10⁶ (1.0×MIC 50 ). Notably, at 2.0×MIC 50 no viability of bacteria was observed in the presence of DKG (Fig. 4 E). Dant-Kanti-Gandush inhibited yeast-to-hypha conversion and biofilm formation in C. albicans Yeast to hyphae transition and biofilm formation are key factors imparting pathogenicity to Candida albicans . DKG exposure for 6 h and 24 h notably inhibited the budding and hyphal germination in a dose-dependent manner (Fig. 5 A). C. albicans biofilm formation also observed a significant inhibition (Fig. 5 B). The density of the biofilm and subsequent surface area covered significantly reduced by ~ 45–50% (Fig. 5 C). Ergosterol is an essential component of fungal cell wall. Disruption in ergosterol biosynthesis or downregulation in ergosterol levels indicate compromised cell membrane integrity 21 , 22 . In order to test whether DKG could exert similar effect on C. albicans , ergosterol levels were evaluated through UHPLC method. The ergosterol content significantly decreased from 100.00 ± 25.27 µg/mg to 33.39 ± 30.67 µg/mg and 19.36 ± 7.50 µg/mg when were exposed to at 2.0× MIC 50 and 4.0× MIC 50 , respectively (Fig. 5 D-E). Dant-Kanti-Gandush inhibited biofilm formation of S. mutans and C. albicans in mono-and co-culture conditions Dental plaques are tight-structured, multi-species, cross-kingdom biofilms. Their formation not only drives dental caries and other periodontal diseases but also reduces susceptibility to antimicrobial treatments, posing a significant challenge in oral health management. The anti-plaque potential of DKG was evaluated by assessing its efficacy against biofilms formed by S. mutans and C. albicans , both individually and in a co-cultured interkingdom biofilm model. Scanning electron microscopy (SEM) was performed on biofilms formed on coverslips by indicated oral pathogens. The dense biofilm formed by S. mutans showed disrupted and spaced growth in the presence of DKG (Fig. 6 A). Candida albicans showed a dense growth on the coverslip. However, in the presence of DKG, yeast cells exhibited disruption with irregularly shaped yeast structures and even shrinkage. This indicated membrane damage or stress confirming the antifungal effect of DKG in suppressing biofilm formed by C. albicans (Fig. 6 B). The SEM analysis illustrated a stark contrast between the untreated and DKG exposed S. mutans and C. albicans inter-kingdom biofilms (Fig. 6 C). The untreated biofilms illustrate a dense, tight-structured microbial network with extensive interactions between bacterial chains and fungal hyphae. In contrast, DKG exposure severely disrupted the microbial density, fragmented hyphal structures and disorganized bacterial aggregation. Dant-Kanti-Gandush prevented the cross-kingdom biofilm formation by S. mutans and C. albicans on Orthodontic fixtures Orthodontic fixtures provide an ideal niche for microbial adhesion and growth, particularly in the presence of oral saliva 11 , 23 , 24 . S. mutans and C. albicans culture were inoculated on the sterile orthodontic brackets and elastomeric ligature in the presence of their respective growth media. Scanning electron micrographs of elastomeric ligature and orthodontic brackets demonstrated a thick microbial growth, which upon further zooming in showed dense meshwork of hyphal growth by the yeast. S. mutans cells appear embedded within the exopolysaccharide (EPS) matrix, reinforcing microbial co-aggregation and resilience. DKG exposure, on the contrary prevented the formation of dense architecture and EPS matrix, thereby inhibiting microbial adherence and biofilm formation. The microbial growth was drastically reduced, with C. albicans fragmented hyphae and deformed yeast cells. S. mutans appearing sparsely distributed, lysed and ruptured on the surface of both elastomeric ligature (Fig. 7 A) and orthodontic brackets (Fig. 7 B). DISCUSSION Essential oils (EOs) are volatile secondary metabolites produced by plants, responsible for imparting typical aroma, flavor, or both. Essential oils (EOs) have been extensively studied for their therapeutic potential across various diseases. Their pharmacological properties encompass antimicrobial, anti-inflammatory, antitumor, and antioxidant activities 25 . The current study has explored EOs as alternative or adjunctive agents in oral healthcare. Dant-Kanti-Gandush (DKG) oil consists of a 98.0% fixed oil blend of coconut and sesame oil, with the remaining 2.0% comprising essential oils from clove, peppermint, eucalyptus, prickly ash, and basil. DKG is recommended for oil pulling, an ancient Ayurvedic practice, that has recently gained popularity for its natural, cost-effective and health benefits. The process involves swishing oil in the mouth for about 10–20 minutes. Traditionally, sesame oil or coconut oils are preferred over other edible oils. Sesame oil, known for its antimicrobial properties and plaque-removing ability, contains lignans such as sesamin, sesamolin, and sesaminol, which possess strong antioxidant activity 26 , 27 . Coconut oil, rich in lauric acid, plays a scientifically recognized role in oral hygiene by exhibiting antimicrobial and antibiofilm activity against plaque-causing and cariogenic bacteria. Additionally, it possesses antioxidant and anti-inflammatory properties 28 , 29 .However, the combination of five essential oils in DKG serves as an enhancement to the traditional oil-pulling formulation. The present study elucidates the antimicrobial efficacy of this essential oil combination in inhibiting growth, attenuating virulence factors, and disrupting biofilm formation in key oral pathogens, Streptococcus mutans and Candida albicans . The 2.0% composition of DKG consists of essential oils containing a substantial amount of eucalyptol, D-limonene, menthol, linalool, and eugenol, as identified through GC-MS/MS analysis. Interestingly, MIC determination revealed that this essential oil mix exhibited a remarkably low effective concentration of 0.45% (v/v), 0.29% (v/v), 0.10% (v/v), and 0.11% (v/v) against S. mutans , P. mirabilis , S. pyogenes , and C. albicans , respectively. All pathogens under the in vitro conditions are inhibited at < 0.5% (v/v) of essential oil mix present in Dant-Kanti-Gandush (DKG). When treated above MIC 50 , the essential oil mix significantly inhibited growth dynamics, leading to delayed and reduced exponential growth of these pathogens. S. mutans are the key pathogen contributing significantly to dental caries and plaque formation. With strong adherence, acid enduring (acidurity) and producing (acidogenicity) properties, S. mutans create an environment that promotes enamel demineralization, bacterial colonization, biofilm maturation and persistence 5 , 17 , 30 , 31 . DKG markedly inhibited S. mutans biofilm formation and suppressed its aciduric and acidogenic properties, demonstrating its effectiveness in disrupting S. mutans metabolism. By reducing acid production and promoting oral pH balance, DKG plays a crucial role in lowering the risk of dental caries and contributing to overall oral health maintenance. C. albicans , yeast that also plays a crucial role in oral plaque biofilms by interacting with oral bacteria, albeit enhancing antimicrobial resistance and other oral diseases 8 , 32 , 33 . The transition from yeast to hyphal morphology is important for C. albicans to adhere to the host’s surface, enhances its ability to invade the host and impart pathogenicity 20 , 34 . DKG suppressed the hyphal transition ability of C. albicans and even reduced the biofilm formation significantly. The diminishing levels of ergosterol, an important cell membrane component for maintaining membrane integrity, was observed with DKG treatment. Ergosterol also act as a target for many antifungal drugs 35 . Disrupting its levels, DKG compromises the growth and virulence of C. albicans , highlighting mechanistic aspects of DKG offering a promising approach to reducing pathogenicity of the yeast and thereby improving oral health. Several published reports have previously demonstrated that essential oils employ multiple mechanisms to inhibit the bacterial growth 16 , 36 . Mechanistically, either by cell membrane disruption in the bacteria or interfering with the cell-to-cell communication (quorum sensing), essential oils could even suppress the extracellular polymeric substance (EPS) production, matrix crucial for biofilm formation by the bacteria 37 – 39 . However, majority of studies have primarily evaluated the antimicrobial activity of essential oils against planktonic bacterial cultures. Given that biofilms exhibit enhanced resistance due to their protective extracellular matrix, conventional antimicrobial agents often fail to penetrate and eradicate these multi-layers, tightly structured communities. Furthermore, most investigations have focused on single-species biofilms, despite the fact that oral infections commonly involve complex, inter-kingdom interactions. Addressing these gaps, the current study examines the antimicrobial efficacy of essential oils against S. mutans and C. albicans co-cultured biofilms grown on orthodontic brackets and rings, a clinically relevant surface. This approach provides deeper insights into biofilm susceptibility in realistic conditions, reinforcing the potential of essential oils for combating persistent oral biofilms. Orthodontic treatment involves the placement of brackets, archwires and elastomeric ligatures on teeth are susceptible sites for plaque accumulation and biofilm formation, increasing the risk of caries and enamel demineralization. These orthodontic fixtures, being constantly exposed to oral fluids, provide a favorable niche and surface for microbial colonization, making the maintenance of oral hygiene complicated and challenging 11 , 14 , 23 , 24 . Preventive strategies comprising of stringent oral hygiene practices play an important role in mitigating biofilm formation risks and ensure optimal oral health. Oil pulling could be an effective practice for oral health maintenance. Dant Kanti Gandush, containing a combination of essential oils with coconut and sesame oil, offers potential antimicrobial benefits, inhibits the inter-kingdom biofilm formation and thereby supports overall oral hygiene. A 1.0% (v/v) concentration of the essential oil mix was tested on biofilms, which is half the concentration of essential oils present in the oil-pulling formula of DKG. Biofilm formation was analyzed using a scanning electron microscopy (SEM) technique. SEM revealed a dense mesh of hyphae and yeast cells of C. albicans with coccus shaped S. mutans building a complex architecture and extensive extracellular polymeric substance (EPS) matrix. At lower magnification, SEM imaging of the entire bracket and elastomeric ligatures revealed a dense, thick layer of dual-culture biofilm. In contrast, brackets and elastomeric ligatures exposed with DKG exhibited a notably reduced microbial adherence, accompanied by significant structural alterations, cell lysis and scattered cellular debris. The current study was limited to in vitro antimicrobial assessment of DKG, however, does not highlight the complexities of in vivo conditions. Host-pathogen interaction studies and preclinical efficacy determination are part of the future investigations to further substantiate the therapeutic relevance of the present findings. Collectively, the study highlighted the potential of DKG in mitigating microbial adherence and biofilm formation on orthodontic surfaces, promoting oral hygiene and overall health. CONCLUSION DKG serves as a valuable natural intervention for maintaining oral hygiene and preventing biofilm-associated complications even in the presence of orthodontic fixtures, where microbial adhesion and plaque accumulation are heightened. DKG could inhibit multiple oral pathogens, disrupt dual-species biofilms, reduce their adherence on orthodontic fixtures, all highlight DKG as an effective adjunct to conventional oral care, particularly for individuals undergoing orthodontic treatment. Further in vivo studies and randomized clinical trials are needed to validate its therapeutic potential and clinical efficacy in real-world oral healthcare applications. Abbreviations DKG: Dant-Kanti-Gandush, MIC: Minimum inhibitory concentration, SEM: Scanning electron microscopy, EPS: Extracellular polymeric substance; OD: Optical density, GC-MS/MS: Gas chromatography-mass spectrometry, UHPLC: Ultra-High-Performance Liquid Chromatography. Declarations ETHICS APPROVAL Not applicable CLINICAL TRIAL NUMBER Not applicable. ACKNOWLEDGMENTS The authors express appreciation to Mr. Devendra Kumawat for his contributions in designing schematics. The authors acknowledge the invaluable chemistry support of Dr. Sudeep Verma and Dr. Priya Rani M. We thank Dr. Swati Haldar for her scientific guidance. The authors would like to extend sincere appreciation to Dr. Ramakrishna Gupta and Mr. Naresh Bhende for their assistance with SEM analysis. Additionally, the authors acknowledge Mr. Tarun Rajput and Mr. Gagan Kumar for their prompt administrative supports. CONFLICT OF INTEREST STATEMENT The test article, Dant-Kanti-Gandush was sourced from Divya Pharmacy, Haridwar, India. AB is an honorary trustee in Divya Yog Mandir Trust, which governs Divya Pharmacy, Haridwar. In addition, he holds an honorary managerial position in Patanjali Ayurved Ltd., Haridwar, India. Divya Pharmacy and Patanjali Ayurved Ltd commercially manufacture and sell several Ayurvedic products. Other than providing the test article, Divya Pharmacy was not involved in any part of this study. Other authors have no conflict of interest to disclose. DATA AVAILABILITY STATEMENT The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. AUTHOR CONTRIBUTIONS AB: Conceptualization, Supervision, Resources, Writing-review & editing; HJ: Methodology, Data curation, Formal analysis, Writing-original draft; NKN: Methodology, Investigation; YV: Methodology, Data curation, Formal analysis, Writing-original draft; MT: Methodology, Data curation, Formal analysis; MJ: Methodology, Investigation; KS: Methodology, Investigation; PN: Supervision; Writing-review & editing; SL: Visualization, Methodology, Data curation, Formal analysis, Writing-review & editing; AV: Project administration, Conceptualization, Visualization, Supervision; Writing-review & editing. FUNDING This presented work has been conducted using internal research funds from a non-profit Patanjali Research Foundation Trust, Haridwar, India. References Peres, M. A. et al. Oral diseases: a global public health challenge. Lancet 394 , 249–260 (2019). Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I. & Dewhirst, F. E. 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F. & Jabra-Rizk, M. A. Pathogenesis of Candida albicans biofilm. Pathog Dis. 74 , ftw018 (2016). Schaffer, J. N. & Pearson, M. M. Proteus mirabilis and Urinary Tract Infections. Microbiol Spectr 3 , (2015). Zaatout, N. Presence of non-oral bacteria in the oral cavity. Arch. Microbiol. 203 , 2747–2760 (2021). Abutayyem, H., Abdullatif Alshehhi, M. & Alameri, M. Sohail Zafar, M. Microbial adhesion on different types of orthodontic brackets and wires: An in vitro study. Saudi Dent. J. 36 , 1459–1465 (2024). Joshi, P. & Joshi, S. Oil pulling - A natural therapy for oral health stress management. CURRENT Med. DRUG RESEARCH 3 , (2019). Naseem, M. et al. Oil pulling and importance of traditional medicine in oral health maintenance. Int. J. Health Sci. (Qassim) . 11 , 65–70 (2017). Alexa, V. T. et al. Molecular Docking and Experimental Analysis of Essential Oil-Based Preparations on Biofilm Formation on Orthodontic Archwires. Int. J. Mol. Sci. 25 , 13378 (2024). de Galvão, L. C. et al. C. 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The plaque reducing efficacy of oil pulling with sesame oil: a randomized-controlled clinical study. Clin. Oral Investig . 29 , 53 (2025). Li, Z. et al. Antibacterial Effect and Possible Mechanism of Sesamol against Foodborne Pathogens. Foods 13 , 435 (2024). Haron, U. A., Mukhtar, N. I., Omar, M. N. & Abllah, Z. Fatty Acid Evaluation and Antimicrobial Activity of Virgin Coconut Oil and Activated Virgin Coconut Oil on Streptococcus mutans. Archives Orofac. Sci. 14 , 87–98 (2019). M, M. et al. Evaluating the effect of virgin coconut oil pulling on viral load, bacterial load and inflammatory mediator levels in chronic periodontitis – A clinical study. J. Oral Biol. Craniofac. Res. 15 , 153–158 (2025). Lemos, J. A. et al. The Biology of Streptococcus mutans. Microbiol Spectr 7 , (2019). Matsui, R. & Cvitkovitch, D. Acid Tolerance Mechanisms Utilized by Streptococcus Mutans. Future Microbiol. 5 , 403–417 (2010). Janus, M. M., Willems, H. M. E. & Krom, B. P. Candida albicans in Multispecies Oral Communities; A Keystone Commensal? in 13–20 (2016). 10.1007/5584_2016_5 Du, Q. et al. Candida albicans promotes tooth decay by inducing oral microbial dysbiosis. ISME J. 15 , 894–908 (2021). Chow, E. W. L., Pang, L. M. & Wang, Y. From Jekyll to Hyde: The Yeast–Hyphal Transition of Candida albicans. Pathogens 10 , 859 (2021). Suchodolski, J., Muraszko, J., Bernat, P. & Krasowska, A. A Crucial Role for Ergosterol in Plasma Membrane Composition, Localisation, and Activity of Cdr1p and H+-ATPase in Candida albicans. Microorganisms 7 , (2019). Wińska, K. et al. Essential Oils as Antimicrobial Agents—Myth or Real Alternative? Molecules 24 , 2130 (2019). Yap, P. S. X., Yusoff, K., Lim, S. H. E., Chong, C. M. & Lai, K. S. Membrane Disruption Properties of Essential Oils—A. Double-Edged Sword? Processes . 9 , 595 (2021). Rao, J., Chen, B. & McClements, D. J. Improving the Efficacy of Essential Oils as Antimicrobials in Foods: Mechanisms of Action. Annu. Rev. Food Sci. Technol. 10 , 365–387 (2019). Kim, Y. G. et al. Essential Oils and Eugenols Inhibit Biofilm Formation and the Virulence of Escherichia coli O157:H7. Sci. Rep. 6 , 36377 (2016). Additional Declarations Competing interest reported. The test article, Dant-Kanti-Gandush was sourced from Divya Pharmacy, Haridwar, India. AB is an honorary trustee in Divya Yog Mandir Trust, which governs Divya Pharmacy, Haridwar. In addition, he holds an honorary managerial position in Patanjali Ayurved Ltd., Haridwar, India. Divya Pharmacy and Patanjali Ayurved Ltd commercially manufacture and sell several Ayurvedic products. Other than providing the test article, Divya Pharmacy was not involved in any part of this study. Other authors have no conflict of interest to disclose. Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 08 Oct, 2025 Reviews received at journal 06 Oct, 2025 Reviews received at journal 30 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers agreed at journal 13 Sep, 2025 Reviewers agreed at journal 11 Sep, 2025 Reviewers invited by journal 11 Sep, 2025 Editor assigned by journal 28 Jul, 2025 Editor invited by journal 23 Jul, 2025 Submission checks completed at journal 26 Jun, 2025 First submitted to journal 26 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6928179","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":479776449,"identity":"0ae8ae37-576c-11f0-91e4-06cc9d20a69f","order_by":0,"name":"Acharya Balkrishna","email":"","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Acharya","middleName":"","lastName":"Balkrishna","suffix":""},{"id":479776474,"identity":"1244d8fd-576c-11f0-91e4-06cc9d20a69f","order_by":1,"name":"Harshita Jonwal","email":"","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Harshita","middleName":"","lastName":"Jonwal","suffix":""},{"id":479776558,"identity":"1a455454-576c-11f0-91e4-06cc9d20a69f","order_by":2,"name":"Nem Kumar Ngpoore","email":"","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Nem","middleName":"Kumar","lastName":"Ngpoore","suffix":""},{"id":479776593,"identity":"23996931-576c-11f0-91e4-06cc9d20a69f","order_by":3,"name":"Yash Varshney","email":"","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Yash","middleName":"","lastName":"Varshney","suffix":""},{"id":479776594,"identity":"2c717021-576c-11f0-91e4-06cc9d20a69f","order_by":4,"name":"Meenu Tomer","email":"","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Meenu","middleName":"","lastName":"Tomer","suffix":""},{"id":479776595,"identity":"35a8b900-576c-11f0-91e4-06cc9d20a69f","order_by":5,"name":"Monali Joshi","email":"","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Monali","middleName":"","lastName":"Joshi","suffix":""},{"id":479776596,"identity":"3e800146-576c-11f0-91e4-06cc9d20a69f","order_by":6,"name":"Kuldeep Singh","email":"","orcid":"","institution":"Patanjali Ayurved Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kuldeep","middleName":"","lastName":"Singh","suffix":""},{"id":479776597,"identity":"4780c0d8-576c-11f0-91e4-06cc9d20a69f","order_by":7,"name":"Pardeep Nain","email":"","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Pardeep","middleName":"","lastName":"Nain","suffix":""},{"id":479776685,"identity":"4fee8ae2-576c-11f0-91e4-06cc9d20a69f","order_by":8,"name":"Savita Lochab","email":"","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Savita","middleName":"","lastName":"Lochab","suffix":""},{"id":479776836,"identity":"58acf534-576c-11f0-91e4-06cc9d20a69f","order_by":9,"name":"Anurag Varshney","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYPACGwglwWDBA2EZENSSxsDABtYiQbSWwxAtIE0EAf/sHrMPP36dz+Of3/zsgUWNhAyD9OHDHxgK7uDUInHnjPHM3r7bxRLH2MwNJI4BHcaXlibBYPAMtzU3cowZeHtuJzYcYzCTkGADauHhMQP65TBOHfJALYx/e84lzj/G/k1C4h9IC//nD/i0GAC1MPP8OJC44RiPmYRkG9gWYCDg0WJ4I62YWbYhudjwWE6ZhGSfBA8bD5uZRAIeLXI3kjczvvljlyd3+Pg2aYlvNvb8PMyPP3z4g1sLGDC2MSSAaGZQrIDjJwG/BiD4A1HD+IGgylEwCkbBKBiJAAD7Tkq0sVjJ5QAAAABJRU5ErkJggg==","orcid":"","institution":"Patanjali Research Foundation","correspondingAuthor":true,"prefix":"","firstName":"Anurag","middleName":"","lastName":"Varshney","suffix":""}],"badges":[],"createdAt":"2025-06-19 06:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6928179/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6928179/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-31656-0","type":"published","date":"2025-12-10T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85989186,"identity":"53ba1a3d-4b4c-46b8-af74-fae3fce9a211","added_by":"auto","created_at":"2025-07-04 04:28:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":107862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGas Chromatography-tandem mass spectrometry (GC-MS/MS) analysis of Essential Oils of Dant-Kanti-Gandush (DKG). \u003c/strong\u003e(A) Representative chromatograms of DKG and a standard mixture, illustrating the retention times of identified phytometabolites. Major peaks corresponding to key phytocompounds are annotated. (B) Quantitative analysis of predominant phytometabolites in DKG, presented as bar graphs alongside their chemical structures. Values represent the relative percentage composition of each compound.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6928179/v1/64d3a8f48d3f5b9c5d718538.jpg"},{"id":85990118,"identity":"db086d73-2052-4584-8bd1-72c465ce3848","added_by":"auto","created_at":"2025-07-04 04:44:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKG demonstrated remarkable antimicrobial activity against oral pathogens.\u003c/strong\u003e\u003cbr\u003e\n(A) Antimicrobial activity of DKG against different microbial strains, by the disk diffusion method. The inhibition zones correspond to different concentrations of DKG.\u003cbr\u003e\n(B-E) A dose-response curve was plotted using non-linear regression based on data from broth microdilution assays of DKG against \u003cem\u003eS. pyogenes\u003c/em\u003e (B), \u003cem\u003eP. mirabilis\u003c/em\u003e (C), \u003cem\u003eS. mutans\u003c/em\u003e (D) and \u003cem\u003eC. albicans\u003c/em\u003e (E). MIC₅₀ and MIC₉₀ values are marked with arrows, indicating the concentrations required to inhibit 50% and 90% of microbial growth, respectively. Data represents the mean from three independent experiments, with error bars indicating mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6928179/v1/21db742f3bd5d0b6ccb42dc2.jpg"},{"id":85989187,"identity":"e238c33e-62cc-4d4e-ba6d-b14b1bc39a49","added_by":"auto","created_at":"2025-07-04 04:28:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":93365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKG decelerated the exponential growth phase of oral pathogens.\u003c/strong\u003e Growth kinetics of microbial strains in the presence of different concentrations (0.25×, 0.5×, 1.0× and 2.0× MIC\u003csub\u003e50\u003c/sub\u003e) of DKG. Absorbance (OD\u003csub\u003e600\u003c/sub\u003e\u003cbr\u003e\n) was measured over time to assess the inhibitory effects of DKG on (A) \u003cem\u003eS. pyogenes\u003c/em\u003e, (B) \u003cem\u003eP. mirabilis\u003c/em\u003e, (C) \u003cem\u003eS. mutans\u003c/em\u003e and (D) \u003cem\u003eC. albicans\u003c/em\u003e. Each curve represents microbial growth under varying DKG concentrations, with higher concentrations showing stronger inhibition. Data represents mean from triplicate experiments, with error bars indicating mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6928179/v1/eab52e0c781c64e26200b030.jpg"},{"id":85989189,"identity":"b3ba41b9-c085-4ab3-8b22-3051df25ce41","added_by":"auto","created_at":"2025-07-04 04:28:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":185426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKG suppressed cariogenic potential of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. mutans.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(A) Microscopic analysis of bacterial biofilm formation under different concentrations of DKG. The untreated (UT) sample exhibits dense biofilm coverage, while progressive concentrations of DKG (0.5×, 1.0×, and 2.0× MIC₅₀) concomitantly reduce biofilm formation. The highest concentration (2.0× MIC₅₀) results in significant disruption of biofilm structure. Scale bars represent 20 μm. (B) Quantification of biofilm inhibition by DKG at different concentrations. The bar graph shows the percentage of biofilm biomass relative to the untreated control (UT). A dose-dependent reduction in biofilm formation is observed with increasing DKG concentrations (0.5×, 1.0×, and 2.0× MIC₅₀). (C) Glycolytic pH drop assay of \u003cem\u003eS. mutans\u003c/em\u003e. DKG treatment significantly inhibited the glycolytic pH drop at 0.5×, 1.0×, and 2.0× MIC₅₀ concentrations. The decrease in pH values over time was modeled using a one-phase decay fit, and the resulting curve was used to derive τ (tau) values. (D-E) Effect of DKG on the acid tolerance of bacterial cells. Bar graphs depict the survival percentage of bacterial cells exposed to acidic conditions (pH ~5.0) with or without DKG treatment at varying concentrations (0.5×, 1.0×, and 2.0× MIC₅₀). Representative images of bacterial colonies recovered following acid exposure. A dose-dependent reduction in bacterial survival is evident with increasing concentrations of DKG. Error bars represent mean ± SEM; (***) indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) from triplicate experiments.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6928179/v1/7988a9acdbbc91055073ddd7.jpg"},{"id":85989532,"identity":"3220bfd7-e375-4e43-86fe-4afe5e9463b4","added_by":"auto","created_at":"2025-07-04 04:36:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":234070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKG inhibited\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eyeast-to-hypha conversion and biofilm formation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans. \u003c/strong\u003e\u003c/em\u003e(A) Yeast cells treated with DKG at increasing concentrations (0.5×, 1.0×, and 2.0× MIC\u003csub\u003e50\u003c/sub\u003e). Representative phase-contrast microscopy images show untreated (UT) and DKG-treated yeast cells. Scale bars represent 2 μm. (B) Microscopic observation of \u003cem\u003eC. albicans\u003c/em\u003e biofilm strained with crystal violet. The untreated (UT) \u003cem\u003eC. albicans\u003c/em\u003e biofilm shows dense coverage, while DKG treatment (0.5×, 1.0×, and 2.0× MIC\u003csub\u003e50\u003c/sub\u003e) progressively induced disruption of biofilm. Scale bars indicate 40 μm. (C) Subsequent quantification of the surface area covered by biofilm in (B) using ImageJ. (D) Representative chromatogram for ergosterol detection using UHPLC-DAD. The chromatograms displaying peaks corresponding to ergosterol detected in \u003cem\u003eC. albicans\u003c/em\u003e treated with different concentrations of DKG. Quantification performed with reference to the ergosterol standard (blue). Inset provides a magnified view of the main ergosterol peak, highlighting differences in peak shape and intensity across DKG treatments. (E) Quantification of ergosterol content detected in (D) represented through bar graph. Error bars represent mean ± SEM from three independent experiments; significance of data has been represented as, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6928179/v1/9849002c8bfc2875336e49e9.jpg"},{"id":85989191,"identity":"f5ba0bef-3f42-4b7c-8404-69af9b35d02e","added_by":"auto","created_at":"2025-07-04 04:28:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":198457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKG inhibited biofilm formation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. mutans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in mono- and co-culture conditions. \u003c/strong\u003e(A-C) Scanning Electron Microscopy (SEM) images showing biofilm formation of \u003cem\u003eS. mutans\u003c/em\u003e (A), \u003cem\u003eC. albicans\u003c/em\u003e(B) and co-cultured \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e in the presence 1.0% (v/v) and absence of DKG. Left panel depicts a dense biofilm matrix of clustered microbial cells (control), while the right panel shows a disrupted biofilm structure with scattered cells, indicating reduced biofilm formation (treated with 1.0% (v/v) of DKG). Inset in all images provide a visual overview of the biofilms at lower magnification. Scale bars indicate 20.0 µm and 100.0 µm, respectively.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6928179/v1/806e9f585f0050c38cffd695.jpg"},{"id":85990119,"identity":"d55f9145-2b5e-4f3e-92c1-bfc3485e5629","added_by":"auto","created_at":"2025-07-04 04:44:20","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":226450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDKG prevented the cross-kingdom biofilm formation by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. mutans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e on Orthodontic fixtures. \u003c/strong\u003eScanning electron microscopy (SEM) images of \u003cstrong\u003e(\u003c/strong\u003eA) Elastomeric ligature and (B) Orthodontic brackets\u003cstrong\u003e \u003c/strong\u003eshowing biofilms formed by co-cultured \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e. The untreated (UT) samples (pink background) exhibit dense and well-established biofilm structures (panel 1, 2 and 3) characterized by extensive microbial colonization and extracellular matrix production. In contrast, the DKG (1.0% (v/v) treated samples (blue background) show significant biofilm disruption and reduced microbial adhesion (panel 4, 5 and 6), with altered microbial morphology and reduced biofilm density on the surface of the elastomeric ligature and orthodontic bracket.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6928179/v1/b4b3b453611c4e0114f569a7.jpg"},{"id":98244862,"identity":"a6203680-db24-4f04-9208-4d1cb4e7c838","added_by":"auto","created_at":"2025-12-15 16:15:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2715461,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6928179/v1/b78c2811-5edf-4f03-8fc9-4a46b39774b4.pdf"}],"financialInterests":"Competing interest reported. The test article, Dant-Kanti-Gandush was sourced from Divya Pharmacy, Haridwar, India. AB is an honorary trustee in Divya Yog Mandir Trust, which governs Divya Pharmacy, Haridwar. In addition, he holds an honorary managerial position in Patanjali Ayurved Ltd., Haridwar, India. Divya Pharmacy and Patanjali Ayurved Ltd commercially manufacture and sell several Ayurvedic products. Other than providing the test article, Divya Pharmacy was not involved in any part of this study. Other authors have no conflict of interest to disclose.","formattedTitle":"Essential Oils Enriched Dant-Kanti-Gandush (Oil-pulling) Inhibits Inter-kingdom Biofilm Formation on Orthodontic Fixtures and Ameliorates Cariogenic Virulence Factors of Oral Pathogens","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDental caries, commonly known as tooth decay or cavity formation, is one of the most prevalent global health issues, affecting individuals of all ages \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. According to the WHO Global Oral Health Status Report (2022), nearly 3.5\u0026nbsp;billion people are affected by oral diseases, with 3 out of 4 people living in middle-income countries. Specifically, about 2\u0026nbsp;billion people suffer from caries of permanent teeth, and 514\u0026nbsp;million children are affected by caries of primary teeth. Interestingly, oral cavity harbors a complex microbiome, consisting of approximately 700 bacterial species that form biofilms on surfaces like teeth, gingiva, and prosthetic devices \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eStreptococcus mutans\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e, are key contributors to dental caries and contribute to biofilm formation and acid production, which leads to enamel demineralization and development of cavities \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Therefore, maintaining a balanced oral microbiota and practicing good oral hygiene are critical in preventing the formation of dental caries and related infections.\u003c/p\u003e \u003cp\u003eBiofilm formation is a critical factor in the development and progression of dental caries, as it enables pathogenic bacteria and fungi to thrive in the oral cavity. The primary cariogenic bacterium, \u003cem\u003eS. mutans\u003c/em\u003e, initiates biofilm development by adhering to salivary pellicles on tooth surfaces, aided by extracellular polymeric substances (EPS), mainly glucans produced by glucosyltransferases \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These glucans facilitate bacterial aggregation and biofilm maturation, making the biofilm highly resilient and structured. Additionally, \u003cem\u003eCandida albicans\u003c/em\u003e, another key pathogen involved in biofilm formation, produces organic acids such as pyruvate and formate, which lower the pH of the surrounding environment, further promoting enamel demineralization \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This acidic environment created by \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e enhances bacterial survival and accelerates tooth decay \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e has been associated with dental plaque while, \u003cem\u003eProteus mirabilis\u003c/em\u003e, is commonly linked to urinary tract infections, has also been isolated from the oral cavity \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOrthodontic surfaces provide niche for bacterial adhesion and biofilm development, they contribute to increased bacterial load, enamel demineralization, and inflammation of the gingiva \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Therefore, preventing biofilm formation is essential in preventing dental caries and preserving oral health, particularly in patients with orthodontic fixtures, where biofilm control is challenging yet crucial for long-term oral health maintenance.\u003c/p\u003e \u003cp\u003eOil pulling, also known as \u003cem\u003eGandusha\u003c/em\u003e or \u003cem\u003eKavala Graha\u003c/em\u003e in Ayurvedic medicine, is a traditional Indian remedy practiced for centuries to maintain oral hygiene and overall health. The technique involves swishing edible oils such as coconut, sesame, or sunflower oil in the mouth for several minutes to facilitate oral detoxification and enhance oral hygiene. It is believed that oil pulling works by emulsifying and trapping bacteria within the oil, effectively reducing microbial load, dissolving plaque biofilms, and promoting gum health. Unlike commercial mouthwashes, oil pulling is safe, accessible, and free from side effects such as staining, unpleasant aftertaste, or allergic reactions, making it a favorable adjunct to conventional oral hygiene practices \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Coconut oil contains lauric acid, which has shown antimicrobial properties, significantly reducing the levels of \u003cem\u003eStreptococcus mutans\u003c/em\u003e, a key bacterium responsible for dental caries. Studies have demonstrated that oil pulling with antimicrobial oils not only reduces plaque accumulation but also promotes gum health by combating harmful bacteria and supporting oral hygiene. In orthodontic settings, essential oils such as eucalyptol have been shown to reduce biofilm thickness and bacterial colonization on orthodontic fixtures, including brackets and arch wires \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Other essential oil components like limonene and linalool disrupt bacterial cell membranes, inhibiting the growth of \u003cem\u003eS. mutans\u003c/em\u003e, further highlighting their role in managing biofilm-related complications \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe current study demonstrates the antimicrobial activity of essential oils of Dant-Kanti-Gandush (DKG) against oral pathogens, including \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eP. mirabilis\u003c/em\u003e, \u003cem\u003eS. pyogenes\u003c/em\u003e, and \u003cem\u003eC. albicans.\u003c/em\u003e DKG is a combination of essential oil extracted from clove (\u003cem\u003eSyzygium aromaticum\u003c/em\u003e), peppermint (\u003cem\u003eMentha piperita\u003c/em\u003e), eucalyptus (\u003cem\u003eEucalyptus globulus\u003c/em\u003e), prickly ash (\u003cem\u003eZanthoxylum armatum\u003c/em\u003e), and basil (\u003cem\u003eOcimum sanctum\u003c/em\u003e). The oral pathogens showed reduced growth dynamics in a dose-dependent manner upon exposure to DKG. Notably, DKG disrupts the cross-kingdom colonization and biofilm formation of \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e on orthodontic braces. These findings suggest that DKG may serve as a safe, natural adjunct for managing dental caries and biofilm-associated oral infections, reinforcing the role of Ayurveda as a complementary and alternative medicine.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProcurement of test article, microbial strains and growth conditions\u003c/h2\u003e \u003cp\u003eThe test article, a blend of essential oil present in Dant-Kanti-Gandush (Batch# PRF/CHI/0424/0288) was supplied by Herbal Chemistry Department, Patanjali Research Foundation, Haridwar, India. The study employed specific microbial strains, which were sourced from Microbial Type Culture Collection (MTCC), CSIR-Institute of Microbial Technology (Chandigarh, India). The bacterial strains used included \u003cem\u003eStreptococcus mutans\u003c/em\u003e (MTCC 497), \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e (MTCC 442), and \u003cem\u003eProteus mirabilis\u003c/em\u003e (MTCC 1429), as well as the fungal strain \u003cem\u003eCandida albicans\u003c/em\u003e (MTCC 183). Bacterial species were cultured in Brain Heart Infusion (BHI) broth (HiMedia, India), while the yeast was cultured in Yeast Extract Peptone Dextrose (YPD) broth (HiMedia, India). Single isolated colonies of each microorganism were inoculated in their respective media and incubated at 35\u0026ndash;37\u0026deg;C for 24\u0026ndash;48 hours. For routine maintenance, these strains were sub-cultured onto Brain Heart Infusion agar and Yeast Extract Peptone Dextrose agar and preserved as glycerol stocks at \u0026minus;\u0026thinsp;80\u0026deg;C for long-term storage.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGas chromatography-mass spectrometry (GC-MS/MS) Analysis of Essential Oil\u003c/h3\u003e\n\u003cp\u003eGC-MS/MS (7000D GC/MS triple quad with 7890B GC system, Agilent-USA) was performed using mass hunter software to identify, quantify, and characterize chemical compounds present in DKG. Separation was carried out using Agilent HP-5MS capillary column (30 m x 0.25 mm, 0.25 \u0026micro;m) Helium was used as carrier gas at a flow rate of 1 mL/min. The temperature of the split injector was maintained at 280\u0026deg;C and the split ratio was 20:1. The column temperature was initially set at 60\u0026deg;C without hold, then ramped at 5\u0026deg;C/min to 100\u0026deg;C (held for 1 min), followed by an increase at 3\u0026deg;C/min to 160\u0026deg;C (held for 3 minutes), and finally ramped at 10\u0026deg;C/min to 280\u0026deg;C, where it was held for 1 minute. The GC-MS temperature was 230\u0026deg;C and ionization potential was 70 eV.\u003c/p\u003e\n\u003ch3\u003eAntimicrobial effects of Dant-Kanti-Gandush Essential Oil\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of DKG was evaluated using the disk diffusion method. The optical density (OD) of an overnight-grown culture was adjusted to 0.06 at 600 nm, and the bacterial culture was swabbed onto Muller Hinton Agar, while \u003cem\u003eCandida albicans\u003c/em\u003e was swabbed on Yeast Peptone Dextrose agar. Sterile disks carrying varying concentrations of DKG (5% (v/v), 25% (v/v), 50% (v/v), and 100% (v/v)) were placed to the agar plates. A sterile disk impregnated with sterile water was included as a negative control and placed at the center of the plate. The plates were incubated at 37\u0026deg;C for 24 hours.\u003c/p\u003e\n\u003ch3\u003eMicrobroth- dilution assay\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of DKG was assessed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI, 2015) guidelines. The Minimum Inhibitory Concentration (MIC) test was conducted using 96-well tissue culture microplates, each containing 100 \u0026micro;L of Brain Heart Infusion (BHI) medium (Himedia, India). The stock solution of DKG was initially diluted to 10% v/v by adding sterile autoclaved water, and this was transferred to the first well. Two-fold serial dilutions were subsequently performed, resulting in concentrations ranging from 0.02% (v/v) to 5.0% (v/v). The bacterial inoculum (OD 0.06\u0026ndash;0.08 at 600 nm) was added to each well, except for the blank wells, which contained only the medium contain respective DKG concentration. The plates were then incubated at 37\u0026deg;C for 24 h. The experiments were repeated in three biological replicates.\u003c/p\u003e\n\u003ch3\u003eGrowth kinetic assessment in oral pathogens\u003c/h3\u003e\n\u003cp\u003eThe microbial cultures were adjusted to an optical density (OD) 0.06\u0026ndash;0.08 and treated with different concentrations of DKG (0.25\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, 0.50\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, 1.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, 2.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e and 4.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e) in 96-well microtiter plate. Growth kinetics of the bacterial and yeast cultures were recorded every two hours while their incubation at 37\u0026deg;C for 24 hours in Infinite 2000 Pro microplate reader (Tecan Group Ltd., Switzerland). All experiments were performed in independent triplicate to ensure consistency and reproducibility. The recorded absorbance, after blank correction, was plotted using a non-linear regression curve fit function.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Biofilm formation\u003c/h2\u003e \u003cp\u003eBiofilm biomass assessment was performed by crystal violet staining method and scanning electron microscopy. Actively growing \u003cem\u003eS. mutans\u003c/em\u003e culture adjusted at optical density of 0.1 at 600nm in BHI media (supplemented with 1% sucrose) was used to establish biofilm on the sterile cover slips in the 6-well plate. Biofilm in \u003cem\u003eC. albicans\u003c/em\u003e was developed on the sterile cover slips in the 6-well plate in YPD (supplemented with 50 mM glucose). For dual-species co-culturing, equal volumes (1:1) of the above cultures in their respective media were added to sterile coverslips in a 6-well plate. Subsequently, DKG at different concentrations (0.5\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e: 0.25% (v/v), 1\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e: 0.50% (v/v), and 2\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e: 1.0% (v/v)) was added to the \u003cem\u003eS. mutans\u003c/em\u003e culture and allowed to develop biofilm under aerobic incubation in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The supernatant was aspirated after 72 h of incubation and the tightly adhered cells forming biofilms were washed twice with PBS to remove planktonic bacterial cells. Biofilm was fixed with 2.0% formaldehyde for 20 minutes, followed by crystal violet (0.1%) staining for 30 minutes. Once dried, the stained biofilms were photographed under an TS2 inverted brightfield microscope (Nikon, Japan). The surface area covered by the biofilm was evaluated using ImageJ software (US National Institutes of Health, Bethesda, Maryland, USA). The biofilms formed by co-cultured inter-kingdom species, \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e were examined using a scanning electron microscope (FlexSEM1000, Hitachi, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGlycolytic pH drop assay\u003c/h3\u003e\n\u003cp\u003eUsing a pH-drop experiment, the impact of DKG on \u003cem\u003eS. mutans\u003c/em\u003e acidogenicity was assessed. The actively growing \u003cem\u003eS. mutans\u003c/em\u003e culture was harvested, washed and resuspended in the buffer containing 50 mM potassium chloride (KCl) and 1 mM magnesium chloride (MgCl₂) in the presence of DKG at different concentrations (0.25\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, 0.50\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, 1.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, 2.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e and 4.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e). Glucose was added to a final concentration of 55.6 mM, and the initial pH of the mixtures was adjusted to 7.2\u0026ndash;7.4 using 1.0 M potassium hydroxide. Change in the pH was recorded (Laboholic Microprocessor pH meter, India) at 20-minute intervals over a total duration of 100 minutes \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The experiment was conducted in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003ch3\u003eAcid tolerance assay\u003c/h3\u003e\n\u003cp\u003eAcid tolerance is defined as the ability of bacteria to survive in acidic environments, a critical characteristic of \u003cem\u003eS. mutans\u003c/em\u003e, which employ both constitutive and acid-inducible mechanisms (Matsui et al., 2010). The effect of DKG on the acidurity of \u003cem\u003eS. mutans\u003c/em\u003e was assessed by exposing bacteria to an acidic pH of 5.0. Actively growing \u003cem\u003eS. mutans\u003c/em\u003e bacterial cells were harvested to resuspend in TYEG (Tryptone Yeast Extract Glucose) broth (pH 5.0) in the presence of DKG at sub-inhibitory and inhibitory concentrations of DKG (0.5%, 1%, and 2% (v/v)) at 37\u0026deg;C for 6 and 24 hours \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The untreated control group contained no DKG. After incubation, viability of \u003cem\u003eS. mutans\u003c/em\u003e was determined from all treatment conditions by plating the bacterial cells on Brain heart infusion agar plates. The experiment was independently repeated three times to ensure reproducibility (15).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHyphal growth assessment in\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eCandida albicans\u003c/em\u003e cultured in Yeast Peptone Dextrose (YPD) medium supplemented with 10% Fetal Bovine Serum to induce hyphal formation \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Yeast culture adjusted to an optical density of 0.1 at 600 nm was inoculated in hyphal-inducing media (YPD medium containing 10% Fetal Bovine Serum) in the presence of DKG (0.5\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e: 0.05% (v/v), 1\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e: 0.10% (v/v), and 2\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e: 2.0% (v/v)) at 37\u0026deg;C with shaking for 6 h and 24 h. After incubation cell morphology was observed and photographed using a Zeiss Observer.Z1 microscope (Carl Zeiss, Jena, Germany) (16).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eUHPLC-DAD based ergosterol detection\u003c/h2\u003e \u003cp\u003eFrom an overnight-grown culture, a 100 mg pellet of untreated yeast cells and cells treated with DKG (at 0.5x, 1.0x and 2.0x MIC\u003csub\u003e50\u003c/sub\u003e) was dissolved in 0.25 mL of methanol and homogenized by adding sterile stainless-steel balls for 10 minutes. This solution was then centrifuged for 5 min at 10000 rpm and 100 \u0026micro;l of supernatant was transferred in injecting vials and injected into the system. Ergosterol standard (of 1 mg/mL) in methanol was used to prepare 1000 ppm standard solution. 0.1 ml of this standard solution was diluted to 10 ml to prepare 10 \u0026micro;g/mL working stock. Ergosterol content of the treated and untreated \u003cem\u003eC. albicans\u003c/em\u003e was analyzed on Prominence-XR UHPLC system (Shimadzu, Japan) equipped with Quaternary pump (Nexera XR LC-20AD XR), DAD detector (SPD-M20 A), Auto-sampler (Nexera XR SIL-20 AC XR), Degassing unit (DGU-20A 5R) and Column oven (CTO-10 AS VP). Separation was achieved using a Shodex C18-4E (5 \u0026micro;m, 4.6*250 mm) column subjected to isocratic elution with a flow rate of 1.0 mL/min. The mobile phase was used for the analysis consisted of the ratio of methanol: acetonitrile (80:20). 50 \u0026micro;L of standard and test solutions were injected and wavelength was set at 280 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eScanning electron microscopy on orthodontic brackets\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe dual-species biofilms were formed using the same method as described in the above section, with the only difference being that clean orthodontic brackets and rings (Koden orthodontic brackets, provided by Dental Clinic and Research Centre, Patanjali Ayurved Hospital, Haridwar, India) replaced the coverslips in the six-well plate. Subsequently, DKG treatment (1.0% (v/v)) was added to the co-cultures and plates were aerobically incubated at 37\u0026deg;C for 72 hours in 5% CO₂ (17). Orthodontic brackets and rings were thoroughly washed to remove planktonic cells, fixed in formaldehyde and air dried before imaging under SEM scanning electron microscope (FlexSEM1000, Hitachi, Japan) to observe the effect of DKG on dual-species biofilms.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGas Chromatography-tandem mass spectrometry (GC-MS/MS) analysis of Dant-Kanti-Gandush Essential Oils (DKG)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe oil-pulling formulation, DKG consists of 90% (v/v) \u003cem\u003eSesamum indicum\u003c/em\u003e (sesame) oil and 8% \u003cem\u003eCocos nucifera\u003c/em\u003e (coconut) oil. The oil base, constituting 98% of the formulation, is supplemented with a 1.5% essential oil blend from \u003cem\u003eSyzygium aromaticum\u003c/em\u003e, \u003cem\u003eMentha piperita\u003c/em\u003e, \u003cem\u003eEucalyptus globules\u003c/em\u003e, \u003cem\u003eZanthoxylum armatum\u003c/em\u003e and \u003cem\u003eOcimum sanctum\u003c/em\u003e, in a ratio of 2:3:8:1:1, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The essential oil blend of Dant-Kanti-Gandush (hereafter, called as DKG) was subjected to detailed microbiological testing, and GC-MS/MS analysis for phytochemical profiling. The chromatograph generated demonstrated prominent peaks, which were subsequently identified by matching mass fragmentation data with the National Institute of Standards and Technology (NIST, USA) library (MS Search 2.2). Notably, the identified phytometabolites, D-limonene, eucalyptol, linalool, menthol and eugenol were further validated and quantified against their respective reference standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The quantified content percentage (w/w) for D-limonene, eucalyptol, linalool, menthol and eugenol were 5.64%, 21.79%, 3.34%, 5.55%, and 3.32%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of Dant-Kanti-Gandush.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnglish name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eScientific name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlant Part\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eForm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eQty. (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eEssential oils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClove\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSyzygium aromaticum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBuds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePeppermint\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMentha piperita\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEucalyptus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eEucalyptus globules\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrickly ash\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eZanthoxylum armatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSeeds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBasil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eOcimum sanctum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCarrier oils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoconut\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCocos nucifera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEndosperm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSesame\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSesamum indicum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSeeds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDant-Kanti-Gandush demonstrated remarkable antimicrobial activity against oral pathogens\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe study tested the anti-microbial activity of DKG, blend of essential oils, against few common oral pathogens associated with dental caries and periodontal infections, such as \u003cem\u003eStreptococcus mutans\u003c/em\u003e, \u003cem\u003eProteus mirabilis\u003c/em\u003e, \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e. Initial screening by disc diffusion demonstrated the potential antimicrobial activity of DKG, tested at 50% (v/v) and 100% (v/v) against these oral pathogens. DKG against \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e showed a zone of inhibition of 10 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 at 50% (v/v) and 11.5 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 at 100% (v/v). The bacterial lawn of \u003cem\u003eProteus mirabilis\u003c/em\u003e exhibited\u0026thinsp;\u0026lt;\u0026thinsp;10 mm and 12.1 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 zone at 50% (v/v) and 100% (v/v), respectively. DKG against \u003cem\u003eStreptococcus mutans\u003c/em\u003e showed\u0026thinsp;\u0026lt;\u0026thinsp;10 mm and 10.8 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 of clear zone at 50% (v/v) and 100% (v/v), respectively. Most prominent clearance with zone diameters of 11.8 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83, 19 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96 and 25 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28 at 25% (v/v), 50% (v/v) of DKG, respectively was observed against \u003cem\u003eCandida albicans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Carrier oils of Dant Kanti Gandush, sesame oil and coconut oil, were also tested in these experiments with minimal effects (Data not shown).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMinimum inhibitory concentrations (MIC) corresponding to MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e were also determined using broth microdilution method to evaluate the DKG concentrations that inhibited 50% and 90% of visible microbial growth, respectively. For \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, DKG exhibited MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e at 0.10% (v/v) and 0.17% (v/v), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e for DKG in \u003cem\u003eProteus mirabilis\u003c/em\u003e were evaluated to be 0.29% (v/v) and 1.59% (v/v), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e in \u003cem\u003eStreptococcus mutans\u003c/em\u003e were evaluated as 0.45% (v/v) and 1.70% (v/v), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). DKG demonstrated MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e in \u003cem\u003eCandida albicans at\u003c/em\u003e 0.11% (v/v) and 0.64% (v/v), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDant-Kanti-Gandush decelerated the exponential phase of oral pathogens\u003c/h2\u003e \u003cp\u003eThe time-kill assay in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (A-D), demonstrated a time-dependent anti-microbial activity of DKG over a 22 h incubation period. The untreated controls from all four pathogens tested exhibited continuous logarithmic growth up to 22 h, whereas DKG exposure retarted the growth progression profiles, significantly. In \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, a delay in the onset of the exponential phase was evident at 1.0\u0026times; and 2.0\u0026times; of MIC\u003csub\u003e50\u003c/sub\u003e compared to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, in \u003cem\u003eProteus mirabilis\u003c/em\u003e, DKG not only delayed the exponential phase by 2\u0026ndash;4 h, but the pathogen also achieved the stationary phase at half the growth density (0.5 and 0.2 optical density at 1.0\u0026times; and 2.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, respectively) compared to the untreated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The most pronounced inhibition in growth dynamics was observed in \u003cem\u003eStreptococcus mutans\u003c/em\u003e, where exponential growth phase was observed after 12 h at 1.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, compared to 2 h in the untreated. Interestingly, at the highest dose of 2.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, no growth progression was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, in \u003cem\u003eCandida albicans\u003c/em\u003e, the exponential phase initiated after 12\u0026ndash;14 h of growth at 2.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e compared to 4\u0026ndash;6 h in the untreated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Collectively, DKG limited the microbial proliferation rate and even inhibited the pathogens to achieve usual population density and biomass required for achieving stationary phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDant-Kanti-Gandush suppresses the cariogenic properties in\u003c/b\u003e \u003cb\u003eS. mutans\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. mutans\u003c/em\u003e contribute substantially to oral biofilm formation, enamel demineralization posing risks to oral and overall health. DKG was evaluated for its impact on the biofilm forming potential of \u003cem\u003eS. mutans\u003c/em\u003e. Untreated samples exhibited dense biofilm formation, whereas DKG treatment at 0.5\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e and above showed a dose-dependent significant disruption in the biofilms formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). ImageJ analysis revealed a significant reduction in the surface area covered by biofilm in the presence of DKG. The surface area covered reduced by ~\u0026thinsp;30%, ~\u0026thinsp;50% and ~\u0026thinsp;70% at 0.5\u0026times;, 1.0\u0026times; and 2.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e of DKG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of DKG on \u003cem\u003eS. mutans\u003c/em\u003e acidogenicity was determined by monitoring glycolytic pH drop (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In untreated cultures, pH decreased from 7.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 to 3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 within 100 minutes, indicating acid production capabilities of \u003cem\u003eS. mutans\u003c/em\u003e. However, DKG treatment significantly delayed the pH drop, with terminal pH values of 5.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61, 5.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61, and 6.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 at 0.5\u0026times;, 1.0\u0026times;, and 2.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Higher concentrations of DKG notably impeded acid production. An exponential decay curve was generated on the obtained pH values to calculate Tau (τ) constant. The decay rate (τ\u003csup\u003e\u0026minus;1\u003c/sup\u003e) evaluated by fit function for untreated (41.14), 0.5\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e (83.30), 1.0\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e (74.36) and ambiguous results for 2.0\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e indicated inhibited acidogenicity potential in \u003cem\u003eS. mutans\u003c/em\u003e in the presence of DKG targeted. In addition, the acid tolerance capability of S. \u003cem\u003emutans\u003c/em\u003e, when evaluated in the presence of DGK, on the contrary showed notable reduction in the number of viable \u003cem\u003eS. mutans\u003c/em\u003e colonies at 6 h and 24 h under acid stress. At 6 h, viable counts calculated as CFU/mL reduced from 725 \u0026times; 10⁶ (Untreated) to 329 \u0026times; 10⁶ (0.5\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e) 118 \u0026times; 10⁶ (1.0\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e) and 17 \u0026times; 10⁶ (2.0\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Whereas, by 24 h, further reductions were observed from 517 \u0026times; 10⁶ (Untreated) to 189 \u0026times; 10⁶ (0.5\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e) 4 \u0026times; 10⁶ (1.0\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e). Notably, at 2.0\u0026times;MIC\u003csub\u003e50\u003c/sub\u003e no viability of bacteria was observed in the presence of DKG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDant-Kanti-Gandush inhibited yeast-to-hypha conversion and biofilm formation in\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYeast to hyphae transition and biofilm formation are key factors imparting pathogenicity to \u003cem\u003eCandida albicans\u003c/em\u003e. DKG exposure for 6 h and 24 h notably inhibited the budding and hyphal germination in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). \u003cem\u003eC. albicans\u003c/em\u003e biofilm formation also observed a significant inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The density of the biofilm and subsequent surface area covered significantly reduced by ~\u0026thinsp;45\u0026ndash;50% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Ergosterol is an essential component of fungal cell wall. Disruption in ergosterol biosynthesis or downregulation in ergosterol levels indicate compromised cell membrane integrity \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In order to test whether DKG could exert similar effect on \u003cem\u003eC. albicans\u003c/em\u003e, ergosterol levels were evaluated through UHPLC method. The ergosterol content significantly decreased from 100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;25.27 \u0026micro;g/mg to 33.39\u0026thinsp;\u0026plusmn;\u0026thinsp;30.67 \u0026micro;g/mg and 19.36\u0026thinsp;\u0026plusmn;\u0026thinsp;7.50 \u0026micro;g/mg when were exposed to at 2.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e and 4.0\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDant-Kanti-Gandush inhibited biofilm formation of\u003c/b\u003e \u003cb\u003eS. mutans\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003ein mono-and co-culture conditions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDental plaques are tight-structured, multi-species, cross-kingdom biofilms. Their formation not only drives dental caries and other periodontal diseases but also reduces susceptibility to antimicrobial treatments, posing a significant challenge in oral health management. The anti-plaque potential of DKG was evaluated by assessing its efficacy against biofilms formed by \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, both individually and in a co-cultured interkingdom biofilm model. Scanning electron microscopy (SEM) was performed on biofilms formed on coverslips by indicated oral pathogens. The dense biofilm formed by \u003cem\u003eS. mutans\u003c/em\u003e showed disrupted and spaced growth in the presence of DKG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). \u003cem\u003eCandida albicans\u003c/em\u003e showed a dense growth on the coverslip. However, in the presence of DKG, yeast cells exhibited disruption with irregularly shaped yeast structures and even shrinkage. This indicated membrane damage or stress confirming the antifungal effect of DKG in suppressing biofilm formed by \u003cem\u003eC. albicans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The SEM analysis illustrated a stark contrast between the untreated and DKG exposed \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e inter-kingdom biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The untreated biofilms illustrate a dense, tight-structured microbial network with extensive interactions between bacterial chains and fungal hyphae. In contrast, DKG exposure severely disrupted the microbial density, fragmented hyphal structures and disorganized bacterial aggregation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDant-Kanti-Gandush prevented the cross-kingdom biofilm formation by\u003c/b\u003e \u003cb\u003eS. mutans\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003eon Orthodontic fixtures\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOrthodontic fixtures provide an ideal niche for microbial adhesion and growth, particularly in the presence of oral saliva \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e culture were inoculated on the sterile orthodontic brackets and elastomeric ligature in the presence of their respective growth media. Scanning electron micrographs of elastomeric ligature and orthodontic brackets demonstrated a thick microbial growth, which upon further zooming in showed dense meshwork of hyphal growth by the yeast. \u003cem\u003eS. mutans\u003c/em\u003e cells appear embedded within the exopolysaccharide (EPS) matrix, reinforcing microbial co-aggregation and resilience. DKG exposure, on the contrary prevented the formation of dense architecture and EPS matrix, thereby inhibiting microbial adherence and biofilm formation. The microbial growth was drastically reduced, with \u003cem\u003eC. albicans\u003c/em\u003e fragmented hyphae and deformed yeast cells. \u003cem\u003eS. mutans\u003c/em\u003e appearing sparsely distributed, lysed and ruptured on the surface of both elastomeric ligature (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and orthodontic brackets (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eEssential oils (EOs) are volatile secondary metabolites produced by plants, responsible for imparting typical aroma, flavor, or both. Essential oils (EOs) have been extensively studied for their therapeutic potential across various diseases. Their pharmacological properties encompass antimicrobial, anti-inflammatory, antitumor, and antioxidant activities \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The current study has explored EOs as alternative or adjunctive agents in oral healthcare. Dant-Kanti-Gandush (DKG) oil consists of a 98.0% fixed oil blend of coconut and sesame oil, with the remaining 2.0% comprising essential oils from clove, peppermint, eucalyptus, prickly ash, and basil. DKG is recommended for oil pulling, an ancient Ayurvedic practice, that has recently gained popularity for its natural, cost-effective and health benefits. The process involves swishing oil in the mouth for about 10\u0026ndash;20 minutes. Traditionally, sesame oil or coconut oils are preferred over other edible oils. Sesame oil, known for its antimicrobial properties and plaque-removing ability, contains lignans such as sesamin, sesamolin, and sesaminol, which possess strong antioxidant activity \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Coconut oil, rich in lauric acid, plays a scientifically recognized role in oral hygiene by exhibiting antimicrobial and antibiofilm activity against plaque-causing and cariogenic bacteria. Additionally, it possesses antioxidant and anti-inflammatory properties \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.However, the combination of five essential oils in DKG serves as an enhancement to the traditional oil-pulling formulation. The present study elucidates the antimicrobial efficacy of this essential oil combination in inhibiting growth, attenuating virulence factors, and disrupting biofilm formation in key oral pathogens, \u003cem\u003eStreptococcus mutans\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e. The 2.0% composition of DKG consists of essential oils containing a substantial amount of eucalyptol, D-limonene, menthol, linalool, and eugenol, as identified through GC-MS/MS analysis. Interestingly, MIC determination revealed that this essential oil mix exhibited a remarkably low effective concentration of 0.45% (v/v), 0.29% (v/v), 0.10% (v/v), and 0.11% (v/v) against \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eP. mirabilis\u003c/em\u003e, \u003cem\u003eS. pyogenes\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e, respectively. All pathogens under the \u003cem\u003ein vitro\u003c/em\u003e conditions are inhibited at \u0026lt;\u0026thinsp;0.5% (v/v) of essential oil mix present in Dant-Kanti-Gandush (DKG). When treated above MIC\u003csub\u003e50\u003c/sub\u003e, the essential oil mix significantly inhibited growth dynamics, leading to delayed and reduced exponential growth of these pathogens. \u003cem\u003eS. mutans\u003c/em\u003e are the key pathogen contributing significantly to dental caries and plaque formation. With strong adherence, acid enduring (acidurity) and producing (acidogenicity) properties, \u003cem\u003eS. mutans\u003c/em\u003e create an environment that promotes enamel demineralization, bacterial colonization, biofilm maturation and persistence \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. DKG markedly inhibited \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation and suppressed its aciduric and acidogenic properties, demonstrating its effectiveness in disrupting \u003cem\u003eS. mutans\u003c/em\u003e metabolism. By reducing acid production and promoting oral pH balance, DKG plays a crucial role in lowering the risk of dental caries and contributing to overall oral health maintenance. \u003cem\u003eC. albicans\u003c/em\u003e, yeast that also plays a crucial role in oral plaque biofilms by interacting with oral bacteria, albeit enhancing antimicrobial resistance and other oral diseases \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The transition from yeast to hyphal morphology is important for \u003cem\u003eC. albicans\u003c/em\u003e to adhere to the host\u0026rsquo;s surface, enhances its ability to invade the host and impart pathogenicity \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. DKG suppressed the hyphal transition ability of \u003cem\u003eC. albicans\u003c/em\u003e and even reduced the biofilm formation significantly. The diminishing levels of ergosterol, an important cell membrane component for maintaining membrane integrity, was observed with DKG treatment. Ergosterol also act as a target for many antifungal drugs \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Disrupting its levels, DKG compromises the growth and virulence of \u003cem\u003eC. albicans\u003c/em\u003e, highlighting mechanistic aspects of DKG offering a promising approach to reducing pathogenicity of the yeast and thereby improving oral health.\u003c/p\u003e \u003cp\u003eSeveral published reports have previously demonstrated that essential oils employ multiple mechanisms to inhibit the bacterial growth \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Mechanistically, either by cell membrane disruption in the bacteria or interfering with the cell-to-cell communication (quorum sensing), essential oils could even suppress the extracellular polymeric substance (EPS) production, matrix crucial for biofilm formation by the bacteria \u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, majority of studies have primarily evaluated the antimicrobial activity of essential oils against planktonic bacterial cultures. Given that biofilms exhibit enhanced resistance due to their protective extracellular matrix, conventional antimicrobial agents often fail to penetrate and eradicate these multi-layers, tightly structured communities. Furthermore, most investigations have focused on single-species biofilms, despite the fact that oral infections commonly involve complex, inter-kingdom interactions. Addressing these gaps, the current study examines the antimicrobial efficacy of essential oils against \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e co-cultured biofilms grown on orthodontic brackets and rings, a clinically relevant surface. This approach provides deeper insights into biofilm susceptibility in realistic conditions, reinforcing the potential of essential oils for combating persistent oral biofilms.\u003c/p\u003e \u003cp\u003eOrthodontic treatment involves the placement of brackets, archwires and elastomeric ligatures on teeth are susceptible sites for plaque accumulation and biofilm formation, increasing the risk of caries and enamel demineralization. These orthodontic fixtures, being constantly exposed to oral fluids, provide a favorable niche and surface for microbial colonization, making the maintenance of oral hygiene complicated and challenging\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Preventive strategies comprising of stringent oral hygiene practices play an important role in mitigating biofilm formation risks and ensure optimal oral health. Oil pulling could be an effective practice for oral health maintenance. Dant Kanti Gandush, containing a combination of essential oils with coconut and sesame oil, offers potential antimicrobial benefits, inhibits the inter-kingdom biofilm formation and thereby supports overall oral hygiene. A 1.0% (v/v) concentration of the essential oil mix was tested on biofilms, which is half the concentration of essential oils present in the oil-pulling formula of DKG. Biofilm formation was analyzed using a scanning electron microscopy (SEM) technique. SEM revealed a dense mesh of hyphae and yeast cells of \u003cem\u003eC. albicans\u003c/em\u003e with coccus shaped \u003cem\u003eS. mutans\u003c/em\u003e building a complex architecture and extensive extracellular polymeric substance (EPS) matrix. At lower magnification, SEM imaging of the entire bracket and elastomeric ligatures revealed a dense, thick layer of dual-culture biofilm. In contrast, brackets and elastomeric ligatures exposed with DKG exhibited a notably reduced microbial adherence, accompanied by significant structural alterations, cell lysis and scattered cellular debris.\u003c/p\u003e \u003cp\u003eThe current study was limited to \u003cem\u003ein vitro\u003c/em\u003e antimicrobial assessment of DKG, however, does not highlight the complexities of \u003cem\u003ein vivo\u003c/em\u003e conditions. Host-pathogen interaction studies and preclinical efficacy determination are part of the future investigations to further substantiate the therapeutic relevance of the present findings. Collectively, the study highlighted the potential of DKG in mitigating microbial adherence and biofilm formation on orthodontic surfaces, promoting oral hygiene and overall health.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eDKG serves as a valuable natural intervention for maintaining oral hygiene and preventing biofilm-associated complications even in the presence of orthodontic fixtures, where microbial adhesion and plaque accumulation are heightened. DKG could inhibit multiple oral pathogens, disrupt dual-species biofilms, reduce their adherence on orthodontic fixtures, all highlight DKG as an effective adjunct to conventional oral care, particularly for individuals undergoing orthodontic treatment. Further \u003cem\u003ein vivo\u003c/em\u003e studies and randomized clinical trials are needed to validate its therapeutic potential and clinical efficacy in real-world oral healthcare applications.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eDKG: Dant-Kanti-Gandush, MIC: Minimum inhibitory concentration, SEM: Scanning electron microscopy, EPS: Extracellular polymeric substance; OD: Optical density, GC-MS/MS: Gas chromatography-mass spectrometry, UHPLC: Ultra-High-Performance Liquid Chromatography.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCLINICAL TRIAL NUMBER\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors express appreciation to Mr. Devendra Kumawat for his contributions in designing schematics. The authors acknowledge the invaluable chemistry support of Dr. Sudeep Verma and Dr. Priya Rani M. We thank Dr. Swati Haldar for her scientific guidance. The authors would like to extend sincere appreciation to Dr. Ramakrishna Gupta and Mr. Naresh Bhende for their assistance with SEM analysis. Additionally, the authors acknowledge Mr. Tarun Rajput and Mr. Gagan Kumar for their prompt administrative supports.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe test article, Dant-Kanti-Gandush was sourced from Divya Pharmacy, Haridwar, India. AB is an honorary trustee in Divya Yog Mandir Trust, which governs Divya Pharmacy, Haridwar. In addition, he holds an honorary managerial position in Patanjali Ayurved Ltd., Haridwar, India. Divya Pharmacy and Patanjali Ayurved Ltd commercially manufacture and sell several Ayurvedic products. Other than providing the test article, Divya Pharmacy was not involved in any part of this study. Other authors have no conflict of interest to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAB: Conceptualization, Supervision, Resources, Writing-review \u0026amp; editing; HJ: Methodology, Data curation, Formal analysis, Writing-original draft; NKN: Methodology, Investigation; YV: Methodology, Data curation, Formal analysis, Writing-original draft; MT: Methodology, Data curation, Formal analysis; MJ: \u0026nbsp;Methodology, Investigation; KS: Methodology, Investigation; PN: Supervision; Writing-review \u0026amp; editing; SL: Visualization, Methodology, Data curation, Formal analysis, Writing-review \u0026amp; editing; AV: Project administration, Conceptualization, Visualization, Supervision; Writing-review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis presented work has been conducted using internal research funds from a non-profit Patanjali Research Foundation Trust, Haridwar, India.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePeres, M. 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Rep.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 36377 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Oil pulling, Essential oil, Dant-Kanti-Gandush, orthodontic fixtures, S. mutans-C. albicans cross-kingdom biofilms, cariogenic, oral health","lastPublishedDoi":"10.21203/rs.3.rs-6928179/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6928179/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOrthodontic fixtures provide a conducive niche for microbial colonization and inter-kingdom biofilm formation, exacerbating oral hygiene challenges. Conventional mouthwashes, though effective, are associated with adverse effects and potential antimicrobial resistance. Oil pulling is an Indian traditional method of oral detoxification. This study evaluates a blend of six essential oils (referred to as DKG) from \u003cem\u003eSyzygium aromaticum, Mentha piperita, Eucalyptus globulus, Zanthoxylum armatum\u003c/em\u003e, and \u003cem\u003eOcimum sanctum\u003c/em\u003e, mixed with coconut and sesame carrier oils, as a potential oil-pulling formulation. Gas chromatography\u0026ndash;mass spectrometry confirms the phytochemical composition of DKG. Antimicrobial assays demonstrate MIC₅₀ values of DKG ranging from 0.10% (v/v) to 0.45% (v/v) against \u003cem\u003eStreptococcus pyogenes, Streptococcus mutans, Proteus mirabilis\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e, respectively. DKG exposure delays the exponential phase and perturbs the growth of these pathogens. The cariogenic traits of \u003cem\u003eS. mutans\u003c/em\u003e are impaired at \u0026ge;\u0026thinsp;1.0\u0026times; MIC₅₀ DKG, showing reduced biofilm formation, decreased acid production, and lower survival under acidic stress. DKG inhibits \u003cem\u003eC. albicans\u003c/em\u003e biofilms at \u0026ge;\u0026thinsp;1.0\u0026times; MIC₅₀, prevents yeast-to-hyphae transition, and disrupts cell wall integrity by reducing ergosterol. SEM analysis shows reduced microbial density, fragmented hyphae, and disrupted bacterial aggregation. These findings highlight plant-based DKG, an anticariogenic alternative for maintaining oral health in individuals with orthodontic fixtures.\u003c/p\u003e","manuscriptTitle":"Essential Oils Enriched Dant-Kanti-Gandush (Oil-pulling) Inhibits Inter-kingdom Biofilm Formation on Orthodontic Fixtures and Ameliorates Cariogenic Virulence Factors of Oral Pathogens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-04 04:28:15","doi":"10.21203/rs.3.rs-6928179/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-08T14:29:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T10:19:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T13:14:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266062908132455815424578557151135655648","date":"2025-09-16T15:22:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127111677265950234349279027313791231701","date":"2025-09-13T16:32:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279191723696226787476585958284178124607","date":"2025-09-11T13:38:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-11T11:15:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-28T14:42:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-23T05:27:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-26T11:56:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-26T11:52:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"822d30b0-9b21-4ab5-addd-9844b6c1091b","owner":[],"postedDate":"July 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:10:17+00:00","versionOfRecord":{"articleIdentity":"rs-6928179","link":"https://doi.org/10.1038/s41598-025-31656-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-10 15:57:59","publishedOnDateReadable":"December 10th, 2025"},"versionCreatedAt":"2025-07-04 04:28:15","video":"","vorDoi":"10.1038/s41598-025-31656-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-31656-0","workflowStages":[]},"version":"v1","identity":"rs-6928179","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6928179","identity":"rs-6928179","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}