Insisiva Dental Journal: Majalah Kedokteran Gigi Insisiva, 11(2), November 2022, 62-69 62 Research Article Antibacterial Effects of Ethanolic Extract of Bidara (Ziziphus mauritiana Lam) Leaf Against Porphyromonas gingivalis Noor Aziza1, Islamy Rahma Hutami2*, Recita Indraswary3, Suryono4 1Faculty of Dentistry, Sultan Agung Islamic University, Jl. Raya Kaligawe Km.4 Semarang 50112, Indonesia 2Department of Orthodontics, Faculty of Dentistry, Sultan Agung Islamic University, Jl. Raya Kaligawe Km.4 Semarang 50112, Indonesia 3Department of Oral Biology, Faculty of Dentistry, Sultan Agung Islamic University, Jl. Raya Kaligawe Km.4 Semarang 50112, Indonesia 4Department of Periodontics, Faculty of Dentistry, Universitas Gadjah Mada, Jl. Denta 1, Sekip Utara, Yogyakarta 55281 Indonesia Received date: June 26th, 2022; revised date: August 5th, 2022; accepted: October 25th, 2022 DOI: 10.18196/di.v11i2.15222 Abstract The bidara plant (Ziziphus mauritiana Lam) is widely distributed in various Asian countries. Bidara leaves contain secondary metabolites, the main content of which is flavonoids. As a gram-negative anaerobic bacteria, Porphyromonas gingivalis is one of the normal flora of the oral cavity. However, over quantities of this bacteria can promote chronic periodontitis. This research aims to analyze the bidara leaf ethanolic extract as an inhibitory agent of Porphyromonas gingivalis. This research design is experimental laboratory research with a post-test controlled group of Porphyromonas gingivalis inhibition. A total of 25 samples consisted of 5 groups of ethanol extract of bidara leaves at concentrations of 1%, 3%, 9%, positive control betel leaves, and negative control aquadest. Bacteria incubation was held for 48 hours, and the free bacterial zone was analyzed by the One Way ANOVA test. The results of the analysis showed that there was a significant difference between the control group and the treatment group. This study concludes that the ethanol extract of bidara leaves had a strong inhibitory effect on Porphyromonas gingivalis. Keywords: antibacterial; bidara leaf; porphyromonas gingivalis INTRODUCTION The Global Burden of Disease Study (2016) states that periodontal disease is the 11th most common disease globally.1 In Indonesia, the prevalence of periodontal disease in all age groups is still relatively high at 96.58%.2 The most common type of periodontal disease is chronic periodontitis.3 Periodontitis is an inflammation of supporting tissue surrounding the teeth caused by microorganisms and leads to damage to the periodontal ligament, alveolar bone, pocket formation, recession, or.both.4 Periodontitis can be classified into two: aggressive periodontitis and chronic periodontitis.5 Chronic periodontitis is a * Corresponding author, e-mail: rahma.hutami@unissula.ac.id type of periodontitis in which the rate of disease progression is slow to moderate. The main cause of chronic periodontitis is bacteria that accumulate in plaque. Porphyromonas gingivalis is commonly found in dental plaque and can cause pathological changes in periodontal tissues by activating host immune and inflammatory responses. 4 Several types of anaerobic gram- negative bacteria are found in subgingival plaque, such as Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Treponema denticola which can cause and exacerbate periodontal infections.5,6 Based on research on the subject population of 128 people with chronic periodontitis, it Noor Aziza, Islamy Rahma Hutami, Recita Indraswary, Suryono | Antibacterial Effects of Ethanolic Extract of Bidara (Ziziphus mauritiana Lam) Leaf Against Porphyromonas gingivalis 63 is known that the most dominant bacteria in chronic periodontitis is Porphyromonas gingivalis, with a prevalence of about 80.5%.6 Porphyromonas gingivalisis a melanogenic bacterium and belongs to the colonies of black-pigmented gram-negative anaerobes. Porphyromonas gingivalis usually colonizes oral tissues and can be seen in culture media with the characteristics of forming a colony with a diameter of 1-2 mm, convex and smooth, and has a characteristic with the darker color in the center.7,8 Porphyromonas gingivalis can release harmful substances, such as lipopolysaccharide, as a virulence factor that triggers the host inflammatory response, resulting in periodontal tissue destruction in chronic periodontitis.4 Treatment of chronic periodontitis is scalinggandnroottplanning (SRP) and using antibiotics as adjunctive therapy.9 Antibiotics consumed in excess and inappropriately can make Porphyromonas gingivalis resistant. Natural herbal medicine can be another alternative in treating chronic periodontitis, which is considered safer as it has fewer side effects than antibiotics.10 One of the plants that are believed to have many benefits is the bidara tree (Ziziphus mauritiana Lam). The Bidara tree consists of roots, stems, leaves, fruit, and seeds. It is known that bidara leaves have pharmacological effects as it belongs to the group of alkaloids, saponins, flavonoids, tannins, terpenoids, and steroids, which have antibacterial, anti-inflammatory, and antifungal properties.11,14 Studies on the bidara plant as an antibacterial have been discussed by several researchers. Maulana Siregar, 2020 stated that bidara leaves have analgesic, antipyretic and anti-inflammatory properties due to the flavonoid content so they can play an important role in periodontitis.12 In this study, betel leaf extract mouthwash was used as a positive control as it contains active ingredients from herbs and has a secondary metabolite content that is almost the same as bidara leaves as follows: tannins, saponins, and flavonoids.13 Based on the description above, this research was conducted to observe the effectiveness of the antibacterial extract of bidara leaves at concentrations of 1%, 3%, and 9% on the growth of Porphyromonas gingivalis. MATERIALS AND METHODS Analytical, experimental research with observation method using the disk diffusion method was conducted to analyze differences in the antibacterial effectiveness of ethanol, extracts of bidara leaves. A total of 25 samples were divided into five groups. The treatment group with a concentration of 1%, 3%, 9%, negative control of aquadest, and positive control of betel leaves extract mouthwash. This research protocol has been approved by the Research Ethics Commission of the Faculty of Dentistry, Sultan Agung Islamic University, with No.305/B.1-KEPK/SA- FKG/IX/2021. Porphyromonas gingivalis Bacteria Samples Porphyromonas gingivalis ATCC 33277 bacteria were cultured on Mueller Hinton agar media (MHA) with brain heart infusion broth (BHIB). The positive control used mouthwash with betel leaf extract from Mustika Ratu, while the negative control used sterile aquadest. Making Bidara Leaf Extract The fresh green bidara leaves were picked directly from the tree, then wet sorting and dry sorting were carried out, then cut into small pieces and mashed using a blender to obtain bidara leaf powder.11 The initial maceration process was carried out by weighing 300 g of bidara leaf powder. The powder of bidara leaf that had been weighed was then put into a maceration container and macerated with 1 liter of 96% ethanol solvent.11 Bidara leaf Insisiva Dental Journal: Majalah Kedokteran Gigi Insisiva, 11(2), November 2022, 62-69 64 extract (filtrate) was stored in a place protected from sunlight and allowed to stand for 72 hours at room temperature, stirring occasionally. After three days, the filtrate was filtered and macerated using 1 liter of 96% ethanol. Measurements were carried out five times with the same amount of solvent. Each filtrate was filtered using paper Whatman then combined from the maceration and remaceration results.17 After the filtrate was combined, it was evaporated using a rotary evaporator to get a thick extract of bidara leaves.17 The extract was then prepared to a concentration of 1%, 3% and 9% with sterile distilled water as a solvent. Porphyromonas gingivalis Inhibitory Test Preparation of suspension of Porphyromonas gingivalis was carried out by taking the preparation of Porphyromonas gingivalis with a sterile one needle, then inserting it into a test tube containing BHIB and homogenizing it with a vortex. The bacterial turbidity was equalized to 0.5 Mc Farland (approximately 1-2×108 CFU/ml). The next step was to test the effectiveness of the extract. bidara leaf ethanol on growth of Porphyromonas. gingivalis. It was done by spreading the suspension of Porphyromonas gingivalis bacteria using a sterile cotton swab evenly on the surface of the MHA media. Each paper disc dripped 10 µm ethanol extractkofnbidara leaves with concentrations of 1%, 3%, and 9%, respectively. As a negative control, the paper disc was dripped with distilled water and betel leaf extract mouthwash as a positive control.11 It was then incubated at 37°C for 48 hours. After 48 hours, the diameter of the clear zone (free bacterial zone) formed around the paper disc was measured using a caliper.14 The inhibition zone measurement can be conducted using the formula.15 Note: = inhibitory zone Dv = diameter of vertical Dh = diameter of horizontal Statistics This data was processed using the SPSS computer software program. In addition, a parametric statistical test was carried out with the One Way Annova test to analyze the significance of bidara leaves ethanol extract at several concentrations in inhibiting the growth of Porphyromonas gingivalis. RESULT Bidara leaf extract concentrations of 3%, 9%, and positive control mouthwash of betel leaf extract showed antibacterial activity of Porphyromonas gingivalis. In comparison, bidara leaves extract with a concentration of 1% and aquadest did not show any inhibition against Porphyromonas gingivalis. Dv + Dh 2 Noor Aziza, Islamy Rahma Hutami, Recita Indraswary, Suryono | Antibacterial Effects of Ethanolic Extract of Bidara (Ziziphus mauritiana Lam) Leaf Against Porphyromonas gingivalis 65 Picture 1. Inhibitory zone on MHA by the method of disk diffusion Picture 2. Negative control of aquadest Picture 3. Positive control of betel leaves extract mouthwash Picture 4. Bidara leaves extract 1% concentration Picture 5. Bidara leaves extract 3% concentration Picture 6. Bidara leaves extract 9% concentration Vertical diameter (Dv) Horizontal diameter (Dh) Insisiva Dental Journal: Majalah Kedokteran Gigi Insisiva, 11(2), November 2022, 62-69 66 Table 1. Inhibitory effect analysis of bidara leaf extract at several concentrations on the growth of Porphyromonas gingivalis No The concentration of Bidara Leaves Ethanol Extract Control Group 1% 3% 9% Aquadest (negative) Betel Leaf Extract Mouthwash (positive) 1 0 16.8 mm 22.2 mm 0. 27.4 mm. 2 0 16.05 mm 22.mm 0. 27.05 mm. 3 0 14.95 mm 20.8.mm 0. 26.4 mm. 4 0 16.4 mm 21.95 mm 0 27.2 mm 5 0 16.2 mm 22.6 mm 0 27.15 mm Average 0 16.08 mm 21.91 mm 0 27.04 mm Table 2. Analysis of normality and homogeneity among groups of Porphyromonas gingivalis inhibition test of bidara leaves extract (p>0.05) Group Shapiro-Wilk Levene Sig Statistics df Sig. 1% Concentration - 5 - 0.059 3% Concentration 0.9 5 0.42 9% Concentration 0.87 5 0.29 Aquadest Control - 5 - Betel Leaf Extract Mouthwash Control 0.85 5 0.2 Table 3. One-WayyAnnova analysis among groups of Porphyromonas gingivalis inhibition test of bidara leaves extract (p<0.05) Picture 6. Significance of differences between the control and treatment groups * = Significance Group. Mean.±Std. Dev One-way ANOVA(Sig.) 1% Concentration 0.00±0.00 .000 3% Concentration 16.08±0.69 9% Concentration 21.91±0.67 Aquadest Control 0.00±0.00 Betel Leaf Extract Mouthwash Control 27.04±0.37 0 5 10 15 20 25 30 Aquadest Betel Leaf Extract Mouthwash 1% Concentration 3% Concentration 9% Concentration A v e ra g e d ia m e te r o f th e i n h ib it io n z o n e (m m ) Bidara leaf concentration Control group TreatmentControl 0 0 * * ** * * * * * 67 Noor Aziza, Islamy Rahma Hutami, Recita Indraswary, Suryono | Antibacterial Effects of Ethanolic Extract of Bidara (Ziziphus mauritiana Lam) Leaf Against Porphyromonas gingivalis The inhibition test results showed that the ethanol extract of bidara leaves had high inhibitory activity at concentrations of 3% and 9% compared to 1%. Test for normality and homogeneity with Sapphiro- Wilk and Levene p>0.05 showed that the data were normally distributed and homogeneous (Table 2). Further analysis by the One-Way Anova test (Table 3) showed that the p-value on the One-Way Annova test = 0.000 (p<0.05), indicating that there was a significant difference between each group in inhibiting the growth of Porphyromonas gingivalis. Picture 2 shows a significant difference between the control group and the treatment group, namely aquadest with positive control mouthwash of betel leaf extract, aquadest with bidara leaf extract (Ziziphus mauritiana Lam) with a concentration of 3% and 9%, and mouthwash with betel leaf extract with a concentration of 3%. DISCUSSION The antibacterial activity of Porphyromonas gingivalis was shown by measuring the inhibitory zone after 48 hours of incubation at room temperature (37℃). The negative control of sterile distilled water and 1% bidara leaves extract had no antibacterial effect on Porphyromonas gingivalis, which was indicated by the absence of an inhibition zone (0 mm). Positive control of betel leaf extract mouthwash showed the formation of the largest clear zone compared to the three groups of bidara leaves concentration (Table 1). The positive control mouthwash of betel leaf extract had an inhibition zone of 27 mm and was more significant than the aquadest control (Table 3). The 3% bidara leaf extract had an inhibition zone of 16.08 mm and a concentration of 9% of 22 mm (Table 1). However, the positive control of betel leaves extract mouthwash with 9% bidara leaves extract had a significant difference, although both groups had an inhibition zone of >20 mm (Table 3). Thus, it can be concluded that the mouthwash of betel leaf extract with 9% bidara leaves extract is included in the powerful category in inhibiting the growth of Porphyromonas gingivalis bacteria. This result is in accordance with the research conducted by Agrianto (2016) regarding the assessment of inhibitory power using the Davis and Stout categories. It was found that the average inhibition zone of clove flower extract was 13.01 mm, which was classified as having a strong inhibitory effect on the growth of Porphyromonas gingivalis.15 The area of inhibition can be observed by the presence of a clear zone between the growth of bacteria (free bacterial zone). The inhibition zone marked by the clear zone (Figure 1) in the 3% and 9% bidara leaves extract appeared due to antibacterial activity, which was considered to be produced from secondary metabolites, namely alkaloids, saponins, flavonoids, tannins, terpenoids, and steroids which had antibacterial properties, anti-inflammatory, and antifungal agents.16 The antibacterial effect of bidara leaves ethanol extract on the growth of Escherichia coli and showed the presence of an inhibition zone at 9% concentration with an average inhibition zone diameter of 15.66 mm. The antibacterial effectiveness of bidara leaves is due to the active substance. Higher concentrations of bidara extract can increase the number of active ingredients that act as antibacterial agents. Thus, the ability of microbial growth inhibition is more significant.11 The effectiveness of pure 9% bidara leaves extracts actively inhibits the growth of Porphyromonas gingivalis. The betel leaves extracts used in this study contained a mixture of antibacterial ingredients of 0.5% betel leaves and 0.04% mint leaf oil (Mentha piperita oil). In contrast, the bidara leaves extract in this study was pure extract without a mixture of other ingredients for further research. Therefore, it is recommended that bidara leaf extract can be combined with other antibacterial herbs Insisiva Dental Journal: Majalah Kedokteran Gigi Insisiva, 11(2), November 2022, 62-69 68 or other active components to obtain maximum results when used as an antibacterial ingredient. CONCLUSION Bidara leaves extract concentrations of 3% and 9% effectively inhibited the growth of Porphyromonas gingivalis bacteria. In addition, 9% of bidara leaf extract with mouthwash control of betel leaf extract had a diameter of inhibition zone above 20 mm, indicating a potent inhibition against the growth of Porphyromonas gingivalis. REFERENCES 1. Kementrian Kesehatan RI. Faktor Risiko Kesehatan Gigi dan Mulut. 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