Antiplaque and antigingivitis effect of Lippia Sidoides: a double-blind clinical study in humans.

OBJECTIVES: The antiplaque and antigingivitis effect of Lippia Sidoides (LS) was evaluated in this in vivo investigation.
MATERIAL AND METHODS: Twenty-three subjects participated in a cross-over, double-blind clinical study, using 21-day partial-mouth experimental model of gingivitis. A toothshield was constructed for each volunteer, avoiding the brushing of the 4 experimental posterior teeth in the lower left quadrant. The subjects were randomly assigned initially to use either the placebo gel (control group) or the test gel, containing 10% LS (test group).
RESULTS: The clinical results showed statistically significant differences for plaque index (PLI) (p<0.01) between days 0 and 21 in both groups, however only the control group showed statistically significant difference (p<0.01) for the bleeding (IB) and gingival (GI) index within the experimental period of 21 days. On day 21, the test group presented significantly better results than the control group with regard to the GI (p<0.05).
CONCLUSIONS: The test gel containing 10% LS was effective in the control of gingivitis.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

Gingivitis is one of the most frequent periodontal diseases, affecting more than 90% of the population, regardless of age, sex or race21. Brazilian epidemiologic studies have shown a high prevalence of gingival inflammation, ranging from 74% to 100%, although media individual percent of gingival bleeding vary from 28% to 35%16.

Dental plaque can be visible on dental surfaces after 1 or 2 days if oral hygiene procedures are unsatisfactory. The localization and rate of its formation are varied, depending of determinant factors such as diet and salivary flow20. After 10 to 21 days of supragingival dental plaque accumulation, clinical signs of gingivitis appear, such as redness, edema and a tendency to marginal bleeding on gentle probing20.

Chemotherapeutic agents had been used as antiplaque agent, avoiding development of gingivitis13. Chlorhexidine is considered as the gold standard, showing positive results by inhibition or retarding the bacterial proliferation15. However, due to undesirable effects after prolonged use, such as pigmentation and taste disturbance11, phytotherapic agents have been investigated as alternatives4,6,14,16.

Lippia sidoides (LS) is a typical shrub commonly found in the Northeast of Brazil. Its camphorate foliage is indicated as topic antiseptic agent for skin and mucosa surface and also for throat infections9. Essential oil obtained from this phytotherapic is constituted mainly by thymol (56.7%), carvacrol (16.7%) and other substances, such as felandreno, cariofileno, p-cimeno and mirceno3,9. Previous studies indicated that these major components had showed potent antimicrobial activity against fungi and bacteria3,7,9, including species of the genus Streptococcus mutans 3 and reduced the severity of gingivitis, bacterial plaque and histological inflammatory infiltrate in dogs5. In an only controlled short-term clinical study in humans, a LS essential oil mouthrinse reduced bacterial plaque and gingival inflammation4.

Although these initial studies showed positive results, there are no sufficient data about the clinical effects of LS on gingivitis. Thus, a cross-over, double-blind clinical study in humans was conducted to evaluate the in vivo antiplaque and antigingivitis effect of a gel containing LS.

MATERIAL AND METHODS

Subjects

Twenty-six undergraduate dental students from the University of Fortaleza (13 female and 13 male, aged 19 to 25 years) were enrolled in this study. All subjects had at least 20 natural teeth, among which the 4 posterior teeth in the lower left quadrant (experimental teeth). All randomly screened students were informed about the nature of the study and signed an informed consent form in compliance with the guidelines of the Brazilian National Health Council. Participants with medical disorders and under antimicrobial therapy, as well as smokers, pregnant women and individuals presenting a probing depth ≥ 3 mm associated with any mandibular teeth were excluded from the trial. Subjects with retentive factors of dental plaque, such as carious cavity and restoration excess in the test area were not included in this clinical study as well. The protocol was approved by the University's Ethics Committee (Report Coética no. 205/2005, University of Fortaleza) and an ISRCTN register was assigned (no. 26530238).

Toothshield Fabrication

An alginate impression of the experimental teeth was taken and poured in die stone to obtain casts. On each stone cast, a 0.3-mm-thick thermoplastic mouthguard material space was made using a vacuum former. Upon the spacer, an individual toothshield was made of a 2-mm-thick thermoplastic mouthguard material, using the same vacuum former. The toothshield was trimmed 2 mm beyond the gingival margin to assure that gel would be in contact with the gingival margin of the experimental teeth during toothbrushing of the remaining teeth.

Control and Test Products

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

Preparation of the Gels

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 ethylic alcohol (1:9), preparing a 10% mixture. As much as 50 g of carboxymethylcellulose was added to the LS infusion (1000 mL) and the mixture was kept boiling until its complete dissolution to obtain the 10% gel concentration. A glycerin/ethanol mixture (50 mL: 50 mL) was added and the solution was vigorously stirred during 15 min until gel formation. A very small amount of menthol (flavoring) and conserving agent were then added. The control gel had the same formulation except for the LS extract.

Clinical Design

This study was a randomized, double-blind comparison of 2 crossover groups of dental students performed in 2 experimental phases of 21 days each with a 1-month washout interval between them. A partial mouth experimental model was used18. To standardize the groups, the participants were submitted to a meticulous evaluation (pre-experimental phase) to score the Plaque Index (PLI)19, the Gingival Index (GI)10 and the Bleeding Index (BI)1 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 2 groups by random permutation of three and the experimental phase began. On day 0 of both experimental periods, PLI, GI and BI were recorded. A personal "kit" containing a toothshield, a tube with 90 g of control or test gel and a commercial dentifrice with no antiinflammatory properties was given to all students (Sorriso, Kolynos do Brazil Ltda., Osasco, SP, Brazil). During each 21-day experimental period, the participants were instructed to fill the toothshield with the gel prior to insertion in the mouth and seat it over the experimental teeth three times a day for at least 1 min. The students refrained from brushing the test quadrant, while the other teeth were normally brushed three times a day using the commercial dentifrice. In addition to verbal instructions, the students were given written recommendations to follow at home. On the last day of each period (21st day), the indexes were recorded and the teeth were polished with pumice.

Clinical Assessment

All the indexes were recorded by the same examiner on the mesiobuccal, buccal, distobuccal, mesiolingual, lingual and distolingual surfaces of the experimental teeth. The values of six sites of each tooth were recorded to obtain the PLI, GI and BI means. Then, the means for the four experimental teeth were calculated to determine index means of each volunteer. Intra-examiner agreement for all indexes was calculated by repeating the measurements in 10 patients, with at least 1 h of interval2. The Kappa coefficient was used to verify the agreement between the examinations. PLI, GI and BI means were 0.75, 0.83 and 0.86, respectively.

Statistical Analysis

The Mann-Whitney non-parametric test was used to estimate the difference between control and test group on days 0 and 21 (p<0.05). In each group, the mean scores of all indexes were compared between baseline and the end of the trial by the Wilcoxon test (p<0.01). However, for illustration, the results are presented as mean and standard deviation.

RESULTS

Twenty-two subjects completed the clinical trial. Four students were excluded from the study during the experimental phase due to third molar extraction. The test gel had good acceptance and did not show adverse effects, such as abscess, ulcerations or allergic reactions.

On day 0 the control and test groups did not show statistically significant difference from each other with respect to PLI (p=0.9813), BI (p=0.4455) and GI (p=0.4455) means. These results indicated that both groups were well balanced at baseline (Tables 1, 2 and 3). At the 21st day, plaque (p=0.3242) and gingival bleeding (p=0.0707) were present in both groups, but the difference between them was not statistically significant (Tables 1 and 2). However, in this same period, the GI means differed statistically, favoring the test group (p=0.0299) (Table 3).

TABLE 1

Plaque index (PLI) means and standard deviation on day 0 and day 21 for the control and test groups

	Control	Test
Day 0	1.61 + 0.45 A,a	1.71 + 0.37 A,a
Day 21	2.56 + 0.71 A,b	2.39 + 0.74 A,b

Means followed by the same uppercase letters on day 0 and day 21 do not differ statistically (p>0.05). Means followed by different lowercase letters in the same column differ statistically (p<0.01).

TABLE 2

Bleeding Index (BI) means and standard deviation on day 0 and day 21 for the control and test groups

	Control	Test
Day 0	0.02 + 0.05 A,a	0.01 + 0.04 A,a
Day 21	0.13 + 0.14 A,b	0.06 + 0.08 A,a

Means followed by the same uppercase letters on day 0 and day 21 do not differ statistically (p>0.05). Means followed by different lowercase letters in the same column differ statistically (p<0.01).

TABLE 3

Gingival Index (GI) means and standard deviation on day 0 and day 21 for the control and test groups

	Control	Test
Day 0	0.05 + 0.10 A,a	0.03 + 0.09 A,a
Day 21	0.31 + 0.35 A,b	0.13 + 0.16 B,a

Means followed by the same uppercase letters on day 0 and day 21 do not differ statistically (p>0.05). Means followed by different lowercase letters in the same column differ statistically (p<0.01).

Comparing the means between day 0 and day 21 in each group, there was a statistically significant difference in the PLI index – control group (p=0.0003) and test group (p=0.0005) (Table 1). The control group showed statistically significant difference for the BI (p=0.0049) and GI (p=0.0017) indexes (Tables 2 and 3). However, for the test group, BI (p=0.0277) and GI (p=0.0277) means did not differ significantly between baseline and trial end periods (Tables 2 and 3).

DISCUSSION

The inability of adult population to perform adequate mechanical tooth cleaning has stimulated the search for chemotherapeutic agents in mouthrinses or added to dentifrices to improve plaque control and prevent gingivitis13. The gold standard in this scope is the use of chlorhexidine. Studies showed that two diary rinsing with 10 mL of a 0.12% chlorhexidine solution inhibited dental plaque, calculus and gingivitis in humans8,12.

The absence of adverse effects using test gel showed that it was well tolerated, supporting safety for the clinical use. These results were already expected once biocompatibility of LS was reported previously5, although mild and transient burning after using mouthrinse containing this natural agent had been related in the literature4.

An in vitro study showed that LS was effective in inhibiting the growth of oral pathogens3, which allow us to deduce that this phytotherapic could be used as antiplaque agent. However, this antibacterial effect was obtained in vitro conditions, which does not mimic completely the oral environment22. This fact was confirmed in this study in which both groups did not avoided plaque accumulation, although the test group had presented at day 21 less plaque, but not significantly, as compared control group.

Since the composition of the gels differed only for the presence of LS, it can be inferred that this phytotherapic agent had no effect on the bacterial growing in vivo. However, Botelho, et al.4 (2007) showed an antiplaque effect of a 1% LS-based mouthrinse similar to chlorhexidine, using a 7-day treatment regimen. Based on a previous study16, the present work tested a gel containing 10% LS. Perhaps, mouthrinsing per se presented a mechanical effect on dental plaque and the bioavailability of the phytotherapic might be higher than in the gel. The test gel was placed on the toothshield in a non-diluted way and it is possible that a solubilization by saliva or toothbrush's mechanical action in order to have an antibacterial effect would be necessary13. The experimental model used in this present study discarded other factors that could jeopardize the clinical results, such as mechanical plaque control, which could hide the phytotherapic agent's actual effect. However, the efficiency of other dentifrices has been shown using this clinical design13.

In this study, the Turesky, et al.19 (1970) index was used to assess plaque due its sensitivity to detect small deposits of plaque. 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.

Despite the lack of interference of LS in plaque accumulation, the phytotherapic had a positive effect on gingivitis, agreeing with another works4,5. In spite of insufficient data in the literature about the antiinflammatory mechanisms of LS, this property has been highlighted previously4,5. Another explanation for this fact is that the test gel exerted its antigingivitis effect indirectly through the dental plaque pathogenicity. However, this possibility was not confirmed in the literature yet due to complex mechanisms involved in the etiology of gingivitis.

There is no "gold standard" index for assessing the severity of gingivitis. Since the GI has been the most widely used index in studies investigating oral hygiene products, it was employed in the present study to allow for comparison of the present findings to those of other investigations. This index uses a scale in which color changes in the gingival tissues precedes bleeding on probing; however this parameter is not necessarily an accurate indicator of gingivitis17. For this reason, gingival inflammation was evaluated by BI as well.

Finally, more controlled trials using the same concentrations of LS extract are necessary to investigate its action on the treatment of gingivitis, comparing to chlorhexidine. Further studies should be developed to identify the real benefits of LS as a therapeutic and preventive agent for gingivitis, in addition to its common use in popular medicine.

CONCLUSION

The 10% LS gel was not a good antiplaque agent, but it was effective in the control of gingivitis.