Effect of Cocoa Pod Husk Proanthocyanidins Extract on Cyclooxigenase-2 (COX-2) Expression in Gingival Crevicular Fluid of Periodontitis Rats

ABSTRACT


INTRODUCTION
Cocoa pod husk only used as animal feed and if they are not used properly, they will increase the amount of waste from the cacao pod, even though from a medical perspective, the cacao pod husk has many ingredients that can be isolated and utilized. 1 One of the largest ingredients is polyphenols in the form of catechins (37%), anthocyanins (4%), and proanthocyanidins (58%). 2 The polyphenol of cacao pod extract, can be used as an immunomodulatory, anti-cancer, antioxidant, antibacterial, and anti-inflammatory agent. 3 Proanthocyanidin, the largest polyphenol type of cacao pod extract, can be used as a natural alternative for healing inflammation, such as periodontal disease. 4 The damage that occurs in periodontal tissue is caused by various virulence factors of P. gingivalis such as lipopolysacharide, capsule, fimbriae, and gingipains. 5 P. gingivalis is a species that is closely related to the formation process of chronic periodontitis, 85% in periodontal tissue that is inflamed. 6 Virulence factors possessed by P. gingivalis bacteria can cause inflammation by releasing proinflammatory cytokines, interleukin (IL-1, IL-6), and tumor necrosis factor (TNF-α). 7 These proinflammatory cytokines activate transcription of the cyclooxigenase (COX-2) gene. 8 . Proinflammatory cytokines can cause inflammation of the periodontal tissue through the arachidonic acid metabolic pathway. 9 Cyclooxygenase is an enzyme that has an important role in the metabolic process of arachidonic acid to produce inflammatory mediators, especially COX-2. COX-2 is one of the enzymes responsible for the synthesis of inflammatory mediators, thromboxane A2 (TXA2) and prostaglandin E2 (PGE2). COX-2 is not expressed continuously, its expression increases when it receives inflammatory stimuli such as lipopolysaccharides, hormones, growth factors and proinflammatory cytokines, which will cause an increase in inflammatory mediators. Continuous increase of inflammatory mediators can cause tissue damage. 10 COX-2 will be secreted in the gingival crevicular fluid in periodontal inflammation. The resulting of secretions can be used as biomarkers in determining the diagnosis and severity of periodontal disease. Detection of the presence of the COX-2 enzyme is easiest by taking gingival crevicular fluid, in patients with periodontitis the amount of gingival crevicular fluid will increase. 11 The advantages using gingival crevicular fluid as a method of analyzing periodontal inflammation are due to easy, inexpensive, and non-invasive method. 12

Animal preparation
The treatment procedure for experimental animals has received approval (ethical clearance) by the Health Research Ethics Commission of Gajah Mada Faculty of Dentistry, no. 0019/KKEP/FKG-UGM/EC/2019. The experimental animals were adapted first for 7 days in a closed cage and given standard food and drink. This aims to obtain uniformity prior to conducting research to control experimental animals.

Extraction proantocyanidins of cocoa pod husk
The cocoa pod husk (Theobroma cacao L.) was cut and dried in the sun to dry, then shaved and blended to get a fine powder. Powdered cocoa pod husk were freed from fat using n-hexane solvent by maceration with a ratio of powder to solvent (1: 5) three times. The fat-free sample was dried. Powder cocoa pod 100 gram was put into an erlenmeyer containing 700 ml of 70% acetone solution and 300 ml of distilled water, then stirred until homogeneous and put in a waterbath shaker at 50°C for 20 minutes. The extract solution was separated from the supernatant by centrifugation at 2000 rpm for 10 minutes. The extract solution was then put into a rotary evaporator, after which it was transferred to a petri dish and put in the oven. The petri dish was removed from the oven, then the thick part that is at the bottom of the petri dish was removed and placed in a beaker. 14 Chromatography analysize used HPLC method Proanthocyanidins analyze was performed using the column HPLC method which was operated at 25°C. The compounds were detected at 200 and 400 nm wavelengths. The mobile phase of HPLC consisted of 2% (v/v) acetic acid in water (eluent A) and 0.5% acetic acid in water and acetonitrile (50:50, v/v; eluent B). The mass spectrophotometric analysis of the extract was carried out by a mass spectrometer in negative ionization mode. The nebulizer pressure was set to 45 psi and the drying gas flow rate was 5 l/min. The flow rates and temperature of the casing gas were 11 l/min and 350°C, respectively. The mass ranges from 50 m/z to 2000. Chromatographic separation was performed on an ODS C18 analytical column (4.6 × 250 mm), using the Agilent 1290 Infinity HPLC system (Agilent Technologies, USA). About 0.3 ml/min of eluent was introduced into the mass detector. Selecting ion monitoring (SIM) was used to select molecular ion isomers from the procyanidins group in the proanthocyanidin extract of the cacao peel for quantification. The Agilent Mass Hunter Workstation was used for data acquisition and processing. 1 Preparation for Proanthocyanidins of cacao pod husk extract gel 96 ml distilled water was measured with a volumetric flask and poured into the mortar. Then 4 grams CMC-Na were measured by analytical scales and putted into a mortar containing distilled water. Let stand 10-15 minutes, stirring it until expands and forms a yellow gel. The mixture of CMC-Na and distilled water that has become gel was weighed as much as 45 grams and 100% proanthocyanidin of cocoa pod husk extract as much as 5 grams, then it was putted in the mortar and mix until homogeneous to get a gel of proanthocyanidins of cocoa pod extract with 10% concentration. 16

Preparation of P. gingivalis suspention
After making the culture media, one ose of P. gingivalis with 33277 ATCC pure was inoculated in each petridish, then incubated for 2x24 hours. 17 The suspension was made by taking 1 ose P. gingivalis bacteria from the culture preparation and it dissolving in 1 cc of saline/PZ solution in the tube. The reaction after that was homogenized by centrifuge and measured at a concentration of 2x109 CFU/ml according to the Mc Farland 0.5 standard using a spectrophotometer. 13

Applied of P.gingivalis and proanthocyanidins
The negative control, positive control, and treatment groups were injected with 0.05 ml of P. gingivalis ATCC 33277 at a concentration of 2x109 CFU/ml in the distobuccal and distopalatal of gingival sulcus of maxillary first molar. The injection was repeated once every 3 days for 2 weeks. 17 after obtaining the periodontitis rat model, then taking the rat gingival sulcus fluid on day 0 for the negative control and treatment group. Applying placebo gel for the negative control group, metronidazole for the positive control group and proanthocyanidin extract cacao pod 10% for treatment group, daily for 28 days. Gingival crevicular Corresponding Author: Rudy Joelijanto fluid was take on days 7, 14, and 28. Placebo gel and proanthocyanidin extract gel of cocoa pod husk 10% was applied to the distobuccal and distopalatal gingival sulcus area of the maxillary first molar using a plastic filling instrument. 18

Measurement of COX-2 expression
Teeth were cleaned with cotton to control saliva, then gingival crevicular fluid (GCF) samples were taken with paper point number 15 for 30 seconds. The paper point was positioned horizontally in the area of the gingival groove in the distobuccal part of the maxillary first molar. Taking GCF should be done carefully so did not make a injury to the gingival groove area which in turn will cause contamination. Paper points were inserted into 0.5 mL eppendorf tube and stored at -20°C, until the ELISA test was performed. Before the ELISA test, the eppendorf tube was added with 50 µL 0.02 M of Phosphate Buffer Solutions (pH 7.0-7.2), followed by 2000-3000 RPM centrifugation at room temperature 18-25°C for 20 minutes, after that the ELISA test was carried out with the COX-2 ELISA kit. Then the test results are read using an ELISA r eader with a wavelength of 450 nm for a maximum of 30 minutes after giving the stop solution. 19

Statistical analysis
The data obtained were analyzed using the Statistical Package for the Social Sciences (SPSS). Kolmogorov-Smirnov test used to test for normality and Levene's test for homogeneity. One-way analysis of variance (ANOVA) would be carried out to compare the TNF-a expression between each treatment group, followed by Least Significant Different (LSD). Groups differences were significant if the p value was < 0.05.

RESULTS AND DISCUSSION
The expression of COX-2 was presented in Table 1. There were significant difference of COX-2 expression on 0, 7, 14 and 28 days (p= 0.000). On 0 day, the most expression was negative control group (71.740±3.56), proantocyanidins group higher than positive control group (56.577±1.070); on the 7 day, the most expression of COX-2 was negative control group (113.393±12.527), positive control group higher than proantocyanidins group (97.897±6.931); on the 14 day, the most expression of COX-2 was negative control group (194.094±6.592), positive control group higher than proantocyanidins group (78.397±2.134); and on the 28 day, the most expression of COX-2 was negative control group (55.924±1,706), positive control group higher than proantocyanidins group (54.081±2,644). The difference of COX-2 expression between group was presented in Table 2 . On the 0 day, there were difference between negative and positive control group (0.04); on the 7 day, there were difference between negative control and proanthocyanidins group (0.02); between negative control and proanthocyanidins group (0.00), also between positive control and proanthocyanidins group (0.00); on the14 day, there were difference between negative and positive control group (0.00), also between negative control and proanthocyanidins group (0.00) The damage that occurs in periodontal tissues is caused by various virulence factors from P. gingivalis such as lipopolysacharides, capsules, fimbriae, and gingipains. 5 The presence of lipopolysaccharides will stimulate the formation of inflammatory mediators through the cyclooxygenase (COX) pathway. 9 COX is an enzyme that useful for accelerating the synthesis of prostaglandins from arachidonic acid. Arachidonic acid is an unsaturated fatty acid found in the phospholipid bilayer. 20 . The inflammatory stimulus causes the activation of phospholipase A2 which causes the release of arachidonic acid from the cell membrane to the cytosol. Arachidonic acid metabolism through the cyclooxygenase (COX) pathway will produce prostaglandin E2 (PGE2) and thromboxane A2 (TXA2). 21 Prostaglandin E2 causes increased vasodilation and endothelial permeability resulting in increased infiltration of inflammatory cell. PGE2 is the most type prostaglandin in the pathogenesis of periodontitis. 22 Increased proinflammatory cytokines (IL-1 and TNF-α) and PGE2 can stimulate bone resorption by increasing osteoclast formation. Proinflammatory cytokines and PGE2 will also inhibit the formation of osteoprotegerin (OPG) which functions to inhibit osteoclast formation, resulting in increased osteoclast formation and bone resorption. 23 The results of this research showed the treatment group with 10% proanthocyanidin extract gel had the lowest TNF-α and COX-2 expression. This was caused by the proanthocyanidins in the cocoa pod husk extract having antiinflammatory and antibacterial properties. 24 Proanthocyanidin activity in inhibiting COX-2 is probably by inhibiting the activity of proinflammatory cytokines. This was supported by several previous studies showing that proanthocyanidin can inhibit the growth of P. gingivalis and reduce COX-2 expression through inhibition of inflammatory cytokines activity. 25 According to research conducted by La, proanthocyanidin can inhibit the invasion of P. gingivalis and inhibit the activity of virulence factors such as gingipain. Gingipains functions to activate proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β ) and interleukin-6 (IL-6) which can trigger inflammation. Based on research by Lee et al. (2017), proanthocyanidin from grape seeds can also decrease the expression of IL-1β,  Proanthocyanidin from cranberries can decrease the expression of IL-8 and CCL-5 from the oral epithelial cells induced by P. gingivalis. 5 Previous study showed that there was an increase in COX-2 mRNA expression after induced by IL-1β for nine hours and decreased significantly after proanthocyanidins administration. The study also showed that metronidazole gel which was used as a positive control could reduce COX-2 expression and there was a significant difference in the average COX-2 expression on days 7, 14, and 28. The decrease was due to metronidazole gel are bactericid against anaerobic gram-negative bacteria such as P. gingivalis by interfering for bacterial DNA synthesis.
The decreased of IL-1 and TNF-α expression will decrease COX-2 expression due to the signal inhibition of IL-1 and TNF-α for the release of phospholipids from the cell membrane, whereas COX-2 expression in the negative control group is higher. The amount of mRNA and gingival COX-2 protein in subjects with chronic periodontitis was higher than in healthy ones. 28 This is reinforced by the results of Mesa et al. study that COX-2 expression in patients with gingivitis or periodontitis was higher than in healthy gingiva. 23 Research conducted by Mori et al. demonstrated that P. gingivalis gingipain can induce COX-2, wherein COX-2 mRNA levels are greatly increased after 2 hours and can still be detected at 6 hours and 12 hours after exposure by gingipain, lipopolysaccharides, capsules, and fimbriae. P. gingivalis can regulate inflammatory. 24 Tamura et al. showed that cells induced by IL-1β increased the expression of mRNA and protein COX-2 and PGE2.
The changes of COX-2 expression based on time, in the proanthocyanidins group COX-2 increased on day 7 and decreased slowly until day 28. Meanwhile, COX-2 levels in the negative control group continued to increase until day 14 and decreased drastically until day 28. The enhance of COX-2 expression was probably due to the negative control group still experiencing an inflammatory process, where COX-2 is an important enzyme for the synthesis of inflammatory mediator precursors. Based on the research of Paulasilva et al., it was shown that in the periodontal tissue of rats induced by P. gingivalis, there was fluctuation in COX-2 expression, where the highest expression of COX-2 occurred on day 14. 26

CONCLUSIONS
The proanthocyanidins contained in the cocoa pod husk extract (Theobroma cacao L.) potential to reduce COX-2 expression in periodontitis rats. Based on the results of the study, the highest COX-2 expression was in the negative control group, and there was a significant difference in the average COX-2 expression in the negative control group on days 0, 7, 14 and 28.