Kalshiolin A, new lignan from Kalimeris shimadai

Abstract

The Asian plant Kalimeris shimadai has been used as food and ethnologic medicine for over a thousand years. In this study, we isolated and identified one new lignan, kalshiolin A (1), and 12 known lignans (2–13). The structures were characterized by the comprehensive analyses of spectroscopic data (HR-ESI-MS, IR, 1D, and 2D-NMR) and the absolute configuration of 1 was determined from ECD calculations. The new compound 1 was also screened for cytotoxic activity but did not show significant potency (IC₅₀ 35.9–43.3 lM) against A549, MDA-MB-231, MCF7, KB, and KB-VIN cell lines.

Graphical Abstract

1. Introduction

Kalimeris shimadai (family Asteraceae) is widely distributed in eastern and southern China and Taiwan. It is widely and regularly used in daily cooking, due to its nutrient and medical benefits, as well as flavor [1]. Previous phytochemical and medically related reports are limited; however, terpenoids, phenolic derivatives and anthraquinones, sesquiterpenes, and flavonoids have been found in K. shimadi [2–7]. In this study, we report the isolation and structure elucidation of a new lignan, kalshiolin A, along with 12 known lignans (Figure 1).

Figure 1.

Chemical structure of compound 1.

2. Results and discussion

Phytochemical investigation of an ethanol extract of K. shimadai using various chromatographic methods led to the isolation of 13 compounds. Compound 1 was obtained as yellow oil with ${[\alpha ]}_{\text{D}}^{23.7}-8.3(c=0.24;{\text{CH}}_3\text{OH})$. The HR-ESI-MS of 1 displayed a pseudomolecular ion peak [M + K]⁺ at m/z 467.1474, indicating the molecular formula C₂₄H₂₈O₇ with 13 indices of hydrogen deficiency (IHD). The IR spectrum of 1 showed the presence of hydroxyl (3428 cm⁻¹) and carbonyl (1738 cm⁻¹) moieties. The ¹H-NMR spectrum (Table 1) exhibited characteristic signals for five aromatic protons at δ_H 6.89–6.88 and two trans-olefinic protons at δ_H 6.55 (1H, d, J = 15.9 Hz) and 6.17 (1H, m), as the AB part of ABX₂-coupled spin system, as well as two methoxy groups (δ_H 3.90 (3H, s); 3.86 (3H, s)) and one ethoxy group (δ_H 3.55 (2H, q); 1.25 (3H, t, J = 7.1 Hz)). In accordance with the molecular formula, 24 carbon resonances were seen in the ¹³C NMR spectrum (Table 1) and further classified by DEPT and HSQC spectra as four methyl, three methylene, nine methine, and eight quaternary carbons. In addition to the carbon resonances for ethoxy and methoxy groups, the two carbon signals at δ_C 171.0 and 21.0 indicated the presence of an acetoxy group in 1. Furthermore, the key HMBC and ¹H–¹H COSY correlations (Figure 2) of the olefinic side chain fragment (−CH CHCH₂OCH₂CH₃) were H-9/C-5, H-10/C-8, and the methylene of ethoxy group (δ_H 3.55) to C-10, along with ¹H–¹H COSY between H-8/H-9 and H-9/H-10. Based on comparison with literature data, compound 1 is likely an analog of dimeric coniferyl acetate [8] and 10,11-diacetyl dehydrodiconiferyl alcohol [9] but with an ethoxy rather than acetoxy group attached to C-10. Thus, the planar structure of 1 was defined from the aforesaid data. The relative stereochemistry of the dihydrofuran ring was identical to that in 10,11-diacetyl dehydrodiconiferyl alcohol based on NOE experiments (Figure 3) [10]. The strong correlations between H-3 and H-2’/6’ and only weak correlations between H-2 and H-3 indicated a trans configuration between the C-2 and C-3 substituents. The absolute configuration of 1 was determined by the comparison of experimental and theoretical electronic circular dichroism spectra. Using the Gaussian 09 software package, the selected conformers were optimized at B3LYP/6–31G (d,p) level of theory. The theoretical calculation of ECD was performed using time-dependent density functional theory (TDDFT) at B3LYP/6–311G (2d,p) level [11]. As shown in Figure 4, the calculated ECD for 2R,3S matched with the experimental curves and thus determined the absolute configurations at these positions. Therefore, the structure of 1 (kalshiolin A) was characterized completely as ((2R,3S)-5-((E)-3-ethoxyprop-1-enyl)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-yl) methyl acetate (Figure 1).

Table 1.

¹H and ¹³C NMR spectral data for compound 1.

position	δH (J in Hz)	δC	position	δH (J in Hz)	δC
2	5.46 d (7.3)	88.9 CH	1′		132.4 C
3	3.77 m	50.5 CH	2′	6.89–6.88 m	108.7 CH
4	6.89–6.88 m	115.2 CH	3′		146.8 C
5		131.2 C	4′		146.0 C
6	6.89–6.88 m	110.4 CH	5′	6.89–6.88 m	114.4 CH
7		144.5 C	6′	6.89–6.88 m	119.7 CH
8	6.55 d (15.9)	132.4 CH	3a		127.7 C
9	6.17 m	124.5 CH	7a		148.0 C
10	4.12 m	71.4 CH2	3′-OCH3	3.86 s	56.1 CH3
11	4.43 m 4.30 m	65.5 CH2	7-OCH3	3.90 s	56.1 CH3
COCH3	2.02 s	171.0 C 21.0 CH3	OCH2CH3	3.55 q (7.1) 1.25 t (7.1)	65.8 CH2 15.4 CH3

Figure 2.

Key ¹H-¹H COSY and HMBC correlations of compound 1.

Figure 3.

Key ROESY correlations of compound 1.

Figure 4.

Experimental and calculated ECD spectra of 1 (solid line, experimentally recorded in MeOH; dashed line, calculated for 2R,3S configuration in MeOH).

From the NMR and MS data and comparison with the prior literature, the 12 known lignans isolated from the plant were identified as pinoresinol (2) [12], medioresinol (3) [13], syringaresinol (4) [14], episyringaresinol (5) [15], (+)-isolariciresinol (6) [16], ciwujiatone (7) [17], vibruresinol (8) [18], (±)-5’-methoxylariciresinol (9) [19], dehydrodiconiferyl alcohol (10) [20], (−)-simulanol (11) [21], (+)-(1S, 2R)-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (12) [22], and cleomiscosin C (13) [23]. Their structures were characterized by the comprehensive analyses of spectroscopic data. All 13 lignans were isolated from this plant for the first time.

Kalshiolin A was screened for cytotoxic activity against five human tumor cell lines, A549, MDA-MB-231, MCF7, KB and KB-VIN. However, no significant antiproliferative effects were seen with IC₅₀ values ranging from 35.9 to 43.3 µM.

3. Experimental

3.1. General experimental procedures

The IR data were obtained on a Tensor27 FT-IR spectrometer (Bruker, Billerica, MA, USA). The UV spectra were measured on a UV-2401A spectrophotometer (Shimadzu Corp., Kyoto, Japan). The optical rotation was determined on a SEPA 300 polarimeter (Horiba, Kyoto, Japan). 1D and 2D-NMR spectra were recorded on Bruker DRX-500 and AM-400 spectrometers with tetramethylsilane as an internal standard. CD data were obtained using a Chirascan CD spectrometer (Applied Photophysics, Surrey, UK). The HR-ESI-MS data were obtained on a 6200 Q-TOF MS system (Agilent Technologies, Santa Clara, CA, USA). Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used for column chromatography (CC). Preparative HPLC was performed on an Agilent 1260 instrument with an Agilent Zorbax SB-C18 column (5 lm, 9.4 × 150 mm). Fractions were monitored by TLC, and spots were visualized by spraying with 10% H₂SO₄ in EtOH, followed by heating.

3.2. Plant material

Kalimeris shimadai plants were collected from Hefei, Anhui Province of China, on July 2016 and identified by Qing-shan Yang of Anhui University of Chinese Medicine. The voucher specimen (No. 201601) was stored at the Anhui University of Chinese Medicine.

3.3. Extraction and isolation

The air-dried and powdered aerial parts of K. shimadai (20 kg) were heated at reflux with 80% ethanol (2 h × 3). The extract was separated by silica gel CC eluting with a step gradient of CH₂Cl₂/MeOH (100:0–0:100, v/v) to obtain 11 fractions (Fr 1–Fr 11). Fr 5 was chromatographed on a silica gel column eluting with petroleum ether-acetone (v/v, 100:0, 50:1, 10:1, 1:1) to yield five subfractions. Fr 5–4 was chromatographed on a silica gel column eluting with a mixture of CH₂Cl₂ and MeOH (v/v, 100:0, 50:1, 30:1, 10:1, 1:1) to yield four subfractions. Fr 5–4-3 was chromatographed on an ODS column eluting with MeOH/H₂O (20:80 to 100:0). Fr 5–4-3–1 was further purified by Sephadex LH-20 column chromatography (MeOH) and preparative HPLC (CH₃CN/ H₂O, 35:65–55:45, 30 min) to provide compound 1 (9.5 mg). Fr 5–4-3–6 was chromatographed on a silica gel column eluting with petroleum ether-acetone (v/v, 15:1–1:1), and Fr 5–4-3–6-7 was purified by preparative HPLC (CH₃CN/H₂O, 20:80–28:72, 25 min) to afford 2 (8.8 mg). Fr 5–4-3–6-9 was further purified by preparative HPLC (CH₃CN/H₂O, 20:80–25:75, 30 min) to provide 3 (9.8 mg), and Fr 5–4-3–6-9 was further purified by preparative HPLC (CH₃CN/H₂O, 16:84–36:64, 25 min) to provide 4 (11.8 mg). Fr 5–4-3–7 was chromatographed on a silica gel column eluting with petroleum ether-acetone (v/v, 20:1–1:1), further recrystallized to afford 13 (5.5 mg) and purified by preparative HPLC (CH₃CN/H₂O, 15:8–30:70, 25 min) to provide 5 (5.9 mg).

Fr 6 was decolorized using Rp-18 column, eluted with MeOH-H₂O (v/v, 20:80 to 100:0) to yield 11 subfractions. Fr 6–4 was chromatographed on silica gel column eluted with petroleum ether-acetone (v/v, 10:1–8:1), and Fr 6–4-4 was purified by preparative HPLC using (CH₃CN/H₂O, 5:95–20:80, 35 min) to provide 12 (9.5 mg). Fr 6–10 was chromatographed on Sephadex LH-20 column chromatography (MeOH), and Fr 6–10-3 was purified by preparative HPLC (CH₃CN/H₂O, 14:86–24:76, 20 min) to provide 9 (4.8 mg). Fr 6–10-4 was purified by preparative HPLC (CH₃CN/H₂O, 14:86–30:70, 32 min) to provide 6 (2.8 mg), 7 (4.3 mg), and 8 (4.5 mg). Fr 6–11 was chromatographed on Sephadex LH-20 column chromatography (MeOH), and Fr 6–11-2 was purified by preparative HPLC to provide 11 (5.8 mg) (CH₃CN/H₂O, 15:85–20:80, 30 min) and 10 (4.3 mg) (CH₃CN/H₂O, 18:82–30:70, 24 min).

3.3.1. Kalshiolin A (1)

Yellow oil; ${[\alpha ]}_{\text{D}}^{23.7}-8.3(MeOH;c0.24)$; UV (MeOH) λmax (log ε): 203(4.15), 219(3.97), 277(3.70), 299(3.36) nm; IR (KBr) ν_max cm⁻¹: 3428, 2933, 1738, 1607, 1517, 1240, 1122, 1034; ¹H-NMR and ¹³C-NMR (CDCl₃, 400/125 MHz) spectral data see Table 1; HR-ESI-MS: m/z 467.1474 [M + K]⁺ (calcd for C₂₄H₂₈O₇K, 467.1467).

3.4. Cytotoxicity assay

Human lung carcinoma (A549), triple-negative or hormone-responsible breast cancer (MDA-MB-231 or MCF7), originally isolated as epidermoid carcinoma of the nasopharynx (KB), and P-glycoprotein-overexpressing multidrug-resistant KB subline (KB-VIN) cell lines were obtained from ATCC or UNC Lineberger Comprehensive Cancer Center. The cytotoxic activity of test compound was determined by the sulforhodamine B (SRB) colorimetric assay [24]. In brief, the cells (4–11 × 10³ cells/well) were seeded in 96-well plates filled with culture medium containing various concentrations of sample and incubated for 72 h. At the end of the exposure period, the attached cells were fixed in cold 10% trichloroacetic acid for 30 min followed by staining with 0.04% sulforhodamine B (SRB) for 30 min. The bound SRB was solubilized in 10 mM Tris base, and formazan absorbance was assessed by a Bio-Rad microplate reader at 492 nm with Gen5 software. All results were representative of three or more experiments.