Triterpenoids and triterpenoid saponins from Dipsacus asper and their cytotoxic and antibacterial activities

Phytochemistry 162 (2019) 241–249

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Triterpenoids and triterpenoid saponins from Dipsacus asper and their cytotoxic and antibacterial activities

T

Jin-Hai Yua, Zhi-Pu Yua,b, Yin-Yin Wanga, Jie Baoa, Kong-Kai Zhua, Tao Yuanc, Hua Zhanga,∗ a

School of Biological Science and Technology, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan, 250022, China School of Chemistry and Chemical Engineering, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan, 250022, China c Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, China b

ARTICLE INFO

ABSTRACT

Keywords: Dipsacus asper Caprifoliaceae Triterpenoid Triterpenoid saponin Cytotoxicity

Phytochemical investigation of the ethyl acetate soluble part, generated from the ethanol extract of the roots of Dipsacus asper, led to the separation and identification of three undescribed triterpenoids including one arborinane type, one ursane type and one oleanane type, two unreported oleanane type triterpenoid arabinoglycosides, and 18 known analogues. Structures of these compounds were determined by comprehensive spectroscopic analyses, with the absolute configurations of 25-acetoxy-28-dehydroxyrubiarbonone E and 2α,3βdihydroxy-23-norurs-4(24),11,13(18)-trien-28-oic acid being established by evaluation of their experimental and calculated ECD spectra. 25-Acetoxy-28-dehydroxyrubiarbonone E features an oxygenated C-25 that is the first case among arborinane type triterpenoids, while 2α,3β,24-trihydroxy-23-norurs-12-en-28-oic acid incorporates a sp3 C-24 that is a rare structural feature of 23-norursane type triterpenoids. Of these isolates, 2′,4′O-diacetyl-3-O-α-L-arabinopyranosyl-23-hydroxyolea-12-en-28-oic acid and hederagonic acid exhibited moderate antibacterial activity against Staphylococcus aureus with IC50 values of 12.3 and 10.3 μM, respectively, while those with either a feruloyloxy group or an arabinosyl moiety at C-3 displayed potent cytotoxic activities against four tumor cell lines A549, H157, HepG2 and MCF-7.

1. Introduction

phenolic compounds (Hung et al., 2006), etc. A wide spectrum of biological activities associated with the aforementioned ingredients such as anti-inflammatory (Li et al., 2013), antioxidant (Huang et al., 2008; Hung et al., 2006), aldose reductase inhibitory (Koo et al., 2013), Alzheimer's disease inhibitory (Ji et al., 2012; Zhou et al., 2009), antiHIV (Sun et al., 2015), antifungal (Choi et al., 2017), procoagulant (Song et al., 2012), larvicidal (Li et al., 2017) and cytotoxic (Jeong et al., 2008; Tian et al., 2006, 2007; Hung et al., 2005) effects, have also been reported. In our continuing search for cytotoxic compounds from Chinese traditional herbs, the ethyl acetate partition of the ethanol extract from D. asper came to our attention. Subsequent phytochemical investigation resulted in the isolation and characterization of three undescribed triterpenoids (1–3) and two triterpenoid saponins (4 and 5), as well as 18 known related analogues (6–23). Their structures were elucidated by spectroscopic analyses, with the absolute stereochemistries of 1 and 3 being established by evaluation of their experimental and calculated ECD spectra. All of the isolated compounds were evaluated for their cytotoxicity against A549 & H157 (lung), HepG2 (liver) and MCF-7 (breast) and antibacterial activities against Mycobacterium smegmatis ATCC 607, Staphylococcus aureus ATCC 25923,

Dipsacus asper Wall. ex C. B. Clarke (Caprifoliaceae) is a perennial herb which mainly grows in the southern regions of China, such as Hunan and Yunnan Provinces (The Plant List, 2013; Flora of China Editorial Committee, 2011). Its dried roots, named “Xuduan” in traditional Chinese medicine, have been used for hundreds of years as a remedy for arthralgia, lumbago, backache, traumatic hematoma, threatened abortion and bone fractures (Ji et al., 2012; Editorial Committee of the Administration Bureau of Traditional Chinese Medicine, 1999). In recent years, this medicinal plant has become a hot research target and attracted attentions from both natural products chemists and pharmacologists (Sun et al., 2015, 2018; Choi et al., 2017; Li et al., 2013, 2016; Koo et al., 2013; Wang et al., 2013), due to its specialised metabolites with diverse structural scaffolds and bioactivities. Previous chemical studies on the roots of D. asper have led to the discovery of various chemical constituents including iridoid glycosides (Sun et al., 2015; Tian et al., 2006, 2007; Tomita and Mouri, 1996), triterpenoid saponins (Liu et al., 2011; Li et al., 2010; Hung et al., 2005; Jung et al., 1993; Zhang and Xue, 1993), alkaloids (Li et al., 2013),

∗

Corresponding author. E-mail address: [email protected] (H. Zhang).

https://doi.org/10.1016/j.phytochem.2019.03.028 Received 18 October 2018; Received in revised form 13 February 2019; Accepted 30 March 2019 0031-9422/ © 2019 Published by Elsevier Ltd.

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J.-H. Yu, et al.

Table 1 1 H NMR spectroscopic data of compounds 1–5 (600 MHz). 1a

2b

3b

4b

5b

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

1

7.23, d (10.6)

2

6.07, d (10.6)

α 0.88, m β 1.93, m 3.73, m

α 1.12, dd (12.6, 11.1) β 2.25, dd (12.6, 5.2) 3.53, ddd (11.1, 9.0, 5.2)

3.54, dd (9.7, 6.0) 2.20, m 1.34, m α 1.95, m β 1.66, m α 1.66, m β 1.35, m

3.77 br d (9.0)

α 0.96 β 1.61 α 1.87, dt (13.8, 3.3) β 1.72, m 3.61, dd (9.6, 4.8)

α 0.94, m β 1.64, m α 1.84, dt (12.,4.2) β 1.72, m 3.60, dd (11.8, 4.7)

1.76, m α 1.53, m β 1.67, m α 1.44, m β 1.37, m

1.27, m α 1.47, dt (13.2, 3.3) β 1.35, ddd (13.2, 12.5, 3.2) α 1.61, m β 1.27, m

1.27, m α 1.47, dt (12.8, 4.3) β 1.35, td (12.8, 3.3) α 1.61, m β 1.27, dt (13.4, 2.8)

1.61, dd (9.8, 7.9) α 1.35, m β 1.96, m 5.23, m

2.17, dd (3.1, 2.0) 6.49, dd (10.6, 3.1)

1.63, 1.90, 1.90, 5.24,

1.63, 1.90, 1.90, 5.24,

α 1.08, m β 1.95, m α 2.02, td (13.0, 4.0) β 1.64, m 2.22, d (11.2) 1.37, m

α 1.11, β 1.72, α 1.96, β 1.71,

No.

3 4 5 6

1.83, dd (13.8, 2.4) α 2.07, m β 1.71, m 3.95, td (9.8, 6.5)

7 8 9 11

2.16, br d (9.8)

12

α 2.03, dd (2.2, 2.0) β 1.96, dd (6.4, 2.0) α 1.92, m β 1.65, m α 1.52, dd (14.4, 4.1) β 1.65, m 1.65, d (9.5) 4.23, td (9.5, 3.2)

5.58, dt (6.4, 2.2)

15 16 18 19 20 21

α 1.69, m β 1.90, m 1.32, m

22

1.44, m

23 24

1.16, s 1.09, s

25

4.63, 4.16, 1.00, 0.95, 0.81, 0.88, 0.83, 2.00,

26 27 28 29 30 25-OAc 1′ 2′ 3′ 4′ 5′

d d s s s d d s

(10.8) (10.8)

(6.5) (6.5)

0.98, m α β α β

1.30, 1.50, 1.68, 1.63,

m m m m

5.71, dd (10.6, 2.0) m m m m

α 2.55, br d (14.4) β 1.74, br d (14.4) α 1.39, β 1.28, α 1.39, β 2.25,

m m m m

b

m m m m

α 1.08, dt (13.2, 3.4) β 1.78, m α 2.00, m β 1.60, m 2.85, dd (13.9, 4.3) α 1.70, m β 1.14, m

α 1.08, dt (13.6, 3.4) β 1.78, m α 2.00, td (13.8, 2.9) β 1.60, dt (13.6, 3.1) 2.85, dd (13.8, 4.5) α 1.69, t (13.8,13.6) β 1.13, dd(13.8,2.1) α 1.40, td (13.8, 3.4) β 1.21, dt (13.6, 2.7) α 1.54, dt (13.6, 3.2) β 1.74, m 0.59, s 3.33, d (11.3) 3.30, d (11.3) 0.97, s

3.89, dd (11.0, 9.3) 3.58, dd (11.0, 2.0) 0.87, s

5.17, s 4.73, s 0.74, s

α 1.40, m β 1.21, m α 1.54, dt(13.6, 3.2) β 1.74, m 0.59, s 3.33, d (11.3) 3.29, d (11.3) 0.97, s

0.85, s 1.13, s

0.82, s 1.02, s

0.81, s 1.17, s

0.81, s 1.17, s

0.89, d (6.5) 0.96, d (6.0)

0.82, s 0.95, s

0.94, s 0.91, s

0.94, s 0.91, s

4.57, d (7.3) 5.18 dd (9.7, 7.3) 4.89, dd (9.7, 3.4) 4.00, ddd (3.4, 2.9, 1.8) 3.90, dd (12.6, 2.9) 3.63, dd (12.6, 1.8) 2.05, s 2.05, s

4.49, 4.97, 3.83, 5.06, 3.91, 3.63, 2.11,

2′-OAc 3′-OAc 4′-OAc a

m m m m

d (7.8) dd (9.9, 6.9) dd (9.9, 3.7) ddd (3.7, 2.4, 1.3) dd (13.6, 2.4) dd (13.6, 1.3) s

2.14, s

Data were measured in CDCl3. Data were measured in methanol-d4.

absorption bands at 3437 and 1744 cm−1, respectively. In the 1H and 13 C NMR data (Tables 1 and 2), diagnostic signals for a ketone carbonyl (δC 203.4), an acetyl group (δH 2.00; δC 21.0 and 170.9), two double bonds (δC 150.6, 136.8, 127.9, 122.5), two oxymethines (δC 71.5, 70.3), one oxygenated methylene (δC 66.2), two secondary methyls (δH 0.88, 0.83, each 3H, d) and five tertiary methyls (δH 1.16, 1.09, 1.00, 0.95, 0.81, each 3H, s), were observed. Two carbonyls and two double bonds occupied four out of nine indices of hydrogen deficiency, and thus it required five rings in the skeleton of 1. The above mentioned information together with a biogenetic consideration suggested that compound 1 was a pentacyclic triterpenoid, whose arborinane skeleton was finally established by comprehensive analyses of the 2D NMR data. In detail, five isolated spin-spin coupling systems (a–e) as drawn in bold bonds (see Fig. 2A) were easily depicted by analysis of 1H–1H COSY data. Subsequently, the assembly of the whole planar structure was

Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027. Herein, the isolation, structural elucidation and biological evaluations of these compounds are presented (see Fig. 1). 2. Results and discussion Compound 1 was obtained as white solid and displayed a molecular formula of C32H48O5 as deduced from its (+)-HR-ESIMS sodium adduct ion peak at m/z 535.3394 ([M + Na]+, calcd 535.3394) and 13C NMR data, corresponding to nine indices of hydrogen deficiency. A maximum absorption peak at 223 nm in the UV spectrum suggested the presence of an α,β-unsaturated ketone group, which was further confirmed by the strong absorption band at 1672 cm−1 observed from its IR spectrum. Besides this functionality, the IR spectrum also indicated the presence of hydroxyl and ester carbonyl groups as verified by the strong 242

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19-OH α-bonded. Then, the large coupling constant of J5,6β (13.8 Hz) in the chair-like B-ring allowed the assignment of α-direction for H-5. The assignments of α-orientation for H-7, Me-26, H-18 and H-16α were established by the cross-peaks of H-5/H-7, H-7/H3-26 and H3-26/H-16α & H-18, and then the 7-OH was located in β-orientation. Although the correlations of H-21 with other protons were not observed in the 2D NOESY spectrum, excitation of this proton (δH 1.32) in 1D NOESY experiment confirmed its correlations with both H-18 and H-16α (see Fig. 2B), which allowed the assignment of α-direction for it. With an α,β-unsaturated ketone in the structure, compound 1 showed strong Cotton effects in the ECD measurement (Fig. 3), which allowed the assignment of its absolute configuration via computational method based on the time-dependent density functional theory (TDDFT) (Pescitelli and Bruhn, 2016). Firstly, the conformational searches of the enantiomer with (5R, 7S, 8S, 10R, 13R, 14S, 17S, 18S, 19R, 21S) configuration were carried out using MMFFs force field with an energy window of 3.01 kcaL/mol to obtain 12 conformers which were then re-optimized with density functional theory (DFT) at the B3LYP/6-311G(d,p) level in vacuo. Using these re-optimized conformers, the ECD calculations were performed with the method of TDDFT at the B3LYP/6-311G(d,p) level in vacuo. Finally, the Boltzmann-averaged ECD spectrum (see Fig. 3) returned a negative Cotton effect at 217 nm and a positive Cotton effect at 193 nm, well matching the experimental ECD spectrum of 1 with Cotton effects at 214 and 194 nm, respectively (see Fig. 3). The absolute configuration of 1 was then assigned as shown (5R, 7S, 8S, 10R, 13R, 14S, 17S, 18S, 19R, 21S). Thus compound 1 was elucidated as 25-acetoxy-28-dehydroxyrubiarbonone E. Compound 2 was assigned a molecular formula of C29H46O5 as inferred from its (+)-HR-ESIMS data showing a sodium adduct ion peak at m/z 497.3238 ([M + Na]+, calcd 497.3237), as well as the 13C NMR data, corresponding to seven indices of hydrogen deficiency. The existence of hydroxyl groups was evidenced from the strong absorption band at 3431 cm−1 in the IR spectrum. The 13C NMR data (Table 2) displayed 29 carbons whose multiplicities were, with the help of DEPT spectrum, categorized into two secondary methyls, three tertiary methyls, nine methylenes (one oxygenated), nine methines (two oxygenated & one olefinic) and six quaternary carbons (one olefinic & one carboxyl). One double bond and one carboxyl accounted for two indices of hydrogen deficiency, and the remaining five indicated the presence of five rings in the skeleton of 2. The aforementioned observations suggested that 2 was also a pentacyclic triterpenoid, and the subsequent examination of its 2D NMR data (see Fig. 4) finally established the structure of 2, incorporating a 23-norursane type triterpenoid skeleton with a sp3 C-24, which was severely rare in the family of 23-norursane type triterpenoids (Li et al., 2018; Li et al., 2003). The 1H–1H COSY data (see Fig. 4A) enabled the establishment of four proton-bearing fragments (a–d) as drown in bold bonds, which were connected via six quaternary carbons by the key HMBC correlations (see Fig. 4A) from H3-25 to C-1 (δC 48.3), C-5 (δC 47.8), C-9 (δC 49.4) and C-10 (δC 38.9); H3-26 to C-7 (δC 33.8), C-8 (δC 40.9), C-9 and C-14 (δC 43.4); H-12 to C18 (δC 54.5); H3-27 to C-8, C-13 (δC 140.0), C-14 (δC 43.4) and C-15 (δC 29.2); and H2-16 to C-17 (δC 49.2), C-18, C-22 (δC 38.2) and C-28 (δC 182.4). Particularly, the fragment b together with the key HMBC correlations from H-12 (δH 5.23) to C-18 and H3-27 to C-13 (δC 140.0) revealed that the only double bond was located at Δ12. Furthermore, the proton and carbon resonances observed for CH-2 (δH 3.73, δC 69.7), CH3 (δH 3.54, δC 79.8) and CH2-24 (δH 3.89, 3.58, δC 61.9), in combination with the molecular composition, indicted that three hydroxyl groups were attached at C-2, C-3 and C-24, respectively. Comprehensive analysis of the NOESY data (see Fig. 4B) enabled the construction of the relative configuration of 2 as shown. The correlations of H-3 with both H-1 at δH 0.88 and H-5 indicated that they were axially located in the chair-like A-ring and were assigned to be α-oriented. Then H-7 at δH 1.66, H-9, H-16 at δH 2.02, H-19, H-21 at δH 1.30 and Me-27 were also axially located and α-oriented, which was

Table 2 13 C NMR spectroscopic data of compounds 1–5 (150 MHz). No.

1a

2b

3b

4b

5b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 25-OAc 1′ 2′ 3′ 4′ 5′ 2′-OAc 3′-OAc 4′-OAc

150.6 127.9 203.4 43.8 46.4 32.0 70.3 49.1 136.8 44.4 122.5 37.3 37.6 40.0 32.0 36.4 43.9 59.2 71.5 41.2 57.4 30.5 25.8 22.4 66.2 17.3 16.6 15.6 22.1 23.0 21.0/170.9

48.3 69.7 79.8 50.4 47.8 24.7 33.8 40.9 49.4 38.9 24.4 126.4 140.0 43.4 29.2 25.4 49.2 54.5 40.5 40.4 31.9 38.2

47.4 74.2 79.6 151.4 51.1 22.2 32.0 41.8 53.3 39.2 127.4 126.6 137.5 43.4 26.2 33.9 49.3 134.0 41.4 33.4 38.1 36.8

61.9 16.9 17.8 24.1 182.4 17.7 21.6

105.0 16.6 17.1 20.2 180.6 24.6 32.8

39.4 26.5 82.5 43.8 47.7 18.8 33.4 40.5 49.0 37.6 24.5 123.6 145.2 43.0 28.8 24.1 47.6 42.7 47.2 31.6 34.9 33.8 13.5 64.1 16.3 17.8 26.5 181.9 24.0 33.6

39.4 26.5 82.7 43.8 47.7 18.8 33.4 40.5 49.0 37.6 24.5 123.6 145.2 43.0 28.8 24.0 47.6 42.7 47.2 31.6 34.9 33.8 13.5 64.1 16.3 17.8 26.5 181.8 24.0 33.6

103.9 71.2 74.6 67.6 66.7 20.8/171.4 21.0/172.0

104.3 74.1 70.8 72.8 64.8 21.0/171.8

a b

21.2/172.4

Data were measured in CDCl3. Data were measured in methanol-d4.

enabled by connecting these fragments via six quaternary carbons and a ketone carbonyl based on the key HMBC data (see Fig. 2A), where correlations from H2-25 to C-1 (δC 150.6), C-5 (δC 46.4), C-9 (δC 136.8) and C-10 (δC 44.4); H-2 to C-3 (δC 203.4); H3-23(24) to C-3, C-4 (δC 43.8) and C-5; H-7 to C-9; H-11 to C-8 (δC 49.1), C-9 and C-10; H3-26 to C-8, C-13 (δC 37.6), C-14 (δC 40.0) and C-15 (δC 32.0); H3-27 to C-12 (δC 37.3), C-13, C-14 and C-18 (δC 59.2); and H3-28 to C-16 (δC 36.4), C-17 (δC 43.9), C-18 and C-21 (δC 57.4), were evidenced. Especially, the HMBC correlations from H-2 and H3-23(24) to C-3 (δC 203.4), and H225 to C-1, along with the fragment a, indicated that the α,β-unsaturated ketone group was located in A-ring, while the HMBC correlations from H-7 and H2-25 to C-9, and H-11 to C-8 positioned the other double bond at Δ9(11). In addition, the only acetoxy group was attached at C-25 as deduced from the HMBC correlations from H2-25 (δH 4.63, 4.16) to the ester carbonyl (δC 170.9), and thus the remaining oxygenated carbons of C-7 (δC 70.3) and C-19 (δC 71.5) were determined to be connected with hydroxyl groups. Therefore, the establishment of the planar structure of 1 was accomplished, whose structure differed from 28dehydroxyrubiarbonone E (Quan et al., 2016) at C-25 with an acetoxy substitution. Notably, the oxygenation of Me-25 is the first example among reported arborinane type triterpenoids. The relative configuration of 1 was established mainly by examination of the NOESY data (see Fig. 2B). The NOESY correlations of H3-24/H2-25, H2-25/H-6β, H-6β/H-8, H-8/H3-27, H3-27/H-19, H-19/ H3-28 and H3-28/H-20β, revealed that they were on the same side of the molecule and were randomly assigned as β-orientated, thus leaving 243

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Fig. 1. Chemical structures of 1–23.

functionalities together with the 13C NMR data (Table 2) suggested that compound 3 incorporated a 23-noroleanane type scaffold like its analogue 23 (Lai and Dong, 1996) which was also isolated from this plant. The structural establishment of 3 was accomplished by analyses of the 2D NMR data [see Figs. S30–S36, Supplementary material (SM)]. As with 23, two hydroxyl groups were also attached at C-2 (δC 74.2) and C3 (δC 79.6) as verified by their chemical shifts and the HMBC correlations from H2-24 to C-2. Compared to 23, compound 3 incorporated one more double bond. Except the exocyclic terminal double bond which was located at Δ4(24) as that of 23, the other two double bonds were positioned at Δ11 and Δ13(18) as confirmed by the 1H−1H COSY correlations of H-9 with H-11 and the HMBC correlations from H-11 to C-8, C-10, C-12 and C-13; H-12 to C-9, C-14 and C-18; H3-27 to C-13; and H2-19 to C-13 and C-18. The relative configuration of 3 was constructed as shown mainly by analyzing its ROESY data (see Figs. S35 and S36, SM). Specifically, the α- and β-oriented assignments for 2-OH and 3-OH were first defined by the large coupling constant of J2,3 (9.0 Hz) as in 2, which was further confirmed by the ROESY correlations of H-3 with H-5 and H3-25 with H-2. The calculated ECD spectrum

supported by correlations of H-9 with H-5 and H3-27; H3-27 with H-7α and H-19; H-19 with H-16α; and H-16α with H-21α, thus leaving Me-29 and the carboxyl group β-oriented. Accordingly, the correlations of H-2 with H2-24 and H3-25 suggested that H-2, the hydroxymethyl group and Me-25 were also axially located but β-oriented. Moreover, H-6 at δH 1.66, H-11 at δH 1.96, H-15 at δH 1.95, and H-18, H-20, H-22 at δH 1.63 and Me-26 were assigned to be β-oriented, which was supported by the correlations of H3-25 with H-6β and H-11β; H3-26 with H-11β and H15β; H-18 with H-12 and H-20; and H-20 with H-22β. The structure of 2 was thus elucidated as 2α,3β,24-trihydroxy-23-norurs-12-en-28-oic acid. Analysis of the (+)-HR-ESIMS data of 3 which showed a sodium adduct ion peak at m/z 477.2982 ([M + Na]+, calcd 477.2975), together with the 13C NMR data, assigned a molecular formula of C29H42O4 for 3. The 1H NMR data (Table 1) showed diagnostic signals for five tertiary methyls (δH 1.02, 0.95, 0.82, 0.82, 0.74, each 3H), two oxygenated methines (δH 3.53 and 3.77), two double bonds including one exocyclic terminal double bond (δH 4.73, 5.17, each s) and one endocyclic cis-double bond (δH 6.49, 5.71, each d, J = 10.6 Hz). These 244

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Fig. 2. (A) 1H–1H COSY and key HMBC correlations for 1; (B) Selected 1D and 2D-NOESY correlations for 1.

Fig. 4. (A) 1H–1H COSY and key HMBC correlations for 2; (B) Selected NOESY correlations for 2 (For a better view, CH2-24 and Me-25,26,27,29,30 were simplified as one pink ball and only protons described in the main text were shown). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Experimental ECD spectrum of 1 (black) compared with the calculated ECD spectra of 1 (red) and its enantiomer (blue). Calculated spectra were plotted as sums of Gaussians with a 0.35 eV exponential half-width; they were blue-shifted by 1 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. Experimental ECD spectrum of 3 (black) compared with the calculated ECD spectra of 3 (red) and its enantiomer (blue). Calculated spectra were plotted as sums of Gaussians with a 0.25 eV exponential half-width; they were blue-shifted by 7 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

for the enantiomer with [2R, 3R, 5R, 8R, 9R, 10S, 14S, 17S] configuration using TDDFT method at B3LYP/6-311G(d,p) level in vacuo well matched the experimental data with a negative Cotton effect at 250 nm (see Fig. 5), thus assigning the absolute configuration of 3 as shown. The structure of 3 was thus elucidated as 2α,3β-dihydroxy-23-norurs4(24),11,13(18)-trien-28-oic acid.

Compounds 4 and 5 had the same molecular formula of C39H60O10 as determined by their (+)-HR-ESIMS ion peaks both at m/z 711.4078 [M + Na]+ (calcd 711.4079), as well as their respective 13C NMR data. The 1H NMR data (Table 1) clearly indicated the presence of two acetyl groups both in 4 (δH both 2.05, s, each 3H) and 5 (δH 2.11, 2.14, s, each 3H). Comparison of the NMR data of 4 and 5 (Tables 1 and 2) with those of 19 (Chowdhury et al., 2017), a known oleanane-type 245

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hederagonic acid (14) (Wen et al., 2010), 23α-hydroxy-olean-12-en-3one (15) (Peakman et al., 1991), 2α,23α-dihydroxy-3β-O-trans-feruloyloxy-olean-12-en-28-oic acid (16) (Xiong et al., 2013), 2α-hydroxy3β-O-trans-feruloyloxy-olean-12-en-28-oic acid (17) (Siddiqui et al., 1997), maslinic acid (18) (Yamagishi et al., 1988), leontoside A (19) (Chowdhury et al., 2017), 4′-O-acetyl-3-O-α-L-arabinopyranosyl-23-hydroxyolea-12-en-28-oic acid (20) (Youkwan et al., 2005), 3′-O-acetyl-3O-α-L-arabinopyranosyl-23-hydroxyolea-12-en-28-oic acid (21) (Youkwan et al., 2005) 2′-O-acetyl-3-O-α-L-arabinopyranosyl-23-hydroxyolea-12-en-28-oic acid (22) (Frega et al., 1989), and 2α,3β-dihydroxy-23-norolea-4(24),12-dien-28-oic acid (23) (Lai and Dong, 1996), based on spectroscopic analyses and comparison of their NMR data with those reported in the literature. Compounds 1–23 were evaluated for their cytotoxicities against four cancer cell lines of A549 & H157 (lung), HepG2 (liver) and MCF-7 (breast) using the SRB method (Chen et al., 2018). The preliminary assay results at 30 μM (Table S2, SM) revealed that selective ursane and oleanane type triterpenoids displayed significant cytotoxicity, while the arborinane type triterpenoid (1) was inactive. Interestingly, both ursane and oleanane type triterpenoids of basic skeletons displayed very weak activity, while those incorporating feruloyloxy or arabinosyl substituent at C-3 were much more active. In addition, the elimination of Me-23 for both ursane and oleanane type triterpenoids could evidently decrease the cytotoxicity. The oxidation of C-23 into hydroxymethyl in the oleanane type triterpenoids moderately increased the cytotoxicity. The compounds with inhibition ratio above 90% were further tested for their IC50 values (Table 3), based on which further discussion of structure-activity relationship (SAR) was followed. The oleanane type triterpenoids selectively inhibited A549 and H157 cells over HepG2 and MCF-7 cells, while the ursane type triterpenoid (9) displayed no apparent selectivity toward the four cell lines with IC50 values of 12.80, 5.66, 9.50, and 9.36 μM, respectively. For the oleanane type triterpenoid arabinoglycosides (4, 5 and 19–22), it was worth noting that 4 and 5 with a diacetylated sugar unit displayed significant cytotoxicity against A549 and H157 cell lines with IC50 values below 10 μM, while those incorporating a free (19) or monoacetylated (20–22) sugar moiety showed weaker activity with IC50 values above 20 μM. Thus, the discussion of SAR was accomplished as depicted in Fig. 6. In addition, these isolated compounds were also tested for their antibacterial activities against the Gram-positive Mycobacterium smegmatis ATCC 607 and Staphylococcus aureus ATCC 25923, as well as Gram-negative Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027, by the liquid growth inhibition method. Compounds 5 and 14 showed moderate antibacterial activity against Staphylococcus aureus with IC50 values of 12.3 and 10.3 μM, respectively, while 2 and 3 exhibited weaker activity with each of the IC50 values of 29.4 and 34.1 μM.

Table 3 Cytotoxicity of 4, 5, 9 and 19–22 against four human tumor cell lines. compds

4 5 9 19 20 21 22 doxorubicin

cell line (IC50, μM) A549

H157

HepG2

MCF-7

6.67 6.67 12.80 35.24 22.94 34.18 33.14 1.68

9.70 9.57 5.66 36.45 21.21 30.02 27.54 0.85

15.89 16.23 9.50 46.40 27.13 34.35 34.50 1.57

15.08 15.23 9.36 52.06 26.72 37.32 35.66 0.90

triterpenoid arabinoglycoside whose full NMR data in methanol-d4 (Table S1, SM) were unambiguously assigned by the authors based on 2D NMR spectra (see Figs. S66–S68, SM), revealed that they were both arabinoglycosides and possessed the same aglycone of oleanane-type triterpenoid, with the differences being in the arabinose moiety. Compared to 19, the proton resonances of H-2′ and H-3′ in 4 were severely downfield shifted each by ΔδH 1.65 and 1.40 ppm, while the H-2′ and H-4′ resonances in 5 were downfield shifted by ΔδH 1.44 and 1.27 ppm, respectively. This information together with two additional acetyl groups in 4 and 5 revealed that the 2′-OH and 3′-OH in 4 and the 2′-OH and 4′-OH in 5 were acetylated. Such conclusions were further confirmed by examination of their 2D NMR data (see Figs. S42–S48 for 4, S54eS60 for 5, SM), in which the HMBC correlations from H-2′ and H-3′ in 4, and H-2′ and H-4′ in 5 to each of their corresponding acetyl carbonyls were observed. The connections of sugar units in 4 and 5 with their aglycones via ether bonds between C-3 and C-1′ were confirmed by the HMBC correlations from H-3 (δH 3.61 in 4; δH 3.60 in 5) to C-1′ (δC 103.9 in 4; δC 104.3 in 5). The monosaccharide residues of 4 and 5 were both assigned to be L-arabinoses by chiral HPLC analyses of their acid hydrolysis and acetylation products and comparison with standard sugar sample (see Fig. S69, SM), and then the assignments of αanomeric configurations for arabinoses in 4 and 5 were confirmed by the large coupling constants of the anomeric protons (J1′2′ = 7.3 Hz in 4 and 7.8 Hz in 5). Thus the structures of 4 and 5 were depicted as 2′,3′O-diacetyl-3-O-α-L-arabinopyranosyl-23-hydroxyolea-12-en-28-oic acid (4) and 2′,4′-O-diacetyl-3-O-α-L-arabinopyranosyl-23-hydroxyolea-12en-28-oic acid (5), respectively. Eighteen known compounds were identified to be (6α,11α)-6-[(2′-Oacetyl-α-L-arabinopyranosyl)oxy]-3-oxotaraxast-20-ene-11,28-diyl diacetate (6) (Zhu et al., 2009), ursolic acid (7) (Gnoatto et al., 2008), 3βO-trans-feruloyl-2α,23α-dihydroxy-urs-12-en-28-oic acid (8) (Li et al., 2017), 3β-O-trans-feruloyl-2α-hydroxy-urs-12-en-28-oic acid (9) (Haberlein and Tschiersch, 1994), 2α-hydroxyursolic acid (10) (Kuang et al., 1989), 11α,12α-epoxy-3,6β-dihydroxy-24-norurs-3-en-2-on(28 → 13)-olide (11) (Saito et al., 2012), 3β-hydroxy-24-norurs4(23),12-dien-28-oic acid (12) (Wang et al., 2013), 2α,3β-dihydroxy24-norurs-4(23),12-dien-28-oic acid (13) (Nishimura et al., 1999),

3. Conclusion In summary, our phytochemical study on the roots of D. asper

Fig. 6. Graphical depiction of the general SAR for cancer cell cytotoxicity. 246

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resulted in the isolation of five undescribed triterpenoids with three structural skeletons, along with 18 known compounds. The oxidation of Me-25 in 1 was reported for the first time for arborinane type triterpenoids, while the retained sp3 C-24 in 2 was a very rare structural feature among the 23-norursane class. The absolute configurations of 1 and 3 were assigned by comparing their experimental ECD spectra with the calculated ones. Cytotoxic activities of all the compounds were evaluated against four cancer cell lines (A549, HepG2, H157 and MCF7) and their SAR was also discussed. It was worthwhile to mention that feruloyloxylation or glycosylation at C-3 position could significantly increase the cytotoxicity of ursane and oleanane type triterpenoids, which provided new thinking for the development of potential anticancer chemotherapies based on these triterpenoid skeletons.

column, eluted with petroleum ether (PE)-EtOAc (10:1 to 1:2, v/v), to give six subfractions (B1eB6). Fraction B2 (3.81 g) was then chromatographed on a silica gel CC, eluted with CHCl3-MeOH (200:1 to 30:1, v/v), to afford four fractions (B2a–B2d). Fraction B2a was chromatographed by silica gel CC, eluted with PE-EtOAc (10:1 to 3:1, v/v), to obtain one major component which was further purified by semi-preparative HPLC (MeCN-H2O, 65%, v/v) to yield compound 7 (100 mg, tR = 10.2 min). Subsequently, fraction B2b was separated using the same procedure as B2a to yield compounds 1 (5.1 mg) and 15 (3.2 mg) at tR 11.0 and 14.2 min, respectively. Furthermore, fraction B2d yielded 11 (11.4 mg), 12 (3.7 mg) and 14 (23.0 mg) with each of the retention time at tR 7.0, 9.3 and 13.5 min in the semi-preparative HPLC preparation (MeCN-H2O, 65%, v/v). Fraction B4 (5.2 g) was separated by silica gel CC, eluted with CHCl3-MeOH (200:1 to 30:1, v/v), to obtain five fractions (B4a–B4e). Fraction B4b (30 mg) was subjected to silica gel CC, eluted with PE-acetone (7:1 to 3:1, v/v), to give two major elutions (B4b1 and B4b2), each of which was purified by semi-preparative HPLC, using MeCN-H2O (80%, v/v) as the mobile phase, to afford compounds 9 (8.2 mg, tR = 9.0 min) and 17 (4.7 mg, tR = 8.3 min). Using the same procedure as B4b, fraction B4c returned 8 (8.3 mg, tR = 12.3 min) and 16 (3.5 mg, tR = 13.5 min). Fraction B4e was separated by silica gel CC, eluted with CH2Cl2-MeOH (150:1 to 30:1, v/v), to obtain two fractions (B4e1 and B4e2), which were subsequently purified by semi-preparative HPLC (MeOH-H2O, 72%, v/v) to obtain 3 (28 mg, tR = 13.2 min), 13 (3.2 mg, tR = 17.8 min) and 23 (3.6 mg, tR = 18.6 min), respectively. Fraction B5 (1.2 g) was subjected to a silica gel CC, eluted with CHCl3-MeOH (200:1 to 10:1, v/v), to afford five fractions (B5a–B5e). Fraction B5b (370 mg) was separated using repeated silica gel CC and then was purified by semi-preparative HPLC, using MeCN-H2O (80%, v/v) as the mobile phase, to afford compounds 4 (14.2 mg, tR = 14.3 min), 5 (80 mg, tR = 15.3 min), 6 (2.5 mg, tR = 12.6 min), 10 (16.5 mg, tR = 9.2 min) and 18 (1.3 mg, tR = 11.6 min). Fraction B6 (5 g) was subjected to silica gel CC eluted with CHCl3-MeOH (20:1 to 5:1, v/v) to furnish five major fractions (B6a–B6e). Fraction B6b was separated by silica gel CC, eluted with CHCl3-MeOH-H2O (130:13:5, v/v/v), to obtain three fractions and each of them was finally purified by semi-preparative HPLC, using MeCNH2O (70%, v/v) as the mobile phase, to yield compounds 2 (3.6 mg, tR = 9.6 min), 19 (15.0 mg, tR = 6.3 min), 20 (1.1 mg, tR = 11.8 min), 21 (3.5 mg, tR = 12.3 min) and 22 (27.7 mg, tR = 7.3 min) sequentially.

4. Experimental 4.1. General experimental procedures Optical rotations were measured on a Rudolph VI polarimeter (Rudolph Research Analytical, Hackettstown, USA) with a 10 cm length cell. UV and CD spectra were obtained on a Chirascan Spectrometer (Applied Photophysics Ltd, Leatherhead, UK) with 0.1 cm pathway cell. IR spectra were recorded on a VERTEX70 spectrometer (Bruker Optics Inc., Billerica, USA) with KBr disks. NMR experiments were performed on a Bruker Avance DRX600 spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) and referenced to solvent peaks (δC 49.00 and δH 3.31 ppm for CD3OD; δC 77.16 and δH 7.26 ppm for CDCl3). ESIMS analyses were carried out on an Agilent 1260–6460 Triple Quad LC-MS instrument (Agilent Technologies Inc., Waldbronn, Germany). HRESIMS data were acquired on an Agilent 6545 QTOF mass spectrometer (Agilent Technologies Inc., Waldbronn, Germany). All HPLC analyses and separations were performed on Agilent 1260 series LC instruments (Agilent Technologies Inc., Waldbronn, Germany). All solvents used for column chromatography were of analytical grade (Tianjin Fuyu Fine Chemical Co. Ltd., Tianjin, China) and solvents used for HPLC were of HPLC grade (Oceanpak Alexative Chemical. Ltd, Goteborg, Sweden). Agilent SB-C18 (9.4 × 250 mm) column (Agilent Technologies Inc., Santa Clara, USA) was used for HPLC separations, and Chiral MZ(2)-RH (4.6 × 250 mm) chiral column (NuAnalytical-FLM, Torrance, USA) was used for chiral analysis. 4.2. Plant materials

4.3.1. 25-Acetoxy-28-dehydroxyrubiarbonone E (1) White solid; [α]D24 −45.9 (c 0.43, MeOH); UV (MeOH) λmax (log ε) 223 (4.06) nm; ECD (c 0.05, MeOH) 194 (Δε 15.15), 214 (Δε −9.60) nm; IR (KBr) νmax 3437, 2953, 2930, 2870, 1744, 1672, 1472, 1380, 1236, 1038, 756 cm−1; 1H and 13C NMR data (CDCl3) see Tables 1 and 2; (+)-ESIMS: m/z 535.3 [M + Na]+; (+)-HR-ESIMS: m/z 535.3394 [M + Na]+ (calcd for C32H48O5Na, 535.3394).

The roots of Dipsacus asper Wall. ex C. B. Clarke (Caprifoliaceae) were bought in Kunming ‘Juhuayuan’ herbal market (collected in September (autumn) 2016 from An'ning county of Yunnan Province, China) and were authenticated by Prof. Jie Zhou from University of Jinan. The plant was originally identified as Dipsacus asperoides C. Y. Cheng et T. M. Ai in the Chinese version ‘Flora of China’ (Lu and Chen, 1986) and later revised in the emended English version (Flora of China Editorial Committee, 2011) to be consistent with that in ‘The Plant List’ (The Plant List, 2013). A voucher specimen has been deposited at School of Biological Science and Technology, University of Jinan (Accession number: npmc-013).

4.3.2. 2α,3β,24-trihydroxy-23-norurs-12-en-28-oic acid (2) White solid; [α]D24 80.2 (c 0.10, MeOH); IR (KBr) νmax 3431, 2930, 2873, 1698, 1459, 1379, 1058 cm−1; 1H and 13C NMR data (CD3OD) see Tables 1 and 2; (+)-ESIMS: m/z 497.3 [M + Na]+, 971.6 [2M + Na]+; (+)-HR-ESIMS: m/z 497.3238 [M + Na]+ (calcd for C29H46O5Na, 497.3237).

4.3. Extraction and isolation The air-dried roots of Dipsacus asper (10 kg) were smashed into powder and then extracted with 95% EtOH at room temperature for four times (one week per time) to afford a crude extract (1.0 kg). The extract was partitioned between EtOAc (2 L) and H2O (2L) for three times to afford the EtOAc soluble part. After evaporation of the solvent under reduced pressure, the residue (148 g) was subjected to column chromatography (CC) over D101 macroporous absorption resin, eluted with EtOH-H2O (50%, 80% and 95%, v/v) to get three fractions (A, B and C). Fraction B (37 g) was subjected to passage over a silica gel

4.3.3. 2α,3β-dihydroxy-23-norurs-4(24),11,13(18)-trien-28-oic acid (3) White solid; [α]D24 −90.9 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 250 (4.01) nm; ECD (c 0.04, MeOH) 250 (Δε −5.47) nm; IR (KBr) νmax 3427, 2933, 2864, 1699, 1651, 1633, 1454, 1386, 1265, 1059 cm−1; 1 H and 13C NMR data (CD3OD) see Tables 1 and 2; (+)-ESIMS: m/z 477.1 [M + Na]+; (+)-HR-ESIMS: m/z 477.2982 [M + Na]+ (calcd for C29H42O4Na, 477.2975). 247

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4.3.4. 2′,3′-O-diacetyl-3-O-α-L-arabinopyranosyl-23-hydroxyolea-12-en28-oic acid (4) White solid; [α]D24 59.6 (c 0.51, MeOH); IR (KBr) νmax 3453, 2943, 2871, 1742, 1463, 1372, 1237, 1051, 758 cm−1; 1H and 13C NMR data (CD3OD) see Tables 1 and 2; (−)-ESIMS: m/z 687.3 [M − H]−, 723.3 [M + Cl]−; (+)-HR-ESIMS: m/z 711.4078 [M + Na]+ (calcd for C39H60O10Na, 711.4079).

4.7. Antibacterial assays The antibacterial activities were tested using liquid growth inhibition method as we described previously (Bao et al., 2018). Ceftriaxone sodium was used as a positive control (IC50 < 1.0 μM). Conflicts of interest The authors declare no conflict of interest.

4.3.5. 2′,4′-O-diacetyl-3-O-α-L-arabinopyranosyl-23-hydroxyolea-12-en28-oic acid (5) White solid; [α]D24 48.5 (c 0.41, MeOH); IR (KBr) νmax 3499, 2951, 1733, 1467, 1376, 1239, 1055, 753 cm−1; 1H and 13C NMR data (CD3OD) see Tables 1 and 2; (−)-ESIMS: m/z 687.3 [M − H]−, 723.3 [M + Cl]−; (+)-HR-ESIMS: m/z 711.4078 [M + Na]+ (calcd for C39H60O10Na, 711.4079).

Acknowledgements This work was funded by Natural Science Foundation of Shandong Province [No. JQ201721 & ZR2018BB023], the Young Taishan Scholars Program [No. tsqn20161037], National Natural Science Foundation of China [No. 21672082], the Director Foundation of XTIPC, CAS (No. 2015RC015) and Shandong Talents Team Cultivation Plan of University Preponderant Discipline [No. 10027].

4.4. ECD calculation of compounds 1 and 3 The initial conformations of 1 and 3 were established via the MM2 force field in the ChemDraw_Pro_14.1 software. Conformational searches using mixed torsional/Low-mode sampling method with MMFFs force field in an energy window of 3.01 kcaL/mol were carried out by means of the conformational search module in the Maestro 10.2 software, returning 12 conformers for 1 and 4 conformations for 3. The reoptimization and the following TD-DFT calculations of the re-optimized conformations were all performed with Gaussian 09 (Frisch et al., 2010) at the B3LYP/6-311G(d,p) level in vacuo. Frequency analysis was performed as well to confirm that the re-optimized conformers were at the energy minima. Finally, the SpecDis 1.64 software (Bruhn et al., 2013) was used to obtain the Boltzmann-averaged ECD spectra of 1 and 3.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.03.028. References Bao, J., Zhai, H.J., Zhu, K.K., Yu, J.H., Zhang, Y.Y., Wang, Y.Y., Jiang, C.S., Zhang, H., Zhang, X.Y., Zhang, Y., Zhang, H., 2018. Bioactive pyridone alkaloids from a deepsea-derived fungus arthrinium sp. UJNMF0008. Mar. Drugs 16. https://doi.org/10. 3390/md16050174. Bruhn, T., Schaumloeffel, A., Hemberger, Y., Bringmann, G., 2013. SpecDis: quantifying the comparison of calculated and experimental electronic circular dichroism spectra. Chirality 25, 243–249. Chen, Z., Duan, H., Tong, X., Hsu, P., Han, L., Morris-Natschke, S.L., Yang, S., Liu, W., Lee, K.H., 2018. Cytotoxicity, hemolytic toxicity, and mechanism of action of pulsatilla saponin D and its synthetic derivatives. J. Nat. Prod. 81, 465–474. Choi, N.H., Jang, J.Y., Choi, G.J., Choi, Y.H., Jang, K.S., Nguyen, V.T., Min, B.S., Le Dang, Q., Kim, J.C., 2017. Antifungal activity of sterols and dipsacus saponins isolated from Dipsacus asper roots against phytopathogenic fungi Pestic. Biochem. Physiol. 141, 103–108. Chowdhury, M.A., Ko, H.J., Lee, H., Aminul Haque, M., Park, I.S., Lee, D.S., Woo, E.R., 2017. Oleanane triterpenoids from Akebiae caulis exhibit inhibitory effects on Aβ42 induced fibrillogenesis. Arch Pharm. Res. (Seoul) 40, 318–327. Editorial Committee of the Administration Bureau of Traditional Chinese Medicine, 1999. Chinese Materia Medica, vol. 20. Shanghai Science & Technology Press, Shanghai, pp. 581–584. Flora of China Editorial Committee, 2011. Flora of China, vol. 19 Science Press & Missouri Botanical Garden Press, Beijing & St. Louis. http://foc.eflora.cn/content.aspx? TaxonId=242318436. Frega, N., Bonaga, G., Lercker, G., Bortolomeazzi, R., 1989. Triterpenic acids in the epicarp of the olive drupe. Riv. Ital. Sostanze Grasse 66, 107–109. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G., Sonnenberg, J.L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery Jr., J.A., Peralta, J.E., Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Rega, Millam, N.J., Klene, M., Knox, J.E., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R.E., Stratmann, O., Yazyev, A.J., Austin, R., Cammi, C., Pomelli, J.W., Ochterski, R., Martin, R.L., Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P., Dannenberg, J.J., Dapprich, S., Daniels, A.D., Farkas, O., Foresman, J.B., Ortiz, J.V., Cioslowski, J., Fox, D.J., 2010. Gaussian 09, Rev. B.01. (Wallingford CT). Gnoatto, S.C.B., Dassonville-Klimpt, A., Da Nascimento, S., Galera, P., Boumediene, K., Gosmann, G., Sonnet, P., Moslemi, S., 2008. Evaluation of ursolic acid isolated from Ilex paraguariensis and derivatives on aromatase inhibition. Eur. J. Med. Chem. 43, 1865–1877. Haberlein, H., Tschiersch, K.P., 1994. Triterpenoids and flavonoids from Leptospermum scoparium. Phytochemistry 35, 765–768. Hung, T.M., Jin, W., Thuong, P.T., Song, K.S., Seong, Y.H., Bae, K., 2005. Cytotoxic saponins from the root of Dipsacus asper wall. Arch Pharm. Res. (Seoul) 28, 1053–1056. Hung, T.M., Na, M.K., Thuong, P.T., Su, N.D., Sok, D.E., Song, K.S., Seong, Y.H., Bae, K.H., 2006. Antioxidant activity of caffeoyl quinic acid derivatives from the roots of Dipsacus asper wall. J. Ethnopharmacol. 108, 188–192. Huang, T.M., Thuong, P.T., Youn, U.J., Zhang, X.F., Min, B.S., Woo, M.H., Lee, H.K., Bae, K.H., 2008. Antioxidant activities of phenolic derivatives from Dipsacus asper Wall. (II). Nat. Prod. Sci. 14, 107–112.

4.5. Determination of L-arabinoses of 4 and 5 The solutions (3.0 mg each) of 4 and 5 in 1.0 moL/L HCl (3.0 mL) were stirred at 80 °C for 3 h. After removing the excess HCl under reduced pressure, the residual aqueous mixture was filtered to eliminate the aglycone products and was then evaporated under reduced pressure to afford the monosaccharaides, which were acetylated with excess acetic anhydride in pyridine at room temperature for 10 h. The authentic D- and L-arabinoses were also acetylated using the same method. The prepared samples were analyzed by HPLC using a Chiral MZ(2)-RH (4.6 × 250 mm) column (see Figs. S69–S73 in SM). 4.6. Cytotoxic assays The cytotoxic assays were performed using the SRB method described below. The human cancer cell lines of A549 & H157 (lung), HepG2 (liver) and MCF-7 (breast) were cultured in RPMI-1640 medium supplemented with 2.05 mM L-glutamine and 10% FBS (Every Green, Zhejiang Tianhang Biotechnology Co., Ltd., China) in a humidified environment with 5% CO2 at 37 °C. Trypsin-EDTA (Every Green, Zhejiang Tianhang Biotechnology Co., Ltd., China) was used to separate cells from the culture flask. After dilution to 5 × 104 cells mL−1 with the complete medium, 100 μL of the cell suspension was added to each well of 96-well culture plates. The subsequent incubation was carried out at 37 °C in 5% CO2 atmosphere for 24 h. The tested compounds at indicated concentrations were added to each well and then incubated for 48 h. Doxorubicin was used as a positive control. The attached cells were fixed with cold 50% trichloroacetic acid for 30 min, and then stained with 0.04% SRB (Sigma Chemical Co.) for 30 min. After solubilizing the bound SRB in 10 mM Tris-base, the absorbance was measured at 450 nm on a SPARK multimode microplate reader (Tecan, Switzerland). The IC50 is the concentration of tested samples that reduced cell growth by 50% under the experimental conditions. All results were representatives of three independent experiments. 248

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