Clinical applicability of natural product ( s )-containing mouthwashes as adjunctive treatment of biofilm-induced gingivitis : a systematic review

RESUMO: Aplicabilidade clínica de colutórios à base de produtos naturais como tratamento adjuvante da gengivite induzida por biofilme: uma revisão sistemática. Os produtos naturais têm surgido como alternativa eficaz e de baixo custo para o tratamento de várias doenças da cavidade oral. Objetivou-se avaliar, a partir de revisão sistemática da literatura, se há evidências científicas garantindo a utilização segura e eficaz de antissépticos bucais contendo produto(s) natural(is) como tratamento adjuvante da gengivite induzida por biofilme. Foram realizadas buscas nas bases de dados Medline, SciELO, LILACS e Cochrane Library, através de combinações usando as palavras-chave gengivite/produtos naturais/ fitoterápicos/ bochechos, em Inglês, Português e Espanhol. Consideraram-se os estudos publicados até setembro de 2010. Quatro examinadores analisaram separadamente: desenho e fase do estudo, qualidade metodológica (escala de Jadad EJ), produto experimental e a concentração, intervalo de administração e tempo de uso, bem como a análise estatística empregada e os resultados clínicos de interesse. Foram encontrados 503 artigos dos quais 08 foram incluídos na revisão final como sendo ensaios clínicos fase II, controlados, randomizados e cegos, marcando 4 (25%) e 5 (75%) na EJ. Os principais produtos naturais avaliados foram Azadirachta indica, Garcinia mangostana, Lippia sidoides, Salvadora persica e Sesamum indicum, cuja concentração, intervalo de administração, tempo de uso, e efeitos adversos, variaram de acordo com cada estudo. Índice de placa e Índice Gengival foram os mais utilizados, bem como =5% e testes t-pareado, tStudent, Wilcoxon e Mann-Whitney. 62,5% e 50% dos produtos reduziram significativamente a presença de bioflme supragengival e gengivite, respectivamente. Os colutórios contendo o óleo essencial das folhas de L. sidoides (1%) e o extrato das folhas de A. indica (25%) podem ser indicados como tratamento adjuvante da gengivite induzida por biofilme.


INTRODUCTION
Chronic periodontal disease (CPD) has affected a large percentage of the population and has been responsible for a significant number of tooth losses, as well as for influencing the development of systemic changes like diabetes (Mealey & Ocampo, 2007), premature newborns (Davernport et al., 2002), brain lesions (Tiejian et al., 2000) and cardiovascular disorders (Genco et al., 2002).
In the United States, the early stages of CPD have affected about 53.1% of the population, whereas moderate or severe stages have been reported for 12.6% of the people (Albandar et al., 1999).In Brazil, the latest epidemiological survey, undertaken in 2003, indicated that 78.1% of adults between 34 and 44 years old showed diseased gingival tissue (Brazil, 2004).
Chronic periodontal involvement is an infectious, irreversible and destructive disease which has the contribution of the host immune system to be developed, since the production of cytokines such as interleukin-1 (IL-1), Tumor Necrosis Factor (TNF-) and prostaglandins by immune cells promotes bone resorption and destruction of conjunctive tissue by the action of osteoclasts (activated by IL-1 and TNF-) and metalloproteinases (activated by prostaglandins), respectively (Baker, 2000;Koop & Medzhitov, 2003;Teng, 2006;Alnaeeli et al., 2006).
Etiologically, CPD has been recognized to be caused by biofilm-forming microrganisms in the subgingival area of the teeth (Lovegrove, 2004).In a general way, a microbial community initially consisting of Gram-positive facultative anaerobes becomes, according to the disease progression, a community of Gram-negative strict anaerobes.Such ecological alteration is a result of changes in environmental conditions, caused by reduced availability of oxygen, increased temperature in the gingival sulcus fluid, and establishment of an alkaline environment.Primarily, microrganism colonization on the tooth surface promotes an inflammatory reaction in the adjacent gingival tissues, favoring the clinical condition known as gingivitis, which is mainly characterized by spontaneous or induced gingival bleeding (Sbordone et al., 2000).Thus, the prevention of chronic periodontal disease has been known to require constant removal of dental biofilm, either by mechanical (toothbrush) or by chemical means (mouthwashes or other topical methods).
The main chemical agents currently available are: triclosan, cetylpyridinium chloride, chlorhexidine, and natural products (Nogueira-Filho et al., 2002;Andrade et al., 2009;Yévenes et al., 2009).Hence, natural products have been proposed in an attempt to minimize undesirable effects caused by synthetic products, such as tooth staining, imbalance of the resident microbiota and altered taste, besides representing a new antimicrobial possibility that might be used in a situation of microbial resistance (Gjermo et al., 1970;Torres et al., 2000).Nevertheless, some natural products-containing agents have been evidenced to lack scientific basis supporting their use for preventing and/or removing dental biofilm and for treating induced gingivitis.
In this perspective, considering the broad antimicrobial activity provided by natural products, this study aimed to evaluate, through a systematic literature review, if there is scientific evidence ensuring the safe and effective use of natural product(s)-containing mouthwashes as adjunctive treatment of biofilm-induced gingivitis.

MATERIAL AND METHOD
A systematic review of scientific studies was undertaken according to the methodology proposed by Higgins & Green (2008).
Systematic selection of articles was performed by establishing the following criteria: Inclusion criteria -Study design: randomized clinical trials and systematic reviews.
-Sample: all ages and both genders.
-Intervention: product rinsing for any predetermined time period, concentration, quantity and dosing interval.
-Primary Outcome of interest: reduction or not in the biofilm-induced gingival inflammation levels.
Exclusion criteria -Automatically, all the studies which did not match the search strategies were excluded.
-Studies evaluating synthetic products.
-Studies evaluating other dosage forms such as dentifrice, gel etc instead of mouthwash.
-Studies whose clinical condition did not characterize the context of interest, for instance, gingivitis induced by orthodontic appliances.

Search strategies
Identification of papers was performed through systematic searches in the databases MEDLINE (from 1966); SciELO (Scientific Electronic Library Online); LILACS (Latin American and Caribbean Literature on Health Sciences) and Cochrane Library.All existing articles related to the topic and published up to September 26 th 2010 were selected for analysis.
The search strategy in MEDLINE was: (gingivitis AND natural product) OR (gingivitis AND phytotherapy) OR (gingivitis AND mouthwash) OR (gingivitis AND mouthrinse).To refine the search, the filters Humans and Clinical trial were activated.
The search strategy in SciELO, LILACS and Cochrane Library was: (gingivitis AND natural product) OR (gengivite E produto natural) OR (gingivitis AND phytotherapy) OR (gengivite E fitoterapia) OR (gingivitis AND mouthwash) OR (gengivite E enxaguatório) OR (gingivitis AND mouthrinse) OR (gengivite E colutório).Due to shortage of articles in Scielo database, the general descriptor (gingivitis) OR (gengivite) was used and all found studies were assessed.
Another strategy consisted in manual search in reference lists from the identified and selected articles.
Four examiners independently analyzed the methodological quality of the selected articles (title and abstract) to verify if they met the previously established inclusion criteria to be incorporated into the review.
Examiners agreed that in cases that did not allow identifying whether the article should be included or not (only by reading its title and abstract), it would be requested and read in full.The final verdict on which articles would be included in the study was reached by consensus.Finally, the selected studies were evaluated in accordance with an analysis protocol.

Protocol for analysis of included studies
Table 1 demonstrates the variables that were examined in the included studies.

RESULT
According to the previously set strategy, literature searches resulted in 503 articles.From that total, 18 studies met the inclusion criteria and were selected for analysis.Subsequently, after thorough TABLE 1. Protocol followed by examiners for analysis of the studies included in the systematic review.

Preliminary Analysis
Title; Main Author; Country; Language; Journal/Year of publishing

Methodological Analysis
Primary outcome of interest: anti-gingivitis effect 1. Assessment of quality of the trials: Jadad Scale (Jadad et al., 1996) The scale checks the validity of evidence on interventions and evaluates three conditions: Randomization, Blinding and Loss of follow-up.Based on these criteria, scores were assigned to the studies ranging from 0 to 5. Studies reaching a score <3 were considered of poor quality and thus excluded from this review.1. Methodological design 4. Type of blinding and sampling unit allocation 5. Profile and sample size 6.Sample loss and reasons 7. Masking of product color, taste and/or smell 8. Presence and characterization of Placebo or Control Group 9. Comparability between the experimental and control groups at the beginning of the study: group description in order to evaluate the equivalence between them at baseline 10.Citation of a pilot study 11.Quality of outcome's measurement: intra or inter-examiners calibration 12. Indexes used to assess gingival condition and amount of biofilm 13.Statistical analysis and significance level employed 14.Type of clinical trial (Phase I, II, III or IV) according to Lapa et al. (2007) 2.1.Intervention Analysis 1. Experimental product: tincture, essential oil, extract etc. 2. Product concentration 3. Dosing interval: quantity and frequency per day; at which time the product had been used.4. Time of usage (e.g.: 2 weeks) 5. Assessment intervals of gingival condition and amount of biofilm 6. Treatment adherence and adverse effects: patient's complaints about side effects or discomfort caused by the product

Analysis of Results
Verification of accuracy according to the confidence interval and sample size

Analysis of Conclusions
Attesting if the conclusion met the aims of the study

Serfaty et al. (1988)
The study was not found in full after excessive national and international searches by the Bibliographic Commuting Program (COMUT).

Pistorius et al. (2003)
It evaluated subgingival irrigation and does not represent the clinical context of interest.

Willershausen et al. (2004)
It evaluates individuals using orthodontic appliances and did not represent the clinical context of interest.

Al-Teen et al. (2006)
It evaluated orthodontic brushes combined with a natural product and did not represent the clinical context of interest.
In the Cochrane library, no systematic review relating to the topic was found.Nevertheless, 22 records of controlled trials which had already been included from other sources were identified and 02 articles not yet counted in the search were considered for analysis.

Data Synthesis
The included clinical trials were conducted in six countries: India, Nepal, Thailand, USA, New Zealand and Brazil.The years of their publications ranged from 2004 to 2009 (Table 2).
As seen in Table 2, all studies were well designed and predominantly classified as phase II, randomized, blind trials, controlled, placebo-controlled or having a comparative group.All of them were parallel studies, except for the study of Khalessi et al. (2004), designed as crossover.Jadad Scale scores were "4" in 25% of the studies and "5" in 75%, which reflected high methodological quality.
Identification of potentially relevant studiesreading the titles and abstracts -performed by four examiners The size of samples differed substantially in each study, without equivalence in the distribution of TABLE 2. General aspects, methodological design and quality of the studies included in the systematic review.gender and age (ranged from 12 to 69 years old).All studies included men and women in their samples, except the study of Asokan et al. (2009), in which only men were included (Table 3).

Khalessi
It was verified that 50% of the studies had no sample loss, whereas the other ones had a sample loss varying from 4.76% to 29.0% of the total sample size.Table 3 shows the reasons why participants withdrew from the studies.
The investigation of a calibration of the study's examiners in order to verify inter-or intraexaminer agreement indicated that 50% of them did not mention a calibration process; 25% cited an intraexaminer calibration but did not mention the indexvalue and, finally, 25% mentioned an intra-examiner calibration, reaching a k statistics of 0.86.
As regards statistical analysis, significance level was unanimously set at 5% and the mostly used tests were Paired t, Student's t, Wilcoxon and Mann-Whitney (Table 3).
Table 4 shows the intervention profile, including plant species, forms of masking, plaque and gingival indexes employed, assessment intervals, adverse effects and outcomes of interest.
The period of product use varied a lot in each study, oscillating from 10 to 84 days (12 weeks), and assessment intervals were consequently diversified.With respect to evaluation measures, Plaque (Silness & Löe, 1964) and Gingival Index (Löe, 1967) were most employed.
The main adverse effects reported by the participants related to product use were bitter taste, burning sensation and mild nauseas.
Even though the studies of Khalessi et al. ( 2004

DISCUSSION
Systematic reviews have been recommended as the best source of evidence to guide clinical decisions (Lavis et al., 2004).A synthesis by this type of study leads to a comprehensive answer to clinical questions, with least possible systematic errors and with high internal validity (Mickenautsch, 2010).In this perspective, there is a clear need to verify if there is scientific evidence to indicate natural product(s)containing mouthwashes as adjunctive treatment against biofilm-induced gingivitis.
The of antimicrobial mouthrinses as an adjuvant to mechanical methods for controlling dental biofilms and gingival inflammation is well established (Cortelli, 2010;Gunsolley, 2010;Marsh, 2010).In addition, some clinical trials have demonstrated that the clinical value of a mouthrinse may meet or exceed the value of flossing for interproximal cleaning (Sharma et al., 2002;Bauroth, 2003).
A recent systematic review aimed at evaluating the efficacy of anti-plaque and anti-gingivitis mouthrinses and at determining the clinical relevance of this evidence found that the clinical benefits of mouthrinses containing chlorhexidine and essential oils are similar to the benefits of oral prophylaxis and oral hygiene instructions at six-month recall appointments (Gunsolley, 2010).
Among the products that presented significant anti-gingivitis activity are two commercial products containing oil of S. indicum and extract of S. persica and three products prepared by the own researchers or collaborators from extracts of A. indica, G. mangostana and from the essential oil of L. sidoides.
It is noteworthy that the type of plant byproduct (crude extract, essential oil, fractions etc), as well as the used part of the plant, influences the biological activity of the product.Of the studies that showed satisfactory results, only Rassameemasmaung et al. (2007), Botelho et al. (2008) and Botelho et al. (2009) specify which part of the plant was used in the mouthrinse formulation.
Sesame indicum oil is relatively high in unsaponifiable substances.The unsaponifiable fraction, a class of substances not found in other fats (sesamin or sesamolin) can probably protect the oral cavity from infection and inflammation by its antioxidant property (Asokan et al., 2009).By performing a microbiological assessment, the authors found a considerable reduction in the colony counting of aerobic microrganisms promoted by the product (Asokan et al., 2009).
Azadirachta indica presents a vast array of biologically active compounds that are chemically diverse and structurally complex.All parts of the plant -leaves, flowers, seeds, fruits, roots and bark -have been traditionally used for the treatment of inflammation, infections, fever, skin diseases and dental disorders (Subapriya & Nagini, 2005).A study recorded the properties of 150 triterpenoids from A. indica leaves (Akhila & Rani, 1999).
The extract from the leaves of A. indica at 25% was tested, reaching significant reductions in the amount of biofilm and gingivitis when compared to the baseline (Botelho et al. 2008).The same result was not found at a concentration of 1% (Sharma et al., 2007).
Garcinia mangostana L. has many pharmacologically active compounds, especially its pericarp.It is rich in a variety of oxygenated and prenylated xanthones (Peres & Nagem, 1997;Peres et al., 2000).This species has been considered to have antioxidant, anti-inflammatory, cytotoxic, and antifungal activities (Jung, 2006).Recently, three new compounds trivially named garcimangosxanthone A-C (1-3) have been isolated along with fourteen known xanthones from the pericarp of this plant (Zhang et al., 2010).
Besides its anti-gingivitis efficacy, the mouthwash of G. mangostana L. pericarp was also confirmed to significantly reduce the levels of volatile sulfur compounds when compared to the baseline and to a placebo.Thus, such product has also been considered an alternative option in treating malodor (Rassameemasmaung et al., 2007).
A study investigated the chemical composition and the antimicrobial activity of the essential oil from L. sidoides.Thymol has been the main component found and might be associated with the antimicrobial activity presented by that natural product (Fontenelle et al., 2007).Botelho et al. (2009) also demonstrated that the essential oil from the leaves of L. sidoides (1%) is responsible for a decrease in the levels of salivary mutans streptococci.
Limitations could be found in the studies of Khalessi et al. (2004), Southern et al. (2006), Rassameemasmaung et al. (2007) and Asokan et al. (2009) as the concentration of the experimental products were not cited.Thus, it is inappropriate to indicate these products without knowing their concentrations, since the concentration of the product may influence its biological action.Another shortcoming relates to the absence of a standard mouthwash protocol, hindering inter-study comparisons, with regard to the quantity of product to be rinsed, and the rinsing time and frequency.
The names of the herbaria where the voucher specimens were deposited, as well as their registration numbers, were not reported in the methodologies of the reviewed articles, except for the studies of Lauten et al. (2005), Botelho et al. (2008) and Botelho et al. (2009).Such information is important in view of the need of highlighting data of the studied species, once the plant needs to be reliably identified.
Concerning the safety for clinical use, it is emphasized that only the study of Lauten et al. (2005) used phase-I experimental clinical model to verify possible side effects of the product in healthy individuals, as seen in Table 2.This step must be considered indispensable for the development of subsequent clinical trials, such as phase II, guaranteeing thus the development of an effective and safe It is considered that for phase II clinical trials the sample must consist of at least 30 individuals in each group (experimental and control or placebo arms) (Lapa et al., 2007); however, three out of the eight studies did not obey this sample size criterion (Table 3).Ideally, clinical trials should be large enough to reliably detect the smallest possible differences in the primary outcome with treatment that are considered clinically worthwhile (Kirby et al., 2002).
Nonetheless, the criteria for sample exclusion in the studies were well designed, such as individuals who made use of systemic antibiotics or non-steroidal anti-inflammatory drugs or used mouthwashes prior to the study, with complicating systemic conditions or hormonal alteration (e.g.pregnant women), had less than a determined number of dental elements, used orthodontic brackets, among others.This selection becomes crucial since it is important to prevent any type of bias that could influence the results.
Study subjects can modify their behavior or their way of reporting outcomes (including adverse effects) systematically if they are aware of the treatment they are going to receive (Panutti, 2009).Thus, the masking of products as regards color, taste, smell and administration methods is very important.This procedure ensures that neither the subject under study nor the examiner identifies the intervention arm and therefore be biased.In this sense, only the studies of Botelho et al. (2008) and Botelho et al. (2009) masked the products (Table 4).
Besides this possibility of bias, the existence of the Hawthorne effect (Panutti, 2009) should still be considered; it is a form of reactivity whereby subjects improve or modify an aspect of their behavior being experimentally measured simply in response to the fact that they are being studied.In this case, there would be an improvement in the oral hygiene, interfering in the results.This effect was considered by Southern et al. (2006), Rassameemasmaung et al. (2007) and Botelho et al. (2009).
Examiner reliability is a very significant issue in research.Different measures of reliability may be used to assess intra-or inter-examiner agreement.In spite of that, four out of the eight studies did not mention any calibration process in order to assure the reproducibility of measurements.
An inferential statistical analysis to evaluate which product was most effective against gingivitis would be ideal.However, due to those differences in methodological criteria, such as interval and quantity of the product to be rinsed, time of usage, different forms of assessment, among others, equivalence of contexts for analysis could not be detected, which might reflect biases.
Jadad Scale was adopted in this review as it checks the validity of evidence on interventions and evaluates methodological quality, allowing thus an objective assessment (Jadad et al., 1996).Several studies, including systematic reviews, have already embraced this validated evaluation tool (Linde et al., 2001;Hu et al., 2002;Beek et al., 2004;Huntley & Ernst, 2004).
Summarizing, S. indicum, G.mangostana, and S. persica showed reduction in the amount of biofilm and gingivitis levels.Nevertheless, this review cannot indicate with certainty their general use as adjunctive treatment for biofilm-induced gingivitis since the authors did not mention the concentration and/or the used part of the natural product, even though two of these products have already been marketed.
However, there is strong evidence to indicate the essential oil from the leaves of L. sidoides (1%) and the extract from the leaves of A. indica (25%) to significantly reduce biofilm and gingival inflammation levels.
It has been recommended the conduction of other clinical trials in this line of research evaluating different concentrations and time of usage, relating to the products reported in this study, proposing a protocol for standardization of interventions (quantity, frequency and duration of rinsing) and, finally, investigating adverse effects arising from prolonged use on the teeth and oral mucosa, as well as substantivity and inactivation in the gastrointestinal tract.

FIGURE 1 .
FIGURE 1. Flowchart of the search strategy.

TABLE 3 .
Asokan et al. (2009)al.(2007)meemasmaung et al. (2007)andAsokan et al. (2009)did not mention the concentration of the experimental product, mouthwashes containing A. indica, G. mangostana, L. sidoides and S. indicum were capable of reducing both the amount of biofilm and the gingival inflammation levels.Moreover, the alcoholic extract from Salvadora persica was capable of reducing gingivitis but did not hamper biofilm accumulation.Characterization of the study population, sample loss, examiner calibration and statistical analysis of the studies included in the systematic review.

TABLE 4 .
Characterization of products, forms of masking, indexes employed, assessment intervals and outcomes of interest of the studies included in the systematic review.
Statistically significant reduction (P>.05) in the amount of biofilm and / or gingival inflammation levels, respectively, promoted by the experimental product when compared to the initial condition (baseline).The symbol (+) represents a statistically significant reduction, whereas the symbol (-) represents no reduction in the values.AB: Amount of Biofilm; GI: Gingival Inflammation.Rev.Bras.Pl.Med., Botucatu, v.14, n.4, p.700-711, 2012.

TABLE 4 .
Characterization of products, forms of masking, indexes employed, assessment intervals and outcomes of interest of the studies included in the systematic review.Statistically significant reduction (P>.05) in the amount of biofilm and / or gingival inflammation levels, respectively, promoted by the experimental product when compared to the initial condition (baseline).The symbol (+) represents a statistically significant reduction, whereas the symbol (-) represents no reduction in the values.AB: Amount of Biofilm; GI: Gingival Inflammation.