Journal of Indian Society of Pedodontics and Preventive Dentistry

Official journal of the Indian Society of Pedodontics and Preventive

Dentistry

Year : 2020 | Volume : 38 | Issue : 4 | Page : 367--373

Comparative evaluation of antimicrobial efficacy of glass ionomer cement added with

propolis, chitosan, and chlorhexidine against Streptococcus mutans and Lactobacillus
acidophilus: An in vitro study
B Neelima1, J Sharada Reddy2, P Tara Singh3, K Suhasini3, I Hemachandrika3, Shaik Hasanuddin3,
1
Senior Resident, Department of Pedodontics and Preventive Dentistry, Government Dental College and Hospital, Hyderabad, Telangana State, India
2
Professor and Head, Department of Pedodontics and Preventive Dentistry, Government Dental College and Hospital, Hyderabad, Telangana State,
India
3
Associate Professor, Department of Pedodontics and Preventive Dentistry, Government Dental College and Hospital, Hyderabad, Telangana State,
India

Correspondence Address:
Dr. J Sharada Reddy
Professor and Head, Department of Pedodontics and Preventive Dentistry, Room No 305, 2nd Floor, Government Dental College and Hospital,
Afzalgunj, Hyderabad, 500 012; Telangana State
India

Abstract
Context: Glass ionomer cement (GIC) is known for its antimicrobial activity due to its low pH and fluoride release. The fluoride
released has an inhibitory effect on a finite number of bacteria which leads to the risk of recurrent caries. Additives such as
chlorhexidine (CHX) and triclosan have been tried to maximize the antibacterial activity of GIC. Although CHX is known for its
impressive antimicrobial action, it has adverse after effects which include alteration of commensal oral flora, staining of teeth,
etc., Hence, there is a need for a material with improved antimicrobial efficacy with nominal side effects. Aim: The aim of this
study is to assess the antimicrobial efficacy of conventional GIC added with Propolis, Chitosan (CH), and CHX against
Streptococcus mutans and Lactobacillus acidophilus. Materials and Methods: Eighty discs of size 10 × 2 mm are prepared
with Conventional GIC and GIC added with Propolis, CH and CHX (n = 10) and tested against S. mutans and L. acidophilus
using the agar diffusion assay. Zones of inhibition are measured for day 1, 7, and 14, and the data were tabulated and
analyzed. Statistical Analysis: One-way ANOVA test for intragroup and Tukey's post hoc test for intergroup comparison.
Results: The mean value of zone of inhibition (in mm) against S. mutans on day 14 for Group I, II, III, and IV are 11.70 ± 1.49,
16.50 ± 2.23, 19.30 ± 2.87, and 15.60 ± 2.76, respectively. For L. acidophilus, the mean value of the zone of inhibition (in mm)
on day 14 are 8.40 ± 0.97, 9.70 ± 0.68, 16.20 ± 2.04, and 12.50 ± 0.97 for Group I, II, III, and IV, respectively. Conclusion:
Higher antimicrobial activity was shown by GIC with CHX against both strains. GIC with Propolis and GIC with CH were
effective in inhibiting S. mutans and L. acidophilus, respectively.

How to cite this article:

Neelima B, Reddy J S, Singh P T, Suhasini K, Hemachandrika I, Hasanuddin S. Comparative evaluation of antimicrobial efficacy of glass
ionomer cement added with propolis, chitosan, and chlorhexidine against Streptococcus mutans and Lactobacillus acidophilus: An in
vitro study.J Indian Soc Pedod Prev Dent 2020;38:367-373

How to cite this URL:

Neelima B, Reddy J S, Singh P T, Suhasini K, Hemachandrika I, Hasanuddin S. Comparative evaluation of antimicrobial efficacy of glass
ionomer cement added with propolis, chitosan, and chlorhexidine against Streptococcus mutans and Lactobacillus acidophilus: An in
vitro study. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2022 Nov 5 ];38:367-373
Available from: http://www.jisppd.com/text.asp?2020/38/4/367/306219

Full Text

Introduction

Glass ionomer cement (GIC) is a successfully used restorative material in the clinical practice having favorable features such
as chemical bonding to the tooth, fluoride release, and biocompatibility. It exerts an antimicrobial effect due to its low pH and
fluoride release.[1] The effect of fluoride lasts for a brief duration, and it acts on a limited spectrum of bacteria. Owing to these
limitations, additives are added to augment the antibacterial property.

Chlorhexidine (CHX), a bisbiguanide, is a potent antimicrobial agent. It has been proven as an effective anti-plaque agent. Its
antimicrobial effect lasts long due to its prolonged retention in oral tissues.[2],[3]

Chitosan (CH) is a deacetylated derivative from bio-polysaccharide chitin. It is a weak base that is insoluble in water but soluble
in dilute aqueous acid solution such as acetic acid. Its diverse antimicrobial activity is seen against filamentous fungi, yeasts,
and bacteria. It is more active against Gram-positive bacteria than Gram-negative bacteria.[4],[5],[6]

Propolis is a natural derivative, retrieved from the beehive. It exerts numerous biological activities such as antibacterial,
antifungal, antiviral, anti-inflammatory, bio-stimulative properties attributed to compounds such as polyphenols, aromatic acids,
and diterpenic acids. The antibacterial activity is primarily ascribed to flavonoids.[7],[8]

As there has been limited literature which comparatively evaluated the antimicrobial properties of the above said three
materials, a study was planned to assess the antimicrobial efficacy of conventional GIC added with Propolis, CHX, and CH
against Streptococcus mutans and Lactobacillus acidophilus, which are known to be the main etiological agents in dental caries
initiation and progression.

Materials and Methods

The present study consisted of four groups for each strain to be tested, namely S. mutans (MTCC 497) and L. acidophilus
(MTCC 10307) (Institute of Microbial Technology, Chandigarh, India). Each group comprised of 10 specimens to achieve 80%
power to detect the differences among the means at 0.05% significance level. The groups are: Group I: Conventional GIC, Fuji
IX (GC Corporation, Tokyo, Japan); Group II: GIC with Propolis (HiTech Natural Products Ltd., New Delhi, India); Group III: GIC
with CHX (Basic Pharma Life Sciences Pvt. Ltd, Ankleshwar, Gujarat, India); and Group IV: GIC with CH(In Life Pharma Pvt.
Ltd., Hyderabad, India).

Preparation of propolis modified glass ionomer cement

Ethanol soluble liquid Propolis (ESLP) was diluted from 86% to obtain a final concentration of 25%. Ratio used for specimens
preparation-powdered GIC: Liquid GIC: ESLP = 1: 0.75: 0.25. ESLP was added after mixing powder and liquid of GIC.

Preparation of chlorhexidine modified glass ionomer cement

CHX base (0.2 g) was added to 20 ml of acetic acid to obtain a 1% solution of CHX diacetate. From 1% solution of CHX
diacetate, 0.07 ml of liquid was mixed with 6.4 ml of GIC liquid. The powder/liquid ratio recommended for restorative purposes
by the manufacturer was adopted, i.e., one scoop of powder to one drop of liquid.

Preparation of chitosan modified glass ionomer cement

Twenty milligrams of CH was weighed and dissolved in 0.3 N acetic acid and made up to 100 ml with the same acetic acid in a
100 ml standard flask to get 0.2 mg/ml. Then 0.1 ml of 0.2 mg/ml of CH solution was added to 0.9 ml of GIC liquid to get 10%
v/v CH modified glass ionomer solution.

Specimen preparation

Total eighty discs were prepared for four groups using a circular brass mold of dimensions 10 mm × 2 mm.

Assessment of antimicrobial property

The bacterial strain from stock cultures were cultivated in specific culture broths, i.e., brain heart infusion (BHI) broth for the
growth of S. mutans and De Man Rogosa Sharpe (MRS) broth for L. acidophilus for 24 h and 48 h, respectively. The cultures
were then diluted with their respective broths to obtain turbidity equal to 1.5 × 108 CFU/ml equivalent to 0.5 McFarland turbidity
which was confirmed with Spectrophotometer by measuring the absorbance at a wavelength of 600 nm.

Twenty Petri dishes (9 cm diameter) containing 10 ml agar to a thickness of 2 mm were prepared. BHI Agar was used for S.
mutans and MRS Agar was used for L. acidophilus. Using a sterile swab, the entire surface of the agar plate was swabbed to
ensure even distribution of the inoculums. For each Petri dish, four standardized wells with a diameter of 10 mm were punched
into the agar with the agar puncher, and four specimens one from each group were inserted into the wells with sterile forceps.
They were incubated at 37°C ± 1°C for 24 h for S. mutans and 48 h for L. acidophilus. The diameter of the inhibition zones
produced around the specimens (specimens + inhibition zones) was measured in millimeters with a digital caliper at three
different points, and the mean was recorded as the day 1 value. The bacterial population usually dies due to the release of toxic
metabolites if cultures are kept for a long duration. Hence, on day 7 and 14, fresh agar plates were used and cultured, and the
specimens were transferred, and incubated and inhibition zones were calculated.

Results

The data were analyzed using the one-way ANOVA test for intragroup comparison, whereas intergroup comparison was done
by Tukey's post hoc test (SPSS version 20). The mean values of zones of inhibition for Group I, II, III, and IV were 13.90 ± 2.38,
19.70 ± 2.63, 23.00 ± 3.43 , and 19.50 ± 2.68 (in mm) on day 1 and on day 14; they were 11.70 ± 1.49, 16.50 ± 2.23, 19.30 ±
2.87, and 15.60 ± 2.76 (in mm), respectively, for S. mutans [Table 1]. For L. acidophilus, the mean values were 12.40 ± 0.97,
13.70 ± 0.68, 20.30 ± 2.00, and 16.50 ± 0.97 (in mm), on day 1 and 8.40 ± 0.97, 9.70 ± 0.68, 16.20 ± 2.04, and 12.50 ± 0.97 (in
mm) on day 14, respectively, [Table 2].{Table 1}{Table 2}

The intergroup comparison of four groups against S. mutans showed higher mean values of the zone of inhibition for Group III
(GIC with CHX) followed by Group II (GIC with Propolis), Group IV (GIC with CH), and Group I (Conventional GIC, Fuji IX). The
difference was statistically significant at a probability level of <0.001 on day 1, day 7, and day 14 [Table 3], [Table 4], [Table 5],
respectively.{Table 3}{Table 4}{Table 5}

For L. acidophilus, the inhibition zones were higher for Group III followed by Group IV, Group II, and Group I which was
statistically significant (P < 0.001) on day 1, day 7, and day 14 [Table 6], [Table 7], [Table 8], respectively.{Table 6}{Table 7}
{Table 8}

Discussion

GIC is the most widely used restorative material because of its favorable characteristics such as its adhesive effect and fluoride
release. However, the fluoride released is not adequate to alter the growth of bacteria and inhibit them. This leads to the risk of
recurrent caries around the GIC restorations. To overcome this problem, the addition of antibacterial agents can be a
therapeutical advantage.

Agar plate diffusion was employed in this study because set materials can be assayed through this procedure. It is a widely
accepted simple screening assay to assess the antibacterial properties of restorative materials. Moreover, its simplicity, ability to
test many specimens, and relatively low cost are the other advantages of this assay.

All the four groups of restorative materials in the present study showed antimicrobial activity against S. mutans and L.
acidophilus on day 1, 7, and 14 (P < 0.001). The antimicrobial activity exhibited by GIC with CHX was the highest, and
Conventional GIC was the least. Propolis exhibited higher antimicrobial activity against S. mutans than with L. acidophilus. CH
exhibited significant antimicrobial activity against L. acidophilus than with S. mutans.

The mean zone of inhibition of GIC with CHX (Group III) was more against S. mutans than compared to Lactobacillus. The
values reported in this study were a little higher in contrast to those obtained by Mittal et al., Türkün et al., and Takahashi et al.
[2],[9],[10] This difference could be attributed to the liquid form of CHX diacetate, which was used in the present study. Liquid
can diffuse well into the agar and exhibit more antibacterial action. The decline in antibacterial activity from day 1 to day 7 and
day 14 could be attributed to the concomitant decline in the available concentration of CHX with time. The decrease in CHX
may be a result of the loss of material by elution or the decrease in CHX is related to the formation of insoluble salts with GIC.
Although the concentration of CHX is decreased with time, the level of CHX in the micro-environment might be sufficient to
prevent secondary caries for an extended period of time. It was suggested that a decrease in CHX concentration with time
leads to the recolonization of less-sensitive microorganisms on the tooth and prevent S. mutans from re-establishing on the
tooth surface.[11] The concentration used (1%) was optimal to provide antibacterial activities without altering the mechanical
properties, bonding abilities, or setting time of GIC. CHX diacetate is preferable to use over CHX digluconate as it is a stable
material and is not prone to decomposition.

The mean zone of inhibition in Group II (GIC with Propolis) was significant against S. mutans and L. acidophilus (P < 0.001).
The activity of propolis against microorganisms was more related to the synergistic effects of flavonoids than individual
compounds. The components of propolis extract such as flavonoids, caffeic acid, and cinnamic acid influence the microbial
membrane or cell wall sites, resulting in functional and structural defects. The presence of propolis in the matrix of glass
ionomer creates a pathway for the release of fluoride ions which confers an additional advantage to the restorative materials.
The mean values obtained in the present study were 19.70 ± 2.67 mm against S. mutans which was higher to that obtained by
Airen et al.[12] where they used 20% Ethanolic Extract of Propolis (EEP), which exhibited a mean diameter of 12.4 ± 1.46 mm
against S. mutans. The higher values obtained in our study could be due to the difference in the concentration used (25%) or
may be due to the combined antibacterial effect of GIC and Propolis. The effect of concentration on antimicrobial activity is yet
to be concluded since the studies so far done are few and they are contradictory to each other. According to Topcuoglu et al.,
[13] the diameters of zones of inhibition determined against S. mutans were not based upon the concentration of EEP.
Hatunoğlu et al.[14] found that 25% and 50% EEP added to GIC inhibited S. mutans but inhibition was not seen with 10% EEP.
In the well-diffusion test conducted by Elgamily et al.,[15] the diameter of inhibitory zones was 32.60 ± 2.22 mm with 1.25%
Propolis added to GIC, and they concluded that the results were concentration dependent. The difference in the values of
various studies could be attributed to the geographical origin of propolis, variations in the strains used, and the type of agar
used.

Against L. acidophilus the inhibition zones shown by Group II (GIC with Propolis) when compared to Group III (GIC with CHX)
and Group IV (GIC with CH) were smaller but were significantly larger (P < 0.001) when compared to Group I (Conventional
GIC). This indicates that GIC with propolis was effective against L. acidophilus up to some extent but not as effective as that of
GIC with CH and GIC with CHX. These results were closer to that of a study done by Airen et al.[12] These features exhibited
by propolis and CH promises a new ray of hope for their use as an additive into restorative materials as they are effective not
only on S. mutans but also on L. acidophilus.

The mean zone of inhibition shown by Group IV (GIC with CH) was smaller when compared to the other two groups, namely
Group II (GIC with Propolis) and Group III (GIC with CHX) but larger when compared to Group I (Conventional GIC) against S.
mutans [Figure 1]. Higher values were seen in our study in contrast to that of Mishra et al.[1] and Ahmed et al.[16] This
difference could be possibly due to the variation in the method of measurement of the inhibition zone. Debnath et al.[4] found
that modification of conventional GIC with 10% v/v CH improved the antibacterial property of GIC against S. mutans. CH exerts
antimicrobial activity by modifying the electric potential of the cell wall of bacteria and also its acidic nature which prevents the
microbial growth and by promoting the release of fluoride.{Figure 1}

Against L. acidophilus, the zone of inhibition shown by GIC with CH was larger compared to GIC with Propolis and
Conventional GIC but smaller when compared to GIC with CHX [Figure 2]. This suggests that GIC with CH is more effective
than GIC with propolis and conventional GIC, but it is not as effective as with CHX against L. acidophilus.{Figure 2}

Limitations of the study

In the present in vitro study, the antimicrobial efficacy of the study materials was tested against standard strains. However, the
antibacterial effect of these restorative materials in the oral cavity may vary because of the multitude of microorganisms present
in oral biofilm. Hence, further clinical studies should be undertaken to compare these study materials with different
concentrations to explore their antimicrobial activity against diverse oral microflora.

Conclusion

All the four groups of restorative materials exhibited antibacterial activity from day 1 to day 14 among which CHX had shown
significantly larger zones of inhibition against S. mutans and L. acidophilus. All the groups had shown higher antimicrobial
activity against S. mutans than L. acidophilus.

Although CHX showed a large zone of inhibition against both the strains used in the study, it could adversely affect the
commensal microflora and is harmful to pulpal cells when placed in deep cavities. The two natural substances used in the
study, i.e., Propolis and CH were effective against S. mutans and L. acidophilus. With the current trend, “shift to nature,” these
natural derivatives which also have an added advantage of fluoride release can have a novel and promising role in treating
dental caries. This is especially useful in children affected with early childhood caries and also in those exhibiting behavioral
problems in whom cavity cutting and restoration could be challenging.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References
1 Mishra A, Pandey RK, Manickam N. Antibacterial effect and physical properties of chitosan and chlorhexidine-cetrimide-
modified glass ionomer cements. J Indian Soc Pedod Prev Dent 2017;35:28-33.
2 Mittal S, Soni H, Sharma DK, Mittal K, Pathania V, Sharma S. Comparative evaluation of the antibacterial and physical
properties of conventional glass ionomer cement containing chlorhexidine and antibiotics. J Int Soc Prev Community
Dent 2015;5:268-75.
3 Yadiki JV, Jampanapalli SR, Konda S, Inguva HC, Chimata VK. Comparative evaluation of the antimicrobial properties of
glass ionomer cements with and without Chlorhexidine gluconate. Int J Clin Pediatr Dent 2016;9:99-103.
4 Debnath A, Kesavappa SB, Singh GP, Eshwar S, Jain V, Swamy M, et al. Comparative evaluation of antibacterial and
adhesive properties of chitosan modified glass ionomer cement and conventional glass ionomer cement: An in vitro
study. J Clin Diagno Res 2017;11:75-8.
5 Kong M, Chen XG, Xing K, Park HJ. Antimicrobial properties of chitosan and mode of action: A state of the art review. Int
J Food Microbiol 2010;144:51-63.
6 Cheung RC, Ng TB, Wong JH, Chan WY. Chitosan: An update on potential biomedical and pharmaceutical applications.
Mar Drugs 2015;13:5156-86.
7 Libério SA, Pereira AL, Araújo MJ, Dutra RP, Nascimento FR, Monteiro-Neto V, et al. The potential use of propolis as a
cariostatic agent and its actions on mutans group streptococci. J Ethnopharmacol 2009;125:1-9.
8 Koo H, Vacca Smith AM, Bowen WH, Rosalen PL, Cury JA, Park YK. Effects of Apis mellifera propolis on the activities of
streptococcal glucosyltransferases in solution and adsorbed onto saliva-coated hydroxyapatite. Caries Res 2000;34:418-
26.
9 Türkün LS, Türkün M, Ertuğrul F, Ateş M, Brugger S. Long-term antibacterial effects and physical properties of a
chlorhexidine-containing glass ionomer cement. J Esthet Restor Dent 2008;20:29-44.
10 Takahashi Y, Imazato S, Kaneshiro AV, Ebisu S, Frencken JE, Tay FR. Antibacterial effects and physical properties of
glass-ionomer cements containing chlorhexidine for the ART approach. Dent Mater 2006;22:647-52.
11 Ribeiro J, Ericson D. In vitro antibacterial effect of chlorhexidine added to glass-ionomer cements. Scand J Dent Res
1991;99:533-40.
12 Airen B, Sarkar PA, Tomar U, Bishen KA. Antibacterial effect of propolis derived from tribal region on Streptococcus
mutans and Lactobacillus acidophilus: An in vitro study. J Indian Soc Pedod Prev Dent 2018;36:48-52.
13 Topcuoglu N, Ozan F, Ozyurt M, Kulekci G. In vitro antibacterial effects of glass-ionomer cement containing ethanolic
extract of propolis on Streptococcus mutans. Eur J Dent 2012;6:428-33.
14 Hatunoğlu E, Oztürk F, Bilenler T, Aksakallı S, Simşek N. Antibacterial and mechanical properties of propolis added to
glass ionomer cement. Angle Orthod 2014;84:368-73.
15 Elgamily H, Ghallab O, El-Sayed H, Nasr M. Antibacterial potency and fluoride release of a glass ionomer restorative
material containing different concentrations of natural and chemical products: An in vitro comparative study. J Clin Exp
Dent 2018;10:e312-20.
16 Ahmed F, Prashanth ST, Sindhu K, Nayak A, Chaturvedi S. Antimicrobial efficacy of nanosilver and chitosan against
Streptococcus mutans, as an ingredient of toothpaste formulation: An in vitro study. J Indian Soc Pedod Prev Dent
2019;37:46-54.

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