Effects of Herbal Mouthwashes on Plaque and Inflammation Control for Patients with Gingivitis: A Systematic Review and Meta-Analysis of Randomised Controlled Trials

Objective The aim of this study was to evaluate the overall effects of herbal mouthwashes as supplements to daily oral hygiene on plaque and inflammation control compared with placebos and chlorhexidine (CHX) mouthwashes in the treatment of gingivitis. Methods PubMed, EMBASE, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, and grey literature databases were searched. Only randomised controlled trials (RCTs) comparing herbal mouthwashes with placebos or CHX in the daily oral hygiene of patient with gingivitis were included to compare the effect of different mouthwashes on plaque and inflammation control. Results A total of 13 studies satisfied the eligibility criteria, and 11 studies were included in meta-analyses. Significant differences were observed in favour of herbal mouthwashes compared with placebos in both plaque- and inflammation-related indices (Quigley-Hein Plaque Index, QHPI: WMD = −0.61, 95% CI (−0.80, −0.42), P < 0.001; Gingival Index, GI: −0.28 (−0.51, −0.06), P=0.01; Modified Gingival Index, MGI: −0.59 (−1.08, −0.11), P=0.02; Gingival Bleeding Index, GBI: −0.06 (−0.09, −0.04), P < 0.001). No significant difference was found between herbal and CHX mouthwashes. Conclusions Herbal mouthwashes have potential benefits in plaque and inflammation control as supplements to the daily oral hygiene of patients with gingivitis. Although no difference was observed between herbal and CHX mouthwashes in the selected studies, further high-quality RCTs are needed for more firm support before advising patients with gingivitis about whether they can use herbal mouthwashes to substitute for CHX mouthwashes or not (PROSPERO registration number: CRD42019122841).


Introduction
Gingivitis, which has a direct association with dental plaque [1,2], affects the oral health of 70%-100% of the population across the world [3][4][5]. Gingivitis is reversible through plaque control; however, it may progress with inadequate oral care and eventually affect the entire periodontal attachment apparatus of the involved teeth, resulting in further harmful consequences, such as periodontitis, tooth loss, and worse quality of life [6].
us, effective plaque control plays a crucial role in resolving and preventing gingivitis and related conditions [7].
Currently, toothbrushing is the most popular self-performed oral hygiene method to mechanically remove dental plaque. However, this mechanical approach by most individuals is often not sufficiently effective [8], suggesting that a chemical plaque control by mouthwashes could be beneficial as a supplement to daily oral care [9]. Chlorhexidine (CHX), a broad-spectrum antiseptic, is considered as the gold standard for chemical dental plaque control [2], but CHX mouthwashes can lead to the staining of teeth and tongue, taste disturbance, and adverse effects on the oral mucosa after prolonged use [10,11]. ose undesirable side effects limit the long-term use and the patient acceptability of CHX mouthwashes. us, the search for alternatives continues, and the focus shifted toward biogenic agents.
Herbal medicines, derived from botanical sources, have been applied in dentistry for a long history to inhibit microorganisms, reduce inflammation, soothe irritation, and relieve pain [12][13][14]. It has been recently reported that a considerable number of herbal mouthwashes have achieved encouraging results in plaque and gingivitis control [15,16]. Herbal mouthwashes are designed and prepared with extracts and essential oils from phytotherapeutic plants, containing a mixture of active agents such as catechins, tannins, and sterols [17,18]. e mixture of natural compounds inside the herb-or plant-derived substances usually performs gentle remedial effects. Compared with the antimicrobial mechanisms by synthetic chemicals, herbal mouthwashes can have additional anti-inflammatory and antioxidant properties, which could further benefit gingival health [19].
Numerous herbal mouthwashes have been introduced and tested; however, the results of existing literature are inconsistent regarding the clinical effects of herbal mouthwashes on both dental plaque and gingival inflammation control compared with placebo or CHX [20][21][22], and there is a scarcity of meta-analytical evidence highlighting the overall effects of herbal mouthwashes as adjuncts to the daily self-performed oral hygiene of patients with gingivitis. Without this information, it is not possible to provide comprehensive evidence-based advice to the patients and practitioners. erefore, the aim of this study was to conduct a systematic review and a meta-analysis of randomised controlled trials (RCTs) only to compare the overall effects of herbal mouthwashes as supplements to the daily oral hygiene on both plaque and inflammation control with either negative placebos or CHX mouthwashes in the treatment of gingivitis.

Materials and Methods
e current systematic review was reported following the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA) guidelines [23]. A detailed protocol was developed a priori and registered in the International Prospective Register of Systematic Reviews (http://www.crd.york. ac.uk/PROSPERO/) (registration number: CRD42019122841).

Participants-Interventions-Comparisons-Outcomes-Study Design (PICOS) Question.
is systematic review was performed to answer the focused question "How are the effects of herbal mouthwashes for plaque and inflammation control as a supplement to daily oral hygiene when compared to placebos or CHX-containing mouthwashes among patients with gingivitis?" according to the following elements: Participants: systematically healthy participants with gingivitis Interventions: the application of herbal mouthwashes from botanical sources Comparisons: the application of negative placebos without any active agents or positive CHX-containing mouthwashes Outcomes: the clinical effects of mouthwashes as a supplement to daily oral hygiene (i.e., toothbrushing) on plaque and inflammation control Study design: RCTs only 2.2. Eligibility Criteria. Based on the PICOS question, a study must fulfil the following inclusion criteria: (i) Studies with participants who had gingivitis and were otherwise systemically healthy during the test period (ii) e intervention group(s) must use a mouthwash containing extract(s) or essential oil(s) from plants (iii) e comparison group(s) must comprise a negative placebo solution or a CHX-containing mouthwash (iv) All the interventions should be applied as a supplement to daily self-performed oral hygiene routine (v) Studies should include at least one plaque-or gingivitis-related indices as an outcome measure (vi) RCTs It should be noted that only RCTs were included to synthesise high-quality evidence which would then enable inferences to be drawn with confidence. Studies that had participants with periodontitis or participants who were undertaking orthodontic treatment; studies which only reported outcomes relating to the specific oral bacteria (such as the Streptococcus mutans count); and in vitro, animal or cadaver studies, case studies, letters, and historical reviews were excluded. Further, to replicate the "real-life" circumstances of patients, studies with professional mechanical prophylaxis during or in three months before the trials were excluded. OR gingivit * OR plaque * OR biofilm * OR microorganism * OR microflora * OR "gingival pocket" OR "gingival pockets" OR "pseudo pocket" OR "pseudo pockets" OR pseudopocket * OR ((gingiv * OR papilla * OR sulc * ) AND (hemorrha * OR bleed * OR inflam * )) (2) Interventions-("Botany" [ (2) For "grey" literature, the ClinicalTrials.gov and the International Clinical Trials Registry Platform were searched for unpublished clinical studies or registries; the ProQuest Dissertation Abstracts and esis database was searched for related dissertations and theses; the Conference Proceedings Citation Index-Science was searched for conference proceedings; additionally, some other online resources such as the System for Information on Gray Literature in Europe database were also searched as a supplement. Subsequently, a manual search was carried out based on the reference lists of selected trials and related reviews.

Study Selection.
Duplicates were removed, and the titles and abstracts were screened by two independent reviewers (HC and JC) against the inclusion/exclusion criteria. After the initial screening, full texts of the potentially relevant articles were retrieved. When the articles [16,[24][25][26] were unavailable online, then the corresponding authors or the editors of the journals were contacted to request full-text articles, and two full-text articles [16,24] were received. Once all the full-text articles were collected, they were independently screened by the same two authors. If any article failed to meet a single criterion, then it was immediately excluded. If there was disagreement regarding inclusion/ exclusion of a study, this was resolved through discussion or by a third reviewer (XL).

Data Collection and Data Items.
A standard data extraction sheet was developed a priori, and the data were extracted and cross-checked for accuracy by two independent reviewers (HC and JC). Detailed data pertaining to author; year of publication; sample size; age range; number of males and smokers; active pharmaceutical ingredients, detailed dosage, and putative chemical properties of applied herbal mouthwashes; comparisons; relevant plaque-and/or gingivitis-related measures; follow-up; loss to follow-up; and effects and side effects of herbal mouthwashes reported in the selected studies were extracted.

Risk of Bias in Individual Trials.
e risk of bias was independently graded by two reviewers (HC and JC) using the Cochrane Collaboration's tool for assessing risk of bias [28]. e risk of bias within studies was estimated as "low," "unclear," or "high" through seven aspects of criteria including random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias.

Summary Measures.
e clinical outcomes were mainly evaluated with Plaque Index (PI) [29] and Turesky modification of Quigley-Hein Plaque Index (QHPI) [30] for supragingival plaque, and Gingival Index (GI) [31] and Modified Gingival Index (MGI) [32] were used for visual signs of gingival inflammation. Gingival bleeding was also reported in a few studies as another sign for gingival inflammation via varied measures, such as Bleeding Index (BI) [33], Sulcus Bleeding Index (SBI) [34], and Gingival Bleeding Index (GBI) [35]. Means and standard deviations (SDs), as well as data in other formats (e.g., standard errors, medians, and interquartile ranges) reported for these measures, were collected for further meta-analyses.

Synthesis of Results.
To compare the antimicrobial and anti-inflammatory properties of herbal mouthwashes to negative placebos and positive "gold standard" CHX-containing mouthwash and to increase the accuracy of the overall estimate of the effect size, the data were pooled from individual studies with the same comparison and outcome measure to perform a meta-analysis. Due to the random allocation in individual studies, the mean and SD of each index at the endpoint of study in each group (herbal, placebo, and CHX) were obtained to estimate the weighted mean differences (WMDs) and their 95% confidential intervals (95% CIs), comparing the effects between different mouthwashes. Some studies reported other data formats and when possible were converted into the desired format (i.e., means and SDs) to include in the analyses [36]. e missing SDs in some articles were estimated by the average of SDs from other studies in the same meta-analysis [37]. e extracted data were analysed using RevMan version 5.3 software ( e Cochrane Collaboration, Copenhagen, Denmark). Heterogeneity across trials was investigated using the Chi-squared test and I 2 statistics [38]. Considering the sample size of individual studies and the heterogeneity across trials, either a fixed effects model or a random effects model [39]
e most commonly used herb-or plant-derived substance reported in the selected studies was green tea extract [17,21,42,47], followed by neem [20,42,46] and marigold [18,21,22]. e commercial essential oil-containing mouthwashes were also used in some studies [24,43,46], which usually consist of eucalyptol, menthol, methyl salicylate, and thymol. In these selected studies, all the natural ingredients in the herbal mouthwashes were expected to have antibacterial, anti-inflammatory, or antioxidant properties. However, few possible active compounds and relevant molecular mechanisms inside the antiplaque/ antigingivitis effects of herbal extracts were studied in these clinical trials. e follow-ups within studies between herbal and CHX were always less than four weeks as the long-term use of CHX mouthwashes is not recommended due to its side effects [48], while the studies between herbal mouthwashes and placebos could have a longer follow-up up to 24 weeks [43]. Most herbal mouthwashes were demonstrated to have potential benefits in reducing plaque and inflammation for gingivitis patients as supplements to daily oral hygiene.
Four studies [17,21,24,46] addressed the side effects during herbal mouthwashes rinsing and reported some degree of hypogeusia [24], lightheadedness [21], and an unpleasant taste [17,46]. In the other nine studies, five [18,22,[41][42][43] reported that herbal mouthwashes were well tolerated with no side effects, and the remaining studies [20,44,45,47] made no comment on side effects or adverse events. [18, 20-22, 24, 41-46] were of low and unclear risk of bias, and two studies [17,47] included a high risk of bias (Figure 2). Six studies [21,41,42,44,46,47] had a low risk of bias for random sequence generation, and five [18,21,24,42,46] provided clear information in terms of allocation concealment. Two studies [17,47] failed to blind the participants and personnel (Figure 2), which introduced a high risk of bias and were then excluded from the meta-analyses to raise the quality of evidence in this study [49]. us, all the studies included in meta-analyses were without any high risk of bias.

Results of Individual Studies and Synthesis of Results.
e focus of this study was mainly to compare the overall clinical effects between herbal mouthwashes and placebo or CHX mouthwashes on plaque and inflammation control. Five included studies [18,20,22,24,43] showed significantly greater reductions in plaque, gingivitis, and bleeding indices after the use of herbal mouthwashes compared with placebos, while two studies [17,21] reported that no difference was observed between herbal mouthwashes and placebos. Five studies [20,22,41,44,45] revealed that there was no difference between herbal mouthwashes and CHX for plaque or inflammation control, and two studies [42,47] even showed greater reductions in gingival inflammation after the intervention of herbal mouthwashes. However, in another study, no significant changes were observed in either the plaque index or gingivitis index after the use of herbal mouthwash, while CHX showed significant effects on both the two indices [46].

Discussion
e present study is the first systematic review and metaanalysis to investigate the clinical effects of herbal mouthwashes as supplements to the self-performed oral hygiene of patients with gingivitis. It is found that herbal mouthwashes are effective in reducing plaque and gingivitis, and there was no difference between herbal and CHX mouthwashes. e effects of herbal mouthwashes were intended to be studied in a "real-life" condition, and hence the professional dental prophylaxis was excluded from this study to make sure that    6 Evidence-Based Complementary and Alternative Medicine  8 Evidence-Based Complementary and Alternative Medicine  Herbal medicines used in oral hygiene is popular due to their antimicrobial, anti-inflammatory, and antioxidant properties [50,51].

Evidence-Based Complementary and Alternative Medicine
ere is a dynamic and rapid accumulation of clinical trials showing that mouthwashes containing herbal exacts, such as Camellia sinensis (green tea) and Azadirachta indica (neem), and essential oils can probably be an effective treatment to reduce dental plaque and gingival inflammation [18,52,53]. Based on the evidence of relevant RCTs, it is indicated that herbal mouthwashes have potential antiplaque and antigingivitis effects compared with placebos. e natural botanical products seem to be a promising field for the treatment of gingivitis, and further studies are needed to seek for more effective herbal mouthwashes with either the traditional medicine formulations or new herbal preparations. However, the exact mechanisms of the antiplaque and antigingivitis actions of herbal mouthwash treatments were not clear due to the inherent complex nature of botanical products. e possible mechanisms can probably be that the natural compounds, such as catechins, tannins, sterols, and oils, may inhibit the growth and adhesion of oral microorganisms and subside inflammatory cytokines such as interleukin-6 and tumour necrosis factor-α [17,18,41,54]. Also, herbal mouthwashes may protect the gingiva from infection and inflammation by their antioxidant property [41]. Further studies are required to clarify the biological mechanisms inside the antiplaque and antigingivitis effect of those herbal mouthwashes. With the literature indicating that herbal mouthwashes are superior to placebo as a supplement to daily oral hygiene [18,52,53], practitioners would be better informed about the potential choices if the comparisons could be conducted directly between herbal mouthwashes and one "good standard" antiseptic.
CHX has been considered as the primary agent for chemical plaque control for more than four decades. However, CHX has several side effects, such as tooth staining [55]. CHX-containing mouthwashes may result in the local colour precipitation between tooth-bound CHX and the chromogens in the daily diet, causing unpleasant brownish staining or discolouration [56,57]. Also, there can be an idiosyncratic reaction of oral mucosal desquamations and soreness after the use of CHX [55]. Mouthwashes containing herbal extracts were investigated to have less or even no staining effect [58,59]. Based upon the existing evidence, no significant differences in the effects on plaque or gingivitis were observed between herbal and CHX mouthwashes. Notably, some specific herbal mouthwashes, such as neem and green tea, were reported to have an even better effect than CHX on gingiva [42]. erefore, based on the findings of this current study, herbal mouthwashes seem to be possible alternatives to CHX-containing mouthwashes as part of the daily oral hygiene of patients with gingivitis, especially for the longterm use. However, due to the limited studies included in this review, it is not sufficient to advise patients with gingivitis about whether they can use herbal mouthwashes to substitute for CHX mouthwashes or not. Further highquality RCTs are needed to get more firm evidence for clinical decision-making. e studies selected for this review also reported a few side effects during herbal mouthwash rinsing, such as hypogeusia, bitter taste, and lightheadedness in individual participants. However, the relevant evidence was insufficient, and the safety of herbal mouthwash still warrants more careful examination.
e evidence of this study should be viewed considering the limitations of the study. High heterogeneity was observed in some analyses, possibly due to the variation in the baseline indices across studies. However, as all the included Asokan (2009) Random sequence generation (selection bias) Allocation concealment (selection bias) Blinding of particioants and personnel (performance bias) Blinding of outcome assessment (detection bias) Incomplete outcome date (attrition bias) Selective reporting (reporting bias) Other bias Balappanavar (2013) Charles (2013) Charles (2015) Chatterjee (2011) Jenabian (2012) Khairnar (2013) Lauten (2005) Mahyari (2015) Mamgain (   provide clear information in respect of the loss to followup, it was not possible to verify the validity of the evidence within individual studies. Future studies should clearly report the number of participants who enter and exit the studies to enable the calculation of loss to follow-up and state findings accordingly. Moreover, two RCTs [17,47] were found with a high risk of bias in blinding of participants and personnel by reporting a single-blinded trial without participants blinded, which might result in a drop of the internal validity of the study [49]. To minimise the likelihood of performance bias and raise the quality of evidence within this meta-analysis, the two studies were excluded from meta-analysis and were described for characters only. Additionally, the limited number of included studies implied that there might be a lack of sufficient evidence and robust assessments of publication biases for most of the outcomes, compromising the validity of analysis to some extent. erefore, further high-quality RCTs are needed for stronger support of clinical decisionmaking.