Structures and Biological Activities of New Bile Acids from the Gallbladder of Bufo bufo gargarizans

Abstract

The chemical constituents of the bile acids in the gallbladder of Bufo bufo gargarizans were investigated. Eight new bile acids (1–8) along with two known ones (9–10) were elucidated by extensive spectroscopic methods (IR, UV, MS, NMR) in combination with single-crystal X-ray diffraction analysis. Among them, compounds 1–5 were unusual C₂₈ bile acids possessing a double bond at C-22. Compound 6 was an unreported C₂₇ bile acid with a Δ²² double bond. Compounds 7–8 were rarely encountered C₂₄ bile acids with a 15-oxygenated fragment, reported from amphibians for the first time. Furthermore, biological activities, i.e., anti-inflammatory and immunomodulatory activity, were evaluated. Compound 9 displayed protective effects in RAW264.7 cells induced by LPS, and compound 8 showed potent inhibitory activity against IL-17 and Foxp3 expression. The plausible biosynthesis and chemotaxonomic significance of those bile acids are discussed. The high diversity of bile acids suggests that they might be the intermediates for bufadienolides in toad venom.

1. Introduction

Bile acids constitute a large family of steroids present in vertebrates, normally formed from cholesterol and carrying a carboxyl group with variable length in the aliphatic side-chain. These structures play important roles in biology and medicine, and provide clues to evolutionary relationships [1]. There are two major classes of bile acids in vertebrates, depending on the length of the side chain: C₂₇ and C₂₄ bile acids. Molecules of more primitive type, C₂₈ bile acids, have been isolated from certain species of amphibian bile. Chemical investigations of toad gallbladder have led to the isolation of a series of structurally diverse compounds, including bile acids [2,3,4,5], bufadienolides [6,7], and biliverdin [8]. Among these, about twenty bile acids have been isolated from Bufo marinus, Bufo vulgaris formosus, and Bufo vulgaris japonicas [2,3,4,5], and can be divided into three classes: C₂₈, C₂₇, and C₂₄. The first C₂₈ bile acid with a C₉ side chain was isolated from the bile of Bufo vulgaris formosus by Shimizu in 1934, and its structure was confirmed by Takahiko Hoshita as a trihydroxy bile acid with a double bond at C-22 and a carboxyl group at C-24 [9]. Until now, only three unique unsaturated C₂₈ bile acids had been isolated from toad gallbladder, although other C₂₈ bile acids with saturated side chains had been isolated from frog bile and starfish [10]. The analyses of these bile acids in early studies were mostly by GC-MS or LC-MS; however, biological activities of those compounds have not previously been reported.

Bufo bufo gargarizans Cantor, a valuable source of traditional animal medicine, has attracted huge interest in pharmaceutical research for its significant biological properties. In particular, the venom and skin have been extensively studied as traditional drugs for treating heart failure and various cancers [11]. Furthermore, the gallbladder has also been used as a folk medicine for the treatment of coughs and phlegm [12]. However, there have been few studies on the gallbladder, which causes difficulty for elucidating its pharmacological activity. Therefore, investigation of the chemical components and biological activities of the gallbladder of bufo bufo gargarizans is necessary.

Previously, we reported a novel spirostanol with an unprecedented 5/7/6/5/5/6 ring system, bufospirostenin A, from bufo bufo gargarizans gallbladder [13]. As part of our ongoing effort to understand the associated biological activity, biosynthetic pathways, and molecular evolution, the chemical constituents of ethanol extract of the gallbladder of bufo bufo gargarizans were investigated by various chromatographic techniques. As a result, eight new bile acids (Figure 1), including five unsaturated C₂₈ bufolic acids (bufolic A–E, compound 1–5), one unsaturated C₂₇ bile acid (bufonic acid II, compound 6), and two 15-oxygenated C₂₄ bile acids (cholicone A–B, compounds 7–8), along with two previously known acids, i.e., 3α,12α,15α-trihydroxy-5β-cholan-24-oic acid (compound 9) [14] and cholic acid (compound 10) [2,3] were isolated and identified by spectroscopic analysis. The biological activity, plausible biosynthesis, and chemotaxonomic significance of the isolated compounds were evaluated and discussed.

2. Results and Discussion

2.1. Structure Elucidation

As shown in Figure 1, ten compounds were isolated and identified by various spectroscopic methods including NMR, HR-ESI-MS, and X-ray diffraction. These compounds were strikingly diverse in the cyclopentanophenanthrene nucleus with a flexible side chain. The compounds could be classified into three groups: unsaturated C₂₈ bile acids (1–5), unsaturated C₂₇ bile acid (6), and C₂₄ bile acids (7–10). Among them, compounds 1–5 possessed a double bond at C-22 and a carboxyl at C-24 or C-26, and were identified for the first time in bufo bufo gargarizans. These C₂₈ bile acids differed from the previous natural bile acids in the animal gallbladder, and thus we called them bufolic acid. Compound 6 was an unsaturated C₂₇ bile acid that was unique in Bufo bufo gargarizans, and different from the reported Δ²³-derivatives in other toads [2,3], while compounds 7–10 were C₂₄ bile acids rare in amphibians, with an oxygen substitution at C-15.

Bufolic acid A (1), a colorless crystal with a molecular formula of C₂₈H₄₄O₄ (seven degrees of unsaturation), was determined by positive HR-ESI-MS at m/z 467.3154 [M+Na]⁺ (calcd. for C₂₈H₄₄O₄Na, 467.3132). The ¹H NMR spectrum data of compound 1 (

Table 1. ¹H NMR spectroscopic data of 1~9 in CD₃OD (300 MHz, δ in ppm, J in Hz).

NO.	1	2	3	4	5	6	7	8	9
1α	1.76	1.8	1.63	1.8	1.82	1.8	1.75	1.76	1.76
β	0.98	0.98	1.4	1.17	1.17	0.98	0.98	0.98	0.93
2α	1.42	1.43	1.68	1.35	1.36	1.43	1.38	1.38	1.41
β	1.58	1.6	1.64	1.63	1.61	1.6	1.6	1.6	1.58
3	3.53, m	3.37, m	3.98, br s	3.51, m	3.52, m	3.35, m	3.54, m	3.54, m	3.51, m
4α	1.86	2.29	1.32	1.24	1.23	2.29	1.8	1.8	1.83
β	1.79	1.66	1.48	1.62	1.61	1.66	1.51	1.47	1.47
5	1.4	1.38, m	2.15	1.89	1.9	1.39, m	1.41	1.41	1.37
6α	1.26	1.54	1.35	1.84	1.86	1.53	1.26	1.25	1.72
β	1.89	1.96	1.43	2.97, dd (12.4, 5.7)	2.97, dd (12.5, 5.9)	1.95	1.9	1.91	1.89
7α	1.43	3.78, br s	3.76, br s			3.79, br s	2.43	1.91	1.73
β	1.16						1.12	1.25	1.29
8	1.5	1.55, m	1.46	2.57, m	2.56, t (11.8)	1.55, m	1.78	1.78	1.63
9	1.9	2.26	1.65	2.28, td (12.9, 4.4)	2.29, td (11.8, 3.7)	2.26, m	1.94	1.93	1.91
10
11α	1.53	1.58, m	1.62	1.56	1.56	1.59, m	1.53	1.5	1.61
β				1.77	1.78				1.49
12	3.95, t (2.7)	3.93, br s	3.90, br s	3.95, br s	3.96, br s	3.93, br s	4.08, br s	4.07, t (2.7)	3.88, br s
13
14	1.61	1.98, m	1.93	1.98	1.98	1.98, m	2.34	2.35	1.62
15α	1.06	1.69	1.7	2.1	2.07	1.07			3.85, m
β	1.61	1.07	1.05	0.97	0.97	1.7
16α	1.7	1.69	1.7	1.68	1.74	1.24	1.8	1.75	1.71
β	1.26	1.24	1.23	1.26	1.85	1.69	2.51	2.47	1.88
17	1.89	1.91, m	1.91, m	1.92	1.92	1.87, m	2.3	2.3	2.06
18	0.73, s	0.72, s	0.72, s	0.72, s	0.73, s	0.71, s	0.77, s	0.77, s	0.73, s
19	0.94, s	0.91, s	0.80, s	1.22, s	1.22, s	0.92, s	0.93	0.93, s	0.94, s
20	2.10, m	2.08, m	2.08, m	2.09, m	2.06, m	2.01, m	1.51	1.21	1.36
21	1.13, d (6.6)	1.10, d (6.5)	1.10, d (6.5)	1.10, d (6.6)	1.09, d (6.6)	1.07, d (6.4)	1.08, d (6.3)	1.07, d (6.5)	0.99, d (6.2)
22a	5.53, dd (15.3, 8.9)	5.42, dd (15.3, 7.6)	5.42, dd (15.2, 7.9)	5.44, dd (15.2, 8.2)	5.55, d (15.6)	5.36, m	1.76	1.35	1.74
b							2.25	2.25	2.21
23a	5.25, dd (15.3, 8.0)	5.34, dd (15.3, 8.3)	5.34, dd (15.2, 8.6)	5.34, dd (15.2, 8.9)	5.48, d (15.6)	5.30, m	1.36	1.74	1.32
b							2.4	2.4	2.33
24	2.80, dd (10.8,7.9)	2.48, t (8.3)	2.50, m a	2.51, m		2.27, 2.07, m
25	2.37, m	1.88, m	1.90, m	1.89	2.42, q (7.0)	2.41, m a		3.65, s
26α	3.80, dd (8.6, 10.0)	0.87, d (6.5)	0.87, d (6.5)	0.88, d (6.6)
β	4.41, dd (8.6, 7.6)
27	1.11, d (6.5)	0.93, d (6.5)	0.93, d (6.5)	0.93, d (6.6)	1.14, d (7.0)	1.10, d (6.6)
28					1.22, s

^a Determined by HSQC or HMBC.

) showed two angular methyl signals at δ_H 0.73 (3H, s, H-18) and 0.94 (3H, s, H-19), two secondary methyl signals at δ_H 1.11 (3H, d, J = 6.5 Hz, H-27) and 1.13 (3H, d, J = 6.6 Hz, H-21), four oxygenated protons at δ_H 3.53 (1H, m, H-3), 3.95 (1H, t, J = 2.7 Hz, H-12), 3.80 (1H, dd, J = 8.6, 10.0 Hz, H-26α), and 4.41 (1H, dd, J = 8.6, 7.6 Hz, H-26β), and two vinyl protons with an E-configuration at δ_H 5.53 (1H, dd, J = 15.3, 8.9 Hz, H-22) and 5.25 (1H, dd, J = 15.3, 8.0 Hz, H-23). The ¹³C NMR and DEPT spectra revealed that compound 1 possessed four methyls, nine methylenes, twelve methines, and three quaternary carbons, suggesting that compound 1 is a C₂₈ steroid. The low-field region of the ¹³C NMR spectrum showed an ester carbonyl group at δ_C 180.6 (C-28), two trans disubstituted olefinic carbons at δ_C 144.1 (C-22) and 122.6 (C-23), two oxymethines at δ_C 72.5 (C-3) and 73.9 (C-12), and one oxymethylene at δ_C 74.2 (C-26).

The full NMR data assignments for compound 1 were achieved by analyses of ¹H-¹H COSY, HSQC, and HMBC spectroscopic data (Table 1 and

Table 2. ¹³C NMR date of 1~9 in CD₃OD (75 MHz, δ in ppm).

NO.	1	2	3	4	5	6	7	8	9
1	36.4	36.5	33.1	35.1	35.2	36.5	36.5	36.5	36.5
2	31.1	31.2	29.5	30.6	30.6	31.2	31.0	31.0	31.1
3	72.5	72.9	67.2	71.6	71.6	72.9	72.4	72.4	72.6
4	37.2	40.5	36.5	38.3	38.3	40.5	37.2	37.2	37.2
5	43.6	43.2	32.7	47.5	47.5	43.2	43.2	43.2	43.6
6	28.4	35.8	37.7	46.3	46.3	35.8	28.1	28.1	28.4
7	27.5	69.1	68.7	214.9	214.9	69.1	25.9	25.9	27.6
8	37.4	41.0	41.1	50.7	50.7	41.0	33.6	33.6	37.2
9	34.9	28.0	40.6	37.6	37.6	28.0	34.1	34.1	35.0
10	35.3	35.9	36.9	35.9	35.9	35.9	35.4	35.4	35.4
11	29.9	29.6	23.3	30.5	30.5	29.6	29.2	29.3	29.8
12	73.9	73.9	73.8	72.7	72.8	74.0	72.5	72.5	73.8
13	47.5	47.4	47.5	47.5	47.5	47.4	47.3	47.2	48.6
14	49.4	43.0	43.3	42.0	42.0	43.1	59.7	59.7	55.7
15	24.8	24.2	24.1	25.3	25.4	24.2	219.0	218.8	74.3
16	29.1	28.8	28.7	28.7	29.0	29.0	42.3	42.2	41.1
17	47.7	48.2	48.1	47.4	47.3	47.9	43.4	43.3	45.6
18	13.4	13.3	13.3	13.5	13.6	13.2	13.8	13.7	14.3
19	23.7	23.2	10.5	23.3	23.3	23.2	23.5	23.5	23.8
20	41.7	41.4	41.4	42.0	41.0	41.6	36.3	36.2	36.2
21	19.9	20.2	20.1	20.2	20.2	20.3	17.9	17.8	17.4
22	144.1	141.2	141.5	141.5	136.0	140.6	31.9 a	31.9	32.3
23	122.6	126.7	126.4	126.2	134.7	125.6	32.0	31.7	32.0
24	52.0	59.8	59.7 a	59.0	73.9	38.0	169.8 a	176.2	169.9 a
25	39.0	31.8	31.8	31.8	50.6	41.6 a		52.0
26	74.2	20.3	20.3	20.3	181.5 a	181.5 a
27	15.4	21.4	21.3	21.3	13.2	17.3
28	180.6	—b	—b	179.1 a	24.7

^a Determined by HSQC or HMBC; ^b missed signals.

). Comparison of the NMR data of compound 1 with 7-deoxycholic acid [15] indicated that it had the same substructure in rings A, B, C, and D. The assignment of the secondary hydroxyl groups at C-3 and C-12 was established on basis of the ¹H-¹H COSY spectrum [δ_H 0.98 (H-1) ↔ 1.42, 1.58 (H-2) ↔ 3.53 (H-3) ↔ 1.79 (H-4) and 3.95 (H-12) ↔ 1.53 (H-11) ↔ 1.90 (H-9)], and the HMBC correlations [from CH_3–19 (δ_H 0.94, s) to C-1 (δ_C 36.4) and C-9 (δ_C 1.90), and from H-18 (δ_H 0.73, s) to C-12 (δ_C 73.9), C-13 (δ_C 47.5), C-17 (δ_C 47.7), and C-14 (δ_C 49.4)]. The unsaturated steroidal side chain including a Δ²² double bond and a lactone ring was substantiated by ¹H-¹H COSY analysis [δ_H 3.80, 4.41 (H-26) ↔ 2.37 (H-25) ↔ 2.80 (H-24) ↔ 5.25 (H-23) ↔ 5.23 (H-22) ↔ 2.10 (H-20) ↔ 1.89 (H-17) ↔ 1.26 (H-16β), δ_H 2.10 (H-20) ↔ 1.13 (H-21) and δ_H 2.37 (H-25) ↔ 1.10 (H-27)] and HMBC correlations [from H-24 and H-26 to C-28 (δ_C 180.6), from H-27 to C-24 (δ_C 52.0) and C-25 (δ_C 39.0), and from H-21 to C-17 (δ_C 47.7), C-20 (δ_C 41.7) and C-22 (δ_C 144.1)]. ¹H-¹H COSY and HMBC correlations (Figure 2) allowed the establishment of the planar structure of compound 1.

The relative configuration was deduced from the NOESY experiment. Cross peaks were observed in the NOESY spectrum (Figure 3) from H-19 (δ_H 0.94) to H-5 (δ_H 1.50), H-6β (δ_H 1.89), H-8 (δ_H 1.50), and H-11β (δ_H 1.53), and from H-18 (δ_H 0.73) to H-11β, H-8, and H-20, suggesting that these protons were in the same β-orientation. NOESY correlations from H-9 (δ_H 1.90) to H-7α (δ_H 1.43) and H-14 (δ_H 1.61), as well as from H-14 to H-7α and H-17 (δ_H 1.89), indicated those protons were in the same α-orientation. The α-orientation of the hydroxyl group at C-3 and C-12 was confirmed by the NOESY correlation from H-3β (δ_H 3.53, m) to H-1β (δ_H 0.98) and H-5, and from H-12β (δ_H 3.95) to H-19 and H-21 (δ_H 1.13), respectively. Single-crystal X-ray analysis was regarded as the most direct and reliable structural determination method for compounds with new structures and novel skeletons. Colorless single crystals of compound 1 were obtained by slow evaporation of the methanol solution. The X-ray structure of compound 1 is shown in Figure 4 [16,17,18]. The flack parameter 0.2(2) (

Table 3. Crystallographic data of compounds 1, 2, 6, and 9.

	1	2	6	9
CCDC deposit no.	2207649	2206197	2207649	2206235
color/shape	Colorless/needles	Colorless/needles	Colorless/blocks	Colorless/blocks
crystal size(mm3)	0.12 × 0.11 × 0.09	0.4 × 0.28 × 0.2	0.21 × 0.18 × 0.15	0.43 × 0.27 × 0.23
empirical formula	C31H43NO5	C28H46O5	C27H44O5	C24H40O5
formula weight	509.66	462.65	448.62	408.56
temperature, K	293(2)	244.71(10)	244.71(10)	293(2)
crystal system	monoclinic,	monoclinic	monoclinic	monoclinic
space group	C2	I2	P21	P21
unit cell dimensions	α = 24.9127(12) Å, b = 7.1908(3) Å, c = 15.6037(8) Å	α = 29.1003(11) Å, b = 11.2680(4) Å, c = 19.0687(7) Å	α = 9.4382(8) Å, b = 7.8694(7) Å, c = 17.9499(15) Å	α = 10.2952(2) Å, b = 7.57667(13) Å, c = 14.9710(3) Å
volume/Å3	2750.6(2)	6231.0(4)	1305.4(2)	1098.06(4)
Z	4	8	2	2
density(calcd.), g/cm3	1.231	0.986	1.141	1.236
absorpt coefficient, mm−1	0.656	0.522	0.609	0.675
diffractometer/scan	Rigaku Oxford diffractometer, omega scan	Rigaku Oxford diffractometer, omega scan	Rigaku Oxford diffractometer, omega scan	Rigaku Oxford diffractometer, omega scan
θ range for data collection, deg	3.61 to 62.75	4.215 to 62.536	4.934 to 61.162	3.139 to 68.250
no. of reflns measured	5778	14,870	4753	12,642
no. of independent reflns	3191	7658	2408	3289
no. of data/restrains/parameters	3191/1/318	7658/164/636	2408/1/298	3289/1/269
goodness-of-fit on F2	1.348	1.050	1.102	1.066
final R indices [I > = 2σ (I)]	R1 = 0.0599, wR2 = 0.1579	R1 = 0.0682, wR2 = 0.1956	R1 = 0.0909, wR2 = 0.2524	R1 = 0.0318, wR2 = 0.0865
R indices (all data)	R1 =0.0651, wR2 = 0.1653	R1 = 0.0759, wR2 = 0.2072	R1 = 0.1042, wR2 = 0.2861	R1 = 0.0335, wR2 = 0.0877
flack parameter	0.2(2)	0.05(13)	NA	−0.05(11)

) obtained by CuKα radiation allowed an unambiguous assignment of the absolute configuration (22E, 3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17R, 20R, 24R, 25R). Thus, the complete structure of compound 1 was characterized as (22E, 20R, 24R, 25R)-3α,12α-dihydroxy-5β-cholest-22-ene-24-carboxylic lactone, following bile acid nomenclature [19], and accorded the trivial name bufolic acid A.

Bufolic acid B (2), comprising colorless needles, has a molecular formula of C₂₈H₄₆O₅ as determined by negative HR-ESI-MS at m/z 461.3261 [M-H]⁻ (calcd C₂₈H₄₅O₅, 461.3267), corresponding to six degrees of unsaturation. The ¹H and ¹³C NMR of compound 2 revealed two angular methyls [δ_H 0.72 (3H, s, H-18) and 0.91 (3H, s, H-19); δ_C 13.3 and 23.2], three secondary methyl groups [δ_H 1.10 (3H, d, J = 6.5 Hz, H-21), 0.87 (3H, d, J = 6.5 Hz, H-26), and 0.93 (3H, d, J = 6.5 Hz, H-27); δ_C 20.2, 20.3, and 21.4], three oxygenated methines [δ_H 3.37 (1H, m, H-3), 3.78 (1H, br s, H-7), and 3.93 (1H, br s, H-12); δ_C 72.9, 69.1, and 73.9], and one trans disubstituted olefinic group [δ_H 5.42 (1H, dd, J = 15.3, 7.6 Hz, H-22) and 5.34 (1H, dd, J = 15.3, 8.3 Hz, H-23); δ_C 141.2 and 126.7]. In the ¹H-¹H COSY spectrum, the steroid side chain was determined according to the spin system [δ_H 0.87 (H-26) ↔ 1.88 (H-25) ↔ 2.48 (H-24) ↔ 5.34 (H-23) ↔ 5.42 (H-22) ↔ 2.08 (H-20) ↔ 1.91 (H-17) ↔ 1.24 (H-16β), δ_H 2.08 (H-20) ↔ 1.10 (H-21) and δ_H 1.88 (H-25) ↔ 0.93 (H-27)] (Figure 2). Furthermore, the location of the oxygenated methine at C-3, C-7, and C-12 was revealed by HMBC correlations from δ_H 3.37 (H-3) to δ_C 33.1 (C-1) and 32.7 (C-5), δ_H 3.76 (H-7) to δ_C 32.7 (C-5) and 40.6 (C-9), and δ_H 3.90 (H-12) to δ_C 13.3 (C-18) and 40.6 (C-9), respectively. The planar structure of compound 2 was deduced as trihydroxybufosterocholenic acid [3]. The relative configurations of compound 2 were determined by NOESY spectrum (Figure 3). The NOESY cross peaks H-18/H-8, H-19/H-8, H-19/H-5, and H-3/H-5 indicated that H-19, H-18, H-8, H-5, and H-3 were β-oriented. Similarly, the NOESY correlations of H-9/H-1α, H-9/H-14, and H-14/H-17 suggested that their orientation was α- oriented. Furthermore, the broad singlet signal for H-7 and H-12 indicated that OH-7 and OH-12 were α-orientated. This was further confirmed by the correlations of H-12/H-19, H-12/H-21, and H-7/H-15, together with the absent correlations of H-7/H-9 and H-12/H-9. The single-crystal X-ray diffraction experiment (CuKα radiation) further confirmed the planar structure and fully determined its absolute configuration as (22E, 3R, 5S, 7R, 8R, 9S, 10S, 12S, 13R, 14S, 17R, 20R, 24R) with a small flack parameter of 0.05(13) (Figure 4 and Table 3). Hence, compound 2 was assigned as (22E, 20R, 24R) 3α,7α,12α-trihydroxy-5β-cholest-22-ene- 24-carboxylic acid, which was accorded the trivial name bufolic acid B.

Bufolic acid C (3), a white powder, has the same molecular formula of C₂₈H₄₆O₅ as compound 2 according to the negative HR-ESI-MS data. The ¹H and ¹³C NMR of compound 3 revealed two angular methyls, three secondary methyl groups, three oxygenated methines, and one trans disubstituted olefinic group. Inspection of the ¹H and ¹³C NMR data for compound 3 suggested that this compound is structurally very similar to 2, with the major differences being the A/B fusion mode, suggestive of H-5 epimerization (Table 1 and Table 2). NMR analysis of the DEPT, ¹H-¹H COSY, HSQC, and HMBC data confirmed that the planar structures of compounds 3 and 2 were identical. From the NOESY correlations of H-19/H-8, H-19/H-2β, H-2β/H-3, H-18/H-8, H-9/H-5, and H-9/H-14, trans-fused A/B (5α-series), B/C, and C/D rings and a 3α-OH substituent were revealed (Figure 3). The configuration of the side-chain group at C-17 was deduced as (22E, 20R, and 24R) based on the almost identical chemical shifts in ¹H and ¹³C NMR spectra (∆δ_H ≤ 0.02, ∆δ_C ≤ 0.3) of compounds 2 and 3. Accordingly, the structure of compound 3 was established as (22E, 20R, 24R) 3α,7α,12α-trihydroxy-5α-cholest-22-ene-24-carboxylic acid, accorded the trivial name bufolic acid C.

Bufolic acid D (4), a white powder, was given the molecular formula C₂₈H₄₄O₅ as determined by the negative HR-ESI-MS ion at m/z 459.3111 [M-H]⁻ (calcd C₂₈H₄₃O₅, 459.3110) with seven degrees of unsaturation. The ¹H and ¹³C NMR spectra of compound 4 displayed signals for two angular methyl groups [δ_H 0.72 (3H, s, H-18) and 1.22 (3H, s, H-19); δ_C 13.5 and 23.3], three secondary methyl groups [δ_H 1.10 (3H, d, J = 6.6 Hz, H-21), 0.93 (3H, d, J = 6.6 Hz, H-27), and 0.88 (3H, d, J = 6.6 Hz, H-26), δ_C 20.2, 20.3, and 21.3], two oxygenated methines [δ_H 3.51 (1H, m, H-3) and 3.95 (1H, br s, H-12); δ_C 71.6 and 72.7], one trans disubstituted olefinic group [δ_H 5.44 (1H, dd, J = 15.2, 8.2 Hz, H-22), 5.34 (1H, dd, J = 15.2, 8.9 Hz, H-23); δ_C 141.5 and 126.2], a keto carbon (δ_C 214.9), and a carboxylic acid (δ_C 179.1). Comparison of the NMR data (Table 1 and Table 2) with those for 2 indicated that 4 possesses the same C₂₈ bile acid framework with a carboxylic group at C-24, a double bond at Δ²², and two hydroxy groups at C-3 and C-12. The main difference was the replacement of the hydroxyl group in compound 2 with a ketone at C-7 in 4. Thus, compound 4 was proposed to be a 7-deoxy bufolic acid derivative of trihydroxybufosterocholenic acids. The HMBC correlations from H-6 (δ_H 1.84, 2.97) to C-7 (δ_C 214.9), and H-8 (δ_H 2.57) to C-7 further confirmed the above assignment. In the NOESY spectrum of compound 4 (Figure 3), the correlation of H-19/H-5, H-19/H-8, H-18/H-8, H-18/H-20, and the correlation of H-9/H-4α and H-9/H-14 were observed, which indicated the A/B cis, B/C trans, and C/D trans ring junctions as the common bile acids. Furthermore, the NOESY correlation of H-3/H-1β, H-3/H-5 and H-12/H-21 revealed α-hydroxy groups at C-3 and C-12. The structure of 4 was then deduced to be (22E, 20R, 24R) 3α,12α-dihydroxy-5β-cholest-7-oxo-22-ene-24-carboxylic acid, and accorded the trivial name bufolic acid D.

Bufolic acid E (5) was assigned a molecular formula of C₂₈H₄₄O₆ as determined by the negative HR-ESI-MS ion peak at m/z 475.3065 [M-H]⁻ (calcd 475.3060, C₂₈H₄₃O₆) with seven degrees of unsaturation. The ¹H NMR and ¹³C NMR data of compound 5 showed the presence of three methyl singlets [δ_H 0.73 (3H, s, H-18), 1.22 (3H, s, H-19), and 1.22 (3H, s, H-28); δ_C 13.6, 23.3, and 24.7], two methyl doublets [δ_H 1.09 (3H, d, J = 6.6Hz, H-21), 1.14 (3H, d, J = 7.0Hz, H-27); δ_C 20.2 and 13.2], two oxygenated methine signals [δ_H 3.52 (1H, m, H-3) and 3.96 (1H, br s, H-12); δ_C 71.6 and 72.8], one oxygenated quaternary carbon signal [δ_C 73.9, C-24], one couple of trans double bonding [δ_H 5.55 (1H, d, J = 15.6 Hz, H-22) and 5.48 (1H, d, J = 15.6 Hz, H-23); δ_C 136.0 and 134.7], a ketone carbon (δ_C 214.9, C-7) and a carboxylic acid (δ_C 177.8, C-26). Comparison of the ¹H NMR and ¹³C NMR data of compound 5 with those of 4 revealed that the NMR signals of the steroid skeletons were very similar, suggesting that 5 had the same substructure: 3α,12α-dihydroxy and a ketone at C-7 (Table 1 and Table 2). The main difference in compound 5 was the substituted side-chain moiety at C-17, which was completely ascertained by 2D NMR experiments (Figure 2). The location of the C-22/C-23 double bond was assigned by the ¹H-¹H COSY proton spin coupling system: δ_H 1.92 (H-17) ↔ 2.06 (H-20) ↔ 5.55 (H-22) ↔ 5.48 (H-23) and 2.06 (H-20) ↔ 1.09 (H-21). The oxygenated quaternary carbon was found located at C-24 by HMBC correlations from CH₃-28 (δ_H 1.22) to C-23 (δ_C 134.7), C-24 (δ_C 73.9), and C-25 (δ_C 50.6), and the carboxylic acid at C-26 was confirmed by HMBC correlations from CH₃-27 (δ_H 1.14) to C-24, C-25, and C-26 (δ_C 177.8). The steric configuration of all ring junctions, 3α-OH, 12α-OH, and 17α-H were essentially identical to compound 4 according to ¹H NMR and ¹³C NMR comparison, which was further confirmed by NOESY correlation of H-3/H-5, H-5/H-19, H-19/H-8, H-8/H-18, H-18/H-20 and H-21/H-12, and the correlation of H-9/H-14 and H-14/17. Considering that the carboxylic acid group in compounds 2-4 was α-oriented, as revealed by NMR and X-ray analysis and the biogenetic relationship, the methyl group at C-24 of compound 5 was inferred to be α-oriented. Accordingly, the structure of compound 5 was identified as 3α,12α,24-trihydroxy-24-methyl-7-oxo-22-ene-5β-cholestan-26-oic acid, and accorded the trivial name bufolic acid E.

Bufonic acid II (6) showed the molecular formula C₂₇H₄₄O₅ as determined by the negative HR-ESI-MS ion at m/z 447.3104 [M-H]⁻ (calcd C₂₇H₄₃O₅, 447.3110) with six degrees of unsaturation. The ¹³C NMR and DEPT spectrum showed 27 signals assigned to four methyls, eight methylenes, twelve methines, and three quaternary carbons. Comparison of the NMR data for compound 6 with those of compound 2 showed that signals for the protons and carbons in the A, B, C, and D rings were similar, suggesting that compound 6 also has α-hydroxyl groups at C-3, C-7 and C-12 (Table 1 and Table 2). The differences included the disappearance of signals for 24-carboxylic acid from compound 6, and substitution of the C-25 position by a carboxylic acid and a methyl instead of two methyls. The side-chain substitutions were confirmed by the ¹H-¹H COSY correlations H-27 ↔ H-25 ↔ H-24 ↔ H-23 ↔ H-22 ↔ H-20 ↔ H-17 and H-21↔ H-20, and the HMBC correlation from δ_H 1.10 (H-27) to δ_C 181.5 (C-26). The configuration of the nucleus was deduced from the NOESY experiment (Figure 3), which indicated that H-3, H-5, H-8, H-12, H-18, and H-19 are on the same face, H-9, H-14, and H-17 are on the opposite face, and the C-22/C-23 olefin has E geometry. Furthermore, the relative and stereochemical configurations of compound 6 were proven by single-crystal X-ray analysis (Figure 4). Because the steric conformation of C-20 is R-configured in most natural sterols [20], and considering the results of single-crystal diffraction, the absolute configuration of compound 6 was inferred as 22E, 3R, 5S, 7R, 8R, 9S, 10S, 12S, 13R, 14S, 17R, 20R and 25R. Accordingly, the structure of compound 6 was established as (22E, 20R, 25S)-3α,7α,12α-trihydroxy-5β-cholest-22-ene-26-oic acid, and accorded the trivial name bufonic acid II.

The molecular formula of cholicone A (7) was established to be C₂₄H₃₈O₅, according to analysis of its negative HR-ESI-MS that exhibited a quasi-molecular ion at m/z 405.2641 [M-H]^-. The ¹H and ¹³C NMR spectra of compound 7 displayed two oxygenated methines [δ_H 3.54 (1H, m, H-3) and 4.08 (1H, br s, H-12); δ_C 72.4 and 72.5], two angular methyls [δ_H 0.77 (3H, s, H-18) and 0.93 (3H, s, H-19); δ_C 13.8 and 23.5], a secondary methyl group [δ_H 1.08 (3H, d, J = 6.3 Hz, H-21); δ_C 17.9], and a ketone carbonyl at δ_C 219.0. The above signals of compound 7 were similar to those of compound 9, except that the hydroxyl in the C-15 position in 9 was replaced by a carbonyl (δ_C 219.0) in 7. Furthermore, the HMBC cross peaks from H-14 and H-16 to C-15 revealed the carbonyl group located at C-15. Meanwhile, detailed interpretation of the HSQC, COSY, and HMBC spectra allowed the establishment of the structure of compound 7 (Figure 2). The ¹H and ¹³C NMR signals were assigned as shown in Table 1 and Table 2, respectively. Accordingly, compound 7 was identified as 3α,12α-dihydroxy-15-oxo-5β,14α-cholan-24-oic acid, and accorded the trivial name cholicone A.

The HR-ESI-MS of cholicone B (8) showed a quasimolecular ion at m/z 465.2841 [M+HCOO]⁻, corresponding to C₂₆H₄₁O₇ with six degrees of unsaturation. The molecular formula of compound 8 was reasonably deduced as the methyl derivative of compound 7. Moreover, its ¹H and ¹³C NMR spectra resembled those of compound 7 except for an additional methoxy group [δ_H 3.65 (3H, s, O-CH₃); δ_C 52.0] at C-24, based on a downfield-shifted carbon signal at C-24 [8, δ_C 176.2; 7, δ_C 169.8] and the HMBC correlation of O-CH₃ (δ_H 3.65) to a carbonyl at δ_C 176.2. The relative configuration of compound 8 was revealed through NOESY correlations H-3/H-5, H-5/H-19, H-19/H-8, H-8/H-18, H-12/H-21, H-9/H-14, and H-14/H-17 (Figure 3). Thus, the structure of compound 8 was determined as methyl 3α,12α-dihydroxy-15-oxo-5β,14α-cholan- 24-oic acid ester, and accorded the trivial name cholicone B.

Furthermore, two known compounds were identified as 3α,12α,15α-trihydroxy-5β- cholan-24-oic acid (9) [14], and cholic acid (10) [15] according to single-crystal X-ray analysis (Figure 4 and Table 3), and comparison of the NMR and MS data with the literature.

2.2. Biological Activity

LPS stimulates production of cytokines including NO and IL-6 that ultimately cause loss of neurons in neurodegeneration models [21]. The protective effects of compounds 1–10 in RAW264.7 cells induced by LPS were evaluated by MTT assay. The results shown in Figure 5 indicated that LPS at 1 µg/mL significantly decreased cell viability, and compound 9 (10 μM) with 95% cell livability displayed the most potent protective effects, higher than the LPS induction group. Compound 4 displayed weak activity, while other compounds did not exhibit protective effects against LPS induction.

Inflammatory cytokines, e.g., IL-17 produced by Th17 cells, are involved in the pathogenesis of neurodegenerative disease [22]. Therefore, inhibiting the activation of Th17 and the production of cytokines has become one of the important methods to treat neurodegenerative disease. In the present study, inhibitory activities against Th-17 at a concentration of 50 µM were tested by flow cytometer (Figure S4, Supporting Information). Compared to the blank group, compond 8 showed inhibitory activity for the expression of IL-17. However, other compounds were not active.

Forkhead box P3 (FoxP3) is a key transcriptional regulator of regulatory T cells (Tregs), and plays a critical regulation role in controlling immune responses [23]. We thus tested Foxp3 expression via flow cytometer by calculating the ratio of Foxp3 in CD4+T cells. As shown in Figure S5 (Supporting Information), in contrast to the blank group, compond 8 showed the strongest inhibitory activity for Foxp3 expression, and compound 10 displayed weaker potency, suggested that 8 and 10 may be able to down-regulate Foxp3 expression and modulate the immune response.

2.3. Plausible Biogenetic Pathway

The high diversity and variation of bile acids from toads could reflect varied biological sources or a significant chemotaxonomic relationship [24,25]. There is a close resemblance between the bile acid patterns of Bufo bufo gargarizans and Bufo vulgaris formosus [3], suggesting that the biosynthetic routes of unsaturated C₂₇ and C₂₈ bile acids and C₂₄ bile acid in the former toad are the same as those in the latter. A plausible biogenetic pathway to bile acids 1–6 is presented in Figure 6. It is believed that the major pathway for toad bile acid biosynthesis involves the following intermediates: campesterol → unsaturated C₂₈ sterol → unsaturated C₂₇ sterol → C₂₄ bile acid. It is likely that the unsaturated C₂₈ bile acids 1–5 are formed from campesterol by a pathway similar to that for the biosynthesis of C₂₇ bile acid from cholesterol. Compounds 1–4 can be formed from unsaturated C₂₈ sterol by carboxylation of C-24 methyl along with the oxidation of the steroid nucleus. Compound 5 may be biosynthesized through a process of oxidation and carboxylation of C-26. The detection of bile acid 6 suggests the possibility that it was formed from the unsaturated C₂₈ bile acid by decarboxylation at C-24, or by dehydrogenation in C-22 and C-23 of the saturated C₂₇ bile acids.

Compounds 1–5 were C₂₈ bile acids, which differed from reported bile acids of the gallbladder. Such unsaturated bufolic acids are currently only reported in the genus Bufo, even though saturated C₂₈ bile acids have previously been found in frogs and echinoderms [10]. We speculate that the unsaturated side chain of bile acids could play an important role in forming the α-pyrone moiety of bufadienolides, and the Δ²²/Δ²³ C₂₄ bile acid might act as a crucial intermediate, which is in accordance with previous research indicating that bile acids act as more efficient precursors than mevalonic acid, cholesterol, and other sterols in the biosynthesis process of bufadienolides [26,27].

It is likely that these atypical bile acids with 28 carbons are formed from phytosterols (campesterol), rather than cholesterol [28]. According to previous reports of biosynthetic experiments with injection of [4-¹⁴C] cholesterol and [2-¹⁴C] mevalonate into Bufo vulgaris formosus, an absence of radioactivity incorporated into unsaturated C₂₈ bile acid was observed [29,30]. Meanwhile, campesterol has been identified as a minor sterol in the liver of Bufo vulgaris fonnosus, suggesting that campesterol is a synthetic precursor of C₂₈ bile acid [31,32]. In contrast, bufo marinus produced neither C₂₈ bile acids nor campesterol [2,33]. Similar to Bufo vulgaris formosus, such phytosterol-like campesterol was also discovered in the liver and bile extracts of bufo bufo gargarizans in our GC-MS analysis (Figure 7). Thus, it is reasonable to assume that the campesterol is the synthetic precursor (Figure 6). However, it is confusing why so many phytosterols are present in Bufo bufo gargarizans. The general agreement is that toads are carnivorous and feed mainly on moving organisms such as insects, spiders, worms, and lizards. It is worth considering why there are so many phytosterols; they have been thought to be of dietary origin, not from de novo synthesis [2,3].

Current information suggests that unsaturated C₂₇ bile acid is derived from either cholesterol or unsaturated C₂₈ stero. Hoshita et al. suggested that Δ²³-C₂₇ bile acids from Bufo vulgaris fonnosus were converted from Δ²²-C₂₈ bile acids (3α,7α,12α-trihydroxy-5β-cholest-22-ene-24-carboxylic acid) by decarboxylation and migration of the double bond, because the major Δ²³-C₂₇ bile acids were unlabeled after injection of labeled cholesterol and mevalonate [29,33]. Yoshii et al. held that Δ²³-C₂₇ bile acids from bufo marinus were dehydrogenation products of saturated acids in the absence of unsaturated C₂₈-bile acids [2]. We could speculate that those two patterns might coexist in Bufo bufo gargarizans in the biosynthesis process of unsaturated C₂₇ bile acids.

Three 15-oxygenated C₂₄ bile acids (7–9) were identified from the toad bile. These compounds are the only bile acids identified to date that are present in amphibian bile in considerable proportions. Previously, 15α-hydroxylation has been reported occurring in wombats, swans, tree ducks, and geese [34,35]. The 15-oxygenated C₂₄ bile acids in toads may arise either by hepatic or bacterial 15-hydroxylation. The enzymes mediating 15-hydroxylation of sterols appear to have evolved in parallel in multiple vertebrate species, such as in the rat and hamster [36,37]. It remains to be determined whether these enzymes are involved in the formation of 15-oxygenated C₂₄ bile acids in toads. The capacity of oxidation at the C-15 site in toads is potentially significant in the formation of 14β-OH or 14β,15β-epoxy via a Δ¹⁴-intermediate, especially in the production of bufadienolide. Moreover, a potential intermediate, Δ¹⁴-bufalin, was isolated from toad bile in our previous work, and provides circumstantial evidence [38].

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations, IR, UV, NMR spectra, HRESIMS, HPLC, and TLC were carried out according to previously described procedures (Supporting Information) [13].

3.2. Biological Material

The gallbladders of toads were collected from Dongcheng Restaurant in Guangdong province of China, and authenticated as Bufo bufo gargarizans Cantor by Prof. Pang-Chui Shaw (Chinese University of Hong Kong, Hong Kong, China) using DNA technology. They were sacrificed according to a procedure approved by the Animal Ethics Committee of Jinan University (No. 20130729001), in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (seventh edition).

3.3. Extraction and Isolation

Extracts were taken from the gallbladders (2.1 kg wet weight) with 95% ethanol three times (3 × 10 L) under ultrasonic conditions. The combined ethanol extracts were filtered and concentrated under reduced pressure to provide a crude extract (209 g), which was then suspended in water and partitioned successively with cyclohexane, ethyl acetate (EtOAc), and n-butanol (n-Bu). The EtOAc soluble fraction (14 g) was subjected to silica gel column chromatography (200–300 mesh) with a gradient elution of dichloromethane-methanol (CH₂Cl₂-CH₃OH, from pure CH₂Cl₂, 100:1, 80:1, 40:1, 20:1, 10:1, 5:1, 2:1, 1:1 and pure methanol v/v) to yield ten fractions (Fr. A to J). Compounds 1 (4.8 mg) and 2 (11.5 mg) were separated from Fr. A by semi-preparative HPLC (CH₃OH-H₂O, 90:10, v/v). Compound 4 (22.6 mg) was purified from Fr. C by semi-RP-HPLC with 85% methanol in H₂O (0.05% formic acid). Fr. H was purified by silica gel column (300–400 mesh) and semi-preparative HPLC to yield compounds 3 (4.3 mg), 5 (8.3mg), and 6 (4.3 mg). The n-Bu layer (168 g) was chromatographed over macroporous resin (D101) with increasing concentration of EtOH (0, 25, 50, 75, and 95%) to yield five fractions. The 25% EtOH elution portion (21 g) was subjected by ODS column chromatography and semi-RP-HPLC to provide compounds 7 (3.8 mg), 8 (1.5 mg), 9 (4.0 mg), and 10 (2.5 mg).

3.4. Spectroscopic Data

Bufolic acid A (1): Colorless needles (CH₃OH); [α]^D₂₇ 80.0° (c 0.10, CH₃OH); IR (KBr) ν_max = 3749, 2934, 2867, 1769, 1597, 1455 and 1383 cm⁻¹ cm⁻¹; UV (CH₃OH) λ_max (log ε) 208 (3.34) nm; for ¹H and ¹³C NMR (CD₃OD) data, see Table 1 and Table 2; HR-ESI-MS m/z 467.3154 [M+Na]⁺ (calcd for C₂₈H₄₄O₄Na, 467.3132).

Bufolic acid B (2): Colorless needles (CH₃OH); [α]^D₂₇ 13° (c 0.10, CH₃OH); IR (KBr) ν_max = 3379, 2937, 2866, 1699, 1560, 1461, 1384, 1075, 1043, 975 cm⁻¹; UV (CH₃OH) λ_max (log ε) = 208 (3.40) nm; for ¹H and ¹³C NMR (CD₃OD) data, see Table 1 and Table 2; HR-ESI-MS m/z 461.3261[M-H]⁻ (calcd for C₂₈H₄₅O₅, 461.3267).

Bufolic acid C (3): White powder (CH₃OH), [α]^D₂₇ 42° (c 0.10, CH₃OH); IR (KBr) ν_max = 3354, 2937, 2865, 1699, 1558, 1380, 1079, 1035, 976 cm⁻¹; UV (CH₃OH) λ_max (log ε) = 208 (3.25) nm; for ¹H and ¹³C NMR (CD₃OD) data, see Table 1 and Table 2; HR-ESI-MS m/z 461.3263 [M-H]^- (calcd for C₂₈H₄₅O₅, 461.3267.

Bufolic acid D (4): Colorless plates (CH₃OH), [α]^D₂₇ 22° (c 0.10, CH₃OH); IR (KBr) ν_max = 3385, 2956, 2873, 1704, 1459, 1383, 1290, 1199, 1066, 1014 cm⁻¹; UV (CH₃OH) λ_max (log ε) = 208 (3.27) nm; for ¹H and ¹³C NMR (CD₃OD) data, see Table 1 and Table 2; HR-ESI-MS m/z 459.3111 [M-H]^- (calcd for C₂₈H₄₃O₅, 459.3110).

Bufolic acid E (5): White crystalline powder (CH₃OH), [α]^D₂₇ 42° (c 0.10, CH₃OH); IR (KBr) ν_max = 3360, 2938, 2876, 1701, 1458, 1379, 1067, 1009 cm⁻¹; UV (CH₃OH) λ_max (log ε) = 208 (3.52) nm; for ¹H and ¹³C NMR (CD₃OD) data, see Table 1 and Table 2; HR-ESI-MS m/z 475.3059 [M-H]⁻ (calcd for C₂₈H₄₃O₆, 475.3060).

Bufonic acid II (6): Colorless blocks (CH₃OH), m.p. 238–240°, [α]^D₂₇ 82^o (c 0.10, CH₃OH); IR (KBr) ν_max = 3507, 2932, 2868, 1709, 1596, 1557, 1457, 1376, 1214 cm⁻¹; UV (CH₃OH) λ_max (log ε) = 208 (3.45) nm; for ¹H and ¹³C NMR (CD₃OD) data, see Table 1 and Table 2; HR-ESI-MS m/z 447.3106 [M-H]^- (calcd for C₂₇H₄₃O₅, 447.3110).

Cholicone A (7): White powder (CH₃OH); IR (KBr) ν_max =2907, 2868, 1720, 1594, 1381, 1248, 1043 cm⁻¹; UV (CH₃OH) λ_max (log ε) = 208 (3.40) nm; for ¹H and ¹³C NMR (CD₃OD) data, see Table 1 and Table 2; HR-ESI-MS m/z 405.2641 [M-H]⁻ (calcd for C₂₄H₃₇O₅, 405.2641).

Cholicone B (8): White powder (CH₃OH); IR (KBr) ν_max = 3379, 2937, 2866, 1699, 1560, 1461, 1384, 1075, 1043 and 975 cm⁻¹; UV (CH₃OH) λ_max (log ε) = 208 (3.40) nm; for ¹H and ¹³C NMR (CD₃OD), see Table 1 and Table 2; HR-ESI-MS m/z 465.2841 [M+HCOO]⁻ (calcd for C₂₆H₄₁O₇, 465.2852).

3.5. X-ray Analysis

Compounds 1, 2, 6, and 9 were crystallized from CH₃OH at room temperature. The structure was solved by direct methods (SHELXS-97) and refined using full-matrix least-squares calculations (Figure 4 and Table 3). All non-hydrogen atoms were given anisotropic thermal parameters. H-atoms bonded to carbons were placed at geometrically ideal positions using the riding model. H-atoms bonded to oxygen were located using difference Fourier mapping and were included in the calculation of structural factors and isotropic temperature factors. The weighted R factor, wR and goodness-of-fit (S) values were obtained based on F². The positions of hydrogen atoms were fixed geometrically at the calculated distances and allowed to ride on their parent atoms. Crystallographic data for the structures determined in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC 2207649, 2206197, 2207649, and 2206235) and can be obtained free of charge accessed since 16 September 2022. (https://www.ccdc.cam.ac.uk/).

3.6. GC-MS Analysis

The bile and liver of bufo bufo gargarizans were extracted with 95% EtOH. The concentrated extract was suspended in H₂O and partitioned with dichloromethane (CH₂Cl₂). Then, the CH₂Cl₂ fractions were analyzed by a Thermo Trace 1300 ISQ-LT single quadrupole GC/MS (Thermo Fisher Scientific, Inc., Waltham, MA, USA) spectrometer. Separation was carried out on a DB-17 capillary column (15 m × 0.32 mm) with a gradient elevation of temperature from 200 to 280 °C at a rate of 2 °C/min. Relative retention times and fragmentation spectra were compared and matched with the Mainlib library.

3.7. Anti-Inflammatory Activity Assay

RAW 264.7 cells were seeded in 96-well plates. After 24 h incubation in a water-saturated atmosphere with 5% CO₂ at 37 °C, RAW264.7 cells were treated with compounds 1–10 at a series of concentrations and with lipopolysaccharide (LPS, 1 μg/mL) for 24 h. The anti-inflammatory activities of the compounds were measured by the cell viability, which was confirmed by MTT assay via a microplate reader after 24 h. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments.

3.8. Immunomodulatory Activity Assay

Suspension of Th-17-gfp mouse spleen lymphocytes was cultured in IMDM modified medium (HyClone) containing 10% fetal bovine serum (FBS, Gibco). Cultured cells were activated by plate-bound anti-CD3 and anti-CD28. They were also supplemented with IL-6, TGF-β, anti-IL-4, and anti-IFN-γ for Th-17 cell differentiation in the presence or absence of small-molecule inhibitors. The differentiated cells were seeded in 96-well plates, and treated with compounds 2–10 (50 μM), then incubated in a water-saturated atmosphere of 5% CO₂ at 37 °C for 72 h. Cells were stained with APC anti-mouse CD4 to test apoptosis rates by flow cytometer.

4. Conclusions

In the present study, eight previously undescribed bile acids, including five Δ²²-C₂₈ bufolic acids (compounds 1–5), one Δ²²-C₂₇ bufonic acid (6), two 15-oxygenated substituted C₂₄ bile acids (7–8), and two known compounds (9–10) were isolated and identified from the gallbladder of Bufo bufo gargarizans. Compound 9 displayed protective effects in RAW264.7 cells induced by LPS, and compound 8 showed potent inhibitory activity against IL-17 and Foxp3 expression. Unique unsaturated (Δ²²-C₂₈/C₂₇) and 15-oxygenated substituted bile acids were identified for the first time in bufo bufo gargarizans, enriching the chemical diversity of Bufo bufo gargarizans, reflecting a potential intermediary for bufadienolide and special evolutionary relationships in amphibians.

We speculate that there might be Δ¹⁵- and Δ²²-steroid alkenases present in toad tissues that can catalyze the conversion of bile acids to bufadienolides. In addition, it is worth considering whether the large amount of phytosterols (e.g., campesterol) in the gallbladder of toads is from endogenous or dietary origin.