Effect of Roselle Extract in Expression of Matrix Metallo Proteinase-8 ( MMP-8 ) in Gingival Creviculer Fluid ( GCF )

Background: This study is performed to determine the efrect of roselle exhact gel l07o toward the changing of MMP 8 mRNA expression in Gingival Crevicu.ler Fluid (GCF) in patient with gingivitis cxperience post acrylic crown insertion. Methods: This study is a pre and post test experimental research. Research subjccts were 9 patients who expericnce gingivitis post acrylic crown inscrtion and divided in three groups (tcatrncnt vr'ith rosellc gel, negativc control, and positive contol). GCF samples were takcn with paper strips before and seven days after application of ros€lle €xtract gel l0lo. The change of MMP-6 mRNA expression is tested using Real Time-Polymerase Chain Reaction (RT-PCR) method. The results thar statistically testcd using paired T test to sce thc effect of ros€lla extract toward MMP-8 activity. Result: The MMP 8 mRNA expression in gingivitis post acrylic crown insertion crown in the cxperimcntd groq is significandy dccrcascd after application ofroselle extract gel 10%, which also seen in the positive control group, but no changes obsewed in the negative control goup. Conclusions: Roselle extract g€l l0% is proven to be effectire in reducing the activity of MMP 8 in GCF.


l. Introduction
Thc usc of the natural substances in thc world of health tends to increasc from year by year, which also occur in dcntistry field. Thc main benefit of using natural ingredients in medication is minimal side effects which made them safer to use. One ofthe herbs that is widely used as beveragc food and rnedicine is Hibiscus Sabdanlla (Roselle). This flower is known for anti-bacterial cffect, containing authocyanin pigments that act as atrtioxidants, and also has vitamins and minerals which are useful for th€ body. Rescarch by Pacome et al found that pctals oflose/le, such as anthocyanins, flavonoids and phenolic acid contribute to th€ antioxidativ€ activity. This finding provide evidence that the petal extract of/L sabdaifa is apote!'tial sowca of natural antioxidants, and this justifu it application in folkloric medicincs. [] The artificial crown is ons altcmative restoratioo that is made for aesthetic rehabilitation dental cades. The common materials used are acrylic, porcelain or combination of metal and porcelain tbat resemble a veit with forms and colors is adfrtcd !o the natural tooth color. According to Oginni €t al (2flX), distribution and treatmcnt fiequancy using artificial crojm bosed on most Aequent age is between 20-29 years old on vital (24,7%o) and on non yital teeth (4 5,2%). l2l The charactcristic of restorative material such as surface rougbness, can affect adhesion of bacterial coating which is caused bv availabiliw of surfaces for bacterial attachment l-enni Ind.idi et al: Efrect ofRoselle Extract ir Expression ofMatrix Metallo  in Gingival Creviculer Fluid (GCF) and proiection of bacteria colonization. [3,4] The effects of crowns or Fixed Partial Dentures (FPDs) on gingival inflammatiol, probing depth, and bone loss were evaluat€d based on accuracy and reliability of measuroment, and,/or appropriateness of data analysis. Campbell and Knoernschild (2000) found tltat Crowns and FPDs increased the progressive gingival inflammation incident that occurred after restorations, especially if r€storations is using intracrevicular finish line placement with impoverished marginal adaptation, or irregular surfaces. Clinically, both defective and acceptable restorations can play a role in gingival infl ammation. [5] Ababnaeh stu<ly in 201I justilies the lolg held cooccpt dut restorations put under the gingival margin are harmful to gingival and periodonbl well being. This study also proposes that in t€eth with subgingival restorations, the increased loss of attachment started slowly and might be clinically discovered I to 3 years after the restorations procedure.
Crowns, bridge abutments (especially acrylic and nonprecious metals) and Class II amalgam restorations appear to be associated with periodontal breakdown. [6] Matrix metalloproteinase-8  or collagenase-2 has been identified as the central biotnarker in cor rective tissue injury that is caused by periodontitis [7] and is also known to have diagnostic value. Matrix m€talloproteinase cause the increase of the blood barrier permeability by damaging extracellullar matrix, basal lamin and endothelial binding tissue, with tbe final result of acute inflammatory destruction. [8] Experiment conducted by Sorsa et al (2010), using immuno-fluorecentric assay technique and dentoanalyzen, detects MMP-8 in GCF samples. [9] Nowadays,23 MMPs have been identified. MMP-I,   A total of 9 (3 males and 6 females) individuals in the age range of 20-25 years who have gingivitis experience after insertiof of acrylic crown. They divided in three groups: Group I: Eeatment with roselle extract gel l0%, group II: negative conhol (base gel), and group III: positive control (ffeatment with Povidon lodine).

The Exeact of Roselle
The identification ofroselle petals is conducted to know the distinctiveness of crude drugs that are going to be used h the experimeot. Four hurdred graurs ofrosella peial was proccssed in the form of powder. Then 100 gram of simplisia powder was extracted by maceration using I L of ethanol which was placed in a glass jar for tbree days, filtered, and into the residue 1.125 L of ethanol was added, stired and left inside a closed vessel for two days. The residue and sediment wore ssparated fiom the filtrat€ using filt€r paper, the result of the fih:dte I and II were mixed and decanted for two da)rs and ther vr'ere concenhated using a rotary evaporator to obtain a thick extrsct.

Preparation ofthe Roselle Exftact Gel
The roselle gen contain Aquadest (min€ral water), Mcthyl paraben, Hydroxyiethyl cellulose, Carboxfmethyl cellulose, Glycerin, Sodium cyclamate, and rosella extract. Eighty thr€€ ml of aquades ri/as heated at 70-90"C, Methyl parab€n and cyclamates were added into aquades until they dissolve and then Hydroxyiethyl cellulose and Carboxyimethyl cellulose were added, then homogenized at the speed of 1000 rpm until it a clear gel rnass was formed. ln this forq glycerin was added while still homogenized. Then, the homogeneous gel base was left until 30-40'C. The last step was adding roselle exhact into the gel while homogenized until it was mixed uniformly into the gel base.

Procedure Gingival crcvicular Fluid Collection b'ith
The research materials were derived GCF of the acrylic crown us€rs that were taken before and 7 days after application of roselle extract gel l0%. GCF was obtained by isolating the gingival area that was using acrylic crowns.
GCF was aspirated using filter paper or paper strip with dimension of 15 mm x 3 mm. The filter paper was left for l0 minutes at the gingival crevice in order to filter the GCF optimally. Once finished, the filter paper was inserted into a srnall tubc that contain liquid L6.
Then this mixture was centrifuged at 12.000 rpm for 10 minutes. The concentrated sediment sample was homogenized for 30 minutes. Before adding the diatom suspension, the mixtu€ of buffer L6 which already contains RNA from the extract was centrifuged for 2-3 minutes at 12.000 rpm, I,ith the aim of RNA extracted s€ttles at th€ botlom of the diatom tube. Twenty miclo liter of diatom was added into the tube, the diatom suspense should always be rotated and stirrcd using a gyratory shaker, 100 rpm for l0 minutes. L5 buffcr and diatom mixtured was rotated again using eppendorf miuocentrifuge at 12.000 rpm for 15 seconds. Supematant that \rras formed fiom each tube was separated using a suction tube which was made of a Pasteur pipette that was connected with a vacuum pnmp to prevent loss of diatoms in the suspense. Ten milliliters of the suspension was res€rved.
Supernatant was washed two times using I ml of L2 wasb buffcr. Onc millilitcr of L2 wash buffcr was addcd, rotatcd and centrifuged in 12.000 rym for 15 seconds, theu the supematant was discarded. The precipitate was washed again with I ml of 70% ethanol twice, and then rotated and centrifugcd at 12.000 rpm for 15 seconds. Th€ supematant was discarde{ the precipitatc was washed again by I ml aceton€, rotated and centrifirged at 12.000 rpm for 15 secoods. Therr the supel[atallt was discalded agail. Acctone rcmainin8 in the sediment was evapotated by opening the lid of the tube and heated in an oven at 50 -55"Cfor l0 minutes. After the sediment was dried.60 mL of TE buffer elution was added, then rotated evenly so that the sediment and the suspension can be dissolved. Then, the tube was incubated in an ovclt at 56'C for l0 minutes. The mixture is centrifuged at 12.000 rpm for 30 s€conds. Forty to fifty microliters of supematant was carefully obtained aod put into a n€\ , tube. The result of the cxtraction were stored in -80"C. Quantitativc RT -PCR using 516r qRT -PCR grcen master mix kit, one step. This protocol was optimized for MX 4000 instrument. Custorrized protocols was adjusted using the insFument by changing the dye dilution based on the instruction manual and followed the recommended factory instumsnt for RT -PCR cycle programme. The passive rcference dy€ was put into reaction, diluted l: 500.
The solution that contains dy€ was kept away Aom [ght.
Dilute 2 x SYBR Green qRT -PCR oastor mixed and stored on top of ica. Fr.rllow the master oix ilitial liquefactiorq the unused portion was stored at 4"C with thc notc: avoid the repcated cycles of Aeeze-liquid.
Thc reaction was mixed gently to prevent bubbles formation (not rotated), then the mixture was distributed into experiment tube. We then added x lrl RNA experiments in each test tubc. The reactioD was mixed slowly to prevent bubbles formation (not rotated). The reactions were centrifuged in a short time. The rcactions wele put into instrumeut and the PCR programme as ready to be run.

2, E. Stotisticol Anslysis
Thc dala analysis was performed to investigate the change MMP-8 nRNA from pretest to posttest for each group and we compared those change tsing paired r /es, (P<0,05).

Results
The analysis results of the cbange of exprcssion of MMP -8 mRNA in GCF, in each group beforc gel application (pretest) ( To evaluate the efI€ct of the gel application of Roselle extract I 0% toward the expression of MMP-8 nRNA in GCF as the standard heatment of gingivitis post artificial crown insertion, we pertbrmed analysis using paired t test in each group (tablc 2).

Discussion
Thc artificial crou,n is one altemative restoration that is made for aesthetic rehabilitation on dental caries. The Onc prcvious rcscarch showcd MMP-E gcnc cxprcssion changes aftcr rcstoration installation which was performed by Peng (2011) who found that thc level of MMP-8 at one month after crown plac€ment in subgingival group was higher than the baseline (P <0. 05) and continued to hcrease for three months after crown placement. The level of MMP-8 at the sixth months after crown placem€nt in subgingival group wns decreased, but was higher than ttre baseline. !2] ln this research, we found that the expression of  GCF early in all tkee groups, namely heatment group, regative coutrol, and positive control (Povidott Iodhe) did not differ significant (  [7] In antimioobial testing that has been done in this study by using bacteria P gigivalrs and ,L sangzis, and we found that inhibition zons of rosella extract looks more effective in inhibiting lhe developme of,t rdnguit. In this study also used povidon iodine (positiv€ control) as the ingredient that is often used ir dre treaiment of girgivitis.
Iu this study, we found that povidon iodine is also able to reduce MMP -8 and the rezults are not much different from the rosella extract gel l0% (Tahle 2). However, povidon iodine is antiseptic chemicals that bas side effects which can cause sensitivity, local erythema, pain, mucosal erosion, and major risks associated with thyroid function. Some findings about the side effects of povidon iodine was reported, but no serious danger happens.
Based on the box plot in Fig. l. gingivitis was occurred in post insertion of acrylic artifrcial crown and the efectiveness of roselle extract gel l0% has been proven can reduce the activity of MMP -8 in GCF.

Conclusion
Evaluation of the effectiveness of roselle extract gel l0olo using RT PCR method showed significant change in expression of MMP-8 mRNAr and this bring us to conclusion that there is a relationship betw€€n effectiveness of roselle extract gel l0% and changes in the expression of MMP E mRNA in GCF after application ofthe gel Roselle exhact 10plo in inflammation areas of the gingiva post acrylic crown insstion.