PH AND ANTIBIOFILM ANALYSIS OF ELEPHANT GINGER MOUTHWASH FORMULATIONS AGAINST Streptococcus mutans – An in vitro Study

Zoliflodacin (ZFD) is a new type of antibiotic whose anticaries potential through controlling biofilms remains unknown. This study aimed to explore the effects of ZFD on the conditional formation of cariogenic Streptococcus mutans (S. mutans) biofilms. Crystal violet analysis, colony forming unit (CFU) counting, and MTT assays were used to determine biofilm biomass, viable bacterial cell counts, and bacterial metabolic viability, respectively, while acid generation by biofilms was monitored by measuring pH and lactic acid production. The anthrone-sulfuric acid method and polysaccharide/bacterial staining were used to investigate extracellular polysaccharide (EPS) production. The microstructure of S. mutans biofilms was observed using scanning electron microscopy (SEM). Growth curves were used to visualize bacterial growth, while live/dead bacterial staining was used to determine microbial activity. Furthermore, the cytotoxicity of ZFD was evaluated by live/dead cellular staining together with the CCK-8 assay, while drug resistance was induced using bacterial reduplicative exposure to ZFD. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to calculate gene expression levels. In addition, checkerboard microdilution assays were performed to evaluate potential synergistic or antagonistic interactions of ZFD in combination with NaF or chlorhexidine (CHX) against bacterial growth and biofilm formation. We found ZFD significantly inhibited S. mutans biofilm formation, decreasing overall biofilm biomass, viable bacterial cell counts within biofilms, and biofilm metabolic viability. ZFD not only markedly suppressed S. mutans cariogenic virulence involving acid and EPS synthesis, but also affected bacterial morphology within biofilms. Furthermore, ZFD inhibited bacterial growth without significantly suppressing the survival rate of bacterial cells within biofilms. Moreover, ZFD demonstrated no significant cytotoxicity and did not trigger drug resistance in S. mutans after 20 passages. qRT-PCR analysis revealed significant differences in the expression of virulence-, quorum sensing-, and oxidative stress protection-associated genes. Indifferent effects were found when ZFD was combined with NaF or CHX on S. mutans growth and biofilm formation. In summary, ZFD is a potential antibacterial agent for caries control because of its antibiofilm effects, inconspicuous cytotoxicity, low risk of resistance induction and antibacterial activity against S. mutans.

BackgroundOral diseases like periodontitis and mucositis often require home care using topical agents in the form of mouthwashes. Many herbal mouthwashes are found to be beneficial; however lack proper scientific evidence to support their use. ObjectivesStudy 1 evaluated clinical efficacy of herbal mouthwash in the management of chronic periodontitis in comparison with chlorhexidine mouthwash. Study 2 aimed at assessment of herbal mouthwash in patients of radiation-induced mucosititis. MethodsThe novel herbal mouthwash used in the present study wa prepared from extracts of five plants namely Emblica Officinalis, Terminalia chebula, Terminalia bellerica, Glycyrrhiza glabra, and Azadirachta indica. 50 periodontitis patients were randomly allocated to two groups. As per allocation, they were instructed to use either herbal mouthwash or chlorhexidine mouthwash twice daily for two weeks after nonsurgical periodontal therapy. Similarly, patients with radiation-induced mucositis were randomly given herbal mouthwash and soda saline mouthwash. Intergroup and intragroup comparisons of continuous variables were conducted using paired and unpaired t-tests. Categorical variables were compared using the chi-square test. ResultsSignificant reductions in gingival bleeding, plaque accumulation, and pocket depth were noticed in periodontitis patients in both groups. Patients reported acceptable taste, freshness, and satisfaction after the use of herbal mouthwash. The herbal mouthwash group noticed a significant reduction in the severity of radiation-induced mucositis and analgesic requirements. The intensity of pain, dryness of mouth, oral hygiene, and need for the use of antibiotic and antifungal during radiotherapy was not significant among the groups. ConclusionThe results of this preliminary clinical trial support the use of the tested herbal formulation mouthwash as an adjunct in the treatment of periodontitis as well as radiation-induced mucositis. Clinical trial registration NumberFor Study 1: CTRI/2019/10/021574, Study 2: CTRI/2020/04/024851.

Medicinal plant or herbs are considered to have rich source of ingredients which can be used in drug development. The formulation of herbal mouthwash is a natural approach to oral hygiene that utilizes plant-based ingredients to create a safe and effective mouthwash. Herbal mouthwash is intended to provide a range of benefits, including anti-bacterial, anti-inflammatory, and anti-plaque effects, as well as freshening breath and promoting healthy gums and teeth. The formulation of herbal mouthwash offers an alternative to chemical-based oral hygiene products that can sometimes cause irritation and sensitivity. With the growing interest in natural remedies and alternative health practices, the formulation of herbal mouthwash offers a promising solution for those seeking a more natural approach to oral hygiene. The main objective of this literature work is to prepare and evaluate a herbal mouthwash and check its effectiveness against microorganisms of oral cavity. The prepared mouthwash was evaluated against pathogens and was found effective for controlling and demolishing microbial growth in mouth. Further physiochemical properties were tested for mouthwash and it possesses a good antibacterial activity. Next, studies on physical evaluation, pH measurement, stability, viscosity and antibacterial activity were conducted.  

Senyawa 1,5-bis(3’-etoksi-4’-hidroksifenil)-1,4-pentadien-3-on (EHP) merupakan analog kurkumin yang memiliki aktivitas antimikroba. Pada penelitian ini, dilakukan formulasi dan evaluasi larutan pencuci mulut dengan EHP sebagai bahan aktif antimikroba. Penelitian ini bertujuan untuk membuat formula dan menguji kestabilan formula obat kumur dengan bahan aktif EHP. Formulasi larutan pencuci mulut dibuat dengan variasi konsentrasi senyawa EHP 3, 6, 12, 16 dan 18 bpj. Evaluasi larutan pencuci mulut meliputi uji organoleptik, pH, kejernihan, bobot jenis dan aktivitas antimikroba terhadap Staphylococcus aureus, Steptococcus mutans, dan Candida albicans. Uji antimikroba dilakukan dengan metode diffusi agar. Hasil evaluasi sediaan obat yang didapat dari pengujian organoleptik antara lain obat kumur berupa cairan dengan warna biru jernih, aroma mint dan rasa segar; memiliki rentang pH 6,15 - 6,74; dan bobot jenis 1,0419 - 1,0561 g/cm3. Evaluasi larutan pencuci mulut menunjukkan bahwa larutan pencuci mulut stabil dalam penyimpanan selama 1 bulan pada suhu 40°C. Uji antimikroba menunjukkan diameter daya hambat terhadap bakteri Staphylococcus aureus (ATCC 6538); Streptococcus mutans (ATCC 31987) dan Candida albicans (ATCC 10231) berkisar antara 6,2-8,4 mm pada konsentrasi senyawa EHP 18 bpj. Hasil ini menunjukkan bahwa senyawa EHP sangat potensial digunakan sebagai bahan antimikroba dalam formula larutan pencuci mulut.

The role of Streptococcus mutans in the initiation of caries is related to its acidogenicity, aciduricity, and polysaccharides extracellular layer production by glucosyltransferases in dental biofilms. Therefore, inhibition of glucosyltransferase activity impairs the virulence of cariogenic biofilms, which can be used to prevent dental caries. We evaluated the anti-bacterial, anti-biofilm, and anti-glucosyltransferases effects of nanoliposomal herbal aqueous extracts of Liquorice (Glycyrrhiza glabra; G. glabra), Ginger (Zingiber officinale; Z. officinale), Pomegranate (Punica granatum; P. granatum), and Rose (Rosa damascene; R. damascene) via minimum bactericidal concentration and minimum inhibitory concentration against Streptococcus mutans strain ATCC 35,668. An anti-biofilm assay was performed using a minimum biofilm inhibitory concentration test. Among herbs, only P. granatum showed an antibacterial effect. Therefore, a nanoliposomal formulation of P. granatum was developed and characterized. Its effect on S.mutans glucosyltransferases was assessed by measuring glucan amount. The nanoliposomal formulation of P.granatum showed a significantly higher anti-biofilm effect than P. granatum aqueous extract. Their similar potential in blocking glucosyltransferases showed that the nanoliposomal formulation of P.granatum blocked other pathways rather than blocking glucosyltransferases for its anti-biofilm effect. Collectively, the nanoliposomal formulation of P.granatum, due to its anti- Streptococcus mutans characteristics, would be a production which open a new horizon for the oral pharmaceutical industry.

Oral pathogens contribute to dental caries and other infections affecting oral health. To address concerns related to the adverse effects of chemical mouthwashes like chlorhexidine (CHX), this study introduces a novel herbal mouthwash formulated with green-synthesized zinc oxide nanoparticles (ZnONPs) infused with Cymbopogon citratus essential oil (CEO). Unlike traditional formulations, this herbal mouthwash aims to offer a safer, eco-friendly alternative. Four formulations (F1-F4) with varying concentrations of CEOZnONPs were evaluated for in vitro antibacterial and anti-biofilm activities against Streptococcus mutans, Staphylococcus aureus, and Escherichia coli. The F4 formulation demonstrated superior antibacterial efficacy, with inhibition zones of 64.2 ± 2.6mm for S. mutans, 43.6 ± 1.0mm for S. aureus, and 31.8 ± 1.5mm for E. coli, and achieved 97.12% biofilm inhibition of S. mutans. Additionally, in vivo toxicity was assessed using embryonic zebrafish, showing a high survival rate of 93% for F1 and 82% for F4after 24 hours, compared to 65% for CHX, indicating lower toxicity. This preclinical study shows that the F4 formulation exhibited promising antibacterial and safety profiles, highlighting its potential as a safe, natural oral care product targeting diverse oral pathogens with reduced toxicity. Further research, including in vivo and clinical trials, is needed to validate its effectiveness and safety for regular dental care use. The herbal mouthwash formulated with CEOZnONPs demonstrates strong antibacterial and anti-biofilm effects with lower toxicity, making it a promising natural alternative for oral hygiene. Its safer profile compared to CHX may reduce side effects in patients, potentially enhancing compliance in oral care regimens.

Objectives: To investigate the effect of various probiotic mouthwash formulations on the quantity of oral pathogenic microorganisms. Methods: This was a laboratory-based study testing four different mouthwash formulations: (1) 10% probiotic supernatant mouthwash, (2) 10% probiotic supernatant / 5% propolis mouthwash, (3) 10% probiotic supernatant / 1% cannabidiol mouthwash, and (4) 10% probiotic supernatant / 5% propolis / 1% cannabidiol mouthwash. A positive control (chlorhexidine mouthwash) and a negative control (0.9% saline solution) were also included. The inhibitory efficacy of these formulations was tested against two microorganisms: Candida albicans and Streptococcus mutans. Quantitative data were collected by counting colonies before and after treatment (CFU/mL). Descriptive statistics and One way ANOVA test were used for analysis (p<0.05). Results: The results showed that each mouthwash formulation could inhibit both pathogenic microorganisms to varying degrees, with a greater effect observed on Streptococcus mutans than on Candida albicans. When the quantitative data from colony counts were subjected to statistical analysis, no statistically significant differences were found among the mouthwash formulations (p>0.05) in inhibiting both types of microorganisms. Notably, the mouthwash containing 10% probiotic supernatant, 5% propolis and 1% cannabidiol demonstrated a statistically significant inhibitory effect on Streptococcus mutans (p<0.05). Conclusions: All four mouthwash formulations showed a tendency to inhibit both pathogenic microorganisms, although this was not statistically significant in all cases. The formulation containing 10% probiotic supernatant, 5% propolis, and 1% cannabidiol had the most favorable effect in inhibiting the cariogenic bacterium Streptococcus mutans.

The leaves of Pongamia pinnata was extracted with 800 ml of 70% ethanol and 30% water (V/V) mixture for 48 hr. The hydroalcoholic extract of Pongamia pinnata leaves was subjected to preliminary phytochemical analysis. The presence of alkaloids, flavonoids, phenolic compounds, tannins and terpenoids was confirmed by preliminary phytochemical screening. The chemical composition of hydroalcoholic extract was determined by GC-MS analysis. The GC-MS investigation revealed the presence of sixteen different bioactive compounds in the Pongamia pinnata leaves extract. The least amount of constituents present in the hydroalcoholic extracts was dioctyl phthalate has the peak area 0.84 and maximum 38.17 peak area % was recorded for 3-Methoxy-1,2-propanediol. Three formulations (F-1), (F-2) and (F-3) of herbal mouthwash containing hydroalcoholic leaves extract of Pongamia pinnata were prepared. Then antibacterial activity of all three formulations of mouthwash were tested against Streptococcus mutans (oral pathogen). The results obtained were compared with standard mouthwash (Chlorhexidine). The findings revealed that prepared herbal mouthwash formulation 2 (F-2) showed significant zone of inhibition compared with standard Chlorhexidine.

This study aimed to develop and evaluate a herbal mouthwash formulation containing an ethanolic extract of sappan wood (Caesalpinia sappan L.) as a potential natural alternative to chlorhexidine-based products. The extract and derived mouthwash were assessed for antibacterial activity against Streptococcus mutans Clarke and Pseudomonas aeruginosa (Schroeter) Migula using the agar well diffusion and minimum inhibitory concentration (MIC) methods at concentrations of 1 %, 3 % and 5 %. Positive controls included 0.2 % chlorhexidine and a commercial mouthwash, while negative controls consisted of mouthwash without extract and 96 % ethanol. The stability of the mouthwash was evaluated through a six-cycle cycling test by monitoring organoleptic properties, homogeneity, pH, sedimentation and redispersion. The mouthwash exhibited a maximum inhibition zone of 21.5 mm and 15.2 mm against S. mutans and P. aeruginosa, respectively, with MIC values of 1 % and 3 %. It remained physically stable during six storage cycles with consistent pH (4.6–6.5) and homogeneous dispersion. These results demonstrate that sappan wood extract possesses strong antibacterial activity and good formulation stability, supporting its potential as a safe and effective herbal mouthwash. The study highlights the novelty of exploring a plant-based mouthwash as a promising natural alternative to chlorhexidine, showing comparable in vitro antibacterial activity with potentially fewer adverse effects.

Formulation of Mouthwash Using Combined Herbal Extracts to Control the Predominant Oral Pathogens and Biofilm

Herbal composite preparation was studied with the aim of inhibiting the virulence factors of two dental pathogens: Prevotella intermedia and Porphyromonas gingivalis. A novel herbal composite was developed using the herbal extracts of Wrightia tinctoria and Bauhinia variegata. During the study, the following observations were noted. The minimal inhibitory concentration of Wrightia tinctoria and Bauhinia variegata composites (WBc) was obtained for the test concentration of 20 μg/ml (16 ± 0.57 mm and 15 ± 0.75 mm of inhibitory zones against Prevotella intermedia and Porphyromonas gingivalis, respectively). Biofilm inhibition assay results revealed about 0.51 ± 1.25 mg/ml and 0.53 ± 0.57 mg/ml of minimal biofilm eradication concentration (MBEC) against Prevotella intermedia and Porphyromonas gingivalis, respectively. The effect of WBc on lactic acid production showed that 200 μg/ml and 400 μg/ml concentrates reduced up to 80% and 70% in Prevotella intermedia and Porphyromonas gingivalis, respectively. Formulated herbal mouthwash showed good stability under all three different test conditions (5°C, 25°C, and 40°C) as the color, odor, phase separation, and homogeneity were not changed for the period of 3 months. The anti-bacterial activity of formulated mouthwash (30 μg/ml) exhibited maximum inhibitory zones of about 18 ± 0.75 mm and 19 ± 1.05 mm against the respective test bacteria – Prevotella intermedia and Porphyromonas gingivalis. Amplification of mfa1 and clpB genes showed 246 bp and 294 bp fragments of P. gingivalis and 238 bp and 280 bp fragments of P. intermedia during agarose electrophoretic analysis. The docking report revealed -5.84 Kcal/Mol binding energy and found three hydrogen bonding between the quercetin and target protein, mfa1 of Porphyromonas gingivalis. The target protein, clpB of Prevotella intermedia, and quercetin had -6.72 Kcal/Mol binding energy and found four hydrogen bonds between them. The developed composite could be optimized in future to develop a novel and biocompatible herbal mouthwash for the prevention of different dental caries and gingival inflammation associated with dental biofilm formation.

Formation of a dental biofilm by Streptococcus mutans can cause dental caries, and remains a costly health problem worldwide. Recently, there has been a growing interest in the use of peptidic drugs, such as peptide p1025, analogous to the fragments 1025–1044 of S. mutans cellular adhesin, responsible for the adhesion and formation of dental biofilm. However, peptides have physicochemical characteristics that may affect their biological action, limiting their clinical performance. Therefore, drug-delivery systems, such as a bioadhesive liquid-crystalline system (LCS), may be attractive strategies for peptide delivery. Potentiation of the action of LCS can be achieved with the use of bioadhesive polymers to prolong their residence on the teeth. In line with this, three formulations – polyoxypropylene-(5)-polyoxyethylene-(20)-cetyl alcohol, oleic acid, and Carbopol C974P in different combinations (F1C, F2C, and F3C) were developed to observe the influence of water in the LCS, with the aim of achieving in situ gelling in the oral environment. These formulations were assessed by polarized light microscopy, small-angle X-ray scattering, rheological analysis, and in vitro bioadhesion analysis. Then, p1025 and a control (chlorhexidine) were incorporated into the aqueous phase of the formulation (F + p1025 and F + chlorhexidine), to determine their antibiofilm effect and toxicity on epithelial cells. Polarized light microscopy and small-angle X-ray scattering showed that F1C and F2C were LCS, whereas F3C was a microemulsion. F1C and F2C showed pseudoplastic behavior and F3C Newtonian behavior. F1C showed the highest elastic and bioadhesive characteristics compared to other formulations. Antibiofilm effects were observed for F + p1025 when applied in the surface-bound salivary phase. The p1025-loaded nanostructured LCS presented limited cytotoxicity and effectively reduced S. mutans biofilm formation, and could be a promising p1025-delivery strategy to prevent the formation of S. mutans dental biofilm.

Streptococcus mutans (S. mutans) is recognized as the primary oral pathogen responsible for dental caries. The formation of biofilms on tooth surfaces is a crucial virulence factor for S. mutans. This study aimed to investigate the antimicrobial and anti-biofilm effects of silicon dioxide nanoparticles (SiO2 NPs) and zinc oxide/zeolite nanocomposites (ZnO/Zeolite NCs) on S. mutans gene expression and biofilm formation. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), sub-MIC, safranin staining, growth curve analysis, and MTT assays were performed to evaluate the anti-biofilm properties of the nanoparticles. The expression levels of the ftf, gtfB, vicR, and gbpB genes were evaluated by real-time PCR. Cytotoxic effects of the nanoparticles were measured by the MTT assay with human gingival fibroblast (HGF2PI2) cells. Both SiO2 NPs and ZnO/Zeolite NCs effectively inhibited S. mutans biofilm formation, with MTT assay results showing a 79% inhibition by SiO2 NPs and 95% by ZnO/Zeolite NCs. Additionally, both nanoparticles reduced the transcription levels of the ftf, gtfB, vicR, and gbpB genes, with no toxic effects observed on HGF2PI2 cells at a concentration of 32 mg/ml. These findings suggest that SiO2 NPs and ZnO/Zeolite NCs are promising agents against S. mutans biofilms, with potential applications in oral care products. SiO2 NPs and ZnO/Zeolite NCs show significant potential for preventing biofilm formation by S. mutans, representing effective and cost-efficient antibacterial options for oral health.

The aim of this study was to prepare a polyherbal mouthwash and evaluate its antimicrobial and anti-inflammatory efficacy against commercially available herbal mouthwash. The objective was to signify whether the novel herbal combination (extracts of Zingiber officinale [ginger], Curcuma longa [turmeric], and Syzygium aromaticum [clove] 5% v/w) could be a better alternative to commercially available herbal mouthwashes. An in vitro study was undertaken in which extracts of Z. officinale (ginger), C. longa (turmeric), and S. aromaticum (clove) 5% v/w were used. Seven different concentrations were prepared and tested against Streptococcus mutans, Enterococcus faecalis, Candida albicans, and Staphylococcus aureus in Mueller-Hinton agar medium. Plates were incubated aerobically at 37° C for 48 h, and the zone of inhibition was measured using a vernier caliper. Commercially available herbal mouthwash (Hiora) was used as a control group. The data were analyzed by descriptive analytics. Results showed that the efficacy of novel polyherbal mouthwash had comparatively less significant antimicrobial properties against the microorganisms as compared to the commercially available herbal mouthwash. The minimum inhibitory concentration was also found to be very high, that is, 100 μg/mL. There was no significant antimicrobial and anti-inflammatory effects for the polyherbal mouthwash as compared to commercially available herbal mouthwash (Hiora). Because this combination is readily available, it can be a cost-effective alternative to commercially available herbal mouthwashes.

Effect of the antimicrobial decapeptide KSL on the growth of oral pathogens and Streptococcus mutans biofilm