A HISTOPATHOLOGIC STUDY OF DIRECT PULP-CAPPING TREATMENT WITH PROPOLIS-FLAVONOIDS EXTRACT Ardo Sabir*

ABSTRACT Propolis is a resinous material collected by honey bees from various plants. Flavonoid is the main of chemical substance in propolis that has inhibit bacterial growth and reduce the release of free radicals, suggesting that this substance has anti-bacterial and anti-inflammatory properties. Therefore, the aim of the present study was to assess the response of rat dental pulp to direct pulp capping with flavonoids-propolis extract. Crude propolis was obtained from South Sulawesi Province. Flavonoid materials was purified from an ethanol extract of propolis using thin layer chromatography method. A Class I cavity was prepared on the occlusal surface of the right maxillary first molar in Sprague-Dawley rats. The dental pulp was exposed and then capped directly with zinc oxide-based filler as a control (group I) or with flavonoids-propolis extract (group II). The animals were sacrificed at week 1, 2 or 4, biopsy samples were obtained, and these were stained and viewed by light microscopy. The results of the present study showed that pulp inflammation occurred in groups I as early as week 1. No reparative dentin formation was seen in this group. In contrast, there was no evident inflammatory response in group II at week 1. However, mild and moderate pulp inflammation in this group occurred at 2 and 4 weeks after treatment, respectively. Partial reparative dentin formation was seen in group II at week 4. In conclusion, direct pulp capping treatment with flavonoids-propolis extract in rats dental pulp may delay dental pulp inflammation and stimulate reparative dentin formation. Keywords : Flavonoid, Propolis, Direct pulp capping, Reparative dentin, Rat.

* Department of Conservative Dentistry, Faculty of Dentistry, Hasanuddin University

Jl. Perintis Kemerdekaan Km 10 Tamalanrea, Makassar

STUDI HISTOPATOLOGIS PERAWATAN KAPING PULPA LANGSUNG DENGAN EKSTRAK FLAVONOID PROPOLIS Ardo Sabir*
ABSTRAK Propolis adalah bahan resin yang dikumpulkan oleh lebah madu dari berbagai jenis tumbuhan. Flavonoid adalah senyawa kimia utama dalam propolis yang menghambat pertumbuhan bakteri dan mengurangi terlepasnya radikal bebas, sehingga diduga senyawa ini memiliki sifat anti bakteri dan anti-inflamasi. Oleh karena itu, tujuan penelitian ini adalah untuk menilai respons pulpa gigi tikus terhadap ekstrak flavonoid propolis pada perawatan kaping pulpa langsung. Propolis diperoleh dari Provinsi Sulawesi Selatan. Flavonoid dipurifikasi dari ekstrak etanol propolis menggunakan metode kromatografi lapis tipis. Suatu kavitas Klas I dibuat pada permukaan oklusal gigi molar kanan rahang atas tikus Sprague-Dawley. Dilakukan perforasi ruang pulpa dan segera dilakukan penutupan dengan bahan zinc oxide sebagai kontrol (kelompok I) atau dengan ekstrak flavonoid propolis (kelompok II). Hewan coba dikorbankan pada minggu ke-1, 2 atau 4 setelah perlakuan. Sediaan diperoleh, dilakukan pewarnaan dan diamati dengan mikroskop cahaya. Hasil penelitian ini menunjukkan bahwa inflamasi pada pulpa terjadi pada kelompok I pada minggu ke-1. Tidak terlihat pembentukan dentin reparatif pada kelompok ini. Sebaliknya, tidak terjadi respons inflamasi pada kelompok II minggu ke-1. Namun demikian, inflamasi ringan dan moderat terjadi pada kelompok ini 2 dan 4 minggu setelah perlakuan. Pembentukan dentin reparatif parsial terlihat pada kelompok II pada minggu ke-4. Sebagai kesimpulan, perawatan kaping pulpa langsung dengan ekstrak flavonoid propolis pada pulpa gigi tikus mampu menghambat inflamasi pulpa dan menstimulus terbentuknya dentin reparatif. Kata kunci : Flavonoid, Propolis, Kaping pulpa langsung, Dentin reparatif, Tikus.

* Bagian Ilmu Konservasi Gigi, Fakultas Kedokteran Gigi, Universitas Hasanuddin Jl.
Perintis Kemerdekaan Km 10 Tamalanrea, Makassar.

Introduction Direct pulp capping with materials such as adhesive resins is a technique to seal the dental pulp that may be exposed due to mechanical and/or chemical factors as well as bacterial activities and stimulate reparative dentin formation 1. Propolis, a resinous material collected by honey bees from various plants, has been used as a traditional medicine for anti-inflammation and anti-bacteria for many centuries. Of interest, previous studies demonstrated that propolis is not toxic to dental pulp fibroblasts
2

and

may, in fact, stimulate dental pulp collagen formation and reduce both pulp inflammation and degeneration 3. Propolis (bee glue, or royal jelly) is a natural substance based on the resin of pines, collected by bees. The term 'propolis' derives from 'pro' (Greek = before), and 'polis' (city) based on the fact that honeybees use propolis to narrow the opening to their hives. Propolis is a complex entity, containing about 55% resinous compounds and balsam, 30% beeswax, 10% ethereal and aromatic oils, and 5% bee pollen. Contained chemicals include amino acids; flavanoids including flavones, flavonols and flavanones; terpenes; vanillin; tetochrysin; isalpinin pinocembrin chrysin galangin; ferulic acid; caffeic acid; caffeic acid phenethyl ester; cinnamic acid and cinnamyl alcohol 4. Among of propolis constituents found, flavonoids regulate the immune response, reduce free radical release, and inhibit bacterial and fungal growth, suggesting that this component has natural anti bacterial, anti-inflammatory and immunoregulatory properties
5,6

. Therefore, the aim of the present study was to assess the response of rat

dental pulp tissue to direct pulp capping with flavonoids-propolis extract.

Materials and methods This study was an experimental laboratory research with a random post test only control group design. The study was conducted at Biology Pharmacy Laboratory, Faculty of Pharmacy; Experimental Animal Breeding Unit, Faculty of Veterinary; and Histology and Cell Biology Laboratory, Faculty of Medicine, Gadjah Mada University Yogyakarta. Propolis Trigona Sp was collected from honeycombs in Bulukumba regency, South Sulawesi in early monsoon season. Dried propolis was submitted to exhaustive maseration, filtered using aqueous ethanol and concentrated by rotary evaporator. The residue was separated using using toluene solution to yield flavonoid fraction, which then chromatographed on silica gel GF254 precoated plate with n-butanol : acetic acid : water (3:1:1 v/v) as mobile phase. Examination under ultraviolet light at max 254 nm and 366 nm showed that the flavonoids from propolis contain flavones, flavonoles, flavanols and chalcone 7. Twelve to sixteen week-old Sprague-Dawley rats (weight 200 g - 250 g) were divided into 2 groups, each group consisted of 9 animals. Group I as a control was direct pulp capped with a zinc oxide-based filler (Dentorit, Dentoria, French). Meanwhile the dental pulp of group II was directly capped with propolis-derived flavonoids extract. Briefly, animals were anaesthetized intramuscularly with ketamine (Ketaral, Warner Lambert, Ireland) (65 mg kg-1 body weight) and xylazine HCl (Rompun, Lerkusen, Germany) (7 mg kg-1 body weight). A Class I cavity was prepared on the occlusal surface of right maxillary first molars using a low-speed tapered diamond round bur (Intensiv, Swizerland) (0.84 mm in diameter). The pulps were then exposed by a dental explorer (Martin, Germany) (0.35 mm in diameter of the tip) on the cavity floor and directly pulp-capped with zinc oxide-based filler (Dentorit, Dentoria

France) or propolis flavonoid (0.5 mg). The cavities were then air-dried and filled with glass ionomer cement (Fuji IX, GC corp., Tokyo, Japan). The experiment protocol has been approved by the ethical committee, School of Medicine, Gadjah Mada University, Yogyakarta. Animals were sacrificed at week 1, 2, or 4. The teeth and the surrounding bone were resected, fixed in 10% neutral formalin buffer, decalcified with 10% EDTA for 30 days, embedded in paraffin and sectioned serially at 6 m. The sections were stained with hematoxylin-eosin and viewed under a light microscope. Histological evaluation of the slides was made according to the Recommended Standard Practices for Biological Evaluation of Dental Materials 8. Inflammation was evaluated according to the presence or absence of polymorphonuclear leukocytes (PMNL) and/or macrophages and was graded as follows: 0 = No infiltration of inflammatory cells; 1 = Infiltration by a few PMNL, and/or macrophages (mild inflammation); 2 = Infiltrated by a moderate number of PMNL, and/or macrophages (moderate inflammation); and 3 = Massive infiltration of PMNL and/or macrophages (severe inflammation). Meanwhile evaluation for reparative dentin formation based on absent the formation of reparative dentin was graded as follows: 0 = No reparative dentin; 1 = Incomplete/partial reparative dentin; and 2 = complete reparative dentin. Results The response of inflammation on rats dental pulp after application of zinc oxidebased filler or propolis-derived flavonoids extract can be seen in Table 1.

Table 1. Response of inflammation on rats dental pulp after application with zinc oxide-based filler or propolis-derived flavonoids extract Time period (weeks) 1 2 4 Number of specimen (n) 3 3 3 3 3 3 Inflammation Response None 3 2 Mild 2 1 1 Moderate 1 2 1 1 Severe 1 2 1

Groups I (zinc oxide) II (flavonoid) I (zinc oxide) II (flavonoid) I (zinc oxide) II (flavonoid)

From Table 1, it seems that in group I, mild and moderate inflammation was evident in pulp chamber as early as week 1, and the levels of inflammatory response tend to increase at week 2 and 4 (moderate and severe inflammation). In contrast, in group II, no inflammation was evident in propolis-derived flavonoid-treated animals at week 1. However, a mild inflammatory response occurred in 1 out of 3 animals at week 2. At week 4, 1 moderate and 1 severe inflammation were seen in pulp chamber from this group. Interestingly, partial reparative dentin formation beneath the pulp-capping material in 1 out of 3 animals from group II could be seen at week 4 (Table 2). No evidence of necrotic pulp tissue was seen in both of groups throughout the study. Table 2. Reparative dentin formation on rat dental pulp after direct pulp capping with zinc oxide-based filler or propolis-derived flavonoids extract Time period (weeks) 1 2 4 Groups I (zinc oxide) II (flavonoid) I (zinc oxide) II (flavonoid) I (zinc oxide) II (flavonoid) Number of specimen (n) 3 3 3 3 3 3 Reparative Dentin Formation None 3 3 3 3 3 2 Incomplete 1 Complete -

For the sake of brevity and clarity, photomicrographs are only shown from group II of animals at week 1 and 4 (Figure.1).

RD

Figure 1. Rat dental pulp response following direct pulp capping with propolis-derived flavonoids. (A) No inflammatory cells could be found in dental pulp chamber at week 1; (B) Numerous of inflammatory cells (arrows) and partial reparative dentin formation (RD) could be seen in dental pulp chamber at week 4. Hematoxylin-eosin stain, original magnification 40x.

Discussion Application of direct pulp-capping materials on the exposed pulp direct pulp capping may induce inflammatory reaction beneath the materials. The results of the present study showed that direct pulp capping with flavonoid materials extracted from propolis reduced inflammatory response at week 1. The present results are not surprising, since flavonoids from propolis on inflammatory response are known to have anti-infllammatory properties, perhaps, via suppression of immune cell activation, macrophage-derived nitric oxide and cytokine production, and neutrophil activation 5. Alternatively, flavonoids from propolis might inhibit bacterial growth in the rat pulp chamber, thereby reducing the host response to bacterial antigens 5. Furthermore, the exact mechanism by which persistent inflammatory reaction at week 4 could still be observed in the dental pulp directly capped with flavonoids from propolis needs to be further clarified, however. Several possibilities may be put forward to explain these results. This persistent inflammation at week 4 might be due to oral bacterial microleakage
9

and such infection might not have been completely eliminated by

propolis-derived flavonoid. Alternatively, both anti-inflammatory and anti-bacterial properties of flavonoids from propolis at week 4 might have been considerably reduced due to metabolism of these materials 10. Of interest, 4 weeks after direct pulp capping with propolis-derived flavonoids, partial reparative dentin formation in one of the experimental animals could be observed. This histological finding suggests that flavonoids from propolis may stimulate reparative dentinogenesis. Dentin formation following pulp capping is known to involve differentiation of odontoblast-like cells forming reparative dentin and biosynthesis activity of surrounding primary odontoblasts, phenomena that require interaction

between extracellular matrix molecules and growth factors such as transforming growth factor (TGF)-1 (11). Indeed, propolis are also capable to stimulating the production of (TGF)-1 (12), a growth factor known to be important for odontoblast-like cell differentiation 9, and the synthesis of collagen by dental pulp cells 3. Whether or not stimulatory effects of propolis-derived flavonoids on reparative dentin formation as seen in the present study might be operated by this pathway remains to be further assessed. In conclusion, the present study showed that direct pulp capping with propolisderived flavonoids inhibited rat dental pulp inflammatory response at week 1 and stimulated both moderate inflammation and reparative dentin formation at week 4. In contrast, slight inflammation at week 1 but increased inflammation at week 4 without detectable reparative dentin formation could be found after direct pulp capping with a temporary filler. Therefore, the results of the present study suggest that direct pulp capping with flavonoids from propolis may reduce dental pulp inflammation and stimulate reparative dentin in rats. Acknowledgements The author gratefully thank to Prof. S. Pramono (Faculty of Pharmacy, Gadjah Mada University) for assisting in propolis preparation. References 1. de Souza Costa CA, Hebling J, Hanks CT. Current status of pulp capping with dentin adhesive systems; a review. Dent Mat 2000; 16:188-97. 2. Al-Shaher A, Wallace J, Agarwal S, Bretz W, Baugh D. Effect of propolis on human fibroblasts from the pulp and periodontal ligament. J Endod 2004; 30:359-61. 3. Scheller S, Ilewics L, Luciak M, Skrobidurska D, Stoijko A, Matuga W. Biological properties and chemical application of propolis. IX. Experimental observation on the influence of the ethanol extract of propolis (EEP) on dental pulp regeneration. Arzneim-Forsch 1978; 28:289-91.

4. Scully C. Propolis: a background. Br Dent J 2006; 200: 359-60. 5. Burdock GA. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol 1998; 36:347-63. 6. Kosalec I, Pepeljnjak S, Bakmaz M, Vladimir-Knezevi S. Flavonoid analysis and antimicrobial activity of commercially available propolis products. Acta Pharm 2005; 55(4): 423-30. 7. Sabir A. Identifikasi golongan flavonoid dalam propolis Trigona sp dari Kabupaten Bulukumba Sulawesi Selatan yang digunakan pada perawatan kaping pulpa langsung. Maj Ked Gigi (Edisi Khusus TIMNAS III) 2003; 36: 59-63. 8. Kirk EEJ, Lim KC, Khan MOG. A comparison of dentinogenesis on pulp capping with calcium hydroxide in paste and cement form. Oral Surg Oral Med Oral Pathol 1989; 68:210-19. 9. Murray PE, Hafez AA, Windsor LJ, Smith AJ, Cox CF. Comparison of pulp responses following restoration of exposed and non-exposed cavities. J Dentistry 2002; 30:213-22. 10. Havsteen BH. The biochemistry and medical significance of flavonoids. Pharmacol Ther 2002; 96:67-202. 11. Tziafas D. The future role of a molecular approach to pulp-dentinal regeneration. Caries Res 2004; 38:314-20. 12. Ansorge A, Reinhold D, Lendeckel U. Propolis and some of its constituents downregulate DNA synthesis and inflammatory cytokine production but induce TGF-1 production of human immune cells. Z Naturforsch (C) 2003; 58:580-9.

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