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Assessment of in Vitro and in Vivo antimicrobial activities of selected Nigerian tootpastes and mouth washes on some oral pathogens.


Dental plaque is the film of microorganisms formed on the tooth surface embedded in a matrix of polymers of salivary and bacterial origin. Dental plaque develops naturally on the surface and forms part of the host defenses of the mouth by acting as barrier to colonization by exogenous microorganisms [11]. Although dental plaque forms naturally on teeth, in the absence of adequate oral hygiene, it can accumulate beyond levels that are compatible with dental health, and at a susceptible site, dental caries or periodontal diseases can occur.

In many individuals, the customary oral hygiene method of tooth brushing is, by itself, usually insufficient over a long period to provide a level of plaque control consistent with oral health. Consequently, the incorporation of chemical agents with anti-plaque or antimicrobial activity into dental products has been proposed as a potential prophylactic method of reducing plaque-mediated disease [6,10,1]. In a similar development, Killoy [8] submitted that a recent trend is to employ local antimicrobial delivery in the treatment of periodontitis. Alkaloid extract of Sanguinaria canadensis incorporated into various dentifrices and oral rinses have been shown to possess broad-spectrum in vitro activity against a wide variety of microorganisms [4]. Similarly, a recent report has shown that extract of Dichrostachys cinerea could be used in the development of dentifrice [9].

Antimicrobial agents might control plaque by reducing or preventing the formation and development of plaque, by selectively inhibiting only those bacteria directly associated with disease or by inhibiting the expression of virulence determinants [2]. The laboratory assessment of antimicrobial agents for use in dentistry is often based on results from relatively simple tests, particularly the determination of the minimum inhibitory concentration (MIC) that prevents growth of an organism. However, Marsh [12] had earlier opined that such tests might have limitations and can give misleading impressions of efficacy. From the foregoing, this present paper presents an in vitro and in vivo assessment of antimicrobial activities of some commonly employed toothpastes and mouthwashes in Nigeria against some oral pathogens such as Streptococcus mutans, Enterococcus faecalis and Candida albicans

Materials and methods

Sampling and Preparation of Samples

Two hundred people were randomly selected in a survey conducted by structured questonaires on the campus of The Polytechnic, Ibadan, Nigeria. The survey was aimed at knowing the brands of toothpastes and mouthwashes that are mostly used. As a result, seven toothpastes and three mouthwashes were selected for assessment of their in vitro and in vivo antimicrobial activities. They were purchased from local markets in Ibadan, South West, Nigeria. Different concentrations (50%, 25%, 12.5%, 6.25% and 3.175%) of the selected toothpastes and mouthwashes were prepared with sterile saline (0.9%); these and undiluted samples were immediately used for the in vitro testing.


Pure cultures of Streptococcus mutans, Enterococcus faecalis and Candida albicans isolated from patients with dental diseases were obtained from the Medical Microbiology Department of the University College Hospital (UCH) Ibadan, Nigeria. Bacterial cultures were maintained on Nutrient agar slants and the fungus on Potato dextrose agar slants, both at 6-8[degrees]C

Preparation of inoculum and in vitro testing of samples

Prior to the in vitro testing, the cultures were transferred into McCartney bottles containing 5ml of prepared Brain-Heart Infusion broth. The inoculated media were incubated at 37[degrees]C until a turbidity of 0.5 0 Mcfarland standard was achieved or exceeded. Mueller-Hinton chocolate agar was prepared according to standard procedure [14].

Six cups were bored on each of the dried Mueller-Hinton chocolate agar plate with the aid of the cork borer (Number 4) and each of the microbial suspensions prepared was thereafter swabbed on them. Cup 1 of each of the sets of plates was filled with 0.2ml undiluted paste/mouthwash, while cups 2,3,4,5 and 6 were filled with two drops of 50%, 25%, 12.5%, 6.25% and 3.175% (w/v or v/v) of toothpastes or mouthwashes respectively. The plates were then left undisturbed for thirty minutes to allow for proper diffusion of the samples introduced into their respective plates. The plates were incubated at 37 C for 24 hours. However, Potato dextrose agar 0 was used for Candida albicans and the incubation conditions of 28[degrees]C and 48 hours of incubation were 0 used.

After incubation, the plates were examined and the diameters of zones of complete inhibition were measured with the aid of transparent ruler. The results obtained were recorded and interpreted according to NCCLS standards [15].

In vivo testing of the samples

The study involved ten groups of people, with each group consisting of five volunteers. Each of the volunteer was made to rinse his/her mouth with sterile saline alone after an overnight fast. The mouth rinse obtained from each volunteer was collected into properly labeled sterile bottles. Immediately after, each of them was made to wash his/her teeth with a sterile toothbrush and toothpaste or mouthwash of the particular group he/she belongs to; and their respective mouth rinses were collected into other properly labeled sterile bottles. After 30, 60, 90 and 120 minutes, mouth rinses were collected from each volunteer following the same procedure. The obtained mouth rinses were serially diluted and the appropriate dilutions plated on Mueller-Hinton chocolate agar. The inoculated plates were incubated at 37 C for 24 0 hours. After incubation, the colonies were counted and colony forming unit per milliliter (cfu/ml) of the original mouth rinse estimated.

Statistical analysis

Data obtained were expressed as means. The statistical significance of differences was assessed using analysis of variance. A two-tailed P value of less than 0.05 was considered to be statistically significant. Values that were significantly different were separated using Duncan's Multiple Range test using SPSS for Windows ver. 11.0 statistical package.

Results and discussion


Table 1 shows the in vitro antimicrobial activity of the tested toothpastes and mouthwashes against Streptococcus mutans. Growth inhibitions were dependent on both the sample used and the concentrations tested. ANOVA test revealed that there were significant differences (P<0.05) between the exhibited in vitro antibacterial activities of the investigated mouth care products. In the undiluted state, sample I (a mouthwash) had the highest in vitro antibacterial activity while sample H (also a mouthwash) had the least bioactivity. However, at 50% concentration samples I and B had the highest anti-streptococcal activities and samples D and H the least. At [less than or equal to] 12.5% concentrations, the anti-streptococcal activities of all the toothpastes except sample G had been lost.

The anti-enterococcal activities of the investigated mouth care products are shown in Table 2. In a manner similar to the inhibition of S. mutans, the anti-enterococcal activities were both dependent on the sample used and the tested concentrations. In this regards, sample I exhibited higher growth inhibition in vitro; this was followed by samples F and A. However, the loss of anti-enterococcal activities of all the toothpastes (except sample G) was observed at lower concentrations of 6.25 and 3.175%.

The in vitro growth inhibition of Candida albicans by the investigated dental care products is presented in Table 3. Results obtained again revealed that growth inhibition was dependent on the tested samples and the concentration used. At 100% concentration, sample F had the highest anti-candidal activity and sample J the least. However, at 50% concentration sample I had the highest activity. In a trend similar to the inhibition of Streptococcus mutans, anti-candidal activities of most of the toothpastes were lost at [less than or equal to] 12.5% concentrations.

Results of the in vivo assessment of the investigated oral care products is shown in Table 4. Sample J exhibited the highest immediate reduction of viable count of the oral microflora in the volunteers. This was closely followed by sample F while sample E demonstrated the least reduction. Of all the investigated dental care products, samples I and A were notable for their abilities to sustain the microbial load reduction earlier observed immediately after mouth washing. Paradoxically, sample J was prone to loosing its in vivo antimicrobial activity two hours after initial mouth washing compared to other tested dental products.


Moran et al. [13] stated that potential antimicrobial agents present in both past and presently available toothpastes could have a beneficial effect in the prevention of plaque and gingivitis. Data from the present study is in support of this assertion as all the investigated dental care products exhibited both in vitro and in vivo antimicrobial activities; a feature that may have been largely due to their antimicrobial active ingredients (Table 5). Among all the investigated toothpastes, the exceptional ability of sample G to retain its in vitro antimicrobial activity at lower concentrations of [less than or equal to] 12.5% is notable. This might be connected with the presence of 0.1% triclosan in its formulation. This becomes more plausible as the utility and effectiveness of a 1% triclosan formulation in health care industry has been reviewed by Jones et al[7].

Results emanating from the assessment of the in vitro antimicrobial activities of the tested dental care products in the present study depict that there was uniqueness in the pattern of sensitivity of the tested indicator organisms to the different investigated oral/dental care products. Two insights are consequent upon this. Firstly, it is a challenge to further search for powerful antimicrobials with a sufficiently broad spectrum activity. Secondly, it is further invigorating the fact that any supposed efficacy of a given dental care product must be consequent upon its assessment against broad spectrum of oral organisms.

The activities of oral microflora being responsible for mouth odour and most oral diseases is not in doubt. The need to keep these oral organisms to a level consistent with oral health by antimicrobial agent inclusion in dentifrice has been stressed [12,8]. In this regard, data from the present in vivo study generates a lot of concern, as many of the assessed oral care products were not able to sustain their immediate reduction in viable counts of oral flora in the volunteers for a reasonably long period; a situation that may encourage plaque formation. This discovery may be possibly supporting Bradtke's expression of despair on fluoride-"And where did the controversial fluoride get us? Holes everywhere" [3].

As a possible way out of the fluoride saga, studies have shown the efficacy of dentifrice containing herbal extracts in maintaining good oral health[17,3]. Reports have also shown that sanguinarine (a plant extract) with high in vitro antimicrobial activity against oral pathogens did not reproduce that activity clinically, possibly due to limited oral retention [18,5]. This fact may be responsible for the unimpressive performance of sample B in the present study. To this end, the development of efficacious dentifrice containing herbal extracts needs a collaborative approach involving specialists in pharmaceutical and basic sciences.

Conclusively, result from this study has shown that the regulatory body need to reappraise the efficacy of the available dental care products in Nigeria so as to ensure that the consumers get the expected value for their money.


[1.] Addy, M., 1990. Chemical plaque control. In: Kieser JB (Ed). Periodontics: A practical approach. London. Wright, 527-534.

[2.] Bradshaw, D.J., P.D. Marsh, G.K. Watson and D. Cummins, 1993. The effects of triclosan and Zinc citrate, alone and in combination, on a community of oral bacteria grown in vitro. J. Dent. Res., 72(1): 25-30.

[3.] Bradtke, B., 2008. Neem Toothpaste, Dental Care and Gum Health. html.

[4.] Dzink, J.L., S.S. Socransky, 1985. Comparative in vitro activity of sanguinarine against oral microbial isolates. Antimicrobial Agents Chemotherapy, 27: 663-665.

[5.] Goodson, J.M., 1989. Pharmacokinetic principles controlling efficacy of oral therapy. J. Dent. Res., 68(Spec Iss): 1625-1632.

[6.] Hull, P.S., 1980. Chemical inhibition of Plaque. J. Clin. Periodontol., 7: 431-442.

[7.] Jones, R.D., B.S. Jampani, B. Hanuman, J.L. Newman and A.S. Lee, 2000. Triclosan: A review of effectiveness and safety in health care settings. American J. of Infection Control, 28(2): 184-196.

[8.] Killoy, W., 1998. Chemical treatment of periodontitis: local delivery of antimicrobials. International Dental Journal, 48 (Suppl 1): 305-315.

[9.] Kolapo, A.L., M.B. Okunade, J.A. Adejumobi and M.O. Ogund iya , 2008. In Vitro Antimicrobial activity and Phytochemical Composition of Dichrostachys cinerea. Medicinal and Aromatic Plant Science and Biotechnology, 2(2): 131-133.

[10.] Korman, K.S., 1986. The role of supragingival plaque in the prevention and treatment of periodontal diseases. A review of current concepts. J. periodont. Res., 21(16 Suppl): 5-22.

[11.] Marsh, P.D., 1989. Host defenses and microbial homeostatis: role of microbial interactions. J. Dent. Res., 68: 1567-1575.

[12.] Marsh, P.D., 1992. Microbial Aspects of the Chemical Control of Plaque and Gingivitis. J. Dent. Res., 71(7): 1431-1438.

[13.] Moran, J., M. Addy and R. Newcombe, 1988. The antibacterial effect of toothpastes on the salivary flora. J. Clin. Periodontol., 15(3): 193-199

[14.] National Committee for Clinical Laboratory Standards, 2000. Approved Standard: M2-A7. Performance Standards for antimicrobial disk susceptibility tests. 7th ed. NCCLS, Wayne, Pa.

[15.] National Committee for Clinical Laboratory Standards, 2002. Performance Standards for antimicrobial susceptibility testing, twelfth informational supplement, M100-S12(M2). NCCLS, Wayne, Pa.

[16.] Pair, M.R., L.D. Acharya and N. Udupaa, 2004. Evaluation of antiplaque activity of Azadirachta indica leaf extract gel- a 6 weeks clinical study. J. Ethnopharmacol., 90(2-3): 99-103.

[17.] Southard, G.L., R.T. Boulware, D.R. Walborn, W.J. Groznic, S.L. Senior and S.L. Yankell, 1984. Sanguinarine-a new antiplaque agent. Am. J. Dent. (Suppl), 5: 572-575.

[18.] van der Ouderaa, F.J.G., 1991. Anti-plaque agents. Rationale and prospects for prevention of gingivitis and periodontal diseases. J. Clin. periodontal., 18: 447-454.

[19.] van der Ouderaa, F. and D.Cummins, 1991. Antiplaque dentifrices: current status and prospects. Int. Dent. J., 41: 117-123.

(1) Adejumo O.E, (2) Olubamiwa A.O., (2) Ogundeji B.A., and (2) Kolapo A.L.

(1) Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Olabisi Onabanjo University, 1 Sagamu, Ogun State, Nigeria.

(2) Biology Department, The Polytechnic, Ibadan. Oyo state. Nigeria. 2

Corresponding Author

Adejumo O.E., Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, E.mail:<>
Table 1: Zones of inhibition (mm) of Streptococcus mutans by
different concentrations of selected toothpastes and mouthwashes.

 Concentration (%)

Sample 100 50 25 12.5 6.25 3.175

A 12 (d) 11 (ab) 9 (b) -- -- --
B 14 (bc) 12 (a) 9 (b) -- -- --
C 14 (bc) 9 (cd) 8 (c) -- -- --
D 12 (d) 8 (d) 8 (c) -- -- --
E 13 (cd) 9 (cd) 8 (c) -- -- --
F 15 (b) 9 (cd) 9 (b) -- -- --
G 15 (b) 10 (bc) 9 (b) 8 (b) 8 (a) 8 (a)
H 8 (f) 8 (d) 8 (c) 8 (b) 8 (a) 8 (a)
I 21 (a) 12 (a) 12 (a) 10 (a) 8 (a) 8 (a)
J 10 (c) 9 (cd) 8 (c) 8 (b) 8 (a) 8 (a)

Values are means of quintuplicate determinations.

Within column, values with different superscripts are significantly
different (P<0.05)

A-G are toothpaste samples while H-J are mouthwashes

Table 2: Zones of inhibition (mm) of Enterococcus faecalis by
different concentrations of selected toothpastes and mouthwashes.

 Concentration (%)

Sample 100 50 25 12.5 6.25 3.175

A 13 (bc) 11 (b) 9 (b) 8 (b) -- --
B 10 (cf) 8 (c) 8 (c) 8 (b) -- --
C 11 (dc) 9 (c) 9 (b) 8 (b) -- --
D 12 (cd) 8 (c) 8 (c) 8 (b) -- --
E 9 (cf) 9 (c) 8 (c) 8 (b) -- --
F 14 (b) 12 (b) 9 (b) 9 (ab) -- --
G 8 (f) 8 (c) 8 (c) 8 (b) 8 (a) 8 (a)
H 8 (f) 8 (c) 8 (c) 8 (b) 8 (a) 8 (a)
I 17 (a) 14 (a) 12 (a) 10 (a) 8 (a) 8 (a)
J 9 (cf) 8 (c) 8 (c) 8 (b) 8 (a) 8 (a)

Values are means of quintuplicate determinations.

Within column, values with different superscripts are significantly
different (P<0.05)

A-G are toothpaste samples while H-J are mouthwashes

Table 3: Zones of inhibition (mm) of Candida albicans by different
concentrations of selected toothpastes and mouthwashes.

 Concentration (%)

Sample 100 50 25 12.5 6.25 3.175

A 13 (ab) 9 (bc) 8 (b) -- -- --
B 12 (bc) 10 (b) 8 (b) -- -- --
C 14 (ab) 10 (b) 9 (a) 9 (a) -- --
D 13 (ab) 9 (bc) 8 (b) -- -- --
E 10 (cd) 9 (bc) 8 (b) -- -- --
F 15 (a) 9 (bc) 8 (b) -- -- --
G 12 (bc) 8 (c) 8 (b) 8 (a) 8 (a) 8 (a)
H 8 (d) 8 (c) 8 (b) 8 (b) 8 (a) 8 (a)
I 14 (ab) 12 (a) 9 (a) 8 (b) 8 (a) 8 (a)
J 8 (d) 8 (c) 8 (b) 8 (b) 8 (a) 8 (a)

Values are means of quintuplicate determinations.

Within column, values with different superscripts are significantly
different (P<0.05)

A-G are toothpaste samples while H-J are mouthwashes

Table 4: Viable counts (log cfu-ml) of oral micro-flora obtained from
volunteers before and after using different toothpastes and

Sample Before Mouth
 washing Time after Washing(min)

 Immediately 30 60 90 120

A 13.15 12.76 12.83 12.94 12.74 12.84
B 13.15 12.36 12.76 13.27 13.15 13.11
C 13.64 11.69 12.69 11.69 11.26 12.97
D 13.08 11.69 12.11 12.52 13.11 12.44
E 13.00 12.93 13.18 13.38 13.33 13.18
F 12.92 10.69 12.63 12.89 12.88 12.60
G 11.39 11.11 11.30 12.18 12.74 12.30
H 13.28 12.58 13.08 13.15 13.36 12.76
I 13.00 12.81 13.00 12.83 12.76 12.86
J 12.74 10.40 12.90 12.11 11.88 12.40

Values are means of quintuplicate determinations.

A-G are toothpaste samples while H -J are mouthwashes

Table 5: Active ingredients of selected toothpastes and mouthwashes

Sample Active ingredients

A Sodium monofluorophosphate (0.76%) and Sodium
 fluoride (0.1%)
B 0.01% Basil oil, 5% Herbal extract, Sodium Lauryl
 sulphate, Ginger, Spearmint, Lemon oil
C Sodium monofluorophosphate (0.76%)
D Sodium fluoride (0.30%)
E Sorbitol, Silica, Sodium Lauryl sulphate, Sodium
F Sodium m onofluorophosphate (0.76%)
G Sodium fluoride (0.32%), Triclosan (0.1%)
H Thym ol (0.47%), Ethanol
I Hydrogen peroxide
J Phenol (0.175%), Halogenated phenol (0.68%), Sodium
 salicylate (0.052%)
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Title Annotation:Original Article
Author:Adejumo, O.E.; Olubamiwa, A.O.; Ogundeji, B.A.; Kolapo, A.L.
Publication:Advances in Medical and Dental Sciences
Article Type:Report
Geographic Code:6NIGR
Date:Sep 1, 2008
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