C19-Norditerpenoid Alkaloids from Aconitum szechenyianum.

Three new C19-norditerpenoid alkaloids (1⁻3), along with two known C19-norditerpenoid alkaloids (4,5), have been isolated from Aconitum szechenyianum. Based on extensive spectroscopic techniques (1D, 2D-NMR, IR, and MS) and chemical methods, their structures were established as szechenyianine D (1), szechenyianine E (2), szechenyianine F (3), 8-O-methyl-14-benzoylaconine (4), and spicatine A (5). The immunosuppressive effects of compounds 1⁻5 were studied using a ConA-induced or LPS-induced splenocyte proliferation model. In vitro tests showed that Compounds 2, 4, and 5 suppressed ConA-induced or LPS-induced splenocyte proliferation in a concentration-dependent manner. The CC50/IC50 values of 2, 4, and 5 suggested that these compounds were potential immunosuppressive agents for the treatment of autoimmune diseases characterized by arthritis, such as rheumatoid arthritis.

1. Introduction

The roots of Aconitum szechenyianum Gay. and n class="Species">A. flavum Hand.-Mazz., which belong to the Aconitum genus of Ranunculaceae, are widely used in folk medicine in Shaanxi province in China [1]. C19- and C20-diterpenoid alkaloids possessing aconitine-type, 7,17-secoaconitine-type, and napeline-type skeletons, which are the main components of A. szechenyianum [2,3,4,5], possess anti-inflammatory, analgesic, anticancer, anti-epileptiform, and antiparasitic activities [6,7,8]. A. szechenyianum has been preliminarily studied; in that study, several norditerpenoid alkaloids were obtained with an aconitine or 7,17-secoaconitine skeleton, and these skeleton were demonstrated to have anti-inflammatory activities in a dose-dependent manner [9]. This paper reports a new investigation on A. szechenyianum, conducted to explore more bioactive lead compounds, and three new, along with two known, C19-norditerpenoid alkaloids—szechenyianine D (1), E (2), F (3), 8-O-methyl-14-benzoylaconine [10] (4), and spicatine A [11] (5)—were isolated in different fractions from the previous study (Figure 1). The previous study shows that the active ingredients of A. flavum exhibit immunosuppressive effects [12,13], which had not been previously reported from A. szechenyianum. Therefore, the immunosuppressive effects of Compounds 1–5 were evaluated in vitro through ConA- or LPS-induced splenocyte proliferation models. Three compounds inhibited ConA- or LPS-induced splenocyte proliferation, revealing for the first time that the roots of A. szechenyianum possess immunosuppressive activities.

Figure 1

Structures of Compounds 1–5.

2. Results

Szechenyianine D (1) was isolated as a white amorphous powder and showed a positive reaction with Dragendorff′s reagent. Its molecular formula n class="Chemical">C31H43NO10 was derived from the protonated molecular ion peak at m/z 590.2979 [M + H]+ (calcd. 590.2965) in the HR-ESI-MS spectrum. The 1H-NMR spectrum (Table 1) of 1 showed the presence of five aromatic proton signals due to a monosubstituted benzene at δH 8.00 (2H, d, J = 7.4 Hz), 7.54 (1H, t, J = 7.4 Hz), and 7.44 (2H, t, J = 7.4 Hz), five OMe protons at δH 3.80(3H, s), 3.37 (3H, s), 3.29 (over-lapped), 3.29 (over-lapped), and 3.21 (3H, s), and two strongly shielded protons at δH 3.32 (1H, s) and δH 4.29 (1H, s). The 13C-NMR spectrum (Table 1) displayed 31 carbon resonances. Among them, resonances at δC 166.4, 133.4, 130.0, 130.0 (C × 2), and 128.7 (C × 2) were attributed to a benzoyloxy group; δC 62.2, 59.3, 59.0, 55.4, and 50.7 were attributed to five OMe groups; δC 74.7 and 76.4 were attributed to two oxygenated carbons associated with hydroxyl groups. Out of the 10 oxygen atoms in 1, 9 are associated with five methoxy groups, two hydroxyl groups, and one benzoyl group, and the remaining one may be a hydroxyl group or an internal ether. The NMR features of the remaining 19 resonances were characteristic of an aconitine-type alkaloid, where the δC 64.4 and 70.0 resonances were attributed to the two carbons associated with an internal ether. The deduction was confirmed by the chemical shift of C-7 (δC 64.4) and C-17 (δC 70.0) to downfield in 13C-NMR spectra of 1 compared with C-7 (δC 49.6) and C-17 (δC 60.6) of szechenyianine A [9], the signals of which are shielded by oxygen atom. In the HMBC spectrum (Figure 2), correlations of H-5 (δH 2.41) and H-6 (δH 4.12) to C-7 (δC 64.4), and H-1 (δH 3.45) to C-17 (δC 70.0), suggested the involvement of an internal ether bond. The correlation of H-14 (δH 4.84) to the carbonyl carbon signal of the benzoyl group (δC 166.4) suggested that the benzoyl group was located at C-14. The correlations of OCH3 (δH 3.37) to C-1 (δC 80.3), OCH3 (δH 3.29) to C-6 (δC 82.2), OCH3 (δH 3.21) to C-8 (δC 83.3), OCH3 (δH 3.80) to C-16 (δC 93.2), and OCH3 (δH 3.29) to C-18 (δC 76.6) suggested that five methoxyl groups were linked at C-1, C-6, C-8, C-16, and C-18. The correlations of H-12 (δH 1.84, 2.24) and H-14 (δH 4.84) to C-13 (δC 74.7), and H-16 (δH 3.22) to C-15 (δC 76.4), suggested that two hydroxyl groups were linked at C-13 and C-15. Thus, the planar structure of 1 was deduced as 14-benzoyloxy-13, 15-dihydroxy-1, 6,8,16,18-pentamethoxyl-7(17)-oxide-aconitane. In the ROESY spectrum (Figure 2) of 1, the NOE correlations of H-1/H-3, H-3/H-5, H-5/H-10, H-10/H-9, H-10/H-14, H-14/H-9, and H-9/H-6 indicated β-orientation of H-1, H-5, H-6, H-9, H-10, and H-14, and α-axial configurations of 1-OCH3, 6-OCH3, and 14-benzoyloxy. NOE correlations of H-6/H-5 and H-5/H-18 revealed β-orientation of H-18 and 18-OCH3; NOE correlations of H-17/H-7, H-15/16-OCH3 revealed α-axial orientation of H-16, H-17, and 15-OH and β-orientation of 16-OCH3, 13-OH, and 8-OCH3. Moreover, the NOE correlations of H-1/H-3 and H-5 and the lack of correlation between H-2 and H-5 indicated that ring A (C-1, C-2, C-3, C-4, C-5, and C-11) in 1 was in the chair conformation. Thus, Compound 1 was assigned the name (A-c)-14α-benzoyloxy-13β,15α-dihydroxy-1α,6α,8β,16β,18β-pentamethoxy-7(17)-oxide-aconitane.

Table 1

1H-NMR and 13C-NMR spectral data for Compounds 1–5.

NO.	1		2		3		4	5
	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δC
1	80.3	3.45 (d, 7.6)	82.4	3.07 (d, 8.3)	80.6	3.40 (m)	82.8	82.8
2	29.6	1.22 (m, H-2a)	25.3	1.43 (m, H-2a)	20.6	1.40 (m, H-2a)	33.6	33.6
		2.41 (m, H-2b)		1.87 (m, H-2b)		1.72 (m, H-2b)
3	29.9	1.22 (m, H-3a)	33.4	2.61 (m, H-3a)	25.5	1.94 (m)	72.0	71.9
		1.40 (m, H-3b)		2.71 (m, H-3b)
4	43.5		37.8		48.6		43.3	43.2
5	42.3	2.41 (d, 6.7)	47.6	2.29 (d, 6.7)	38.4	2.46 (m)	46.2	45.9
6	82.2	4.12 (d,6.7)	83.3	4.04 (d, 6.7)	24.6	1.82 (m, H-6a)2.20 (m, H-6b)	83.6	83.7
7	64.4	3.32 (s)	50.9	2.63 (s)	45.1	2.22 (m)	45.4	43.3
8	83.3		91.3		72.3		82.6	82.5
9	44.5	2.59 (t, 5.8)	43.5	2.84 (t, 5.8)	53.8	2.47 (m)	42.7	45.4
10	40.8	2.26 (m)	41.1	2.19 (m)	38.3	2.01 (m)	41.7	41.6
11	50.9		50.0		51.2		50.8	50.8
12	35.5	2.24 (m, H-12a)	35.6	2.80 (m, H-12a)	27.8	2.04 (m, H-12a)1.26 (m, H-12b)	36.5	36.5
		1.84 (m, H-12b)		2.19 (m, H-12b)
13	74.7		74.3		43.3	1.96 (m)	75.0	75.0
14	78.8	4.84 (d, 5.8)	78.8	4.89 (d, 5.8)	75.0	4.21 (t, 4.9)	79.7	79.8
15	76.4	4.65 (d, 5.4)	79.0	4.49 (dd, 2.9, 5.4)	39.5	2.27 (m, H-15a)	78.0	78.7
						2.40 (m, H-15b)
16	93.2	3.22 (d, 5.4)	90.4	3.30 (d, 5.4)	81.5	3.45 (m)	93.6	93.6
17	70.0	4.29 (s)	56.7	4.11 (s)	68.1	3.79 (s)	62.7	61.4
18	76.6	3.54(d, 8.2, H-18a)	80.0	3.78(d,8.2,H-18a)	73.4	3.71 (2H, m)	77.2	77.2
		3.46 (d,8.2, H-18b)		3.06(d,8.2,H-18b)
19	50.3	3.62(d,11.9,H-19a)	173.3		179.2	9.19 (s)	49.2	49.2
		3.72(d,11.9,H-19b)
20			45.5	3.04 (m, H-20a)
				3.92 (m, H-20b)
21			33.7	1.51 (m, H-21a)
				1.62 (m, H-21b)
22			25.0	1.59 (m, H-22a)
				1.68 (m, H-22b)
23			29.6	1.30 (m, H-23a)
				1.23 (m, H-23b)
24			31.9	1.30 (m, H-24a)
				2.26 (m, H-24b)
25			22.9	1.30 (2H, m)
26			14.3	0.86 (3H, t,6.2)
8-OAc			172.5
			21.6	1.35 (s)
8-OCH2CH3								57.4
8-OCH2CH3								15.5
1-OCH3	55.4	3.37 (s)	55.5	3.19 (s)	56.7	3.16 (s)	56.1	56.1
6-OCH3	59.3	3.29 (s)	58.0	3.12 (s)			59.4	58.8
8-OCH3	50.7	3.21 (s)					50.1
16-OCH3	62.2	3.80 (s)	61.5	3.75 (s)	56.9	3.36 (s)	61.4	62.6
18-OCH3	59.0	3.29 (s)	59.4	3.30 (s)	59.8	3.38 (s)	59.3	59.3
N-CH2CH3					56.3	4.01 (dq, 13.9, 7.2)	47.6	47.6
						4.42 (dq, 13.9, 7.2)
N-CH2CH3					14.0	1.51 (t, 7.2)	13.6	13.5
ArC=O	166.4		166.2				166.5	166.4
ArC-1′	130.0		129.9				130.4	130.6
3′, 5′	128.7	7.44 (t, 7.4)	128.9	7.45 (t, 7.5)			128.6	128.6
2′, 6′	130.0	8.00 (d, 7.4)	129.8	8.01 (d, 7.5)			129.9	129.9
4′	133.4	7.54 (t, 7.4)	133.6	7.56 (t, 7.5)			133.1	133.1

δ in CDCl3, in ppm from TMS; coupling constants (J) in Hz; 1H-NMR at 400 MHz and 13C-NMR at 100 MHz for Compounds 1, 3, 4, and 5, and 1H-NMR at 600 MHz and 13C-NMR at 150 MHz for Compound 2.

Figure 2

Key HMBC (H→C) and ROESY (H↔H) correlations of Compound 1.

Szechenyianine E (2) was isolated as a white amorphous powder and showed a positive reaction with Dragendorff′s reagent. Its molecular formula C39H55n class="Chemical">NO11 was derived from the protonated molecular ion peak at m/z 714.3840 [M + H]+ (calcd.714.3853) in the HR-ESI-MS spectrum. The 1H-NMR spectrum (Table 1) of 2 showed the presence of five aromatic proton signals due to a monosubstituted benzene at δH 8.01 (2H, d, J = 7.5 Hz), 7.56 (1H, t, J = 7.5 Hz), 7.45 (2H, t, J = 7.5 Hz), four OMe protons at δH 3.75 (3H, s), 3.30 (3H, s), 3.19 (3H s), and 3.12 (3H, s); one acetoxyl proton at δH 1.35 (3H, s), and one methylic proton of the hydrocarbon chain at δH 0.86 (3H, t, J = 6.2 Hz). The 13C-NMR spectrum (Table 1) displayed 39 carbon resonances. Among them, the resonances at δC 166.2, 133.6, 129.9, 129.8 (C × 2), and 128.9 (C × 2) were attributed to a benzoyloxy group; δC 61.5, 59.4, 58.0, and 55.5 were attributed to four OMe groups, δC 172.5 and 21.6 were attributed to one acetoxyl group; δC 173.3 was attributed to C=O, δC 74.3 and 79.0 were attributed to two carbons associated with the hydrocarbon chain, and δC 14.3 was attributed to one CH3 group. The assignments of the NMR signals associated with 2 were derived from HSQC, HMBC, and ROESY experiments. In the HMBC spectrum (Figure 3), correlations of H-14 (δH 4.89) to the carbonyl carbon signal of the benzoyl group (δC 166.4) suggested that the benzoyl group was located at C-14. Correlations of OCH3 (δH 3.19) to C-1 (δC 82.4), OCH3 (δH 3.12) to C-6 (δC 83.3), OCH3 (δH 3.75) to C-16 (δC 90.4), and OCH3 (δH 3.30) to C-18 (δC 80.0) suggested that four methoxyl groups were linked at C-1, C-6, C-16, and C-18, respectively. Correlations of CH3 (δH 1.32) to 8-OAc (δC 172.5) suggested that one acetoxyl was linked at C-8, and correlations of H-3 (δH 2.61, 2.71), H-17 (δH 4.11), and H-20 (δH 3.04, 3.92) to C-19 (δC 173.3) suggested that C=O was linked at C-19. Correlations of H-20 (δH 3.04, 3.92) to C-17 (δC 56.7) and C-21 (δC 33.7), H-21 (δH 1.51, 1.62) to C-22 (δC 25), and H-22 (δH 1.59, 1.68), H-25 (δH 1.30, 2H, m), and H-26 (δH 0.86, 3H, t) to C-24 (δC 31.9) suggested the presence of an N-heptyl group. Correlations of H-12 (δH 2.19, 2.80), H-14 (δH 4.89), and H-16 (δH 3.30) to C-13 (δC 74.3), and H-16 (δH 3.30) to C-15 (δC 79.0) suggested that two hydroxyl groups were linked at C-13 and C-15, respectively. This compound differed from the known compound (A-c)-8β-acetoxy-14α-benzoyloxy-N-ethyl-13β,15α-dihydroxy-1α,6α,16β,18β-tetramethoxy-19-oxo-aconitane [14] only in terms of the substituents on the N atom. According to the ROESY (Figure 3) spectrum, NOE correlations of H-6/H-5 and H-5/H-18 revealed β-orientation of H-18 and 18-OCH3, α-axial orientation of 6-OCH3; NOE correlations of H-7/H-15, H-17/H-16 revealed α-axial orientation of H-16, H-17, and 15-OH and β-orientation of 16-OCH3, 13-OH, and 8-OAc. Moreover, the NOE correlations of H-3/H-1/H-10/H-9/H-6/H-5 and the lack of correlation between H-2 and H-5 indicated that Ring A (C-1, C-2, C-3, C-4, C-5, and C-11) in 2 was in the chair conformation, the relative configuration of this compound was confirmed. Thus, the planar structure of 2 was assigned the name (A-c)-14α-benzoyloxy-8β-acetoxyl-N-heptyl-13β,15α-dihydroxy-1α,6α,16β,18β-tetramethoxy-19-oxo-aconitane.

Figure 3

Key HMBC (H→ C) and ROESY (H↔H) correlations of Compound 2.

Szechenyianine F (3) was isolated as a white amorphous powder and showed a positive reaction with Dragendorff′s reagent. Its molecular formula n class="Chemical">C24H38NO5+ was derived from the ion peak at m/z 421.2782[M]+ (calcd.421.2823) in the HR-ESI-MS spectrum. The 1H-NMR spectrum (Table 1) of 3 showed the presence of a methine proton due to one N=CH group at δH 9.19 (1H, s), one N–CH2CH3 group at δH 1.51 (t, J =7.2 Hz), 4.01 (dq, J =7.2, 13.9 Hz ), and 4.42 (dq, J =7.2, 13.8 Hz), and three OMe resonances at δH 3.38 (3H, s), 3.36 (3H, s), and 3.16 (3H, s). The 13C-NMR spectrum (Table 1) displayed 24 carbon resonances. Among them, the resonances at δC 59.8, 56.9, and 56.7 were attributed to three OMe groups, δC 179.2 was attributed to one N=CH group, and δC 14.0 and 56.3 were attributed to one N–CH2CH3 group. Comparison of the NMR data of N–CH2CH3 and C-19 with those of the known compound 11 in [15] indicated the existence of the ＋N=CH group. The assignments of the NMR signals associated with 3 were based on HSQC, HMBC, and ROESY experiments. In the HMBC spectrum (Figure 4), correlations of H-5 (δH 2.46) and H-17 (δH 3.79) to C-19 (δC 179.2) suggested that C-19 was involved in the N=CH group. Correlations of OCH3 (δH 3.18) to C-1 (δC 80.6), OCH3 (δH 3.36) to C-16 (δC 81.5) and of OCH3 (δH 3.38) to C-18 (δC 73.4) suggested that three methoxyl groups were linked at C-1, C-16, and C-18, respectively. Correlations of H-6 (δH 1.82, 2.20), H-7 (δH2.22), H-9 (δH 2.47), and H-10 (δH 2.01) to C-8 (δC 72.3) and of H-16 (δH 3.45) to C-14 (δC 75.0) suggested that two hydroxyl groups were linked at C-8 and C-14, respectively. Thus, the planar structure of 3 was deduced as 8,14-dihydroxy-1,16,18-trimethoxy-19-en-aconitane. In the ROESY spectrum (Figure 4) of 3, the NOE correlations of H-1/H-5, H-1/H-10, and H-10/H-14 indicated β-orientation of H-1, H-9, H-10, and H-14, and α-axial configurations of 1-OCH3, 14-OH. NOE correlations of H-5/H-18 indicated β-orientation of H-18 and 18-OCH3. NOE correlations of H-17/H-12, H-12/H-16, and H-15/H-16 indicated α-axial configurations of H-16, H-17, and 16-OCH3 and β-orientation of 8-OH. Thus, Compound 3 was assigned the name 8β,14α-dihydroxy-1α,16β,18β-trimethoxy-19-en-aconitane.

Figure 4

Key HMBC (H→C) and ROESY (H↔H) correlations of Compound 3.

The roots of A. szechenyianum have long been used to treat rheumatic diseases, in which n class="Disease">inflammation and suppressive immunoreaction are involved in the pathophysiological process. The immunosuppressive effects of Compounds 1–5 were evaluated in vitro by ConA-induced or LPS-induced splenocyte proliferation, which was suppressed in a concentration-dependent manner by 2, 4, and 5 (Figure 5b,c), with IC50 values of 5.780 ± 1.12 μm, 3.151 ± 0.52 μm, and 2.644 ± 0.77 μm (ConA-induced), or 4.293 ± 3.20 μm, 3.852 ± 1.57 μm, and 2.283 ± 1.28 μm (LPS-induced), respectively. These three compounds showed low cytotoxic effect (Figure 5a), with CC50 values of 422.85 ± 66.4 μm, 176.35 ± 69.65 μm, and 188 ± 84.15 μm, respectively. The CC50/IC50 values of 2, 4, and 5 suggested that these compounds are potential immunosuppressive agents.

Figure 5

Cytotoxicity on splenocytes and inhibition on ConA- or LPS-induced splenocyte proliferation of Compounds 1–5. (a) Cytotoxicity of Compounds 1–5 on BALB/c mice splenocytes. (b) Inhibition of Compounds 1–5 on ConA-induced splenocyte proliferation. (c) Inhibition of Compounds 1–5 on LPS-induced splenocyte proliferation. Results are mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001, treatment group versus control.

These three compounds had a certain immunosuppressive effects, but low cytotoxic effects compared with n class="Chemical">cyclosporin A. We will conduct further experiments in vivo using the arthritis model in rat induced by adjuvant and arthritis model in mice induced by collagen, to obtain more lead compounds to treat rheumatoid arthritis.

3. Materials and Methods

3.1. General Information

Optical rotation indices were determined in methanol on a Rudolph Autopol II digital polarimeter (Rudolph, Hackettstown, NJ, USA). ESI-MS analysis was n class="Chemical">performed on a Quatro Premier instrument (Waters, Milford, MA, USA). HR-ESI-MS spectra were recorded on an Agilent Technologies 6550 Q-TOF (Santa Clara, CA, USA). 1D- and 2D-NMR spectra were recorded on Bruker-AVANCE 400 (Bruker, Rheinstetten, Germany) and Bruker-AVANCE 600 instrument (Bruker, Rheinstetten, Germany) using TMS as an internal standard. Analytical HPLC was performed on a Waters e2695 Separations Module system coupled with a 2998 Photodiode Array Detector and an Accurasil C-18 column (4.6 mm × 250 mm, 5 μm particles, Ameritech, Chicago, IL, USA). Semipreparative HPLC was performed on a system comprising an LC-6AD pump equipped with an SPD-20A UV detector (Shimadzu, Kyoto, Japan) and an Ultimate XB-C18 (10 mm × 250 mm, 5 μm particles) or YMS-Pack-ODS-A (10mm × 250 mm, 5 μm particles) column. Silica gel was purchased from Qingdao Haiyang Chemical Group Corporation (Qingdao, China).

3.2. Plant Material

The roots of A. szechenyianum Gay. were collected from the Xi Mountains in Gansu Province of China in July 2014 and identified by senior experimentalist Jitao Wang. A voucher sn class="Chemical">pecimen (herbarium No. 20140728) has been deposited in the Medicinal Plants Herbarium (MPH), Shaanxi University of Chinese Medicine, Xianyang, China.

3.3. Extraction and Isolation

The air-dried and powdered underground parts of A. szechenyianum (5.0 kg) were extracted with 80% EtOH at 80 °C (3 × 40 L; 1.5 h). After the removal of n class="Chemical">EtOH under reduced pressure, the extract (2 L) was dispersed in water (1.5 L), adjusted to pH 0.8 with 9% HCl solution, and extracted with petroleum ether (PE). The acidic water solution was alkalized to pH 10.26 with 25% ammonia solution, extracted with CHCl3 three times, and evaporated under pressure to give crude alkaloids (50 g). The crude alkaloids (47 g) were loaded on a silica gel column and eluted with a gradient solvent system (PE/acetone/diethylamine, 50:1:0.1–1:1:0.1) to yield 12 fractions (Fr.1–Fr.12). Fr.3 (2.5 g) was purified by HPLC (YMC-Pack-ODS-A, 10 × 250 mm, 5 μm particles, flow rate of 1.0 mL·min−1) with CH3OH/H2O (83:17) as the mobile phase to obtain 1 (6 mg, tR = 45 min). Fr.4 was purified by HPLC with CH3OH/H2O (75:25) as the mobile phase to obtain 4 (60 mg, tR = 46 min) and 5 (40 mg, tR = 58 min). Fr.7 was purified by HPLC with CH3OH/H2O (65:35) as the mobile phase to obtain 2 (7 mg, tR = 50 min) and 3 (7 mg, tR = 56 min). More details of the spectra are provided in the Supplementary Material.

(A-c)-14α-benzoyloxy-8β-acetoxyl-13β,15α-dihydroxy-1α,6α,8β,16β,18β-n class="Chemical">pentamethoxyl-7(17)–oxide-aconitane (szechenyianine D): A white amorphous powder, [α-9.3 (c 0.043, MeOH), IR (KBr) νmax: 3495, 2914, 1719, 1277, 1099, 1031 and 712 cm−1; 1H-NMR (400 MHz, CDCl3) and 13C-NMR (100 MHz, CDCl3) spectral data, see Table 1; m/z 590.2979 [M + H]+ (calcd. 590.2965) for C31H43NO10.

(A-c)-14α-benzoyloxy-8β-acetoxy-N-nonyl-13β,15α-dihydroxy-1α,6α,16β,18β-tetramethoxy-19-oxo-aconitane (n class="Chemical">szechenyianine E): A white amorphous powder, [α + 12.1 (c 0.033, MeOH), IR (KBr) νmax: 3471, 2933, 2822, 1717, 1453, 1278, 1098, and 712 cm−1; 1H-NMR (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDCl3) spectral data, see Table 1; m/z 714.3840 [M + H] + (calcd.714.3853) for C39H55NO11.

8β,14α-dihydroxy-1α,16β,18β-trimethoxy-19-en-aconitane (szechenyianine F): A white amorphous powder, [α-17.4 (c 0.013, MeOH), IR (KBr) νmax: 3381, 2933, 1630, 1455, 1376, 1096, and 1030 cm−1; n class="Chemical">1H-NMR (400 MHz, CDCl3) and 13C-NMR (100 MHz, CDCl3) spectral data, see Table 1; m/z 421.2782 [M]+ (calcd.421. 2823) for C24H38NO5+.

3.4. MTT Assay

Splenocytes (4 × 105 cells/well) were incubated in triplicate at 37 °C in a humidified incubator with 5% CO2 and 95% air. The assay was n class="Chemical">performed in a 96-well format, and different concentration of Compounds 1–5 (0.16–100 μm) and CsA（2μm）were added. The cells cultured with media alone were used as controls. Approximately 48 h later, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/mL, Sigma) was added to each well. The plates were then incubated for another 5 h. Approximately 150 μL of DMSO (Sigma) was then added to each well. Optical density was measured at 570 nm (BioTek, PowWave XS2, VT, USA). The IC50 and CC50 values were calculated according to the dose curves generated by plotting the percentage of viable cells against the test concentration on a logarithmic scale by using SPSS 15.0. CsA (Cyclosporine A, Sigma, Chicago, IL, USA) was used as a positive control.

3.5. ConA- and LPS-Induced Assay

Splenocytes (4 × 105 cells/well), different concentration of Compounds 1–5 (0.16–100 μm), and n class="Chemical">CsA（2 μm）in 96-well plates at 37 °C in a 5% CO2 atmosphere were cultured in triplicate for 48 h using ConA (2 μg/mL, Sigma) or LPS (1 μg/mL, Sigma). The cells cultured with media alone were used as controls. The cells were pulsed at 0.25 μCi/well of [3H]-thymidine for 8 h before the end of the culture period and then harvested onto glass fiber filters. [3H]-thymidine incorporation was measured using a beta scintillation counter (MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA, USA).