Evaluating the effects of chlorhexidine and vitamin c mouthwash on oral health in non-surgical periodontal therapy: a randomized controlled clinical trial

Abstract

Chlorhexidine (CHX) is the most commonly used mouthwash with proven antiplaque and antibacterial activity. The aim is to evaluate the effect of vitamin C (VitC) in CHX mouthwash on plaque accumulation and gingivitis, and to compare it with CHX alone mouthwash and antiseptic phenol-containing mouthwashes. This study conducted as a multicenter, randomized, controlled, double-blind, parallel design clinical study. Sixty patients were included, randomly divided into three groups. 1. Antiseptic phenol agent (P, Phenol), 2. Antiseptic bisbiguanide agent CHX-only (CHX) and 3. Antiseptic bisbiguanide agent CHX + VitC mouthwash (CHX + VitC). The study assessed oral hygiene and periodontal health status, followed by scaling and root planning (SRP) and subsequent polishing. After using the mouthwash for 60 s twice daily along 14 days, patients recalled for evaluation of plaque index (PI), gingival index (GI), bleeding on probing (BOP) and staining. One-way analysis of variance (ANOVA) was used to compare the differences formed between groups and Tukey multiple comparison analysis was used to determine groups that showed the differences. Statistical significance was determined using a p-value threshold of 0.05. There were no significant differences between the groups regarding baseline PI, GI and BOP (p > 0.05). Changes at 14th day in PI, GI and BOP in all groups were similar and no significant differences were observed (p > 0.05). Regarding ‘all surfaces’, staining density of two mouthwashes containing CHX was significantly higher than that of P mouthwash. CHX, CHX + VitC, and P mouthwashes appeared to exhibit comparable effects as oral hygiene adjuncts to periodontal mechanical treatment, with the exception of staining, which was more noticeable in the CHX groups compared to the P group. Adding vitamin C did not enhance the effects of CHx alone.

Introduction

Dental plaque is defined as a specific biofilm resulted from an interaction between saliva layer and microbial deposits embedded in the extracellular polysaccharide matrix^1,2. Accumulation and maturation of bacterial biofilm at the gingival margin considered as a primary etiological factor in progression of gingivitis. The initial changes that formed during transition from healthy to diseased periodontal status cannot be observed clinically; however, as the disease progressed, the clinical signs and symptoms become more obvious. Erythema, edema, bleeding and tenderness are the clinical symptoms of gingivitis. Regardless of the age, poor oral hygiene considered as an important predictor of the periodontal diseases³. Thus, prevention of the periodontal diseases should be achieved by adequate plaque control and removal of the microbial biofilm from the tooth surface⁴.

It is not the mechanical approach only which can achieve the bacterial control. Using antimicrobial agents as an adjunctive treatment with scaling and root planning (SRP) can also serve the same purpose. Oral mouthwashes are used for supragingival plaque control in addition to mechanical oral hygiene methods⁵.

Phenolic compounds, recognized for their potent bactericidal properties and their notable ability to reduce inflammatory processes in the periodontium, are commonly used in oral mouthwashes. The clinical effectiveness of a mouthwash composed of phenolic compounds (P, Phenol), is attributed to its bactericidal properties⁶. In a review article aimed at evaluating the short- and long-term effects of P mouthwash on improving oral health, its effectiveness was highlighted in promoting healthy gingival tissue and reducing plaque⁷.

Chlorhexidine (CHX) is a bisbiguanide antiseptic antimicrobial agent that has an effective role against gram-positive and gram-negative bacteria, facultative anaerobes and aerobs, yeast and viruses and it is the most commonly used product for plaque control and gingivitis⁸. CHX mouthwashes are effective in reducing plaque formation and in controlling gingivitis and dental caries⁹. It is considered as a “Gold Standard" for oral hygiene due to its antimicrobial spectrum and its permanent effect. CHX may present different effects regarding its concentration. While playing a bacteriostatic agent role with low concentration, it could act as a bacteriocidal with high concentration^10,11. CHX can bind on teeth surfaces, oral mucosa, pellicle and plaque in the mouth. It has been shown that the effect of CHX on oral surfaces and salivary bacteria lasts for 12 h⁸. CHX can be used to maintain oral hygiene in situations like surgical procedures that impact plaque control. Studies with CHX as an antiplaque agent have been proved that rinsing with 0.2%

CHX, 10 ml for one minute twice daily can demonstrate effective results regarding the plaque control¹².

Vitamine C (VitC) is a water-soluble nutrient that is essential for human’s diet. Studies have shown that VitC plays a role in reducing oxidative damage of the tissues by neutralizing the free radicals and removing hydroxyl radicals¹³. VitC is one of the few nutrients associated with periodontal disease. Historically it was known that severe VitC deficiency causes a periodontal syndrome called “Scorbutic Gingivitis” which causes tooth loss due to ulcerative gingivitis and periodontal pathological pocket formation¹⁴. It has been long time that VitC has been considered as a candidate for being an adjunct to periodontal treatment, but the effect of VitC deficiency in periodontitis has not been fully known yet. In addition of this fact, supplemental VitC is needed as long as presence of the infectious diseases and during the period of tissue regeneration and wound healing. VitC deficiency is primarily associated with impaired collagen synthesis, leading to undesirable wound healing and capillary rupture¹⁵. It has also been shown that VitC deficiency causes collagen formation deficiency by increasing endotoxin permeability through oral mucosa and affecting proline hydroxylation¹⁶.

Indigenously prepared 4% ethanolic extract of Citrus sinensis (orange peel) mouthwash was suggested as a short-term alternative to 0.2% chlorhexidine mouthwash for reducing plaque and gingival inflammation, with no potential side effects¹⁷. Adding VitC to a CHX-containing mouthwash may allow for the beneficial effects of VitC on oral and periodontal health. Chlorhexidine gluconate 0.05% combined with ascorbic acid (vitamin C) was reported to be statistically effective in maintaining low levels of volatile sulfur compounds compared to other commercial solutions and the placebo, even after a single rinse¹⁸.

The null hypothesis tested in this study was that "no difference will be observed in the periodontal health status of subjects using CHX plus VitC mouthwash compared to those using a phenolic mouthwash or CHX alone. The aim of this study is to evaluate the potential of adding VitC in CHX mouthwash on plaque accumulation and gingivitis.

Materials and methods

This study was conducted as a multicenter, randomized, controlled, double-blind, parallel design clinical study. A total number of sixty patients, admitted for treatment of the periodontal diseases to Department of Periodontology in two different centers (Ordu University and Gazi University) were included in this study. Ethical approval was taken (25901600—95Y IP) and written informed consents were obtained from each participant or their legal guardians. This study adhered to the ethical principles set forth in the 1964 Declaration of Helsinki. It has been reviewed and approved by Thailand Clinical Trials Register (TCTR) Committee on 10 June 2024. The TCTR identification number is TCTR20240610004 (https://www.thaiclinicaltrials.org/show/TCTR20240610004) and followed the CONSORT reporting guidelines.

Beside the ability to give informed consent and the willingness to participate in the study, the criteria of the patients included in the study were as following;

• Presence of at least 20 teeth in mouth,

• No usage of any orthodontic or prosthetic apparatus,

• Patients diagnosed with gingivitis,

• No history of periodontitis,

• No allergies,

• Without any systemic diseases that may affect the study outcomes and wound healing.

The criteria of patients excluded from the study were as following;

• Smokers (active, passive and/or former smokers), users of other forms of nicotine and tobacco, such as vaping and tobacco in chewable forms,

• Presence of any uncontrolled systemic disease,

• History of radiotherapy in head and neck region,

• Current chemotherapy treatment,

• Current corticosteroid usage,

• Presence of metabolic or systemic bone disease,

• Pregnancy and breast feeding.

Clinical evaluation

Sixty patients that included in this study were randomly divided into three groups. Patients who refused to follow the instructions provided prior to the study or used any oral care tools such as toothbrushes or toothpaste during the evaluation period (nine patients) were excluded from the study. Three groups arranged as following: group 1, Antiseptic phenol agent P, 0.060 % (Listerine Original mouthwash, Johnson and Johnson, Inc., New Jersey, USA) (P; n = 13), group 2, Antiseptic bisbiguanide agent 0.12%, CHX-only mouthwash (Signal Professional, Unilever, Istanbul, Türkiye) (CHX; n = 19) and group 3, Antiseptic bisbiguanide agent, 0.12% CHX+Vit.C mouthwash (Signal Professional C, Unilever, Istanbul, Türkiye) (CHX+Vit.C; n= 19). The participants were randomly assigned to one of the three mouthwash groups. The group assignment was done using random number generators. A simple randomization with three groups involved assigning participants to Group 1, Group 2, or Group 3 using a random number generator. Each participant was assigned a number between 1 and 3 using a random number generator. Participants with the number 1 are assigned to Group 1, those with the number 2 to Group 2, and those with the number 3 to Group 3. Sixty participants in total, they were distributed randomly among the three groups, ensuring no bias in group allocation. This method maintains equal chances for participants to be assigned to any group. Clinical evaluation of oral hygiene and examination of periodontal health status were performed after detailed systemic and dental history assessment; (Fig. 1)

Fig. 1.

Flow chart of the study.

Plaque index¹⁹. (PI, Silness and Löe 1964),

PI is defined as follows: 0=no plaque accumulation; 1= A thin film of plaque adhering to the free gingival margin and adjacent tooth; 2= Moderate accumulation of microbial plaque within the gingival pocket and/or on the tooth surface visible to the naked eye; 3= Abundant microbial plaque within the gingival pocket and/or on the tooth and gingival margin

Gingival index²⁰. (GI, Löe and Silness 1963),

GI is defined as follows: 0 = normal gingiva; 1 = mild inflammation, characterized by slight changes in color and texture, with no bleeding upon probing; 2 = moderate inflammation, with noticeable glazing, redness, edema, and bleeding upon probing; 3 = severe inflammation, marked by pronounced redness and edema, with a tendency toward spontaneous bleeding and ulceration.

• Modified stain index^21,22

• Bleeding was performed by probing (BOP) measurements²³. Bleeding was recorded as present or absent. Bleeding was calculated as a percentage of affected sites.

Clinical measurements were performed from 6 points (mesio-buccal, mid-buccal, disto- buccal, mesio-lingual\palatal, mid-lingual\palatal and disto-lingual\palatal) from each tooth by independent calibrated examiners (AE, MT and HS), blinded to the type of mouthwash investigated in this study using a 0.5 mm diameter Williams type periodontal probe (Nordent Mfg Inc. Elk Grove Village, IL). The value of periodontal measurements for each participant was determined as follows:

SRP and polishing were performed after completion of the clinical measurements. Both examiner and patients were blinded to the assignment of the study group. The mouthwashes were masked by being provided in bottles without labels, marked only with the patient’s number. So, both the patient and the examiner were blind to the type of mouthwash. The patients were asked to rinse with the allocated mouthwash only without any type of mechanical tooth brushing twice daily for 60 seconds in the morning upon waking up and at night before bed for duration of 14 days. The quantity of mouthrinse that was prescribed to be used in each event of rinsing the mouth during the duration of study was 15 ml. It was same for all three groups. No additional oral hygiene maintenance strategies were implemented throughout the study. Mechanical toothbrushing was not employed, nor were electronic brushing methods or gum flossing techniques used. They were recalled to control appointment for evaluation of PI, GI, BOP and staining. As at the baseline, PI, GI and BOP were evaluated and recorded. 11,12, 13, 21, 22, 23 for the upper jaw and 31, 32, 33, 41, 42 and 43 for the lower jaw were determined as index teeth, thus evaluation of staining performed on these teeth. Lobene stain index²¹ was used to evaluate staining. In this index, the scoring of stain accumulations on the buccal/labial and lingual/palatinal surfaces of the index teeth was made by dividing the tooth surfaces into 4 different regions as gingival (G), mesial (M), distal(D) and body (B)²². Gingival (G): section parallel 2 mm to the gingival margin. This section ends at interdental papillae region. Mesial (M)-Distal (D): section between the line angle of the tooth and the adjacent tooth. Line angle, is the part where the two surfaces meet, the part where vestibular surface is slightly inclined. Body (B): part of tooth that stays out from other three areas and extended to incisal region of the tooth. Two different parameters were evaluated for tooth staining, density and region.

Density:

(0) No stains, normal tooth color (1) Pale stain (2) Clearly visible stain, orange or brown (3) Dark staining, dark brown or black.

Regional scoring was performed in cases where the intensity score was 2 or 3. Regional scoring for proximal “Mesial-Distal” and “Gingival” areas:

(1) Thin line, can be continuous (2) Thick line or band (3) Covering whole area.

Regional scoring for “Body” area as following:

Score 1; For buccal / labial surfaces, staining of pits and fissures. For lingual / palatal surfaces, staining 1/3 of the area. Score 2; For buccal / labial surfaces, staining %10 of the area regardless pits and fissures. For lingual / palatal surfaces, staining 1/3 to 2/3 of the area. Score 3; For buccal / labial surfaces, staining more than %10 of the area regardless pits and fissures. For lingual / palatal surfaces, staining more than 2/3 of the area.

Data analysis

The interexaminer reliability kappa scores was 0.75 for both PI and GI. According to PI, the mean values on 14th day between groups, when the effect size was accepted as 0.5, the primary type error amount as (α) 0.05 and the power of test as (1-β) 0.85; number of subjects required to be in the study was determined to be 49 for %85 power. With a sample size of 60 patients, the power of the study is approximately 91.3%, assuming the original effect size and significance level remain the same.

SPSS (Statistical Package fort the Social Sciences) (Windows ver. 22.0) package program was used for statistical analysis. The assumption of data normality was verified using the Shapiro-Wilk test, alongside visual assessments of histograms and Q-Q plots. Regarding to index scores, One-way analysis of variance (ANOVA) was used to compare the differences formed between groups and Tukey multiple comparison analysis was used to determine groups that showed the differences. Statistical significance was determined using a p-value threshold of 0.05.

Results

Out of the originally intended 60 patients, 51 were included in the study. Patients who did not comply with the study protocol and used oral care tools were excluded from the study. Among these, group 1, Antiseptic phenol agent mouthwash (P; n = 13), group 2, Antiseptic bisbiguanide agent CHX-only mouthwash (CHX; n = 19) and group 3, Antiseptic bisbiguanide agent CHX+Vit.C mouthwash (CHX+Vit.C; n = 19) completed the primary endpoint and provided demographic data. Age and baseline index scores that belong to all the groups presented in Table 1. Ages of patients found similar in all study groups. There were no significant differences between groups regarding the baseline PI, GI and BOP (p > 0.05).

Table 1.

Comparison of the study groups regarding patient’s age and periodontal parameters at baseline.

	Phenol (n = 13)	CHX-only mouthwash (n = 19)	CHX + VitC mouthwash (n = 19)	P value
Age	23.40 ± 12.22	16.66 ± 2.91	22.00 ± 3.00	p > 0.05
PI	1.10 ± 0.47	1.30 ± 0.45	1.23 ± 0.47	p > 0.05
GI	1.07 ± 0.19	1.42 ± 0.43	1.15 ± 0.25	p > 0.05
BOP	0.49 ± 0.20	0.61 ± 0.29	0.66 ± 0.22	p > 0.05

*Results were dedicated as average ± standard error. *p < 0.05.

Changes at the 14th day in PI, GI and BOP according to the baseline values were summarized at Table 2. Accordingly, changes in all groups were similar and no significant differences were observed (p > 0.05).

Table 2.

Comparison of periodontal parameters of the study groups with periodontal measurements at 14th day.

	Phenol (n = 13)	CHX-only mouthwash (n = 19)	CHX + VitC mouthwash (n = 19)	p value
PI	0.28 ± 0.59	0.08 ± 0.23	0.13 ± 0.21	p > 0.05
GI	0.19 ± 0.18	0.04 ± 0.41	0.06 ± 0.11	p > 0.05
BOP	0.05 ± 0.06	0.01 ± 0.19	0.03 ± 0.05	p > 0.05

*Results were dedicated as average ± standard error.

*p < 0.05 accepted as a meaningful value.

Table 3 showed staining intensities of the facial and lingual surfaces at the end of 14 days of mouthwash use. Accordingly, the staining of the gingival and body regions of the the facial surface was found similar in all three groups (p > 0.05). There was a significant difference in density scores between P and CHX+Vit.C mouthwash at ‘mesial / distal’ surfaces (p < 0.05). When ‘all surfaces’ were taken into consideration, staining density of the two mouthwashes containing CHX was significantly higher than that of P mouthwash. There was no significant difference observed regarding staining effects between the three types of mouthwashes at the lingual surface. The staining regions of the facial and lingual surfaces at end of the 14 days usage of mouthwash could be seen at Table 4. Regarding the facial surface ‘body’, ‘mesial / distal’ and ‘all surfaces’, P group was found to have significantly lower staining degree than both types of CHX groups (p <0.05); while no differences were found at ‘gingival’ region. There was no difference between the groups in ‘mesial/distal’ regions of the lingual surface; however, at ‘gingival’, ‘body’ and ‘all surfaces‘, P group has statically significant lower staining degree than the other two CHX groups (p <0.05).

Table 3.

Comparison the staining density on the facial and lingual surfaces between groups on the 14th day using the Lobene stain index (17).

	Phenol (n = 13)	CHX-only mouthwash (n = 19)	CHX + VitC mouthwash (n = 19)
Facial
All regions	17.67 ± 16.86a	46.33 ± 6.34,b	30.89 ± 21.92 b
Gingival	4.66 ± 1.52a	11.44 ± 3.47a	7.56 ± 5.27a
Body	4.67 ± 9.07a	11.33 ± 3.81a	7.00 ± 5.02a
Mesial/distal	8.33 ± 10.60a	24.89 ± 3.10b	16.11 ± 12.71a,b
Lingual
All regions	36.00 ± 9.00a	42.13 ± 15.64a	41.11 ± 29.84a
Gingival	8.67 ± 3.06a	9.63 ± 4.50a	8.89 ± 6.47a
Body	8.00 ± 7.64a	10.88 ± 6.88a	10.22 ± 7.43a
Mesial/distal	19.67 ± 11.59a	24.00 ± 6.46a	22.33 ± 16.32a

*Results were dedicated as average ± standard error.

*One way variance analysis (ANOVA),

*There is no statistical difference between those parameters who shared the same letter, different letters show statistical differences. Tukey multiple comparison test (p  < 0.05).

Table 4.

Comparison the staining patterns on the facial and lingual surfaces among groups on the 14th day using the Lobene stain index (17).

	Phenol (n = 13)	CHX-only mouthwash (n = 19)	CHX + VitC mouthwash (n = 19)
Facial
All regions	2.54 ± 1.40a	4.89 ± 4.70b	3.11 ± 6.27b
Gingival	0.50 ± 0.24a	0.88 ± 1.36a	1.22 ± 2.10a
Body	0.33 ± 0.44a	0.88 ± 1.69b	0.88 ± 2.01b
Mesial/distal	1.20 ± 0.6a	3.00 ± 3.50b	2.11 ± 5.62b
Lingual
All regions	4.00 ± 7.50a	7.00 ± 8.57b	8.11 ± 11.22b
Gingival	0.55 ± 0.65a	1.25 ± 2.12b	1.22 ± 2.10b
Body	1.67 ± 1.34a	3.38 ± 4.81b	2.11 ± 3.10b
Mesial/distal	1.67 ± 5.95a	2.25 ± 3.24a	5.11 ± 6.97a

* Results were dedicated as average ± standard error.

*One way variance analysis (ANOVA),

*There is no statistical difference between those who shared the same letter, different letters show statistical differences. Tukey multiple comparision test (p < 0.05).

Discussion

Microbial dental plaque is the primary role factor in pathogenesis of the periodontal diseases. It is a mixture of co-aggregated bacteria which considered as a causative agent of the periodontal diseases^24,25. Therefore, the primary and the most important point in the development of periodontal diseases is the prevention of accumulation of dental plaque. Thus, it has been suggested to use chemical plaque control agents in addition with mechanical cleaning.

CHX is currently the most effective anti-plaque agent and is frequently used in both medicine and dentistry²⁶. In dentistry, CHX is available in various forms, including mouthwashs, gels, sprays, and varnishes. Challenges may arise when delivering CHX in gel or toothpaste form, as it can bind to ingredients in these products, diminishing its effectiveness²⁷. Mouthwashs with CHX hold an advantage due to their long-standing use in oral health and widely accepted as part of a standard oral hygiene routine, typically used after regular mechanical tooth cleaning. CHX mouthwash is commonly available in concentrations of 0.1%, 0.12%, or 0.2% chlorhexidine digluconate, as well as in a lower concentration rinse of 0.06% or less. In a study by Cheng et al.²⁸ on Down syndrome patients with chronic periodontitis, patients were asked to use CHX twice daily either by brushing with a gel form or rinsing with a mouthwash form and evaluation of the clinical parameters was performed. After 12 months of non-surgical mechanical periodontal treatment, positive results such as decrease in pocket depth values and BOP scores with clinical attachment gain were observed in that study. In a recent review article, the effectiveness of CHX mouthwash as an addition to mechanical oral hygiene practices for controlling gingivitis and plaque, compared to mechanical oral hygiene alone or combined with a placebo/control mouthwash was evaluated²⁹. Fifty-one, parallel group designed studies were included to that study and similar to our findings, there was insufficient data reported to assess the reduction in gingivitis associated with CHX mouthwash use in individuals with mean GI scores of 1.1–3. Our subjects in all intervention groups have moderate GI scores. Moreover, there was no evidence to suggest that any specific concentration of CHX rinse was more effective than others in reducing gingivitis and plaque²⁹. In our study, 0.12% CHX mouthwash formulation alone and with VitC supplementation was used and any statistical significant difference was not found in GI, PI and BOP between these formulations.

Brown staining of teeth is the most significant side effect of long-term usage of CHX- containing mouthwashes³⁰. The mechanism of CHX staining has been shown to be the cleavage of the CHX molecule to release para-chloroaniline, protein denaturation by metal sulfide formation, and precipitation of anionic dietary chromogens^31,32. In our study, it was observed that CHX containing mouthwashes caused more staining compared to P mouthwash. However, it was observed that the addition of VitC had no further staining effect, thus both CHX-containing mouthwashes caused the same staining degree on the teeth. There was moderate-quality evidence indicating that CHX rinse leads to substantial extrinsic tooth staining when used for 4 weeks or more. Comparisons between subgroups with different CHX concentrations did not show statistically significant differences²⁹. Although our study period was 14 days, the same level of staining was observed with both CHX formulations. To overcome the adverse effects of CHX including staining of teeth and restorations, mucosal irritation, microbial resistance, and alterations in taste sensation, mouthwashes containing polyhexamethylene biguanide or polyhexanide and chlorine dioxide offer viable alternatives to CHX. Studies have demonstrated that polyhexamethylene biguanide antimicrobial effects are comparable to those of CHX, while chlorine dioxide antimicrobial effects surpass those of CHX³³. It may be possible to remove extrinsic pigmentations caused by CHX using toothpastes that contain micro-particles of activated charcoal and micro-cleaning crystals, offering a potential solution for managing staining after CHX use³⁴.

Welk et al.³⁵ compared the antibacterial and antiplaque effects of CHX, phenol, octenidol and placebo mouthwashes and found similar antiplaque effects of CHX and phenol. Abou Sulaiman and Shehadeh³⁶ reported that supplying of additional VitC did not provide an additional benefit to non-surgical periodontal treatment. A placebo mouthwash was not employed as a control group in our study. This decision was made to investigate whether incorporating VitC into CHX mouthwash results in clinical distinctions when compared to CHX-only mouthwash, especially considering the well-established favorable effects of CHX-only mouthwash. In this study, a 14-day experimental gingivitis study model was used. Due to many complications with prolonged usage of CHX mouthwash, it was preferred to use it for 14 days. This model is a non-invasive method to evaluate the onset and recovery of inflammation that caused by excessive bacterial accumulation in humans³⁷. The baseline periodontal index scores of the individuals in all groups were statistically similar, so that the efficacy of the mouthwashes compared more accurately. In the evaluation after 14 days, it was found that both the mean PI and GI scores were higher in P group compared to both mouthwashes containing CHX groups. However, this difference was not statistically significant. The topical application of VitC has its benefits in reduction of gingival bleeding³⁸. In our study, even PI and GI scores were found lower in both CHX groups than P group, there were no statistically significant differences in both parameters between groups.

While there was a limitation in number of studies regarding topical application of VitC, its systemic application in many of studies have been proved as providing a positive effect in regards to the clinical parameters^38–42. In a study by Kuzmanova et al.⁴⁰ on 21 individuals with untreated periodontal disease and 21 healthy individuals, plasma polymorphonuclear leukocytes and mononuclear cells of peripheral blood were investigated for different VitC concentrations; it was observed that patients with periodontitis had lower levels of VitC than the healthy individuals. Staudte et al.⁴¹ compared plasma VitC levels and inflammatory status in patients with periodontal diseases and healthy individuals. They indicated that plasma VitC levels were lower in the diseased group than the healthy subjects. They reported an increase in VitC levels after two weeks of its supplementation and also a significant decrease in bleeding index. Amaliya et al.⁴² found that VitC deficiency contributes to increased severity of periodontal damage thus a negative relationship between plasma VitC levels and clinical attachment loss had been reported. In a study of Alyahya et al.³⁸, the topical application of VitC was performed with cotton rolls at gingival margins once a week for 1 month resulted in a reduction of gingival bleeding index scores. A recent review concluded that the efficacy of herbal and chlorhexidine-based mouthwashes in maintaining oral hygiene during fixed orthodontic treatment remains inconclusive due to mixed results and varying levels of evidence across studies⁴³.

In our study, it was aimed to evaluate whether an adding of VitC to CHX mouthwash, which is the most commonly used mouthwash in routine with proven antiplaque and antibacterial activity, has an additional effect on the clinical outcomes. At the end of the study, the efficacy of CHX + Vit.C mouthwash on periodontal health was similar to that of CHX-only mouthwash, we also observed that it caused the same degree of staining on the teeth. In the light of these results, it can be said that VitC mouthwash has clinically no additional positive effect on periodontal health status. In regard of applying VitC topically via mouthwash, the duration of vitamine in mouth is too short to conclude about the synergistic effect of CHX and VitC. However, further studies are needed in the future with long-term different topical administration routes on larger populations which can be accomplished not by determining the clinical measurements only, but also by supporting the results with microbiological and biochemical analysis.

The main limitations of the current study include the short follow-up period. Additionally, the variability in study methodologies and participant demographics may affect the generalizability of the findings. The limited duration of follow-up restricts the ability to assess long-term effects and outcomes. Another limitation of this study is that mouthwash was typically recommended for patients with severe gingivitis (GI > 2 or BI > 50%) or severe periodontitis to enhance healing, particularly in those with limited manual dexterity. Additionally, in cases of mild gingivitis, professional cleaning alone is often sufficient to achieve the desired clinical outcomes, as indicated in the findings of this study. Therefore, future research should focus on evaluating these mouthwashes in patients with periodontitis, where changes in pocket depth would be clinically significant. A key limitation of home treatment using mouthwash in research is the difficulty in ensuring participant compliance and adherence to the prescribed regimen. Self-reporting introduces potential bias, while the lack of direct supervision means improper use or variability in technique may affect outcomes.

The conclusion that VitC presence with CHX does not appear to offer a significant advantage to periodontal health raises important questions regarding the experimental conditions, particularly the concentration of VitC used in the mouth rinse. Empirical evidence from the literature highlights the positive impact of VitC on periodontal health, primarily due to its antioxidant properties, ability to promote collagen synthesis, and role in reducing oxidative stress and inflammation in periodontal tissues. However, the efficacy of VitC in combination with CHX might depend heavily on its concentration and bioavailability.

A suboptimal concentration of VitC in the mouth rinse could fail to achieve the desired therapeutic effects, potentially overshadowing its benefits. Furthermore, interactions between VitC and CHX may alter the stability or effectiveness of either component. Future studies could explore whether modifying the concentration of VitC or adjusting the formulation could enhance its synergistic effects with CHX. Additionally, evaluating the long-term effects of such combinations on clinical outcomes and comparing them with single-agent therapies may provide more comprehensive insights. For future research, it is recommended to explore alternatives to chlorhexidine as a single treatment rather than in combination with other agents^44,45.

Conclusion

Usage of 14 days period of CHX + Vit.C mouthwash has similar outcomes on periodontal health status regarding clinical parameters compared to CHX-only mouthwash. The two CHX mouthwash forms (CHX-only and CHX + VitC) caused higher staining density compared to P mouthwash. Adding VitC to CHX mouthwash did not appear to provide any additional benefit compared to CHX alone in terms of periodontal health.

Author contributions

A.E. worked on the drafting the article, data collection, and interpretation of data. S.C.I. worked on interpretation of data, drafting the article. C.G. worked on conception and design of the study and approval of the version to be published. M.T. and H.S. worked on Data collection. G.A. worked on data analysis. S.A.S. worked on data collection. N.O. worked on interpretation of data, conception and design of the study, analysis, and approval of the version to be published. All authors declare that they have no conflict of interests.

Funding

Ordu university, scientific research project. Project No: AR-1414.

Data availability

The datasets used and or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

Ethical no: 25901600— 95Y IP Ordu University.

Informed consent

A written informed consents were obtained from each participant.