Capsisteroids A–F, withanolides from the leaves of Solanum capsicoides

Abstract

A known withanolide steroid cilistol G (1) and six new withanolides, capsisteroids A–F (2–7), were isolated from the EtOAc extract of the leaves of Solanum capsicoides. The structures of compounds 1–7 were elucidated by extensive spectroscopic analysis, including 2D NMR spectroscopy (COSY, HSQC, HMBC, and NOESY). The structure of 1 was further confirmed by a single-crystal X-ray diffraction analysis. These compounds were found not to be cytotoxic toward a limited panel of cancer cell lines. Further, the anti-inflammatory activity of compounds 1–7 was studied by measuring their ability to suppress superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils.

---

Introduction

Withanolides are a group of structurally diverse C₂₈ ergostane-type steroids that are mostly encountered in certain genera of the family Solanaceae.^1–18 Also, a few withanolides have been discovered from marine organisms.^19–21 These compounds possess diversified structures and the structural diversity is responsible for the wide array of biological activities such as phytotoxicity,¹ anti-inflammatory,^7,11,14,21 cytotoxic,^{12,18,20–28} antimicrobial,^29–31 immunosuppresive,^32,33 and chemopreventive³⁴ activities. Although many compounds have been reported from various species^3–6 of this genus, very few investigations on chemical constituents of Solanum capsicoides (syn. Solanum aculeatissimum) have been reported.³⁵

The present study describes the isolation and structural elucidation of a known C₂₈ ergostane-derived steroid cilistol G (1)³ and six new withanolides, capsisteroids A–F (2–7), from the leaves of S. capsicoides. Compound 1 has been previously reported as an epimeric mixture at C-26, whereas we found that 1 was initially obtained as the 26S diastereomer and its structure was further confirmed by X-ray crystallography.

Upon prolonged standing, compound 1 was gradually epimerized to 26S/R diastereomers, with the 26S isomer being the major component. The cytotoxicities of metabolites 1–7 against a variety of human tumor cell lines including human erythro myeloblastoid leukemia (K562), human acute lymphoblastic leukemia (Molt-4) and human promyelocytic leukemia (HL-60) have been studied. The abilities of 1–7 to inhibit superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils were also evaluated.

Results and discussion

The EtOAc extract of the leaves of S. capsicoides was subjected to silica gel column chromatography, which rendered 20 fractions. The fractions were further purified by a series of normal and reversed-phase HPLC to afford pure compounds 1–7.

The HRESIMS spectrum of 1 exhibited a quasimolecular ion peak [M + Na]⁺ at m/z 497.2874 corresponding to a molecular formula of C₂₈H₄₂O₆, possessing eight degrees of unsaturation. The NMR data of 1 were found to be structurally similar to that of cilistol G isolated from S. cilistum,³ whereas we found that 1 existed initially as the 26S diastereomer from its ¹H NMR spectrum (Fig. S1 in the ESI†). This is different from the previous report, in which compound 1 was demonstrated to be an C-26 epimeric mixture.³ However, 26S conformer of 1 is not stable for prolonged periods of time, and was gradually epimerized to 26R and finally reached to a 3 : 1 ratio of 26S/R disatereomers, as shown in the ¹H NMR spectrum (ESI S9†), which revealed the characteristic H-22 epimeric protons resonating at δ 4.32 and 4.99. Moreover, both of the ¹³C signals due to C-22 and C-26 appeared as split pairs; in this case signals appeared at δ 98.4 and 70.1 ppm for (26S)-1 and δ 99.1 and 74.3 ppm for (26R)-1. Nevertheless, the major 26S isomer was crystallized from MeOH–H₂O (4 : 1), and its structure was confirmed by a single crystal X-ray analysis as depicted in Fig. 1.

Fig. 1 Molecular structure of 1 based on X-ray analysis.

Capsisteroid A (2) was obtained as colorless gum and its molecular formula was established as C₃₀H₄₄O₇ based on HRESIMS, implying nine degrees of unsaturation. The ¹H and ¹³C NMR data of 2 closely resembled those of 1, except that the hydroxyl group at C-24 in 1 was replaced by acetate in 2. This was confirmed by the upfield chemical shift of C-24 (δ_C 77.4) of 1, relative to that of 2 (δ_C 89.0). In the NOESY spectrum of 2, the correlations between H-8 with H₃-19 and H₃-18; H-20 with H₃-18 and H-22; H-23β (δ 2.58) with H-22 and H₃-28 as well as H₃-28 with H-26 suggested that H-8, H₃-18, H₃-19, H-20, H-22, and H₃-28 are β-orientated. Besides, correlations of H-23α (δ 1.76) with H₃-21 and H₃-27 revealed that H₃-21 and H₃-27 are α-oriented. Since the absolute structure of 1 has been determined,³ therefore, the structure of compound 2 was elucidated as (22R,24R,25S,26S)-1-oxo-22,26-epoxy-24-acetoxy-17α,24,25,26-tertrahydroxyergost-2,5-diene.

The HRESIMS spectrum of 3 exhibited a pseudomolecular ion peak at m/z 497.2876 [(M + Na)⁺], consistent with a molecular formula of C₂₈H₄₂O₆, indicating the presence of eight degrees of unsaturation. The spectroscopic data of 3 (IR, ¹H and ¹³C NMR) were similar to those of 1, but showed a difference in the double bond position. The HMBC (Fig. 2) correlations from H-2α/H-2β (δ 2.65/3.37) to C-1 (δ 213.3) and H-3 (δ 5.62) to C-1 (δ 213.3) indicated that there was a Δ^3,4 double bond in 3 (δ_H 5.62; δ_C 122.7 and δ_H 6.06; δ_C 130.5), instead of the Δ^2,3 double bond in compound 1. The structure of 3 was unambiguously determined by extensive analysis of COSY and HMBC (Fig. 2) correlations. A structurally-related metabolite, capsisteroid C (4), was also isolated as a colorless gum with a molecular formula of C₃₀H₄₄O₇, implying nine degrees of unsaturation. The NMR data of compound 4 differed from those of 3, only by the absence of the hydroxy group at C-24 and the presence of additional signals of an acetate functionality (δ_H 2.03; δ_C 22.1, and 173.2). This was supported by the upfield shift of C-24 (δ_C 75.2) and H₃-28 (δ_H 1.32) of 3 relative to that of 4 (δ_C 88.9, C-24; δ_H 1.62, H₃-28) and the HMBC correlations from H₃-28 (δ 1.62, s) to C-23 (δ 38.7, CH₂), C-24 (δ 88.9, C), and C-25 (δ 76.5, C). A detailed analysis of the ¹H and ¹³C NMR spectroscopic data and the detected 2D correlations in the COSY and HMBC spectra led to the establishment of the molecular framework of 4.

Fig. 2 COSY and HMBC correlations for 2, 3 and 5–7.

The molecular formula of capsisteroid D (5) was established as C₂₈H₄₄O₈ by HRESIMS (m/z 531.2931 [M + Na]⁺) which showed the molecule to have seven degrees of unsaturation. Comparison of the NMR data of 5 with those of 1 revealed that in 5 two hydroxy groups have been added across the Δ^5,6 double bond in 1. This was further evidenced by the HMBC correlations observed from H-3 (δ_H 6.64) and H₃-19 (δ_H 1.30) to C-5 (δ_C 78.4) and COSY correlations from H-6 (δ 3.52) to H₂-7 (δ 1.73/1.55). Thus, the planar structure of 5 was established. In the NOESY spectrum of 5 (Fig. 3), the NOE correlations between H₃-19 with H-8 and H-4β (δ 3.32) suggested that H-8 and H₃-19 are β-orientated. Also, correlations of H-6 with H-4α (δ 2.04) and H-7α (δ 1.73); H-9 with H-7α suggested that 5-OH, H-6 and H-9 are α-oriented. Further analysis of the NOE interactions revealed that 5 possessed the same relative configurations at C-13, C-14, C-17, C-20, C-22, C-24, C-25, and C-26 as those of 1.

Fig. 3 Key NOESY correlations of 5.

Capsisteroid E (6) was obtained as a white powder with a pseudomolecular ion peak at m/z 513.2820 [M + Na]⁺ in the HRESIMS spectrum, corresponding to a molecular formula of C₂₈H₄₂O₇. Comparison of the NMR data of 6 with those of 1 (Tables 1–3) indicated that the Δ^5,6 double bond in 1 was migrated to Δ^4,5 and the presence of an additional oxygen-bearing methine (δ_H 4.54; δ_C 74.7) in 6. The HMBC correlations of H-4 (δ 6.23) to C-6 (δ_C 74.7) along with the COSY cross peaks of H-3/H-4 and H-6/H-7 enabled the establishment of the Δ^4,5 double bond and C-6 hydroxy group, respectively. The relative configuration of 6 was determined from the NOESY spectrum, in which correlations of H-6 with H-7α (δ 1.22) and H-7α with H-9 suggested that H-6 is α-orientated. In addition, the NOE cross peaks of H-8 with H₃-19 and H-7β (δ 2.02) with H-8 suggested the β-orientation of H-8. Thus, the structure of 6 was established as shown.

Table 1 ¹³C-NMR spectral data for compounds 1–7

Position	1^a	1^b	2^a	3^a	4^a	5^a	6^a	7^a
a 100 MHz in methanol-d4.b 100 MHz in pyridine-d5.c Multiplicities deduced by DEPT.d Chemical shift data correspond to the major epimer (26S). Distinct resonances for the 26R epimer observed in the spectrum of the epimeric mixture are shown in brackets.
1	206.9, C^c	203.9, C	207.0, C	213.3, C	213.0, C	207.6, C	208.1, C	204.4, C
2	128.3, CH	127.9, CH	128.4, CH	40.7, CH2	40.7, CH2	129.1, CH	126.7, CH	133.4, CH
3	148.1, CH	145.8, CH	148.3, CH	122.7, CH	122.8, CH	143.8, CH	142.9, CH	145.3, CH
4	34.4, CH2	33.6, CH2	34.6, CH2	130.5, CH	130.4, CH	36.6, CH2	118.6, CH	71.3, CH
5	137.3, C	136.4, C	137.5, C	142.6, C	142.6, C	78.4, C	160.7, C	64.9, C
6	125.8, CH	124.9, CH	126.0, CH	128.1, CH	128.1, CH	75.3, CH	74.7, CH	61.4, CH
7	32.0, CH2	31.3, CH2	32.2, CH2	32.2, CH2	32.2, CH2	34.2, CH2	42.2, CH2	32.8, CH2
8	34.9, CH	33.7, CH	35.0, CH	33.2, CH	33.3, CH	31.6, CH	32.2, CH	31.6, CH
9	44.4, CH	43.3, CH	44.5, CH	42.3, CH	42.3, CH	42.3, CH	51.1, CH	45.6, CH
10	51.8, C	50.8, C	51.9, C	53.5, C	53.4, C	53.0, C	55.8, C	49.8, C
11	37.7, CH2	37.4, CH2	37.8, CH2	37.7, CH2	37.7, CH2	37.7, CH2	37.7, CH2	37.9, CH2
12	33.5, CH2	32.9, CH2	33.7, CH2	33.4, CH2	33.4, CH2	33.9, CH2	33.3, CH2	33.2, CH2
13	49.2, C	48.2, C	49.3, C	49.4, C	49.4, C	49.8, C	49.5, C	49.8, C
14	51.6, CH	50.7, CH	51.8, CH	51.6, CH	51.6, CH	51.0, CH	51.3, CH	51.7, CH
15	24.6, CH2	24.0, CH2	24.8, CH2	24.6, CH2	24.6, CH2	24.6, CH2	22.2, CH2	22.3, CH2
16	24.7, CH2	24.2, CH2	24.8, CH2	23.4, CH2	23.4, CH2	24.3, CH2	24.8, CH2	24.9, CH2
17	86.6, C	85.0, C	86.6, C	86.6, C	86.4, C	86.7, C	86.2, C	86.5, C
18	15.4, CH3	15.3, CH3	15.6, CH3	15.4, CH3	15.6, CH3	15.8, CH3	15.5, CH3	15.2, CH3
19	19.5, CH3	19.0, CH3	19.3, CH3	20.8, CH3	20.8, CH3	16.2, CH3	19.1, CH3	17.0, CH3
20	44.8, CH	44.2, CH	45.0, CH	44.8, CH	44.8, CH	44.9, CH	44.8, CH	44.9, CH
21	10.3, CH3	10.7, CH3	10.3, CH3	10.3, CH3	10.1, CH3	10.3, CH3	10.3, CH3	10.4, CH3
22	74.9, CH	70.1 [74.3]^d CH	74.4 [69.9] CH	74.9 [70.6] CH	74.3 [69.9] CH	74.9 [69.4] CH	74.8 [70.2] CH	75.0 [70.6] CH
23	40.8, CH2	41.1, CH2	38.8, CH2	40.7, CH2	38.7, CH2	40.8, CH2	40.7, CH2	40.9, CH2
24	77.4, C	77.3 [75.3] C	89.0, C	75.2, C	88.9, C	77.4, C	77.4, C	77.6, C
25	75.2, C	77.3 [74.4] C	76.6, C	77.5, C	76.5, C	75.2, C	75.2, C	75.3, C
26	97.7, CH	98.4 [99.1] CH	97.2 [99.8] CH	97.7 [99.3] CH	97.1 [99.2] CH	97.7 [99.2] CH	97.7 [99.2] CH	97.9 [99.2] CH
27	14.9, CH3	16.2, CH3	15.7, CH3	14.9, CH3	15.6, CH3	14.9, CH3	14.9, CH3	15.0, CH3
28	22.7, CH3	22.6 [23.8] CH3	19.3, CH3	22.7, CH3	19.1, CH3	22.7, CH3	22.7, CH3	22.8, CH3
24-OAc			173.4, C		173.2, C
			22.3, CH3		22.1, CH3

---

Table 2 ¹H-NMR spectral data for compounds 1–3

Position	1^a	1^b	2^a	3^a
a Spectra recorded at 400 MHz in methanol-d4 at 25 °C.b Spectra recorded at 400 MHz in pyridine-d5 at 25 °C.c J values in Hz in parentheses.d Chemical shift data correspond to the major epimer (26S). Distinct resonances for the 26R epimer observed in the spectrum of the epimeric mixture are shown in bracket.
2	5.80, d (10.0)^c	5.98, dd (9.6, 2.4)	5.82, dd (10.0, 2.8)	3.37, d (20.0)
				2.65, dd (20.0, 4.8)
3	6.89, br d (10.0)	6.73, br d (9.6)	6.92, ddd (10.0, 5.2, 2.8)	5.62, dt (8.8, 4.8)
4	3.33, br d (20.8)	3.22, br d (21.2)	3.36, br d (22.0)	6.06, d (8.8)
	2.86, br d (20.8)	2.72, br d (21.2)	2.88, dd (22.0, 5.2)
6	5.61, br s	5.49, br s	5.62, br d (6.0)	5.67, br d (3.6)
7	2.00, m	1.87, m	2.00, m	2.19, m
	1.57, m	1.45, m	1.59, m	1.65, m
8	1.47, m	1.35, m	1.46, m	1.57, m
9	1.55, m	1.78, m	1.55, m	1.73, m
11	2.03, m	2.09, m	2.17, m	2.03, m
	1.68, m	1.96, m	1.70, m	1.69, m
12	1.77, m	2.22, m	1.76, m	1.76, m
	1.64, m	1.81, m	1.63, m	1.63, m
14	1.74, m	2.07, m	1.72, m	1.76, m
15	1.55, m	2.52, m	1.53, m	1.69, m
	1.16, m	1.55, dd (12.8, 3.2)	1.18, m	1.17, m
16	2.18, m	1.66, m	2.00, m	1.78, m
	1.71, m	1.11, dd (12.0, 5.6)	1.59, m	1.39, m
18	0.82, s	0.83, s [0.82, s]^d	0.82, s	0.82, s
19	1.24, s	1.23, s	1.24, s	1.38, s
20	2.09, m	2.44, m [2.41, m]	2.12, qd (7.2, 2.8)	2.09, m
21	1.01, d (6.4)	1.40, d (7.2)	1.00, d (7.2)	1.00, d (6.8)
22	3.82, d (11.2)	4.32, d (12.4) [4.99 d (12.0)]	3.81, br d (12.4)	3.82, d (12.0) [4.60, d (12.0)]
23	1.80, m	2.54, m	2.58, ddd (13.2, 12.4, 1.6)	1.83, m
	1.71, m	2.37, m	1.76, d (13.2)	1.69, m
26	4.61, s	5.49, s [5.59, s]	4.63, s	4.61, s
27	1.21, s	1.87, s [1.93, s]	1.22, s	1.21, s
28	1.32, s	2.13, s [2.02, s]	1.62, s	1.32, s
24-OAc			2.04, s

---

Table 3 ¹H-NMR spectral data for compounds 4–7

Position	4^a	5^a	6^a	7^a
a Spectra recorded at 400 MHz in methanol-d4 at 25 °C.b J values in Hz in parentheses.c Chemical shift data correspond to the major epimer (26S). Distinct resonances for the 26R epimer observed in the spectrum of the epimeric mixture are shown in brackets.
2	3.37, d (19.6)^b, 2.65, dd (19.6, 4.4)	5.77, dd (10.0, 2.8)	5.98, d (9.6)	6.17, d (10.0)
3	5.62, dd (9.6, 4.4)	6.64, ddd (10.0, 5.2, 2.0)	7.09, dd (9.6, 6.0)	7.06, dd (10.0, 6.5)
4	6.06, d (9.6)	3.32, dt (20.0, 2.4), 2.04, dd (20.0, 5.2)	6.23, d (6.0)	3.65, d (6.5)
6	5.67, br d (3.6)	3.52, br s	4.54, br s	3.16, br s
7	2.19, m	1.73, m	2.02, m	2.12, m
	1.69, m	1.55, m	1.22, m	1.34, m
8	1.59, m	1.79, m	2.08, m	1.43, m
9	1.72, m	1.78, m	1.66, m	0.83, m
11	2.03, m	2.00, m	2.03, m	2.01, m
	1.70, m	1.68, m	1.68, m	1.68, m
12	1.77, m	1.76, m	1.66, m	1.58, m
	1.62, m	1.57, m	1.55, m	1.52, m
14	1.76, m	1.81, m	1.64, m	1.59, m
15	1.72, m	2.24, m	1.81, m	1.69, m
	1.22, m	1.69, m	1.54, m	1.23, m
16	1.78, m	1.35, m	1.68, m	1.49, m
	1.38, m	1.19, m	1.23, m	1.15, m
18	0.82, s	0.84, s	0.88, s	0.78, s
19	1.38, s	1.30, s	1.46, s	1.37, s
20	2.12, m	2.10, m	2.09, m	2.06, m
21	1.00, d (6.8)	1.01, d (7.2)	0.99, d (6.0)	0.98, d (7.0)
22	3.81, d (12.4) [4.32, d (11.9)]^c	3.83, d (12.0) [4.33, d (12.0)]	3.81, dt (12.0, 2.8) [4.30, d (12.1)]	3.80, m [4.30, m]
23	2.58, ddd (14.0, 12.4, 2.4)	1.84, m	1.69, m	1.78, m
	1.75, m	1.70, m	1.79, m	1.68, m
26	4.63, s	4.61, s	4.60, s	4.60, s
27	1.22, s	1.22, s	1.21, s	1.20, s
28	1.62, s	1.33, s	1.32, s	1.32, s
24-OAc	2.03, s

---

Capsisteroid F (7), was also isolated as a white powder with a molecular formula of C₂₈H₄₂O₈, as revealed from its HRESIMS (m/z 529.2773 [M + Na]⁺), implying eight degrees of unsaturation. The NMR spectroscopic data of 7 (Tables 1 and 3) indicated that it is structurally similar to 1, with the difference being the presence of an epoxide in 7, rather than a Δ^5,6 double bond in 1. In addition, a hydrogen atom at C-4 in 1 was replaced by a hydroxy group. This suggested structure was further confirmed by the HMBC correlations of H-2 (δ 6.17) to C-4 (δ 71.3), H₃-19 (δ 1.37), H-6 (δ 3.16) to C-5 (δ 64.9), C-8 (δ 31.6) and C-10 (δ 49.8), and H-4 (δ 3.65) to C-5, along with the COSY correlations of H-3 (δ 7.06) and H-4, and the epoxy proton H-6 and H-7 (δ 2.12). In the NOESY spectrum, correlations of H-8/H₃-19 and H-7β (δ 2.12)/H-8 suggested that H-8 and H₃-19 are β-orientated. Besides, NOE correlations of both H-4 and H-6 with H-7α (δ 1.34) suggested that H-4 and H-6 are α-oriented. ¹H and ¹³C NMR spectroscopic data for compound 7 were assigned from the above results and the supporting evidence from DEPT, HSQC, and HMBC spectral studies.

Compounds 2–7 showed characteristic H-22 epimeric protons in the ¹H NMR spectrum and the pair of signals observed for C-22 and C-26 in the ¹³C NMR spectrum revealed that all of these compounds appeared as C-26 epimeric mixtures, similar to that of 1.

The cytotoxicity of compounds 1–7 against the proliferation of a limited panel of cancer cell lines, including human erythro myeloblastoid leukemia (K562), human acute lymphoblastic leukemia (Molt-4) and human promyelocytic leukemia (HL-60), was evaluated. However, none of the compounds showed any appreciable cytotoxicity at 20 μM. The in vitro pro-inflammatory activities of compounds 1–7 were evaluated by suppressing N-formyl-methionyl-leucyl-phenyl-alanine/cytochalasin B (fMLP/CB)-induced superoxide anion (O₂⁻˙) generation and elastase release in human neutrophils. As shown in Table 4, compounds 1, 2 and 6 were found to inhibit superoxide anion generation (20.9 ± 2.3, 21.1 ± 6.8 and 34.0 ± 3.1%, respectively) at a concentration of 10 μM. On the other hand, compounds 1, 2, 5 and 6 showed some inhibition toward elastase release in the same fMLP/CB-stimulated cells at the same concentration.

Table 4 Inhibitory effects of compounds 1–7 on superoxide anion generation and elastase release by human neutrophils

Compound	Superoxide anion		Elastase release
	Inh^a%		Inh%
a Percentage of inhibition (Inh%) at 10 μM concentration. Results are presented as mean ± S.E.M. (n = 3 or 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the value.b Positive control.
1	20.9 ± 2.3	***	19.2 ± 5.3	*
2	21.1 ± 6.8	*	15.8 ± 3.7	*
3	13.2 ± 2.9	**	10.3 ± 5.1
4	11.2 ± 5.1		11.5 ± 6.1
5	7.4 ± 3.8		26.9 ± 6.6	*
6	34.0 ± 3.1	***	15.6 ± 5.5	*
7	7.9 ± 0.3	***	11.1 ± 3.0	*
LY294002^b	100.3 ± 2.5%	***	90.6 ± 5.1%	***

---

Conclusions

A known withanolide steroid cilistol G (1) and six new withanolides, capsisteroids A–F (2–7), were discovered from the leaves of S. capsicoides. Compounds 1, 2, and 6 showed anti-inflammatory activity to inhibit superoxide anion generation and elastase release in human neutrophils. This study also demonstrates that S. capsicoides could be a source of bioactive natural products. Further chemical investigation of this plant might lead to the discovery of other metabolites with stronger bioactivities.

Experimental section

General experimental procedures

Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer. NMR spectra were recorded on a Varian 400 MR FT-NMR instrument at 400 MHz for ¹H and 100 MHz for ¹³C. LRMS and HRMS were obtained by ESI on a Bruker APEX II mass spectrometer. Silica gel (Merck, 230–400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. High-performance liquid chromatography was performed on a Hitachi L-7100 HPLC apparatus with a Supelco C18 column (250 × 21.2 mm, 5 μm).

Plant material

The fresh leaves of S. capsicoides were collected during July 2013 from Pingtung County, southern Taiwan. This plant material was identified by H.-Y. Liu, Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan. A voucher specimen (SC-20130701) was deposited in the Department of Marine biotechnology, National Sun Yat-sen Univeristy, Kaohsiung, Taiwan.

Extraction and separation

The air-dried leaves (132.1 g) were powdered and extracted with EtOAc at room temperature. The filtrate was evaporated under reduce pressure to give a residue (22.0 g), which was loaded onto a silica gel column and the column was eluted with a gradient of a n-hexane–acetone (0–100%), which afforded 20 fractions. Fractions 10 and 11, eluting with n-hexane–acetone (1 : 1), were rechromatographed over a reversed-phase RP-18 column using MeOH and H₂O (9 : 1) as the mobile phase to afford four subfractions (A1–A4). Subfractions A2 and A3 were separated by reversed-phase HPLC (MeOH–H₂O, 5 : 2) to afford compounds 1 (100.1 mg), 2 (1.5 mg), 3 (10.3 mg), and 4 (1.3 mg), respectively. Fraction 15, eluted with acetone (100%), was rechromatographed over a reversed-phase RP-18 column using MeOH and H₂O (3 : 1) as the mobile phase to afford four subfractions (B1–B8). Subfraction B4 was separated by reversed-phase HPLC (MeOH–H₂O, 2 : 3 and 1 : 1) to afford compounds 5 (2.5 mg), 6 (8.4 mg), and 7 (1.1 mg), respectively.

Cilistol G (1). Yellow oil; [α]²³_D = −9 (c 0.0012, MeOH); IR (neat) ν_max 3460 cm⁻¹; ¹³C and ¹H NMR data, see Tables 1 and 2; ESIMS m/z 497 [M + Na]⁺; HRESIMS m/z 497.2874 [M + Na]⁺ (calcd for C₂₈H₄₂O₆Na, 497.2874).

Capsisteroid A (2). Yellow oil; [α]²³_D = +49 (c 0.004, MeOH); IR (neat) ν_max 3458 and 1735 cm⁻¹; ¹³C and ¹H NMR data, see Tables 1 and 2; ESIMS m/z 539 [M + Na]⁺; HRESIMS m/z 539.29817 [M + Na]⁺ (calcd for C₃₀H₄₄O₇Na, 539.29792).

Capsisteroid B (3). Colorless oil; [α]²³_D = +21 (c 0.0071, MeOH); IR (neat) ν_max 3479 cm⁻¹; ¹³C and ¹H NMR data, see Tables 1 and 2; ESIMS m/z 497 [M + Na]⁺; HRESIMS m/z 497.2876 [M + Na]⁺ (calcd for C₂₈H₄₂O₆Na, 497.2874).

Capsisteroid C (4). Colorless oil; [α]²³_D = +7 (c 0.0037, MeOH); IR (neat) ν_max 3446 and 1733 cm⁻¹; ¹³C and ¹H NMR data, see Tables 1 and 3; ESIMS m/z 539 [M + Na]⁺; HRESIMS m/z 539.29768 [M + Na]⁺ (calcd for C₃₀H₄₄O₇Na, 539.29792).

Capsisteroid D (5). Colorless oil; [α]²³_D = +20 (c 0.7, MeOH); IR (neat) ν_max 3404 cm⁻¹; ¹³C and ¹H NMR data, see Tables 1 and 3; ESIMS m/z 531 [M + Na]⁺; HRESIMS m/z 531.2931 [M + Na]⁺ (calcd for C₂₈H₄₄O₈Na, 531.2928).

Capsisteroid E (6). Colorless oil; [α]²³_D = −75 (c 0.3, MeOH); IR (neat) ν_max 3383 cm⁻¹; ¹³C and ¹H NMR data, see Tables 1 and 3; ESIMS m/z 513 [M + Na]⁺; HRESIMS m/z 513.2820 [M + Na]⁺ (calcd for C₂₈H₄₂O₇Na, 513.2823).

Capsisteroid F (7). Colorless oil; [α]²³_D = +18 (c 0.34, MeOH); IR (neat) ν_max 3395 cm⁻¹; ¹³C and ¹H NMR data, see Tables 1 and 3; ESIMS m/z 529 [M + Na]⁺; HRESIMS m/z 529.2773 [M + Na]⁺ (calcd for C₂₈H₄₂O₈Na, 529.2771).

Cytotoxicity testing

Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of compounds 1–7 were performed using the Alamar Blue assay.^36,37

Preparation of human neutrophils

Blood samples were obtained from healthy volunteers aged 20–30 years old after written informed consent was obtained. The study protocol was investigated and approved by the Institutional Review Board at Chang Gung Memorial Hospital. The methods were carried out in accordance with the approved guidelines. Neutrophils were isolated by standardized procedures that included dextran sedimentation, gradient centrifugation using Ficoll-Hypaque, and hypotonic lysis of erythrocytes. Purified neutrophils were more than 98% viable as calculated by the trypan blue exclusion technique. The cells were suspended in ice cold Ca21-free Hank’s balanced salts solution (HBSS).

Superoxide anion generation and elastase release by human neutrophils

Human neutrophils were obtained using dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were performed according to previously described procedures.^38,39 LY294002, a phosphatidylinositol-3-kinase inhibitor, was used as a positive control for inhibition of superoxide anion generation and elastase release with IC₅₀ 1.4 ± 0.1 (inhibition, 100.3 ± 2.5% in 10 μg ml⁻¹) and 5.5 ± 0.8 μM (inhibition, 90.6 ± 5.1% in 10 μg ml⁻¹).⁴⁰

X-ray diffraction analysis of cilistol G (1)

A suitable colorless crystal (0.5 × 0.3 × 0.03 mm³) of 1 was grown by slow evaporation of the MeOH–H₂O (5 : 1) solution. Diffraction intensity data were acquired with a CCD area detector with graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). Crystal data for 1: C₂₈H₄₇O_8.5 (molecular weight 519.65), approximate crystal size, 0.5 × 0.3 × 0.03 mm³, orthorhombic, space group, P2₁2₁2₁ (# 19), T = 100(2) K, a = 7.5227(4) Å, b = 9.6240(6) Å, c = 37.396(2) Å, V = 2707.4(3) ų, D_c = 1.275 Mg m⁻³, Z = 4, F(000) = 1132, μ_(CuKα) = 0.758 mm⁻¹. A total of 13 445 reflections were collected in the range 2.363° < θ < 66.257°, with 4384 independent reflections [R(int) = 0.0520], completeness to θ_max was 92.0%; psi-scan absorption correction applied; full-matrix least-squares refinement on F², the number of data/restraints/parameters were 4384/3/344; goodness-of-fit on F² = 1.113; final R indices [I > 2σ(I)], R₁ = 0.0936, wR₂ = 0.2351; R indices (all data), R₁ = 0.0943, wR₂ = 0.2355, largest difference peak and hole, 0.416 and −0.443 e Å⁻³. The structure was refined as a two component inversion twin and that the absolute configuration could not be determined from the X-ray crystallographic data.⁴¹

Acknowledgements

Financial support awarded to J.-H. S was provided by the National Science Council of Taiwan (NSC-100-2320-B-110-001-MY2), and NSYSU-KMU JOINT RESEARCH PROJECT (NSYSUKMU 02C030117) from National Sun Yat-sen University and Kaohsiung Medical University.

Notes and references

1. V. E. Nicotra, N. S. Ramacciotti, R. R. Gil, J. C. Oberti, G. E. Feresin, C. A. Guerrero, R. F. Baggio, M. T. Garland and G. Burton, J. Nat. Prod., 2006, 69, 783–789.
2. E. J. Kennelly, C. Gerhäuser, L. L. Song, J. G. Graham, C. W. W. Beecher, J. M. Pezzuto and A. D. Kinghorn, J. Agric. Food Chem., 1997, 45, 3771–3777.
3. X.-H. Zhu, M. Takagi, T. Ikeda, K. Midzuki and T. Nohara, Phytochemistry, 2001, 56, 741–745.
4. X.-H. Zhu, J. Ando, M. Takagi, T. Ikeda, A. Yoshimitsu and T. Nohara, Chem. Pharm. Bull., 2001, 49, 1440–1443.
5. X.-H. Zhu, J. Ando, M. Takagi, T. Ikeda and T. Nohara, Chem. Pharm. Bull., 2001, 49, 161–164.
6. R. Niero, I. T. da Silva, G. C. Tonial, B. D. S. Camacho, E. Gacs-Baitz, G. Delle Monache and F. Delle Monache, Nat. Prod. Res., 2006, 20, 1164–1168.
7. B.-Y. Yang, R. Guo, T. Li, J.-J. Wu, J. Zhang, Y. Liu, Q.-H. Wang and H.-X. Kuang, Steroids, 2014, 87, 26–34.
8. S.-T. Fang, J.-K. Liu and B. Li, Steroids, 2012, 77, 36–44.
9. H. Zhang, C.-M. Cao, R. J. Gallagher, V. W. Day, G. Montenegro and B. N. Timmermann, Phytochemistry, 2014, 98, 232–235.
10. B.-N. Su, R. Misico, E. J. Park, B. D. Santarsiero, A. D Mesecar, H. H. S. Fong, J. M. Pezzuto and A. D. Kinghorn, Tetrahdron, 2002, 58, 3453–3466.
11. B. Jayaprakasam and M. G. Nair, Tetrahdron, 2003, 59, 841–849.
12. L. Ma, M. Ali, M. Arfan, L.-G. Lou and L.-H. Hu, Tetrahdron Lett., 2007, 48, 449–452.
13. A. M. Cirigliano, A. S. Veleiro, R. I. Misico, M. C. Tettamanzi, J. C. Oberti and G. Burton, J. Nat. Prod., 2007, 70, 1644–1646.
14. I. -ul-Haq, U. J. Youn, X. Chai, E.-J. Park, T. P. Kondratyuk, C. J. Simmons, R. P. Borris, B. Mirza, J. M. Pezzuto and L. C. Chang, J. Nat. Prod., 2013, 76, 22–28.
15. M. Kuroyanagi, M. Murata, T. Nakane, O. Shirota, S. Sekita, H. Fuchino and Z. K. Shinwari, Chem. Pharm. Bull., 2012, 60, 892–897.
16. H. Zhang, H. Motiwala, A. Samadi, V. Day, J. Aubé, M. Cohen, K. Kindscher, R. Gollapudi and B. Timmermann, Chem. Pharm. Bull., 2012, 60, 1234–1239.
17. C.-M. Cao, H. Zhang, R. J. Gallagher, V. W. Day, K. Kindscher, P. Grogan, M. S. Cohen and B. N. Timmermann, J. Nat. Prod., 2014, 77, 631–639.
18. H. Zhang, J. Bazzill, R. J. Gallagher, C. Subramanian, P. T. Grogan, V. W. Day, K. Kindscher, M. S. Cohen and B. N. Timmermann, J. Nat. Prod., 2013, 76, 445–449.
19. M. B. Ksebati and F. J. Schmitz, J. Org. Chem., 1988, 53, 3926–3929.
20. C.-Y. Huang, C.-C. Liaw, B.-W. Chen, P.-C. Chen, J.-H. Su, P.-J. Sung, C.-F. Dai, M. Y. Chiang and J.-H. Sheu, J. Nat. Prod., 2013, 76, 1902–1908.
21. C.-H. Chao, K.-J. Chou, Z.-H. Wen, G.-H. Wang, Y.-C. Wu, C.-F. Dai and J.-H. Sheu, J. Nat. Prod., 2011, 74, 1132–1141.
22. S. Minguzzi, L. E. S. Barata, Y. G. Shin, P. F. Jonas, H.-B. Chai, E. J. Park, J. M. Pezzuto and G. A. Cordell, Phytochemistry, 2002, 59, 635–641.
23. P.-W. Hsieh, Z.-Y. Huang, J.-H. Chen, F.-R. Chang, C.-C. Wu, Y.-L. Yang, M.-Y. Chiang, M.-H. Yen, S.-L. Chen, H.-F. Yen, T. Kübken, W.-C. Hung and Y.-C. Wu, J. Nat. Prod., 2007, 70, 747–753.
24. K. Vermillion, F. Omar Holguin, M. A. Berhow, R. D. Richins, T. Redhouse, M. A. O’Connell, J. Posakony, S. S. Mahajan, S. M. Kelly and J. A. Simon, J. Nat. Prod., 2011, 74, 267–271.
25. V. Roumy, M. Biabiany, T. Hennebelle, E. M. Aliouat, M. Pottier, H. Joseph, S. Joha, B. Quesnel, R. Alkhatib, S. Sahpaz and F. Bailleul, J. Nat. Prod., 2010, 73, 1313–1317.
26. H. Zhang, A. K. Samadi, R. J. Gallagher, J. J. Araya, X. Tong, V. W. Day, M. S. Cohen, K. Kindscher, R. Gollapudi and B. N. Timmermann, J. Nat. Prod., 2011, 74, 2532–2544.
27. G. V. Subbaraju, M. Vanisree, C. V. Rao, C. Sivaramakrishna, P. Sridhar, B. Jayaprakasam and M. G. Nair, J. Nat. Prod., 2006, 69, 1790–1792.
28. R. P. Machin, A. S. Veleiro, V. E. Nicotra, J. C. Oberti and J. M. Padrón, J. Nat. Prod., 2010, 73, 966–968.
29. J. Bravo B, M. Sauvain, A. Gimenez T, E. Balanza, L. Serani, O. Laprévote, G. Massiot and C. Lavaud, J. Nat. Prod., 2001, 64, 720–725.
30. S. A. Jamal, S. Qureshi, S. N. Ali and M. Choudhary, Khim. Geterotsikl. Soedin., 1995, 9, 1200–1213.
31. B.-N. Su, R. Misico, E. J. Park, B. D. Santarsiero, A. D. Mesecar, H. H. S. Fong, J. M. Pezzuto and A. D. Kinghorn, Tetrahedron, 2002, 58, 3453–3466.
32. J. G. Luis, F. Echeverri, F. Garcia and M. Rojas, Planta Med., 1994, 60, 348–350.
33. B. Shohat, I. Kirson and D. Lavie, Biomedicine, 1978, 28, 18–24.
34. B.-N. Su, E. J. Park, D. Nikolic, B. D. Santarsiero, A. D. Mesecar, J. S. Vigo, J. G. Graham, F. Cabieses, R. B. van Breemen, H. H. S. Fong, N. R. Farnsworth, J. M. Pezzuto and A. D. Kinghorn, J. Org. Chem., 2003, 68, 2350–2361.
35. R. Saijo, C. Fuke, K. Murakami, T. Nohara and T. Tomimatsu, Phytochemistry, 1983, 22, 733–736.
36. G. R. Nakayama, M. C. Caton, M. P. Nova and Z. Parandoosh, J. Immunol. Methods, 1997, 204, 205–208.
37. J. O’Brien, I. Wilson, T. Orton and F. Pognan, Eur. J. Biochem., 2000, 267, 5421–5426.
38. T.-L. Hwang, C.-C. Wang, Y.-H. Kuo, H.-C. Huang, Y.-C. Wu, L.-M. Kuo and Y.-H. Wu, Biochem. Pharmacol., 2010, 80, 1190–1200.
39. T.-L. Hwang, Y.-L. Leu, S.-H. Kao, M.-C. Tang and H.-L. Chang, Free Radical Biol. Med., 2006, 41, 1433–1441.
40. Y.-N. Lee, C.-J. Tai, T.-L. Huang and J.-H. Sheu, Mar. Drugs, 2014, 12, 1148–1156.
41. ESI†.

---

Footnote

† Electronic supplementary information (ESI) available: NMR spectra data for new compounds 1–7. CCDC 1057315. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra12014d

---