New Neo-Lignan From Acanthopanax senticosus and the Cytotoxic Effects on Human Cancer Cell Lines

Abstract

A new neo-lignan named (7ʹS,8ʹR)-4ʹ,5ʹ,9ʹ-trihydroxy-5-methoxy-4,8ʹ-oxyneolign-7-en-9-al (1), together with 5 known compounds (2-6) were isolated from the ethylacetate-soluble extract of Acanthopanax senticosus. The structure of new neo-lignan was elucidated with spectroscopic and physicochemical analyses. All the isolates were evaluated for in vitro cytotoxicity against 4 human cancer lines including HepG2, A549, Hela, and MCF-7. Among them, compounds 1 and 3 showed the potent antiproliferative activities against the HepG2 cancer cells with half-maximal inhibitory concentration (IC₅₀) values of 9.8 ± 1.6 and 15.0 ± 1.1 μM, respectively. In addition, compound 6 exhibited moderate inhibitory activity on MCF-7 cancer cells with an IC₅₀ value of 18.3 ± 1.5 μM.

Acanthopanax senticosus (Rupr. & Maxim.) Harms (Araliaceae), is a shrub found widely distributed in the northeast of Asia such as China, Korea, Japan, and Russia. The stems and roots of A. senticosus were named as “Ci-Wu-Jia” in China, and it was traditionally used for treating acute rheumatism, ¹ hydroncus, ² hypopiesia, ² and chloasma. ³ Pharmacological experiments showed that A. senticosus possesses anti-inflammatory, ⁴ antidiabetes, ⁵ anticancer, ⁶ hepatoprotective, ⁶ antioxidant, ⁷ hypocholesterolemic, ⁷ and other biological activities. And previous chemical studies of A. senticosus revealed the presence of flavonoids, ⁸ lignans, ⁸ triterpene saponins, ⁹ and aromatic glycosides. ¹⁰ During our course of identifying more interesting and bioactive compounds from A. senticosus, a new neo-lignan named (7’S,8’R)−4’,5’,9’-trihydroxy-5-methoxy-4,8’-oxyneolign-7-en-9-al (1), together with 5 known compounds (2-6), were isolated from A. senticosus. Herein, we report the isolation and structural elucidation of six compounds, and the evaluation of their cytotoxicity on HepG2, A549, Hela, and MCF-7 cell lines.

Results and Discussion

Compound 1 was obtained as a brown amorphous powder, and the molecular formula was established as C₁₉H₂₀O₇ based on high-resolution electrospray ionization mass spectrometry (HREIMS) at m/z 360.1024 for the [M]⁺. The ¹H nuclear magnetic resonance (NMR) spectrum (Supplemental Figure S1; Table 1) of 1 showed a characteristic aldehyde proton at δ _H 9.60 (1H, d, J = 8.0 Hz, H-9), 2 olefinic protons at δ _H 7.58 (1H, d, J = 16.0 Hz, H-7) and 6.69 (1H, dd, J = 16.0, 8.0 Hz, H-8), 2 set of ABX-type aromatic protons at δ _H 7.29 (1H, d, J = 2.0 Hz, H-6), 7.18 (1H, dd, J = 8.5, 2.0 Hz, H-2), and 7.09 (1H, d, J = 8.5 Hz, H-3); and 7.04 (1H, d, J = 2.0 Hz, H-6′), 6.86 (1H, dd, J = 8.5, 2.0 Hz, H-2′), and 6.78 (1H, d, J = 8.5 Hz, H-3′), 2 oxymethines and a oxygen-bearing methylene at δ _H 4.90 (1H, d, J = 5.5 Hz, H-7′), 4.51 (1H, m, H-8′) and 3.55 (1H, d, J = 12.0, 5.5 Hz, H-9′)/3.79 (1H, d, J = 12.0, 3.5 Hz, H-9′), and a methoxy group at δ _H 3.91. The ¹³C NMR spectrum of 1 (Supplemental Figure S2) revealed 19 carbons including 12 aromatic carbons, an aldehyde carbon at δ _C 196.3 (C-9), 2 olefinic carbons at δ _C 155.6 (C-7) and 127.9 (C-8), a methoxyl carbon at δ _C 56.5, and 3 oxygenated carbons at δ _C 86.2 (C-8′), 74.0 (C-7′) and 62.2 (C-9′). In particular, the nuclear magnetic resonance (NMR) data of 1 were in close agreement with those of erythro-guaiacylglycerol-β-coniferyl aldehyde ether, ¹¹ and the key difference between them is an extra methoxy group in erythro-guaiacylglycerol-β-coniferyl aldehyde ether. The heteronuclear multiple bond correlations (HMBC) (Supplemental Figure S3; Figure 1) of δ _H 7.58 (H-7) with δ _C 129.5 (C-1), 124.7 (C-2), and 112.8 (C-6), and of δ _H 6.69 (H-8) with δ _C 129.5 (C-1), 155.6 (C-7), and 196.3 (C-9), indicating the propenal moiety attached to C-1. In addition, the location of methoxy group could be deduced by the key HMBC of δ _H 3.91 with δ _C 151.8 (C-5), and of δ _H 7.18 (H-2) with δ _C 129.5 (C-1), 116.0 (C-3) 153.1 (C-4), and 112.8 (C-6). And the key nuclear Overhauser effect spectroscopy (NOESY) correlations of δ _H 3.91 with δ _H 7.29 (H-6) could also confirm this conclusion. The erythro-configuration of H-7′ and H-8′ was determined by the coupling constant J _7ʹ8ʹ = 5.5 Hz and the NOESY correlations (Supplemental Figure S4; Figure 1) between H-7′ with H_α− 9′. The absolute configuration of C-8′ of 1 was established on the basis of the circular dichroism spectroscopic evidence (Supplemental Figure S5). And the 7′S,8′R-configuration was confirmed by a negative Cotton effect near 229 nm and some study of related system. ^12,13 Thus, the structure of compound 1 was assigned as (7′S,8′R)-4′,5′,9′-trihydroxy-5-methoxy-4,8′-oxyneolign-7-en-9-al.

Table 1

Nuclear Magnetic Resonance Data of Compound 1 in Deuterated Methanol (¹H: 500 MHz, ¹³C: 125 MHz).

No.	δ C	δ H
1	129.5
2	124.7	7.18 dd (2.0, 8.5)
3	116.0	7.09 d (8.5)
4	153.1
5	151.8
6	112.8	7.29 d (2.0)
7	155.6	7.58 d (16.0)
8	127.9	6.69 dd (8.0, 16.0)
9	196.3	9.60 d (8.0)
1′	133.9
2′	120.8	6.86 dd (2.0, 8.0)
3′	117.3	6.78 d (8.0)
4′	148.2
5′	146.6
6′	111.8	7.04 d (2.0)
7′	74.0	4.90 d (5.5)
8′	86.2	4.51 m
9′	62.2	3.55 dd (5.5, 12.0)3.79 dd (3.5, 12.0)
OCH3	56.5	3.91 s

Figure 1

Two-dimensional nuclear magnetic resonance correlations of compound 1 . HMBC, heteronuclear multiple bond correlation; NOESY, nuclear Overhauser effect spectroscopy.

Moreover, 5 known compounds (2-6) were obtained from A. senticosus, and identified as (7S, 8R)-5-methoxyl-balanophonin (2), ¹⁴ balanophonin (3), ¹⁵ curcasinlignan B (4), ¹⁶ curcasinlignan C (5), ¹⁶ and 1-(4-hydroxy-3-methoxyphenyl)−2-{4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy}-propane-1,3-diol (6) ¹⁷ based on NMR data and literatures (Figure 2).

Figure 2

The structure of compounds 1-6.

All compounds were evaluated for their cytotoxicity against 4 cell lines including HepG2, A549, Hela, and MCF-7, and the doxorubicin was used as the positive control. Compounds 1-3 and 6 showed different levels of cytotoxicity on 4 cancer cell lines (Table 2). Among these isolates, compound 1 showed the highest cytotoxicity on HepG2 and A549 cells with half-maximal inhibitory concentration (IC₅₀) values of 9.8 ± 1.6, 19.5 ± 1.3 µM, respectively. And compound 3 also exhibited significant cytotoxicity toward HepG2 cells with an IC₅₀ value of 15.0 ± 1.1 µM. Furthermore, the comparison of cytotoxicity on HepG2 cells between 2 (41.5 ± 1.9 µM) and 3 (15.0 ± 1.1 µM) indicated that the cytotoxicity might be associated with the methoxy group which linked with C-3 of 2. In addition, compound 6 showed moderate cytotoxicity on MCF-7 cells with an IC₅₀ value of 18.3 ± 1.5 µM.

Table 2

Cytotoxicities of Compounds 1-6 on 4 Cell Lines.

Compounds	IC50 (μM)
	HepG2	A549	Hela	MCF-7
1	9.8 ± 1.6	19.5 ± 1.3	>50	32.0 ± 1.1
2	41.5 ± 1.9	>50	47.1 ± 1.2	>50
3	15.0 ± 1.1	38.8 ± 1.4	>50	45.8 ± 0.8
4	.>50	>50	>50	>50
5	>50	>50	>50	>50
6	32.5 ± 1.0	>50	>50	18.3 ± 1.5
Doxorubicin	3.9 ± 0.8	3.5 ± 1.0	2.1 ± 1.2	4.6 ± 1.7

IC₅₀, half-maximal inhibitory concentration.

IC₅₀ values were determined by regression analyses and expressed as means ± SD of 3 replicates.

^aPositive control.

Experimental

General

Optical rotations were determined by a JASCO P-2000 polarimeter (JASCO, Tokyo, Japan). Ultraviolet (UV) spectra were recorded on the diode-array detector Agilent SL1100 series (Agilent, Santa Clara, CA, USA). Infrared (IR) spectra were recorded with a JASCO FT-IR 620 spectrophotometer (JASCO, Tokyo, Japan). NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer with tetramethylsilane (TMS) as an internal standard (Bruker, Karlsruhe, Germany). Mass spectra were obtained from an API QSTAR Pulsar mass spectrometer (Thermo, Wythenshawe, UK). Column chromatography was conducted using silica gel 60 (200 μm particle size, Yantai Xinde Chemical Co., Ltd, Yantai, China). Thin-layer chromatography was performed with precoated silica gel GF₂₅₄ glass plates (Qingdao Marine Chemical Co., Ltd). High-performance liquid chromatography (HPLC) was carried out using a Shimadzu LC-6AD instrument with an SPD-20A detector (Shimadzu, Kyoto, Japan) and a C-18 column (YMC-Pack ODS-A, 10 × 250 mm, 5 µm particle size, Shiseido Fine Chemicals, Tokyo, Japan).

Plant Material

The stems of A. senticosus were collected in Jilin, Jilin province, China, and authenticated by Professor Xiaohui Chen (College of Pharmacy, Zhengzhou University). A voucher specimen of the plant (No. 20180828) was deposited at the College of Pharmacy, Zhengzhou University, Henan, China.

Extraction and Isolation

Dried stems of A. senticosus (5.0 kg) was extracted with ethanol aqueous and then concentrated under vacuum at 45°C to obtain a residue (713.3 g). This extract was suspended in water (H₂O), partitioned successively with petroleum ether (PE), ethyl acetate (EtOAc) and n-butanol. The EtOAc fraction (123.4 g) was chromatographed over a silica gel column using a gradient of dichloromethance–methanol (MeOH) (from 100:1 to 1:1) and was separated into 15 fractions (Fr.1-Fr.15). Fr.7 (8.9 g) was fractionated by column chromatography on silica gel using a gradient of PE-EtOAc (from 100:1 to 0:1) and was separated into 12 fractions (Fr.7.1-Fr.7.12). Furthermore, Fr.7.4 (1.2 g) was subjected to the RP-18 column and using the elution of MeOH–H₂O (from 3:7 to 1:0) to afford 9 fractions (Fr.7.41- Fr.7.4.9). Fr.7.4.4 (100.3 mg) was applied to the C₁₈ reversed-phase HPLC column and eluted with a gradient of 55%-65% MeOH in H₂O at a flow rate of 3.0 mL/min over 80 minutes. This resulted in the isolation of compound 1 (2.2 mg, t _R = 30.8 minutes), compound 4 (4.3 mg, t _R = 37.6 minutes), and compound 5 (4.8 mg, t _R = 40.4 minutes). The Fr.7.4.6 (70.6 mg) was separated by HPLC, using an gradient solvent system 50%-70% MeOH in H₂O over 60 minutes, yielded compounds 2 (7.2 mg, t _R = 28.3 minutes), 3 (7.0 mg, t _R = 33.5 minutes), and 6 (5.9 mg, t _R = 45.1 minutes).

(7′S,8′R)-4′,5′,9′-trihydroxy-5-methoxy-4,8′-oxyneolign-7-en-9-al (1). A brown amorphous powder; ${\left[\alpha \right]}_D^{25}$ : −4.9 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 274 (3.04), 224 (3.31) nm; IR (potassium bromide disc) ν _max 3504, 2954, 2836, 1620, 1607, 1523, 1474 cm^{−1; 1}H (500 MHz) and ¹³C NMR (125 MHz) spectral data in deuterated methanol, see Table 1; HREIMS: m/z 360.1024 [M]⁺ (calcd for C₁₉H₂₀O₇, 360.1209).

Cytotoxicity Assay

All cancer cell lines were obtained from the Beyotime Biotechnology Company. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the proliferation inhibition of compounds 1-6 against these 4 cell lines according to the method that previously described. ¹⁸ Each cancer cell line was cultured in Roswell Park Memorial Institute Medium medium 1640 supplemented with 10% fetal bovine serum, 100 unit/mL penicillin, and 100 µg/mL streptomycin in a humidified 37°C incubator supplied with 5% carbon dioxide (CO₂). Cells were harvested by trypsin and dispensed into 96-well microtiter assay plates at the concentration of 5 × 10⁴ cells/mL and then incubated for 12 hours at 37°C with 5% CO₂. Compounds 1-6 were dissolved in dimethylsulfoxide (DMSO) to obtain a series of final concentrations. Cells were treated and continuously exposed to different concentrations of compounds for 48 hours. The absorbance of each well was measured at 570 nm with liquid scintillation counting (Tecan Infinite Pro, Tecan, Switzerland). DMSO was used as the negative control and doxorubicin as positive control.

Supplemental Material

Figure S1 - Supplemental material for New Neo-Lignan From Acanthopanax senticosus and the Cytotoxic Effects on Human Cancer Cell Lines

Supplemental material, Figure S1, for New Neo-Lignan From Acanthopanax senticosus and the Cytotoxic Effects on Human Cancer Cell Lines by Zhi-Chao Feng, Sheng Wang, Jun Li and Jun-Sheng Wang in Natural Product Communications