Oral health plays a vital role in overall well-being and is influenced by various factors, including genetic predisposition, environmental factors, and oral hygiene practices (1). Dental plaque is the primary etiological factor for several oral diseases, such as dental caries, gingivitis, and periodontitis. It is defined as a structurally complex microbial biofilm that adheres to tooth surfaces and restorations, which cannot be removed by simple rinsing with water (2,3). If left undisturbed, plaque matures and leads to gingival inflammation, eventually progressing to periodontitis, which causes irreversible damage to periodontal structures (4).

Plaque control is essential in preventing gingival and periodontal diseases. Mechanical plaque control methods, including tooth brushing and flossing, are the most commonly recommended approaches. However, these methods require proper technique, manual dexterity, and patient compliance, which may be challenging for individuals with physical disabilities, malpositioned teeth, or geriatric patients (5,6). To overcome these limitations, chemical plaque control agents, such as mouthwashes, have gained popularity as adjuncts to mechanical oral hygiene measures (7).

Chlorhexidine (CHX) is considered the gold standard among antiplaque agents due to its broad-spectrum antimicrobial properties against Gram-positive and Gram-negative bacteria, aerobes, anaerobes, yeast, and fungi (8,9). Its effectiveness is attributed to its cationic nature, which allows it to bind to bacterial cell membranes, leading to bacteriostatic or bactericidal effects depending on its concentration (10). However, prolonged use of CHX is associated with various side effects, including tooth discoloration, altered taste perception, oral mucosal irritation, and increased supragingival calculus formation (11,12). These adverse effects have led to an increased interest in natural alternatives, particularly herbal mouthwashes.

Ayurvedic medicine has been used for centuries in oral health care, with various plant extracts demonstrating antimicrobial and anti-inflammatory properties. Herbal mouthwashes, such as HiOra (Himalaya Drug Company, India), contain a combination of medicinal plant extracts, including Salvadora persica (Miswak), Terminalia bellerica (Bibhitaki), Piper betel (Nagavalli), and Gaultheria fragrantissima (Gandhapura taila), among others (13). These natural ingredients exhibit antibacterial, antifungal, analgesic, and antioxidant properties, making them a potential alternative to CHX for maintaining oral hygiene (14).

Although herbal mouthwashes have gained attention for their potential benefits, limited clinical evidence is available to establish their efficacy compared to CHX. Therefore, this randomized crossover clinical trial was conducted to compare and evaluate the effectiveness of Ayurvedic (HiOra) and allopathic (0.2% CHX gluconate) mouthwashes in reducing plaque accumulation and gingival inflammation among young adults.

Study Design and Ethical Approval

This study was designed as a randomized controlled crossover clinical trial to compare and assess the efficacy of allopathic (0.2% Chlorhexidine gluconate) and Ayurvedic (HiOra) mouth rinses in reducing plaque accumulation and gingival inflammation. Ethical clearance for the study was obtained from the Institutional Ethical Committee of I.T.S Dental College, Hospital, and Research Centre, Greater Noida, Uttar Pradesh, India. Written informed consent was obtained from all study participants before enrollment in the study.

Study Population and Sampling Method

The study was conducted among students aged 18–25 years from educational institutions in Greater Noida. A convenience sampling method was used to select participants based on the inclusion and exclusion criteria. (Figure 1)

Inclusion and Exclusion Criteria

Inclusion Criteria:

• Participants aged 18–25 years.
• Presence of a minimum of 20 teeth.
• Gingival index score between 1.1 and 3.0.
• Plaque index score greater than 1.

Exclusion Criteria:

• History of smoking.
• Presence of systemic diseases.
• History of antibiotic therapy or periodontal treatment within the past three months.

Study Protocol

A pilot study was conducted from May to July 2017 on 10 subjects (5 in each group) to determine the feasibility of the study. These participants were excluded from the final study. The main trial was conducted from October 2017 to April 2018.

The study followed a randomized crossover design with a 4-week washout period. Participants were randomly allocated to two groups using a coin toss method:

• Group A: Chlorhexidine mouthwash (0.2% CHX gluconate).
• Group B: Ayurvedic mouthwash (HiOra).

Each participant used the assigned mouthwash for three weeks, followed by a four-week washout period, after which they switched to the other mouthwash for another three weeks.

Blinding and Dispensing of Mouthwashes

To ensure blinding, both mouthwashes were dispensed in identical-looking, coded plastic bottles by a third party (pharmacist). Participants and the investigator were unaware of the group allocations until the study concluded. Each participant received 420 mL of mouthwash to be used over three weeks, with instructions to rinse 10 mL twice daily for one minute, 30 minutes after brushing. Compliance was assessed by checking the remaining volume of the mouthwash at follow-up visits.

Clinical Examination and Data Collection

Clinical assessments were performed at baseline, after three weeks, after the washout period, and after three weeks of crossover.

Plaque Assessment
Plaque levels were evaluated using the Turesky et al. modification of the Quigley-Hein Plaque Index (1970):

• A disclosing agent was applied to all tooth surfaces.
• Plaque accumulation was scored on a scale of 0 to 5, where higher scores indicated more plaque.
• The total score was divided by the number of surfaces examined to obtain an individual plaque index score.

 Gingival Inflammation Assessment
Gingival status was assessed using the Loe and Silness Gingival Index (1963):

• Gingiva was examined for redness, swelling, and bleeding on probing.
• Scoring was done from 0 (normal) to 3 (severe inflammation with spontaneous bleeding).
• The average score for each participant was calculated.

 Microbiological Analysis
Plaque samples were collected before and after the intervention for microbiological evaluation.

• Colony Forming Unit (CFU) Assessment:
• 1 mg of supragingival plaque was collected using a sterile curette.
• The sample was transferred into a sterile vial containing 1 mL of phosphate-buffered saline (PBS).
• The plaque suspension was diluted (10⁻¹ to 10⁻⁵), and 50 µL of each dilution was inoculated on Muller-Hinton agar plates.
• After 48 hours of incubation at 37°C, bacterial colonies were counted and expressed as CFU/mL (Figure 2,3,4)

Figure 1: Armamenterium Used

Figure 2: Swabbing of culture plate

Figure 3: Mean Colony Forming Unit at Start of Study

Figure 4: Mean Colony Forming Unit after 3 weeks

Zone of Inhibition Measurement:

• Bacteria were streaked on Muller-Hinton agar plates using a sterile cotton swab.
• The plates were divided into two sections labeled C (Chlorhexidine) and H (HiOra).
• Filter paper discs soaked in the respective mouthwash were placed on the agar.
• After incubation at 37°C for 24 hours, the diameter of the inhibition zone was measured using a divider and ruler.

Statistical Analysis

Data were compiled and analyzed using SPSS version 21.

This randomized crossover clinical trial evaluated and compared the efficacy of Ayurvedic (HiOra) and allopathic (0.2% Chlorhexidine gluconate) mouth rinses in reducing plaque accumulation and gingival inflammation among young adults.

Colony Forming Unit (CFU) Reduction

The mean CFU values were significantly reduced after three weeks of intervention in both groups. At the start of the study, the mean CFU count was 18.93 ± 4.24 in the Chlorhexidine group and 19.78 ± 4.16 in the HiOra group. After three weeks, the CFU count dropped to 2.70 ± 1.03 in the Chlorhexidine group and 2.68 ± 0.87 in the HiOra group, with a percentage reduction of 85.73% ± 4.62% and 86.41% ± 3.50%, respectively. The intragroup comparison showed a statistically significant difference (p=0.001), indicating the effectiveness of both mouth rinses in reducing microbial load. However, the intergroup comparison between the two groups showed no statistically significant difference (p=0.941), suggesting similar antimicrobial efficacy (Table 1).

Gingival Index Score Reduction

At baseline, the mean gingival index score was 1.92 ± 0.25 in the Chlorhexidine group and 1.94 ± 0.26 in the HiOra group. After three weeks of mouthwash use, the scores reduced to 1.07 ± 0.22 in the Chlorhexidine group and 1.08 ± 0.29 in the HiOra  group. The percentage reduction was 44.64% ± 6.33% for Chlorhexidine and 45.11% ± 9.29% for HiOra. The reduction in gingival inflammation was statistically significant within both groups (p=0.001). However, intergroup comparison at three weeks showed no statistically significant difference (p=0.892), indicating that both mouthwashes had comparable effects on gingival health (Table 2).

 Plaque Index Score Reduction

The baseline plaque index score was 3.26 ± 0.37 for the Chlorhexidine group and 3.30 ± 0.31 for the HiOra group. After three weeks, the plaque index scores reduced to 2.18 ± 0.30 in the Chlorhexidine group and 2.18 ± 0.23 in the HiOra group. The percentage reduction in plaque accumulation was 33.23% ± 3.35% for Chlorhexidine and 33.78% ± 3.22% for HiOra. The intragroup comparisons showed significant reductions in plaque scores within both groups (p=0.001). However, the intergroup comparison did not show any statistically significant difference (p=0.895), confirming that both mouthwashes had similar antiplaque efficacy (Table 3).

Table 1: Intragroup and Intergroup Comparison of Mean CFU at Baseline and After 3 Weeks

(*Paired t-test, p≤0.05 significant)

Table 2: Intragroup and Intergroup Comparison of Mean Gingival Index Score at Baseline and After 3 Weeks

(*Paired t-test, p≤0.05 significant)

Table 3: Intragroup and Intergroup Comparison of Mean Plaque Index Score at Baseline and After 3 Weeks

(*Paired t-test, p≤0.05 significant)

• Both mouthwashes significantly reduced CFU, plaque index, and gingival index after three weeks of use (p=0.001).
• There was no statistically significant difference between Chlorhexidine and HiOra in terms of antimicrobial efficacy (p=0.941), plaque reduction (p=0.895), and gingival inflammation reduction (p=0.892).

The findings suggest that Ayurvedic mouthwash (HiOra) is equally effective as the gold-standard allopathic mouthwash (Chlorhexidine) in maintaining oral hygiene

The present randomized crossover clinical trial compared the antiplaque and anti-gingivitis efficacy of allopathic (0.2% Chlorhexidine gluconate) and Ayurvedic (HiOra) mouthwashes among young adults. The results demonstrated that both mouthwashes significantly reduced plaque accumulation, gingival inflammation, and microbial load, with no statistically significant difference between the two groups. These findings align with previous studies that have evaluated herbal mouthwashes as alternatives to Chlorhexidine for maintaining oral hygiene (1-3).

Plaque accumulation is a major risk factor for gingival and periodontal diseases, making effective plaque control essential for oral health (4). In the current study, both mouthwashes led to significant plaque reduction after three weeks (Chlorhexidine: 33.23% ± 3.35%, HiOra: 33.78% ± 3.22%, p=0.895). These results are consistent with studies conducted by Narayan et al. (5) and Shetty et al. (6), which found that herbal mouthwashes, including those containing Piper betel and Salvadora persica, were as effective as Chlorhexidine in reducing plaque.

Herbal mouthwashes achieve plaque control primarily through their antimicrobial and anti-inflammatory properties. Salvadora persica, a key ingredient in HiOra, has been reported to possess strong antibacterial and plaque-inhibiting properties similar to Chlorhexidine (7). Additionally, the presence of Triphala and Gaultheria fragrantissima in HiOra may contribute to its antiplaque activity by reducing bacterial adhesion to tooth surfaces (8).

Gingival inflammation is an early indicator of periodontal disease and is closely associated with plaque accumulation (9). In this study, both mouthwashes significantly reduced gingival index scores (Chlorhexidine: 44.64% ± 6.33%, HiOra: 45.11% ± 9.29%, p=0.892), demonstrating their effectiveness in controlling gingival inflammation. These findings align with studies conducted by Gomes et al. (10) and Priya et al. (11), which showed no significant difference in gingival inflammation reduction between Chlorhexidine and herbal mouthwashes.

The anti-inflammatory effects of HiOra may be attributed to its natural components, including Piper betel, which has been shown to reduce gingival inflammation by modulating pro-inflammatory cytokines (12). Furthermore, the presence of Bibhitaki (Terminalia bellerica) enhances antioxidant activity, reducing oxidative stress and promoting gingival healing (13). This mechanism may explain the comparable efficacy of HiOra to Chlorhexidine in controlling gingival inflammation.

The study also assessed microbial load using colony-forming unit (CFU) analysis. Both mouthwashes significantly reduced CFU counts after three weeks (Chlorhexidine: 85.73% ± 4.62%, HiOra: 86.41% ± 3.50%, p=0.941). These results are consistent with findings by Bagchi et al. (14), who reported that herbal mouthwashes exhibited antimicrobial activity comparable to Chlorhexidine against Streptococcus mutans and Porphyromonas gingivalis.

The antimicrobial effect of Chlorhexidine is well established, primarily due to its cationic nature, which disrupts bacterial cell membranes and inhibits plaque biofilm formation (15). Similarly, the active ingredients in HiOra, such as Syzygium aromaticum (clove) and Cinnamomum zeylanicum (cinnamon), possess strong antimicrobial properties that inhibit oral pathogens (16). Additionally, Ajwain (Trachyspermum ammi) has demonstrated significant antibacterial activity against oral biofilms (17). These findings suggest that HiOra may serve as an effective alternative to Chlorhexidine in reducing oral microbial load.

Several studies have compared herbal and allopathic mouthwashes, with varying conclusions. Manipal et al. (18) conducted a meta-analysis and found that five out of eleven studies reported no significant difference in efficacy between Chlorhexidine and herbal mouthwashes. Similarly, a study by Ashraf et al. (19) concluded that herbal mouthwashes, particularly those containing neem, tulsi, and triphala, were equally effective in plaque and gingivitis control. However, Malhotra et al. (20) reported that Chlorhexidine was slightly more effective than herbal alternatives, though the difference was not clinically significant.

Although Chlorhexidine remains the gold standard for plaque control, its long-term use is associated with side effects such as staining, altered taste perception, and increased supragingival calculus formation (21). The present study suggests that Ayurvedic mouthwashes like HiOra offer comparable efficacy with minimal adverse effects, making them a promising alternative for long-term use. Moreover, herbal formulations are biocompatible, well-accepted by patients, and cost-effective, making them suitable for widespread use, particularly in resource-limited settings (2).

The findings of this study indicate that Ayurvedic (HiOra) and allopathic (Chlorhexidine) mouthwashes exhibit comparable efficacy in reducing plaque accumulation, gingival inflammation, and microbial load. Given its natural composition and minimal side effects, HiOra presents a viable alternative to Chlorhexidine for long-term plaque control and gingival health maintenance. However, further long-term clinical trials are warranted to validate these findings and explore the full potential of herbal mouthwashes in preventive dentistry.

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