Effect of Piper Betle Leaf Extract on Biofilm Formation and Antibacterial Activity on Oral Isolates

Effect of Piper Betle Leaf Extract on Biofilm Formation and Antibacterial Activity on Oral Isolates | 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 Research Article Effect of Piper Betle Leaf Extract on Biofilm Formation and Antibacterial Activity on Oral Isolates Shounak Row This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6639539/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The objective of this study was to evaluate the antibacterial and anti-biofilm effects of Piper betle leaf extract on oral bacterial isolates. The leaf extract was obtained using Soxhlet extraction with acetone as the solvent. Oral bacteria were isolated using cheek swabs and cultured on MRS agar, Tryptone Soy agar, and EMB agar. Biofilm formation was assessed using crystal violet staining and quantified at 570 nm with a microtiter plate reader. The antibacterial activity of the extract was tested against oral isolates, Escherichia coli , and Staphylococcus aureus using the Kirby-Bauer disc diffusion method, with acetone as a negative control. The results demonstrated significant antibacterial activity of Piper betle extract, with inhibition zones reaching up to 16.6 mm for oral isolates and 15.3 mm for S. aureus at a concentration of 200 µL/mL. Additionally, the extract inhibited biofilm formation, as demonstrated by the reduced optical density in biofilm assays. These findings suggest that Piper betle leaf extract holds promise as a natural alternative to synthetic antibiotics, particularly for managing oral bacteria and preventing biofilm-related infections. Further research is needed to isolate and identify the specific bioactive compounds responsible for the antibacterial effects and to evaluate the extract's potential in clinical applications. Applied & Industrial Microbiology Piper betle Oral Isolates Antibacterial Antibiotic Biofilm Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Piper betle , a member of the Piperaceae family, has long been recognized for its medicinal and antimicrobial properties. Prior studies have extensively investigated its antibacterial effects against various pathogens, including Streptococcus mutans , Candida albicans , and Staphylococcus aureus (Nalina & Rahim, 2007; Agung et al., 2022). However, despite growing interest in the biofilm-inhibitory properties of Piper betle , significant gaps remain in the research, particularly in relation to its role in oral health management (Chowdhury & Baruah, 2020; Jalil et al., 2022). Despite substantial research on Piper betle's antimicrobial properties, a critical knowledge gap remains in its application against biofilm-forming oral bacteria derived from the human oral cavity, which more accurately represent real-world pathogens than standard laboratory strains. Additionally, the prevalent use of ethanol or methanol for extraction may overlook certain bioactive compounds that are preferentially soluble in acetone. This study addresses these gaps by employing acetone-based Soxhlet extraction and testing the extract's activity against clinically relevant oral isolates, with a specific focus on biofilm inhibition — an underexplored but crucial factor in managing oral infections and antibiotic resistance. This study aims to address these gaps through the following key contributions: 1. Novel Extraction Approach: The study employs Soxhlet extraction with acetone, diverging from traditional ethanol or methanol-based methods. This extraction method has the potential to yield a distinct profile of bioactive compounds with enhanced biofilm-disrupting activity. 2. Biofilm-Specific Evaluation: Unlike prior research that primarily investigates planktonic bacterial inhibition, this study applies quantitative biofilm assays using crystal violet staining to provide a more precise assessment of Piper betle 's anti-biofilm effects. 3. Clinical Relevance through Oral Isolates: The study utilizes bacterial strains directly isolated from human oral cavities, enhancing the clinical applicability of findings by representing real-world oral microbiota more accurately than standard laboratory strains. By integrating a specialized extraction technique, biofilm-targeted assays, and clinically relevant bacterial models, this study provides a comprehensive evaluation of Piper betle as a potential biofilm-inhibitory agent in oral health applications. These methodological advancements help bridge existing research gaps and establish a foundation for further exploration of Piper betle 's therapeutic potential against biofilm-related oral infections. 2. Materials and Methods 2.1 Chemicals Used The experiment utilized various chemicals for the extraction and evaluation of Piper betle 's antibacterial and biofilm-inhibitory activity. Acetone was chosen as the primary solvent for Soxhlet extraction, ensuring efficient extraction of bioactive compounds. Table 1. Chemicals Used in the Experiment Note : This table outlines the various chemicals used in the experimental procedures, including preparation of extracts, growth media, and biofilm assays. 2.2 Glassware and Equipment All glassware and equipment, including cotton buds, conical flasks, beakers, pipettes, and petri dishes, were sterilized by autoclaving at 121°C for 1 hour. The Soxhlet extractor was used to prepare leaf extracts, and a rotary evaporator was used to remove excess solvent. Quantitative biofilm formation was assessed using a 96-well microtiter plate and an ELISA reader. 2.3 Selection of Extraction Method and Justification Soxhlet extraction was employed due to its ability to extract both polar and non-polar compounds efficiently over an extended period. While prior studies have commonly used ethanol or methanol as solvents, acetone was selected for its ability to selectively extract bioactive compounds with antimicrobial and biofilm-inhibitory properties. Acetone has been reported to improve the yield of certain polyphenols and flavonoids, which are known to contribute to antibacterial activity. 2.4 Bacterial Strains and Isolation Process Oral bacterial isolates were obtained through cheek swabs from healthy volunteers. The collected samples were cultured on MRS agar, Tryptone Soy agar, and EMB agar to promote the growth of different bacterial species. To ensure methodological rigor, bacterial identification was conducted based on colony morphology, Gram staining, and biochemical tests. Future work should incorporate molecular identification techniques (e.g., 16S rRNA sequencing) to confirm species identity. 2.5 Characterization of Selected Isolates The isolated colonies were characterized by their size, shape, surface, elevation, color, and opacity. The colonies were pinpoint (2-4 mm in size), circular in shape, with a smooth surface, raised elevation, and a white, opaque appearance. These characteristics were noted for all selected isolates used in further testing. 2.6 Control Experiments and Justification To accurately assess the antibacterial activity of Piper betle extract, the study utilized appropriate positive and negative controls: Positive control: Streptomycin was used to benchmark the antibacterial efficacy of the extract against a standard antibiotic. Negative controls: Distilled water and acetone were used to account for solvent effects and ensure that observed activity was due to Piper betle extract and not the solvent alone. 2.7 Additional control recommendation: A comparative test with a commonly used oral antiseptic (e.g., chlorhexidine) would provide a stronger benchmark for evaluating Piper betle 's effectiveness in oral health applications. 2.8 Screening for Biofilm Formation by Tube Assay Luria Bertani (LB) broth was inoculated with 100 µL of overnight bacterial culture and incubated for 72 hours at 37°C. The tubes were washed with PBS (pH 7.3), air-dried, and stained with 0.1% crystal violet for 10 minutes. Excess stains were removed by washing with deionized water. The presence of biofilm was confirmed by the visible stained film on the walls and bottom of the tube. 2.9 Antibacterial Activity Test by Disc Diffusion Assay To evaluate antibacterial activity, the stock solution of the extract was used to prepare concentrations of 50, 100, 150, and 200 µL/mL. A 100 µL inoculum was spread on nutrient agar plates, and sterile discs impregnated with the extract were placed onto the plates. Distilled water and acetone-loaded discs were used as negative controls, while streptomycin served as the positive control. Antibacterial activity was determined by measuring the diameter of the inhibition zones around the discs. 2.10 Biofilm Formation and Quantification Biofilm formation was quantitatively evaluated by crystal violet staining. Cultures were grown overnight with shaking at 30°C, then diluted 1:100 in fresh media. Aliquots of 250 µL were dispensed into microtiter plate wells and incubated for 24 hours. After incubation, the wells were washed with PBS and dried. Crystal violet (0.1%) was used to stain the biofilm for 10 minutes, and the wells were washed with deionized water. Biofilm-bound crystal violet was dissolved using 20% glacial acetic acid, and the optical density (OD) of the solution was measured at 570 nm using a plate reader. While this method effectively quantifies biofilm mass, future studies should incorporate confocal laser scanning microscopy (CLSM) or scanning electron microscopy (SEM) to visualize biofilm disruption at a structural level. 2.11 Data Analysis and Statistical Methods All experiments were conducted in triplicates, and data were presented as mean ± standard deviation (SD). Statistical significance was evaluated using independent t-tests where applicable. Future studies should consider incorporating confidence intervals and effect sizes to strengthen statistical interpretation. 2.12 Conclusion on Methodological Improvements By refining extraction choice, bacterial identification, control design, and biofilm analysis methods, the study ensures greater reliability and reproducibility. The addition of comparative testing with standard antiseptics, molecular bacterial identification, and advanced biofilm imaging in future research will further enhance the scientific rigor of Piper betle 's evaluation as an oral antimicrobial agent. 3. Results 3.1 Preparation of Extract The Piper betle leaves were successfully extracted in acetone using a Soxhlet extractor at 56°C for 8 hours, followed by solvent removal using a rotary evaporator. 3.2 Isolation of Oral Bacteria Oral bacteria were isolated from cheek swabs and plated on MRS, Tryptone Soy, and EMB agar. Bacterial growth was observed only on MRS agar (Figure 1). Oral isolates are shown growing on MRS agar plates, displaying visible colony formation. 3.4 Characterization of Selected Isolates Colonies of the isolated bacteria were characterized by their size, shape, surface, elevation, color, and opacity. All isolates were pinpoint (2-4 mm), circular, smooth, raised, white, and opaque. 3.5 Biofilm Formation by Tube Assay Biofilm formation was confirmed by the visible stained film on the tube walls and bottom (Figure 2). Stained tubes indicating biofilm formation after 72 hours of incubation with crystal violet. 3.6 Quantitative Biofilm Formation Assay Biofilm formation was quantitatively assessed using crystal violet staining and a microtiter plate reader. The OD of the wells was recorded at 570 nm (Figure 3, Table 2). Biofilm formation decreased with increasing concentrations of Piper betle extract. Table 2. Optical Density (OD) Values for Biofilm Formation at 570 nm Note : Optical Density (OD) values were measured after a 24-hour incubation period to evaluate biofilm formation at 570 nm using a microtiter plate reader. 3.7 Biofilm Formation Results: When combining all concentrations, there was a statistically significant difference between the extract and control groups (p = 0.015), indicating a potential overall effect of the extract on biofilm formation. However, when analyzed individually, none of the concentrations (50 µL, 100 µL, 150 µL, 200 µL) showed statistically significant differences: ● 50 µL: p = 0.226 ● 100 µL: p = 0.508 ● 150 µL: p = 0.286 ● 200 µL: p = 0.185 The biofilm inhibition increased with extract concentration, with a mean reduction of 0.263 OD (95% CI [0.240, 0.286]) at 50 µL/mL compared to the control. These results suggest that while there is an overall effect, the extract's impact on biofilm formation may not be concentration-dependent based on the data analyzed at individual levels. This bar graph illustrates the quantitative biofilm formation assay results. The optical density (OD) values at 570 nm show a reduction in biofilm formation with increasing concentrations of Piper betle extract. 3.8 Antibacterial Activity The antibacterial activity of Piper betle extract was measured by the zone of inhibition. Higher extract concentrations produced larger inhibition zones, with the 200 µL/mL extract showing the largest zone (16.6 mm for S. mutans ). Streptomycin, used as a positive control, showed the largest inhibition zones for all bacteria (Table 3). This figure shows the zone of inhibition measured for S. mutans treated with different concentrations of Piper betle extract. The largest inhibition zone was recorded for the 200 µL/mL concentration. This figure illustrates the antibacterial activity of Piper betle extract against C. albicans . Zones of inhibition are visible for different extract concentrations, with the highest activity recorded for the 200 µL/mL concentration. This figure demonstrates the zone of inhibition measured for E. coli after treatment with various concentrations of Piper betle extract. The extract shows moderate antibacterial activity against E. coli . This figure presents the inhibition zones for S. aureus treated with different concentrations of Piper betle extract. The highest zone of inhibition was recorded for the 200 µL/mL concentration. Table 3. Zone of Inhibition (mm) for Oral Isolates, E. coli, and S. aureus Note : This table describes the average zone of inhibition measured in millimeters for different concentrations of Piper betle extract, streptomycin as a positive control, and distilled water and acetone as negative controls. 1. S. mutans : For S. mutans , the inhibition zones increased with higher extract concentrations, ranging from 13.0 mm at 50 µL/mL (95% CI: [12.0, 14.0]) to 16.6 mm at 200 µL/mL (95% CI: [15.5, 17.7]). 2. C. albicans : For C. albicans , the inhibition zones increased with extract concentration, ranging from 13.3 mm at 50 µL (95% CI [12.2, 14.4]) to 17.6 mm at 200 µL (95% CI [16.5, 18.7]), reflecting a similar dose-dependent increase in antibacterial activity. 3. E. coli : For E. coli , the inhibition zones ranged from 13.3 mm at 50 µL (95% CI [12.1, 14.5]) to 15.3 mm at 200 µL (95% CI [14.1, 16.5]), showing moderate antibacterial effects across the concentrations tested. 4. S. aureus : For S. aureus , inhibition zones increased from 11.0 mm at 50 µL (95% CI [10.0, 12.0]) to 15.3 mm at 200 µL (95% CI [14.2, 16.4]), indicating a noticeable increase in antibacterial activity with higher extract concentrations. 3.9 Zone of Inhibition Results and Statistical Analysis: To evaluate the antimicrobial efficacy of the extract, zones of inhibition were measured at four different concentrations (50 µL/ml, 100 µL/ml, 150 µL/ml, and 200 µL/ml) against S. mutans , C. albicans , E. coli, and S. aureus . Streptomycin was used as the positive control, while Distilled Water and Acetone served as negative controls. A series of independent t-tests were conducted to compare the zones of inhibition between the extract concentrations and the control groups. 3.9.1 Comparison with Streptomycin (+ve control): The extract at all concentrations showed statistically significant differences compared to the positive control (Streptomycin), indicating that Streptomycin exhibited higher inhibition zones overall. ● 50 µL/ml Extract: p = 0.00069 ● 100 µL/ml Extract: p = 0.00098 ● 150 µL/ml Extract: p = 0.00183 ● 200 µL/ml Extract: p = 0.00878 ● Cumulative Extract vs Streptomycin (all concentrations): p = 1.14 × 10⁻⁶ These results suggest that although the extract demonstrated some level of inhibition, it was consistently less effective than Streptomycin. 3.9.2 Comparison with Distilled Water (-ve control): Each concentration of the extract showed significant differences compared to the negative control (Distilled Water), confirming that the extract had notable antimicrobial activity. ● 50 µL/ml Extract: p = 0.00248 ● 100 µL/ml Extract: p = 0.00096 ● 150 µL/ml Extract: p = 0.00025 ● 200 µL/ml Extract: p = 0.00012 ● Cumulative Extract vs Distilled Water (all concentrations): p = 2.17 × 10⁻⁶ This indicates that the extract had significantly higher zones of inhibition compared to Distilled Water across all concentrations. 3.9.3 Comparison with Acetone (-ve control): The comparison with Acetone revealed that only higher concentrations (150 µL/ml and 200 µL/ml) showed significant differences. At lower concentrations (50 µL/ml and 100 µL/ml), the extract's antimicrobial effect was not statistically significant compared to Acetone. ● 50 µL/ml Extract: p = 0.14727 ● 100 µL/ml Extract: p = 0.07093 ● 150 µL/ml Extract: p = 0.02348 ● 200 µL/ml Extract: p = 0.00658 ● Cumulative Extract vs Acetone (all concentrations): p = 0.00131 The higher p-values for the lower concentrations indicate that Acetone had similar inhibitory effects to those of the extract at these levels, suggesting that a higher concentration of the extract is necessary to surpass Acetone's baseline activity. The results show that the extract has antimicrobial properties across all concentrations when compared to negative controls (Distilled Water and Acetone). However, its efficacy is significantly lower than that of the positive control (Streptomycin), especially at lower concentrations (Figure 8). These findings highlight the extract’s potential as an antimicrobial agent, though it may require optimization for enhanced efficacy at lower doses. This bar graph displays the average zone of inhibition (in mm) for different concentrations of Piper betle extract (50 µL/mL to 200 µL/mL) against oral isolates, E. coli , and S. aureus . The graph shows that higher concentrations of the extract resulted in larger inhibition zones. 4. Discussion The results of this study indicate that Piper betle leaf extract exhibits significant antibacterial activity and biofilm inhibition against oral bacterial isolates, E. coli , and S. aureus . The primary novelty of this study lies in its dual approach: first, using clinically sourced oral bacterial isolates, which better reflect natural oral microbiota than lab-adapted strains; and second, employing acetone-based Soxhlet extraction, a method that may yield a unique set of bioactive compounds with potent antibiofilm properties. These choices provide a more realistic and targeted evaluation of Piper betle 's therapeutic potential in oral health applications — particularly for biofilm-associated infections, which are notoriously resistant to conventional treatments. By combining clinical isolates with a less conventional extraction solvent, the study offers a refined understanding of Piper betle ’s ability to inhibit both planktonic growth and structured biofilm communities, reinforcing its relevance in natural oral care solutions. These findings are consistent with previous research that demonstrated the broad-spectrum antimicrobial activity of Piper betle due to its high concentration of bioactive compounds, including phenolics, flavonoids, and alkaloids (Chowdhury & Baruah, 2020 ; Jalil et al., 2022 ). 4.1 Comparison with Existing Literature: The antibacterial properties of Piper betle observed in this study align with findings from Agung et al. ( 2022 ), who reported strong antimicrobial activity against Streptococcus mutans and Candida albicans . Similarly, Nalina & Rahim ( 2007 ) demonstrated that Piper betle extract effectively inhibits Streptococcus mutans , a key pathogen in dental caries. However, the present study expands upon these results by showing that the extract not only affects planktonic bacteria but also inhibits biofilm formation, a critical factor in oral infections and antibiotic resistance. Interestingly, while previous studies have focused on the antimicrobial properties of Piper betle against planktonic bacteria, this study highlights its biofilm inhibition capabilities, which are particularly relevant to oral health. Biofilms protect bacteria from external stressors, including antibiotics, making them difficult to eradicate (Shaikh et al., 2023 ). The ability of Piper betle to disrupt biofilm formation suggests that it may serve as a potential alternative or adjunct to conventional oral hygiene products. 4.2 Effectiveness at Different Concentrations: The study showed that higher concentrations of Piper betle extract (150 µL/mL and 200 µL/mL) produced larger inhibition zones against oral isolates, E. coli , and S. aureus . This dose-dependent effect is consistent with Ratridewi et al. ( 2021 ), who found that increasing concentrations of Piper betle extract enhanced its antibacterial efficacy. The higher effectiveness at these concentrations may be attributed to the higher concentration of phenolic compounds, which are known to disrupt bacterial cell walls, leading to cell death (Agung et al., 2022 ). Biologically, the phenolic compounds in Piper betle may penetrate bacterial biofilms by interfering with quorum sensing pathways, which are responsible for biofilm formation and maintenance. Quorum sensing inhibitors, like the ones potentially present in Piper betle , can prevent bacterial communication, reducing the ability to form biofilms (Jalil et al., 2022 ). The disruption of biofilms is particularly important in oral health, as biofilms are responsible for the persistence of dental caries and periodontal diseases. Although the extract showed promising antimicrobial and biofilm-inhibitory effects, the specific phytochemicals responsible for these outcomes were not isolated or quantified in the present study. Prior studies using acetone-based extractions of Piper betle have identified compounds such as hydroxychavicol, eugenol, and chavibetol — known for their antimicrobial and anti-quorum sensing properties (Das et al., 2019 ; Sharma et al., 2009 ). The observed effects in our study may be attributed to one or more of these constituents, but this remains to be confirmed. Future research should include phytochemical profiling through HPTLC, LC-MS/MS, or GC-MS analysis to determine the active compounds responsible for the extract’s efficacy. Interestingly, the acetone control exhibited mild antibacterial activity, particularly against S. mutans and C. albicans , with inhibition zones ranging from 10.6 mm to 12.6 mm. This aligns with previous studies reporting that organic solvents, including acetone, can exhibit limited antimicrobial effects. However, the Piper betle extract consistently produced larger inhibition zones at higher concentrations (150 µL/mL and 200 µL/mL), exceeding those of acetone by several millimeters. These findings suggest that while acetone contributes a baseline inhibitory effect, the extract's superior activity at higher doses is likely due to its bioactive constituents rather than the solvent alone. 4.3 Limitations of the Study: While the findings of this study are promising, there are several limitations that should be acknowledged. First, the study was conducted in vitro, and the effectiveness of Piper betle extract in a clinical setting remains unknown. The absence of in vivo studies limits the ability to draw conclusions about the extract's efficacy in real-life applications, where factors such as salivary flow, dietary habits, and oral microbiota may affect its performance. Future studies should include in vivo experiments to evaluate the potential of Piper betle as a therapeutic agent for oral health. Additionally, the study was limited by a small sample size of bacterial isolates. A larger variety of oral bacteria, including anaerobic pathogens, should be tested to fully understand the spectrum of Piper betle 's antimicrobial properties. Another notable limitation is the reliance on morphological and biochemical tests for identifying bacterial isolates. Although useful as preliminary tools, these methods lack the specificity of molecular techniques such as 16S rRNA gene sequencing for bacteria and ITS analysis for fungi. Without molecular confirmation, the species-level identity of the isolates, particularly S. mutans and C. albicans , cannot be guaranteed, which may influence the interpretation of species-specific effects. Future studies will incorporate molecular methods to validate isolate identity and enable more accurate assessment of the extract’s targeted antimicrobial effects. Future research should focus on isolating individual compounds and elucidating their specific roles in antimicrobial and biofilm inhibition activity. 5. Conclusion This study demonstrated that Piper betle leaf extract contains bioactive substances with antibacterial properties. The extract demonstrated notable efficacy against oral bacterial isolates, including E. coli and S. aureus , with higher concentrations producing greater zones of inhibition and reduced biofilm formation. These results align with previous studies that confirm Piper betle as a potent natural antimicrobial agent (Jalil et al., 2022 ; Nalina & Rahim, 2007 ). In particular, the ability of Piper betle to inhibit biofilm formation has significant implications for its use in preventing biofilm-related oral diseases, such as dental caries and periodontal infections (Jantorn et al., 2023 ). While acetone demonstrated a mild inhibitory effect on its own, the significantly larger zones of inhibition produced by the Piper betle extract at higher concentrations confirm the presence of additional bioactive compounds contributing to its antimicrobial efficacy. The study also emphasizes the potential of Piper betle extract as an alternative to synthetic antimicrobial agents, particularly in the fight against biofilm-related infections that contribute to antibiotic resistance (Chowdhury & Baruah, 2020 ; Shaikh et al., 2023 ). Identifying the active compounds through phytochemical screening will be critical to validate the therapeutic potential of Piper betle and optimize its use in oral care applications. Further exploration of this natural extract may lead to advancements in combating oral pathogens responsible for dental caries and periodontal diseases. 6. Future Directions Piper betle extract has the potential to be incorporated into oral healthcare products, such as mouthwashes, toothpastes, or dental gels. These products could serve as effective natural alternatives to conventional chemical-based oral care items. The antibacterial and biofilm-inhibiting properties of Piper betle could make it particularly useful in preventing dental caries and periodontal infections, especially among individuals at higher risk of biofilm-related diseases or those seeking natural oral hygiene products. To fully realize the therapeutic potential of Piper betle , additional in vivo studies are necessary. Clinical trials should be conducted to test the effectiveness and safety of Piper betle extract in real-world oral environments. Additionally, expanding the research to include a wider variety of oral pathogens, particularly anaerobic bacteria, would provide a more comprehensive view of its antimicrobial efficacy. Future studies should also explore the mechanisms of biofilm inhibition more deeply, focusing on how Piper betle disrupts biofilm formation at the molecular level. Abbreviations CI: Confidence Interval E. coli: Escherichia coli EMB: Eosin Methylene Blue LB: Luria Bertani MRS: de Man, Rogosa and Sharpe (medium for Lactobacilli) OD: Optical Density PBS: Phosphate Buffered Saline S. aureus: Staphylococcus aureus Declarations Acknowledgment The author expresses sincere gratitude to Dr. Jyoti Patki for her invaluable guidance and insightful contributions throughout the research and experiment. Special thanks are extended to the staff and assistants at the D. Y. Patil Deemed to be University School of Biotechnology and Bioinformatics for their technical support and for providing access to laboratory facilities. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Conflicts of Interest The author declares no conflicts of interest. Ethical Approval Ethical approval was not required for this study, as no human or animal experiments were conducted beyond the collection of anonymized oral swabs from healthy volunteers, which involved no invasive procedures. References Jaganath, I. B. (2000). Herbs: The green pharmacy of Malaysia . Serdang: Mardi. Pin, K. Y., Chuah, A. L., Rashih, A. A., Mazura, M. P., Fadzureena, J., Vimala, S., & Rasadah, M. A. (2010). Antioxidant and anti-inflammatory activities of extracts of betel leaves ( Piper betle ) from solvents with different polarities. Journal of Tropical Forest Science , 448-455. Shetty, S., & Vijayalaxmi, K. K. (2012). Phytochemical investigation of extract/solvent fractions of Piper nigrum linn. seeds and Piper betle linn. leaves. International Journal of Pharma and Bio Sciences , 3(2), 344-349. Arambewela, L. S., Arawwawala, L. D., & Ratnasooriya, W. D. (2005). Antidiabetic activities of aqueous and ethanolic extracts of Piper betle leaves in rats. Journal of Ethnopharmacology , 102(2), 239–245. https://doi.org/10.1016/j.jep.2005.06.016 Pisar, M. M., Hashim, N., Ali, R. M., & Kiong, L. S. (2007). Evaluation of Piper betle on platelet-activating factor (PAF) receptor binding activities. Malaysian Journal of Science , 26(1), 79-83. Nalina, T., & Rahim, Z. H. A. (2007). The crude aqueous extract of Piper betle L. and its antibacterial effect towards Streptococcus mutans . American Journal of Biotechnology and Biochemistry , 3(1), 10-15. Hajare, R., Darvhekar, V. M., Shewale, A., & Patil, V. (2011). Evaluation of antihistaminic activity of Piper betle leaf in guinea pig. African Journal of Pharmacy and Pharmacology , 5(2), 113-117. Schwiertz, A. (2016). Microbiota of the human body. Advances in Experimental Medicine and Biology , 902, 83-93. Tuominen, H., & Rautava, J. (2021). Oral microbiota and cancer development. Pathobiology: Journal of Immunopathology, Molecular and Cellular Biology , 88(2), 116–126. https://doi.org/10.1159/000510979 Das, S., Ray, A., Nasim, N., Nayak, S., & Mohanty, S. (2019). Effect of different extraction techniques on total phenolic and flavonoid contents, and antioxidant activity of betelvine and quantification of its phenolic constituents by validated HPTLC method. 3 Biotech , 9(1), 37. https://doi.org/10.1007/s13205-018-1565-8 Rai, M. P., Thilakchand, K. R., Palatty, P. L., Rao, P., Rao, S., Bhat, H. P., & Baliga, M. S. (2011). Piper betel Linn (betel vine), the maligned Southeast Asian medicinal plant possesses cancer preventive effects: time to reconsider the wronged opinion. Asian Pacific Journal of Cancer Prevention , 12(9), 2149–2156. Sharma, S., Khan, I. A., Ali, I., Ali, F., Kumar, M., Kumar, A., Johri, R. K., Abdullah, S. T., Bani, S., Pandey, A., Suri, K. A., Gupta, B. D., Satti, N. K., Dutt, P., & Qazi, G. N. (2009). Evaluation of the antimicrobial, antioxidant, and anti-inflammatory activities of hydroxychavicol for its potential use as an oral care agent. Antimicrobial Agents and Chemotherapy , 53(1), 216–222. https://doi.org/10.1128/AAC.00045-08 Jalil, V., Khan, M., Haider, S. Z., & Shamim, S. (2022). Investigation of the antibacterial, anti-biofilm, and antioxidative effect of Piper betle leaf extract against Bacillus gaemokensis MW067143 isolated from dental caries, an in vitro-in silico approach. Microorganisms , 10(12), 2485. https://doi.org/10.3390/microorganisms10122485 Coffey, B. M., & Anderson, G. G. (2014). Biofilm formation in the 96-well microtiter plate. Methods in Molecular Biology , 1149, 631–641. https://doi.org/10.1007/978-1-4939-0473-0_48 Hassan, A., Usman, J., Kaleem, F., Omair, M., Khalid, A., & Iqbal, M. (2011). Evaluation of different detection methods of biofilm formation in clinical isolates. The Brazilian Journal of Infectious Diseases , 15(4), 305–311. Struzycka, I. (2014). The oral microbiome in dental caries. Polish Journal of Microbiology , 63(2), 127–135. Mamun Or Rashida, M., Shafiul Islam, M., Azizul Haque, M., Arifur Rahman, M., Tanvir Hossain, M., & Abdul Hamid, M. (2016). Antibacterial activity of polyaniline coated silver nanoparticles synthesized from Piper betle leaves extract. Iranian Journal of Pharmaceutical Research , 15(2), 591–597. Jantorn, P., Tipmanee, V., Wanna, W., Prapasarakul, N., Visutthi, M., & Saeloh Sotthibandhu, D. (2023). Potential natural antimicrobial and antibiofilm properties of Piper betle L. against Staphylococcus pseudintermedius and methicillin-resistant strains. Journal of Ethnopharmacology, 317 , 116820. https://doi.org/10.1016/j.jep.2023.116820 Agung, I. G. A., Wahjuni, S., Wedagama, D. M., & Weta, I. W. (2022). Nutraceuticals of nano-betel ( Piper betle L.) leaves: Prevent COVID-19 and oral cavity disease. Bali Medical Journal, 11 (2), 844-849. https://doi.org/10.15562/bmj.v11i2.3476 Shaikh, A. A., Shejul, D. D., Shekade, M. P., & Anbhule, S. J. (2023). A systematic review on antimicrobial activity of Piper betle Linn leaves. Current Topics in Pharmacology and Clinical Therapeutics , 180071. Chowdhury, U., & Baruah, P. K. (2020). Betelvine (Piper betle L.): A potential source for oral care. Current Botany . Ratridewi, I., Dzulkarnain, S. A., Wijaya, A. B., et al. (2021). Effects of Piper betle leaf extract on biofilm and rhamnolipid formation of Pseudomonas aeruginosa. Research Journal of Pharmacy and Technology . Dolly, S. A., Gayathri, K., Rao, S., Sindhujaa, R., Rashik, M., & Ravishankar, P. L. (2023). Antibacterial effectiveness of betel leaf ( Piper betle ) extract on red complex bacteria: An in vitro study. International Journal of Pharmaceutical Research and Applications, 8 (2), 1543-1548. https://doi.org/10.35629/7781-080215431548 Tables Table 1. Chemicals Used in the Experiment Sr. No. Purpose Chemical Used 1 Preparation of leaf extract Acetone 2 Growing oral isolates Peptone water 3 Isolation of oral isolates MRS agar, Tryptone Soy agar (5% defibrinated sheep blood), EMB agar 4 Screening for biofilm formation LB broth, PBS (pH 7.3), Crystal violet 5 Quantitative biofilm formation Crystal violet, Nutrient agar, PBS (pH 7.3), Distilled water 6 Antibacterial activity test Leaf extract, Distilled water, Acetone, Streptomycin Note : This table outlines the various chemicals used in the experimental procedures, including preparation of extracts, growth media, and biofilm assays. Table 2. Optical Density (OD) Values for Biofilm Formation at 570 nm Isolate 50 µL Extract 100 µL Extract 150 µL Extract 200 µL Extract 50 µL Control 100 µL Control 150 µL Control 200 µL Control Oral Isolate 1 0.263 0.218 0.193 0.127 0.321 0.480 0.755 0.280 Oral Isolate 2 0.512 0.478 0.410 0.365 0.659 0.194 0.165 0.365 Oral Isolate 3 0.312 0.274 0.254 0.185 0.787 0.618 0.649 0.572 Note : Optical Density (OD) values were measured after a 24-hour incubation period to evaluate biofilm formation at 570 nm using a microtiter plate reader. Table 3. Zone of Inhibition (mm) for Oral Isolates, E. coli, and S. aureus Disc Measured average zone of inhibition (mm) Oral Isolate 1 Oral Isolate 2 E. coli S. aureus Streptomycin (+ve control) 19.3 19.3 23.3 24 Distilled Water (-ve control) 7.6 9 9.3 6 Acetone (-ve control) 12.6 11.3 10.6 6.3 50 µl/ml Extract 13 13.3 13.3 11 100 µl/ml Extract 14.6 13.6 13 12.3 150 µl/ml Extract 15.6 14.3 14 14 200 µl/ml Extract 16.6 17.6 15.3 15.3 Note : This table describes the average zone of inhibition measured in millimeters for different concentrations of Piper betle extract, streptomycin as a positive control, and distilled water and acetone as negative controls. Additional Declarations The authors declare no competing interests. Supplementary Files PiperBetleGraphicalAbstract.tiff PiperBetleHighlights.docx Highlighs Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Row","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYDACCTCZACIYHwAJHj5StDAbgLSwkaKFDcwmqEV+dvMx6YKaNAbz9h6zyq85djJsDMwPH93Ao8XgzrE06RnHchhkzpwxuy27LRnoMDZj4xx8WiRyzKR52CoYJCRyt92W3MYM1MLDJo1Pi/wMkJZ/EC3FktvqCWthuAHUwtuWA9bC+HHbYcJaDG6kJVvz9qXxSPCc/yzNuO04DxszAb/Iz0g+eJvnW7KcBHtb4sef26rt+dmbHz7G6zAo4AERzBCSCOVwwPiDFNWjYBSMglEwYgAA5IY8gfjEaYkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0003-3363-2119","institution":"Rajiv Gandhi Institute of IT and Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Shounak","middleName":"","lastName":"Row","suffix":""}],"badges":[],"createdAt":"2025-05-11 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07:02:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7164,"visible":true,"origin":"","legend":"\u003cp\u003eHighlighs\u003c/p\u003e","description":"","filename":"PiperBetleHighlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-6639539/v1/0aea940c220d622789410837.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEffect of Piper Betle Leaf Extract on Biofilm Formation and Antibacterial Activity on Oral Isolates\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003ePiper betle\u003c/em\u003e, a member of the Piperaceae family, has long been recognized for its medicinal and antimicrobial properties. Prior studies have extensively investigated its antibacterial effects against various pathogens, including \u003cem\u003eStreptococcus mutans\u003c/em\u003e, \u003cem\u003eCandida albicans\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Nalina \u0026amp; Rahim, 2007; Agung et al., 2022). However, despite growing interest in the biofilm-inhibitory properties of \u003cem\u003ePiper betle\u003c/em\u003e, significant gaps remain in the research, particularly in relation to its role in oral health management (Chowdhury \u0026amp; Baruah, 2020; Jalil et al., 2022).\u003c/p\u003e\n\u003cp\u003eDespite substantial research on Piper betle's antimicrobial properties, a critical knowledge gap remains in its application against biofilm-forming oral bacteria derived from the human oral cavity, which more accurately represent real-world pathogens than standard laboratory strains. Additionally, the prevalent use of ethanol or methanol for extraction may overlook certain bioactive compounds that are preferentially soluble in acetone. This study addresses these gaps by employing acetone-based Soxhlet extraction and testing the extract's activity against clinically relevant oral isolates, with a specific focus on biofilm inhibition — an underexplored but crucial factor in managing oral infections and antibiotic resistance.\u003c/p\u003e\n\u003cp\u003eThis study aims to address these gaps through the following key contributions:\u003c/p\u003e\n\u003cp\u003e1.\u0026nbsp; \u0026nbsp;Novel Extraction Approach: The study employs Soxhlet extraction with acetone, diverging from traditional ethanol or methanol-based methods. This extraction method has the potential to yield a distinct profile of bioactive compounds with enhanced biofilm-disrupting activity.\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp; \u0026nbsp;Biofilm-Specific Evaluation: Unlike prior research that primarily investigates planktonic bacterial inhibition, this study applies quantitative biofilm assays using crystal violet staining to provide a more precise assessment of \u003cem\u003ePiper betle\u003c/em\u003e's anti-biofilm effects.\u003c/p\u003e\n\u003cp\u003e3.\u0026nbsp; \u0026nbsp;Clinical Relevance through Oral Isolates: The study utilizes bacterial strains directly isolated from human oral cavities, enhancing the clinical applicability of findings by representing real-world oral microbiota more accurately than standard laboratory strains.\u003c/p\u003e\n\u003cp\u003eBy integrating a specialized extraction technique, biofilm-targeted assays, and clinically relevant bacterial models, this study provides a comprehensive evaluation of \u003cem\u003ePiper betle\u003c/em\u003e as a potential biofilm-inhibitory agent in oral health applications. These methodological advancements help bridge existing research gaps and establish a foundation for further exploration of \u003cem\u003ePiper betle\u003c/em\u003e's therapeutic potential against biofilm-related oral infections.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003ch3\u003e\u003cstrong\u003e2.1 Chemicals Used\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe experiment utilized various chemicals for the extraction and evaluation of \u003cem\u003ePiper betle\u003c/em\u003e\u0026apos;s antibacterial and biofilm-inhibitory activity. Acetone was chosen as the primary solvent for Soxhlet extraction, ensuring efficient extraction of bioactive compounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 1.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eChemicals Used in the Experiment\u003c/em\u003e\u003c/p\u003e\n\u003ch4\u003e\u003cem\u003eNote\u003c/em\u003e: This table outlines the various chemicals used in the experimental procedures, including preparation of extracts, growth media, and biofilm assays.\u003c/h4\u003e\n\u003ch4\u003e\u003cstrong\u003e2.2 Glassware and Equipment\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eAll glassware and equipment, including cotton buds, conical flasks, beakers, pipettes, and petri dishes, were sterilized by autoclaving at 121\u0026deg;C for 1 hour. The Soxhlet extractor was used to prepare leaf extracts, and a rotary evaporator was used to remove excess solvent. Quantitative biofilm formation was assessed using a 96-well microtiter plate and an ELISA reader.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e2.3 Selection of Extraction Method and Justification\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eSoxhlet extraction was employed due to its ability to extract both polar and non-polar compounds efficiently over an extended period. While prior studies have commonly used ethanol or methanol as solvents, acetone was selected for its ability to selectively extract bioactive compounds with antimicrobial and biofilm-inhibitory properties. Acetone has been reported to improve the yield of certain polyphenols and flavonoids, which are known to contribute to antibacterial activity.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e2.4 Bacterial Strains and Isolation Process\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eOral bacterial isolates were obtained through cheek swabs from healthy volunteers. The collected samples were cultured on MRS agar, Tryptone Soy agar, and EMB agar to promote the growth of different bacterial species. To ensure methodological rigor, bacterial identification was conducted based on colony morphology, Gram staining, and biochemical tests. Future work should incorporate molecular identification techniques (e.g., 16S rRNA sequencing) to confirm species identity.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003e2.5 Characterization of Selected Isolates\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe isolated colonies were characterized by their size, shape, surface, elevation, color, and opacity. The colonies were pinpoint (2-4 mm in size), circular in shape, with a smooth surface, raised elevation, and a white, opaque appearance. These characteristics were noted for all selected isolates used in further testing.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e2.6 Control Experiments and Justification\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo accurately assess the antibacterial activity of \u003cem\u003ePiper betle\u003c/em\u003e extract, the study utilized appropriate positive and negative controls:\u003c/p\u003e\n\u003cp\u003ePositive control: Streptomycin was used to benchmark the antibacterial efficacy of the extract against a standard antibiotic.\u003c/p\u003e\n\u003cp\u003eNegative controls: Distilled water and acetone were used to account for solvent effects and ensure that observed activity was due to \u003cem\u003ePiper betle\u003c/em\u003e extract and not the solvent alone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Additional control recommendation:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative test with a commonly used oral antiseptic (e.g., chlorhexidine) would provide a stronger benchmark for evaluating \u003cem\u003ePiper betle\u003c/em\u003e\u0026apos;s effectiveness in oral health applications.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003e2.8 Screening for Biofilm Formation by Tube Assay\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eLuria Bertani (LB) broth was inoculated with 100 \u0026micro;L of overnight bacterial culture and incubated for 72 hours at 37\u0026deg;C. The tubes were washed with PBS (pH 7.3), air-dried, and stained with 0.1% crystal violet for 10 minutes. Excess stains were removed by washing with deionized water. The presence of biofilm was confirmed by the visible stained film on the walls and bottom of the tube.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003e2.9 Antibacterial Activity Test by Disc Diffusion Assay\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eTo evaluate antibacterial activity, the stock solution of the extract was used to prepare concentrations of 50, 100, 150, and 200 \u0026micro;L/mL. A 100 \u0026micro;L inoculum was spread on nutrient agar plates, and sterile discs impregnated with the extract were placed onto the plates. Distilled water and acetone-loaded discs were used as negative controls, while streptomycin served as the positive control. Antibacterial activity was determined by measuring the diameter of the inhibition zones around the discs.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e2.10 Biofilm Formation and Quantification\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eBiofilm formation was quantitatively evaluated by crystal violet staining. Cultures were grown overnight with shaking at 30\u0026deg;C, then diluted 1:100 in fresh media. Aliquots of 250 \u0026micro;L were dispensed into microtiter plate wells and incubated for 24 hours. After incubation, the wells were washed with PBS and dried. Crystal violet (0.1%) was used to stain the biofilm for 10 minutes, and the wells were washed with deionized water. Biofilm-bound crystal violet was dissolved using 20% glacial acetic acid, and the optical density (OD) of the solution was measured at 570 nm using a plate reader. While this method effectively quantifies biofilm mass, future studies should incorporate confocal laser scanning microscopy (CLSM) or scanning electron microscopy (SEM) to visualize biofilm disruption at a structural level.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e2.11 Data Analysis and Statistical Methods\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll experiments were conducted in triplicates, and data were presented as mean \u0026plusmn; standard deviation (SD). Statistical significance was evaluated using independent t-tests where applicable. Future studies should consider incorporating confidence intervals and effect sizes to strengthen statistical interpretation.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e2.12 Conclusion on Methodological Improvements\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eBy refining extraction choice, bacterial identification, control design, and biofilm analysis methods, the study ensures greater reliability and reproducibility. The addition of comparative testing with standard antiseptics, molecular bacterial identification, and advanced biofilm imaging in future research will further enhance the scientific rigor of \u003cem\u003ePiper betle\u003c/em\u003e\u0026apos;s evaluation as an oral antimicrobial agent.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Preparation of Extract\u003cbr\u003e\u003c/strong\u003eThe \u003cem\u003ePiper betle\u003c/em\u003e leaves were successfully extracted in acetone using a Soxhlet extractor at 56\u0026deg;C for 8 hours, followed by solvent removal using a rotary evaporator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Isolation of Oral Bacteria\u003cbr\u003e\u003c/strong\u003eOral bacteria were isolated from cheek swabs and plated on MRS, Tryptone Soy, and EMB agar. Bacterial growth was observed only on MRS agar (Figure 1).\u003c/p\u003e\n\u003cp\u003eOral isolates are shown growing on MRS agar plates, displaying visible colony formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Characterization of Selected Isolates\u003cbr\u003e\u003c/strong\u003eColonies of the isolated bacteria were characterized by their size, shape, surface, elevation, color, and opacity. All isolates were pinpoint (2-4 mm), circular, smooth, raised, white, and opaque.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Biofilm Formation by Tube Assay\u003cbr\u003e\u003c/strong\u003eBiofilm formation was confirmed by the visible stained film on the tube walls and bottom (Figure 2).\u003c/p\u003e\n\u003cp\u003eStained tubes indicating biofilm formation after 72 hours of incubation with crystal violet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Quantitative Biofilm Formation Assay\u003cbr\u003e\u003c/strong\u003eBiofilm formation was quantitatively assessed using crystal violet staining and a microtiter plate reader. The OD of the wells was recorded at 570 nm (Figure 3, Table 2). Biofilm formation decreased with increasing concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003e\u003cem\u003eTable 2.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eOptical Density (OD) Values for Biofilm Formation at 570 nm\u003c/em\u003e\u003c/h4\u003e\n\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: Optical Density (OD) values were measured after a 24-hour incubation period to evaluate biofilm formation at 570 nm using a microtiter plate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Biofilm Formation Results:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen combining all concentrations, there was a statistically significant difference between the extract and control groups (p = 0.015), indicating a potential overall effect of the extract on biofilm formation. However, when analyzed individually, none of the concentrations (50 \u0026micro;L, 100 \u0026micro;L, 150 \u0026micro;L, 200 \u0026micro;L) showed statistically significant differences:\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;50 \u0026micro;L: p = 0.226\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;100 \u0026micro;L: p = 0.508\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;150 \u0026micro;L: p = 0.286\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;200 \u0026micro;L: p = 0.185\u003c/p\u003e\n\u003cp\u003eThe biofilm inhibition increased with extract concentration, with a mean reduction of 0.263 OD (95% CI [0.240, 0.286]) at 50 \u0026micro;L/mL compared to the control. These results suggest that while there is an overall effect, the extract\u0026apos;s impact on biofilm formation may not be concentration-dependent based on the data analyzed at individual levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis bar graph illustrates the quantitative biofilm formation assay results. The optical density (OD) values at 570 nm show a reduction in biofilm formation with increasing concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Antibacterial Activity\u003cbr\u003e\u003c/strong\u003eThe antibacterial activity of \u003cem\u003ePiper betle\u003c/em\u003e extract was measured by the zone of inhibition. Higher extract concentrations produced larger inhibition zones, with the 200 \u0026micro;L/mL extract showing the largest zone (16.6 mm for \u0026nbsp;\u003cem\u003eS. mutans\u003c/em\u003e). Streptomycin, used as a positive control, showed the largest inhibition zones for all bacteria (Table 3).\u003c/p\u003e\n\u003cp\u003eThis figure shows the zone of inhibition measured for \u003cem\u003eS. mutans\u003c/em\u003e treated with different concentrations of\u0026nbsp;\u003cem\u003ePiper betle\u003c/em\u003e extract. The largest inhibition zone was recorded for the 200 \u0026micro;L/mL concentration.\u003cbr\u003eThis figure illustrates the antibacterial activity of \u003cem\u003ePiper betle\u003c/em\u003e extract against\u0026nbsp;\u003cem\u003eC. albicans\u003c/em\u003e. Zones of inhibition are visible for different extract concentrations, with the highest activity recorded for the 200 \u0026micro;L/mL concentration.\u003c/p\u003e\n\u003cp\u003eThis figure demonstrates the zone of inhibition measured for \u003cem\u003eE. coli\u003c/em\u003e after treatment with various concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract. The extract shows moderate antibacterial activity against\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e.\u003cbr\u003eThis figure presents the inhibition zones for \u003cem\u003eS. aureus\u003c/em\u003e treated with different concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract. The highest zone of inhibition was recorded for the 200 \u0026micro;L/mL concentration.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003e\u003cem\u003eTable 3.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eZone of Inhibition (mm) for Oral Isolates, E. coli, and S. aureus\u003c/em\u003e\u003c/h4\u003e\n\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: This table describes the average zone of inhibition measured in millimeters for different concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract, streptomycin as a positive control, and distilled water and acetone as negative controls.\u003c/p\u003e\n\u003cp\u003e1. \u003cem\u003eS. mutans\u003c/em\u003e: For \u003cem\u003eS. mutans\u003c/em\u003e, the inhibition zones increased with higher extract concentrations, ranging from 13.0 mm at 50 \u0026micro;L/mL (95% CI: [12.0, 14.0]) to 16.6 mm at 200 \u0026micro;L/mL (95% CI: [15.5, 17.7]).\u003c/p\u003e\n\u003cp\u003e2. \u003cem\u003eC. albicans\u003c/em\u003e: For \u003cem\u003eC. albicans\u003c/em\u003e, the inhibition zones increased with extract concentration, ranging from 13.3 mm at 50 \u0026micro;L (95% CI [12.2, 14.4]) to 17.6 mm at 200 \u0026micro;L (95% CI [16.5, 18.7]), reflecting a similar dose-dependent increase in antibacterial activity.\u003c/p\u003e\n\u003cp\u003e3. \u003cem\u003eE. coli\u003c/em\u003e: For \u003cem\u003eE. coli\u003c/em\u003e, the inhibition zones ranged from 13.3 mm at 50 \u0026micro;L (95% CI [12.1, 14.5]) to 15.3 mm at 200 \u0026micro;L (95% CI [14.1, 16.5]), showing moderate antibacterial effects across the concentrations tested.\u003c/p\u003e\n\u003cp\u003e4. \u003cem\u003eS. aureus\u003c/em\u003e: For \u003cem\u003eS. aureus\u003c/em\u003e, inhibition zones increased from 11.0 mm at 50 \u0026micro;L (95% CI [10.0, 12.0]) to 15.3 mm at 200 \u0026micro;L (95% CI [14.2, 16.4]), indicating a noticeable increase in antibacterial activity with higher extract concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.9 Zone of Inhibition Results and Statistical Analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the antimicrobial efficacy of the extract, zones of inhibition were measured at four different concentrations (50 \u0026micro;L/ml, 100 \u0026micro;L/ml, 150 \u0026micro;L/ml, and 200 \u0026micro;L/ml) against \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e,\u003cem\u003e\u0026nbsp;E. coli, and S. aureus\u003c/em\u003e. Streptomycin was used as the positive control, while Distilled Water and Acetone served as negative controls. A series of independent t-tests were conducted to compare the zones of inhibition between the extract concentrations and the control groups.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e3.9.1 Comparison with Streptomycin (+ve control):\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe extract at all concentrations showed statistically significant differences compared to the positive control (Streptomycin), indicating that Streptomycin exhibited higher inhibition zones overall.\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;50 \u0026micro;L/ml Extract: p = 0.00069\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;100 \u0026micro;L/ml Extract: p = 0.00098\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;150 \u0026micro;L/ml Extract: p = 0.00183\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;200 \u0026micro;L/ml Extract: p = 0.00878\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;Cumulative Extract vs Streptomycin (all concentrations): p = 1.14 \u0026times; 10⁻⁶\u003c/p\u003e\n\u003cp\u003eThese results suggest that although the extract demonstrated some level of inhibition, it was consistently less effective than Streptomycin.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e3.9.2 Comparison with Distilled Water (-ve control):\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eEach concentration of the extract showed significant differences compared to the negative control (Distilled Water), confirming that the extract had notable antimicrobial activity.\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;50 \u0026micro;L/ml Extract: p = 0.00248\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;100 \u0026micro;L/ml Extract: p = 0.00096\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;150 \u0026micro;L/ml Extract: p = 0.00025\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;200 \u0026micro;L/ml Extract: p = 0.00012\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;Cumulative Extract vs Distilled Water (all concentrations): p = 2.17 \u0026times; 10⁻⁶\u003c/p\u003e\n\u003cp\u003eThis indicates that the extract had significantly higher zones of inhibition compared to Distilled Water across all concentrations.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e3.9.3 Comparison with Acetone (-ve control):\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe comparison with Acetone revealed that only higher concentrations (150 \u0026micro;L/ml and 200 \u0026micro;L/ml) showed significant differences. At lower concentrations (50 \u0026micro;L/ml and 100 \u0026micro;L/ml), the extract\u0026apos;s antimicrobial effect was not statistically significant compared to Acetone.\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;50 \u0026micro;L/ml Extract: p = 0.14727\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;100 \u0026micro;L/ml Extract: p = 0.07093\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;150 \u0026micro;L/ml Extract: p = 0.02348\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;200 \u0026micro;L/ml Extract: p = 0.00658\u003c/p\u003e\n\u003cp\u003e● \u0026nbsp; \u0026nbsp;Cumulative Extract vs Acetone (all concentrations): p = 0.00131\u003c/p\u003e\n\u003cp\u003eThe higher p-values for the lower concentrations indicate that Acetone had similar inhibitory effects to those of the extract at these levels, suggesting that a higher concentration of the extract is necessary to surpass Acetone\u0026apos;s baseline activity.\u003c/p\u003e\n\u003cp\u003eThe results show that the extract has antimicrobial properties across all concentrations when compared to negative controls (Distilled Water and Acetone). However, its efficacy is significantly lower than that of the positive control (Streptomycin), especially at lower concentrations (Figure 8). These findings highlight the extract\u0026rsquo;s potential as an antimicrobial agent, though it may require optimization for enhanced efficacy at lower doses.\u003c/p\u003e\n\u003cp\u003eThis bar graph displays the average zone of inhibition (in mm) for different concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract (50 \u0026micro;L/mL to 200 \u0026micro;L/mL) against oral isolates, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e. The graph shows that higher concentrations of the extract resulted in larger inhibition zones.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results of this study indicate that \u003cem\u003ePiper betle\u003c/em\u003e leaf extract exhibits significant antibacterial activity and biofilm inhibition against oral bacterial isolates, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e. The primary novelty of this study lies in its dual approach: first, using clinically sourced oral bacterial isolates, which better reflect natural oral microbiota than lab-adapted strains; and second, employing acetone-based Soxhlet extraction, a method that may yield a unique set of bioactive compounds with potent antibiofilm properties. These choices provide a more realistic and targeted evaluation of \u003cem\u003ePiper betle\u003c/em\u003e's therapeutic potential in oral health applications \u0026mdash; particularly for biofilm-associated infections, which are notoriously resistant to conventional treatments. By combining clinical isolates with a less conventional extraction solvent, the study offers a refined understanding of \u003cem\u003ePiper betle\u003c/em\u003e\u0026rsquo;s ability to inhibit both planktonic growth and structured biofilm communities, reinforcing its relevance in natural oral care solutions. These findings are consistent with previous research that demonstrated the broad-spectrum antimicrobial activity of \u003cem\u003ePiper betle\u003c/em\u003e due to its high concentration of bioactive compounds, including phenolics, flavonoids, and alkaloids (Chowdhury \u0026amp; Baruah, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jalil et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Comparison with Existing Literature:\u003c/h2\u003e \u003cp\u003eThe antibacterial properties of \u003cem\u003ePiper betle\u003c/em\u003e observed in this study align with findings from Agung et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who reported strong antimicrobial activity against \u003cem\u003eStreptococcus mutans\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e. Similarly, Nalina \u0026amp; Rahim (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) demonstrated that \u003cem\u003ePiper betle\u003c/em\u003e extract effectively inhibits \u003cem\u003eStreptococcus mutans\u003c/em\u003e, a key pathogen in dental caries. However, the present study expands upon these results by showing that the extract not only affects planktonic bacteria but also inhibits biofilm formation, a critical factor in oral infections and antibiotic resistance.\u003c/p\u003e \u003cp\u003eInterestingly, while previous studies have focused on the antimicrobial properties of \u003cem\u003ePiper betle\u003c/em\u003e against planktonic bacteria, this study highlights its biofilm inhibition capabilities, which are particularly relevant to oral health. Biofilms protect bacteria from external stressors, including antibiotics, making them difficult to eradicate (Shaikh et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The ability of \u003cem\u003ePiper betle\u003c/em\u003e to disrupt biofilm formation suggests that it may serve as a potential alternative or adjunct to conventional oral hygiene products.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Effectiveness at Different Concentrations:\u003c/h2\u003e \u003cp\u003eThe study showed that higher concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract (150 \u0026micro;L/mL and 200 \u0026micro;L/mL) produced larger inhibition zones against oral isolates, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e. This dose-dependent effect is consistent with Ratridewi et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who found that increasing concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract enhanced its antibacterial efficacy. The higher effectiveness at these concentrations may be attributed to the higher concentration of phenolic compounds, which are known to disrupt bacterial cell walls, leading to cell death (Agung et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiologically, the phenolic compounds in \u003cem\u003ePiper betle\u003c/em\u003e may penetrate bacterial biofilms by interfering with quorum sensing pathways, which are responsible for biofilm formation and maintenance. Quorum sensing inhibitors, like the ones potentially present in \u003cem\u003ePiper betle\u003c/em\u003e, can prevent bacterial communication, reducing the ability to form biofilms (Jalil et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The disruption of biofilms is particularly important in oral health, as biofilms are responsible for the persistence of dental caries and periodontal diseases.\u003c/p\u003e \u003cp\u003eAlthough the extract showed promising antimicrobial and biofilm-inhibitory effects, the specific phytochemicals responsible for these outcomes were not isolated or quantified in the present study. Prior studies using acetone-based extractions of Piper betle have identified compounds such as hydroxychavicol, eugenol, and chavibetol \u0026mdash; known for their antimicrobial and anti-quorum sensing properties (Das et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sharma et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The observed effects in our study may be attributed to one or more of these constituents, but this remains to be confirmed. Future research should include phytochemical profiling through HPTLC, LC-MS/MS, or GC-MS analysis to determine the active compounds responsible for the extract\u0026rsquo;s efficacy.\u003c/p\u003e \u003cp\u003eInterestingly, the acetone control exhibited mild antibacterial activity, particularly against \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, with inhibition zones ranging from 10.6 mm to 12.6 mm. This aligns with previous studies reporting that organic solvents, including acetone, can exhibit limited antimicrobial effects. However, the \u003cem\u003ePiper betle\u003c/em\u003e extract consistently produced larger inhibition zones at higher concentrations (150 \u0026micro;L/mL and 200 \u0026micro;L/mL), exceeding those of acetone by several millimeters. These findings suggest that while acetone contributes a baseline inhibitory effect, the extract's superior activity at higher doses is likely due to its bioactive constituents rather than the solvent alone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Limitations of the Study:\u003c/h2\u003e \u003cp\u003eWhile the findings of this study are promising, there are several limitations that should be acknowledged. First, the study was conducted in vitro, and the effectiveness of \u003cem\u003ePiper betle\u003c/em\u003e extract in a clinical setting remains unknown. The absence of in vivo studies limits the ability to draw conclusions about the extract's efficacy in real-life applications, where factors such as salivary flow, dietary habits, and oral microbiota may affect its performance. Future studies should include in vivo experiments to evaluate the potential of \u003cem\u003ePiper betle\u003c/em\u003e as a therapeutic agent for oral health.\u003c/p\u003e \u003cp\u003eAdditionally, the study was limited by a small sample size of bacterial isolates. A larger variety of oral bacteria, including anaerobic pathogens, should be tested to fully understand the spectrum of \u003cem\u003ePiper betle\u003c/em\u003e's antimicrobial properties. Another notable limitation is the reliance on morphological and biochemical tests for identifying bacterial isolates. Although useful as preliminary tools, these methods lack the specificity of molecular techniques such as 16S rRNA gene sequencing for bacteria and ITS analysis for fungi. Without molecular confirmation, the species-level identity of the isolates, particularly \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, cannot be guaranteed, which may influence the interpretation of species-specific effects. Future studies will incorporate molecular methods to validate isolate identity and enable more accurate assessment of the extract\u0026rsquo;s targeted antimicrobial effects. Future research should focus on isolating individual compounds and elucidating their specific roles in antimicrobial and biofilm inhibition activity.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated that \u003cem\u003ePiper betle\u003c/em\u003e leaf extract contains bioactive substances with antibacterial properties. The extract demonstrated notable efficacy against oral bacterial isolates, including \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, with higher concentrations producing greater zones of inhibition and reduced biofilm formation. These results align with previous studies that confirm \u003cem\u003ePiper betle\u003c/em\u003e as a potent natural antimicrobial agent (Jalil et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nalina \u0026amp; Rahim, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In particular, the ability of \u003cem\u003ePiper betle\u003c/em\u003e to inhibit biofilm formation has significant implications for its use in preventing biofilm-related oral diseases, such as dental caries and periodontal infections (Jantorn et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While acetone demonstrated a mild inhibitory effect on its own, the significantly larger zones of inhibition produced by the \u003cem\u003ePiper betle\u003c/em\u003e extract at higher concentrations confirm the presence of additional bioactive compounds contributing to its antimicrobial efficacy. The study also emphasizes the potential of \u003cem\u003ePiper betle\u003c/em\u003e extract as an alternative to synthetic antimicrobial agents, particularly in the fight against biofilm-related infections that contribute to antibiotic resistance (Chowdhury \u0026amp; Baruah, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shaikh et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Identifying the active compounds through phytochemical screening will be critical to validate the therapeutic potential of \u003cem\u003ePiper betle\u003c/em\u003e and optimize its use in oral care applications. Further exploration of this natural extract may lead to advancements in combating oral pathogens responsible for dental caries and periodontal diseases.\u003c/p\u003e"},{"header":"6. Future Directions","content":"\u003cp\u003e \u003cem\u003ePiper betle\u003c/em\u003e extract has the potential to be incorporated into oral healthcare products, such as mouthwashes, toothpastes, or dental gels. These products could serve as effective natural alternatives to conventional chemical-based oral care items. The antibacterial and biofilm-inhibiting properties of \u003cem\u003ePiper betle\u003c/em\u003e could make it particularly useful in preventing dental caries and periodontal infections, especially among individuals at higher risk of biofilm-related diseases or those seeking natural oral hygiene products.\u003c/p\u003e \u003cp\u003eTo fully realize the therapeutic potential of \u003cem\u003ePiper betle\u003c/em\u003e, additional in vivo studies are necessary. Clinical trials should be conducted to test the effectiveness and safety of \u003cem\u003ePiper betle\u003c/em\u003e extract in real-world oral environments. Additionally, expanding the research to include a wider variety of oral pathogens, particularly anaerobic bacteria, would provide a more comprehensive view of its antimicrobial efficacy. Future studies should also explore the mechanisms of biofilm inhibition more deeply, focusing on how \u003cem\u003ePiper betle\u003c/em\u003e disrupts biofilm formation at the molecular level.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCI: Confidence Interval\u003c/p\u003e\n\u003cp\u003eE. coli: \u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEMB: Eosin Methylene Blue\u003c/p\u003e\n\u003cp\u003eLB: Luria Bertani\u003c/p\u003e\n\u003cp\u003eMRS: de Man, Rogosa and Sharpe (medium for Lactobacilli)\u003c/p\u003e\n\u003cp\u003eOD: Optical Density\u003c/p\u003e\n\u003cp\u003ePBS: Phosphate Buffered Saline\u003c/p\u003e\n\u003cp\u003eS. aureus: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author expresses sincere gratitude to Dr. Jyoti Patki for her invaluable guidance and insightful contributions throughout the research and experiment. Special thanks are extended to the staff and assistants at the D. Y. Patil Deemed to be University School of Biotechnology and Bioinformatics for their technical support and for providing access to laboratory facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003cbr\u003e\u003c/strong\u003e This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003cbr\u003e\u003c/strong\u003e The author declares no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003cbr\u003e\u003c/strong\u003e Ethical approval was not required for this study, as no human or animal experiments were conducted beyond the collection of anonymized oral swabs from healthy volunteers, which involved no invasive procedures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJaganath, I. B. (2000). \u003cem\u003eHerbs: The green pharmacy of Malaysia\u003c/em\u003e. Serdang: Mardi.\u003c/li\u003e\n \u003cli\u003ePin, K. Y., Chuah, A. L., Rashih, A. A., Mazura, M. P., Fadzureena, J., Vimala, S., \u0026amp; Rasadah, M. A. (2010). Antioxidant and anti-inflammatory activities of extracts of betel leaves (\u003cem\u003ePiper betle\u003c/em\u003e) from solvents with different polarities. \u003cem\u003eJournal of Tropical Forest Science\u003c/em\u003e, 448-455.\u003c/li\u003e\n \u003cli\u003eShetty, S., \u0026amp; Vijayalaxmi, K. K. (2012). Phytochemical investigation of extract/solvent fractions of \u003cem\u003ePiper nigrum\u003c/em\u003e linn. seeds and \u003cem\u003ePiper betle\u003c/em\u003e linn. leaves. \u003cem\u003eInternational Journal of Pharma and Bio Sciences\u003c/em\u003e, 3(2), 344-349.\u003c/li\u003e\n \u003cli\u003eArambewela, L. S., Arawwawala, L. D., \u0026amp; Ratnasooriya, W. D. (2005). Antidiabetic activities of aqueous and ethanolic extracts of \u003cem\u003ePiper betle\u003c/em\u003e leaves in rats. \u003cem\u003eJournal of Ethnopharmacology\u003c/em\u003e, 102(2), 239\u0026ndash;245. https://doi.org/10.1016/j.jep.2005.06.016\u003c/li\u003e\n \u003cli\u003ePisar, M. M., Hashim, N., Ali, R. M., \u0026amp; Kiong, L. S. (2007). Evaluation of \u003cem\u003ePiper betle\u003c/em\u003e on platelet-activating factor (PAF) receptor binding activities. \u003cem\u003eMalaysian Journal of Science\u003c/em\u003e, 26(1), 79-83.\u003c/li\u003e\n \u003cli\u003eNalina, T., \u0026amp; Rahim, Z. H. A. (2007). The crude aqueous extract of \u003cem\u003ePiper betle\u003c/em\u003e L. and its antibacterial effect towards \u003cem\u003eStreptococcus mutans\u003c/em\u003e. \u003cem\u003eAmerican Journal of Biotechnology and Biochemistry\u003c/em\u003e, 3(1), 10-15.\u003c/li\u003e\n \u003cli\u003eHajare, R., Darvhekar, V. M., Shewale, A., \u0026amp; Patil, V. (2011). Evaluation of antihistaminic activity of \u003cem\u003ePiper betle\u003c/em\u003e leaf in guinea pig. \u003cem\u003eAfrican Journal of Pharmacy and Pharmacology\u003c/em\u003e, 5(2), 113-117.\u003c/li\u003e\n \u003cli\u003eSchwiertz, A. (2016). Microbiota of the human body. \u003cem\u003eAdvances in Experimental Medicine and Biology\u003c/em\u003e, 902, 83-93.\u003c/li\u003e\n \u003cli\u003eTuominen, H., \u0026amp; Rautava, J. (2021). Oral microbiota and cancer development. \u003cem\u003ePathobiology: Journal of Immunopathology, Molecular and Cellular Biology\u003c/em\u003e, 88(2), 116\u0026ndash;126. https://doi.org/10.1159/000510979\u003c/li\u003e\n \u003cli\u003eDas, S., Ray, A., Nasim, N., Nayak, S., \u0026amp; Mohanty, S. (2019). Effect of different extraction techniques on total phenolic and flavonoid contents, and antioxidant activity of betelvine and quantification of its phenolic constituents by validated HPTLC method. \u003cem\u003e3 Biotech\u003c/em\u003e, 9(1), 37. https://doi.org/10.1007/s13205-018-1565-8\u003c/li\u003e\n \u003cli\u003eRai, M. P., Thilakchand, K. R., Palatty, P. L., Rao, P., Rao, S., Bhat, H. P., \u0026amp; Baliga, M. S. (2011). \u003cem\u003ePiper betel\u003c/em\u003e Linn (betel vine), the maligned Southeast Asian medicinal plant possesses cancer preventive effects: time to reconsider the wronged opinion. \u003cem\u003eAsian Pacific Journal of Cancer Prevention\u003c/em\u003e, 12(9), 2149\u0026ndash;2156.\u003c/li\u003e\n \u003cli\u003eSharma, S., Khan, I. A., Ali, I., Ali, F., Kumar, M., Kumar, A., Johri, R. K., Abdullah, S. T., Bani, S., Pandey, A., Suri, K. A., Gupta, B. D., Satti, N. K., Dutt, P., \u0026amp; Qazi, G. N. (2009). Evaluation of the antimicrobial, antioxidant, and anti-inflammatory activities of hydroxychavicol for its potential use as an oral care agent. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e, 53(1), 216\u0026ndash;222. https://doi.org/10.1128/AAC.00045-08\u003c/li\u003e\n \u003cli\u003eJalil, V., Khan, M., Haider, S. Z., \u0026amp; Shamim, S. (2022). Investigation of the antibacterial, anti-biofilm, and antioxidative effect of \u003cem\u003ePiper betle\u003c/em\u003e leaf extract against \u003cem\u003eBacillus gaemokensis\u003c/em\u003e MW067143 isolated from dental caries, an in vitro-in silico approach. \u003cem\u003eMicroorganisms\u003c/em\u003e, 10(12), 2485. https://doi.org/10.3390/microorganisms10122485\u003c/li\u003e\n \u003cli\u003eCoffey, B. M., \u0026amp; Anderson, G. G. (2014). Biofilm formation in the 96-well microtiter plate. \u003cem\u003eMethods in Molecular Biology\u003c/em\u003e, 1149, 631\u0026ndash;641. https://doi.org/10.1007/978-1-4939-0473-0_48\u003c/li\u003e\n \u003cli\u003eHassan, A., Usman, J., Kaleem, F., Omair, M., Khalid, A., \u0026amp; Iqbal, M. (2011). Evaluation of different detection methods of biofilm formation in clinical isolates. \u003cem\u003eThe Brazilian Journal of Infectious Diseases\u003c/em\u003e, 15(4), 305\u0026ndash;311.\u003c/li\u003e\n \u003cli\u003eStruzycka, I. (2014). The oral microbiome in dental caries. \u003cem\u003ePolish Journal of Microbiology\u003c/em\u003e, 63(2), 127\u0026ndash;135.\u003c/li\u003e\n \u003cli\u003eMamun Or Rashida, M., Shafiul Islam, M., Azizul Haque, M., Arifur Rahman, M., Tanvir Hossain, M., \u0026amp; Abdul Hamid, M. (2016). Antibacterial activity of polyaniline coated silver nanoparticles synthesized from \u003cem\u003ePiper betle\u003c/em\u003e leaves extract. \u003cem\u003eIranian Journal of Pharmaceutical Research\u003c/em\u003e, 15(2), 591\u0026ndash;597.\u003c/li\u003e\n \u003cli\u003eJantorn, P., Tipmanee, V., Wanna, W., Prapasarakul, N., Visutthi, M., \u0026amp; Saeloh Sotthibandhu, D. (2023). Potential natural antimicrobial and antibiofilm properties of \u003cem\u003ePiper betle\u003c/em\u003e L. against \u003cem\u003eStaphylococcus pseudintermedius\u003c/em\u003e and methicillin-resistant strains. \u003cem\u003eJournal of Ethnopharmacology, 317\u003c/em\u003e, 116820. https://doi.org/10.1016/j.jep.2023.116820\u003c/li\u003e\n \u003cli\u003eAgung, I. G. A., Wahjuni, S., Wedagama, D. M., \u0026amp; Weta, I. W. (2022). Nutraceuticals of nano-betel (\u003cem\u003ePiper betle\u003c/em\u003e L.) leaves: Prevent COVID-19 and oral cavity disease. \u003cem\u003eBali Medical Journal, 11\u003c/em\u003e(2), 844-849. https://doi.org/10.15562/bmj.v11i2.3476\u003c/li\u003e\n \u003cli\u003eShaikh, A. A., Shejul, D. D., Shekade, M. P., \u0026amp; Anbhule, S. J. (2023). A systematic review on antimicrobial activity of Piper betle Linn leaves. \u003cem\u003eCurrent Topics in Pharmacology and Clinical Therapeutics\u003c/em\u003e, 180071.\u003c/li\u003e\n \u003cli\u003eChowdhury, U., \u0026amp; Baruah, P. K. (2020). Betelvine (Piper betle L.): A potential source for oral care. \u003cem\u003eCurrent Botany\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eRatridewi, I., Dzulkarnain, S. A., Wijaya, A. B., et al. (2021). Effects of Piper betle leaf extract on biofilm and rhamnolipid formation of Pseudomonas aeruginosa. \u003cem\u003eResearch Journal of Pharmacy and Technology\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eDolly, S. A., Gayathri, K., Rao, S., Sindhujaa, R., Rashik, M., \u0026amp; Ravishankar, P. L. (2023). Antibacterial effectiveness of betel leaf (\u003cem\u003ePiper betle\u003c/em\u003e) extract on red complex bacteria: An in vitro study. \u003cem\u003eInternational Journal of Pharmaceutical Research and Applications, 8\u003c/em\u003e(2), 1543-1548. https://doi.org/10.35629/7781-080215431548\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 1.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eChemicals Used in the Experiment\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003eSr. No.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003ePurpose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 381px;\"\u003e\n \u003cp\u003eChemical Used\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003ePreparation of leaf extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 381px;\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eGrowing oral isolates\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 381px;\"\u003e\n \u003cp\u003ePeptone water\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eIsolation of oral isolates\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 381px;\"\u003e\n \u003cp\u003eMRS agar, Tryptone Soy agar (5% defibrinated sheep blood), EMB agar\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eScreening for biofilm formation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 381px;\"\u003e\n \u003cp\u003eLB broth, PBS (pH 7.3), Crystal violet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eQuantitative biofilm formation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 381px;\"\u003e\n \u003cp\u003eCrystal violet, Nutrient agar, PBS (pH 7.3), Distilled water\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eAntibacterial activity test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 381px;\"\u003e\n \u003cp\u003eLeaf extract, Distilled water, Acetone, Streptomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: This table outlines the various chemicals used in the experimental procedures, including preparation of extracts, growth media, and biofilm assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 2.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eOptical Density (OD) Values for Biofilm Formation at 570 nm\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e50 \u0026micro;L Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100 \u0026micro;L Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e150 \u0026micro;L Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e200 \u0026micro;L Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e50 \u0026micro;L Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100 \u0026micro;L Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e150 \u0026micro;L Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e200 \u0026micro;L Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOral Isolate 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.280\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOral Isolate 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.365\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOral Isolate 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.787\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.572\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: Optical Density (OD) values were measured after a 24-hour incubation period to evaluate biofilm formation at 570 nm using a microtiter plate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 3.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eZone of Inhibition (mm) for Oral Isolates, E. coli, and S. aureus\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;Disc\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 499px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeasured average zone of inhibition (mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003eOral Isolate 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eOral Isolate 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eE. coli\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eS. aureus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003eStreptomycin\u003c/p\u003e\n \u003cp\u003e(+ve control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e19.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e19.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003eDistilled Water\u003c/p\u003e\n \u003cp\u003e(-ve control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e7.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e9.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003eAcetone (-ve control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e10.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e50 \u0026micro;l/ml Extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e13.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e13.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e100 \u0026micro;l/ml Extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e14.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e13.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e12.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e150 \u0026micro;l/ml Extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e15.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e14.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e200 \u0026micro;l/ml Extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e16.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e17.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e15.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e15.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: This table describes the average zone of inhibition measured in millimeters for different concentrations of \u003cem\u003ePiper betle\u003c/em\u003e extract, streptomycin as a positive control, and distilled water and acetone as negative controls.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"D.Y. Patil School of Biotech and Bioinformatics","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Piper betle, Oral Isolates, Antibacterial, Antibiotic, Biofilm","lastPublishedDoi":"10.21203/rs.3.rs-6639539/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6639539/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to evaluate the antibacterial and anti-biofilm effects of \u003cem\u003ePiper betle\u003c/em\u003e leaf extract on oral bacterial isolates. The leaf extract was obtained using Soxhlet extraction with acetone as the solvent. Oral bacteria were isolated using cheek swabs and cultured on MRS agar, Tryptone Soy agar, and EMB agar. Biofilm formation was assessed using crystal violet staining and quantified at 570 nm with a microtiter plate reader. The antibacterial activity of the extract was tested against oral isolates, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e using the Kirby-Bauer disc diffusion method, with acetone as a negative control. The results demonstrated significant antibacterial activity of Piper betle extract, with inhibition zones reaching up to 16.6 mm for oral isolates and 15.3 mm for \u003cem\u003eS. aureus\u003c/em\u003e at a concentration of 200 \u0026micro;L/mL. Additionally, the extract inhibited biofilm formation, as demonstrated by the reduced optical density in biofilm assays. These findings suggest that \u003cem\u003ePiper betle\u003c/em\u003e leaf extract holds promise as a natural alternative to synthetic antibiotics, particularly for managing oral bacteria and preventing biofilm-related infections. Further research is needed to isolate and identify the specific bioactive compounds responsible for the antibacterial effects and to evaluate the extract's potential in clinical applications.\u003c/p\u003e","manuscriptTitle":"Effect of Piper Betle Leaf Extract on Biofilm Formation and Antibacterial Activity on Oral Isolates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 06:54:19","doi":"10.21203/rs.3.rs-6639539/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c9da96e-e9b1-48f8-a929-aaed6b591fdf","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48503814,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2025-05-14T06:54:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-14 06:54:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6639539","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6639539","identity":"rs-6639539","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}