C19-Norditerpenoid Alkaloids from Aconitum Szechenyianum

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Article C19-Norditerpenoid Alkaloids from Aconitum szechenyianum

Bei Song 1,2,†, Bingliang Jin 3,†, Yuze Li 1,†, Fei Wang 4, Yifu Yang 3, Yuwen Cui 5, Xiaomei Song 2, Zhenggang Yue 2,* and Jianli Liu 1,* 1 The College of Life Sciences, Northwest University, Xi’an 710069, China; [email protected] (B.S.); [email protected] (Y.L.) 2 Shaanxi Collaborative Innovation Center of Chinese Medicinal Resource Industrialization, School of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang 712046, China; [email protected] 3 Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; [email protected] (B.J.); [email protected] (Y.Y.) 4 Shaanxi Institute for Food and Drug Control, Xi’an 710065, China; [email protected] 5 Department of Pharmacy, Xi’an Medical University, Xi’an 710021, China; [email protected] * Correspondence: [email protected] (J.L.); [email protected] (Z.Y.); Tel.: +86-136-0929-8392 (J.L.); +86-029-3818-2209 (Z.Y.) † These authors contribute equally to this work.

Received: 5 March 2018; Accepted: 25 April 2018; Published: 8 May 2018

Abstract: 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.

Keywords: Aconitum szechenyianum;C19-norditerpenoid alkaloids; immunosuppressive effects

1. Introduction The roots of Aconitum szechenyianum Gay. and 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 [2–5], possess anti-inflammatory, analgesic, anticancer, anti-epileptiform, and antiparasitic activities [6–8]. 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 (Figure1). 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

Molecules 2018, 23, 1108; doi:10.3390/molecules23051108 www.mdpi.com/journal/molecules Molecules 2018, 23, x 2 of 11 Molecules 2018, 23, 1108 2 of 9 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 throughthrough ConA- ConA- or or LPS-induced LPS-induced splenocyte splenocyte proliferationproliferation models. models. Three Three compounds compounds inhibited inhibited ConA- ConA- or LPS-inducedor LPS-induced splenocyte splenocyte proliferation, proliferation, revealing revealing for the first ﬁrst time time that that the the roots roots of ofA.A. szechenyianum szechenyianum possesspossess immunosuppressive immunosuppressive activities. activities.

FigureFigure 1. 1.Structures Structures of of CompoundsCompounds 1–5..

2. Results 2. Results

1 13 Szechenyianine DTable (1) 1. was H-NMR isolated and asC-NMR a white spectral amorphous data for Compounds powder 1– and5. showed a positive 0 reaction with Dragendorff1 s reagent. Its molecular 2 formula C31H43NO10 3 was derived 4 from the 5 protonatedNO. molecular ion peak at m/z 590.2979 [M + H]+ (calcd. 590.2965) in the HR-ESI-MS spectrum. The 1H-NMRδ spectrumC δH (Table (J in Hz)1) of 1 showed δC the δH presence(J in Hz) of five δC aromatic δH (J protonin Hz) signals δC due δ toC a monosubstituted1 80.3 benzene 3.45 (d, at 7.6)δH 8.00 (2H, 82.4 d, J = 3.07 7.4 (d, Hz), 8.3) 7.54(1H, 80.6 t, J = 3.40 7.4 (m) Hz), and 7.44 82.8 (2H, 82.8 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), 1.22 (m, H-2a) 1.43 (m, H-2a) 1.40 (m, H-2a) 13 and 3.21 (3H, s), and two strongly shielded protons at δH 3.32 (1H, s) and δH 4.29 (1H, s). The C-NMR 2 29.6 25.3 20.6 33.6 33.6 spectrum (Table1) displayed2.41 (m, 31 H- carbon2b) resonances.1.87 Among (m, H-2b) them, resonances1.72 at(m,δ HC-2b)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 attributed1.22 to(m, five H-3a) OMe groups; δ2.6174.7 (m, andH-3a) 76.4 were attributed to two oxygenated 3 29.9 33.4 C 25.5 1.94 (m) 72.0 71.9 carbons associated with1.40 hydroxyl (m, H-3b) groups. Out2.71 of the(m, H 10-3b) oxygen atoms in 1, 9 are associated with five methoxy groups, two hydroxyl groups, and one benzoyl group, and the remaining one may 4 43.5 37.8 48.6 43.3 43.2 be a hydroxyl group or an internal ether. The NMR features of the remaining 19 resonances were characteristic5 42.3 of an aconitine-type2.41 (d, 6.7) alkaloid,47.6 where2.29 the (d, δ6.C7)64.4 and38.4 70.0 resonances2.46 (m) were46.2 attributed 45.9 to the two carbons associated with an internal ether. The deduction was confirmed by the chemical 1.82 (m, H-6a) shift6 of C-7 (δ 82.264.4) and 4.12 C-17 (d,6.7) (δ 70.0) 83.3 to downfield 4.04 (d, in 6.7)13 C-NMR 24.6 spectra of 1 compared83.6 with C-7 83.7 C C 2.20 (m, H-6b) (δC 49.6) and C-17 (δC 60.6) of szechenyianine A [9], the signals of which are shielded by oxygen atom.7 In the HMBC64.4 spectrum3.32 (s)(Figure 2),50.9 correlations 2.63 of (s)H-5 ( δH45.12.41) and2.22 H-6 (m) (δ H 4.12)45.4 to C-7 (43.3δC 64.4), and H-1 (δ 3.45) to C-17 (δ 70.0), suggested the involvement of an internal ether bond. 8 83.3 H C 91.3 72.3 82.6 82.5 The correlation of H-14 (δH 4.84) to the carbonyl carbon signal of the benzoyl group (δC 166.4) suggested9 that44.5 the benzoyl2.59 (t, group 5.8) was43.5 located at2.84 C-14. (t, 5.8) The correlations53.8 of2.47 OCH (m) 3 (δH 3.37)42.7 to C-145.4 δ δ δ δ δ δ ( C 1080.3), OCH40.83 ( H 3.29)2.26 to (m) C-6 ( C 82.2),41.1 OCH2.193 ( (m)H 3.21) to38.3 C-8 ( C 83.3),2.01 (m) OCH 3 ( 41.7H 3.80) 41.6 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 (δ 1.84, 2.24) and H-14 (δ H H 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, Molecules 2018, 23, 1108 3 of 9

15-dihydroxy-1, 6,8,16,18-pentamethoxyl-7(17)-oxide-aconitane. In the ROESY spectrum (Figure2) 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.

1 2 3 4 5 NO. 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 1.22 (m, H-2a) 1.43 (m, H-2a) 1.40 (m, H-2a) 2 29.6 25.3 20.6 33.6 33.6 2.41 (m, H-2b) 1.87 (m, H-2b) 1.72 (m, H-2b) 1.22 (m, H-3a) 2.61 (m, H-3a) 3 29.9 33.4 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 2.24 (m, H-12a) 2.80 (m, H-12a) 2.04 (m, H-12a)1.26 (m, 12 35.5 35.6 27.8 36.5 36.5 1.84 (m, H-12b) 2.19 (m, H-12b) 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 2.27 (m, H-15a) 15 76.4 4.65 (d, 5.4) 79.0 4.49 (dd, 2.9, 5.4) 39.5 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 3.54(d, 8.2, H-18a) 3.78(d,8.2,H-18a) 18 76.6 80.0 73.4 3.71 (2H, m) 77.2 77.2 3.46 (d,8.2, H-18b) 3.06(d,8.2,H-18b) 3.62(d,11.9,H-19a) 19 50.3 173.3 179.2 9.19 (s) 49.2 49.2 3.72(d,11.9,H-19b) 20 3.04 (m, H-20a) 45.5 3.92 (m, H-20b) 21 1.51 (m, H-21a) 33.7 1.62 (m, H-21b) 22 1.59 (m, H-22a) 25.0 1.68 (m, H-22b) 23 1.30 (m, H-23a) 29.6 1.23 (m, H-23b) 24 1.30 (m, H-24a) 31.9 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 4.01 (dq, 13.9, 7.2) N-CH2CH3 56.3 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-10 130.0 129.9 130.4 130.6 30, 50 128.7 7.44 (t, 7.4) 128.9 7.45 (t, 7.5) 128.6 128.6 20, 60 130.0 8.00 (d, 7.4) 129.8 8.01 (d, 7.5) 129.9 129.9 40 133.4 7.54 (t, 7.4) 133.6 7.56 (t, 7.5) 133.1 133.1 1 13 δ in CDCl3, in ppm from TMS; coupling constants (J) in Hz; H-NMR at 400 MHz and C-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. Molecules 2018, 23, x 5 of 11

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, MoleculesCompound2018, 23 ,1 1108 was assigned the name (A-c)-14α-benzoyloxy-13β,15α-dihydroxy-1α,6α,8β,16β,18β4- of 9 pentamethoxy-7(17)-oxide-aconitane.

FigureFigure 2. 2.Key Key HMBC HMBC (H(H→→C) and ROESY (H (H↔↔H)H) correlations correlations of of Compound Compound 1. 1.

Szechenyianine E (2) was isolated as a white amorphous powder and showed a positive reaction withSzechenyianine Dragendorff′s E reagent. (2) was Its isolated molecular as formula a white C amorphous39H55NO11 was powder derived and from showed the protonated a positive 0 reactionmolecular with ion Dragendorff peak at m/z s714.3840 reagent. [M Its+ H] molecular+ (calcd.714.3853) formula in Cthe39H HR-ESI-MS55NO11 was spectrum. derived The from 1H- the protonatedNMR spectrum molecular (Table ion peak 1) of at2 mshowed/z 714.3840 the presence [M + H]+ of(calcd.714.3853) five aromatic in proton the HR-ESI-MS signals due spectrum. to a 1 ThemonosubstitutedH-NMR spectrum benzene (Table at δ1H) 8.01 of 2 (2H,showed d, J = the7.5 Hz), presence 7.56 (1H, of ﬁve t, J = aromatic 7.5 Hz), 7.45 proton (2H, signals t, J = 7.5 due Hz), to a monosubstitutedfour OMe protons benzene at δH 3.75 at δ H(3H,8.01 s), (2H, 3.30 d,(3H,J = s), 7.5 3.19 Hz), (3H 7.56 s), (1H,and 3.12 t, J = (3H, 7.5 Hz),s); one 7.45 acetoxyl (2H, t, protonJ = 7.5 at Hz), 13 fourδH OMe1.35 (3H, protons s), and at oneδH methylic3.75 (3H, proton s), 3.30 of (3H,the hydrocarbon s), 3.19 (3H chain s), and at δ 3.12H 0.86 (3H, (3H, s); t, J one = 6.2Hz). acetoxyl The protonC- C at δNMRH 1.35 spectrum (3H, s), (Table and one 1) displayed methylic 39 proton carbon of resonances. the hydrocarbon Among chain them, at theδH resonances0.86 (3H, t,at Jδ= 166.2, 6.2 Hz). 133.6,13 129.9, 129.8 (C × 2), and 128.9 (C × 2) were attributed to a benzoyloxy group; δC 61.5, 59.4, 58.0, The C-NMR spectrum (Table1) displayed 39 carbon resonances. Among them, the resonances at δC and 55.5 were attributed to four OMe groups, δC 172.5 and 21.6 were attributed to one acetoxyl group; 166.2, 133.6, 129.9, 129.8 (C × 2), and 128.9 (C × 2) were attributed to a benzoyloxy group; δC 61.5, δC 173.3 was attributed to C=O, δC 74.3 and 79.0 were attributed to two carbons associated with the 59.4, 58.0, and 55.5 were attributed to four OMe groups, δC 172.5 and 21.6 were attributed to one hydrocarbon chain, and δC 14.3 was attributed to one CH3 group. The assignments of the NMR signals acetoxyl group; δC 173.3 was attributed to C=O, δC 74.3 and 79.0 were attributed to two carbons associated with 2 were derived from HSQC, HMBC, and ROESY experiments. In the HMBC spectrum associated with the hydrocarbon chain, and δC 14.3 was attributed to one CH3 group. The assignments (Figure 3), correlations of H-14 (δH 4.89) to the carbonyl carbon signal of the benzoyl group (δC 166.4) of the NMR signals associated with 2 were derived from HSQC, HMBC, and ROESY experiments. suggested that the benzoyl group was located at C-14. Correlations of OCH3 (δH 3.19) to C-1 (δC 82.4), In the HMBC spectrum (Figure3), correlations of H-14 ( δ 4.89) to the carbonyl carbon signal of OCH3 (δH 3.12) to C-6 (δC 83.3), OCH3 (δH 3.75) to C-16 (δC 90.4),H and OCH3 (δH 3.30) to C-18 (δC 80.0) thesuggested benzoyl group that four (δC 166.4) methoxyl suggested groups that were the linked benzoyl at C-1, group C-6, was C-16, located and at C-18, C-14. respectively. Correlations δ δ δ δ δ δ of OCHCorrelations3 ( H 3.19) of CH to3 (δ C-1H 1.32) ( C to82.4), 8-OAc OCH (δC 3172.5)( H 3.12)suggested to C-6 that ( Cone83.3), acetoxyl OCH was3 ( linkedH 3.75) at toC-8, C-16 and ( C 90.4),correlations and OCH of3 H-3(δH (δ3.30)H 2.61, to C-182.71), (H-17δC 80.0) (δH 4.11), suggested and H-20 that (δ fourH 3.04, methoxyl 3.92) to C-19 groups (δC were173.3) linkedsuggested at C-1, C-6,that C-16, C=O and was C-18, linked respectively. at C-19. Correlations Correlations of H-20 of CH(δH 33.04,(δH 3.92)1.32) to to C-17 8-OAc (δC 56.7) (δC 172.5) and C-21 suggested (δC 33.7), that oneH-21 acetoxyl (δH 1.51, was 1.62) linked to C-22 at C-8,(δC 25), and and correlations H-22 (δH 1.59, of 1.68), H-3 (H-25δH 2.61, (δH 1.30, 2.71), 2H, H-17 m), and (δH H-264.11), (δ andH 0.86, H-20 (δH3H,3.04, t) to 3.92) C-24 to (δC C-19 31.9) ( δsuggestedC 173.3) suggestedthe presence that of an C=O N-heptyl was linkedgroup. atCorrelations C-19. Correlations of H-12 (δH of2.19, H-20 (δH2.80),3.04, H-14 3.92) (δ toH 4.89), C-17 and (δC H-1656.7) (δ andH 3.30) C-21 to C-13 (δC (δ33.7),C 74.3), H-21 and H-16 (δH 1.51, (δH 3.30) 1.62) to toC-15 C-22 (δC( 79.0)δC 25), suggested and H-22 (δHthat1.59, two 1.68), hydroxyl H-25 groups (δH 1.30, were 2H, linked m), at and C-13 H-26 and (C-15,δH 0.86, respectively. 3H, t) to This C-24 compound (δC 31.9) differed suggested from the the known compound (A-c)-8β-acetoxy-14α-benzoyloxy-N-ethyl-13β,15α-dihydroxy-1α,6α,16β,18β- presence of an N-heptyl group. Correlations of H-12 (δH 2.19, 2.80), H-14 (δH 4.89), and H-16 (δH tetramethoxy-19-oxo-aconitane [14] only in terms of the substituents on the N atom. According to the 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 wereROESY linked (Figure at C-13 3) spectrum, and C-15,NOE respectively. correlations Thisof H-6/H-5 compound and H-5/H-18 differed revealed from the β-orientation known compound of H- 18 and 18-OCH3, α-axial orientation of 6-OCH3; NOE correlations of H-7/H-15, H-17/H-16 revealed (A-c)-8β-acetoxy-14α-benzoyloxy-N-ethyl-13β,15α-dihydroxy-1α,6α,16β,18β-tetramethoxy-19-oxo- α-axial orientation of H-16, H-17, and 15-OH and β-orientation of 16-OCH3, 13-OH, and 8-OAc. aconitane [14] only in terms of the substituents on the N atom. According to the ROESY (Figure3) Moreover, the NOE correlations of H-3/H-1/H-10/H-9/H-6/H-5 and the lack of correlation between spectrum, NOE correlations of H-6/H-5 and H-5/H-18 revealed β-orientation of H-18 and 18-OCH , 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 3 α-axialconformation, orientation the of relative 6-OCH configuration3; NOE correlations of this compound of H-7/H-15, was confirmed. H-17/H-16 Thus, revealed the planarα-axial structure 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. Molecules 2018, 23, x 6 of 11

Moleculesof 2 2018was, 23 , assigned1108 the name (A-c)-14α-benzoyloxy-8β-acetoxyl-N-heptyl-13β,15α-dihydroxy-5 of 9 1α,6α,16β,18β-tetramethoxy-19-oxo-aconitane.

Figure 3. Key HMBC (H→ C) and ROESY (H↔H) correlations of Compound 2. Figure 3. Key HMBC (H→ C) and ROESY (H↔H) correlations of Compound 2.

SzechenyianineSzechenyianine F F (3 ()3 was) was isolated isolated asas aa white amorphous amorphous powder powder and and showed showed a positive a positive reaction reaction 0 + + withwith Dragendorff Dragendorff′ss reagent. reagent. ItsIts molecular formula formula C C24H2438HNO38NO5 was5 wasderived derived from the from ion the peak ion at peak m/z at m/z421.2782[M]421.2782[M]+ (calcd.421.2823)+ (calcd.421.2823) in in the the HR-ESI-MS HR-ESI-MS spectrum. spectrum. The The 1H-NMR1H-NMR spectrum spectrum (Table (Table 1) of1) of3 3 showedshowed the the presence presence of of a methinea methine proton proton due due toto oneone N=CHN=CH group group at at δδHH 9.199.19 (1H, (1H, s), s), one one N–CHN–CH2CH2CH3 3 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 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 resonances at δH 3.38 (3H, s), 3.36 (3H, s), and 3.16 (3H, s). The 13C-NMR spectrum13 (Table 1) displayed OMe resonances at δH 3.38 (3H, s), 3.36 (3H, s), and 3.16 (3H, s). The C-NMR spectrum (Table1) 24 carbon resonances. Among them, the resonances at δC 59.8, 56.9, and 56.7 were attributed to three displayed 24 carbon resonances. Among them, the resonances at δC 59.8, 56.9, and 56.7 were attributed OMe groups, δC 179.2 was attributed to one N=CH group, and δC 14.0 and 56.3 were attributed to one to three OMe groups, δC 179.2 was attributed to one N=CH group, and δC 14.0 and 56.3 were attributed N–CH2CH3 group. Comparison of the NMR data of N–CH2CH3 and C-19 with those of the known to one N–CH CH group. Comparison of the NMR data of N–CH CH and C-19 with those of the compound 211 in3 [15] indicated the existence of the ＋N=CH group.2 The3 assignments of the NMR known compound 11 in [15] indicated the existence of the +N=CH group. The assignments of the signals associated with 3 were based on HSQC, HMBC, and ROESY experiments. In the HMBC 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 spectrum (Figure4), correlations of H-5 ( δ 2.46) and H-17 (δ 3.79) to C-19 (δ 179.2) suggested that C-19 was involved in the N=CH group. CorrelationsH of OCH3 (δH H 3.18) to C-1 (δC C80.6), OCH3 (δH 3.36) δ δ δ C-19to wasC-16 involved(δC 81.5) and in theof OCH N=CH3 (δH group. 3.38) to Correlations C-18 (δC 73.4) of suggested OCH3 ( Hthat3.18) three to methoxyl C-1 ( C 80.6), groups OCH were3 ( H 3.36)linked to C-16 at C-1, (δC C-16,81.5) and and C-18, of OCH respectively.3 (δH 3.38) Correlations to C-18 (δC of73.4) H-6 suggested (δH 1.82, 2.20), that H-7 three (δ methoxylH2.22), H-9 groups (δH were2.47), linked and H-10 at C-1, (δH C-16, 2.01) andto C-8 C-18, (δC 72.3) respectively. and of H-16 Correlations (δH 3.45) to of C-14 H-6 (δ (Cδ H75.0)1.82, suggested 2.20), H-7 that (δ twoH2.22), H-9hydroxyl (δH 2.47), groups and H-10 were ( linkedδH 2.01) at to C-8 C-8 and (δ C C-14,72.3) respectively. and of H-16 Thus, (δH 3.45) the planar to C-14 structure (δC 75.0) of suggested 3 was thatdeduced two hydroxyl as 8,14-dihydroxy-1,16,18-trimethoxy-19-en-aconitane. groups were linked at C-8 and C-14, respectively. In the ROESY Thus, spectrum the planar (Figure structure 4) of of 3, wasthe NOE deduced correlations as 8,14-dihydroxy-1,16,18-trimethoxy-19-en-aconitane. of H-1/H-5, H-1/H-10, and H-10/H-14 indicated β-orientation In the ROESYof H-1, H-9, spectrum H- (Figure10, and4) ofH-14,3, the and NOE α-axial correlations configurations of H-1/H-5, of 1-OCH H-1/H-10,3, 14-OH. NOE and correlations H-10/H-14 of indicated H-5/H-18β indicated-orientation 3 and of H-1,β-orientation H-9, H-10, of H-18 and andH-14, 18-OCH and α-axial. NOE conﬁgurations correlations of of H-17/H-12, 1-OCH3 , H-12/H-16, 14-OH. NOE correlationsH-15/H-16 of indicated α-axial configurations of H-16, H-17, and 16-OCH3 and β-orientation of 8-OH. Thus, H-5/H-18 indicated β-orientation of H-18 and 18-OCH3. NOE correlations of H-17/H-12, H-12/H-16, Compound 3 was assigned the name 8β,14α-dihydroxy-1α,16β,18β-trimethoxy-19-en-aconitane. 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. Molecules 2018, 23, x 7 of 11

Figure 4. KeyFigure HMBC 4. Key(H HMBC→C) (H→ andC) and ROESY ROESY (H (H↔H)↔ correlationsH) correlations of Compound of 3 Compound. 3. The roots of A. szechenyianum have long been used to treat rheumatic diseases, in which inflammation and suppressive immunoreaction are involved in the pathophysiological process. The The roots ofimmunosuppressiveA. szechenyianum effects of Compoundshave long 1–5 beenwere evaluated used in to vitro treat by ConA-induced rheumatic or diseases,LPS- in which inﬂammation andinduced suppressive splenocyte proliferation, immunoreaction which was suppressed are involvedin a concentration-dependent in the pathophysiological manner by 2, process. 4, and 5 (Figures 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.

These three compounds had a certain immunosuppressive effects, but low cytotoxic effects compared with 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.

Molecules 2018, 23, x 7 of 11

Figure 4. Key HMBC (H→C) and ROESY (H↔H) correlations of Compound 3. Molecules 2018, 23, 1108 6 of 9 The roots of A. szechenyianum have long been used to treat rheumatic diseases, in which 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- The immunosuppressiveinduced splenocyte effects proliferation, of Compounds which was suppressed1–5 were in a concentration-dependent evaluated in vitromanner by 2, ConA-induced or 4, and 5 (Figures 5b,c), with IC50 values of 5.780 ± 1.12 μm, 3.151 ± 0.52 μm, and 2.644 ± 0.77 μm (ConA- LPS-induced splenocyteinduced), proliferation, or 4.293 ± 3.20 μm, 3.852 which ± 1.57 wasμm, and suppressed 2.283 ± 1.28 μm (LPS-induced), in a concentration-dependent respectively. These manner by 2, 4, and 5 (Figure5threeb,c), compounds withIC showed50 valueslow cytotoxic of effect 5.780 (Figure± 5a),1.12 withµ CCm,50 values 3.151 of 422.85± 0.52 ± 66.4 µμm,m, 176.35 and 2.644 ± 0.77 µm ± 69.65 μm, and 188 ± 84.15 μm, respectively. The CC50/IC50 values of 2, 4, and 5 suggested that these (ConA-induced), orcompounds 4.293 ± are3.20 potentialµm, immunosuppressive 3.852 ± 1.57 agents.µm, and 2.283 ± 1.28 µm (LPS-induced), respectively.

These three compoundsThese showed three compounds low cytotoxic had a certain effect immunosuppressive (Figure5a), effects, with but CC low cytotoxic50 values effects of 422.85 ± 66.4 µm, 176.35 ± 69.65 µm,compared and 188 with± cyclosporin84.15 A.µm, We will respectively. conduct further experiments The CC in vivo/IC using valuesthe arthritis of model2, 4, and 5 suggested in rat induced by adjuvant and arthritis model in mice induced by50 collagen,50 to obtain more lead that these compoundscompounds are potentialto treat rheumatoid immunosuppressive arthritis. agents.

Molecules 2018, 23, x 8 of 11 a

Figure 5. CytotoxicityFigure 5. Cytotoxicity on splenocytes on splenocytes and and inhibition inhibition on on ConA- ConA- or LPS-induced or LPS-induced splenocyte proliferation splenocyte of proliferation of CompoundsCompounds1–5.(a) 1 Cytotoxicity–5. (a) Cytotoxicity of of Compounds Compounds 1–5 on BALB/c1–5 miceon splenocytes. BALB/c (b)mice Inhibition splenocytes. of Compounds (b) Inhibition 1–5 on ConA-induced splenocyte proliferation. (c) Inhibition of Compounds 1–5 on LPS-induced splenocyte of Compoundsproliferation.1–5 on ConA-induced Results are mean ± S.D. splenocyte* p < 0.05, ** p < 0.01, proliferation. *** p < 0.001, treatment (c )group Inhibition versus control. of Compounds 1–5 on LPS-induced splenocyte proliferation. Results are mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001, 3. Materials and Methods treatment group versus control. 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 performed on a Quatro Premier

Molecules 2018, 23, 1108 7 of 9

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 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 specimen (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 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-pentamethoxyl-7(17)–oxide-aconitane 23.1 (szechenyianine D): A white amorphous powder, [α]D -9.3 (c 0.043, MeOH), IR (KBr) νmax: 3495, 2914, −1 1 13 1719, 1277, 1099, 1031 and 712 cm ; H-NMR (400 MHz, CDCl3) and C-NMR (100 MHz, CDCl3) + spectral data, see Table1; 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 23.0 (szechenyianine E): A white amorphous powder, [α]D + 12.1 (c 0.033, MeOH), IR (KBr) νmax: 3471, Molecules 2018, 23, 1108 8 of 9

−1 1 13 2933, 2822, 1717, 1453, 1278, 1098, and 712 cm ; H-NMR (600 MHz, CDCl3) and C-NMR (150 MHz, + CDCl3) spectral data, see Table1; 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 22.9 −1 powder, [α]D -17.4 (c 0.013, MeOH), IR (KBr) νmax: 3381, 2933, 1630, 1455, 1376, 1096, and 1030 cm ; 1 13 H-NMR (400 MHz, CDCl3) and C-NMR (100 MHz, CDCl3) spectral data, see Table1; 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 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 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).

Supplementary Materials: The spectra of Compounds 1–3 are available online at http://www.mdpi.com/1420- 3049/23/5/1108/s1. Author Contributions: Every author participated in the research and contributed to the article: B.S. and B.J. conducted the experiments and collected the data; Y.L., F.W., and Y.C. coordinated the experiments; Z.Y. analyzed the data; X.S. and Y.Y. designed the study; J.L. planned and oversaw the research project and drafted the paper. Finally, all authors read and approved the final manuscript. Funding: This research was funded by the National Natural Science Foundations of China (grant No. 81102805, 81373978), the Open Projects Program of the Key Laboratory of Tibetan Medicine Research, Chinese Academy of Sciences (grant No. 2014-TMR-01), the Program of Shannxi Provincial Department of Education (grant No. 15JK1204), and the Innovative Research Team in TCM Material Foundation and Key Preparation Technology (grant No. 2012KCT-20). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 4–5 are available from the authors.

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