RESULTS

Yield of the ethanol extract of Miana leaves

Maceration of Miana leaves using 96% ethanol produced a thick, dark-green extract with a semisolid consistency. A total of 28.4 g of concentrated extract was obtained from 200 g of dried leaf powder, corresponding to an extraction yield of 14.2% (w/w). The extract exhibited a characteristic aromatic odor and a sticky texture at 25°C.

Bioactive compounds identified in the ethanol extract of Miana leaves by LC–HRMS

Untargeted LC–HRMS metabolomic analysis identified 243 compounds in the ethanol extract of Miana leaves. The total ion chromatogram obtained in positive ionization mode is presented in Figure 1, with major peaks detected at retention time (RT, min) of 0.79, 6.64, 11.95, 16.24, and 22.92 min. Based on mass accuracy, molecular formula assignment, and spectral database matching, 20 major bioactive compounds were annotated with high confidence. These compounds were selected according to their signal intensities and a Metabolomics Standards Initiative confidence level of ≥3. The complete annotated dataset for all 243 compounds detected in both positive and negative ionization modes, including RT, measured m/z values, adduct types, molecular formulas, and confidence levels, is provided in Supplementary Table S1.

Figure 1: Total ion chromatogram of the ethanol extract of Miana leaves obtained by liquid chromatography–high-resolution mass spectrometry.

Table 1 summarizes the 20 major secondary metabolites identified in the ethanol extract of Miana leaves based on signal intensity and biological relevance. Flavonoids constituted the predominant class of metabolites, including apigenin (270.05257 Da; RT, 8.356 min), kaempferol (286.04726 Da; RT, 7.566 min), apigetrin (432.10570 Da; RT, 6.428 min), and cynaroside (448.10024 Da; RT, 5.287 min). Organic acids detected in appreciable amounts included caffeic acid (180.04198 Da; RT, 6.666 min), stearidonic acid (276.20867 Da; RT, 13.641 min), and α-eleostearic acid (278.22417 Da; RT, 14.124 min). The identified terpenoid compounds comprised lupeol (426.38582 Da; RT, 17.695 min), (+)-nootkatone (218.16658 Da; RT, 12.543 min), β-ionone (192.15123 Da; RT, 12.283 min), and (+)-ar-turmerone (216.15111 Da; RT, 12.862 min). Multiple coumarin derivatives (146.03664 Da) were detected at RT of 7.306, 7.086, 6.071, 5.910, 5.738, 5.365, and 4.366 min, indicating the presence of coumarin isomers or structurally related derivatives.

Additional bioactive diterpenoids, including triptolide (360.15679 Da; RT, 11.560 min), carnosol (330.18260 Da; RT, 6.212 min), and kahweol (314.18782 Da; RT, 15.172 min), were also identified. Among the detected metabolites, betaine (RT, 0.79 min), coumarin (RT, 6.64 min), kaempferol (RT, 11.95 min), and lupeol (RT, 22.92 min) exhibited the highest relative signal intensities in the chromatogram. Flavonoid glycosides, including apigetrin and cynaroside, eluted earlier than their corresponding aglycones, apigenin and kaempferol, because of their greater polarity. In contrast, highly hydrophobic terpenoid compounds, such as lupeol, eluted during the final phase of chromatographic separation (RT, 17.695 min), consistent with their physicochemical properties.

No. | Compound | Molecular formula | Structure | Calculated MW (Da) | RT (min) | Compound group
1 | Apigenin | C₁₅H₁₀O₅ |  | 270.05257 | 8.356 | Flavonoid
2 | Kaempferol | C₁₅H₁₀O₆ |  | 286.04726 | 7.566 | Flavonoid
3 | Caffeic acid | C₉H₈O₄ |  | 180.04198 | 6.666 | Phenolic acid
4 | Apigetrin | C₂₁H₂₀O₁₀ |  | 432.10570 | 6.428 | Flavonoid glycoside
5 | Cynaroside | C₂₁H₂₀O₁₁ |  | 448.10024 | 5.287 | Flavonoid glycoside
6 | Lupeol | C₃₀H₅₀O |  | 426.38582 | 17.695 | Triterpenoid
7 | Stearidonic acid | C₁₈H₂₈O₂ |  | 276.20867 | 13.641 | Unsaturated fatty acid
8 | α-Eleostearic acid | C₁₈H₃₀O₂ |  | 278.22417 | 14.124 | Unsaturated fatty acid
9 | Triptolide | C₂₀H₂₄O₆ |  | 360.15679 | 11.560 | Diterpenoid
10 | (+)-Nootkatone | C₁₅H₂₂O |  | 218.16658 | 12.543 | Sesquiterpenoid
11 | β-Ionone | C₁₃H₂₀O |  | 192.15123 | 12.283 | Terpenoid
12 | (+)-ar-Turmerone | C₁₅H₂₀O |  | 216.15111 | 12.862 | Sesquiterpenoid
13 | Kahweol | C₂₀H₂₆O₃ |  | 314.18782 | 15.172 | Diterpenoid
14 | Carnosol | C₂₀H₂₆O₄ |  | 330.18260 | 6.212 | Diterpenoid
15 | Coumarin | C₉H₆O₂ |  | 146.03664 | 7.306 | Coumarin
16 | Diosmetin | C₁₆H₁₂O₆ |  | 300.06328 | 8.514 | Flavonoid
17 | Tangeretin | C₂₀H₂₀O₇ |  | 372.12029 | 10.721 | Methylated flavonoid
18 | Erucamide | C₂₂H₄₃NO |  | 337.33370 | 17.204 | Fatty acid amide
19 | Methyl palmitate | C₁₇H₃₄O₂ |  | 270.25566 | 17.048 | Fatty acid ester
20 | Betaine | C₅H₁₁NO₂ |  | 117.07902 | 0.801 | Alkaloid

Antimicrobial activity of the ethanol extract of Miana leaves

The ethanol extract of Miana leaves exhibited both antifungal and antibacterial activities against the tested pathogens (Table 2). Against C. albicans, the extract produced an MIC of 62,500 ppm (62.5 mg/mL) and an MFC of 62,500 ppm (62.5 mg/mL), yielding an MFC/MIC ratio of 1.0, indicating fungicidal activity. Visual examination of the microplates revealed no turbidity at extract concentrations of 250,000, 125,000, and 62,500 ppm, whereas visible turbidity was observed at concentrations of ≤31,250 ppm. Subculture on SDA confirmed the absence of fungal colony growth at concentrations of ≥62,500 ppm (Figure 2A).

The extract also demonstrated antibacterial activity against N. gonorrhoeae, with both the MIC and MBC determined to be 100,000 ppm (100 mg/mL), yielding an MBC/MIC ratio of 1.0 and confirming bactericidal activity. No visible turbidity was observed at extract concentrations of 200,000 and 100,000 ppm. At 50,000 ppm, one replicate showed complete inhibition of bacterial growth, whereas the second replicate exhibited visible turbidity. In accordance with CLSI recommendations, the higher concentration (100,000 ppm) was therefore designated as the MIC. At the reported MIC values (62,500 ppm for C. albicans and 100,000 ppm for N. gonorrhoeae), duplicate wells produced concordant results with no visible turbidity. Bacterial growth was clearly observed at concentrations of ≤25,000 ppm. Subculture on chocolate agar confirmed the absence of bacterial colony growth at concentrations of ≥100,000 ppm (Figure 2B).

Pathogen | Sample | MIC (ppm/mg/mL) | MFC/MBC (ppm/mg/mL) | Ratio | Activity type
Candida albicans | Miana extract | 62,500/62.5 | 62,500/62.5 | 1.0 | Fungicidal
 | Nystatin* | 2/0.002 | 2/0.002 | 1.0 | Fungicidal
Neisseria gonorrhoeae | Miana extract | 100,000/100 | 100,000/100 | 1.0 | Bactericidal
 | Vancomycin† | 0.0625/0.0000625 | 0.0625/0.0000625 | 1.0 | Bactericidal

Figure 2: Confirmation of the antimicrobial activity of the ethanol extract of Miana leaves on solid media. (A) Growth of Candida albicans on Sabouraud dextrose agar following treatment with various concentrations of the Miana leaf extract and nystatin (positive control). (B) Growth of Neisseriagonorrhoeae on chocolate agar following treatment with various concentrations of the Miana leaf extract and vancomycin (positive control). MO = Medium only (negative medium control); BO = Microorganism only (growth control). No colony growth was observed at extract concentrations of ≥62,500 ppm for C. albicans and ≥100,000 ppm for N. gonorrhoeae.

The positive control nystatin inhibited C. albicans at an MIC and MFC of 2 ppm, whereas vancomycin inhibited N. gonorrhoeae at an MIC and MBC of 0.0625 ppm. Compared with the positive controls, the antimicrobial activity of the Miana leaf extract was approximately 31,250-fold lower than that of nystatin and 1,600,000-fold lower than that of vancomycin. Nevertheless, the identical MFC/MIC and MBC/MIC ratios (1.0) observed for both the extract and the positive controls indicate that they produced fungicidal and bactericidal effects, respectively, at their corresponding MICs.

The negative medium control remained free of contamination throughout the experiment, whereas the microbial growth control demonstrated satisfactory viability during the incubation period.

Antioxidant activity of the ethanol extract of Miana leaves

The ethanol extract of Miana leaves demonstrated free radical scavenging activity in both antioxidant assays (Figure 3). In the DPPH assay, the extract exhibited an IC₅₀ value of 43.68 ppm (95% CI: 30.98–60.90 ppm), indicating very strong antioxidant activity. The nonlinear regression model generated the equation:

Y = 100/[(43.68/X)1.199 + 1]

with a coefficient of determination (R²) of 0.9707. The antioxidant activity of the extract was greater than that of the positive control (ascorbic acid). Pearson's correlation analysis demonstrated a significant positive association between extract concentration and percentage radical inhibition (r = 0.8106, p = 0.03), indicating that radical scavenging activity increased with increasing extract concentration.

In the ABTS assay, the extract exhibited an IC₅₀ of 128.7 ppm (95% CI: 99.75–164.30 ppm), indicating moderate antioxidant activity. The nonlinear regression equation was:

Y = 100/[(128.7/X)1.767 + 1]

with an R² value of 0.9754. The antioxidant activity exceeded that of the positive control (Trolox). Pearson's correlation analysis revealed a strong positive relationship between extract concentration and radical inhibition (r = 0.9640, p < 0.001), confirming a consistent concentration-dependent response.

The dose–response curves for both assays exhibited the characteristic sigmoidal pattern of radical-scavenging activity, with inhibition increasing with increasing extract concentration. Comparison of the two assays demonstrated that the extract scavenged DPPH radicals more effectively than ABTS radicals, as indicated by an ABTS/DPPH IC₅₀ ratio of 2.95 (Figure 3).

Figure 3: Antioxidant activity of the ethanol extract of Miana leaves. (A) 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. (B) 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay. Data are presented as the mean of three independent replicates.