MKG Vol 42 No 2 April 2009.indd 82 Vol. 42. No. 2 April–June 2009 Research Report The effect of watermelon frost on prostaglandin E2 (PGE2) in inflamed pulp tissue (in vitro study) Dennis and Trimurni Abidin Department of Conservative Dentistry Faculty of Dentistry, University of North Sumatera Medan - Indonesia ABSTRACT Background: Pulp inflammation can be marked by the increase of prostaglandin E2 (PGE2) level compared to normal pulp. The increase of PGE2 may lead to vasodilatation, increase of vascular permeability, pain and bone resorption. Watermelon frost has been well known in Chinese society for pain relief and inflammation in oral cavity and teeth. Purpose: The aim of this study was to investigate that watermelon frost can be used to decrease the PGE2 level. Method: 27 samples of pulp tissues used in this in-vitro study, were extirpated from the patients’ teeth with symptomatic irreversible pulpitis referred to clinic of Conservative Dentistry, RSPGM Faculty of Dentistry, USU. Trial materials were applied to 27 samples i.e. watermelon frost as a trial material and commercial watermelon frost and eugenol to observe their effect on PGE2. PGE2 level of each material was detected through ELISA method by measuring and comparing the absorbance reading of the wells of the samples against standards with a micro plate reader at W1 = 650 nm and W2 = 490 nm. Result: The result showed the biggest effect was found in the third group (eugenol), mean 4.6933, followed by the first group (watermelon frost as a trial material), mean 18,1578 then the second group (commercial watermelon frost), mean 82,2689. One- Way ANOVA revealed that there were significant differences among all trial materials (p < 0.001) on PGE2 level. Conclusion: This study demonstrated that watermelon frost can be used to decrease the PGE2 level in inflamed pulp tissue and led to the acceptance of traditional medicine and natural products as an alternative form of dental care. Key words: watermelon frost, prostaglandin-E2, inflammation, pain Correspondence: Dennis, c/o: Bagian Konservasi Gigi, Fakultas Kedokteran Gigi Universitas Sumatera Utara. Jl. Alumni No.2, Kampus USU Medan 20155, Indonesia. E-mail: dennis_dionisius@yahoo.co.uk INTRODUCTION Pulp inflammation is a case which is often found in dental practice. This inflammation has caused pain to the patients. The inflamed pulp will cause pain that is obviously seen by the increase of prostaglandin E 2 (PGE2) if it is compared with normal pulp. The increase of PGE2 will trigger pain on teeth, vein vasodilatation, vascular permeability, and bone resorption.1-7 Hospitals and dental clinics with patients of symptomatic irreversible pulpitis and periodontitis need intradental pain relief to decrease inflammation. Generally, the pain relief, which is often used, is eugenol (clove oil) as sedative. Apparently, most of Indonesians have used clove oil as traditional medicine to relieve intradental pain for a long time. They unconsciously have known eugenol as local sedative but they have not precisely figured out how much eugenol should be consumed.8 A study by Olsson et al.,9 showed that any materials containing eugenol had more serious reaction of tissues than those without eugenol.10 Another pain relief commonly used is pulperyl containing creosate, procaine, phenol and chloroform. Several corticosteroids can be used to reduce intradental pain for acute pulpitis patients. Practically their use is combined with polyantibiotic. Because polyantibiotic is imported, its market price is expensive and it is not widely sold. Watermelon frost, a traditional medicine for therapy, has been widely known by the Chinese community for ages. Clinical experiment has proven that watermelon frost affects inflammation treatment of oral cavity, tonsilitis, laryngitis, 83Dennis: The effect of watermelon frost pharyngitis, dental pain caused by high fever, inflamed gums, scald, etc.11,12 This indicates that inflammation can be reduced or healed after being given watermelon frost as traditional medicine. Research dealing with watermelon frost which will be used as pain relief should be increased more because Indonesia is enriched by tropical fruit like watermelon. In endodontic treatment the materials used should be biocompatible (nontoxic in human tissues). However, concerning the use of watermelon frost as pain relief, a question arises as the following: Can watermelon frost that has been widely known for medical treatments of oral cavity, pharyngitis, laryngitis, gingivitis be used as intradental pain relief? The objective of the study was to figure out how watermelon frost affects PGE2 which is one of inflammatory mediators. It is expected that the study can be seen as one consideration to develop the use of watermelon frost as traditional medicine for the inflammation and the pain relief besides eugenol, miswak and corticosteroid.13,14 Furthermore, the study can familiarize the use of watermelon frost so that people can utilize watermelon which is not expensive. In addition, the study will encourage primary health services to provide economical material used as pain relief which is nontoxic and biocompatible enough in dental tissues. MATERIALS AND METHODS Type of this study was comparative experimental research. It involved watermelon frost as the material. The steps to make watermelon frost were: chose a watermelon weighing 2.5 kg, sliced its top, and part of the flesh was removed. Put 500 grams of glauber’s salt (sodium sulfate decahydrate) on it (Figure 1). Then a small stick of bamboo to retained it and put the top back so the hole was covered. Afterwards the watermelon was hung in fridge (not a part of freezer) approximately 7–10 days or until frost (white powder) was formed in outer part of watermelon rind. This frost has a therapeutic effect in reducing pain and inflammation.15 Watermelon as a trial material produced white powder (frost) by adding glauber’s salt (Na2SO4.10H2O) for several days and frost was dissolved in aquadest (Figure 2). Figure 2. Watermelon frost found in the rind surface of watermelon. Sample was divided based on the materials: Group I (9 samples) was given watermelon frost with concentration 50% (dissolved with aquadest), Group II (9 samples) was given watermelon frost with commercial 50% (content based on the supply of factory) and Group III (9 samples) was given eugenol 50% (dissolved with dimethyl sulfoxide/ DMSO). Pulp tissues were obtained from the teeth which were chosen by opening the cavity and extirpating pulp with extirpating needle (barbed-broach). The teeth rooted with more than one pulp were gained from the largest root canal. Then, the pulp was placed in Eppendorf containing 0.5 ml of phosphate buffer saline (PBS) and 10–3 M of nimesulide; centrifuge was applied at 2400 rpm for 10 minutes; after that, it was restored at temperature –23° C. Each Eppendorf was given watermelon frost as trial material, commercial watermelon frost and eugenol. Analysis of PGE2 quantitative level was seen by using Enzyme-Linked Immunosorbent Assay kit (Neogen, USA) through an equipment called ”ELISA reader” that showed the absorbance level of color at the wave length W1 = 650 nm and W2 = 490 nm. RESULT The result from reading the absorbance level of color on each sample by ELISA reader showed the largest decrease of PGE2 level in eugenol group then followed by watermelon as trial material. Meanwhile, the group of Figure 1. Watermelon which is given glauber’s salt. 84 Dent. J. (Maj. Ked. Gigi), Vol. 42. No. 2 April–June 2009: 82−85 commercial watermelon frost showed the smallest decrease of PGE2 level (Table 1). Table 1. The PGE2 level from watermelon frost as trial material, commercial watermelon frost and eugenol (ng/mL) Number Sample Concentration of PGE2 (ng/mL) Group I (Watermelon frost as trial material) Group II (Commercial watermelon frost) Group III (Eugenol) 1. 17.87 86.62 4.30 2. 14.96 98.95 6.51 3. 28.35 68.99 12.27 4. 17.87 108.05 5.68 5. 15.95 68.65 3.76 6. 15.80 71.25 2.92 7. 13.59 83.50 3.80 8. 26.15 92.78 1.95 9. 12.88 61.63 1.05 Table 2. The average level and standard of deviation from the decrease of PGE2 level (ng/mL) Type of Materials n X SD Watermelon frost as trial material 9 18.16 5.45 Commercial watermelon frost 9 82.27 15.74 Eugenol 9 4.69 3.31 Total 27 35.04 35.75 Table 2 indicated that the average level and standard of deviation at group of watermelon frost as trial material were 18.16 ± 5.45 ng/mL. At commercial watermelon frost group, the average level and standard of deviation were 82.23 ± 15.74 ng/mL. At eugenol group, they were 4.69 ± 3.31 ng/mL. These implied that effect of eugenol in reducing the PGE2 level is higher than that in watermelon frost (as trial material and commercial). In the meantime, the effect of watermelon as trial material in reducing the PGE2 level is higher than that of commercial watermelon frost. To see the difference of PGE2 level in watermelon frost as trial material, commercial watermelon frost and eugenol, One-Way ANOVA was applied with α = 0.001 (Table 3). The result of ANOVA showed that there was statistically significant difference (p < 0.001) among groups of watermelon frost as trial material, commercial watermelon frost and eugenol in the decrease of PGE2 level. DISCUSSION In this study, the effect of watermelon frost as trial material was compared to the effect of commercial watermelon frost and eugenol. Of those three materials, it was eugenol that had the greatest effect on PGE2. This was followed by watermelon frost as trial material and commercial watermelon frost. However, the decrease of PGE2 in commercial watermelon frost was lower than in watermelon frost as trial material. This was probably caused by the content of pure watermelon frost in commercial product as much as 50% and the mixture with other materials; Rhizoma Belamcandae (5%), Bulbus Fritillariae (15%), Radix Sophorae Tonkinensis (10%), Mentholum (5%). Indigo Naturalis (5%) and Borneolum (10%). The content of watermelon frost as much as 50% has caused the decrease of PGE2 lower than the watermelon frost as trial material where the content is pure. Besides, other contents found in commercial watermelon frost can influence the effectiveness of watermelon frost in reducing the PGE2 level. According to Cohen et al. cit Hashimoto et al.,8 the PGE2 level on the tissue of symptomatic inflamed pulp was significantly higher than that of asymptomatic inflamed pulp. Isett J et al.17 in his study stated that higher PGE2 level is found in irreversible pulpitis and it leads to vein vasodilatation, the increase of vascular permeability, chemotaxis, pain, and bone resorption.16 The tables in this study implied that each group had several samples showing higher PGE2 level (out layer) even though it was given trial material. In table 1, it was seen that even though the pulp tissue was taken from symptomic irreversible pulpitis, the PGE2 level in each case could be different. In severe inflammation, the PGE2 level would be high. Consequently, in group I the PGE2 level of the third sample was 28.55 ng/mL; in group II the fourth sample was 108.05 ng/mL; in group III the third sample was 12.27 ng/mL. These cases are possibly caused by the PGE2 level that was higher than that found on other samples. Eugenol has the greatest capability to reduce the PGE2 level. It was shown from a study by Dewhirst and Hirafuji cit. Hashimoto et al.,8 where eugenol can inhibit biosynthesis of PGE2 through in vitro. In contrary, a study by Prashar et al.18 showed that eugenol is very cytotoxic in fibroblast and endothelium cells. The toxicity effect has caused the cell inactive through apoptosis and necrosis. Finally, if the inflamed cells in the pulp tissue are inactive, the PGE2 level will be significantly decreased. Table 3. Analysis of variant for the groups: watermelon frost as trial material, commercial watermelon frost, and eugenol the PGE2 level (ng/mL) df F P. Between Groups 2 160.821 .000* Within Groups 24 Total 26 Explanation : P: ANOVA test * : Significance F : Frequency df : degree of freedom 85Dennis: The effect of watermelon frost According to the study by Ho et al.,10 it was mentioned that eugenol can inhibit the growth and proliferation of osteoblastic cell line U2OS. Therefore, eugenol has significant role on periapical toxicity and destroys the cell in the pulp tissue. Watermelon frost is a traditional/herbal medicine which has been empirically known by the Chinese community as one of therapies against inflammation. The use of watermelon frost has been approved by Chinese Drugs Administration Department. Watermelon frost is obtained from watermelon by using glauber’s salt. Since watermelon frost is naturally gained from watermelon, it is biocompatible enough in oral tissues. As consequence, the cells in the pulp tissue including the inflamed cells after being given watermelon frost are probably vital.19 From the data analysis and the discussion, it can be concluded that there were differences among the decrease of PGE2 level found in commercial watermelon frost, watermelon frost as trial material, and eugenol. Based on the analysis above, it can be seen that eugenol has bigger effect of the PGE2 decrease 4 times than watermelon frost as trial material and it is bigger 17 times than commercial watermelon frost. Meanwhile the effect of the PGE2 decrease is bigger 5 times than commercial watermelon frost. Eugenol is very cytotoxic in fibroblast and endothelium cells. The effect of cytotoxicity can lead to the death of cells through apoptosis and necrosis. If the inflamed cells in the pulp tissue are inactive, the PGE2 level will be significantly decreased. Because watermelon frost is naturally gained from the watermelon, watermelon frost is biocompatible and nontoxic in oral tissues, so the cells in the pulp tissue including the inflamed cells after being given watermelon frost are vital. To identify the active substance which functions as anti PGE2 in watermelon frost, fraction and characterization tests need to be carried out. The tests refer to the physical and chemical characteristics of watermelon frost. Besides, cell culture needs to be applied to observe the condition of cells in the pulp tissue after being given watermelon frost. REFERENCES 1. Waterhouse PJ, Nunn JH, Whitworth JM. Prostaglandin E2 and treatment outcome in pulp therapy of primary molars with carious exposures. International Journal of Paediatric Dentistry 2002; 12:116–23. 2. Waterhouse PJ, Whitworth JM, Nunn JH. Development of a method to detect and quantify prostaglandin E2 in pulpal blood from cariously exposed, vital primary molar teeth. Int Endodon J 1999; 32:381–7. 3. Nakano K, Ohishi M, Ogawa Y, Ohba T, Kido J, Miyake Y, Nagata T. Prostaglandin E2 inhibits Alveolar bone resorption in experimental periodontitis in hamster. Dentistry in Japan 1998; 34:108–11. 4. Trowbridge HO. History of pulpal inflammation. In: Seltzer, Bender, editors. Dental pulp. IL: Quintessance Publishing Co, Inc; 2002. p. 227–45. 5. Fouad AF. Molecular Mediators of pulpal inflammation. In: Seltzer and Bender. Dental Pulp. IL: Quintessance Publishing Co, Inc; 2002. p. 247–79. 6. Isett J, Reader A, Gallatin E, Beck M, Padgett D. Effect of an intraosseous injection of Depo-Medrol on pulpal concentrations of PGE2 and IL-8 in untreated irreversible pulpitis. J Endodon 2003; 29:268–71. 7. Ohnishi T, Suwa M, Oyama T, et al. Prostaglandin E2 predominantly induces production of hepatocyte growth factor/scatter factor in Human dental pulp in acute inflammation. J Dent Res 2000; 79:748–55. 8. Hashimoto S, Uchigama K, Maeda M, et al. In vivo and in vitro effects of zinc oxide eugenol (ZOE) on biosynthesis of cyclooxygenase products in rat dental pulp. J Dent Res 1988; 67:1092–109. 9. Ollson B, Sliwkoarski A, Largeland K. Subcutaneous implantation for the biological evaluation of endodontic materials. J Endodon 1981; 7:355–65. 10. Ho YC, Huang FM, Chang YC. Mechanisms of cytotoxicity of eugenol in human osteoblastic cells in vitro. Int Endodon J 2006; 39:389–93. 11. Fang YC, Huang HC, Chen HH, Juan HF, TCM gene DIT: A database for associated traditional chinese medicine, gene and disease information using text mining BioMed Central Ltd 2008; 8(58): 1–11. 12. Zhang, Yi Fang. Comprehensive methods for preventing and treating influenza. J Chinese Medicine 2001; 65:6–10. 13. Sulaiman MI, Al-khateeb TL. The analgesic effects of Miswak. The Saudi Dent J 1996; 8:140–4. 14. Al-Samh DA, Al-Nazhan S. In vitro study of the cytotoxixity of the miswak ethanolic extract. The Saudi Dent J 1997; 9:125–32. 15. Yin-Fang, Dai, Cheng-Jun, Liu. Terapi buah. Adi Loka Sujono, editor. Jakarta: Prestasi Pustaka Publisher; 2002. p. 32–4. 16. Nakano K, Ohishi M, Ogawa Y, et al. Prostaglandin E2 inhibits Alveolar bone resorption in experimental periodontitis in hamster. Dentistry in Japan 1998; 34:108–11. 17. Isett J, Reader A, Gallatin E, Beck M, Padgett D. Effect of an intraosseous injection of Depo-Medrol on pulpal concentrations of PGE2 and IL-8 in untreated irreversible pulpitis. J Endodon 2003; 29:268–71. 18. Prashar A, Locke IC, Evans CS. Cytotoxicity of clove (Syzygium aromaticum) oil and its major components to human skin cells. Cell Prolif 2006; 39:241–8. 19. Zong-Chang, Xiu. Traditional chinese medical therapies: Melons, fruits and vegetables as medicine. 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