INTRODUCTION:

Teeth are hard tissues that serve several functions in daily life. Unfortunately, these teeth can fall out at any moment during a person's life due to a variety of circumstances. Tooth loss causes functional, cosmetic, and social deficits that can have an impact on a person's quality of life. Because of its high prevalence, it has become a serious public health hazard, even though the World Dental Federation (FDI) and WHO were projecting Global Oral Health Goals 2020, which stated that each population should have a proper functioning dentition in the 35–44 and 65–74 age groups¹. The major goal of practicing dentistry is to restore the patient's normal state so that they may function normally in daily life. Based on these primary goals, dental implants are one of the best solutions for replacing lost teeth².

Dental implants have a 98% success rate, and their surface properties play a key role in their success and lifespan. Certain factors that an implant must have in order to accommodate osteointegration are: (i) biological compatibility in order to be non-toxic to the surrounding hard and soft tissues; (ii) mechanical compatibility in order to smoothly transfer stress between the implanted root and hard tissues; and (iii) morphological compatibility in order to accommodate surface rugophilicity and promote bone cell growth². However, studies have indicated that 12%–66% of individuals with dental implants will develop peri-implantitis after 5-10 years³.

Peri-implantitis is an infection of the soft tissue around the implant that is characterized by bone resorption, reduced osseointegration, and enlarged pockets. Peri-implantitis develops as a result of an interaction between the host immune system and the pathogenic bacteria present in dental implants. Polymicrobial infection will result in the formation of a biofilm composed of perioidiopathic bacteria such as Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Prevotella nigrescens (Pn), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn)⁴.

Non-surgical and surgical techniques can be used to treat peri-implantitis. First and foremost, the patient will be urged to maintain proper dental hygiene and antibiotic use.

If the initial therapy fails to offer appropriate access to debridement mechanisms and chemical cleansing of the implant surface, the doctor may consider surgical treatment⁵.

However, the use of systemic antibiotics may result in comorbidities as well as the spread of antimicrobial resistance, leading to the superinfection of opportunistic infections that are difficult to prevent due to their capacity to interact with other medications. The possibility of bacterial resistance arises from insufficient biofilm removal⁶.

Graptophyllum pictum L. Griff has been used to cure hemorrhoids and speed up wound healing, as well as as an antipyretic and analgesic⁷. Previous studies found bioactive substances such as flavonoids, tannins, alkaloids, saponins, steroids, and glycosides in G. pictum L. Griff. The majority of these chemicals have antibacterial activities that target the bacterial cell wall, causing cell lysis. Furthermore, flavonoids and alkaloids are primary antioxidants that may stabilize free radical molecules by contributing H atoms, whereas saponins are secondary antioxidants that create hydroperoxides. These chemicals can also decrease the inflammatory process by decreasing prostaglandin production⁸. Further, this study aims to investigate the effectiveness of G. pictum L. Griff extract as a complementary therapy for peri-implantitis by means of in vitro and in silico studies examining the antibacterial, antioxidant, and anti-inflammatory activities.

MATERIALS AND METHODS:

The G. pictum L. Griff. utilized in this study was collected from Tawangmangu, East Java. Ethanol, aquadest, magnesium powder, hydrochloric acid, ammonia, chloroform, reagens (Dragendorff, Wagner, Mayer), sulfuric acid, ferric chloride acid, anhydrous acetic acid, ethyl acetate, methanol, and DPPH powder were used for extraction, phytochemical testing, and antioxidant testing. ATCC bacteria such as Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn), BHIB media, nutritional agar, and doxycycline are used in the MIC and MBC tests. Ethylcholest and octadeactrienoic ligand information (ID, sdf file, SMILE canonical), protein ligand 3D structure, ID, weight, and target chain are the materials for the in-silico test.

A set of laboratory glassware, blender, oven, extractor, rotary vacuum evaporator, centrifuge, analytical balance, micropipette, dropper, Whatman No. 41, measuring flask, syringe, waterbath, rubber bulb, petri dish, ose inoculum UV-vis spectrophotometer, and infrared spectrophotometer were used in this study. Several software packages were employed in the insilico tests, including PyMol ver. 2.5, OpenBabel v2.3.1, PyRx 0.9.9, Discovery Studio ver. 2016, Swiss ADME and ProTox-II servers, Pubchem, and RCSB PDB databases.

Ethical Approval:

The protocol for this study was accepted by the Faculty of Dental Medicine at Universitas Airlangga's ethical committee (Registration ID: 338/HRECC.FODM/VI/2022).

Extraction:

Extraction was performed using 150.3458 grams of simplicia powder macerated in 1500 ml of 70% ethanol for 24 hours, with the solution being agitated for 1 hour with a magnetic stirrer, after which it was soaked for another 18 hours at room temperature. While the pulp is macerated back into a clear-colored solution, the extract is filtered using Whatman No. 41 filter paper⁹. To get concentrated ethanol extract, the macerate is evaporated using a rotating vacuum evaporator at 60°C⁸.

Phytochemical Test:

Flavonoids Test:

In a test tube, two ml of extract were mixed with magnesium powder and 8–9 drops of HCl. The presence of flavonoids was shown by the development of yellow, purple, red, or brown¹⁰.

Alkaloids Test:

One milliliter of extract was cooked in a water bath for 30–60 minutes before being mixed with one milliliter of 28% ammonia and one milliliter of chloroform. Four tubes were filled with the solution. For comparison, 0.5% HCl was applied to tube 1. 2-3 drops of Dragendorff's reagent were put in Tube 2. Tube 3 received 2-3 drops of Mayer's reagent, whereas tube 4 received 2-3 drops of Wagner's reagent. The presence of alkaloid was revealed by an orange mark on tube 2, a yellowish white tint in tube 3, and a brown precipitate in tubes 4^8,10.

Saponins Test:

5cc of water was used to extract a dry sample (1g), which was then cooked in a water bath for 5 minutes. The extract was divided into two tubes, the first of which was rapidly agitated for 10minutes, and the second of which contained 1ml of 10% sulfuric acid and was placed in a water bath for 5 minutes. Saponins are indicated by the presence of honeycomb-like foam in both tubes⁸.

Tannins Test:

Three drops of 5% ferric chloride were added to one to two ml of extract. Green color formation showed the presence of gallotannins, whereas brown color formation suggested the presence of pseudotannins⁸.

Steroid Test:

1mL of chloroform extract was introduced gently from the side of the test tube wall, followed by 1mL of sulfuric acid. The presence of steroids was revealed by the creation of a red or bluish-green ring⁸.

Terpenoid Test:

One milliliter of extract was mixed with 0.5 milliliters of anhydrous acetic acid. The solution was progressively dripped with 2cc of sulfuric acid from the test tube wall. The presence of terpenoids was shown by the creation of a brownish or violet ring between two solvents¹⁰.

Quinone Test:

A dry sample (0,5g) was combined with 10ml of boiling water and cooked for 30 seconds. Following that, three drops of natrium hydroxide were applied to the sample. The presence of a red precipitate suggested the presence of quinone¹¹.

Antioxidant Test:

Antioxidant Activity Evaluation

Each dark tube received 2.5ml of G. pictum L. Griff. extract (20, 40, 60, 80, 100, and 120ppm) and 2.5 ml of DPPH 35g/ml. The mixture was agitated and incubated for 30 minutes before measuring the absorbance at maximum absorbance with a UV-Vis spectrophotometer. Using the method below, the absorbance data from each concentration may be used to get the absorbance percentage value¹².

Control absorbance-sample absorbance

% Absorbance = ------------------------------------ x 100 %

Control absorbance

The regression curve was generated using y = bx + an equation with the concentration of extract in pm as the x-axis and the value of absorbance % as the y-axis. The EC50 value was then computed. The EC50 value is a metric used to calculate the effective concentration required to block 50% of free radicals. According to the linear regression equation, the lower the value, the higher the antioxidant activity^13,14.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Tests

Bacteria Sample Preparation:

Brain Heart Infusion Broth (BHIB) medium was used to cultivate Aa, Pg, Pi, and Fn. It included 3 grams of BHIB powder and 100mL of distilled water. Bacterial culture was performed by inoculating 1 ose of each bacterium from the pure culture into BHIB medium and incubating for 24hours at 37°C¹⁵.

MIC and MBC Test Preparation and Determination:

Except for one tube containing 100% G. pictum L. Griff. extract, each microbe was placed in a test tube, and 5ml of BHIB was added. Different concentrations of G. pictum L. Griff. extract was obtained (100%, 75%, 50%, 25%, and 12.5%). The positive control test tube included BHIB medium containing 0.05mL of bacteria suspension and doxycycline, while the negative control test tube had BHIB medium containing no bacteria suspension and G. pictum L. Griff. extract. All test tubes were incubated in an anaerobic incubator at 37°C for 24 hours. After incubation, the production of precipitate and the appearance of cloudiness show that bacterial growth happened. The MIC value is the test tube with the lowest concentration that looked clear.

To calculate the MBC, all test tubes were subcultured on nutritional agar medium, and 0.01ml of each bacterial culture on BHIB media was collected using a micropipette and inoculated on a petri dish using the spread plate technique. All petri dishes were placed in an anaerobic jar and incubated for 24hours at 37°C. The number of colonies generated after 24 hours was counted using the Total Plate Count (TPC) technique. Petri dishes were separated into four quadrants, and each quadrant's colonies were marked with a marker from the rear of the petri dish. A turbidity test using a UV-Vis spectrophotometer was also used to determine the number of bacteria.

Molecular docking, in silico study:

Ligand-protein preparation:

G. pictum L. Griff chemical compounds include ethylcholest and octadecatrienoic acid. The ID, file in sdf format, and Canonical SMILE were obtained from Pub Chem (https://pubchem.ncbi.nlm.nih.gov), and then OpenBabel v2.3.1 was used to perform ligand reduction. The proteins used as ligand binding targets consisted of Nuclear Factor Kappa Beta (NF-κB), Tumor Necrosis Factor Alpha (TNF-α), Receptor Activator Nuclear Kappa Beta and its ligand (RANKL-RANK), Interleukin (IL)-6, IL-10, runt related transcription factor-2 (RUNX2), Osteoprotegrin (OPG), Osteocalcin, Nuclear Factor Associated T-cells 1 (NFATc1), Tartate Resistant Acid Phosphatase (TRAP), Peptidoglycan, Flagellin, Dectin, Heat Shock Protein (HSP)-70, and Hsp10. 3D structure, protein ID, experimental method type, resolution, sequence length, weight, and chain, obtained via RCSB PDB (https://www.rcsb.org/). The protein sterilization procedure is then carried out using PyMol 2.5 version software by removing water molecules from the protein chain^16,17.

ADMET Analysis:

In this investigation, ADMET prediction on ethylcholest and octadecatrienoic was performed using the Swiss ADME server (http://www.swissadme.ch/) and ProTox-II (https://tox-new.charite.de/protox_II/). The goal of ADMET prediction is to discover physicochemical features, water solubility, and drug-like molecules in query compounds, with the toxicity value of query compounds assumed to be in low categories such as class IV and V^17,18.

Docking Computational:

The interactions between ligand molecules and proteins were studied using molecular docking simulations. NF-B, TNF-, RANKL-RANK, IL-6, IL-10, RUNX2, RANKL-OPG, Osteocalcin, NFATC1, TRAP, Peptidoglycan, Flagellin, Dectin, Hsp70, and Hsp10 are the targets of Daun Ungu compounds. PyRx 0.9.9 was used in this study to run molecular docking simulations and compare the activity of the two ligands when bound to the target domain²⁰. The binding affinity value is mentioned in the results of the molecular docking simulation. Binding affinity is the amount of energy released when ligands and proteins combine to form molecular complexes; a negative binding affinity value decreases the ligand's capacity to activate the target protein²¹.

Chemical Interaction:

Molecular interaction analysis on the docking complex from G. pictum L. Griff with all target proteins was carried out using the Discovery Studio 2016 version. The identified molecular interactions consist of Van der Waals, hydrophobic, electrostatic, hydrogen, and pi, these interactions are weak bonds and play a role in triggering protein activity²².

Molecular Visualization:

This work visualizes molecular complexes using a 3D structure using PyMol 2.5 version software, a single staining selection approach on protein chains and ligands, and then structural selection. Sticks, cartoons, and surfaces make up the 3D structure represented by the PyMol 2.5 version program²³.

Statistics Analysis:

A one-way analysis of variance (ANOVA) was used to analyze the data, with a significance threshold of 0.05 set. The Tukey post-hoc test was used to identify groups with significant differences. As a non-parametric variance analysis alternative, the Kruskal-Wallis technique was utilized, followed by the Dunnett T3 post-hoc test.

RESULT:

Phytochemical Test:

The concentrated ethanol extract of G. pictum L. Griff was subjected to phytochemical testing to see what bioactive compounds it contained. The results of the phytochemical test are presented in Table 1, which shows that G. pictum L. Griff extract contains flavonoids, alkaloids, saponins, tannins, steroids, and terpenoids.

Antioxidant Activity Test:

The results of antioxidant percentage of vitamin C and Wungu Leaf (G. pictum L. Griff) extract were used to calculate the EC₅₀ value. The calculation was carried out by creating the linear regression curve between sample concentration and the percentage of antioxidant so that a linear regression equation is obtained, namely y = bx + a, where x is the Wungu leaf extract concentration and y is the percentage of EC₅₀. The results are shown in Figures 1 and Figure 2. Based on the data, the Wungu leaf extract (Graptophyllum pictum L. Griff) EC₅₀ value was 161.002mg/mL (Figure 1), while the EC₅₀value of vitamin C as a comparison was 2.4123mg/mL (Figure 2).

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Tests:

From the MIC and MBC test results, it was shown that 25% Wungu leaf extract (G. pictum L. Griff) concentration is the minimum concentration that has the potential to inhibit the growth of periodontopathogen bacteria: Aa, Pg, Pi, and Fn (Figure 3). The following figures will show the average amount of bacteria after being given various concentrations of Wungu leaf extract (Graptophyllum pictum L. Griff) against periodontopathogenic bacteria, namely Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. The results were obtained based on the total plate count method (Figure 4A) and spectrophotometer (Figure 4B). Bacteria colony count’s normality test data shows that the data is normally distributed except for Fusobacterium nucleatum bacteria, followed by the homogeneity test using the Levene statistic test, where the data obtained were not homogeneous. We conducted a variance analysis test to see the difference in each bacterium; the results can be seen in Table 2.

The normality test results for spectrophotometry data using the Shapiro Wilk test show that the data is normally distributed except for Aggregatibacter actinomycetemcomitans bacteria. Following the homogeneity test using the Levene statistic test, the data obtained were homogenous except for the Prevotella intermedia bacteria. Therefore, we conducted a variance analysis test to see the difference in each bacterium; the results can be seen in Table 3. Based on the result, both colony count and spectrophotometer data were significantly different with a p value < 0.05, it can be concluded that there are significant differences in each concentration group. To see which treatment group gave the best results in inhibiting the bacteria's growth, we carried out a post-hoc test. The results of the post-hoc test showed that the best concentrations that could inhibit the bacteria were 100% and 75% Wungu leaf extract, because they both produced the highest yields compared to other Wungu leaf extracts. Even so, all concentrations are effective in inhibiting bacterial growth.

In Silico Test:

In silico testing was carried out to see the interaction mechanism between ethylcholest and octadecatrienoic ligands and the target, which was performed through molecular docking simulation. The molecular docking simulation results show that ethylcholest has a binding affinity value on the target domain that is more negative than octadecatrienoid, which can be seen in Table 4. The 3D structures in this study were visualized using PyMol 2.5 version software with the selection mechanism shown in Figure 5. The identification of molecular interactions and binding positions on the docked protein-ligand complex showed that the binding of ethylcholest compounds to all target proteins resulted in non-covalent bond interactions consisting of Van der Waals, pi, and hydrogen. The results can be seen in Table 5.

Table 1: Phytochemical Tests Result

No.	Bioactive compound	Reagents	Color Changes	Result
1	Flavonoids	Wilstater	Brown	+
2	Alkaloids	Dragendorff’s, Mayer’s, and Wagner’s reagents	Yellowish spot and brown	+
3	Saponins	Hot water	Honeycomb-like foam	+
4	Terpenoids	Anhydrous acetic acid and sulfuric acid	Brown ring	+
5	Tannins	Ferric chlorid	Green	+
6	Quinones	Natrium hydroxide	Precipitate was not found	-
7	Steroids	Sulfuric acid	Green ring	+

Notes:

(+): positive (in the sample)

(-): negative (not in the sample)

Figure 1. Linier Regression Curve of (A) Wungu Leaf Extract.

Figure 2: Linier Regression Curve of Vitamin C.

Table 4: The Autogrid Positions and Binding Affinity From Molecular Docking Simulation

Protein	Autogrid						Binding Affinity (kcal/mol)
	Center (Å)			Dimensions (Å)			Ethylcholest	Octadecatrienoic
	X	Y	Z	X	Y	Z	Ethylcholest	Octadecatrienoic
TNF-α	19.968	49.675	39.930	80.739	58.243	58.256	-6.0	-5.2
NF-κB	42.464	14.683	38.036	90.709	67.390	51.935	-6.7	-5.0
RANKL-RANK	8.830	-0.536	17.364	47.953	57.932	51.740	-8.3	-5.5
IL-6	2.675	-20.084	8.908	58.092	62.897	43.139	-7.1	-4.6
IL-10	13.263	21.832	5.096	58.882	38.528	85.481	-7.8	-6.1
RUNX2	-50.956	39.759	-15.612	64.939	22.064	47.175	-5.7	-3.6
RANKL-OPG	-2.998	-3.671	23.788	95.207	98.636	77.806	-8.3	-5.0
Osteocalcin	8.075	25.301	22.863	31.622	16.932	12.741	-4.5	-4.1
NFATC1	15.501	-7.918	1.696	68.461	53.410	57.713	-7.7	-5.6
TRAP	68.142	-24.322	17.856	33.318	38.191	40.670	-6.7	-5.1
Peptidoglycan	37.648	37.735	21.932	64.278	40.527	45.926	-7.5	-4.6
Flagellin	-23.829	37.749	33.866	149.906	40.586	98.210	-7.0	-3.8
Dectin	43.337	20.890	45.579	55.338	39.093	31.835	-6.8	-4.4
Hsp70	17.349	28.677	16.059	81.800	54.749	57.266	-8.7	-6.3
Hsp10	-40.185	60.186	24.034	107.037	65.608	86.197	-8.4	-5.6

Table 5: The Result of Chemical interaction

Ligan-Protein	Chemical Interaction
Ethylcholest_TNF-α	Van der Waals: Ser133, Ala134, Asp45, Asn137, Glu135, Gln25, Gln27 Alkyl: Ile136, Leu26, Pro139 Unfavorable: Asn46
Ethylcholest_NF-κB	Van der Waals: Arg198, Glu152, Lys149, Lys148, Thr205, Met208 Alkyl: Phe151, Lys147, Leu202, Val150, Lys206
Ethylcholest_RANKL-RANK	Hydrogen: Tyr217, Ala166 Van der Waals: Gly309, Ala310, Phe165, Gln163, Lys312, Val 313, Lys195 Alkyl: Tyr215, Phe311, Tyr307, His167
Ethylcholest_IL-6	Hydrogen: Ser107, Glu106, Glu42, Lys46 Van der Waals: Ser108, Phe105, Thr43, Ser47, Gln156, Asp160, Arg104 Alkyl: Met49, Trp157
Ethylcholest_IL-10	Van der Waals: Met77, Leu65, Ile69, Leu101, Lys34 Alkyl: Arg27, Tyr72, Met68, Leu48, Leu98, Phe56, Leu94, Leu26, Phe30, Arg27
Ethylcholest_RUNX2	Hydrogen: Arg193 Van der Waals: Gly192, Val219, Thr220, Asp222, Pro224, Pro227 Alkyl: Arg190, Val221, His129 Unfavorable: Arg225
Ethylcholest_RANKL-OPG	Hydrogen: Lys312 Van der Waals: Lys195, Gly309, Ala310, Ala166, Phe165, Gln163 Alkyl: Tyr307, His167, Phe311, Tyr215 Unfavorable: Tyr217
Ethylcholest_Osteocalcin	Van der Waals: Gln39, Gly47, Tyr46 Alkyl: Ile48, Tyr42, Arg43
Ethylcholest_NFATC1	Hydrogen: Asp69, Thr67 Van der Waals: Leu73, Gly66, Arg74, Ala68, Asp70, Arg148, Arg71, Leu73, Ser8, His9 Alkyl: Leu6, Trp4, Pro7, Ala168, Val167, Val150 Unfavorable: Gln166
Ethylcholest_TRAP	Hydrogen: Ile55 Van der Waals: Asp8, Phe9, Thr65, Lys56, Ser72, Glu73, Glu71 Alkyl: Phe48, Val10, His67, Arg66, Ile63, Ile70, Val57
Ethylcholest_Peptidoglycan	Van der Waals: Gln163, Gln61 Alkyl: Lys62, Leu159, Leu126, Leu122, Ile147, Tyr65
Ethylcholest_Flagellin	Van der Waals: Asp379, Gln176, Thr382, Thr117, Asn174, Asn393, Gln113, Asp110, Glu383, Met320, Ala335, Tyr334, Gly333, Gly332, Tyr322 Alkyl: Ala114, Leu375, Ala344, Lys381
Ethylcholest_Dectin	Hydrogen: Trp187, Glu132 Van der Waals: Phe181, Glys186, Gln123, Thr185, Gly188, Asn124, Glu120, Gln128, Glu132, Phe181 Alkyl: Val127, Phe163
Ethylcholest_Hsp70	Hydrogen: His227 Van der Waals: Glu231, Asp69, Arg72, Asp86, Pro91, Arg264, Val59 Alkyl: Arg261, Val260, Lys257, Phe68, Trp90, His89
Ethylcholest _Hsp10	Hydrogen: Gln66 Van der Waals: Ala58, Asp57, Thr56, Val64, Pro65, Leu14, Asp96, Asp16 Alkyl: Ala58, Phe55, Val63, Phe55, Met15

DISCUSSION:

Based on the study, we found that the Wungu leaf extract (G. pictum L. Griff) contains several bioactive compounds, including flavonoids, alkaloids, saponins, terpenoids, tannins, and steroids, after passing the phytochemical test. The results may vary in different color changes after being given their respective reagents. These bioactive compounds have their respective functions, but broadly speaking, these components have a pharmacological effect as antioxidants, antibacterials, anti-inflammatory agents, immune modulators, and antidiabetic agent²⁴. Previous study found the same contents in Wungu leaf extract (G. pictum L. Griff) with several more compounds such as glycoside, chlorophyll, formic acid, and pectin⁷. Other studies showed that the compounds contained in Wungu leaf extract (G. pictum L. Griff) analyzed by UV-Vis, FTIR, and GCMS are stigmasta-5,22-dien-ol and stigmast-5-en-3-ol-acid¹².

The antioxidant activity of the Wungu leaf extract (G. pictum L. Griff) can be seen in the results of antioxidant tests. The EC₅₀is calculated using a regression linear curve with the concentration of the extract as the x-axis and the value of the absorbance percentage as the y-axis.The EC₅₀ value is a parameter used to determine the effective concentration to inhibit 50% of free radicals. From the results obtained, the Wungu leaf extract (G. pictum L. Griff) EC₅₀value is 161.002 µg, whereas the EC₅₀ value of vitamin C as a comparison is 2.4123µg. Specifically, antioxidants are categorized as very strong if the EC₅₀ value is less than 50ppm, strong if the value is in the 100–150ppm range, and weak if the EC₅₀ value is in the 150–200ppm range. A smaller EC₅₀ value indicates higher antioxidant activity¹⁷. Based on the data, it shows that Wungu leaf extract (G pictum L. Griff) is classified as a weak antioxidant compound. However, Wungu leaf (G. pictum L. Griff) is a rich source of antioxidants, which could be beneficial to support or prevent the body from free radical overproduction⁸. This study used six kinds of fractions, which will support their research. Ethyl acetate fractions have the best antioxidant activity. Previous study also support that ethanol, ethyl acetate, and n-butanol fractions have good antioxidant activity¹².

From the data obtained, the MIC and MBC of Wungu leaf extract (G. pictum L. Griff) against the bacteria were 25%. The bacterial colony count data shows a significant difference between concentrations according to the result of the Kruskal-Wallis method. It can be interpreted that there was a significant difference in the number of colonies among each concentration group. Therefore, we also conducted a post hoc test to see the significant group as we compared each treatment group with others. The results showed that the concentrations with the best results in reducing bacteria growth on Aa, Pg, Pi, and Fm were 100%, 75%, 50%, or 25% Wungu leaf extract (G. pictum L. Griff) concentrations because those four concentrations had a number of colonies that were not significantly different from each other but significantly different from 12.5% Wungu leaf extract (G. pictum L. Griff) and the negative control. The result of the spectrophotometer data shows a significantly different result among each group. We continued the post-hoc test, and the results showed that the Wungu leaf extract (G. pictum L. Griff) concentrations with the best results in reducing the bacteria are 100%, 75%, and 50% (A. actinomycetemcomitans), 100% and 75% (P. gingivalis), 100% (F. nucleatum), and 100%, 75%, and 50% (P. intermedia). Tannins, flavonoids, alkaloids, and glycosides play a major role in inhibiting the growth of bacteria. Tannins have an antibacterial effect by inhibiting glucosyltransferase formation, so sucrose cannot be converted to glucose for the bacteria's energy sources. The effect of crude extract of wungu leaf (G. pictum L. Griff) against Streptococcus mutans and, based on the observation results obtained, found that 0,09% concentration did not cause any bacterial growth in nutrient agar²⁵. There was a study that compared the betadine gurgle with a 90% concentration of wungu leaf extract as a mouth wash, and this study resulted in the finding that wungu leaf extract had the effect of decreasing saliva viscosity and lowering saliva pH, thus preventing the growth of Streptococcus mutans⁷.

To prove the pharmacological activity of Wungu leaf extract (G. pictum L. Griff), the molecular interaction between Wungu leaf compounds and target proteins consisting of of NF-κB, TNF-α, RANKL-RANK, IL-6, IL-10, RUNX2, RANKL-OPG, osteocalcin, NFATC1, TRAP, peptididoglycan, flagellin, dectin, Hsp70, and Hsp10 was done. Ethylcholest and octadecatrienoic acids from Wungu leaf extract (G. pictum L. Griff) were used in this investigation because those two chemicals could match the drug likeness criteria. The interaction mechanism of ethylcholest and octadecatrienoic ligands with the target is simulated using molecular docking. According to the molecular docking simulation results, ethylcholest has a lower binding affinity value on the target domain than octadecatrienoic. Structure interaction between Wungu leaf extract (G. pictum L. Griff) chemicals and target proteins in 3D the identification of molecular interactions and binding locations on the docked protein-ligand complex revealed that ethylcholest compounds bound to all target proteins via non-covalent bond interactions, including Van der Waals, pi, and hydrogen. Overall, weak binding interactions can lead to the creation of persistent ligand-protein complexes and induce activity responses such as enhancement and inhibition of target proteins. Hydrogen bonding, hydrophobicity, Van der Waals interactions, and pi all play roles in the docking complex in triggering the creation of certain biological functions²⁶. The function of Wungu leaf extract (G. pictum L. Griff), Ethylcholest substances permit them to be anti-inflammatory by inhibiting the regulation or decreasing the activity of pro-inflammatory proteins such as TNF-α, NF-κB, RANKL-RANK, and IL-6, which can subsequently induce the upregulation of anti-inflammatory proteins such as IL-10. Ethylcholest activity can increase osteoblast and osteoclast activity via RUNX2, RANKL-OPG, osteocalcin, NFATC1, and TRAP; it also has antibacterial properties via inhibition of peptidoglycan, flagellin, and dectin activity; and it can also be an antioxidant via upregulation of Hsp70 and Hsp10.

CONCLUSION:

Considering the results of the studies, it can be concluded that Wungu leaf extract (G. pictum L. Griff) may be effective as a complementary therapy for peri-implantitis. The antibacterial, anti-inflammatory, and antioxidant activities of Wungu leaf are several pharmacological effects given by the bioactive compound that will support the healing of peri-implantitis.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The author would like to thank Widya Mandala Catholic University, Brawijaya University, and Research Center Airlangga University for supporting this study. This study obtained research funding from Penelitian Unggulan Fakultas (PUF) in 2022 fiscal year of Faculty of Dental Medicine, Universitas Airlangga.

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Received on 18.04.2023 Modified on 09.09.2023

Research J. Pharm. and Tech 2024; 17(4):1517-1526.

DOI: 10.52711/0974-360X.2024.00240

Bacteria	p	Description
Aggregatibacter actinomycetemcomitans^A	0,000*	Significantly Different
Porphyromonas gingivalis^A	0,000*	Significantly Different
Fusobacterium nucleatum^K	0,000*	Significantly Different
Prevotella intermedia^A	0,000*	Significantly Different

Bacteria	*P value*	Description
Aggregatibacter actinomycetemcomitans^K	0,001*	Significantly Different
Porphyromonas gingivalis^A	0,000*	Significantly Different
Fusobacterium nucleatu^A	0,000*	Significantly Different
Prevotella intermedia^A	0,000*	Significantly Different