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Abstract

A new aurone, (2Z)-2-[(4′-hydroxyphenyl) methylene]-6-methoxy-7-prenyl-3(2H)-benzofurane (1), together with 6 known compounds (2-7), was isolated from the EtOAc-soluble extract of the seeds of Psoralea corylifolia. Their structures were elucidated on the basis of spectroscopic methods. All compounds were evaluated for in vitro inhibitory activity against a human colon cancer cell line (SW620). Compounds 1 to 3 showed moderate cytotoxicity against the SW620 cells with IC₅₀ values ranging from 24.31 to 28.72 μM. Among these compounds, 1 showed higher cytotoxicity against SW620 cells with an IC₅₀ value of 24.31 μM.

Psoralea corylifolia L. (Leguminosae; Substance SID of PubChem:135313809) is an annual herbaceous plant which is predominantly distributed in Southeast Asia. The seeds of this species are known as “Bu-Gu-Zhi” in China, and they are used as an important folk medicine to treat asthma,¹ bacterial infections,² osteoporosis,² spermatorrhea,² and gynecological bleeding.³ Previous studies have showed that the major constituents of P. corylifolia are coumarins,⁴ flavonoids,⁵ and meroterpenoids of the monoterpene phenol class.⁶ Several of these compounds have displayed potential biological activities such as anti-inflammatory,⁷ antioxidant,⁸ antibacterial,⁹ and anticancer properties.^10
-12 During the course of our search for natural compounds with anticolon cancer activity from P. corylifolia, a new aurone (1), together with 6 known compounds (2-7), was isolated from the seeds. Herein, we report the isolation and structural elucidation of a new aurone along with 6 known compounds; all were evaluated for their cytotoxicity on the SW620 human colon cancer cell line.

Results and Discussion

Compound 1, obtained as a light yellow amorphous powder, showed the molecular formula C₂₁H₂₀O₄, based on the [M]⁺ ion in the HREIMS (High Resolution Electron Impact Mass Spectrometry) at m/z 336.1358 (supplemental Figure S4). The ¹H nuclear magnetic resonance (NMR) spectrum (supplemental Figure S1; Table 1) of 1 showed signals for a tetrasubstituted benzene ring at δ _H 7.37 (1H, d, J = 8.5 Hz, H-4) and 6.75 (1H, d, J = 8.5 Hz, H-5); a p-substituted benzene ring at δ _H 7.72 (2H, d, J = 8.5 Hz, H-2′,6′) and 6.92 (1H, dd, J = 8.5 Hz, H-3′,5′); an olefinic proton at δ _H 6.56 (1H, s, H-10); a prenyl group at δ _H 3.48 (2H, d, J = 7.5 Hz, H-1″), 5.33-5.34 (1H, m, H-2″), 1.80 (3H, s, H-4″), and 1.62 (3H, s, H-5″); and a methoxy proton at δ _H 3.83 (3H, s). Twenty-one carbons were observed in the ¹³C NMR spectrum, including 2 sets of benzene rings, a carbonyl carbon, a prenyl group, and a methoxy group (supplemental Figure S2). The above NMR data of 1 were very similar to those of licoagroaurone, indicating the same aurone skeleton.¹³ In addition, the prenyl group which linked with C-7 could be deduced by the key HMBC correlations of δ _H 3.48 (H-1″) with δ _C 122.4 (C-2″), 133.0 (C-3″), and 113.2 (C-7) (supplemental Figure S3). The HMBC (Heteronuclear Multiple Bond Correlation) spectrum also showed key correlations of δ _H 3.83 to 166.9 (C-6), which revealed that the methoxy was located at C-6 (Figure 1). The Z-stereochemistry of the double bond at C-10 in 1 was determined by the chemical shift of C-10 (δ _C 112.1).¹³ Thus, the structure of 1 was elucidated as (2Z)-2- [(4′-hydroxyphenyl) methylene]-6-methoxy-7-prenyl-3(2H)-benzofurane.

Table 1

¹H and ¹³C Nuclear Magnetic Resonance Spectral Data of Compound 1 in Acetone-d ₆ (¹H: 500 MHz and ¹³C: 125 MHz).

Position	δ _C	δ _H (Multiplicity)
2	147.6
3	183.2
4	125.7	7.37 (1H, d, 8.5)
5	113.0	6.75 (1H, d, 8.5)
6	166.9
7	113.2
8	164.0
9	115.1
10	112.1	6.56 (1H, s)
1′	127.7
2′	131.0	7.72 (1H, d, 8.5)
3′	117.1	6.92 (1H, d, 8.5)
4′	161.0
5′	117.1	6.92 (1H, d, 8.5)
6′	131.0	7.72 (1H, d, 8.5)
1″	22.8	3.48 (2H, d, 7.5)
2″	122.4	5.33-5.34 (1H, m)
3″	133.0
4″	18.3	1.80 (3H, s)
5″	26.0	1.62 (3H, s)
OCH₃	56.2	3.83 (3H, s)

Figure 1

The structures of compounds 1-7.

Moreover, 6 known compounds (2-7) were obtained from the seeds of P. corylifolia and identified as coryaurone A (2),² bavachinone B (3),² bavacoumestan C (4),² bavacoumestan B (5),² corylifols E (6),¹⁴ and corylifols D (7)¹⁴ by comparing their NMR data with those in the literature (Figure 2).

Figure 2

The key HMBC correlations of compound 1.

All the isolates were evaluated for their cytotoxicity against the SW620 cell line; 5-fluorouracil was used as the positive control. Compounds 1 to 3 showed moderate cytotoxicity on the SW620 cell line with IC₅₀ values ranging from 24.31 to 28.72 µM (Table 2). Among these isolates, compound 1 showed the higher cytotoxicity on the SW620 cell line with an IC₅₀ value of 24.31 µM; compounds 4 to 7 did not exhibit cytotoxicity toward the SW620 cell line.

Table 2

The Cytotoxicity of Compounds 1 to 7 (µM) for SW620 Cells Line (Expressed as IC₅₀ Values).

Compounds	SW620
1	24.31
2	28.72
3	27.43
4	>30
5	>30
6	>30
7	>30
5-Fluorouracil^a	3.19

^aPositive control.

Experimental

General Experimental Procedures

Optical rotations were determined on a Jasco P-2000 polarimeter (Daklia Trading Corporation, Mandaluyong, Philippines). UV spectra were recorded in MeOH using a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Tokyo, Japan). IR (Infrared Radiation) spectra were recorded with a JASCO FT-IR 620 spectrophotometer. Nuclear magnetic resonance spectra were obtained from a Varian Unity Inova 500 MHz spectrometer (Varian Unity Inova, Phoenix, United States) using TMS (Tetramethylsilane) as the internal standard. Mass spectra were obtained on a QTOF2 (Quadrupole Time of Fight) high-resolution mass spectrometer (Micromass, Wythenshawe, United Kingdom). Column chromatography was conducted using silica gel 60 (200 μm particle size, Yantai Xinde Chemical Co., Ltd, Yantai, China) and RP-18 (150-163 μm particle size, Merck, Darmstadt, Germany). For thin-layer chromatography (TLC), precoated TLC silica gel 60 F₂₅₄ plates from Merck were used. HPLC (High Performance Liquid Chromatography) was carried out using a Shimadzu System LC-10AD pump equipped with a model SPD-10Avp UV detector (Shimadzu, Tokyo, Japan) and an Optima Pak C₁₈ column (10 × 250 mm, 10 µm particle size, Shiseido Fine Chemicals, Tokyo, Japan).

Plant Material

The seeds of P. corylifolia were collected in Xishuangbanna, Yunnan province, Republic of China, and authenticated by Professor Jia Zhao (College of Pharmacy, Shenyang Pharmaceutical University). A voucher specimen of the plant (No. 20180515) was deposited at Liaoning University of Traditional Chinese Medicine, Shenyang, China.

Extraction and Isolation

The dried seeds of P. corylifolia (5.0 kg) were extracted 3 times with 95% aqueous EtOH at room temperature. The combined solutions were concentrated to obtain a crude extract (748.3 g), which was suspended in H₂O and partitioned successively with light petroleum, EtOAc, and n-BuOH to afford petroleum, EtOAc, and n-BuOH soluble fractions, respectively. Part of the EtOAc-soluble fraction (100.0 g) was chromatographed over a silica gel column using a gradient of CH₂Cl₂-MeOH (from 0:1 to 0:1), and separated into 12 fractions (Fr.1-Fr.12). Fr.4 (7.3 g) was subjected to a silica gel column and eluted with CH₂Cl₂-MeOH (from 0:1 to 0:1) to obtain 10 fractions (Fr.4.1-Fr.4.10). Fr.4.4 (2.43 g) was chromatographed on a RP-18 column eluted with MeOH/H₂O (2:8 to 10:0) to afford 16 fractions. Fr.4.4.5 (182.1 mg) was then separated by HPLC, using a gradient solvent system of 40% to 65% MeOH in H₂O over 70 minutes, yielding compounds 1 (5.4 mg, t _R = 37.7 minutes) and 2 (12.5 mg, t _R = 50.2 minutes). Further purification of Fr.4.4.6 (223.5 mg) via semipreparative HPLC using a gradient solvent system of 50% to 75% MeOH in H₂O over 80 minutes yielded compounds 3 (12.1 mg, t _R = 35.7 minutes), 4 (18.7 mg, t _R = 52.3 minutes), 5 (14.8 mg, t _R = 48.2 minutes), and 6 (15.2 mg, t _R = 75.2 minutes).

(2Z)-2-[(4′-Hydroxyphenyl)methylene]-6-Methoxy-7-Prenyl-3(2H)-Benzofurane (1)

Light yellow amorphous powder; UV (MeOH) λ _max (nm) (log ε): 260 (3.92), 402 (4.34); IR (KBr) ν _max3424, 1768, 1654, 1611 cm⁻¹; ¹H (500 MHz) and ¹³C NMR (125 MHz) data in acetone-d ₆, see Table 1; HREIMS m/z 336.1358 [M]⁺ (calcd for C₂₁H₂₀O₅, 336.13621).

Cytotoxicity Screening Assays

The MTT (Methyl Thiazolyl Tetrazolium) assay was modified from that previously described.¹⁵ An adherent SW620 cell line was cultured in RPMI (Roswell Park Memorial Institute) medium 1640 as adherent monolayers in flasks supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified 37°C incubator supplied with 5% CO₂. Briefly, cells were harvested with trypsin and dispensed into 96-well microtiter assay plates at 2000 cells/well for the SW620 cell line and then incubated for 18 hours at 37°C with 5% CO₂. The test compounds were dissolved in 1% DMSO (Dimethyl Sulfoxide) in PBS (Phosphate Buffer Saline) (v/v) and aliquots (10 µL) tested over a series of final concentrations ranging from 10 nM to 30 µM. Control wells were treated with 1% aqueous DMSO. After 48 hours incubation at 37°C with 5% CO₂, an aliquot (20 µL) of MTT in PBS (5 mg/mL) was added to each well (final concentration of 0.5 mg/mL), and the microtiter plates incubated for further 4 hours at 37°C with 5% CO₂. After this final incubation, the medium was aspirated and the precipitated formazan crystals dissolved in DMSO (100 µL/well). The absorbance of each well was measured at 580 nm with a liquid scintillation counter (ALOKA LSC-8000, Aloka, Inc., Tokyo, Japan). All experiments were performed in duplicate and 5-fluorouracil was used as a positive control.

Supplemental Material

Supplementary material - Supplemental material for Anticolon Cancer Potential of (2Z)-2-[(4′-Hydroxyphenyl) Methylene]-6-Methoxy-7-Prenyl-3(2H)-Benzofurane, a New Aurone From Psoralea corylifolia

Supplemental material, Supplementary material, for Anticolon Cancer Potential of (2Z)-2-[(4′-Hydroxyphenyl) Methylene]-6-Methoxy-7-Prenyl-3(2H)-Benzofurane, a New Aurone From Psoralea corylifolia by Ai-Hua Liu, Li-Hua Liu and Chun-Ying Liu in Natural Product Communications

Footnotes

Acknowledgment

We thank Dr Huangxiu Wang in the Research Center for Chemistry, Shenyang Pharmaceutical University.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID ID

Chun-Ying Liu

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