Alkaloid Identification

A total of 0.5 g of Miana leaf extract was placed into a test tube, followed by the addition of 1 mL of 2% HCl. Then, 9 mL of distilled water was added, and the solution was heated for 2 minutes. After cooling, it was filtered. The filtrate was divided into three portions, and each portion was treated with Mayer's, Wagner's, and Dragendorff’s reagents (Setyowati et al., 2014).

Flavonoid, Tannin, and Saponin Identifications

Flavonoid, the ethanol extract of Miana leaves was dissolved in hot methanol, followed by the addition of 0.1 g of magnesium powder and 1 mL of concentrated HCl (Setyowati et al., 2014). Tannin, the ethanol extract of Miana leaves was dissolved in 10 mL of distilled water and filtered. The filtrate was then treated with 1% FeCl₃ (Setyowati et al., 2014). Saponin, the ethanol extract of Miana leaves was dissolved in 10 mL of hot water and shaken for 10 seconds (Setyowati et al., 2014).

Bacterial Culture Preparation

Pure cultures of S.pyogenes and S.mutans were taken using a sterile inoculating loop from the bacterial stock. The bacteria were inoculated into sterile Nutrient Agar (NA) plates under aseptic conditions inside a Laminar Air Flow (LAF) cabinet. The inoculated bacteria were then incubated at 37°C for 24 hours (Mulyadi et al., 2017).

Bacterial Suspension Preparation

A single colony of S. pyogenes and S. mutans (24-hour-old culture) was suspended in 5 mL of sterile 0.9% physiological NaCl. The suspension was homogenized using a vortex mixer, and its turbidity was compared to the 0.5% McFarland standard, equivalent to a bacterial concentration of 9 × 10⁸ CFU/ml (Syafriana et al., 2020).

Test solutions were prepared at concentrations of 5%, 10%, 20%, and 40%. This was done by weighing 0.05 g, 0.1 g, 0.2 g, and 0.4 g of Miana leaf ethanol extract, which were then dissolved in DMSO to a final volume of 1 mL. Positive control (amoxicillin) and negative control (DMSO) were also prepared (Wangkanusa et al., 2016). Mueller-Hinton Agar (MHA) medium was sterilized using an autoclave at 121°C for 20 minutes. After cooling, the agar was poured into sterilized petri dishes (Sidoretno, 2022). The bacterial suspensions of S.pyogenes and S.mutans were inoculated onto the solidified Mueller-Hinton Agar medium by swabbing the surface with a sterile cotton swab. Sterile filter paper discs were soaked for 15 minutes in the Miana leaf extract solutions at different concentrations to ensure complete absorption of the extract. The discs were then placed onto the bacterial-inoculated MHA plates. The plates were incubated at 37°C for 24-48 hours (Intan et al., 2021).

Data analysis was performed using SPSSIBM version 27. The data analysis aimed to assess the sensitivity of S.pyogenes and S.mutans to different concentrations of Miana leaf extract The statistical tests performed included One-Way ANOVA, Post-HocLSD, and Shapiro-Wilk tests to determine whether there was any antibacterial activity of Miana leaf extract against S.pyogenes and S.mutans.

Phytochemical screening was conducted to identify the secondary metabolites present in the ethanol extract of Miana leaves that have antibacterial potential. The results showed that Miana leaf ethanol extract contains flavonoids, tannins, and saponins (Figure 1).

These findings are supported by research conducted by (Fati et al., 2020), which stated that Miana leaves contain active compounds such as saponins, flavonoids, and tannins, which play a role as antibacterial agents. However, alkaloid testing yielded negative results. Differences in phytochemical screening results may be influenced by the type of solvent, extraction method, compound concentration, plant growth location, plant age, and differences in test method sensitivity

Based on Table 2, Miana leaf ethanol extract at 5%, 10%, 20%, and 40% concentrations was able to inhibit the growth of S.pyogenes with varying inhibition zone diameters. The mean inhibition zone for S. pyogenes at 5% concentration was 9.83mm (moderate), 10% concentration was 10.76mm (moderate), 20% concentration was 12.42mm (strong), and 40% concentration was 15.66mm (strong). Based on Table 3, Miana leaf ethanol extract at 5%, 10%, 20%, and 40% concentrations was also able to inhibit the growth of S.mutans. The mean inhibition zone for S. mutans at 5% concentration was4.73mm (weak), 10% concentration was 6.54mm (moderate), 20% concentration was 7.66mm (moderate), and 40% concentration was 11.21 mm (strong).

The results indicate that increasing the concentration of Miana leaf ethanol extract enhances the inhibition zone diameter. This is due to the higher concentration of active antibacterial compounds at higher extract concentrations. Additionally, increased concentration enhances the penetration of active compounds into bacterial cells, which disrupts cellular metabolism and leads to bacterial cell death (Lingga et al., 2015). Differences in inhibition zones between S.pyogenes and S.mutans may be due to bacterial type, as each bacterium exhibits different sensitivities to the extract concentrations.

Factors influencing the antibacterial activity of a substance include bacterial density, disc placement time, incubation temperature, incubation duration, medium thickness, bacterial suspension turbidity, and media composition (Hastuty et al., 2018). TThe positive control (amoxicillin) produced a larger inhibition zone compared to the Miana leaf ethanol extract for both S.pyogenes and S.mutans. The average inhibition zone for amoxicillin was 28.56 mm for S. pyogenes (very strong inhibition) and 17.30 mm for S. mutans (strong inhibition). Amoxicillin is a broad-spectrum antibiotic with high oral bioavailability. The β-lactam ring in amoxicillin exerts antibacterial activity by targeting susceptible Gram-positive and Gram-negative bacteria (Sofyani et al., 2018). The mechanism of action of amoxicillin involves inhibiting the final stage of bacterial cell wall synthesis, causing cell rupture and preventing bacterial cell wall formation by binding to one or more penicillin-binding proteins (Pratiwi et al., 2019).

The antibacterial activity of Miana leaf ethanol extract against S.pyogenes and S.mutans is attributed to its secondary metabolite content, including flavonoids, tannins, and saponins. These bioactive compounds exhibit antibacterial properties. Flavonoidsinhibit DNA replication and bacterial membrane function, leading to bacterial cell damage and death.

The mechanism of flavonoids enhances cytoplasmic membrane permeability in Streptococcus pyogenes and Streptococcus mutans, thereby exerting an inhibitory effect on Gram-positive bacteria (Charmelya et al., 2023). Tannins act as antibacterial agents by forming stable complexes with proteins, resulting in bacterial protoplasm coagulation (Rijayanti, 2014). They target polypeptides in the bacterial cell wall, leading to cell lysis due to osmotic or physical pressure, ultimately causing bacterial death (Setiawan et al., 2017). Saponins reduce water surface tension, leading to foam formation. They act by increasing cell membrane permeability, resulting in hemolysis and bacterial cell leakage (Putri et al., 2023).

The antibacterial activity of Miana leaves observed in this study is consistent with previous research, although inhibition zone diameters vary depending on the extract concentration, solvent used, and target bacterial strains. Effectiveness against S.mutans a study focusing on oral pathogenic bacteria found that Miana leaf ethanol extract inhibited S. mutans with an average zone of 9.80 mm at a 25% concentration (Putri et al., 2023). Comparison with other Gram-Positive Bacteria Miana extract has shown even stronger activity against other Gram-positive bacteria like Staphylococcus aureus. Research using a 5% concentration of Miana extract in a solid soap formulation produced an inhibition zone of 18.4 mm (Asjur et al., 2024).

The Shapiro-Wilk normalitytestshowed a p-value > 0.05, indicating that the data were normally distributed. The homogeneity test also yielded a p-value > 0.05, confirming data homogeneity. The One-Way ANOVA testproduced a p-value (sig)= 0.000 < 0.05, indicating significant antibacterial activity and differences in inhibition zone diameters at 5%, 10%, 20%, and 40% ethanol extract concentrations against S.pyogenes and S. mutans.