Prenylflavonoids from the Twigs of Artocarpus nigrifolius

Abstract

Two new prenylated flavones, artocarnin A (2) and carpachromenol (12), together with 13 known prenylflavonoids (1, 3–11, 13–15) were isolated from the twigs of Artocarpus nigrifolius for the first time. Their structures were elucidated by high resolution-electrospray ionization (HR-ESI)-MS, NMR spectroscopic analysis, and in comparison with the reported data. Compounds 1–15 were evaluated for their antiproliferative effects against SiHa and SGC-7901 human cancer cell lines in vitro. The most active compound, eleocharin A (10), showed significant cytotoxicity on SiHa cells (IC₅₀=0.7±0.1 µM) and inhibitory activity against SGC-7901 cells (IC₅₀=8.3±0.2 µM) and could be considered as potential lead compound for further development of novel anti-tumor agents.

The genus Artocarpus (Moraceae) contains about 50 species distributed throughout the tropical and subtropical regions of Asia.¹⁾ Some members of this genus have been widely used as traditional folk medicines in Southeast Asia and China.^2–4) Previous phytochemical research on plants of this genus led to diverse prenylated flavonoids and stilbenoids with interesting biological activities such as cytotoxic,^5–10) antiinflammatory,^11–15) antioxidant,^16,17) antimalarial,^18,19) and antifungal activities,²⁰⁾ as well as tyrosinase inhibitory activities^21,22) and 5α-reductase inhibitory activities.²³⁾ As part of our ongoing efforts to discover novel potential anticancer leads from Moraceous plants,²⁴⁾ two new prenylated flavones (2, 12) along with 13 known compounds (1, 3–11, 13–15) have been isolated from the twigs of Artocarpus nigrifolius C. Y. WU for the first time. In this paper, we describe the structural elucidation of these new compounds, as well as the antiproliferative properties against human cancer cells of all isolates.

Results and Discussion

The air-dried powder of twigs of A. nigrifolius was extracted with 95% EtOH three times at room temperature. The EtOAc-soluble fraction of the EtOH extract was purified using various column chromatographies to afford 15 phenolics (1–15) (Fig. 1). The known compounds were identified as cycloheterophyllin (1),¹³⁾ artocarmin B (3),²¹⁾ artocarmin C (4),²¹⁾ gemichalcone B (5),²⁵⁾ artocarpusin A (6),²⁶⁾ gemichalcone A (7),²⁵⁾ gemichalcone C (8),²⁷⁾ isogemichalcone B (9),²⁵⁾ eleocharin A (10),²⁸⁾ 5,4′-dihydroxy-3′-methoxy-(6 : 7)-2,2-dimethylpyranoflavone (11),²⁹⁾ 2,4,2′,4′-tetrahydroxy-3-(3-methyl-2-butenyl)-chalcone (13),³⁰⁾ 6-prenyl-4′,5,7-trihydroxyflavone (14),³¹⁾ and artocarpesin (15)³²⁾ by analysis of their spectroscopic data and comparison with literature data.

Fig. 1. Chemical Structures of Compounds 1–15

Artocarnin A (2), a yellow amorphous solid, was obtained as a pair of inseparable mixture of two isomers in a ration of approximately 1 : 1.2, as determined by ¹H-NMR spectroscopy. Its molecular formula was deduced to be C₃₀H₃₀O₈ from high resolution electrospray ionization (HR-ESI)-MS (m/z 563.1923 [M+HCOO]⁻, Calcd 563.1912). Analysis of the ¹H-NMR data (Table 1) indicated the presence of one diagnostic exchangeable proton at δ_H 13.33 (1H, s, 5-OH), two isolated aromatic protons at δ_H 7.35/7.39 (1H, s) and 6.47 (1H, s), and one 2, 2-dimethyl-2H-pyran ring at δ_H 6.67 (1H, d, J=10.2 Hz), 5.75 (1H, d, J=10.2 Hz) and 1.50 (6H, s), together with one terminal double bond [δ_H 4.83/4.84 (1H, s) and 4.71 (1H, s)], one oxygenated methine [δ_H 4.48/4.44 (1H, t, J=6.9 Hz)] and another three methyls at δ_H 1.68 (3H, s), 1.94 (3H, s), and 1.88 (3H, s), respectively. While the ¹³C-NMR (Table 1) and heteronuclear single quantum coherence (HSQC) data showed 30 carbon signals attributable to one ketone carbonyl (δ_C 179.5), 14 sp² quaternary carbons, one oxygenated quaternary carbon (δ_C 78.8), five sp² methines (δ_C 128.9, 122.1/122.0, 116.1, 110.3/110.2, and 105.3), two oxymethines (δ_C 75.6/75.3 and 69.9), one sp² methylene (δ_C 110.7/110.6), one methylene (δ_C 30.4), and five methyls (δ_C 28.6, 28.4, 25.9, 18.7, and 18.0). Both the ¹H- and ¹³C-NMR spectrum of 2 closely resembled that of cycloheterophyllin (1),¹³⁾ a major concomitant component, except that 2 had a 2-hydroxy-3-methyl-3-butenyl^33,34) moiety instead of a prenyl group at C-8 of 1, as deduced from the NMR signals at δ_C 30.4 (C-1″), 75.6/75.3 (C-2″), 149.0/148.9 (C-3″), 110.7/110.6 (C-4″), and 18.0 (C-5″) and signals at δ_H 3.07 (1H, dd, J=13.1, 6.9 Hz), 3.06 (1H, dd, J=13.1, 6.9 Hz), 4.48/4.44 (1H, t, J=6.9 Hz), 4.83/4.84 (1H, s), 4.71 (1H, s), and 1.88 (3H, s). Moreover, the heteronuclear multiple bond connectivity (HMBC) correlations (Fig. 2) of H₂-1″ to C-7, C-8, and C-9 further proved that the 2-hydroxy-3-methyl-3-butenyl unit was attached to C-8. In the ¹H-NMR spectrum of 2, the occurrence of pairs of H-2″, H-4″ and H-6′ signals indicated it was a mixture of two C-2″ epimers. As far as we know, no literatures hitherto had reported the absolute configuration on C-11 of cycloheterophyllin (1) and its analogues, which may be determined by comparison of experimental and calculated electronic circular dichroism spectra³⁵⁾ (ECD). However, we have made so far no attempt to establish the absolute configuration at C-11 of 2, due to limited research facilities. Finally, the structure of artocarnin A (2) was elucidated as shown in Fig. 1 by detailed two dimensional (2D)-NMR spectroscopic analysis (Fig. 2).

Table 1. ¹H- and ¹³C-NMR Data for Compounds 2 and 12 in Acetone-d₆ (δ in ppm, J in Hz)^a)

No.	2		12
	δH	δC	δH	δC
2		156.8		165.2
3		110.0/109.9	6.66, s, 1H	104.0
4		179.5		183.2
5		155.6		157.9
6		105.7		106.1
7		157.7/157.6		160.4
8		106.2/106.0	6.47, s, 1H	95.6
9		155.3		157.2
10		105.8		105.8
11	6.15, d (9.0), 1H	69.9
12	5.50, d (9.0), 1H	122.1/122.0
13		138.4
14	1.68, s, 3H	25.9
15	1.94, s, 3H	18.7
16	6.67, d (10.2), 1H	116.1
17	5.75, d (10.2), 1H	128.9
18		78.8
19	1.50, s, 3H	28.4
20	1.50, s, 3H	28.6
1′		107.9/107.8		123.1
2′		152.5/152.4	7.95, d (9.0), 1H	129.3
3′	6.47, s, 1H	105.3	7.03, d (9.0), 1H	116.9
4′		152.1		162.1
5′		141.4	7.03, d (9.0), 1H	116.9
6′	7.35/7.39, s, 1H	110.3/110.2	7.95, d (9.0), 1H	129.3
1″	3.07, dd (13.1, 6.9), 1H	30.4	6.75, d (10.2), 1H	117.4
	3.06, dd (13.1, 6.9), 1H
2″	4.48/4.44, t (6.9), 1H	75.6/75.3	5.73, d (10.2), 1H	126.3
3″		149.0/148.9		82.0
4″	4.83/4.84, s, 1H	110.7/110.6	3.67, d (12.0), 1H	68.7
	4.71, s, 1H		3.62, d (12.0), 1H
5″	1.88, s, 3H	18.0	1.43, s, 3H	23.6
OH-5	13.33, s		13.40, s

a) Recorded at 600 MHz (¹H) and 150 MHz (¹³C).

Fig. 2. ¹H–¹H COSY (Bold Lines) and Key HMBC (Arrows) Correlations of 2 and 12

Carpachromenol (12) was isolated as a pale yellow amorphous solid and shown to have the molecular formula C₂₀H₁₆O₆ deduced from its positive HR-ESI-MS (m/z 353.1026 [M+H]⁺, Calcd for C₂₀H₁₇O₆, 353.1020) and negative HR-ESI-MS (m/z 351.0874 [M−H]⁻, Calcd for C₂₀H₁₅O₆, 351.0874). The ¹H-NMR (Table 1) spectrum of 12 revealed a para-disubstitued benzene ring at δ_H 7.95 (2H, d, J=9.0 Hz) and 7.03 (2H, d, J=9.0 Hz), a deshielded singlet at δ_H 13.40, an isolated aromatic proton at δ_H 6.47 (1H, s), and an isolated olefinic proton at δ_H 6.66 (1H, s), together with two cis olefinic protons at δ_H 6.75 (1H, d, J=10.2 Hz) and 5.73 (1H, d, J=10.2 Hz), a hydroxymethyl at δ_H 3.67 (1H, d, J=12.0 Hz) and 3.62 (1H, d, J=12.0 Hz), and a methyl group at δ_H 1.43 (3H, s). The NMR data (Table 1) for 12 closely resembled those for carpachromene,³⁶⁾ except for the absence of signals for one of the methyls at C-3″ and appearance of one hydroxymethyl. Moreover, the chemical shift of C-8 at δ_C 95.6 indicated the connectivity between C-6 and the pyran ring.^37,38) The dextro rotatory optical activity of 12 ([α]^D₂₀+14.29, c=0.10, CHCl₃) suggested it had an 3″S-configuration.³⁷⁾ From all above evidence, the structure of compound 12 was deduced as depicted, named carpachromenol, which was further corroborated by ¹H–¹H correlation spectroscopy (COSY) (Fig. 2), HSQC, distortionless enhancement by polarization transfer (DEPT), and HMBC (Fig. 2) experiments.

Compounds 1–15 were evaluated for their in vitro antiproliferative effects against human cervical cancer cells SiHa and human gastric cancer cells SGC-7901. As indicated in Table 2, eleocharin A (10) was the most potent compound, which exhibited inhibitory activity against SGC-7901 (IC₅₀=8.3±0.2 µM) and significant cytotoxic effect on SiHa (IC₅₀=0.7±0.1 µM), respectively. While the inhibitory activities of two structurally similar analogues, 11 and 12, declined sharply against both two cell lines, which revealed the 3′-methoxyl group was unfavorable but the 3′-hydroxyl maybe was crucial for the antiproliferative activity. Compound 5 showed inhibitory activity against SiHa cells with an IC₅₀ value of 8.7±0.2 µM. Compounds 9 and 11 exhibited weak activity against SiHa cells (10 µM<IC₅₀<20 µM). Compound 14 showed inhibitory activity against SGC-7901 (IC₅₀=9.6±0.9 µM) and weak activity against SiHa (IC₅₀=13.3±0.4 µM), and compound 7 showed weak activity against SGC-7901 cells (IC₅₀=11.9±0.1 µM). The other compounds showed inactivity against both two cell lines (IC₅₀>20 µM). Based on above results, compound 10 could be considered as potential lead compound for further development of novel anti-tumor agents.

Table 2. Cytotoxicity of Compounds 1–15 against Human Cancer Cell Lines (IC₅₀ ±S.D., µM)

Samples	SiHa	SGC-7901	Samples	SiHa	SGC-7901
1	35.2±0.3	68.4±1.1	9	11.1±1.3	54.7±1.0
2	48.7±0.1	55.0±0.7	10	0.7±0.1	8.3±0.2
3	>100	>100	11	17.8±0.7	25.1±1.4
4	>100	>100	12	20.9±0.5	21.3±0.1
5	8.7±0.2	38.3±0.4	13	21.0±0.2	22.4±0.7
6	33.0±0.5	23.3±0.2	14	13.3±0.4	9.6±0.9
7	40.6±2.2	11.9±0.1	15	73.1±0.3	>100
8	22.6±1.7	42.0±1.5	Doxorubicina)	0.2±0.1	0.3±0.1

a) Used as positive control.

Experimental

General Experimental Procedures

The 1D and 2D-NMR were recorded on a Bruker AVANCE 600 NMR spectrometer with tetramethylsilane (TMS) as an internal reference. All HR-ESI-MS spectra were measured on an AB Sciex 5600 LC/MS/MS system. Optical rotations were measured on an Anton Paar MCP-200 polarimeter. UV spectra were obtained on a Shimadzu UV-2550 Spectrophotometer. IR spectra were made in thin polymer films on a Shimadzu FTIR-8400 spectrometer. Silica gel (300–400 mesh, Qingdao Marine Chemical Plant, Qingdao, P. R. China), C₁₈ reversed-phase silica gel (150–200 mesh, Merck), MCI gel (CHP20P, 75–150 µM, Mitsubishi Chemical Industries Ltd., Japan), and Sephadex LH-20 gel (75–150 µM, GE Healthcare, U.S.A.) were used for column chromatography. Precoated silica gel GF254 plates (Qingdao Marine Chemical Plant) were used for TLC. Semipreparative HPLC was performed on an Agilent 1200 system equipped with a VWD G1314B detector and a Zorbax SB-C₁₈ column (250×10 mm, 5 µm). All solvents used were of analytical grade (Xilong Chemical Reagent Co., Ltd., Guangdong, P. R. China).

Plant Material

The twigs of A. nigrifolius was collected at Mengla county, Yunnan Province, P. R. China, in August 2014, and identified by Professor You-kai Xu of the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, P. R. China. A voucher specimen (AN201408) has been deposited in School of Pharmacy, Nanchang University.

Cell Culture

Human cervical cancer cell lines SiHa and human gastric cancer cell lines SGC-7901 were all purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and were cultured in RPMI 1640 medium (HyClone) supplemented with 10% fetal calf serum (HyClone), 2 mM L-glutamine, 100 U/mL Penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO₂. The cells were used between passages 10 and 20. The complete medium was refreshed every 24 h.

Extraction and Isolation

The dry powder of twigs (5.0 kg) of A. nigrifolius was extracted with 95% EtOH three times at ambient temperature to yield a crude extract (250 g), which was suspended in water and then extracted with EtOAc. The EtOAc extract (51.2 g) was subjected to silica gel column chromatography eluted with petroleum ether–acetone (20 : 1 to 1 : 1, v/v) to give nine fractions, fr. 1–fr. 9. Fr. 7 (4.4 g) was purified by silica gel column chromatography (petroleum ether–acetone, 5 : 1 to 2 : 1, v/v) and a column of C₁₈ reversed-phase (RP-18) silica gel (MeOH–H₂O, 9 : 1–1 : 0) to afford 1 (5.0 mg) and 2 (2.3 mg). Fr. 9 (6.5 g) was fractionated on a silica gel column eluted with petroleum ether–acetone (4 : 1 to 1 : 1, v/v) to give three subfractions fr. 9a–fr. 9c. Subfraction fr. 9b was purified by Sephadex LH-20 (MeOH) and semi-preparative HPLC (CH₃CN–H₂O, 45 : 55) to afford 3 (t_R=43.0 min, 3.0 mg), 4 (t_R=29.2 min, 3.0 mg), 5 (t_R=35.3 min, 2.0 mg), and 15 (t_R=22.4 min, 2.3 mg). Subfraction fr. 9c was subjected to a column of C₁₈ reversed-phase (RP-18) silica gel (MeOH–H₂O, 9 : 1–1 : 0) and semi-preparative HPLC (CH₃CN–H₂O, 45 : 55) to afford 6 (t_R=39.8 min, 2.0 mg), 7 (t_R=30.7 min, 1.5 mg), 8 (t_R=29.5 min, 1.8 mg), 9 (t_R=29.2 min, 1.5 mg), and 13 (t_R=52.1 min, 3.2 mg). Subfraction fr. 9a (3.2 g) was chromatographed over a column of C₁₈ reversed-phase (RP-18) silica gel (MeOH–H₂O, 9 : 1–1 : 0) and semi-preparative HPLC (CH₃CN–H₂O, 40 : 60) to afford 11 (t_R=24.9 min, 3.1 mg), 12 (t_R=23.4 min, 2.4 mg), and 14 (t_R=31.5 min, 2.0 mg). Fr. 8 (1.3 g) was purified by a column of C₁₈ reversed-phase (RP-18) silica gel (MeOH–H₂O, 9 : 1–1 : 0) and semi-preparative HPLC (CH₃CN–H₂O, 65 : 35) to afford 10 (t_R=31.7 min, 2.1 mg).

Artocarnin A (2)

Yellow amorphous solid; [α]^D₂₀+1.67 (c=0.15, CHCl₃). UV λ_max (MeOH) nm (log ε): 233.1 (3.71), 272.0 (3.74), 301.2 (3.92), 400.0 (3.83); IR (Film) cm⁻¹: 3400, 1650, 1625, 1420; ¹H- and ¹³C-NMR data: see Table 1; (−) HR-ESI-MS m/z 563.1923 [M+HCOO]⁻ (Calcd for C₃₁H₃₁O₁₀, 563.1912).

Carpachromenol (12)

Pale yellow amorphous solid; [α]^D₂₀+14.29 (c=0.10, CHCl₃). UV λ_max (MeOH) nm (log ε): 231.9 (3.82), 305.9 (4.02), 344.9 (3.88); IR (Film) cm⁻¹: 3372, 1650, 1608, 1400; ¹H- and ¹³C-NMR data: see Table 1; (+) HR-ESI-MS m/z 353.1026 [M+H]⁺ (Calcd for C₂₀H₁₇O₆, 353.1020); (−) HR-ESI-MS m/z 351.0874 [M−H]⁻ (Calcd for C₂₀H₁₅O₆, 351.0874).

Cytotoxicity Assay

Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, SiHa human cervical cancer cells and SGC7901 human gastric cancer cells (1×10⁴ cells/mL) were seeded into 96-well plates. After 24 h of incubation, the cells were treated with the compounds 1–15 (100, 1000, 2000, and 5000 µg/L), and incubated for further 48 h. Cultured media were then removed and replaced with fresh media containing 0.5 mg/mL MTT and incubated for 2 h at 37°C. The violet crystal was dissolved by dimethyl sulfoxide (DMSO), and the optical density was measured at 570 nm by using SpectraMax Paradigm (Molecular Devices, LLC, U.S.A.). The IC₅₀ value was calculated as concentration of all testing compounds, under which 50% inhibition of cell growth occurs compared to nontreated controls. DMSO and Doxorubicin were used as negative and positive control, respectively.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Nos. 21362023 and 30901855), the Natural Science Foundation of Jiangxi Province, China (Nos. 20142BAB215021 and 20151BAB205083), and the Project of Jiangxi Provincial Education Department (No. 14097).

Conflict of Interest

The authors declare no conflict of interest.

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