DISCUSSION

Overview of the principal findings

This study comprehensively characterized the secondary metabolite profile of the ethanol extract of Miana leaves using LC–HRMS, evaluated its antibacterial activity against Neisseria gonorrhoeae and antifungal activity against C. albicans by determining the MIC and MFC values, and assessed its antioxidant activity using the DPPH and ABTS radical scavenging assays. Untargeted LC–HRMS analysis identified 243 annotated metabolites, including 20 major bioactive compounds belonging to the flavonoids (apigenin, kaempferol, apigetrin, cynaroside, diosmetin, and tangeretin), phenolic acids (caffeic acid), unsaturated fatty acids (stearidonic acid and α-eleostearic acid), terpenoids (lupeol, (+)-nootkatone, β-ionone, and (+)-ar-turmerone), diterpenoids (triptolide, kahweol, and carnosol), coumarins (coumarin), and alkaloids (betaine). Collectively, these findings demonstrate that the ethanol extract possesses a chemically diverse phytochemical composition that may contribute to its biological activities.

Antioxidant activity of the extract

The ethanol extract of Miana leaves exhibited marked antioxidant activity in both radical scavenging assays. The DPPH assay yielded an IC₅₀ of 43.68 ppm, indicating very strong antioxidant activity, whereas the ABTS assay yielded an IC₅₀ of 128.7 ppm, corresponding to moderate antioxidant activity. The higher antioxidant activity observed in the DPPH assay, as reflected by an ABTS/DPPH IC₅₀ ratio of 2.95, suggests that the extract has greater affinity for lipophilic radicals than for hydrophilic radicals. This selectivity is likely attributable to the predominance of relatively lipophilic phytochemicals, particularly flavonoid aglycones such as apigenin and kaempferol and terpenoid compounds including lupeol and (+)-nootkatone, which exhibit strong hydrogen-donating and electron-transfer capacities toward DPPH radicals [40–42]. Furthermore, the significant positive Pearson correlation between extract concentration and radical-scavenging activity confirmed a concentration-dependent antioxidant response, indicating that the observed antioxidant activity was attributable to the extract's phytochemical constituents rather than analytical artifacts [43].

Beyond direct radical scavenging, the extract's antioxidant properties may enhance its antimicrobial effects through complementary biological mechanisms. Phenolic compounds, including caffeic acid and flavonoids, are known to modulate oxidative balance during microbial infection by scavenging excessive free radicals while simultaneously disrupting microbial redox homeostasis [44, 45]. Pakadang et al. [20] similarly reported that Miana leaf extract exhibits both antioxidant and antibacterial activities, supporting the present findings that these biological activities arise from the complex interactions among multiple phytochemical constituents rather than from a single active compound. Because oxidative stress contributes substantially to tissue injury, inflammation, and impairment of mucosal defense mechanisms during genital infections, the combination of antimicrobial and antioxidant activities may provide therapeutic advantages by simultaneously reducing microbial burden and limiting oxidative tissue damage [19, 27]. Likewise, Ślusarczyk et al. [15] reported that Coleus species exhibit high antioxidant activity associated with abundant flavonoids and phenolic compounds, which is consistent with the present LC–HRMS analysis, which demonstrates appreciable quantities of kaempferol, apigenin, caffeic acid, and related antioxidant metabolites.

Antimicrobial activity of the extract

The ethanol extract demonstrated antifungal activity against C. albicans, with MIC and MFC values of 62,500 ppm (62.5 mg/mL), and antibacterial activity against N. gonorrhoeae, with MIC and MBC values of 100,000 ppm (100 mg/mL). The identical MFC/MIC and MBC/MIC ratios of 1.0 indicate that the extract exerted fungicidal and bactericidal activities at their respective MICs. Although these findings confirm the dual antimicrobial potential of the extract, the MIC values were considerably higher than those generally regarded as indicative of potent antimicrobial activity for crude plant extracts (MIC <0.1 mg/mL, highly active; 0.5–1.0 mg/mL, moderately active; 1.0–8.0 mg/mL, weakly active; >8–10 mg/mL, low biological relevance) [46, 47]. Therefore, despite demonstrating measurable antimicrobial activity, the crude ethanol extract exhibited relatively weak potency against the two target pathogens when compared with established antimicrobial agents.

The relatively high MIC values may be explained by several biological and physicochemical factors. First, crude plant extracts contain complex mixtures in which active constituents are substantially diluted by inactive metabolites, thereby reducing the apparent antimicrobial potency of the preparation. Second, the outer lipooligosaccharide membrane of N. gonorrhoeae represents an effective permeability barrier that limits the penetration of hydrophobic phytochemicals. Likewise, the cell wall architecture of C. albicans restricts the diffusion of many polar macromolecular constituents, thereby reducing intracellular accumulation of active compounds [48, 49]. Experimental artifacts were minimized by maintaining the final DMSO concentration below 1% (v/v), confirming complete solubility of the extract before inoculation, and demonstrating the absence of microbial inhibition in the vehicle controls [50, 51]. These methodological controls support the conclusion that the observed antimicrobial effects resulted from genuine biological activity rather than nonspecific physicochemical influences. Nevertheless, the relatively high effective concentrations indicate that bioassay-guided fractionation and purification of active constituents will be necessary before therapeutic application can be considered [52].

Novelty and significance of the study

The present study provides several important advances over previous investigations of Miana. To our knowledge, this is the first study to combine comprehensive untargeted LC–HRMS metabolomic profiling with standardized CLSI-guided antimicrobial susceptibility testing against both N. gonorrhoeae and C. albicans. In addition to confirming dual antimicrobial and antioxidant activities, untargeted metabolomic analysis identified several bioactive metabolites, including triptolide, tangeretin, and betaine, that have not been previously reported in Miana. Furthermore, the use of an Orbitrap LC–HRMS platform with Metabolomics Standards Initiative-compliant compound annotation at a mass accuracy of <5 ppm provides substantially greater metabolomic coverage than earlier studies relying primarily on thin-layer chromatography or spectrophotometric phytochemical analyses [21]. From a microbiological perspective, the application of CLSI-guided broth microdilution with CO₂-supplemented incubation and confirmation on chocolate agar for N. gonorrhoeae represents a rigorous methodological approach that has rarely been applied in antimicrobial investigations of medicinal plant extracts. Consequently, this study establishes a robust phytochemical and microbiological reference for future investigations aimed at developing standardized phytopharmaceutical products targeting genital pathogens.

Biological significance of the identified bioactive compounds

The predominance of flavonoids in the ethanol extract is consistent with the findings of Kueakulpattana et al. [10], who identified flavonoids as the principal bioactive constituents of Coleus spp. with antimicrobial activity mediated through inhibition of bacterial DNA gyrase and topoisomerase IV. Apigenin has been reported to exhibit broad-spectrum antibacterial activity by disrupting bacterial membrane integrity and inhibiting biofilm formation [53]. Similarly, kaempferol exhibits antifungal activity by inhibiting ergosterol biosynthesis in fungal cell membranes, a mechanism comparable to that of azole antifungal agents but with lower reported toxicity [19, 54, 55]. The detection of flavonoid glycosides, including apigetrin and cynaroside, suggests that these compounds function as storage and transport forms of flavonoids within plant tissues. Following hydrolysis by microbial enzymes or under acidic physiological conditions, these glycosides may release the corresponding aglycones, thereby enhancing the oral bioavailability and biological activity of the extract [40–42, 44]. Bismelah et al. [28] identified quercetin as the predominant constituent of P. scutellarioides and reported antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa at substantially lower MIC values than those observed in the present study, likely attributable to differences in extraction methods, phytochemical composition, and the susceptibility of the target microorganisms.

Among the phenolic constituents, caffeic acid is recognized for its antimicrobial activity through induction of oxidative damage to microbial lipids, proteins, and nucleic acids by modulating intracellular ROS production [43, 45]. The unsaturated fatty acids stearidonic acid and α-eleostearic acid, detected at RT of 13.641 and 14.124 min, respectively, have been reported to disrupt phospholipid bilayers, thereby increasing membrane permeability and ultimately causing osmotic cell lysis [56–58]. Desbois and Smith [56] demonstrated that long-chain unsaturated fatty acids effectively inhibit N. gonorrhoeae through hydrophobic interactions with the outer membrane of Gram-negative bacteria, providing further support for the antibacterial activity observed in the present investigation.

Terpenoid constituents identified in the extract may also contribute substantially to its antimicrobial properties. Lupeol has been reported to disrupt fungal cell membrane fluidity and inhibit the dimorphic transition of Candida spp. from yeast to invasive hyphal forms [59–62]. Likewise, (+)-nootkatone and β-ionone are volatile terpenoids that rapidly penetrate microbial cells, disrupt mitochondrial function, deplete intracellular adenosine triphosphate, and ultimately induce cell death [63]. The identification of these compounds supports the hypothesis that multiple phytochemical classes collectively contribute to the observed antimicrobial activity through complementary mechanisms of action.

The diterpenoids identified in the present study further expand the biological relevance of the metabolomic profile. Triptolide has been reported to exhibit potent antifungal activity against fluconazole-resistant Candida spp. by inhibiting the calcineurin signaling pathway and inducing apoptosis in fungi [60, 64]. Carnosol exerts antimicrobial effects through a dual mechanism: inhibition of microbial efflux pumps and modulation of quorum-sensing pathways, thereby suppressing the expression of bacterial virulence factors [64–66]. Kahweol possesses a characteristic furan-diterpenoid structure that exhibits selective antibacterial activity against Gram-positive bacteria and moderate activity against Gram-negative organisms by inhibiting peptidoglycan biosynthesis [67, 68]. In addition, the repeated detection of coumarin peaks at multiple RT values suggests the presence of structurally related coumarin derivatives that interfere with microbial DNA replication through DNA intercalation and inhibition of topoisomerase enzymes [69]. Betaine, which exhibited one of the highest signal intensities, functions primarily as an osmoprotectant and methyl donor and may indirectly contribute to antimicrobial activity by modulating microbial oxidative stress responses [70].

Differential antimicrobial susceptibility of the test microorganisms

The ethanol extract exhibited greater antifungal activity against C. albicans than antibacterial activity against N. gonorrhoeae, as reflected by MIC values of 62,500 and 100,000 ppm, respectively. This difference can be explained by fundamental structural differences between fungal and bacterial cells. The fungal cell membrane contains ergosterol, which has a relatively high affinity for hydrophobic flavonoids and terpenoids such as lupeol and apigenin, thereby facilitating intracellular accumulation of these bioactive compounds [71]. In contrast, _N. _gonorrhoeae, as a Gram-negative bacterium, possesses an outer lipooligosaccharide membrane that restricts penetration of hydrophobic phytochemicals, thereby reducing antimicrobial efficacy and increasing the concentration required for growth inhibition [72].

The identical MIC and MFC or MBC values obtained for both microorganisms (MFC/MIC or MBC/MIC ratio = 1.0) indicate that the extract exerted cidal rather than static antimicrobial effects at the MIC. Such cidal activity is advantageous because complete elimination of viable microorganisms may reduce the likelihood of persistent infection and the emergence of antimicrobial resistance [37, 39]. Bismelah et al. [28] reported that P. scutellarioides extract exhibited an MFC/MIC ratio of 2.0 against periodontal bacteria, which differs from the ratio observed in the present study. These differences are likely associated with variations in microbial susceptibility, the geographical origin of the plant material, the harvest season, extraction procedures, and the resulting phytochemical composition.

Previous investigations of Miana and P. scutellarioides have consistently reported lower MIC values against gram-positive and periodontal bacteria than against N. gonorrhoeae and C. albicans. This discrepancy may be attributed to three principal factors: the intrinsic permeability barrier of gram-negative bacteria [25, 28], differences in the susceptibility of the target microorganisms, and the use of crude extracts in the present study compared with partially purified fractions evaluated in previous investigations [28]. Importantly, the present study provides the first CLSI-standardized quantitative MIC and MFC values for these two clinically important genital pathogens together with comprehensive untargeted metabolomic profiling of Miana, thereby providing a valuable reference for future phytopharmaceutical research.

Clinical relevance and future prospects

Compared with the positive controls, the crude ethanol extract exhibited substantially lower antimicrobial potency, being approximately 31,250-fold less active than nystatin against C. albicans and approximately 1,600,000-fold less active than vancomycin against N. gonorrhoeae. Nevertheless, these comparisons should be interpreted with caution because the extract is a complex mixture of phytochemicals rather than purified antimicrobial compounds (Table 3). Previous studies by Nguyen and Bhattacharya [74] and Vijayakumar et al. [75] demonstrated that flavonoids such as quercetin may act synergistically with conventional antibiotics by inhibiting microbial efflux pumps and modulating antimicrobial resistance mechanisms. Consequently, Miana leaf extract or its purified constituents may have greater potential as adjunctive therapies than as standalone antimicrobial agents.

Reference | Extract | Target microorganism | MIC (mg/mL) | Method | Major compounds identified | Principal findings
Present study | 96% ethanol (maceration) | Neisseria gonorrhoeae (ATCC 43069P), Candida albicans (ATCC 10231) | 100; 62.5 | CLSI broth microdilution | 243 metabolites identified by LC–HRMS, including triptolide, carnosol, kahweol, kaempferol, and apigenin | Dual antimicrobial activity; DPPH IC₅₀ = 43.68 ppm; ABTS IC₅₀ = 128.7 ppm
[28] | Ethanol extract of P. scutellarioides leaves | Streptococcus oralis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans | 1.56–3.13 | Agar diffusion and MIC | Quercetin | Antibacterial activity reported
[17] | Ethyl acetate extract of Coleus spp. | Staphylococcus aureus | Not quantified | Disk diffusion | Flavonoids (qualitative) | Antibacterial activity reported
[73] | Ethanol extract of Miana leaves | C. albicans | In vivo only | Murine vulvovaginal model | Not characterized | Antifungal activity reported
[20] | Miana extract | Bacteria (unspecified) | Reported | Nonstandardized | Not characterized | Antibacterial activity reported
[15] | Coleus amboinicus extracts | Not evaluated | — | HPLC | Flavonoids and terpenoids | High antioxidant activity reported

The dual activity of the extract against N. gonorrhoeae and C. albicans is of particular clinical interest because these pathogens frequently occur as coinfections that require simultaneous antibacterial and antifungal treatment [6, 73]. Although the observed cidal activity provides an encouraging pharmacological basis for further investigation, cytotoxicity toward mammalian cells and determination of the selectivity index (SI = CC₅₀/MIC) are essential before therapeutic applications can be proposed [76]. Bioassay-guided fractionation represents the most immediate strategy for concentrating active constituents and improving antimicrobial potency.

Future investigations should focus on isolation and structural characterization of the principal bioactive compounds, evaluation of synergistic interactions with ceftriaxone against N. gonorrhoeae and fluconazole against C. albicans using checkerboard and time-kill assays, and validation of antimicrobial efficacy using appropriate in vivo infection models [75]. Additional studies should include cytotoxicity assessment in vaginal epithelial or Vero cells, determination of the selectivity index, comparative LC–HRMS fingerprinting of Miana collected from different geographical regions, development of topical formulations such as creams, gels, and vaginal ovules, and ultimately phase I–II clinical trials to establish safety and therapeutic efficacy. Furthermore, proteomic and transcriptomic approaches may provide valuable insights into the molecular mechanisms underlying the antimicrobial activity of the identified phytochemicals.

Study limitations

Several limitations should be considered when interpreting the findings of this study. First, the antimicrobial activity was evaluated using a crude ethanol extract, in which the concentrations of individual bioactive compounds were relatively low because of dilution within a complex phytochemical matrix. Consequently, the MIC and MFC/MBC values obtained may underestimate the antimicrobial potential of the purified active constituents. Second, although comprehensive untargeted LC–HRMS profiling successfully identified 243 metabolites, the study did not isolate or quantify the contribution of individual compounds to the observed antimicrobial and antioxidant activities. Therefore, the biological activities reported are likely attributable to synergistic interactions among multiple phytochemicals rather than to single constituents.

Third, the study was limited to in vitro antimicrobial and antioxidant assays. Although these findings provide valuable preliminary evidence, they do not fully represent the complex physiological environment encountered in vivo, where host immune responses, tissue penetration, metabolism, and pharmacokinetic behavior influence therapeutic efficacy. Furthermore, cytotoxicity toward mammalian cells was not evaluated. At the MIC values obtained (62.5 mg/mL for C. albicans and 100 mg/mL for Neisseria gonorrhoeae), potential nonselective effects on host cells cannot be excluded. Therefore, determination of the half-maximal cytotoxic concentration (CC₅₀) and the selectivity index (SI = CC₅₀/MIC) using appropriate mammalian cell lines, such as vaginal epithelial or Vero cells, is essential before therapeutic applications can be proposed [76].

Another limitation is that antimicrobial activity was evaluated only against standard reference strains. Clinical isolates, particularly multidrug-resistant strains, may exhibit different susceptibility profiles. In addition, although methodological measures were implemented to minimize physicochemical artifacts, the relatively high MIC values observed indicate that further purification of the extract is necessary to improve antimicrobial potency and facilitate therapeutic application.

Future research perspectives

Future investigations should focus on bioassay-guided fractionation to isolate and characterize the individual phytochemicals responsible for the extract's antimicrobial activity. Purification of these compounds may substantially reduce the effective inhibitory concentrations while improving selectivity toward pathogenic microorganisms. Synergistic interactions between purified compounds and conventional antimicrobial agents, including ceftriaxone against N. gonorrhoeae and fluconazole against C. albicans, should be evaluated using checkerboard assays, fractional inhibitory concentration indices, and time-kill curve analyses to determine their potential as adjunctive therapies against antimicrobial-resistant pathogens [75].

Comprehensive preclinical investigations are also warranted, including cytotoxicity studies, pharmacokinetic analyses, pharmacodynamic evaluations, and efficacy testing in appropriate in vivo infection models. Comparative LC–HRMS metabolomic fingerprinting of Miana accessions collected from different geographical regions would improve phytochemical standardization and facilitate identification of chemical biomarkers for quality control of phytopharmaceutical products [25]. Development of advanced drug delivery systems, including polymeric nanoparticles, liposomes, and other nanoformulations, may improve mucosal penetration, prolong retention at the site of infection, and enhance antimicrobial efficacy while reducing the required therapeutic dose [77, 78]. Ultimately, well-designed clinical trials will be necessary to establish the safety, efficacy, and clinical applicability of standardized Miana-based formulations for the management of gonorrhea, vulvovaginal candidiasis, and mixed genital infections.

The identification of reproducible metabolomic fingerprints by LC–HRMS also provides an important platform for standardizing herbal preparations and establishing quality control parameters to ensure batch-to-batch consistency. Integration of metabolomic, transcriptomic, and proteomic approaches would further clarify the molecular mechanisms of action of the identified phytochemicals and facilitate the rational development of evidence-based phytopharmaceutical products.