The Stain Reduction and Whitening Efficacy of an Enzyme-Enhanced Mouthwash: A Comparative Study

Introduction

Dental staining represents a prevalent and significant aesthetic issue in modern dental practice, motivating a large proportion of individuals to pursue corrective or whitening interventions.¹ According to prevailing classifications, stains may originate from extrinsic mechanisms – where chromogenic agents deposit upon the enamel surface or within the acquired pellicle – or from intrinsic processes, where chromogens are incorporated into the tooth structure (enamel or dentin) during development or secondary to systemic, local or traumatic events.^2,3 Extrinsic stains typically derive from environmental sources such as dietary chromogens (eg, coffee, tea, wine), tobacco, and poor biofilm control, whereas intrinsic staining may result from systemic conditions (eg, fluorosis, porphyria), medication (eg, tetracycline), endodontic treatment or aging‐related dentin changes.^4,5 The biochemical processes responsible for dental discoloration involve the adsorption of various organic and inorganic chromogenic compounds – such as tannins, furfurals, and transition metal ions – onto or within the tooth substrate, accompanied by pigment generation within the acquired pellicle or biofilm through Maillard-type nonenzymatic browning reactions. Considering the strong cultural and psychosocial value placed on an attractive smile, individuals affected by dental discoloration demonstrate a heightened inclination toward pursuing safe, effective, and minimally invasive methods to achieve tooth shade improvement.^6,7 However, such interventions are challenged by several factors: extrinsic stains may recur rapidly if causal habits persist and may become progressively more tenacious with pellicle maturation; intrinsic stains are inherently more resistant to superficial prophylaxis and may demand invasive chemical or restorative approaches. Furthermore, some whitening materials carry risks of enamel demineralization, dentin sensitivity or gingival irritation.^8,9

Current clinical and commercial approaches for the management of dental discoloration primarily rely on oxidative bleaching agents, predominantly hydrogen peroxide (H₂O₂) and its derivative carbamide peroxide (CP), which remain the cornerstone of both professional and home-based whitening systems.^10,11 In professional settings, in-office bleaching typically employs high-concentration hydrogen peroxide formulations (ranging from 25% to 40%) often activated by light or heat, whereas take-home regimens incorporate lower concentrations of carbamide peroxide (10–16%), which decomposes to release hydrogen peroxide during application.^12,13 Extensive clinical data confirm that these agents yield significant and quantifiable improvements in tooth coloration over relatively brief treatment periods, thereby substantiating their therapeutic efficacy.^14,15 For instance, randomized clinical evaluations have demonstrated that 10% and 16% CP gels achieve comparable bleaching efficacy following three weeks of nightly application, leading to widespread adoption in aesthetic dentistry.¹⁶ Nevertheless, despite their bleaching effectiveness, significant biological and structural safety concerns persist.¹⁷ Both in vitro and in vivo investigations have revealed that hydrogen peroxide at concentrations exceeding 10% can induce microstructural alterations in enamel, such as interprismatic dissolution, reduced microhardness, and increased surface porosity – effects that compromise enamel’s mineral integrity and resistance to wear.¹⁸ Extended or repeated exposure to concentrated peroxide formulations may also elicit adverse pulpal and soft-tissue responses, including transient pulpitis, mucosal burns, and gingival irritation, with potential exacerbation under improper technique or excessive contact time.^19,20 Furthermore, over-the-counter whitening products – strips, gels, and mouth trays – often lack professional supervision and standardization in peroxide dosage, leading to variable and sometimes unpredictable clinical outcomes.²¹ Systematic reviews have also highlighted the paucity of long-term data on relapse rates, durability of color stability, and cumulative risks associated with chronic peroxide exposure.²² Therefore, although peroxide-based bleaching systems continue to serve as the clinical standard for managing extrinsic tooth discoloration, the intersection of their proven efficacy with concerns regarding chemical aggressiveness and patient safety highlights the imperative for biocompatible, evidence-based alternatives that can deliver equivalent esthetic benefits without jeopardizing the integrity of hard or soft oral tissues.

Enzyme-based whitening technologies have emerged as an important adjunct in modern oral-care formulations, leveraging catalytic systems that target and degrade the organic matrices responsible for pellicle formation, stain retention, and biofilm maturation.²³ Proteolytic enzymes such as papain and bromelain have been incorporated into dentifrices and rinses for their ability to cleave peptide-rich pellicle substrates, thereby reducing the binding affinity of chromogenic compounds to enamel and enhancing stain removal without relying on oxidative bleaching pathways.²⁴ Similarly, carbohydrate-degrading enzymes, most notably glucose oxidase and amyloglucosidase, have been used in enzymatic oral-care systems to generate low, sustained levels of hydrogen peroxide in situ, contributing to mild whitening and antimicrobial activity while minimizing the high-concentration peroxide exposure typically associated with sensitivity.²⁵ Lysozyme, lactoperoxidase, and related salivary-mimetic enzymes further contribute by disrupting bacterial cell walls, inhibiting plaque accumulation, and enhancing the host’s natural oral-defense pathways.²⁶ Collectively, these enzymatic mechanisms can reduce pellicle thickness, interfere with early biofilm formation, and decrease the adherence of dietary chromogens, ultimately improving both whitening outcomes and overall oral health.²⁷ Clinical and laboratory studies have shown that enzyme-enriched formulations may lower plaque indices, reduce gingival inflammation, limit halitosis-causing volatile sulfur compounds, and diminish the recurrence of extrinsic stains compared with non-enzymatic rinses and pastes.^28–30 These benefits highlight the growing relevance of enzyme-based oral-care products as biocompatible alternatives capable of supporting whitening goals while reinforcing biological homeostasis within the oral cavity.

Beyond their capacity to disrupt pellicle-associated chromogenic deposits, enzymatic systems used in oral-care formulations have demonstrated a broad spectrum of additional biological effects that warrant continued investigation. Enzymes such as lactoperoxidase, lysozyme, and glucose oxidase participate in antimicrobial pathways that suppress pathogenic bacterial loads, reduce volatile sulfur compound production, and support a more balanced oral microbiome, contributing to improvements in gingival health and reductions in plaque accumulation.^29,31,32 Proteolytic enzymes may further aid in degrading extracellular polymeric substances within established biofilms, potentially enhancing mechanical plaque removal and limiting biofilm resilience.³³ Enzyme-enriched rinses have also been associated with mitigation of halitosis, modulation of local inflammatory mediators, and reinforcement of the natural salivary defense system, suggesting therapeutic value beyond esthetic enhancement.^34,35 Given this expanding evidence base, evaluating enzyme-containing mouthwash formulations not only for their whitening potential but also for their broader functional performance has become clinically relevant.

The primary objective of the present study was to determine the efficacy of GuruNanda™ enzyme-enriched mouthwash formulations, one with 6-month-aged enzyme and the other with fresh-enzyme, in reducing extrinsic tooth stains and improving the shade of the tooth, comparing them with a placebo mouthwash and a commercially available comparator whitening mouthwash. Our null hypothesis is that the stain removing efficacy of the GuruNanda™ enzyme-enhanced whitening mouthwashes will not be significantly different from those of a placebo and a commercially available comparator whitening mouthwash.

Materials and Methods

Sample Preparation

Following the approval (TR202500000010) by the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee (Animal Welfare Assurance Number: A3345-01), freshly extracted bovine teeth were procured from an abattoir (Animal Technologies, Tyler, TX, USA; Lot# 8-210519). The care and use of animal organ (teeth) in this study was in accordance with the National Research Council (NRC) Publication, as revised in 2011, “Guide for the Care and Use of Laboratory Animals,” and other applicable Federal regulations. A total of 80 bovine incisor teeth without any anomalies and prior staining were selected for this study. Using a water-cooled diamond wire saw (WELLs Walter Ebner Le Locle) Diamond Wire Saws SA, Le Locle, Switzerland), a tooth block (approximately 12 mm length × 10 mm width × 2 mm thick) was produced from the facial surface of each tooth. Each tooth block was then embedded in auto-polymerizing polymethyl methacrylic (PMMA) resin in a custom-made plastic mold customized to fit into the tooth-brushing machine, with the facial surface facing upwards and the dentin surface embedded into the PMMA resin (Figure 1). All produced PMMA base with the embedded tooth block were stored in distilled water at room temperature for 24 hours until use.

Figure 1 (A) Tooth sample embedded in auto-polymerizing polymethyl methacrylic resin in a custom-made plastic mold customized to fit into the tooth-brushing machine. (B) A V8 automated toothbrushing machine.

Baseline (Original) Tooth Shade Measurement

A single examiner performed the digital tooth color assessments in a controlled (color-corrected, 5000 K) lighting room with no windows. The original shade (color) of each tooth was measured digitally, using a calibrated Vita EasyShade Advance 4.0 Spectrophotometer (VITA North America, Brea, CA 92821) (Figure 2), which captured the teeth shade scores according to the VITA^® 3D master shade guide, which is an internationally recognized shade guide that integrates 16 levels of VITA Classical shade guide ranging from the whitest A1 (1) to the darkest D4 (16). This classification is based on the recommendations of the American Dental Association, which formed the basis for the new definition of the lightness levels. During measurement, the device automatically generates the shade score the VITA Classical tooth shade (A1-D4) with the corresponding numerical values (1–16). This device works best on a flat surface, so the measurements with this device were performed on the flattest portion of the tooth surface, with the probe tip placed perpendicular and flush to the tooth surface. The probe tip was placed on this position on every measurement occasion to standardize the measurement position.

Figure 2 Vita EasyShade Advance 4.0 Spectrophotometer.

Tooth Staining and Post-Staining Tooth Shade Measurement

Following teeth color measurement, the enamel surfaces of the 80 prepared tooth blocks were stained as follows. The enamel surface of each tooth block was lightly etched with 32% phosphoric acid etchant (Bisco Inc., Schaumburg, IL, USA) applied for 5 seconds to facilitate stain uptake. After etching, the surface of the tooth blocks was subjected to a cyclic protocol of acquired salivary pellicle formation and stain immersions. Acquired salivary pellicle was formed on the surface of each block by 1-hour incubation in a pasteurized human whole saliva in an incubator at 37°C on a platform laboratory rocker (Labnet Rocker 35 EZ, The Lab Depot, CA, USA) at a 120 rpm. The saliva was voluntarily donated by two members of the research team after obtaining a written informed consent from each donor. The donors contributed the saliva by chewing on a gum base and spitting intermittently into plastic vials partially buried in ice blocks. The pellicle formation was followed by staining by a similar protocol of 2-hour incubation of the tooth blocks in a stain solution containing tea, coffee, red wine, turmeric and tobacco, under same conditions as the pellicle formation. This cyclic regimen was performed for 12 hours with final storage in stain solution overnight for the rest of the 24 hours. Varied levels of stain intensity were achieved among the tooth blocks. Following staining the shade of the tooth blocks were re-measured to record the post-staining shade scores as described for the baseline (original) tooth shade measurement.

Study Grouping

The 80 stained tooth blocks were randomly assigned to one of the following four experimental groups (20 blocks/group) shown in Table 1: Gurunanda™ 6-month-aged Enzyme mouthwash (6M-GN-ENZ), GuruNanda™ Fresh-Enzyme mouthwash (GN-ENZ), Comparator Whitening mouthwash, and Placebo mouthwash. The randomization was based on the post-stain shade score of the blocks, such that the mean values of the shade score for the four groups did not differ statistically significantly.

Table 1 Compositions of Experimental Mouthwashes

Treatment Procedure

All four mouthwashes were applied with the same regimen of swishing with 20 mL of the mouthwash for 2 minutes on each occasion and twice a day in the morning and evening, in accordance with the manufacturers’ instruction. In the present in vitro study, swishing was simulated with a laboratory Shaker with rotary, elliptical, and reciprocating motion (Labnet Rocker 35 EZ, The Lab Depot, CA, USA) at 200 rpm. Each episode of 2-minute swishing is followed by 2 minutes of toothbrushing with de-ionized distilled water (DDW) using a V8 brushing machine (Sabri Dental Enterprises, Inc., Downers Grove, Illinois, USA), which enabled a standardized brushing condition (Figure 1).³⁶ This cyclic regimen of alternating swishing mouthwash and brushing was sequentially performed to simulate 1, 4, and 8 weeks of mouthwash use. At each point, the post-treatment tooth shade was measured.

Sample Size Calculation

Prior to the study, a power analysis was conducted using G*Power statistical software 3.1.9.7 for Windows (Heinrich-Heine-Universität, Düsseldorf, North Rhine-Westphalia, Germany)³⁷ to determine the appropriate sample size required to detect statistical significance between the changes in stain reduction (baseline to endpoint) in the enzyme-enriched Gurunanda™ mouthwashes and the placebo mouthwash was determined. Based on effect sizes reported in previous studies,³⁸ it was calculated that a sample size of 80 tooth blocks (20 per group) will be sufficient, assuming normal distribution, and a single comparison, detecting a difference in stain removal (change in stain scores between baseline to endpoint) of 1 point with a standard deviation of 1.5, type 1 error of 5% and power of 80%.

Statistical Analysis

Statistical analyses were conducted in SPSS v28. An alpha level of 0.05 was adopted to define statistical significance for all analyses. Preliminary analyses were conducted to explore the dataset and to check for assumptions violations. Specific assumptions tests including tests for independence, normality and extreme outliers were performed. The normality assumption was tested using histogram, Q-Q plot and the Shapiro–Wilk’s test from the tests of normality table A one-way between-products ANOVA was conducted to compare the stain intensity of the product groups after staining. Due to the violation of the assumption of normality and homogeneity of variances, a non-parametric equivalent of ANOVA, the Kruskal–Wallis test, was employed to compare the stain intensity of the groups at each measurement time point. Post-hoc comparisons using Dunn’s method with a Bonferroni correction for multiple tests were used to compare the mean rank of stain intensity among the product groups. Repeated measures ANOVA with Greenhouse-Geisser correction were conducted to compare the average stain intensity reduction from after-staining to 8 weeks after the application of each product.

Results

Following staining, ANOVA indicated there was no significant difference [F(3, 47) = 0.06, p = 0.98] in the mean stain intensity among the product groups; 6M-GN-ENZ (8.90 ± 4.33), GN-ENZ (8.33 ± 4.12), Comparator 8.67 ± 4.20), Placebo (8.29 ± 4.25) as shown in Figure 3. After simulated one week of exposure to oral rinses, the Kruskal–Wallis test indicated that there was a significant difference [χ² (3, N = 51) = 9.37, p = 0.025] in stain intensity across the four products. The stain intensity mean rank for the products was 6M-GN-ENZ (20.5), GN-ENZ (25.42), Comparator (21.33), and Placebo (35.43). Post-hoc comparisons using Dunn’s method with a Bonferroni correction for multiple tests indicated that the mean rank of stain intensity for placebo was significantly higher (p < 0.05) than that of 6M-GN-ENZ (p = 0.011), GN-ENZ (p = 0.012), and Comparator (p = 0.007). Similar trend was observed after 4 weeks, there was a significant difference [χ² (3, N = 51) = 13.18, p = 0.004] in stain intensity across the four products. The stain intensity mean rank for products was 6M-GN-ENZ (16.7), GN-ENZ (23.83), Comparator (24.37), and Placebo (36.25). Also, post-hoc comparisons using Dunn’s method with a Bonferroni correction indicated that the mean rank of stain intensity for placebo was significantly higher than that of 6M-GN-ENZ (p < 0.001), GN-ENZ (p = 0.02), and Comparator (p = 0.019). At 8 weeks, there was also a significant difference [χ² (3, N = 51) = 15.53, p = 0.002] in stain intensity across the four products, with the stain intensity mean rank for products being 6M-GN-ENZ (14.8), GN-ENZ (22.25), Comparator (27.8), and Placebo (35.29). The mean rank of stain intensity for 6M-GN-ENZ was significantly lower than that of placebo (p < 0.001), and Comparator (p = 0.019). GN-ENZ also has a significantly lower mean rank than placebo (p = 0.015).

Figure 3 Comparison of the Stain Intensity of products at each measurement time point after staining.

When the mean stain intensity reduction from after-staining through 8 weeks of oral rinse treatment was compared for each product using a repeated measures ANOVA with Greenhouse-Geisser correction (Table 2 and Figure 3), the result determined that for 6M-GN-ENZ, the mean stain intensity reduction differed significantly between the time points F(1.12, 10.09) = 16.93, p = 0.002). Post hoc analysis with a Bonferroni adjustment revealed that the average stain intensity was statistically significantly reduced from after-staining to after 1 week [4.2 (95% CI [0.49, 7.91])], from after-staining to 4 weeks [5.0 (95% CI [0.93, 9.07])], from after-staining to 8 weeks [5.3 (95% CI [1.32, 9.28])] and from 1 week to 8 weeks [1.1 (95% CI [0.19, 0.17])], but not from 1 week to 4 weeks and from 4 weeks to 8 weeks (p > 0.05). Similar analysis for GN-ENZ also determined that the mean stain intensity differed significantly between the time points (F(1.09, 12.0) = 13.05, p = 0.003), while Post hoc analysis with a Bonferroni adjustment indicated that the mean stain intensity was statistically significantly reduced from after-staining to 1 week [2.92 (95% CI [1.22, 4.62])], from after-staining to 4 weeks [4.0 (95% CI [1.64, 6.32])], from after-staining to 8 weeks [4.3 (95% CI [1.69, 6.81])] and from 1 week to 4 weeks [1.1 (95% CI [0.25, 1.92])], one week to 8 weeks [1.3 (95% CI [0.27, 2.39])] but not from 4 weeks to 8 weeks (p > 0.05). For Comparator, the mean stain intensity differed significantly between the time points F(1.54, 21.53) = 16.01, p < 0.001). Post hoc analysis with a Bonferroni adjustment revealed that the average stain intensity was statistically significantly reduced from after-staining to 1 week [3.73 (95% CI [0.87, 6.6])], from after-staining to 4 Weeks [4.3 (95% CI [1.40, 7.26])], from after-staining to 8 weeks [4.3 (95% CI [1.40, 7.26])]. However, no other pair was significant at the alpha level of 0.05 (p > 0.05). However, similar analysis revealed that the mean stain intensity did not differ significantly between the time points (Table 2 and Figure 3).

Table 2 Descriptive Statistics of the Stain Intensity Score of the Product Groups Over Time

When comparing the percentage stain reduction achieved by the products after one week using Kruskal–Wallis test, there was a significant difference [χ² (3, N = 51) = 29.88, p < 0.001] in percentage stain intensity reduction across the four products (Figure 4). The percentage stain reduction mean rank for products was 6M-GN-ENZ (35.75), GN-ENZ (29.63), Comparator (32.93), and Placebo (8.5). Post-hoc comparisons using Dunn’s method with a Bonferroni correction for multiple tests indicated that the mean rank of percent reduction in stain intensity for Placebo was significantly higher than that of GN-ENZ (p < 0.001), 6M-GN-ENZ (p < 0.001), and Comparator (p < 0.001). After 4 weeks of rinse treatments, a significant difference [χ² (3, N = 51) = 15.81, p = 0.001] was observed in percentage stain intensity reduction across the four products (Figure 4). The percentage stain reduction mean rank for the products was 6M-GN-ENZ (35.25), GN-ENZ (28.17), Comparator (29.63), and Placebo (13.64). Post-hoc comparisons using Dunn’s method with a Bonferroni correction for multiple tests indicated that the mean rank of percentage reduction in stain intensity for Placebo was significantly higher than that of GN-ENZ (p = 0.01), 6M-GN-ENZ (p < 0.001), and Comparator (p = 0.003). At 8 weeks, a significant difference [χ² (3, N = 51) = 11.74, p = 0.008] in percent stain intensity reduction across the four products (Figure 4). The percent stain reduction mean rank was 6M-GN-ENZ (35.85), GN-ENZ (28.08), Comparator (27.1), and Placebo (16.0). Post-hoc comparisons using Dunn’s method with a Bonferroni correction for multiple tests indicated that the mean rank of percent reduction in stain intensity for Placebo was significantly higher than that of GN-ENZ (p = 0.033), 6M-GN-ENZ (p < 0.001), and Comparator (p = 0.038).

Figure 4 Comparison of the percentage reduction of stain intensity over time.

When repeated measures ANOVA were employed to compare the mean percentage stain reduction from 1 week to 8 weeks for 6M-GN-ENZ (Figure 4), the mean percentage reduction differed significantly (F(2, 18) = 11.23, p < 0.001) between the time points. The mean percentage reduction is statistically significantly lower for 1 week than for 4 weeks (−8.6 (95% CI [−15.71, −1.52])), and 8 weeks (−13.39 (95% CI [−19.68, −7.09]). There was no significant difference between 4 and 8 weeks (p < 0.05). For GN-ENZ (Figure 4), a repeated-measures ANOVA with Greenhouse-Geisser correction determined that the mean percentage reduction differed significantly between the time points (F(1.06, 11.65) = 8.83, p = 0.011). Post hoc analysis with a Bonferroni adjustment revealed that the average percentage reduction is statistically significantly lower for 1 week than for 4 weeks (−9.36 (95% CI [−16.11, −2.61])), and 8 weeks (−11.39 (95% CI [−19.61, −2.95])). There was no significant difference between 4 and 8 weeks (p > 0.05). However, for Comparator (Figure 4), the result showed that the average percentage reduction did not differ significantly between the time points (F(2, 28) = 1.28, p = 0.28), while for Placebo (Figure 4), the average percentage reduction differed significantly between the time points (F(2, 26) = 6.08, p = 0.007). Figure 5 shows the photographic images depicting the gradual stain removal by the application of each product.

Figure 5 Pictures depicting the gradual stain removal by the application of each product.

Discussion

Extrinsic tooth discoloration remains a prevalent esthetic challenge, arising from the continual accumulation of chromogenic agents from various sources such as diets, tobacco exposure, and pellicle-bound pigments that adhere strongly to enamel surfaces and biofilm matrices.^39–41 These stains negatively affect facial appearance, diminish social confidence, and have been shown to reduce oral health-related quality of life, prompting individuals to seek whitening interventions that are both effective and minimally invasive.⁴² Traditional peroxide-based whitening products can provide measurable shade improvement; however, their associated risks, including enamel sensitivity, surface roughening, gingival irritation, and inconsistent patient tolerance, limit their feasibility for long-term or routine use.^3,7,43

Recognizing the limitations of existing approaches, there is growing interest in natural, biocompatible alternatives such as enzyme-based oral rinses.^21,22 Enzyme-based mouthrinses have been widely studied for their effects on dental biofilm and gingivitis based on their established antimicrobial properties.^28,29 However, there has not been a study on their tooth stain removal potential. The present in vitro study was designed to evaluate the efficacy of the GuruNanda™ enzyme-enhanced, 6-month-aged and non-aged, mouthwash formulations in reducing extrinsic stains and improving tooth shade over an eight-week period.⁴⁴ This study employed controlled rinse applications and repeated shade measurements at baseline, 4, and 8 weeks to determine whether the GuruNanda formulations could provide an effective alternative to conventional whitening systems.⁴⁵ The result demonstrated that swishing with GuruNanda™ enzyme-enhanced mouthwashes effectively removed extrinsic stains on teeth surfaces at a comparable rate to Comparator mouthwash but significantly outperforming the placebo.

The present investigation utilized a standardized automatic tooth-brushing machine to ensure consistent and reproducible mechanical cleaning conditions across all tooth blocks.³⁷ Automated brushing systems are being used in dental research labs because they apply a uniform brushing force, stroke path, and frequency, thereby eliminating operator variability that can significantly affect stain removal outcomes.⁴⁶ The programmed mechanical action of these devices allows precise control of parameters such as brushing load, brush angulation, and stroke rate, enabling the isolation of the chemical effects of the oral rinse from variability in manual tooth-brushing technique.⁴⁵ This level of methodological standardization enhances the internal validity of the study and ensures that any observed differences in whitening efficacy arise from the mouthrinse formulations rather than from variations in brushing technique.⁴⁷ Within the context of this study, the automatic brushing machine ensured a consistent mechanical input across all groups, allowing for a reliable comparison of the enzyme-enhanced mouthwashes with the commercial comparator whitening rinse and the placebo.⁴⁸

To ensure standardization and eliminate environmental lighting bias, all shade measurements were performed in a controlled 5000 K color-corrected lighting room without windows, as recommended for objective dental color evaluation.⁴⁹ The shade of each tooth was assessed digitally and objectively using a calibrated VITA EasyShade Advance 4.0 spectrophotometer (VITA North America, Brea, CA 92821), a device widely validated for its high accuracy, repeatability, and clinical reliability in both clinical and in-vitro tooth color assessment.⁵⁰ The instrument captured shade values according to the VITA 3D-Master system, an internationally standardized shade guide incorporating 16 VITA Classical shades, ranging from A1 (rank 1) to D4 (rank 16).⁴⁴ These shade classifications align with guidelines established by the American Dental Association, which emphasize lightness-based categorization to improve reproducibility and standardization in whitening research.⁵¹ During each measurement, the spectrophotometer automatically generated the corresponding VITA Classical shade and numerical value (eg, D4 = 16). This system ensured comprehensive documentation of color change and facilitated precise longitudinal comparison across all evaluation time points.

The findings of this study demonstrated a consistent and progressive reduction in stain intensity among the tested products over the 8-week evaluation period, consistent with prior evidence that whitening agents vary in their performance over time under controlled conditions.⁵² Although all groups demonstrated comparable stain intensity immediately following the initial staining procedure, confirming equivalent baseline conditions, variation in treatment effects became evident as early as week 1, a trend consistent with findings from prior comparative stain-removal studies.^53,54 Across all subsequent time points, 1, 4, and 8 weeks, both GuruNanda formulations and the commercial comparator exhibited significantly higher percentage of stain reduction over time compared to the placebo. This effect can be attributed to the report of previous literature that enzyme-based or surfactant-containing rinses can reduce adherence of hydrophobic chromogens and plaque-associated pigments.⁵⁵ Notably, all three active oral rinses consistently outperformed the placebo, largely due to the presence of enzymatic and surfactant-mediated whitening mechanisms absent in the placebo formulation.

Each active rinse contained glucose oxidase, an enzyme capable of generating low-level hydrogen peroxide through glucose oxidation, thereby facilitating oxidative cleavage of chromogenic molecules adhering to the pellicle.⁵⁶ Enzymatic whitening systems incorporating glucose oxidase are known to support mild oxidative stain breakdown through reactive oxygen species generation.⁵⁷ Furthermore, each of the active rinses contains papain, a cysteine protease capable of hydrolyzing proteinaceous pellicle structures and loosening biofilm matrices, thereby facilitating chromogen release.⁵⁸ In contrast, the placebo consisted only of water, glycerin, polysorbate-20, sodium benzoate, menthol, and potassium sorbate—none of which possess oxidizing, proteolytic, or stain-solubilizing capabilities. Consequently, stain removal in the placebo group depends solely on mechanical brushing forces rather than biochemical stain disruption, and as such, the placebo group demonstrated the least stain reduction, reinforcing the well-documented limitations of mechanical brushing in removing chemically bound extrinsic pigmentations.⁵⁹

It was observed that despite the three active oral rinses containing similar active ingredients, the 6-month-aged-enzyme oral rinse (Gurunanda™ 6-Month-Aged Enzyme mouthwash) demonstrated the greatest reduction in stain compared to other two active oral rinses, suggesting that extended enzymatic maturation may enhance the surfactant-mediated or lipid-mediated disruption of stain-to-enamel interactions, consistent with enzymatic oral-care research.^60,61 The progressively lower mean ranks observed for this 6-month-aged-enzyme oral rinse indicate a sustained and superior capacity for mitigating stain deposition compared with both placebo and the comparator whitening rinse.^29,62 Enzymes such as glucose oxidase and papain can undergo structural stabilization or increased catalytic efficiency when stored in aqueous–glycerol systems, as glycerin acts as a protein-stabilizing polyol that preserves enzymatic activity over time.^63,64 Aging also promotes more complete hydration and improved conformational flexibility, enhancing enzyme–substrate accessibility and catalytic turnover on chromogenic proteinaceous stain components.⁶⁵ Similar patterns have been observed in oral-care enzymatic formulations where prolonged stabilization increases peroxide-generation and proteolytic performance, improving stain-disruption capacity.⁶⁶ Therefore, the enhanced stain-reduction seen with the aged-enzyme-based rinse aligns with known biochemical behavior of stabilized enzyme systems and supports the hypothesis that enzyme maturation contributes to its superior whitening effect. Collectively, these results reinforce the concept that effective whitening requires targeted chemical mechanisms rather than mechanical or dilute oxidizing approaches alone.^67,68

Interestingly, it was observed that the stain removal effect of the Comparator oral rinse started earlier than that of GuruNanda™ fresh-enzyme formulation (Gurunanda™ Fresh-Enzyme mouthwash). The percentage stain reduction achieved with the Comparator™ oral rinse was higher than that of the GuruNanda™ fresh-enzyme formulation at one-week and 4-week time points, but by 8-week, both rinses demonstrated similar levels of stain reduction (Figure 4). This can be attributed to the polyvinylpyrrolidone (PVP) and D-limonene content of the Comparator oral rinse. The PVP is a polymer capable of binding and sequestering hydrophobic stain molecules,⁶⁹ while D-limonene is a terpene solvent effective at dissolving lipid-associated chromogens.⁷⁰ These components, which are absent in the GuruNanda™ fresh-enzyme formulation and placebo, enable more rapid early-stage stain solubilization.

It was not surprising that at any measurement time point, the stain reduction of the placebo oral rinse was significantly lower than that of the three active oral rinses and obviously represents the mechanical stain removal of the toothbrushing. This outcome is consistent with established evidence demonstrating that mechanical brushing alone is insufficient to remove chemically adherent chromogens embedded within the acquired pellicle, particularly those derived from polyphenols, tannins, and dietary pigments, which bind strongly to enamel surface proteins.^71,72 Consequently, the markedly lower stain reduction observed in the placebo arm underscores the necessity of active ingredients, such as enzymes, oxidative systems, or stain-solubilizing solvents – to meaningfully disrupt extrinsic pigmentation beyond what brushing can achieve. Although manual and powered toothbrushing remain fundamental to daily oral hygiene, both are limited in their ability to remove established extrinsic stains or produce clinically perceptible whitening.^73–76 Mechanical brushing predominantly disrupts plaque biofilm but lacks the chemical action necessary to detach chromogenic molecules incorporated into the pellicle or superficial enamel microstructures.⁷⁷ Comparative investigations consistently show that manual and powered brushes yield only modest surface stain reduction and do not generate significant improvements in overall tooth shade.^78,79 Even advanced oscillating–rotating and sonic technologies provide minimal whitening benefit, as the mechanical forces applied are insufficient to dislodge chemically bound pigments without risking enamel abrasion.⁷⁷ Collectively, these findings indicate that toothbrushing alone, regardless of device type, cannot achieve substantive whitening outcomes and must be supplemented with chemotherapeutic interventions to effectively manage extrinsic discoloration.⁸⁰

Although the design of this in vitro study was adequate for its objective, there are certain limitations that are worth mentioning. The findings of this investigation must be interpreted within the constraints of its in-vitro experimental design. Unlike the oral cavity, the laboratory environment does not reproduce essential biological variables such as salivary flow, enzymatic activity, pH fluctuations, or pellicle renewal – factors known to influence stain accumulation, pellicle composition, and whitening efficacy.⁸¹ The absence of microbial colonization further eliminates biofilm-driven modulation of stain adsorption and retention, which plays a significant role in vivo.⁸² Additionally, bovine enamel, although commonly used as a surrogate substrate, differs from human enamel in mineral composition and surface ultrastructure, potentially altering stain uptake and removal behavior.⁸³ The standardized brushing delivered by the automatic brushing machine also removes human variability in brushing force, duration, and technique, limiting ecological validity relative to real-world oral hygiene practices.⁸⁴ Moreover, the controlled application times and the absence of dietary staining cycles do not reflect the dynamic, repetitive exposure to chromogenic beverages and foods that occur clinically.⁸⁵ Collectively, these factors restrict the generalizability of the results and highlight the need for well-controlled in-vivo clinical trials that incorporate natural oral conditions to fully validate the whitening effects of enzyme-based rinses.⁸⁶ For this reason, the authors suggest that in future further large multicenter clinical trial should be conducted to confirm the outcomes of the present in vitro study.

Conclusion

This study demonstrated that the GuruNanda™ enzyme-enriched mouthwashes effectively removed the extrinsic stains on teeth surfaces at a comparable rate to the marketed comparator mouthwash, but significantly more effective than the placebo mouthwash. Furthermore, the study showed that the 6-month-aged GuruNanda™ enzyme-enriched mouthwash removed teeth stains at a faster rate than the freshly made GuruNanda enzyme-enriched mouthwash and Comparator mouthwash.