The effect of watermelon frost on prostaglandin E 2 ( PGE 2 ) in inflamed pulp tissue ( in vitro study )

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. OneWay 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.


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 (PGE 2 ) if it is compared with normal pulp.][3][4][5][6][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. 8A study by Olsson et al., 9 showed that any materials containing eugenol had more serious reaction of tissues than those without eugenol. 10nother 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, 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 PGE 2 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. 13,14urthermore, 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. 15atermelon as a trial material produced white powder (frost) by adding glauber's salt (Na 2 SO 4 .10H 2 O) for several days and frost was dissolved in aquadest (Figure 2).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 PGE 2 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 W 1 = 650 nm and W 2 = 490 nm.

RESULT
The result from reading the absorbance level of color on each sample by ELISA reader showed the largest decrease of PGE 2 level in eugenol group then followed by watermelon as trial material.Meanwhile, the group of commercial watermelon frost showed the smallest decrease of PGE 2 level (Table 1).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 PGE 2 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 PGE 2 level is higher than that of commercial watermelon frost.
To see the difference of PGE 2 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 PGE 2 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 PGE 2 .This was followed by watermelon frost as trial material and commercial watermelon frost.
However, the decrease of PGE 2 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 PGE 2 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 PGE 2 level.
According to Cohen et al. cit Hashimoto et al., 8 the PGE 2 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 PGE 2 level is found in irreversible pulpitis and it leads to vein vasodilatation, the increase of vascular permeability, chemotaxis, pain, and bone resorption. 16he tables in this study implied that each group had several samples showing higher PGE 2 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 PGE 2 level in each case could be different.In severe inflammation, the PGE 2 level would be high.Consequently, in group I the PGE 2 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 PGE 2 level that was higher than that found on other samples.
Eugenol has the greatest capability to reduce the PGE 2 level.It was shown from a study by Dewhirst and Hirafuji cit.Hashimoto et al., 8 where eugenol can inhibit biosynthesis of PGE 2 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 PGE 2 level will be significantly decreased.

Figure 2 .
Figure 2. Watermelon frost found in the rind surface of watermelon.

Table 1 .
The PGE 2 level from watermelon frost as trial material, commercial watermelon frost and eugenol (ng/mL)

Table 2 .
The average level and standard of deviation from the decrease of PGE 2 level (ng/mL)

Table 3 .
Analysis of variant for the groups: watermelon frost as trial material, commercial watermelon frost, and eugenol the PGE 2 level (ng/mL)