1. Introduction

Artocarpus, belonging to the mulberry family Moraceae, is a genus of approximately 60 trees and shrubs distributed in the Southeast Asian and Pacific regions. A. heterophyllus Lam, popularly known as jackfruit, is one of the important trees in the home gardens of tropical India, Bangladesh, Nepal, Sri Lanka, Vietnam, Thailand, Malaysia, Indonesia, Philippines, Africa, and Brazil [1]. It is also an important fruit in southern China, distributed and cultivated in Hainan, Guangdong, Guangxi, and Yunnan provinces [2]. Different parts of this species have been used for medicinal purposes, such as alleviating asthma and fever (the roots), relieving biliousness and diarrhea (the seeds), acting as a sedative for convulsion (the wood), stimulating lactation in women and animals and acting as an antisyphilitic and vermifuge in humans (the leaves), and relieving ulcers and wounds (the leaf ash) [3, 4]. Wood and bark of this plant are rich sources of prenylated flavonoids, stilbenoids, triterpenoids, and steroids [1]. Some of these compounds have exhibited interesting biological activities, such as cytotoxicity [5, 6], antioxidative activity [7], anti-inflammatory activity [8], antimalarial activity [9], inhibition of tyrosinase and melanin biosynthesis [10, 11], and inhibition of 5areductase [12]. In our previous study, 10 chalcones, 8 flavones, and one 2-arylbenzofuran derivative were isolated from the twigs of A. heterophyllus, among which some showed moderate inhibitory activity on the proliferation of the PC-3 and H460 cell lines [6]. In the course of our ongoing study, the 95% EtOH extract of the leaves of A. heterophyllus was investigated and one new 2-arylbenzofuran derivative, artocarstilbene B (1), one new benzaldehyde derivative, (E)-3, 5-dihydroxy-4-(3-methylbut-1-enyl) benzaldehyde (2), as well as 18 known ones (3-20) (Fig. 1) were obtained. Their structures were elucidated on the basis of extensive spectroscopic techniques including 1D and 2D NMR and HR-ESIMS. Herein, we report the structure elucidation as well as the anti-proliferative activity of these compounds to the PC-3 (human prostate cancer), NCI-H460 (human lung cancer), and A549 (human lung adenocarcinoma epithelial) cells.

2. Experimental 2.1. General

MTT was purchased from Sigma Chemical Co., (St. Louis, MO, USA). Doxorubicin was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). Optical rotations were measured on a Perkin-Elmer 241 MC polarimeter. IR spectra were recorded on a Nicolet iN 10 Micro FTIR spectrometer by transmission mode. UV spectra were obtained on a Shimadzu UV-2550 spectrophotometer. NMR spectra were measured on a Bruker Avance DRX-600 spectrometer operating at 600 (¹H) and 150 (¹³C) MHz with TMS as internal standard. HR-ESIMS were carried out on a LTQ-Orbitrap XL mass spectrometer. HPLC was performed on an Agilent 1260 HPLC system equipped with a G1311C isopump, a G1315D DAD detector, and a YMC-Pack ODS-A column (10 mm × 250 mm, 5 μm). All solvents used were of analytical grade (Laiyang Chemical Reagent Co., Ltd., Shandong, China). Silica gel (200-300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), C₁₈ reversed-phase silica gel (YMC ODS-A gel, YMC Co., Ltd.), MCI-gel (CHP20P, 75-150 μm, Mitsubishi Chemical Industries Ltd.), and Sephadex LH-20 (Pharmacia Biotek, Denmark) were used for column chromatography (CC). Thin layer chromatography (TLC) was carried out with high-performance TLC plates precoated with silica gel GF254 (Qingdao Haiyang Chemical Co., Ltd.). Spots of TLC were observed under UV or visualized by spraying with H₂SO₄-EtOH (1:9) followed by heating.

2.2. Plant material

The leaves of A. heterophyllus were collected from Mengla County, Yunnan Province, China, in July 2009. The plant material was identified by Dr. Tao Shen, Shandong University. A voucher specimen (AH02-2009-07) was deposited at the Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University.

2.3. Extraction and isolation

The leaves of A. heterophyllus (5.0 kg), air-dried and powdered, were percolated with 95% EtOH (3× 25 L, each for one week) at room temperature. The combined extracts were concentrated under reduced pressure to afford a dark gum (123.8 g), which was suspended in warm water and partitioned successively with petroleum ether (4× 1.2 L) and EtOAc (4× 1.2 L). The EtOAc soluble part (57.3 g) was subjected to CC of silica gel eluted with petroleum ether-EtOAc (20:1→1:2) to yield 12 fractions (Fr. 1-Fr. 12). Fr. 4 (3.2 g) was crystallized to yield 19 (150.1 mg). Fr. 5 (7.7 g) was subjected to CC of MCI (30%→100% MeOH) to obtain five subfractions (Fr. 5.1-Fr. 5.5). Fr. 5.2 (820.0 mg) was further separated by CC of Sephadex LH-20 (MeOH) and preparative HPLC (MeOH-H₂O, 88:12, 1.8 mL/min) to afford 16 (9.5 mg, t_R=10.61 min), 7 (9.0 mg, t_R=12.31 min), and 2 (1.2 mg, t_R=15.02 min). Fr. 6 (6.3 g) was subjected to CC of MCI (30%→100% MeOH) to obtain six subfractions (Fr. 6.1-Fr. 6.6). Fr. 6.2 (1.1 g) was further separated by CC of Sephadex LH-20 (MeOH) and preparative HPLC (MeOH-H₂O, 80:20, 1.8 mL/min) to afford 12 (3.0 mg, t_R=8.06 min) and 14 (20.9 mg, t_R=10.02 min). Fr. 6.3 (0.9 g) was first separated by CC of silica gel (petroleum ether-acetone, 10:1) to obtain the major part, which was purified by CC of Sephadex LH-20 (MeOH) to afford 13 (1.6 mg). Fr. 6.6 (1.3 g) was first separated by CC of silica gel (petroleum etheracetone, 10:1) to obtain the major part, which was then separated by CC of C₁₈ reversed-phase silica gel (30%→100% MeOH) to obtain 4 (9.5 mg) and 8 (3.0 mg). Fr. 7 (10.3 g) was subjected to CC of MCI (30%→100% MeOH) to obtain seven subfractions (Fr. 7.1-Fr. 7.7). Fr. 7.1 (0.9 g) was further purified by CC of Sephadex LH-20 (MeOH) and HPLC (MeOH-H₂O, 78:22, 1.8 mL/min) to yield 15 (20.9 mg, t_R=13.61 min). Fr. 7.4 was separated by Sephadex LH-20 CC (MeOH) to obtain the major part, which was then separated by HPLC (MeOH-H₂O, 78:22, 1.8 mL/min) to obtain 3 (7.6 mg, t_R=15.90 min) and 5 (6.3 mg, t_R=21.82 min). Fr. 8 (6.1 g) was subjected to CC of MCI (30%→100% MeOH) to obtain six subfractions (Fr. 8.1-Fr. 8.6). Fr. 8.1 (0.7 g) was separated by CC of Sephadex LH-20 (MeOH) and CC of C18 reversed-phase silica gel (30%→100% MeOH) to obtain 9 (4.3 mg) and 11 (6.2 mg). Fr. 8.2 (0.9 g) was separated by preparative HPLC (MeOH-H₂O, 75:25, 1.8 mL/min) to yield 17 (6.0 mg, t_R=19.50 min), 1 (4.0 mg, t_R=20.85 min), 10 (8.1 mg, t_R=22.02 min), and 6 (5.2 mg, t_R=24.02 min). Fr. 9 (2.3 g) was separated by CC of MCI (30%→100% MeOH) to obtain four subfractions (Fr. 9.1-Fr. 9.4). Fr. 9.2 (0.9 g) was separated by CC of C18 reversed-phase silica gel (30%→100% MeOH) to obtain 18 (8.9 mg). Fr. 9.3 was crystallized to yield 20 (5.0 mg).

Artocarstilbene B (1). Yellowish oil. IR (KBr)υ_max: 3265, 2928, 1617, 1564, 1489, 1426, 1365, 1148, 971, 823 cm^-1. UV (MeOH) λ_max (log ε): 226 (4.03) nm. ¹H NMR and ¹³C NMR data, see Table 1. (-)-HR-ESIMS: m/z 323.0917 [M-H]^- (Calcd. for C₁₉H₁₅O₅, 323.0919).

(E)-3, 5-Dihydroxy-4-(3-methylbut-1-enyl) benzaldehyde (2). Yellowish oil. IR (KBr) υ_max: 3298, 2960, 2929, 1684, 1514, 1437, 1391, 1338, 1206, 1042, 842 cm^-1. UV (MeOH) λ_max (log e): 203 (3.77) nm. ¹H NMR and ¹³C NMR data, see Table 1. (-)-HRESIMS: m/z 205.0869 [M-H]^- (Calcd. for C₁₂H₁₃O₃, 205.0865).

2.4. Cell lines and cell culture

PC-3 cells (ATCC CRL-1435 human prostate adenocarcinoma), NCI-H460 cells (ATCC HTB 177 human lung carcinoma), and A549 cells (ATCC CCL-185 human lung adenocarcinoma epithelial) were cultured in RPMI-1640 medium (HyClone, Thermo Fisher Scientific Inc., Waltham, MA, USA). The medium was supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Carlsbad, CA, USA), 100 μg/mL penicillin and 100 μg/mL streptomycin. Cells were cultured in a humidified atmosphere of 5% CO₂ at 37 ℃.

2.5. Cytotoxicity assay

The tetrazolium-based colorometric assay (MTT assay) was used to determine cell viability. The cancer cells were all cultured under standard culture conditions. The test compounds (1-20) or vehicle control (dimethyl sulfoxide, DMSO) were added to appropriate wells and the cells were incubated for 72 h. Then MTT solution was added into the assay plates (final concentration, 0.5 mg/mL). After shaking for 10 s, plates were returned to incubator and kept for 4 h. The supernatants were removed carefully, followed by the addition of 100 μL of DMSO to each well to dissolve the precipitate. Then, the absorbance was measured at 570 nm with a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA). The percent viability was expressed as absorbance in the presence of test compound as a percentage of that in the vehicle control. Doxorubicin and DMSO were used as positive and negative controls.

3. Results and discussion

Compound 1 was obtained as a yellowish oil. The highresolution ESI-MS showed the pseudo molecular ion [M-H]^- peak at m/z 323.0917, corresponding to the molecular formula C₁₉H₁₆O₅ (Calcd. for C₁₉H₁₆O₅, 323.0919). The IR spectrum displayed absorption bands for hydroxyl (3265 cm^-1) and benzene ring (1684 cm^-1 and 1564 cm^-1). The ¹H NMR (Table 1) showed resonances for one 1, 3, 4, 5-tetrasubstituted benzene ring (δ_H 6.82, br. s and 6.74, br. s), one substituted benzofuran ring (δ_H 7.14, s, H-3; 7.38, d, J=8.5 Hz, H-4; 6.74, d, J=8.5 Hz, H-5; and 6.92, br. s, H-7), one double bond (δ_H 6.64, d, J=10.0 Hz and 5.63, d, J=10.0 Hz), one methyl singlet (δ_H 1.29, s, 3H), and one oxygenated methylene (δ_H 3.45, m, 2H). The ¹³C NMR showed resonances for 19 carbons, which included one methyl, one oxygenated methylene, eight methine (all sp²), and nine quaternary (one sp³ and eight sp²) carbons as discerned from the HSQC spectrum. The NMR data (Table 1) for 1 was very similar to those of moracin D (16) [13]. The only difference was absence of one methyl in 16 and instead, one oxygenated methylene (δ_H 3.45, m, 2H; δ_C 67.0) was observed in 1, indicating oxidation of a methyl to a hydroxymethyl in 1. In the HMBC of 1 (Fig. 2), the methylene protons were observed to correlated with C-5" (δ_C 23.2), C-3" (δ_C 79.2), and C-2" (δ_C 126.6), which also supported the above conclusion. Thus, compound 1 was determined as 2-[5-hydroxy-(2-hydroxymethyl-2-methyl) benzopyran]-1-benzofuran-5-ol, and named artocarstilbene B.

Compound 2 was also obtained as a yellowish oil. The molecular formula was deduced as C₁₂H₁₄O₃ from the HRESIMS of 2, which showed a pseudo molecular ion [M-H]^- at m/z 205.0869 (Calcd. for C₁₂H₁₃O₃, 205.0865). The IR spectrum showed absorption bands for hydroxy (3298 cm^-1) and benzene (1684 and 1514 cm^-1) groups. The ¹H NMR (Table 1) showed signals for an aldehyde (δ_H 9.72, s), a symmetrical 1, 2, 3, 5-tetrasubstituted benzene ring (δ_H 6.83, s, H-2 and H-6), a double bond (δ_H 6.58, d, J=16.0 Hz and 6.82, dd, J=16.0, 6.0 Hz), and one isopropyl (δ_H 2.40, m; 1.04 and 1.05, each d, J=6.2 Hz, each 3H). The 13C NMR showed signals for 12 carbons, including a symmetrically substituted benzene ring at δ_C 134.9 (C-1), 107.7 (C-2, 6), 157.0 (C-3, 5), and 118.4 (C-4), as well as an aldehyde (δ_C 192.9) and a double bond (δ_C 117.9 and 143.5). The above information indicated that compound 2 was a substituted benzaldehyde. In the HMBC of 2 (Fig. 2), correlations of H-1'/C-3(C-5); H-2'/C-4, C-3', and C-5'; and H-7(δ_H 9.72)/C-1, C-2, and C-3 verified the structure of 2 to be (E)-3, 5-dihydroxy-4-(3-methylbut-1-enyl) benzaldehyde.

The known compounds were identified by comparison of their ¹H NMR, ¹³C NMR and MS data with those reported in the literatures as one chalcone, artocarmitin B (3) [14], five flavones, artocarpin (4) [15], cudraflavone C (5) [16], albanin A (6) [17], cudraflavone (7) [18], and brosimone I (8) [18], two flavanones, norartocarpanone (9) [19] and euchrenone a7 (10) [20], six 2-arylbenzofuran derivatives, moracin M (11) [21], moracin C (12) [13], albafuran B (13) [22], artoindonesianin B-1 (14) [23], demethylmoracin I (15) [24], and moracin D (16) [13], one stilbenoid, artocarbene (17) [25], one lignan, 2, 6, 20, 60-tetramethoxy-4, 4'-bis (2, 3-epoxy-1-hydroxy-propyl) biphenyl (18) [26], one triterpenoid, griffithine A (19) [27], and one steroid, 5α, 6α-epoxy-24(R)-methylcholesta-7, 22-dien-3β-ol (20) [28] (Fig.S1 in Supporting information).

Compounds 1-20 were evaluated for the in vitro inhibition of cell proliferation against the PC-3, NCI-H460, and A549 cancer cell lines using the MTT method. Compounds with IC50 value < 50 μmol/L were listed in Table 2. In our previous study [6], moderate to weak cytotoxicity was found for compounds 3-5 against the PC-3 and NCI-H460 cells, and in this report they showed similar results. Besides this, 3-5 also exhibited cytotoxicity against the A549 cells. Compounds 10, 14, and 16 also showed weak inhibitory activity (IC₅₀ < 20 μmol/L) to some or all of the three cancer cell lines. The other compounds were inactive in our study (IC₅₀ > 20 μmol/L).

4. Conclusion

In summary, 20 compounds including one new 2-arylbenzofuran derivative, artocarstilbene B (1) and one new benzaldehyde derivative, (E)-3, 5-dihydroxy-4-(3-methylbut-1-enyl) benzaldehyde (2) were isolated from the leaves of A. heterophyllus. Compounds 3-5, 10, 14, and 16 showed inhibitory activity against the proliferation of the PC-3, NCI-H460, and/or A549 cancer cell lines.

Acknowledgments

Financial supports from National Natural Science Foundation of China (No. 31500280) and Shandong Provincial Natural Science Foundation, China (No. ZR2015PC006) are gratefully acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.06.024.