healthmedicinet

This study was approved by the ethics committee of Tokyo Dental College (No. 386).

Peptides

The amino acid sequences and properties of CL(14-25) used in this study are summarized
in Table 1. The sequence of CL(14-25) is identical to that of CH peptide reported by Taiyoji
et al. 10]. Chemically synthesized CL(14-25) was obtained from Hokkaido System Science Co.,
Ltd. (Sapporo, Japan). Synthetic peptides were purified to 95 % by reversed-phase
high-performance liquid chromatography (RP-HPLC). Molecular weights of purified CL
peptides were confirmed by matrix-assisted laser/desorption ionization–time-of-flight
mass spectroscopy (MALDI–TOF-MS).

Table 1. The amino acid sequence and properties of CL(14-25)

Study population

Participants were recruited from periodontally healthy individuals, between November
2013 and December 2013. Written informed consent was obtained from all participants.
To qualify, subjects were required to have a minimum of 20 natural teeth. Systemic
exclusion criteria were the presence of cardiovascular and respiratory diseases, systemic
inflammatory conditions, such as diabetes mellitus, immunodeficiency, and current
pregnancy or lactation. Current smokers and those with history of allergic reactions
to rice, several mouthrinse components or medications were also excluded. The participants
received no medication that could affect their periodontal conditions, such as antimicrobial
agents or anti-inflammatory drugs, for at least 3 months prior to the microbiological
testing.

Mouthrinse formulation and preparation

Two formulations were compared. Test mouthrinse was prepared with 0.4 % CL(14-25)
and 99.6 % sterilized distilled water [CL(14-25) 4.0 mg/ml]. No flavorants or preservatives
were added. Distilled water was used as control (placebo) rinse. Preservation condition
of the mouthrinse formulations was assessed by a standard microbial limit test using
spread plate method according to the method specified in the Japanese Pharmacopoeia.
It was found that the mouthrinse formulations can be safely stored at both 4 °C and
room temperature during the study period.

Dental plaque regrowth study

The main outcome variable was plaque score assessed by the modified Volpe’s method
14], 15] reported by Suzuki et al. 16]. After disclosing the plaque, a periodontal probe was used to measure the height
of the plaque accumulation from the gingival margin to the nearest 0.5 mm at six sites
per Ramfjord index teeth (#16, 21, 24, 36, 41, 44) 17] considering both buccal and lingual surfaces. Clinical examiners were trained in
study procedures prior to commencement of the study. The plaque score of a given tooth
was expressed as the absolute value from the sum of all six measurements 18].

In Fig. 1, flow diagram of the study is presented. Within 7 days of the screening examination,
eligible participants received oral hygiene instructions and professional tooth cleaning
in order to standardize oral hygiene procedures. Through computer-aided randomization,
the participants were divided in two groups of the same size; Group 1 and Group 2.
A person not directly involved in the research project carried out the allocation
of test or control products. A plaque regrowth study design, with a crossover design
19], 20], was used. The selected mouthrinse formulation was given to participants in Group
1 and the placebo mouthrinse was given to those in Group 2, along with the usage instructions.
Each participant received a bottle containing 125 ml of mouthrinse. They were instructed
to rinse their mouth with 20 ml of the given mouthrinse for 1 min twice a day for
3 days avoiding any other oral hygiene manoeuvre. To check for compliance, subjects
were asked to note the times of day when they rinsed.

Fig. 1. Flow diagram illustrating the study design

Study participants were asked to fill out a questionnaire regarding their perception
of plaque reduction. Perceived plaque reduction was graded on a 5-point rating scale
with discrete responses, ranging from 0 (insufficient) to 5 (very sufficient). At
the completion of the trial, participants were asked to choose the mouthrinse formulation
with which greater plaque reduction was perceived. All returned mouthrinse bottles
were weighed to calculate the amount of mouth rinse used and to check for compliance.

After this period, plaque score was recorded using a disclosing solution. A washout
period of 7 days separated each treatment phase. Then participants resumed their normal
oral self-care. After this washout period, all participants underwent again a session
of professional tooth cleaning and the protocol continued inverting the mouthrinses
according to a crossover design and with the same timing and mode of use previously
described. In this way, each subject received both treatments sequentially. Plaque
reduction rate was calculated as follows: Plaque reduction (%) = [(plaque score of
the placebo rinse) ? (plaque score of the test rinse)/(plaque score of the placebo
rinse)] × 100.

Statistical analysis

The tooth was the unit of analysis. Data were analyzed using nonparametric statistics,
which are more appropriate when the data show a skewed distribution. Thus, the Wilcoxon
matched-pairs signed-ranks test was used to ascertain the differences between the
individual rinse solutions. Data considering the questionnaire scores were also analyzed
using the same test. Fisher’s exact test was used to compare the association with
the two different score category between groups.

All data were registered electronically using Microsoft Excel, and statistical analyses
were carried out using a statistical package (InStat 3.10, GraphPad Software, La Jolla,
CA, USA). P values less than 0.05 were considered as statistically significant.