The inhibitory effects of D. bulbifera crude ethyl acetate extract and antibacterial fraction on all tested bacteria were demonstrated by the MIC, as presented in Table 1. The MIC values ranged from 0.78 to 1.56 mg/mL and 0.02 to 0.78 mg/mL for the ethyl acetate extract and antibacterial fraction, respectively. The strongest antibacterial effect of the D. bulbifera fraction was observed against S. epidermidis 3547. The D. bulbifera ethyl acetate extract demonstrated considerable antibacterial activity, as indicated by its MIC values. The antibacterial fraction exhibited significantly stronger activity against the tested bacterial strains, with lower MICs across all species. This suggests that a potential concentration of active constituents within this fraction amplifies its antimicrobial effect.

The antimicrobial activity of D. bulbifera against various bacteria has been reported previously. The methanol extract of D. bulbifera bulbils exhibited antimicrobial activities against mycobacteria and multidrug-resistant gram-negative bacteria, such as E. coli, Enterobacter aerogenes, Klebsiella pneumoniae, P. aeruginosa, Mycobacterium smegmatis, and Mycobacterium tuberculosis. Plants of the genus Dioscorea, such as Dioscorea deltoidea, have also demonstrated inhibitory effects against S. aureus, P. aeruginosa, and E. coli [16]. D. bulbifera exhibits strong and broad-spectrum antibacterial effects against various microorganisms, making it a potential effective therapeutic against bacterial infections, including multidrug-resistant strains. Compounds isolated from D. bulbifera, such as bafoudiosbulbin A and B, 8-epidiosbulbin E acetate, vanillic acid, isovanillic acid, and dihydrodioscorine, have demonstrated potent antibacterial activity. Diosgenin, a compound found in D. bulbifera, effectively inhibits the growth of both Gram-positive bacteria (including Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Staphylococcus epidermidis) and Gram-negative bacteria such as E. coli and Salmonella typhi [7]. In addition, the aqueous extracts of D. bulbifera displayed significant antibacterial activity against E. coli, whereas its ethanolic extracts were notably potent against C. albicans and S. aureus [16].

The chemical components and antibacterial activity of the ethyl acetate extract of D. bulbifera were investigated using TLC-bioautography with six different solvent systems: hexane, dichloromethane, ethyl acetate, ethanol, methanol, and water. The results showed that the ethyl acetate extract exhibited distinct inhibition zones against bacteria compared to the methanol and ethanol extracts which only showed inhibition zones at the spot of the extract drop (not shown). Therefore, the ethyl acetate extract of D. bulbifera was used to assess the antibacterial activity and purified by column chromatography to isolate the antibacterial compounds. Figure 2 shows that a compound in the ethyl acetate extract (indicated by the arrow) exhibited antibacterial activity against S. aureus, MRSA, S. epidermidis, and S. pyogenes (lanes C–F). However, no activity was observed against the gram-negative bacterium P. aeruginosa 4739 (lane G). These findings suggest that the ethyl acetate extract of D. bulbifera contains potential antibacterial compounds that should be further investigated for therapeutic applications. However, the difference in antibacterial activity between TLC bioautography and Table 1 results, especially against P. aeruginosa, is likely due to concentration changes post-TLC separation. Such separation might not fully represent the synergistic antibacterial effects seen with the crude mixture. TLC bioautography, which highlights individual compounds, might not fully demonstrate the collective antibacterial strength of the extract.

Chromatographic analysis and bioautography of D. bulbifera ethyl acetate extract extract to evaluate its antibacterial activity against S. aureus, MRSA, S. epidermidis, S. pyogenes, and P. aeruginosa. TLC ethyl acetate extract: visible light (A), under UV 254 nm (B), bioautography; S. aureus 8840 (C), MRSA 20651 (D), S. epidermidis 3547 (E), S. epidermidis 4343 (F), and P. aeruginosa 4739 (G)

The resistance of P. aeruginosa against the ethyl acetate extract of D. bulbifera in the present study may be due to its innate defence mechanisms, such as the presence of an outer membrane and efflux pumps, which can decrease its susceptibility to antibacterial agents. In contrast, a previous study using D. bulbifera methanol extract, fractions, and compounds demonstrated promising results against multiple drug-resistant bacteria, including P. aeruginosa [10]. This discrepancy in outcomes could be attributed to the different compounds extracted using methanol. These compounds may possess broad-spectrum antibacterial activity or be capable of overcoming resistance mechanisms in P. aeruginosa. The differences in solvent polarity between methanol and ethyl acetate likely account for the observed variation in antibacterial efficacy. The higher polarity of methanol enables the extraction of a more diverse set of bioactive compounds, potentially conferring effectiveness against P. aeruginosa. Previous studies have identified certain diterpenoids (bafoudiosbulbins and 2,7-dihydroxy-4-methoxyphenanthrene) as pivotal for enhanced antibacterial activity, and their unique structural attributes may bolster their antibacterial potency [10].

The ethyl acetate extract of D. bulbifera was purified in a three-step process using ethyl acetate. The first two steps involved column chromatography, followed by final purification by PTLC. The fractions collected in the first step were tested for antibacterial activity, and fractions 13 and 14 exhibited such activity. These two fractions were subjected to a second round of column chromatography using the same mobile phase. Antibacterial activity was detected in subfractions 11–17 collected during this step, which were further purified by PTLC. TLC-bioautography of ethyl acetate extract, antibacterial fraction, and active antibacterial components from PTLC was performed against S. aureus DMST 8840 (representative of all tested Staphylococci) (Fig. 3).

Thin layer chromatography (TLC) (A) and bioautography using S. aureus DMST 8840 (B); ethyl acetate extract (1), antibacterial fraction (2), active antibacterial component from PTLC (3)

The antibacterial compound isolated from the D. bulbifera extract was subjected to 1H-NMR spectroscopy to elucidate its chemical structure. The resulting 1H-NMR spectrum, indicative of the pure compound's structure, is depicted in Fig. 4. This spectral analysis confirms the identity of the compound as flavanthrinin. A comparison between the 1H-NMR data of the bioactive compound isolated in the present study and flavanthrinin previously reported in the orchids Bulbophyllum reptans and B. vaginatum [17, 18] revealed similar antibacterial activities (Table 2). This similarity suggests that the bioactive antibacterial compound obtained from D. bulbifera is flavanthrinin. This finding was consistent with the results of TLC-bioautography, which identified flavanthrinin as an active compound against S. aureus, MRSA, and S. epidermidis.

Structure of flavanthrinin elucidated from 1H-NMR spectrum

The constituents of D. bulbifera include diterpenoids, glycosides, steroids, steroidal saponins, and sapogenins [19, 20]. To date, only one study has investigated the antibacterial activity of the active components of D. bulbifera. This study examined the antibacterial activity of the methanolic extract of D. bulbifera and found that the clerodane diterpenoids bafoudiosbulbin A and B demonstrated significant activity against Salmonella species [11]. Phytochemical analysis of D. bulbifera revealed a diverse array of chemical constituents, including saponins, tannins, flavonoids, sterols, polyphenols, glycosides, and steroidal saponins. The specific compounds identified can vary based on the geographical origin of the plant, plant part studied, and solvents used for extraction. Notable compounds include flavonol aglycones, such as kaempferol-3, 5-dimethyl ether, caryatin, and catechin, and steroidal sapogenins, such as diosbulbisins and diosbulbisides in plants from China. Bulbs from India contained 8-epidiosbulbin E acetate, whereas those from Cameroon contained clerodane diterpenoids. Additionally, the flowers of D. bulbifera from Cameroon contained a broad spectrum of the steroidal saponins dioscoreanosides [21]. Our findings on Flavanthrinin's antibacterial activity align with the broader scientific consensus that flavonoids possess potent antimicrobial properties, capable of inhibiting the growth of various bacterial strains, including those resistant to conventional antibiotics. The structure–activity relationship analysis demonstrated the critical role of specific structural modifications, such as hydroxylation patterns and the presence of functional groups, in enhancing the antibacterial efficacy of flavonoids. This correlation between structural features and antimicrobial potency suggested that Flavanthrinin's effectiveness may be attributed to its unique molecular configuration, which warrants further investigation [22].

The cytotoxicity of the ethyl acetate extract and the flavanthrinin-containing fraction against Vero cells yielded CC50 values of 0.24 ± 0.10 mg/mL and 0.41 ± 0.03 mg/mL, respectively (Fig. 5). Notably, the ethyl acetate extract demonstrated cytotoxicity at concentrations lower than its MIC values (0.78–1.56 mg/mL), indicating potential cellular toxicity at its inhibitory concentrations. Conversely, the flavanthrinin-containing fraction, with a CC50 higher than its MIC range (0.04–0.78 mg/mL), suggested a safer profile at effective antibacterial concentrations. This difference underscored the importance of careful consideration of the ethyl acetate extract's cytotoxicity while recognizing the flavanthrinin-containing fraction as a promising candidate for safe antibacterial application.

The 50% cytotoxic concentration (CC50) of ethyl acetate extract and flavanthrinin-containing fraction