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Year : 2013  |  Volume : 24  |  Issue : 5  |  Page : 533-536
Effect of Lippia sidoides in mouthrinses on de novo plaque formation: A double-blind clinical study in humans

1 Departments of Periodontology, School of Dentistry, University of Fortaleza, UNIFOR, Fortaleza, CE, Brazil
2 Department of Organic Chemistry, School of Pharmacology, University of Fortaleza, UNIFOR, Fortaleza, CE, Brazil

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Date of Submission29-Oct-2010
Date of Decision26-Mar-2013
Date of Acceptance18-Apr-2013
Date of Web Publication21-Dec-2013


Aim: The aim of this study was to evaluate the antiplaque effect of Lippia sidoides (LS) by in vivo investigation.
Materials and Methods: Ten healthy volunteers participated in a cross-over, double-blind clinical study, using a 3-day partial-mouth plaque accumulation model. The participants abolished any method of mechanical oral hygiene and they were randomly assigned initially to use just the following mouth rinses: Distilled water (negative control group), 0.12% chlorhexidine digluconate (positive control group) or 10% LS (test group). The plaque index was recorded in the six anterior upper teeth at the end of the trial and the one-way ANOVA and Bonferroni tests were used to estimate the difference among groups.
Results: The clinical results did show statistically significant difference among three groups (P < 0.05), favoring the positive control group and test group, however, no difference in efficacy was found between them (P > 0.05).
Conclusions: The mouth rinses containing 0.12% chlorhexidine digluconate and 10% LS were equally able to inhibit plaque re-growth.

Keywords: c0 hlorhexidine, dental plaque, mouth rinses, plant extracts, prevention

How to cite this article:
Rodrigues IC, de Oliveira DB, de Menezes PB, da Costa FN, Carlos MX, Pereira Sd. Effect of Lippia sidoides in mouthrinses on de novo plaque formation: A double-blind clinical study in humans. Indian J Dent Res 2013;24:533-6

How to cite this URL:
Rodrigues IC, de Oliveira DB, de Menezes PB, da Costa FN, Carlos MX, Pereira Sd. Effect of Lippia sidoides in mouthrinses on de novo plaque formation: A double-blind clinical study in humans. Indian J Dent Res [serial online] 2013 [cited 2021 Jul 28];24:533-6. Available from:
The role of dental plaque in the etiology of periodontal diseases is well recognized. [1] Regular plaque removal by effective mechanical tooth cleaning is the main goal of the prevention of gingival inflammation, however, this concept is difficult to implement by many individuals. [1],[2] Therefore, in addition to mechanical oral hygiene, the use of antiseptics is strongly recommended and performed. [1],[3]

Chlorhexidine is considered the gold standard chemical agent, showing positive results, by inhibition or retarding the bacterial proliferation. [1],[2],[4] However, due to undesirable effects after prolonged use, such as pigmentation and taste disturbance, [1] varied phytotherapic agents had been researched recently showing both encouraging [5],[6],[7] and unsatisfactory results. [8],[9],[10]

Lippia sidoides (LS) is a typical shrub commonly found in the Northeast of Brazil. Its camphoric foliage is indicated as topic antisseptic agent for skin and mucosa surface and also for throat infections. [11] Essential oil obtained from this phytotherapic compound is constituted mainly by thymol and carvacrol and other substances, such as felandreno, cariofileno, p-cimeno and mirceno. [11],[12]

Previous studies indicated that these major components had showed potent antimicrobial activity against fungi and bacteria [11],[12],[13] and reduced the severity of gingivitis, bacterial plaque and histological inflammatory infiltrate in dogs. [14] Recently, controlled short-term clinical studies in humans showed an antigingivitis effect of LS. [5],[15]

Although these initial studies showed positive results, sufficient data about preventive clinical effect of LS on de novo plaque formation are unclear. Thus, a clinical study in humans was conducted to evaluate the in vivo antiplaque effect of a mouth rinse containing LS in comparison to chlorhexidine digluconate.

   Materials and Methods Top


Ten young dental students from the University of Fortaleza (5 female and 5 male aged 19-23 years) were enrolled in this study. All subjects had at least 24 natural teeth, showed no signs of periodontitis, had no caries or extensive dental restorations and had not been exposed to systemic antibiotic treatment during past 6 months. Participants with medical disorders, as well as smokers and pregnant women were excluded from the trial. All students, randomly screened, were informed about the nature of the study and signed an informed consent form in compliance with the guidelines of the Brazilian Health Council and Helsinki Declaration of 1975. The protocol was approved by the University's Ethics Committee (Report Coética no. 300/2007, University of Fortaleza).

Test and control products

The control and test mouth rinses were formulated and packed into bottle in the Pharmaceutics' Laboratory at the University of Fortaleza. The bottles were previously coded to warrant that neither the examiner nor the participants knew their content, which was revealed by the pharmacist after the study was completed. All students used all mouth rinses in alternate periods, according to a ­cross-over study.

Preparation of the mouth rinses

The essential oil from LS was extracted by steam distillation in the Pharmaceutics' Laboratory at the University of Fortaleza. Initially 1 ml of essential oil was diluted in 9 ml of ethyl alcohol (1:9), preparing a 10% mixture (V/V) (test group). [13] A mouth rinse containing just distilled water (negative control) and other containing 0.12% chlorhexidine digluconate (positive control) were formulated too. In all groups, a very small amount of menthol (flavoring), color and conserving agent were added.

Clinical design

This study was a randomized, double-blind comparison of three cross-over groups of dental students performed in three experimental phases of 3 days each with a 1-month washout interval between them until all subjects had rinsed with each formulation. To standardize the groups, the participants were submitted to a meticulous evaluation (pre-experimental phase) to score the plaque index (PLI) [16] of each tooth. All teeth of each subject were polished and flossed by the examiner to eliminate dental plaque remnants. The importance of oral hygiene was strongly reinforced.

Thirty days after the initial phase, the volunteers were randomly assigned to three groups by random permutation of three and the experimental phase began. On day 0 of both experimental periods, PLI was recorded. During each 3-day experimental period, the participants were instructed to abstain from all forms of mechanical oral hygiene. A bottle containing 100 ml mouth rinse was given to all students and they were instructed to rinse 10 ml for 60 s, twice daily (in the morning and in the evening), and then expectorate it. In addition to verbal instructions, the students were given written recommendations to follow at home. On the last day of each period (3 rd day), the PLI was recorded and the teeth were polished with pumice.

Clinical assessment

The PLI was recorded by the same examiner on the mesiobuccal, buccal, distobuccal and lingual surfaces of the experimental six upper teeth. The plaque was disclosed using a 1% erythrosine solution and the values of four sites of each tooth were recorded to obtain the PLI means. Then, the means for the six experimental teeth were calculated to determine mean index of each volunteer. Intra-examiner agreement was calculated by repeating the measurements in 10 patients, obtaining 0.84 Kappa co-efficient.

Statistical analysis

The one-way ANOVA and Bonferroni test were used to estimate the difference among groups on day 3 (P < 0.05). However, for illustration, the results are presented as mean and standard deviation.

   Results Top

All subjects completed the clinical trial. The test mouth rinse had good acceptance and did not show adverse effects, such as abscess, ulcerations or allergic reactions.

At the 3 rd day, plaque were present in all groups, but the difference between them was statistically significant (P = 0.0324), favoring the positive control and test groups [Table 1]; however, the PLI means did not differ statistically between them (P = 0.0867).
Table 1: PLI means and SD on day 3 for the negative control, positive control and test groups

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Comparing means for each dental surface, there was a statistically significant difference in the PLI index for all surfaces favoring the positive control and test groups (P = 0.0023, mesiobuccal site; P = 0.0045, distobuccal site; P = 0.0047, buccal site; P = 0.0016, lingual site); however, the PLI means between them did not differ statistically (P = 0.065) [Table 2].
Table 2: PLI means and SD, by dental surface, on day 3 for the negative control, positive control and test groups

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   Discussion Top

This paper presents the data of a short-term, cross-over, de novo plaque re-growth study, involving the replacement of mechanical plaque control by mouthrinsing, comparing an allopathic substance with herbal agent. The inability of adult population to perform adequate mechanical tooth cleaning has stimulated the search for chemotherapeutic agents in mouth rinses to improve plaque control and prevent gingivitis. [1],[2],[3],[4]

Among a variety of antiseptic agents, chlorhexidine digluconate has been used and tested for many years and its long-term efficacy and safety has been confirmed in several "in vivo" studies. [1],[2],[4],[17] Likewise, the absence of adverse effects using the herbal agent showed that it was well tolerated, supporting safety for the clinical use. These results were already expected once biocompatibility of LS was reported previously, [14] although mild and transient burning after using mouth rinse containing this natural agent had been related in the literature. [5]

The study population comprised dental students, which is an interesting group for this kind of study, [1],[4],[18] taking into consideration that they can be easily controlled in terms of compliance. A point that should not be overlooked is the sample size of the present work. However, studies with similar aims have been published with sample sizes comparable to that of this study. [4],[18]

The purpose of the present work was to study plaque accumulation event and not gingival inflammation, in which longer periods of evaluation is necessary. [1],[4] In this way, based on previous work, [2],[19] a 3-day plaque accumulation model was used. In order to reduce the risk for a carryover effect, a washout period of 1 month was adopted. [20]

In this study, the Quigley and Hein, [16] PLI was used due to its sensitivity to detect small deposits of plaque. [15],[18] However, the cut-off between the scores can be difficult to assess and could interfere in the results, so calibration of examiners was performed to solve this problem assuring the confidence of the results. [15] Other studies recorded plaque accumulation on full mouth, using a similar PLI. [1],[2],[4] This fact could explain the differences of values on plaque accumulation using chlorhexidine or placebo solution in comparison to present work.

In vitro studies showed that LS was effective in inhibiting the growth of oral pathogens, [12],[13] which allow us to deduce that this phytotherapic compound could be used as antiplaque agent. This fact was confirmed in this study in which, starting from a plaque-free dentition, LS presented significantly less plaque accumulation than control group. In addition, the results showed that there was no significant difference in 72 h plaque development whether the subjects rinsed with LS or chlorhexidine. This result is in concordance with the study performed by Botelho et al. [5]

The concentration of LS used in the present work was based in a previous in vitro study, in which solutions containing 2%, 5% or 10% showed inhibition rates similar to 0.12% chlorhexidine. [13] Because in vitro conditions do not completely mimetize the oral environment and a part of mouth rinse is expectorated and due to other factors, [15] a higher concentration was chosen. In spite of this, another clinical study showed an antiplaque effect of 1% LS-based mouth rinse using a 7-day treatment regimen. [5] However, it is interesting to note that this study did not include a negative control group that could induce a bias acting on results.

Other clinical study demonstrated that a gel containing 10% LS was not a good antiplaque agent. [15] Perhaps, the mouth rinse by itself presented a mechanical effect and the bioavailability of the phytotherapic compond might be higher than in the gel. In that study 21-day partial-mouth experimental model of gingivitis was used, in which the test gel was placed on the toothshield in a non-diluted way and it is possible that a solubilization by saliva or mechanical action by toothbrush in order to have an antibacterial effect would be necessary. [8]

Further studies are needed to establish whether LS is effective in the treatment of gingivitis too, comparing to chlorhexidine in longer periods. It will be important to identify the real benefits of LS as a therapeutic and preventive agent for periodontal diseases, in addition to its common use in popular medicine.

   Conclusion Top

The mouthrinse containing 10% LS has an antiplaque effect comparable to that of a 0.12% chlorhexidine digluconate solution.

   References Top

1.Lorenz K, Bruhn G, Heumann C, Netuschil L, Brecx M, Hoffmann T. Effect of two new chlorhexidine mouthrinses on the development of dental plaque, gingivitis, and discolouration. A randomized, investigator-blind, placebo-controlled, 3-week experimental gingivitis study. J Clin Periodontol 2006;33:561-7.  Back to cited text no. 1
2.Van Strydonck DA, Timmerman MF, van der Velden U, van der Weijden GA. Plaque inhibition of two commercially available chlorhexidine mouthrinses. J Clin Periodontol 2005;32:305-9.  Back to cited text no. 2
3.Baehni PC, Takeuchi Y. Anti-plaque agents in the prevention of biofilm-associated oral diseases. Oral Dis 2003;9:23-9.  Back to cited text no. 3
4.Quirynen M, Soers C, Desnyder M, Dekeyser C, Pauwels M, van Steenberghe D. A 0.05% cetyl pyridinium chloride/0.05% chlorhexidine mouth rinse during maintenance phase after initial periodontal therapy. J Clin Periodontol 2005;32:390-400.  Back to cited text no. 4
5.Botelho MA, Bezerra Filho JG, Correa LL, Fonseca SG, Montenegro D, Gapski R, et al. Effect of a novel essential oil mouthrinse without alcohol on gingivitis: A double-blinded randomized controlled trial. J Appl Oral Sci 2007;15:175-80.  Back to cited text no. 5
6.Hebbar SS, Harsha VH, Shripathi V, Hegde GR. Ethnomedicine of Dharwad district in Karnataka, India - Plants used in oral health care. J Ethnopharmacol 2004;94:261-6.  Back to cited text no. 6
7.Pai MR, Acharya LD, Udupa N. Evaluation of antiplaque activity of Azadirachta indica leaf extract gel - A 6-week clinical study. J Ethnopharmacol 2004;90:99-103.  Back to cited text no. 7
8.Salgado AD, Maia JL, Pereira SL, de Lemos TL, Mota OM. Antiplaque and antigingivitis effects of a gel containing Punica granatum Linn extract: A double-blind clinical study in humans. J Appl Oral Sci 2006;14:162-6.  Back to cited text no. 8 Oliveira SM, Torres TC, Pereira SL, Mota OM, Carlos MX. Effect of a dentifrice containing Aloe vera on plaque and gingivitis control. A double-blind clinical study in humans. J Appl Oral Sci 2008;16:293-6.  Back to cited text no. 9
10.Pereira SL, Barros CS, Salgado TD, Filho VP, Costa FN. Limited benefit of copaifera oil on gingivitis progression in humans. J Contemp Dent Pract 2010;11:E057-64.  Back to cited text no. 10
11.Lemos TL, Craveiro AA, Alencar JW, Matos FJ, Clarck AM, Mac-Chesney JD. Antimicrobial activity of essential oil of Brazilian plants. Phytother Res 1990;4:82-4.  Back to cited text no. 11
12.Botelho MA, Nogueira NA, Bastos GM, Fonseca SG, Lemos TL, Matos FJ, et al. Antimicrobial activity of the essential oil from Lippia sidoides, carvacrol and thymol against oral pathogens. Braz J Med Biol Res 2007;40:349-56.  Back to cited text no. 12
13.Rodrigues IS, Tavares VN, Pereira SL, Rocha MM, Costa FN. Antiplaque effect of Lippia sidoides hidroalcholic extract. An in vitro study. Rev Periodontia 2007;17:82-6.  Back to cited text no. 13
14.Girão VC, Nunes-Pinheiro DC, Morais SM, Sequeira JL, Gioso MA. A clinical trial of the effect of a mouth-rinse prepared with Lippia sidoides Cham essential oil in dogs with mild gingival disease. Prev Vet Med 2003;59:95-102.  Back to cited text no. 14
15.Rodrigues IS, Tavares VN, Pereira SL, Costa FN. Antiplaque and antigingivitis effect of Lippia sidoides: A double-blind clinical study in humans. J Appl Oral Sci 2009;17:404-7.  Back to cited text no. 15
16.Quigley GA, Hein JW. Comparative cleansing efficiency of manual and power brushing. J Am Dent Assoc 1962;65:26-9.  Back to cited text no. 16
17.Brecx M, Netuschil L, Reichert B, Schreil G. Efficacy of Listerine, Meridol and chlorhexidine mouthrinses on plaque, gingivitis and plaque bacteria vitality. J Clin Periodontol 1990;17:292-7.  Back to cited text no. 17
18.Franco Neto CA, Parolo CC, Rösing CK, Maltz M. Comparative analysis of the effect of two chlorhexidine mouthrinses on plaque accumulation and gingival bleeding. Braz Oral Res 2008;22:139-44.  Back to cited text no. 18
19.Van der Weijden GA, Timmerman MF, Novotny AG, Rosema NA, Verkerk AA. Three different rinsing times and inhibition of plaque accumulation with chlorhexidine. J Clin Periodontol 2005;32:89-92.  Back to cited text no. 19
20.Newcombe RG, Addy M, McKeown S. Residual effect of chlorhexidine gluconate in 4-day plaque regrowth crossover trials, and its implications for study design. J Periodontal Res 1995;30:319-24.  Back to cited text no. 20

Correspondence Address:
Sérgio Luís da Silva Pereira
Departments of Periodontology, School of Dentistry, University of Fortaleza, UNIFOR, Fortaleza, CE
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.123360

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