Chemical Constituents From the Fruits of Xanthium strumarium and Their Antitumor Effects

Abstract

A new neolignan, (7S,8R)-threo-1′-[3′-hydroxy-7-(4-hydroxy-3,5-dimethoxyphenyl)-8-hydroxymethyl-7,8-dihydrobenzofuran]acrylaldehyde (1), along with 5 known compounds 2-6, were isolated from the fruits of Xanthium strumarium. Their structures were elucidated by extensive spectroscopic methods. All the isolates were evaluated for in vitro cytotoxicity against human cancer cell lines, including human hepatoma cell line (HepG2), human breast cancer cell line (MCF-7), human colon cancer cell line (HCT-116), and human gastric cancer cell line (SGC-7901). Among them, compounds 1 and 3 showed selective cytotoxicity on HepG2 cancer cells with half-maximal inhibitory concentration (IC₅₀) values of 10.2 ± 1.2 and 18.3 ± 1.6 μM, respectively. Moreover, compound 5 also exhibited moderate cytotoxicity against MCF-7 cancer cells with an IC₅₀ value of 20.5 ± 1.3 μM.

Xanthium strumarium (Compositae) is an annual plant widely distributed in eastern China. The fruits of X. strumarium, named as “Cang-Er-Zi” or “Chang-Er-Zi” in China, were traditionally used to treat nasal sinusitis, ¹ headache caused by wind cold, ² urticaria, ² and arthritis. ³ And X. strumarium also exhibited some useful medicinal properties such as diuretic, ⁴ anthelmintic, ⁵ antifungal, ⁶ anti-inflammatory, ⁷ antidiabetes, ⁸ anticancer, ⁹ and other activities. Previous chemical studies of X. strumarium revealed the presence of sesquiterpene lactone, ¹⁰ phenolic acid, ³ diterpene glucoside, ¹¹ lignan, ² and flavonoid. ¹² However, there are not enough investigations to further study the constituents of X. strumarium. In continuation of our ongoing search for bioactive compounds from natural products, the fruits of X. strumarium were investigated. Herein we report the isolation and structural characterization of a new neolignan, together with 5 known compounds 2-6. In addition, the cytotoxic activities against human cancer cell lines, including human hepatoma cell line (HepG2), human breast cancer cell line (MCF-7), human colon cancer cell line (HCT-116), and human gastric cancer cell line (SGC-7901) were also be tested.

Results and Discussion

Compound 1 was obtained as a yellow amorphous powder, and the molecular formula was established as C₂₀H₂₀O₇ based on high-resolution electron ionization mass spectrometry (HRESIMS) (Supplemental Figure S6) at m/z 372.1185 for the [M]⁺. The ¹H nuclear magnetic resonance (NMR) spectrum (Supplemental Figure S1; Table 1) of 1 revealed a propenal moiety at δ _H 9.59 (1H, d, J = 8.0 Hz, H-9′), 7.62 (1H, d, J = 15.5 Hz, H-7′), and 6.69 (1H, dd, J = 15.5, 8.0 Hz, H-8′), a dihydrobenzofuran ring at δ _H 5.61 (1H, d, J = 6.5 Hz, H-7), 3.57 (1H, m, H-8), and 3.86 (2H, m, H-9), 2 sets of tetrasubstituted aromatic rings at δ _H 7.29 (1H, s, H-6′), 7.24 (1H, d, J = 1.5 Hz, H-2′), and 6.68 (2H, s, H-2,6), and 2 methoxy groups at δ _H 3.93 (6H, s). The ¹³C NMR spectrum (Supplemental Figure S2; Table 1) of 1 revealed 20 carbons including 12 aromatic carbons, a propenal moiety at δ _C 196.3 (C-9′), 156.2 (C-7′), and 127.3 (C-8′), and 2 methoxyl carbons at δ _C 57.0. The NMR data of 1 were very similar to that of (7S,8R)-5-methoxyl-balanophonin, which indicated the same neolignan skeleton of 1. ¹³ And combined with the ¹H NMR and ¹³C NMR data, the presence of 1′,3′,4′,5′-tetrasubstituted and 1,3,4,5-tetrasubstituted aromatic ring systems could be deduced. Furthermore, the comparison of NMR data between 1 and (7S,8R)-5-methoxyl-balanophonin showed that the only difference was the presence of a hydroxyl replacing the methoxyl at C-3′ of (7S, 8R)-5-methoxyl-balanophonin. The key heteronuclear multiple bond correlations (HMBC) (Supplemental Figure S3, Figure 1) of δ _H 7.24 (H-2′) with δ _C 129.8 (C-1′), 146.2 (C-3′), and 153.1 (C-4′), and of δ _H 7.29 (H-6′) with δ _C 129.8 (C-1′), 114.5 (C-2′), 153.1 (C-4′), and 133.3 (C-5′) could further confirm this conclusion. In addition, the HMBC correlations of δ _H 7.62 (H-7′) with δ _C 129.8 (C-1′), 114.5 (C-2′), and 120.1 (C-6′), and of δ _H 6.69 (H-8′) with δ _C 156.2 (C-7′) and 196.3 (C-9′) indicating the propenal moiety attached to C-1′. The relative configuration of H-7 and H-8 was determined as trans form by the coupling constant J _7,8 = 6.5 Hz. In addition, it was also confirmed by the cross-peaks between H-7 and H-9, as well as between H-2/H-6 and H-8 in the nuclear Overhauser effect spectroscopy spectrum (Supplemental Figure S4). The absolute configuration was assigned as 7S,8R by the circular dichroism spectrum (Supplemental Figure S5) of a positive Cotton effect near 330 nm and a negative Cotton effect near 295 nm. ¹³ Therefore, compound 1 was assigned as (7S,8R)-threo-1′-[3′-hydroxy-7-(4-hydroxy-3,5-dimethoxyphenyl)-8-hydroxymethyl-7,8-dihydrobenzofuran]acrylaldehyde.

Table 1

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

No.	1
	δ C	δ H
1	131.4
2	104.4	6.68, s
3	136.9
4	138.2
5	149.6
6	104.4	6.68, s
7	90.4	5.61, d, (6.5)
8	54.9	3.57, m
9	64.7	3.86, m
1′	129.8
2′	114.5	7.24, d, (1.5)
3′	146.2
4′	153.1
5′	133.3
6′	120.1	7.29, s
7′	156.2	7.62, d, (15.5)
8′	127.3	6.69, dd, (15.5, 8.0)
9′	196.3	9.59, d (8.0)
OCH3	57.0	3.82, s

Figure 1

Key heteronuclear multiple bond correlation and nuclear overhauser effect spectroscopy correlations of compound 1.

Moreover, 5 known compounds 2-6 were obtained from the fruits of X. strumarium and identified as rel-(2α,3β)-7-O-methylcedrusin (2), ¹⁴ erythro-guaiacylglycerol-β-coniferyl aldehyde ether (3), ² erythro-guaiacylglycerol-8-O-4′-(coniferyl alcohol) ether (4), ² cirsilineol (5), ¹⁵ and isoorientin (6) ¹⁶ based on NMR data and literature (Figure 2).

Figure 2

The structure of compounds 1-6.

All the isolates were evaluated for their cytotoxicity against human cancer cell lines of HepG2, MCF-7, HCT-116, and SGC-7901, using cisplatin as a positive control (Table 2). The primary screening results of compounds 1-6 showed that when the sample concentration was 50 µM, compounds 1-3, 5, and 6 exhibited selective cytotoxicity on 4 cancer cells. Among these isolates, compounds 1 and 3 showed moderate cytotoxicity on HepG2 cells with half-maximal inhibitory concentration (IC₅₀) values of 10.2 ± 1.2 and 18.3 ± 1.6 µM, respectively. And compound 5 also exhibited moderate cytotoxicity toward MCF-7 cells with an IC₅₀ value of 20.5 ± 1.3 µM. Furthermore, compound 3 showed higher cytotoxicity compared with 4, which indicated that the aldehyde group which is located at C-9′ might be responsible for increasing cytotoxicity.

Table 2

Cytotoxicities of Compounds 1-6 and the Positive Control Cisplatin.

Compound	IC50 (μM)
	HepG2	MCF-7	HCT-116	SGC-7901
1	10.2 ± 1.2	29.5 ± 1.7	>50	34.5 ± 1.0
2	47.1 ± 1.2	>50	>50	>50
3	18.3 ± 1.6	>50	45.8 ± 0.8	>50
4	.>50	>50	>50	>50
5	>50	20.5 ± 1.3	>50	>50
6	42.5 ± 1.3	>50	>50	>50
Cisplatin a	1.9 ± 0.7	2.1 ± 1.1	3.0 ± 1.2	1.5 ± 1.7

IC₅₀, half-maximal inhibitory concentration; HepG2, human hepatoma cell line; MCF-7, human breast cancer cell line; HCT-116, human colon cancer cell line; SGC-7901, human gastric cancer cell line.

^aPositive control.

^bIC₅₀ values were determined by regression analyses and expressed as means ± SD of 3 replicates.

Experimental

General

Optical rotations were determined on a Perkin-Elmer polarimeter 341 (Perkin-Elmer, Waltham, MA, USA). Ultraviolet (UV) spectra were recorded on the Jasco-UV-vis spectrophotometer (JASCO, Tokyo, Japan). Infrared (IR) spectra were recorded with a Nicolet Magna FT-IR 750 spectrometer (Nicolet, Madison, WI, USA). NMR spectra were recorded on a Bruker 500 MHz spectrometer with tetramethylsilane as an internal standard (Bruker, Karlsruhe, Germany). Mass spectra were obtained on a Q-TOF Micro LC-MS spectrometer (Waters, Milford, MA, USA). 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). A Waters 2535 high-performance liquid chromatography (HPLC) was carried out using a 2998 Photodiode Array Detector and a C-18 column (Waters Sunfire, 10 × 250 mm, 5 µm particle size).

Plant Material

The fruits of X. strumarium were collected in Zibo, Shandong province, People’s Republic of China, and authenticated by Professor Jing Wang (Xiangya Medical College, Central South University). A voucher specimen of the plant (No. 20180923) was deposited at the Xiangya Medical College, Central South University, Changsha, People’s Republic of China.

Extraction and Isolation

Dried fruits of X. strumarium (5.0 kg) were extracted 3 times with 75% ethanol (EtOH) aqueous to give a crude extract (532.7 g). This extract was suspended in water (H₂O), partitioned successively with petroleum ether (PE), chloroform (CHCl₃), ethyl acetate (EtOAc) and n-butanol (n-BuOH). The EtOAc fraction (88.6 g) was subjected to a silica gel column using a gradient of dichloromethane (CH₂Cl₂)-methanol (MeOH) (from 100:1 to 1:1) to yield 12 fractions (Fr.1-Fr.12). Fr.4 (14.5 g) chromatographed on a silica gel column eluted with a gradient of PE-EtOAc (from 100:1 to 0:1) and was separated into 10 fractions (Fr.4.1-Fr.4.10). In addition, Fr.4.4 (2.2 g) was subjected to the RP-18 column and using the elution of MeOH-H₂O (from 3:7 to 1:0) to afford 12 fractions (Fr.4.4.1- Fr.4.4.12). Fr.4.4.5 (271.5 mg) via preparative HPLC using a solvent system of 60% MeOH in H₂O over 80 minutes yielded compound 1 (3.7 mg, t _R = 31.2 minutes), compound 2 (3.8 mg, t _R = 36.8 minutes), and compound 4 (6.1 mg, t _R = 52.6 minutes). The Fr.4.4.6 (150.3 mg) was separated by HPLC, using an gradient solvent system 50%-60% MeOH in H₂O over 70 minutes, yielded compounds 3 (4.2 mg, t _R = 29.1 minutes), 5 (8.1 mg, t _R = 35.1 minutes), and 6 (7.7 mg, t _R = 47.9 minutes).

(7S,8R)-threo-1′-[3′-hydroxy-7-(4-hydroxy-3,5-dimethoxyphenyl)-8-hydroxymethyl-7,8-dihydrobenzofuran]acrylaldehyde (1)

A yellow amorphous powder; ${\left[\alpha \right]}_D^{25}$ : −10.5 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 341 (3.95), 284 (3.36), 234 (3.68), 204 (4.12) nm; IR (potassium bromide disc) ν _max 3451, 1651, 1604, 1141 cm⁻¹; ¹H (500 MHz) and ¹³C NMR (125 MHz) spectral data in deuterated methanol, see Table 1; HREIMS: m/z 372.1185 [M]⁺ (calcd for C₂₀H₂₀O₇, 372.1209).

Cytotoxicity Assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was chosen to evaluate the cytotoxicity of compounds 1-6 on 4 human cancer cell lines according to the published method. ¹⁷ Briefly, HepG2, MCF-7, HCT-116, and SGC-7901 cells were cultured at 37 °C, harvested by trypsin, and dispensed into 96-well microtiter assay plates for 24 hours. Compounds 1-6 and cisplatin were dissolved in dimethyl sulfoxide (DMSO) and diluted with the medium to different tested concentrations (0.1 µM, 1 µM, 10 µM, 50 µM). Then, they were added to individual wells and incubated for 48 hours with cisplatin as the positive control. After 48 hours, the cells were sequentially incubated with MTT solution (0.5 mg/mL) for 4 hours at 37 °C. The produced formazan crystals were solubilized with DMSO, and the absorbance of each well was measured at 570 nm with Spectra Shell reader (Tecan, Austria).

Supplemental Material

Supplementary Material 1 - Supplemental material for Chemical Constituents From the Fruits of Xanthium strumarium and Their Antitumor Effects

Supplemental material, Supplementary Material 1, for Chemical Constituents From the Fruits of Xanthium strumarium and Their Antitumor Effects by Chao Tong, Ri-Hui Chen, Ding-Cheng Liu, De-Sheng Zeng and Hui Liu in Natural Product Communications