Comparative evaluation of chlorhexidine mouthrinse versus cacao bean husk extract mouthrinse as antimicrobial agents in children.
Abstract: AIM: To compare the antimicrobial efficiency of chlorhexidine and cacao bean husk extract mouthrinses in children. STUDY DESIGN: A randomised comparative study which employed purpose sampling. METHODS: Study was conducted on 50 children of both sexes aged 6-10 years old. A group of 25 children were given 10 ml of 0.2% chlorhexidine mouthrinse and another 25 children were given 10 ml of 0.1% cacao bean extract mouthrinse to rinse twice daily for about 30 seconds. The salivary samples were collected from each child in Dentocult SM vials on day one (pre-rinse) and after 7 days, 1 month and 2 months. The readings were tabulated and subjected to statistical analysis. STATISTICS: Mann-Whitney test and the p-value were used for statistical analysis. RESULTS: There was significant reduction in streptococcus mutans counts in saliva at all follow-up intervals for both mouthrinse groups. However, there was no significant difference in reduction of streptococcus mutans counts in saliva, between chlorhexidine mouthrinse group and cacao bean husk extract mouthrinse group. CONCLUSION: Cacao bean husk extract mouthrinse can be used in children as an alternative to chlorhexidine mouthrinse as it has similar antimicrobial properties and evades the side-effects of the latter.

Key words: Chlorhexidine mouthrinse, cacao bean husk extract mouthrinse, Dentoccult test.
Article Type: Report
Subject: Cacao (Usage)
Cacao (Health aspects)
Chlorhexidine (Usage)
Chlorhexidine (Health aspects)
Chlorhexidine (Comparative analysis)
Dental caries (Care and treatment)
Materia medica, Vegetable (Usage)
Materia medica, Vegetable (Health aspects)
Plant extracts (Usage)
Plant extracts (Health aspects)
Authors: Babu, Venkatesh N.S.
Vivek, D.K.
Ambika, G.
Pub Date: 10/01/2011
Publication: Name: European Archives of Paediatric Dentistry Publisher: European Academy of Paediatric Dentistry Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 European Academy of Paediatric Dentistry ISSN: 1818-6300
Issue: Date: Oct, 2011 Source Volume: 12 Source Issue: 5
Product: Product Code: 0138200 Cocoa Beans NAICS Code: 111998 All Other Miscellaneous Crop Farming SIC Code: 0179 Fruits and tree nuts, not elsewhere classified
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 277106779
Full Text: Introduction

Modern concepts of cariograms describe microorganisms as one of the major aetiological factors in the formation of dental caries. Amongst them, mutans streptococci (MS) have been regarded as one of the main microorganisms [Bowden, 1991]. Amongst the various chemical agents, chlorhexidine gluconate (CHX), a cationic bisguanide, has a broad spectrum antibacterial effect by virtue of its high intraoral substantivity and bactericidal and bacteriostatic activity. The greatest success has been with CHX, which is now considered the gold standard against which other potential anti-plaque agents are measured [Kornmann, 1986; Mandel, 1988; Heasman and Seymour, 1995]. However, side-effects may occur such as discolouration of the teeth or unpleasant after taste when these chemicals are used for an extended period [Greenstein et al., 1986].

The possible protective effect of cocoa on dental caries is receiving increasing attention.

The incorporation of cocoa powder or chocolate into hamster diets was reported to reduce caries [Stralfors, 1967]. Subsequently it was suggested that phenolic substances could be responsible for the observed anti-caries effect of cocoa powder [Kashket et al., 1985]. The cocoa bean husk is a waste material generated in the chocolate industry. It has been shown to possess two types of cariostatic substances, one showing anti-glycosyltranferase activity and the other antibacterial activity [Osawa et al., 2001]. The observed effect in dentistry could be due to the inhibitory action of cocoa water extract on the synthesis of water-insoluble glucans.

Considering the side-effects of CHX mouthrinse and the promising effect of cacao bean husk extract mouthrinse, this study was designed to compare the efficiency of both mouthrinses as antimicrobial agents in children. The aim of the study was to compare the efficacy of 0.1% cacao bean husk extract mouthrinse and 0.2% CHX mouthrinse in reducing MS in saliva of children

Materials and methods

The present study was a randomised comparative study; 50 children of both sexes aged 6-10 years were divided into two groups, each consisting of 25 children. The first group was given 10 ml of 0.2% CHX mouthrinse and the second group 10 ml of 0.1% cacao bean extract mouthrinse to rinse twice daily for 30 seconds. Children who had received any antibiotics during the study or within two months before the study were excluded from participation. Parental consent was taken and ethical committee clearance was previously obtained. The study was carried out in a residential school where monitoring of mouthrinse use by the children was performed twice daily (morning and evening).

[FIGURE 1 OMITTED]

Cacao bean husk extract mouthrinse was prepared from ground husks of cacao beans and was obtained from CAMPCO factory, Puttur, Dakshin Karnataka, (India) according to the method described by Matsumoto et al., [2004]. Children were instructed to chew a piece of paraffin wax before saliva sample collection, which were collected from each child in Dentocult SM vials (Orion Diagnostics, Finland) on four occasions (Figure 1). Sampling was performed on day one before the use of mouthrinse, and after 7 days, 1 month and 2 months of mouthrinse use. For saliva collection the rough surface of the Dentocult SM saliva strip was pressed against the saliva on the tongue. The strip was gently placed in the selective culture broth, with surfaces clipped and attached to the cap.

[FIGURE 2 OMITTED]

Dentocult SM vials were incubated in an upright position at 370C for 48 hrs with the cap opened one quarter of a turn to allow growth of microorganisms. Results obtained by culture in the thermostat were read by the use of the model chart supplied by the manufacturer. The presence of MS was confirmed by the detection of light-blue to dark-blue raised colonies on the inoculated surface of the strip (Figure 2) classified as Class 0-3 as follows:

Inspection of the growth was conducted with the strip held sideways against light and with a magnifying glass. The MS counts in the saliva at baseline and the three follow-up intervals for both mouthrinse groups were compared. Interexaminer agreement was performed for the reproducibility of outcome assessment by the semi-quantitative chair side Dentocult SM test.

Results

Chlorhexidine mouthrinse (0.2%) group showed a highly significant reduction in salivary mutans streptococci (23.2%) at intervals from pre-rinse to 7 days, 1 month and 2 months, and also at intervals from 7 days to 1 and 2 months and also from 1 month to 2 months (p<0.01) (Table 1 and Figure 3).

Cacao bean husk extract mouthrinse (0.1%) showed a statistically significant reduction in MS counts in saliva (22.4%) from pre-rinse to 2 months follow-up, and at intervals from 7 days to 1 and 2 months and from 1 month to 2 months (p<0.001) (Table 2 and Figure 4).

Higher mean MS counts in saliva were observed in CBHE mouthrinse group compared to CHX group at all the time intervals. However, the difference in reduction between the two groups was not statistically significant at any of the time intervals (p>0.05) (Table 3 and Figure 5).

Discussion

A variety of chemotherapeutic agents have been examined for their ability to control oral micro organisms and to affect plaque formation. Chlorhexidine (CHX) digluconate has a 30 year history in dental medicine [Loe and Schiott, 1970]. Unfortunately, studies showed that these positive effects were accompanied by side-effects, the most disturbing being extrinsic tooth staining [Eriksen et al., 1985] and others such as unpleasant taste and burning sensation. There is increasing interest in the effect of natural compounds, especially food extracts, on the resident oral microbial flora, both in terms of their ability to promote the growth of beneficial organisms and by their inhibition of the growth and metabolism of species associated with diseases [Percival et al., 2006].

The study of Kashket and co-workers [1985] showed that the inhibitory effects of cocoa on plaque accumulation and caries formation were due to inhibition of bacterial polysaccharide production. Another study [Osawa et al., 2001] reported that 50% ethanol extract of cacao bean husk (CBH50) inhibited the glucan synthesis of glycosal transferase B by 57% and reduced the colony forming units of MS MT8148R cell suspension by 77% at a concentration of 1mg/ml.

In the present study a comparative evaluation of the efficiency of CHX mouthrinse with cacao bean husk extract mouthrinse in children in terms of reduction in MS counts in saliva of the two mouthrinse groups were performed. The reduction in MS in saliva from pre-rinse to 7 days and 1 month was found to be statistically significant (p<0.01) which was similar to the study conducted by Sari and Birinci [2007] and Beyth et al., [2003] where 0.2% CHX mouthrinse decreased MS levels in saliva after 1 week.

Similarly a statistically significant decrease in MS count in saliva was observed from 7 days to 1 month, from 7 days to 2 months and from 1 month to 2 months. In this study 0.2% CHX mouthrinse showed a highly significant reduction in the MS count (23.2%) in saliva and these observations add to the findings of earlier studies [Spets-Happonen et al., 1985; Hefti and Huber, 1987].

An attempt was made to evaluate cacao bean husk extract mouthrinse as an antimicrobial agent. It was observed that 0.1% of cacao bean husk extract mouthrinse resulted in a statistically significant reduction in MS counts in saliva (p<0.001) at the intervals from pre-rinse to 2 months follow-up. Counted in MS in saliva this group at time intervals of 7 days, 1 month and 2 months were compared with pre-rinse which was found to be statistically significant (p<0.001). A decrease in MS in saliva was observed from 7 days to 1 month, 2 months and from 1 month to 2 months that was statistically significant (p<0.001). There was a reduction in MS counts in saliva (22.4%) which was similar to the study conducted by Srikanth et al., [2008] where there was a 20.9% reduction in mutans streptococci counts in saliva when used as mouthrinse in children.

Comparisons in the MS counts in saliva between the CHX and cacao bean husk extract mouthrinse groups at pre-rinse and the three follow-up intervals were made. Higher mean salivary MS count were observed in the CBHE compared with the CHX mouthrinse group at all the time intervals. However, the difference in reduction of MS counts in saliva between the two groups was not statistically significant at any of the time intervals (p>0.05). Two children dropped out of the study after one week of rinsing due to nauseating effects.

The results of this study showed there was no significant difference between both mouth rinses in terms of antimicrobial properties i.e. reduction in the salivary MS counts

Conclusion

This in vivo study showed that

* There was a significant reduction in the MS count in saliva when CHX mouthrinse was used.

* There was a significant reduction in the MS count in saliva when cacao bean husk extract mouthrinse was used.

* There was no significant difference in reduction of MS count in saliva between CHX and cacao bean husk extract mouthrinse groups.

However considering the side-effects of CHX mouthrinse and the similar antimicrobial properties of cacao bean husk extract mouthrinse it is suggested that cacao bean husk extract mouthrinse is a valuable alternative for children. Further studies are recommended of a longer duration and with a larger group of children.

Acknowledgements

We are grateful to the Campco factory Puttur, Dakshina Kannada, Karnataka, India for the kind supply of cacao bean husk extract. We are thankful to Dr Nataraj Karaba, Associate Professor, University of Agriculture Science, Bangalore for the kind help in preparation of the mouthrinses.

References

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Bowden GHW. Which bacteria are cariogenic in man in markers of high and low risk groups and individuals for Dental Caries (ed. Johnson N.W.) 1991, Cambridge Univ Press PP: 266-286.

Eriksen HM, Nordbo H, Kantanen H, Ellingsen JE. Chemical plaque control and extrinsic tooth discoloration. A review of possible mechanisms. J Clin Periodontol 1985; 12:345-350.

Greenstein G, Berman C, Jaffin R. CHX. An adjunct to periodontal therapy. J Periodontol 1986; 57:370-377.

Hefti A, Huber B. The effect on early plaque formation gingivitis and salivary bacterial counts of mouth washes containing hexidine / zinc amine fluoride / tin or CHX. J Clinic Periodontal 1987; 14: 515-518.

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Kashket, S, Paolino VJ, Lewis DA, Van Houte J. In-vitro inhibition of glucosyltransferase from the dental plaque bacterium streptococcus mutan by common beverages and food extracts. Arch Oral Biol 1985; 30:821-826.

Kornmann KS. Antimicrobial agents. In: Loe H, Klienman DV, eds; Dental plaque control measures and oral hygiene practices. Oxford: IRL Press, 1986: 121-42.

Loe H, Schiott CR. The effect of mouth rinses and topical application of CHX on the development of dental plaque and gingivitis in man. J Periodontal Res 1970; 5:79-83.

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Matsumoto M, Tsuji M, Okuda J et al. Inhibitory effects of cocoa bean husk extract on plaque formation in vitro and in vivo. Eur J Oral Sci 2004; 112:249-252.

Osawa K, Miyazaki K, Shimura S et al. Identification of cariostatic substances in the cacao bean husk: Their anti-glucosyltransferase and antibacterial activities. J Dent Res 2001; 80:2000-2004.

Percival RS, Devine DA, Duggal MS, Chartron S, Marsh PD. The effect of cocoa polyphenols on the growth, metabolism and biofilm formation by MS and streptococcus sanguis; Eur J Oral Sci 2006; 114:343-348.

Sari E, Birinci I. Microbiological evaluation of 0.2% CHX gluconate mouth rinse in orthodontic patients. Angle Orthod. 2007; 77:881-884.

Srikanth RK, Shashikiran ND, Subba Reddy VV. Chocolate mouth rinse: Effect on plaque accumulation and mutans streptococci counts when used by children. J Indian Soc Pedod Prev Dent 2008; 26:67-70.

Spets-Happonen S, Markanen H, Pollanen L, Kauppinen T, Luoma H. Salivary MS count and gingivitis in children after rinsing with a CHX-fluoride solution with or without strontium. Scand J Dent Res 1985; 93: 329-335.

Stralfors, A. Inhibition of Hamster Caries by Substances in Chocolate, Arch Oral Biol 1967; 12:959-962.

Dr. N.S. Venkatesh Babu *, D.K. Vivek *, G. Ambika *

* Department of Paedodontics and Preventive Dentistry, V.S. Dental College and Hospital, Bangalore, India.

Postal address: Dr. N.S.Venkatesh Babu, Dept of Pedodontics and Preventive Dentistry, V.S. Dental College and Hospital, Bangalore, Karnataka, India.

Email: drnsvbabu@yahoo.co.in
Class 0:   <10,000 CFU/ml(CFU-colony forming unit).
Class 1:   <100,000CFU/ml.
Class 2:   100,000-1,000,000 CFU/ml
Class 3:   >1,000,000 CFU/ml


Table 1 Comparison of salivary Streptococcus
mutans in chlorhexidine mouthrinse group at
different time intervals.

Time interval   Mean   Std Dev      Mean        Z      p value
                                 difference

Pre-rinse       1.96    0.65       0.333      -2.714   0.007 *
7 days          1.63    0.74
Pre-rinse       1.96    0.65       0.630      -3.392   0.001 *
1 month         1.33    0.88
Pre-rinse       1.96    0.68       1.240      -4.132   <0.001 *
2 months        0.72    0.79
7 days          1.63    0.74       0.296      -2.138   0.033 *
1 month         1.33    0.88
7 days          1.64    0.76       0.920      -4.065   <0.001 *
2 months        0.72    0.79
1 month         1.32    0.90       0.600      -3.441   0.001 *
2 months        0.72    0.79

* denotes significant difference

Table 2 Comparison of salivary Streptococcus
mutans counts in cocao bean husk extract
mouthrinse group at different time intervals.

Time interval   Mean   Std Dev      Mean        Z      p value
                                 difference

Pre-rinse       2.17    0.78       0.348      -2.828   0.005 *
7 days          1.83    0.78
Pre-rinse       2.23    0.75       0.773      -3.690   <0.001 *
1 month         1.45    0.80
Pre-rinse       2.17    0.78       1.391      -4.021   <0.001 *
2 months        0.78    0.67
7 days          1.86    0.77       0.409      -3.000   0.003 *
1 month         1.45    0.80
7 days          1.83    0.78       1.043      -4.021   <0.001 *
2 months        0.78    0.67
1 month         1.45    0.80       0.636      -3.116    0.002*
2 months        0.82    0.66

* denotes significant difference

Table 3 Comparison of salivary Streptococcus
mutans counts at four follow-up periods between
the chlorhexidine (CHX) and cocao bean husk
extract (CBHE) groups.

Time       Mouthrinse   n    Mean   Std       Mean        Z        p
interval      Used                  Dev    difference            value

Pre-          CBHE      23   2.17   0.78     0.211      -1.081   0.280
rinse         CHX       25   1.96   0.65

7 days        CBHE      23   1.83   0.78     0.196      -0.788   0.431
              CHX       25   1.63   0.74

1 month       CBHE      23   1.45   0.80     0.121      -0.583   0.560
              CHX       25   1.33   0.88

2 months      CBHE      23   0.78   0.67     0.063      -0.447   0.655
              CHX       25   0.72   0.79

Figure 3: Mean salivary Streptococcus mutans (MS) counts at
different time intervals in the chlorhexidine (CHX) mouthrinse group.

Mean SM count in Saliva at different time intervals in CHX
mounthrinse group

SM in Saliva

Pre rinse      1.96
7 days         1.63
1 month        1.33
2 months       0.72

Note: Table made from bar graph.

Figure 4: Mean salivary Streptococcus mutans (SM) counts at different
time intervals in the cocao bean husk extract mouthrinse group.

Mean SM count in Saliva at different time intervals in CBHE
mouthrinse group

SM count in Saliva

Pre rinse      2.17
7 days         1.83
1 month        1.45
2 months       0.78

Note: Table made from bar graph.

Figure 5: Mean salivary Streptococus mutans counts recorded in
the chlorhexidine (CHL) and cocao bean husk extract (CBH) groups
at different time intervals.

Mean streptococci mutanscount in saliva recorded
in the groups at different time intervals

Salivary streptococus mutans

               CBH      CHL

Pre rinse      2.17     1.96
7 days         1.83     1.63
1 month        1.45     1.33
2 months       0.78     0.72

Note: Table made from bar graph.
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