INFLUENCE
OF PROPOLIS ON CARIES - AN IN VIVO
STUDY IN RATS
INFLUÊNCIA
DA PRÓPOLIS SOBRE CÁRIE - ESTUDO IN VIVO EM RATOS
Ota-Tsuzuki C*,
Datte CE**, Souza
DC***, Shimizu MT****
ABSTRACT: Propolis
is well known by its antibacterial and anti-inflammatory properties. The aim of
this study was to evaluate the influence of a hydro-alcoholic suspension of
propolis daily consumed on caries using an infected rats model. With this
purpose, Wistar rats were infected with a suspension
of S. mutans
(CCT1901) strain cells previously to the experiment baseline. Then the animals
were divided in two groups: Control group (n=10) which
were fed with a cariogenic diet (sucrose 56% and
powdered skim milk 28%) and drinking water ad
libitum. Experimental Group (n=10) were fed with
the same diet and water containing propolis (1mg/ml). After 90 days, the
animals were sacrificed and the molar teeth caries lesions were registered and
analyzed (Chi-square test, a=0,05).
Even though the number of caries lesions did not vary between groups, no
lesions with score 3 (partial or total coronal destruction)
in experimental group were observed, against 12 observed in control
group. Although propolis did not avoid caries, it has influenced on caries
process since the number of teeth with score 3 were not observed in
experimental group which received propolis in daily diet.
KEYWORDS: Propolis. Caries. Rats. Streptococcus mutans.
RESUMO: A própolis é conhecida
popularmente pelas suas propriedades antibacterianas e antiinflamatórias. Neste trabalho analisamos a influência
do consumo de um extrato hidro-alcoólico de própolis sobre a cárie em modelo
animal. Com esta finalidade, ratos Wistar foram infectados com a cepa de S.
mutans (CCT1901) previamente ao início dos experimentos. Os ratos foram divididos
em dois grupos: Grupo controle (n=10): alimentados com dieta cariogênica
(contendo 56% de sacarose e 28% de leite em pó) e água ad libitum. Grupo
experimental (n=10): a mesma dieta cariogênica e a água de beber foram
acrescidos de extrato alcoólico de própolis (1mg/ml). Após 90 dias, os animais
foram sacrificados e as lesões de cárie em molares foram analisadas quanto ao
número de lesões e quanto ao grau de severidade (Índice de Mellamby). Os
resultados foram submetidos ao teste Qui-quadrado (a=0,05). O número de lesões cariosas foi semelhante entre
os grupos. Foi observado um número maior de lesões grau 3 (destruição parcial
ou total da coroa dental) no grupo controle (n=12), por outro lado, o grupo
experimental não apresentou nenhuma lesão com este grau de severidade.
Concluímos que embora a própolis não tenha evitado o aparecimento da cárie, foi
observada diferença estatística quanto ao grau de severidade; sendo esta menor
no grupo experimental que recebeu própolis na sua dieta diária.
PALAVRAS-CHAVE: Própolis. Cárie.
Ratos. Streptococcus mutans.
INTRODUCTION
Nowadays, science is looking for
alternative medicine in nature, which cause less side
effects on the human body. Among these substances propolis is in evidence, a
resinous wax-like substance which bees collect from plants and buds used as
glue or putty to line their hives and fill up cracks.1 Propolis is
known for its medicinal properties such as bone tissue regeneration2,
immunological properties3-5, antibacterial6-12, and
antifungal activities on yeasts of Candida genera12,13.
In dentistry, its properties have
been studied for many purposes. Scheller et al.3 studied the pulp regeneration of teeth
treated with propolis; Hernandes & Hernandes14
observed satisfactory results by applying propolis on buccal
ulcers; Silveira et al.15 observed that
patients with gingivitis that received applications of medicine with propolis
had a faster regression of painful symptoms. In 2003, Gebara
et al.16 evaluated the effect of subgingival irrigation with a propolis extract and
concluded that the subgingival irrigation as an
adjuvant to periodontal treatment was more effective than conventional
treatment per se.
The antimicrobial properties of
propolis have been attributed to flavonoids, phenolic acids, diterpenic acids,
flavonoid aglycones
(flavones and flavanones) and coumarins.8,9 According to Takaisi-Kikuni
& Schilder17 propolis inhibits bacterial growth by preventing
cell division and disorganizing the cytoplasm, the cytoplasmic
membrane and the cell wall, causing a partial bacteriolysis,
and inhibiting protein synthesis. Nishio et
al.18 studied an ethanolic extract
of Brazilian propolis and observed its antibacterial activity against 7 strains
of Streptococcus mutans.
Ikeno et al.19 observed that rats which received propolis in their diet had less caries than
the control group fed only with cariogenic diet.
There are studies which found cariostatic activities in desalivated
rats model using ethanolic extracts of total propolis
and some of its components.20,21
Considering the aforementioned properties of propolis, we decided to study the
influence of a hydro-alcoholic solution of propolis on caries using S. mutans infected rats model.
MATERIAL AND METHODS
Propolis extract: Propolis was collected from the Vale do Paraiba
region, in São Paulo State located in the Southeast of Brazil. To obtain the
alcoholic extract, 450g of in natura propolis
were suspended in 1.500 mL of ethanol (Merck). This
suspension was kept away from light and was shaken every day for 15 minutes for
a period of 30 days. After this period, it was decanted for about 48 hours and
subsequently filtered. The alcohol was partially evaporated in order to permit
the lyophilisation process and it was stored in a freezer (-20o C)
until use. For the experiments, the lyophilized propolis was weighed, dissolved
in ethanol (99.5%) and then added to drinking water.
Animals: 20 male rats (Rattus novergicus, Wistar)
were used. Since 17th day old until 21st day old,
streptomycin (2mg/mL) was given in drinking water of animals to depress the
oral flora. The experimental diet described below was given to the litters
since 20th day old. At the age of 20 days, the animals were
inoculated with 0.2mL of 108 CFU/mL suspension of S.mutans (CCT1901), a streptomycin resistant strain, two
days before the diet experiment had already started. The success of inoculation
was checked on the first day (litters are 22 days old) of the experiment
cultivating material collected by oral swabbing.22,23
In summary the rats treatment before starting the experiment followed the
scheme below (Figure 1):
Age Event |
17th day |
18th day |
19th day |
20th day |
21st day |
22nd day |
Streptomycin |
|
|
|
|
|
|
Cariogenic diet |
|
|
|
|
|
|
Inoculation of bacteria |
|
|
|
|
|
|
Starting experiment |
|
|
|
|
|
|
Figure 1 – Scheme of rats
treatment before starting the experiment.
At the 23rd day old, the animals were divided in two groups:
Control group: The animals were fed with high cariogenic
diet (containing 56% of sucrose and 28% of skin milk powder (Molico-Nestlé) and, sterile distilled water ad libitum.24
Experimental Group: The animals were fed with the same diet described
above and propolis was added to the drinking water (1mg/mL). All the bottles of
this group were covered with aluminium foil to avoid the light exposure.
Both
groups received the diet for 90 days, the food was
moistened with sterile distilled water before feeding the animals. The food and
water of all animals were replaced daily. At the 90th day, the animals were killed and their jaws were removed and fixed
in a 10% formaldehyde solution for 48 hours. After this period, the material
was dissected and then dyed with 0.5% fucsin for 4
hours.19
The jaws were sectioned using a diamond impregnated disc (KG-Sorensen). The section was made
from the mesial to the distal area, to study the
depth of the caries lesion.
The material was evaluated according to the degree of severity
of the lesions. Although both halves were evaluated, only one score was
attributed for each tooth, as preconized by Mellanby’s
Index25 (Table 1).
Table 1: Mellanby’s Index25 used to
score caries lesion progression stage.
Score |
Degree of caries |
0 |
Absence
of lesion |
1 |
Enamel
lesion |
2 |
Dentin
lesion |
3 |
Partial
or total coronal destruction |
Statistical analysis: The Chi-square
test was used for two independent samples with a=0.05. The
Standardized Residual was used to observe
which proportion was the major contributor to the significant difference
found with Chi-square test.
RESULTS
During
the experiment no differences in the drinking habits of the animals were
observed, both groups consumed about the same volume of water. Each animal from
the experimental group consumed about 50mg of propolis per day.
After
90 days, the animals were sacrificed and the teeth were examined. The results
are shown in Table 2.
Table
2: Distribution of each severity degree among molar teeth of rats.
SEVERITY DEGREE |
FREQUENCY OF EACH
DEGREE |
|||
Control Group |
Experimental Group |
|||
Maxillary teeth |
Mandibular teeth |
Maxillary teeth |
Mandibular teeth |
|
0 |
31 |
11 |
34 |
14 |
1 |
21 |
9 |
15 |
12 |
2 |
8 |
28 |
11 |
34 |
3 |
0 |
12 |
0 |
0 |
Total |
60 |
60 |
60 |
60 |
It was demonstrated that the
maxillary teeth did not show any difference between experimental and control
groups (Chi-square test; a=0,05).
The results of the mandibular teeth of both groups were analyzed using the
same test and we could verify that the groups were different (Chi-square test;a=0,05). It is
noteworthy that we did not find score 3 lesions in this group (Table 2), the Standardized Residual values revealed that the
major contributors for this difference were the teeth with score 3.
Table 3: Distribution of each severity degree in molar
teeth of rats.
SCORE |
FREQUENCY EXPERIMENTAL GROUP |
FREQUENCY EXPERIMENTAL GROUP |
STANDARDIZED RESIDUAL |
0 |
14 |
11 |
+0.90 |
1 |
12 |
9 |
+1.00 |
2 |
34 |
28 |
+1.30 |
3 |
0 |
12 |
-3.46 |
DISCUSSION
Propolis has attracted a lot of attention in recent years as
a useful substance applied in medicine and cosmetics, even it has been known in
folk medicine since ancient times. The antibacterial, antiviral, antifungal and
antiprotozoan properties of propolis has been widely
studied.26
In previous studies we and others
research groups demonstrated the antibacterial10,11,19,27,28
and antifungal activities12,13 on strains isolated from oral cavity.
Streptococcus mutans called our attention by
its sensitivity to propolis;10,11,28 since
from MS group it is the species most associated with human dental decay.30
So our hypothesis is that propolis extract could have some influence on caries
process. Thus, a Streptococcus mutans infected
rats model was proposed.
In previous experiments, we could not
achieve any high degree of severity lesion with a
experimental period of 4220to 56 days23, even in animals
from the control group which received only the cariogenic
diet. So, the experimental period was extended to 90 days (data not shown).
These differences could be explained by some reasons: a) it was used a Streptococcus
mutans strain instead Streptococcus sobrinus as used by Ikeno et
al.,19 Koo et al.,20 and Koo et
al.21 ; b) it was demonstrated by Soet et
al.30 that for rats S. sobrinus is more cariogenic
than S. mutans, then the caries lesion could
appear earlier in experiments with S. sobrinus; and c) it was not used desalivated rats as used by Koo et al.21 and Koo
et al.20, this procedure cause a low saliva flow causing an increase
of smooth surface caries, because a highly acidogenic
flora was selected.
Concerning to the number of lesions
it was observed almost the same results for the experimental and control groups
but the severity degree was different. Ikeno et al.19 found different results by analyzing only the dentin
caries of the first and second molar teeth. Using the same method of
evaluation, we observed 37% of reduction in number of caries in the
experimental group (data not shown). The different bacterial species used in
our experiments could justify this variation. For animals treated with propolis
it was found only dentin and enamel lesions without total or partial coronal
destruction, on the other hand, animals treated only with cariogenic
diet (control group) had lesions with a higher level of severity.
Besides, after analyzing the quality
of lesions, also considering the enamel lesions (Table 2), the data showed that
there was an inhibition of the caries process, even though it did not prevent
them. These data are in accordance with Koo et al.20
that found either a cariostatic effect of a Brazilian propolis even with a desalivated rat infected with S. sobrinus
model was used. Koo et
al.21 studied two substances, specifically: tt-farnesol and apigenin; common
members of propolis composition and it was demonstrated that both have
cariostatic activity but, substances like fluoride, chlorhexidine
are still more efficient.
Nishio et al.18 showed that propolis could be used to control the caries process, the
authors isolated three cinnamic acids from propolis
and verified that two of them strongly inhibit acid formation from sucrose by S.mutans and, the
third inhibit the synthesis of insoluble glucans by glucosyltransferase. It
is known that these insoluble glucans contribute to
the pathogenic potential of S. mutans and S. sobrinus.31 According to Ikeno et al.19, propolis has
antimicrobial activity and clearly inhibits insoluble glucans
synthesis and partially inhibits glycosiltransferase
activity in some species of MS group: S. mutans,
S. sobrinus and S. cricetus .
Zárate Pereira 32
found interesting results dropping propolis solution every day on the surface
of human teeth enamel, with this procedure the enamel becomes more resistant
when submitted to the Knoop hardness test. They do
not explain what substance of propolis composition is responsible for this
property. Gimalia et al.33
also studied this property by immersing enamel slices in different
concentration solutions of propolis. The Vickers
hardness values increased accompanied by higher concentrations of propolis in
the solution.
In summary, our results show that
propolis possess a cariostatic activity in a S. mutans
infected rat model which is the most common species isolated from human
decay. Probably, the cariostatic activity of propolis
is due to a group of properties that is achieved by the large variety of
substances found in its composition. Properties like: antibacterial activity,
its influence on glycosiltranferase and on the
resistance of human enamel and others. Much more investigations are need to
establish which are the substances and the mechanisms and if they would work
alone or together leading to this cariostatic activity. But there is no doubt that
this material is a source for a variety of substances that should be studied
aiming its use in the clinical dentistry routine, preventing not only caries
but also other oral pathologies.
ACKNOWLEDGEMENT: This research was supported by FAPESP (Grant 94/4865-3).
REFERENCES
1. Dobrowolski JW, Vohora SB, Sharma K, Shah AS, Naqvi SAH, Dandiya PC. Antibacterial, antifungal, antiamoebic, antiinflamatory and antipyretic studies on propolis bee
products. J Ethnopharmacol
1991;35:77-82.
2. Stojko A, Scheller S, Szarnowiecka I, Tustonowski J, Ostach H, Obuszko Z. Biological properties and clinical application
of propolis. VIII. Experimental observation on the influence of ethanol extract
of propolis (EEP) on the regeneration of bone tissue. Arzneim–Forsch Drug Res. 1978;28:35-7.
3. Scheller S, Ilewicz L, Luciak M, Skrobidurska D, Stojko A, Matuga, W. Biological properties and clinical application
of propolis. IX. Experimental observation on the influence of ethanol extract
of propolis (EPP) on dental pulp regeneration. Arzneim-Forch
Drug Res. 1978;28:289-91.
4. Dimov V, Ivanovska N, Manolova N, Bankova, V, Nikolov, N. Immunomodulatory action of propolis. Influence on
anti-infectious protection and macrophage function. Apidologie
1991;22:155-62.
5. Mirzoev OK, Calder PC. The effect of propolis and its components on eicosanoid
production during the inflammatory response. Prostaglandins Leukot
Essent Fatty Acids. 1996;55:441-9.
6. Ghisalberti EL. Propolis:
a review. Bee World 1979;60:59-84.
7. Grange JM, Davey RW. Antibacterial
properties of propolis (bee glue). J R Soc Med. 1990;83:159-60.
8. Aga H, Shibuya T, Sugimoto T, Kurimoto M, Nakajima
S. Isolation and identification of antimicrobial compounds in Brazilian propolis. Biosc Biotech Biochem. 1994;58:945-6.
9. Bankova V, Christov R, Kujumgiev A, Marcucci MC, Popov S. Chemical composition and
antibacterial activity of Brazilian propolis. Z Naturforsch. 1995;50c:167-72.
10. Ota C, Valente PHM, Unterkircher
CS, Shimizu MT. Effects of propolis on bacteria isolated from oral cavity. Lecta. 1998;16:73-7.
11. Park YK, Koo MH, Abreu
JA, Ikegaki M, Cury JA, Rosalen PL. Antimicrobial activity
of propolis on oral microorganisms. Curr Microbiol. 1998;36:24-8.
12. Kujumgiev A, Tsvetkova I, Serkedjieva Y, Bankova V, Christov R, Popov S.
Antibacterial, antifungal and antiviral activity of propolis
of different geographic origin. J Ethnopharmacol. 1999;64:235-40.
13. Ota C, Unterkircher C, Fantinato V, Shimizu MT. Antifungal activity of propolis on
different species of Candida. Mycoses. 2001;44:375-8.
14.
Hernandez NC, Hernandez CM. Efecto del
propoleo sobre las aftas bucales. Informe preliminar. In: Asis M. Investigaciones
cubanas sobre el propoleo. Cons Cient Instit Medic Veterin. 1990;182-190.
15. Silveira GM, Godoy AG, Torriente RO, Oritiz MCP,
Cuéllar MAF. Estudio preliminar sobre los efectos del propolan en el
tratamiento de la gingivitis
cronica y de las ulceras bucales. Rev Cubana Estomatol. 1988;25:36-44.
16. Gebaraa ECE, Pustiglioni AN, de Lima LA, Mayer, MP.
Propolis extract as an adjuvant to
periodontal treatment. Oral Health Prev Dent. 2003;1:29-35.
17. Takaisi-Kikuni, NB, Schilcher H. Electron microscopic and microcalorimetric
investigations of the possible mechanism of the antibacterial action of a
defined propolis provenance. Planta Med. 1994;60:222-7.
18. Nishio M, Tabuchi A, Shibuya T. Anti-dental caries compounds in
Brazilian propolis. Honeybee-Sci. 1996;17:151-4.
19. Ikeno K, Ikeno T, Miyazawa C. Effects of propolis on dental caries
in rats. Caries Res. 1991;25:347-51.
20. Koo H, Rosalen PL, Cury JA, Park YK, Ikegaki M,
Sattler A. Effect of Apis mellifera
propolis from two Brazilian regions on caries development
in desalivated rats. Caries Res. 1999; 33:393-400.
21. Koo H, Pearson SK, Scott-Anne K, Abranches J, Cury JA, Rosalen PL, et al. Effects of apigenin
and tt-farnesol on glucosyltransferase
activity, biofilm viability and caries development in
rats. Oral Microbiol Immunol. 2002;17:337-43.
22. Johansson I, Ericson T, Bowen W, Cole M. The effect of malnutrition on caries development and saliva
composition in the rat. J Dent Res 1985;64:37-43.
23. Schuster GS, Navia JM, Amsbaugh S, Larson RH. Sources of variability in rat caries
studies: microbial infection and caging procedure. J Dent Res. 1978;57(2):355-60.
24. Keyes PH, Jordan HV. Periodontal lesions in the syrian hamster-III. Findings related to
an infectious and transmissible component. Arch Oral Biol. 1964;9:377-400.
25. Chaves MM. Odontologia sanitaria. Org Panamericana
de la Salud: Washington; 1962.
26. Bankova V, Christov R, Kujumgiev A, Marcucci MC, Popov S. Chemical composition and
antibacterial activity of Brazilian propolis. Z Naturforsch [C].
1995;50:167-72.
27. Sforcin JM, Fernandes A Jr, Lopes CA, Bankova
V, Funari SR. Seasonal effect on Brazilian propolis
antibacterial activity. J Ethnopharmacol. 2000;73:243-9.
28. Koo H, Rosalen PL, Cury JA, Ambrosano GM, Murata RM, Yatsuda R, et al. Effect of a new variety of Apis mellifera propolis on mutans Streptococci. Curr Microbiol. 2000;41(3):192-6.
29. Loesche WJ. Role of Streptococcus mutans in human
dental decay. Microbiol Rev. 1986;50(4):353-80.
30. De Soet JJ, Van Loveren C, Lammens AJ,
Pavicić MJ, Homburg CH, Ten Cate JM. Differences in cariogenicity
between fresh isolates of Streptococcus sobrinus and Streptococcus
mutans. Caries Res. 1991;25(2):116-31.
31. Gibbons RJ, Van Houte J.
Bacterial adherence in oral microbial ecology. Ann Rev Microbiol. 1975;29:19-44.
32. Zárate Pereira, P. In situ study about the propolis of Apis mellifera action in dental
caries development and in dental biofilm formation.
[Tese de mestrado]. São Paulo: Faculdade de Odontologia, Universidade de
São Paulo; 2003.
33. Giamalia I, Steinberg D, Grobler S, Gedalia I. The effect of
propolis exposure on
microhardness of human enamel in vitro. J Oral Rehabil. 1999;26:941-3.
* Claudia Ota-Tsuzuki
- Professora Assistente do Programa de Pós-Graduação em Odontologia da
Universidade Guarulhos - UnG; e-mail: ctsuzuki@prof.ung.br
** Carlos Eduardo Datte - Mestre em Odontologia,
área de concentração em Periodontia.
*** Diego de Carvalho Souza - Mestre
e Doutorando em Odontologia, área de concentração em Periodontia
da Universidade Guarulhos. e-mail: diego_souzaperio@yahoo.com.br
**** Mario Tsuznezi
Shimizu - Professor Titular da UNESP-São
José dos Campos.; e-mail: mtshimizu@terra.com.br
1) UnG - Universidade Guarulhos 2) Indexador: Latindex 3) Indexador: Dialnet
Comentários sobre o artigo
por Vitoria Viana (19/05/2019)
por Eduardo Nunes (20/05/2019)
por Ingeborg Mowll (21/05/2019)
por Rafael Montenegro (22/05/2019)
por Reagan Hugh (27/05/2019)
por Harry Pool (27/05/2019)
por Brian Mosby (05/06/2019)
por Wesley Canada (17/06/2019)
por Terrence Crutcher (04/07/2019)
por Kathaleen Tisdall (05/07/2019)
por Agustin Tisdall (05/07/2019)
por Daniele Baldwinson (06/07/2019)
por Lucile McFarland (06/07/2019)
por Garfield Prindle (06/07/2019)
por Alisia Winfrey (06/07/2019)
por Melanie Gutman (06/07/2019)
por Soila Essex (14/07/2019)
por Norman Frith (15/07/2019)
por Madge Tamayo (15/07/2019)
por Chasity Climpson (15/07/2019)
por Claudette Cartledge (15/07/2019)
por Cynthia Whaley (15/07/2019)
por Pedro Caldeira (19/07/2019)
por Magnolia Sasaki (19/07/2019)
por Lawerence Manna (19/07/2019)
por Dee Blackmon (19/07/2019)
por Myrna Bowens (23/07/2019)
por Alena Fredrickson (25/07/2019)
por Benjamin Lombardi (25/07/2019)
por Ronny Caraway (26/07/2019)
por Albertina Creech (27/07/2019)
por Cecelia Shelton (30/07/2019)
por Courtney Ogden (31/07/2019)
por Pat Kuhn (07/08/2019)
por Elouise Womble (07/08/2019)
por Margret Leichhardt (07/08/2019)
por Berry Eudy (07/08/2019)
por Marcos Hartwick (09/08/2019)
por Kasha Polanco (10/08/2019)
por Lien Grills (10/08/2019)
por Margret Trombley (11/08/2019)
por Monroe Pape (20/08/2019)
por Tandy Delatte (21/08/2019)
por Larue Cooley (23/08/2019)
por Maria Ana Clara Caldeira (06/11/2019)
por Brooks Brookfield (06/04/2021)
por Ryan Begay (07/04/2021)
por Bella Mahony (09/04/2021)
por Bella Mahony (09/04/2021)
por Bella Mahony (10/04/2021)
por Brooks Brookfield (18/04/2021)
por Brooks Brookfield (18/04/2021)
por Tobias Theissen (11/03/2024)
por Nolan Kaestner (12/03/2024)
por Joni Beahm (12/03/2024)
por Brenda Haszler (15/03/2024)
por Ollie Lower (16/03/2024)
por Norman Bevill (16/03/2024)
por Stephen Waylen (17/03/2024)
por Isiah Knaggs (17/03/2024)
por Sharron Mill (21/03/2024)
por Lilly Austin (24/03/2024)
por Benjamin Forrester (12/04/2024)