A total of 21 bufadienolides (Fig. 1) was isolated from toad bile and identified by a comprehensive analysis of spectroscopic data, including high-resolution MS (HR-ESI–MS), NMR, UV, and single-crystal X-ray diffraction data. Among them, compounds 1–4 were previously undescribed, and compounds 8, 9 and 11–14 were isolated for the first time from the genus Bufo. According to the configuration of the hydroxyl group at the C-3 position, we categorized these compounds into 3α-OH and 3β-OH bufadienolides. 15 of them are 3α-OH bufadienolides (1–15) and six of them are 3β-OH bufadienolides (16–21). It was noteworthy that a considerable quantity of 3α-OH bufadienolides was identified in toad bile, which differed from the reports of 3β-OH bufadienolides found in toad venom and skin [1, 4, 6]. The unique presence of these 3α-OH bufadienolides in toad bile added a new dimension to our understanding of the chemical diversity in different toad organs and opened new insights for pharmacological investigations, biosynthesis and ecological roles.

3-epi-Bufoliene (1), a colorless crystal, had a molecular formula of C24H32O4 (nine degrees of unsaturation), determined by positive HR-ESI–MS at m/z 385.2382 [M + H]+ (calcd. for C24H33O4, 385.2373). The 1H and 13C NMR spectroscopic data (Tables 1 and 2) suggest that 1 had a bufadienolide steroidal structure with a 2H-pyran-2-one or α-pyrone ring system, supported by the UV absorption at λmax 305 nm. The presence of this characteristic α-pyrone ring was evidenced from the 1H NMR data at δH 6.29 (1H, dd, J = 9.2, 0.9 Hz, H-23), 7.69 (1H, dd, J = 2.5, 0.9 Hz, H-21), and 7.85 (1H, dd, J = 9.2, 2.5 Hz, H-22) and from the 13C NMR spectrum at δC 119.3 (C-20), 152.3 (C-21), 150.7 (C-22), 114.5 (C-23) and 164.7 (C-24). In addition, the NMR data assignment of the tetracyclic steroidal nucleus was carried out by comparing it with the reported bufadienolides [14, 15] and by analyzing the 1H-1H COSY, HSQC, and HMBC data (Fig. 2). Singlet methyl signals at δH 1.03 (δC 23.0) and δH 0.98 (δC 23.6) are typical of steroidal methyl groups and were designated as C-18 and C-19, respectively. These designations are based on their HMBC correlations, with H3-18 (δH 1.03) correlating to C-12 (δC 41.7), C-13 (δC 49.5), C-14 (δC 161.4), and C-17 (δC 58.1), and H3-19 (δH 0.98) correlating to C-1 (δC 36.3), C-5 (δC 43.3), C-9 (δC 42.5), and C-10 (δC 36.0).

Key 1H–1H–COSY (blue bold), HMBC (red arrow) correlations of 1 and 2

The presence of a hydroxyl group in ring A was indicated by the 1H and 13C NMR signals ([δH-3 3.55 (m), δC-3 72.3]. The location of this hydroxyl group at C-3 was determined by the 1H-1H COSY correlations of δH 3.55 (H-3) ↔ 1.28 and 1.64 (H2-2) and δH 3.55 (H-3) ↔ 1.51 and 1.71 (H2-4) and confirmed by the HMBC correlations from H2-1 to C-3. The α-orientation of the hydroxyl group was suggested by the width of the entire signal (24 Hz) because the β-oriented proton H-3, adopting the axial position, was split by two axial protons (H-2α and H-4α) and two equatorial proton (H-2β), and thus the width of J values should be much larger than the α-oriented proton H-3 [18]. Furthermore, the chemical shift of β-oriented proton H-3 (< 4.0 ppm) was normally smaller than the α-oriented proton H-3 (> 4.0 ppm) [19] because the former took the axial position in contrast to the equatorial position of the latter. In addition, α-orientation of the hydroxyl group was further confirmed by the NOE correlations H-3β ↔ H-1β and H-3β ↔ H-5 (Fig. 3). Similarly, the presence of another hydroxyl group in ring D was also indicated by the 1H and 13C NMR signals ([δH-16 4.60, dd (J = 5.9, 2.4 Hz), δC-16 77.4]. The location of this hydroxyl group at C-16 was determined by the 1H-1H COSY correlations of δH 5.50 (H-15) ↔ 4.60 (H-16) ↔ 2.57 (H-17) and confirmed by the HMBC correlations from H-16 to C-13 and C-14. The β-orientation of this hydroxyl group was suggested by the NOE correlation H-16α ↔ H-17 which was α-orientated. By analyzing the 1H-1H COSY correlations of δH 5.50 (H-15) ↔ 4.60 (H-16), as well as the long-range correlations from H-16 to and C-14, and from H-15 to C-14 and C-16 in HMBC spectrum, it was determined that ring D contained the trisubstituted vinyl group between C-14 and C-15. The other 2D-NMR data were similar to the reported bufadienolides [19]. Finally, the structure of 1 was established and defined as (3α,5β,16β)-3,16-dihydroxybufa-14,20,22-trienolide, and 3-epi-bufoliene was suggested as a trivial name.

Key NOE correlations of 1 and 2 (A/B trans, B/C cis and C/D cis)

3-epi-6β-Hydroxycinobufagin (2) was isolated as a white powder, and its molecular formula, C26H34O7, was determined from its HR-ESI–MS data (m/z 459.2408 [M + H]+, calcd. for C26H35O7, 459.2377). Comparing the NMR data (Tables 1 and 2) with the literature values for cinobufagin [20] indicated that compound 2 had the same C24 bufadienolide framework with an acetoxy group at C-16, and an epoxy group at C-14 and C-15. The primary difference in 2 was the presence of an additional hydroxyl at C-6 and the epimerization of 3-OH. The hydroxyl group at C-6 in 2 was deduced from the 1H-1H COSY correlations of δH 1.58 (H-5) ↔ 3.73 (H-6) ↔ 1.33 (H-7α), and the HMBC correlations from δH 3.73 (H-6) to δC 29.6 (C-8) and 35.9 (C-10). Additionally, the steric configuration of 3α-OH was essentially identical to that of compound 1, based on the small chemical shift of proton signal of H-3 at δH 3.50 with a large width of J values (21 Hz). It was further supported by NOE correlation of H-3/H-1β (δH 1.10) and H-3/H-5. The α-orientation of H-6 was confirmed by the NOE correlations of H-6/H-7α (δH 1.64) and H-6/H-9 (δH 1.82). Therefore, the structure of 2 was established as 3-epi-6β-hydroxycinobufagin.

3-epi-Cinobufotalin (3), a white powder, was assigned the molecular formula C26H34O7 as determined by the positive HR-ESI–MS ion at m/z HR-ESI–MS m/z 459.2407 [M + H]+ (calcd. for C26H35O7, 459.2377) with ten degrees of unsaturation. The UV data, with a λmax of 295 nm, was consistent with bufadienolide skeleton. The NMR data of 3 were similar to those of 2 except that the hydroxyl group was at C-5 rather than C-6. This was further confirmed by HMBC cross peak from H3-19 (δH 0.92, s, 3H) to C-5 (δC 75.6). Furthermore, this data showed a high similarity to cinobufotalin (21) [21], with the main difference being that the proton signal at δH 3.94 (large width of multiplet) in 3 shifted downfield to δH 4.13 (broad singlet) in cinobufotalin (21), suggesting an inversion of the C-3 stereocenter, which was consistent with the H-3 proton signatures of other isolated 3α-OH bufadienolides. The 1H and 13C NMR signals were assigned as shown in Tables 1 and 2, respectively. Consequently, compound 3 was identified as 3-epi-cinobufotalin.

3-epi-19-Hydroxyresibufogenin (4), a white powder, was assigned the molecular formula C24H32O5 as determined by the positive HR-ESI–MS ion peak at m/z 423.2159 [M + Na]+ (calcd. for C24H32O5Na, 423.2142) with nine degrees of unsaturation. The 1H NMR and 13C NMR data (Tables 1 and 2) of compound 4 displayed resemblances to those of the known compound 3-epi-resibufogenin (12) [22], except for the replacement of the substitution of the methyl group in C-10 with a hydroxymethyl group [δH-19 3.82 and 3.42 (d, J = 11.2 Hz); δC-19 64.9] in 4. The NOE correlations and relative configurations in 4 and 12 exhibited a high degree of similarity. The β-orientation of the hydroxymethyl in 4 was ascertained through the NOE correlations of H2-19/H-5 and H2-19/H-8. The H-3 signal at δH 3.52 featured with a large width of multiplet, along with the NOE correlation of H-3/H-1β and H-3/H-5, validating the 3α-OH configuration. Consequently, the arrangement of 4 was determined to be 3-epi-19-hydroxyresibufogenin.

Additionally, 17 known compounds were identified (Fig. 1) as 3-epi-arenobufagin (5) [23], 3-epi-ψ-bufarenogin (6) [23], 3-epi-bufalin (7) [22], 3-epi-7β-hydroxybufalin (8) [24], 3-epi-bufotalin (9) [25], 3-epi-gambufotalin (10) [25], 3-epi-desacetylbufotalin (11) [25], 3-epi-resibufogenin (12) [22], 3-epi-desacetylcinobufagin (13) [22], 3-epi-12β-hydroxyresibufogenin (14) [26], 3-epi-argentinogenin (15) [5], argentinogenin (16) [5], bufotalin [20] (17), telocinobufagin (18) [27], hellebrigenin (19) [28], desacetylcinobufotalin (20) [20] and cinobufotalin (21) [29] by comparison of the NMR and MS data with the reported values in literatures. Among them, compounds 8, 9, 11–14 were isolated from Bufo genus for the first time (Fig. 1). In addition, the structures of compounds 5, 7, and 8 were confirmed by X-ray analysis for the first time (Fig. 4).

X-ray crystallographic structures of 5, 7 and 8 with atom labeling scheme. The C and O atoms are drawn as 30% thermal ellipsoids

This study revealed the coexistence of 3α-OH and 3β-OH bufadienolides in the bile of toad, with the 3α-OH configuration being predominant. To explore the potential conversion of 3α-OH and 3β-OH bufadienolides, a tissue incubation approach was employed to investigate the metabolic processes in toad liver and kidney tissues. The outcomes provide valuable insights, elucidating the transformation from 3β-OH to 3α-OH bufadienolides.

In Fig. 5, the HPLC chromatograms showed three compounds (bufalin, 3-epi-bufalin, 3-oxo-bufalin) after a 24-h incubation with toad liver tissue culture. The incubation demonstrated a significant conversion of bufalin and 3-oxo-bufalin into 3-epi-bufalin. However, 3-epi-bufalin did not convert back into bufalin or 3-oxo-bufalin. Further examination of bufalin within liver tissue at different incubation time (Fig. 6) showed that the conversion of bufalin to 3-epi-bufalin increased with extended incubation time, whereas 3-oxo-bufalin initially increased and then declined. After 12-h of incubation, bufalin, 3-oxo-bufalin, and 3-epi-bufalin were simultaneously present. This finding strongly suggested that bufalin could undergo conversion to 3-epi-bufalin through an intermediate 3-oxo-bufalin, and this conversion process appears to be irreversible. It was worth noting that similar results were observed in the kidney tissue of toads (see Additional file 1).

The HPLC chromatogram of bufalin (B3), 3-epi-bufalin (αB3), and 3-oxo-bufalin (B3one) after incubated with toad liver suspension for 24 h. (i) and (ii), standards of B3, αB3 and B3one; (iii) incubation of αB3 with toad liver; (iv) incubation of B3one with toad liver; (v) incubation of B3 with toad liver; (vi) the control group of toad liver. Detailed sample preparation method and HPLC method were shown in Sects. 4.6 and 4.7, respectively

The HPLC chromatogram of bufalin (B3) after incubated with toad liver suspension for different time. (i) and (ii), standards of B3, αB3 (3-epi-bufalin), and B3one (3-oxo-bufalin); (c) ~ (g) B3 incubation with toad liver for 0, 6, 12, 24, 36 h, respectively. Detailed sample preparation method and HPLC method were shown in Sects. 4.6 and 4.7, respectively

It was reasonable to postulate the presence of a group of 3(β → α)-OH epimerase in toad liver and kidney tissues [30, 31]. This epimerase comprised two key enzymatic activities (Fig. 7): a differential stereo-selective 3β-dehydrogenase, which converted bufalin into 3-oxo-bufalin, and a 3-keto-reductase that facilitated the transformation of 3-oxo-bufalin into 3-epi-bufalin. This process followed an irreversible conversion pathway: 3β-OH → 3-oxo → 3α-OH. We proposed that this enzyme was widely distributed in toad tissues and likely played a pivotal role in converting highly toxic 3β-OH bufadienolides into the less toxic 3α-OH counterparts. This assumption was supported by our previous studies [15], which demonstrated that 3β-OH bufadienolides had significantly higher inhibitory activity against Na+,K+-ATPase-α1 than 3α-OH bufadienolides. Furthermore, it was worth noting that predators of toads, such as rats [32] and snakes [33], could also convert 3β-OH bufadienolides into 3α-OH bufadienolides, thus neutralizing their toxicity. Hence, we proposed that this epimerase might play a role in the toad's self-defense mechanisms and was linked to crucial physiological functions. This irreversible enzymatic conversion process provided a new perspective on the ecological importance of bufadienolides in toad defense mechanisms, emphasizing the need for further research.

A schematic depicting the conversion pathway of bufalin into 3-epi-bufalin facilitated by the 3(β → α)-OH epimerase. This reaction involves two key enzymatic processes: a differential stereo-selective 3β-dehydrogenase that transforms bufalin into 3-oxo-bufalin and a 3-keto-reductase that subsequently converts 3-oxo-bufalin into 3-epi-bufalin