MATERIALS AND METHODS

Ethical approval

The study protocol was reviewed and approved by the Health Research Ethics Committee of Bakti Tunas Husada University, Tasikmalaya, Indonesia (approval no. 314-01/E.01/KEPK-BTH/IX/2024; approved on September 25, 2024). As this was a laboratory-based in vitro study using authenticated plant material and reference microbial strains, no human participants or animals were involved, and informed consent was not required. All experimental procedures involving N. gonorrhoeae ATCC 43069P and C. albicans ATCC 10231 were conducted in accordance with institutional biosafety requirements and relevant national and institutional laboratory safety guidelines. The microbial cultures were handled only by trained personnel in controlled laboratory facilities, and all contaminated materials, culture media, and biological waste were sterilized before disposal. The study was conducted in accordance with the approved protocol and did not involve the collection of clinical specimens, patient data, or animal-derived samples.

Study period and location

The study was conducted between January and March 2024 at the Microbiology Laboratory and Phytochemistry Laboratory, Medistra Indonesia College of Health Sciences, Bekasi, West Java, Indonesia. All experimental procedures were performed under controlled laboratory conditions at room temperature (25 ± 2°C) and a relative humidity of 60 ± 5%.

Study design

This laboratory-based experimental study employed an in vitro approach to evaluate the antibacterial, antifungal, and antioxidant activities of ethanol extracts prepared from Miana leaves. Untargeted metabolomic profiling was performed to characterize the extract's phytochemical composition prior to evaluation of biological activity.

Sample preparation and extraction

Fresh Miana leaves were sorted, thoroughly washed with running tap water, and dried in the shade at ambient temperature (25 ± 2°C) for 7 days until a constant weight was reached. The dried leaves were ground using an electric blender and passed through a 60-mesh sieve to obtain a uniform fine powder.

A total of 200 g of powdered leaves was macerated with 2,000 mL of 96% ethanol (1:10, w/v) in a sealed amber glass container at 25°C for 48 h with intermittent stirring. The macerate was filtered through Whatman No. 1 filter paper, and the plant residue was remacerated twice with fresh solvent until the filtrate became colorless [32]. All filtrates were combined and concentrated using a rotary vacuum evaporator (IKA Werke GmbH & Co. KG, Staufen, Germany) at 40°C under a pressure of 175 mbar until a thick extract of constant weight was obtained. The extraction yield was calculated as the ratio of the weight of the concentrated extract to the initial dry weight of the plant material. The concentrated extract was transferred into sealed amber glass containers and stored at 4°C until further analysis.

The extraction process yielded 28.4 g of thick extract from 200 g of dried leaf powder, corresponding to an extraction yield of 14.2% (w/w). No phytochemical marker-based standardization was performed for this extract batch; however, batch consistency will be evaluated in future studies using LC–HRMS chemical fingerprinting.

Materials and equipment

Fresh Miana leaves were collected from a medicinal plant garden in Bekasi, West Java, Indonesia. Botanical authentication was performed at the Bogor Herbarium, National Research and Innovation Agency, Bogor, Indonesia, under voucher specimen no. 017/BH/VI/2024.

The test microorganisms used in this study were N. gonorrhoeae (American Type Culture Collection [ATCC] 43069P) and C. albicans (ATCC 10231), obtained from the ATCC, Manassas, VA, USA.

The chemicals used in this study included 96% ethanol, LC–MS-grade methanol, chocolate agar medium (HiMedia Laboratories Pvt. Ltd., Mumbai, India), Sabouraud dextrose agar (SDA; HiMedia), Sabouraud dextrose broth (SDB; HiMedia), and 0.9% sodium chloride solution (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). McFarland Standard No. 3 (Thermo Fisher Scientific Remel, Lenexa, KS, USA), dimethyl sulfoxide (DMSO), formic acid, and acetonitrile were also used. Vancomycin and nystatin supplied by the Indonesian Food and Drug Authority (BPOM), Jakarta, Indonesia, served as positive controls for antibacterial and antifungal assays, respectively.

The principal instruments included a UV–Vis spectrophotometer (B-ONE, Shanghai, China), a biological safety cabinet (Medfuture, Munich, Germany), a rotary vacuum evaporator (IKA Werke GmbH & Co. KG, Staufen, Germany), an LC–HRMS system comprising a Q Exactive Plus Orbitrap mass spectrometer coupled with a Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific), and an Acclaim RSLC 120 C18 analytical column (2.2 μm × 2.1 × 100 mm; Thermo Fisher Scientific). Additional laboratory equipment included an incubator (Memmert GmbH + Co. KG, Schwabach, Germany), an autoclave (GEA, Guangzhou, China), sterile 96-well microplates, and borosilicate laboratory glassware (Pyrex, Corning, NY, USA).

Metabolomic analysis via LC–HRMS

Untargeted LC–HRMS metabolomic analysis was performed to characterize the secondary metabolite profile of the Miana leaf extract and identify its bioactive constituents [32]. Briefly, 10 mg of concentrated extract was dissolved in 1 mL of LC–MS-grade methanol, sonicated for 15 min, and centrifuged at 24,562 × g for 10 min at 4°C. The supernatant was filtered through a 0.22-μm polytetrafluoroethylene syringe filter before injection into the LC–HRMS system. Three technical replicates of the injection were prepared for each sample. Solvent blanks consisting of LC–MS-grade methanol were processed under identical chromatographic conditions and subsequently subtracted from the chromatograms to eliminate background signals.

Peak processing was performed using Compound Discoverer version 3.3 (Thermo Fisher Scientific). The processing parameters included a minimum peak intensity threshold of 1 × 10⁵, a mass tolerance of 5 ppm, a signal-to-noise ratio of ≥3, and targeted adducts of [M+H]⁺, [M+Na]⁺, and [M+NH₄]⁺ in positive ionization mode and [M−H]⁻ and [M+HCOO]⁻ in negative ionization mode.

Chromatographic separation was achieved using an Acclaim RSLC 120 C18 analytical column (2.2 μm, 2.1 × 100 mm) maintained at 40°C with an injection volume of 5 μL. The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. Gradient elution was programmed as follows: 0–2 min, 5% B; 2–20 min, 5%–100% B; 20–25 min, 100% B; 25–26 min, 100%–5% B; and 26–30 min, 5% B at a flow rate of 0.3 mL/min.

Mass spectrometric analysis was performed in both positive and negative ionization modes over an m/z range of 100–1500. The electrospray ionization source parameters were set as follows: spray voltage, 3.5 kV; capillary temperature, 320°C; sheath gas, 40 arbitrary units; auxiliary gas, 10 arbitrary units; and mass resolution, 70,000 full width at half maximum.

LC–HRMS data were processed using Compound Discoverer version 3.3 (Thermo Fisher Scientific) for peak detection, spectral deconvolution, and compound annotation using the mzCloud, ChemSpider, and Kyoto Encyclopedia of Genes and Genomes databases. Compound identification was based on mass accuracy (<5 ppm), MS/MS fragmentation patterns, and comparisons with published literature. Compounds with a confidence level of ≥3, as defined by the Metabolomics Standards Initiative, were selected for further interpretation [29]. Both positive and negative electrospray ionization modes were used to maximize metabolite coverage. The positive mode predominantly generated [M+H]⁺ and [M+Na]⁺ adducts, whereas the negative mode primarily detected [M−H]⁻ adducts for phenolic and organic acids. Among the 243 annotated compounds, the 20 major compounds listed in Table 1 were selected based on their signal intensities and established biological relevance, with a Metabolomics Standards Initiative confidence level of ≥3. The complete annotated dataset, including m/z values, retention times (RT), adduct types, molecular formulas, and Metabolomics Standards Initiative confidence levels for all detected compounds, is provided in Supplementary Table S1**.**

Preparation of test microorganisms

N. gonorrhoeae cultures were revived on chocolate agar medium and incubated at 37°C in a 5% CO₂ atmosphere for 24 h. C. albicans cultures were revived on SDA and incubated aerobically at 37°C for 48 h. Pure colonies were aseptically collected using a sterile inoculating loop and suspended in sterile 0.9% sodium chloride solution to achieve turbidity equivalent to McFarland Standard No. 3 (3 × 10⁸ colony-forming units [CFU]/mL), as determined spectrophotometrically at 625 nm [33].

The microbial suspension (3 × 10⁸ CFU/mL) was serially diluted in sterile 0.9% sodium chloride solution to obtain a working inoculum of 1 × 10⁶ CFU/mL using a 1:100 dilution. Briefly, 10 μL of the 3 × 10⁸ CFU/mL suspension was transferred into 990 μL of sterile 0.9% sodium chloride solution to produce the working suspension. N. gonorrhoeae cultures were maintained at 37°C under a 5%–7% CO₂ atmosphere with ≥95% relative humidity. The viability of the working inoculum was confirmed by plate counting on the appropriate solid medium before each experiment, and all inocula were within the acceptable target range.

MIC assay

The MIC was determined using the broth microdilution method in sterile 96-well microplates in accordance with CLSI guidelines [34]. For antifungal susceptibility testing, the assay followed the CLSI M27 reference standard using SDB [35]. For antibacterial susceptibility testing against N. gonorrhoeae, broth microdilution was employed as a high-throughput adaptation, and all inhibitory endpoints were confirmed by subculturing onto chocolate agar because agar dilution remains the CLSI reference method (CLSI M07/M100) for this fastidious organism [36]. This methodological adaptation is recognized as a limitation of the present study.

A final inoculum concentration of 3 × 10⁶ CFU/mL was used to increase the sensitivity of crude extract screening. This concentration exceeded the CLSI-recommended inoculum of 5 × 10⁵ CFU/mL for standard broth microdilution and may therefore have contributed to the relatively high MIC values obtained. This limitation should be considered when interpreting the extract's antimicrobial activity. All microplates were sealed with sterile parafilm before incubation to minimize evaporation.

The Miana leaf extract was dissolved in 10% DMSO and serially diluted with SDB to obtain final concentrations of 250,000, 125,000, 62,500, 31,250, 15,625, and 7,812.5 ppm for antifungal testing. For antibacterial testing, concentrations of 200,000, 100,000, 50,000, 25,000, 12,500, and 6,250 ppm were prepared.

Each well received 100 μL of the extract solution and 100 μL of the microbial suspension (final inoculum, 3 × 10⁶ CFU/mL). Nystatin (0.5–4 ppm) served as the positive control for C. albicans, whereas vancomycin (0.5–2 ppm) served as the positive control for N. gonorrhoeae. Negative control wells contained sterile medium without microorganisms, whereas growth control wells contained microbial suspension without extract or antimicrobial agents.

The microplates were incubated at 37°C for 48 h under aerobic conditions for C. albicans and in a 5% CO₂ atmosphere for N. gonorrhoeae. The MIC was defined as the lowest extract concentration showing no visible turbidity compared with the growth control. All experiments were performed in duplicate. When duplicate wells produced concordant results, the lowest concentration showing complete inhibition of visible growth was recorded as the MIC. When discordant results occurred between duplicate wells, the higher (more conservative) concentration was accepted as the MIC in accordance with the CLSI M07-A11 recommendations [37].

To eliminate solvent-related effects, the extract stock solution prepared in 10% DMSO was serially diluted with SDB or chocolate broth so that the final DMSO concentration in each assay well did not exceed 1% (v/v), a concentration reported to have no inhibitory effect on the test microorganisms [38]. At each concentration, the extract solution was visually inspected and examined under light microscopy to verify complete dissolution and the absence of precipitation before inoculation. No visible precipitation was observed at concentrations up to 250,000 ppm (250 mg/mL). In addition, the pH of the medium containing the highest extract concentration differed by no more than ±0.2 units from that of the untreated control, confirming the absence of significant pH-related nonspecific effects. Vehicle control wells containing 1% DMSO alone were included in every assay and demonstrated no inhibition of microbial growth.

MFC assay

The MFC for C. albicans and the minimum bactericidal concentration (MBC) for N. gonorrhoeae were determined by subculturing 10-μL aliquots from all wells showing no visible turbidity in the MIC assay onto solid media using the quadrant streak technique. Antifungal subcultures were inoculated onto SDA, whereas antibacterial subcultures were inoculated onto chocolate agar.

The culture plates were incubated at 37°C for 48 h under the same atmospheric conditions as those used in the corresponding MIC assay. The MFC or MBC was defined as the lowest concentration producing no visible colony growth or a maximum of one to two colonies, corresponding to ≥99.9% killing of the initial microbial inoculum [39].

All determinations were performed in duplicate to confirm reproducibility. The lowest concentration that resulted in complete or near-complete elimination of viable microorganisms was recorded as the MFC for C. albicans and the MBC for N. gonorrhoeae, respectively.

Quality control

Each antimicrobial susceptibility assay included appropriate quality control measures to ensure the reliability and reproducibility of the results. The following controls were included in parallel with the test samples: (a) a growth control containing the test microorganism in culture medium without extract or antimicrobial agent to confirm microbial viability throughout the assay; (b) a sterility control consisting of culture medium only to verify the absence of contamination; (c) a vehicle control containing the test microorganism in culture medium supplemented with the maximum final concentration of DMSO (≤1%, v/v) to confirm that the solvent did not affect microbial growth; and (d) positive controls comprising nystatin (0.5–4 ppm) for C. albicans and vancomycin (0.03–2 ppm) for N. gonorrhoeae to verify assay performance during each experimental run.

Antioxidant activity assay

The antioxidant activity of the Miana leaf extract was evaluated using the ABTS and DPPH radical scavenging assays.

For the ABTS assay, the ABTS•⁺ radical solution was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate (1:1, v/v), then incubated in the dark at 25°C for 16 h. The resulting radical solution was diluted with methanol to obtain an absorbance of 0.70 ± 0.02 at 734 nm. The Miana leaf extract was dissolved in methanol to obtain concentrations of 3.91, 7.81, 15.62, 31.25, 62.5, 125, 250, and 500 ppm. Subsequently, 100 μL of each extract solution was mixed with 1,900 μL of the ABTS•⁺ radical solution, vortexed for 30 s, and incubated at 25°C for 6 min in the dark. The absorbance was measured at 734 nm using the UV–Vis spectrophotometer. Trolox served as the positive control, whereas methanol without extract or standard was used as the blank.

For the DPPH assay, a 0.1 mM DPPH radical solution was prepared in methanol. The Miana leaf extract was prepared at concentrations of 3.91, 7.81, 15.62, 31.25, 62.5, 125, 250, 500, 750, 1,000, and 1,500 ppm. A total of 100 μL of each extract solution was mixed with 3,900 μL of the DPPH solution, vortexed for 30 s, and incubated at 25°C in the dark for 30 min. The absorbance was measured at 517 nm using the UV–Vis spectrophotometer. Ascorbic acid was used as the positive control.

The percentage of radical scavenging activity was calculated using the following equation:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ Radical inhibition (\%)=\frac{{A}_{control}-{A}_{sample}}{{A}_{control}}\times 100 \] \end{document}

The half-maximal inhibitory concentration (IC₅₀) values were determined by nonlinear regression using the inhibitor versus normalized response-variable slope model, consistent with the analytical approach used for the ABTS assay.

Statistical analysis

The MIC, MFC, and MBC values were determined as categorical endpoints based on visual assessment of broth turbidity and confirmation by subculture on solid media. Because these endpoints follow the CLSI broth microdilution methodology, parametric inferential statistical analyses were not applicable, and the antimicrobial susceptibility results are presented descriptively [37]. When duplicate determinations yielded discordant results, the higher (more conservative) concentration was recorded as the MIC.

The antioxidant activities were expressed as IC₅₀ values with corresponding 95% confidence intervals (CI). The relationship between extract concentration and radical-scavenging activity was evaluated using Pearson's correlation coefficient, with statistical significance set at p < 0.05**.**

Microbial growth observations were recorded qualitatively as positive (+) when visible turbidity or colony growth was present and negative (−) when no microbial growth was observed. The inhibitory activities of the Miana leaf extract and the positive controls were compared descriptively according to their effective concentration ranges.

Metabolomic data generated by LC–HRMS were evaluated using chemometric analysis based on principal component analysis. Representative photographs of microbial growth on solid media were captured to document the experimental findings. Dose–response curves for antioxidant activity were generated by nonlinear regression using the inhibitor versus normalized response variable slope model to characterize the relationship between extract concentration and radical-scavenging activity. Statistical significance was accepted at p < 0.05**.**