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 properties^3-5, antibacterial^6-12, and antifungal activities on yeasts of Candida genera^12,13.

In dentistry, its properties have been studied for many purposes. Scheller et al.³studied 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 & Schilder¹⁷propolis 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.¹⁸studied 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 (-20^o 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 17^th day old until 21^st 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 20^th day old. At the age of 20 days, the animals were inoculated with 0.2mL of 10⁸CFU/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	17^th day	18^th day	19^th day	20^th day	21^st day	22^ndday
Streptomycin
Cariogenic diet
Inoculation of bacteria
Starting experiment

Figure 1 – Scheme of rats treatment before starting the experiment.

At the 23^rd 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 90^th 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 antibacterial^{10,11,19,27,28} and antifungal activities^12,13 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.³³also 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.