Abstract
Chromatographic separation of the acetone extracts from the twigs and barks of Artocarpus lakoocha led to the isolation of the one new flavanone, lakoochanone (1), together with eleven known compounds (2–12). Lakoochanone (1) and moracin C (4) exhibited weak antiplasmodial activity against Plasmodium falciparum Dd2 with IC50 values of 36.7 and 33.9 µM, respectively. Moreover, moracin C (4) and sanggenofuran B (5) showed cytotoxic activity against A2780 cell line with the respective IC50 values of 15.0 and 57.1 µM. In addition, cyclocommunin (7) displayed strong antimycobacterial activity against Mycobacterium tuberculosis H37Ra with the minimum inhibitory concentration (MIC) value of 12.3 µM.
Introduction
Artocarpus lakoocha, belonging to the Moraceae family, is locally named Ma Haad. It is distributed in South and Southeast Asia such as Thailand, Myanmar, Vietnam and Indonesia.1) In the course of our ongoing research for bioactive metabolites from the local plants, the acetone extracts from the twigs and barks of A. lakoocha exhibited moderate antiplasmodial activity against Plasmodium falciparum Dd2, cytotoxic activity against the A2780 cell line (human ovarian cancer) and antimycobacterial activity against Mycobacterium tuberculosis H37Ra. There are a few reports on the phytochemical investigation and no chemical investigation with these activities has been reported.2,3) We report herein the isolation and structure elucidation of one new flavonoid (1) together with eleven known compounds (2–12) from these extracts. The cytotoxic antimycobacterial, antiplasmodial activities were evaluated.
Chemical investigation of the acetone extracts of the twigs and barks from A. lakoocha led to the separation and identification of one new flavonoid, lakoochanone (1), along with eleven known compounds, (+)-afzelechin-3-O-α-L-rhamnopyranoside (2),4) (+)-catechin (3),4) moracin C (4),5) sanggenofuran B (5),6) integrin (6),7) cyclocommunin (7),8) oxyresveratrol (8),9) (E)-2-methoxy-4,3ʹ,5ʹ-trihydroxystilbene (9),10) engeletin (10),11) isogemichalcone B (11),12) and morachalcone A (12).12) The structures of compounds 2–12 (Fig. 1) were determined by analysis of their UV, IR, NMR spectra and by comparison with data for previously reported compounds in the literature.
Lakoochanone (1) was obtained as a yellow gum with the molecular formula C29H26O8 by high-resolution liquid chromatography time-of-flight (HRLC-TOF)-MS (m/z 503.1691 [M + H]+). The UV spectrum showed absorption bands at 225, 292 and 311 nm, while the IR spectrum displayed absorption bands for hydroxyl (3365 cm−1), conjugated carbonyl (1690 cm−1) and conjugated olefinic (1605 cm−1) functional groups. The 1H-NMR spectrum (Table 1) displayed signals for a trans-p-coumaroyl moiety (δH 7.57 and 6.28, each d, J = 15.5 Hz, 1H and 7.45 and 6.80, each d, J = 8.0 Hz, 2H), three aromatic protons of a 1,2,4-trisubstituted benzene ring [δH 7.29 (d, J = 8.5 Hz, 1H), 6.34 (dd, J = 8.5, 2.5 Hz, 1H), and 6.32 (d, J = 2.5 Hz, 1H)], two ortho-coupled aromatic protons [δH 7.62 and 6.52, each d, J = 8.5 Hz, 1H], one olefinic proton (δH 5.56, t, J = 7.5 Hz, 1H), one oxymethine proton (δH 5.66, dd, J = 13.2, 3.0 Hz, 1H), two sets of equivalent methylene protons [δH 4.82 (br s, 2H) and 3.44 (m, 2H)], one set of non-equivalent methylene protons [δH 2.97 (dd, J = 17.3, 13.2 Hz, 1H) and 2.75 (dd, J = 17.3, 3.0 Hz, 1H)] and a methyl group (δH 1.73, s, 3H). The 1H-NMR spectroscopic data of 1 were almost identical to those of artopithecin E.13) In addition, the 13C-NMR data and the heteronuclear multiple bond coherence (HMBC) correlations (Table 1) supported the above conclusion. The observed specific rotation of 1, [α]21D −2.3 (c 0.45, MeOH) which correlated well with that of artopithecin E, [α]20D −9.2 (c 0.65, MeOH), indicated that C-2 in 1 has the S configuration, identical to that of artopithecin E. The difference was the configuration of the C2″–C3″ double bond in 1. The configuration of the C2″–C3″ double bond of 1 was identified to be E-configuration, not Z-configuration, according to signal enhancement of H3-5″ upon irradiation of H2-1″ in the nuclear Overhauser effect difference (NOEDIFF) experiment as well as the high field shift of the methyl carbon, C-5″, (1: δC 14.1; artopithecin E: δC 21.6). From this spectroscopic data, the structure of lakoochanone was concluded to be 1.
Table 1. The NMR Data of Lakoochanone (
1) in Methanol-
d4| Position | δH, mult. (J in Hz) | δC (Type) | HMBC | COSY |
|---|
| 2 | 5.66, dd (13.2, 3.0) | 76.2 (CH) | C-3, C-4, C-8a, C-1ʹ | Hab-3 |
| 3 | a: 2.97, dd (17.3, 13.2) b: 2.75, dd (17.3, 3.0) | 44.1 (CH2) | C-2, C-4, C-4a, C-1ʹ | H-2, Hb-3 H-2, Ha-3 |
| 4 | — | 193.8 (C) | — | — |
| 4a | — | 113.8 (C) | — | — |
| 5 | 7.62, d (8.5) | 127.3 (CH) | C-4, C-6, C-7, C-8a | H-6 |
| 6 | 6.52, d (8.5) | 110.5 (CH) | C-4a, C-5, C-8 | H-5 |
| 7 | — | 158.2 (C) | — | — |
| 8 | — | 114.7 (C) | — | — |
| 8a | — | 156.0 (C) | — | — |
| 1ʹ | — | 126.0 (C) | — | — |
| 2ʹ | — | 158.8 (C) | — | — |
| 3ʹ | 6.32, d (2.5) | 103.4 (CH) | C-1ʹ, C-2ʹ, C-4ʹ | H-5ʹ |
| 4ʹ | — | 162.6 (C) | — | — |
| 5ʹ | 6.34, dd (8.5, 2.5) | 107.6 (CH) | C-1ʹ, C-3ʹ, C-6ʹ | H-3ʹ, H-6ʹ |
| 6ʹ | 7.29, d (8.5) | 128.5 (CH) | C-1ʹ, C-2ʹ, C-5ʹ | H-5ʹ |
| 1ʹʹ | 3.44, m | 22.4 (CH2) | C-7, C-8, C-2ʹʹ | H-2ʹʹ |
| 2ʹʹ | 5.56, t (7.5) | 127.8 (CH) | C-1ʹʹ, C-3ʹʹ, C-4ʹʹ, C-5ʹʹ | H-1ʹʹ, H-5ʹʹ |
| 3ʹʹ | — | 130.4 (C) | — | — |
| 4ʹʹ | 4.82, brs | 63.9 (CH2) | C-3ʹʹ, C-9ʹʹʹ | — |
| 5ʹʹ | 1.73, s | 14.1 (CH3) | C-2ʹʹ, C-3ʹʹ, C-4ʹʹ | — |
| 1ʹʹʹ | — | 126.0 (C) | — | — |
| 2ʹʹʹ | 7.45, d (8.0) | 131.2 (CH) | C-1ʹʹʹ, C-3ʹʹʹ, C-7ʹʹʹ | H-3ʹʹʹ |
| 3ʹʹʹ | 6.80, d (8.0) | 116.9 (CH) | C-2ʹʹʹ, C-4ʹʹʹ | H-2ʹʹʹ |
| 4ʹʹʹ | — | 159.1 (C) | — | — |
| 5ʹʹʹ | 6.80, d (8.0) | 116.9 (CH) | C-4ʹʹʹ, C-6ʹʹʹ | H-6ʹʹʹ |
| 6ʹʹʹ | 7.45, d (8.0) | 131.2 (CH) | C-1ʹʹʹ, C-5ʹʹʹ, C-7ʹʹʹ | H-5ʹʹʹ |
| 7ʹʹʹ | 7.57, d (15.5) | 146.7 (CH) | C-1ʹʹʹ, C-8ʹʹʹ, C-9ʹʹʹ | H-8ʹʹʹ |
| 8ʹʹʹ | 6.28, d (15.5) | 115.2 (CH) | C-1ʹʹʹ, C-7ʹʹʹ, C-9ʹʹʹ | H-7ʹʹʹ |
| 9ʹʹʹ | — | 168.7 (C) | — | — |
All of the isolated compounds, except for 2, 6, and 9 which were obtained in insufficient amounts, were tested for cytotoxic activity against the A2780 cell line,14,15) for antimycobacterial activity against Mycobacterium tuberculosis H37Ra,16) and for antiplasmodial activity against Plasmodium falciparum Dd2 parasites17) (Table 2). Compounds 10–12 were inactive in all these assays. Compounds 4 and 5 exhibited weak cytotoxic activity against the A2780 cell line with IC50 values of 15.0 and 57.1 µM, respectively. For antimycobacterial activity, compounds 1, 3, 4 and 8 showed mild activity with minimum inhibitory concentration (MIC) values in the range of 25.6–35.6 µM, while compound 7 displayed stronger activity with an MIC value of 12.3 µM. Finally, compounds 1, 4 and 8 exhibited weak antiplasmodial activity with IC50 values of 36.7, 33.9 and 50.1 µM, respectively.
Table 2. Cytotoxic, Antimycobacterial and Antiplasmodial Activities of Crude Extracts and the Selected Compounds
| Compound | Cytotoxicity (IC50, µM) | Anti-TB (MIC, µM) | Antiplasmodial (IC50, µM) |
|---|
| A2780 | M. tuberculosis | P. falciparum Dd2 |
|---|
| Twig crude extract | 5 µg/mL | 50 µg/mL | 35.6 µg/mL |
| Barks crude extract | 10 µg/mL | — | 65.3 µg/mL |
| 1 | >100 | 35.2 | 36.7 |
| 3 | >100 | 52.1 | >100 |
| 4 | 15.0 | 25.6 | 33.9 |
| 5 | 57.1 | >100 | >100 |
| 7 | >100 | 12.3 | >100 |
| 8 | >100 | 35.6 | 50.1 |
| Paclitaxel a) | 0.013 | NT | NT |
| Isoniazid b) | NT | 0.17–0.34 | NT |
| Artemisinin c) | NT | NT | 6.73 nM |
a) Positive control for cytotoxicity. b) Positive control used in the anti-TB assay. c) Reference compound for antiplasmodial activity. NT = not tested. — = inactive.
Experimental
General Experimental ProceduresShimadzu UV-Vis spectrometer obtained UV spectra, and MIDAC M-series Fourier transform (FT)-IR spectrometer measured IR spectra. 1H- and 13C-NMR spectra were recorded by 500 MHz JEOL Eclipse 500 spectrometer in methanol-d4 with the internal standard, tetramethylsilane (TMS). Agilent 622-LC-TOF-MS mass spectrometer recorded MS. Optical rotation was measured on JASCO P-2000 polarimeter. TLC was performed on silica gel 60 F254 (Merck, Germany) and reversed phase TLC (Merck). Column chromatography (CC) was performed on Sephadex LH-20 (Merck) or on silica diol (230–400 mesh ASTM, Silicycle) or on reversed phase silica gel C-18 (Merck). Preparative HPLC was performed using Shimadzu LC-10AT pumps coupled with a semipreparative Varian Dynamax C-18 column (5 µm, 250 × 10 mm), a Shimadzu SPD M10A diode array detector and a SCL-10A system controller.
Plant MaterialsThe twigs and barks of Artocarpus lakoocha were collected from Mea Rim District, Chiang Mai Province, Thailand in January 2016. A voucher specimen (No. QBG91824) was identified by Dr. Nawong Muangyen, Queen Sirikit Botanic Garden, Chiang Mai, and has been deposited at the herbarium collection of the Queen Sirikit Botanic Garden, Mae Rim, Chiang Mai Province, Thailand.
Extraction, Isolation and PurificationThe air-dried twigs (2.0 kg) and barks (4.0 kg) were extracted twice with 10 L of acetone for 7 d. Each extract was filtered and the acetone was then removed under reduced pressure to give an acetone extract as a brown gum (20.0 g) and a dark brown gum (20.2 g) from the twigs and barks, respectively. A 3.00 g portion of the twig extract was suspended in H2O and then partitioned with dichloromethane to afford 198.5 mg of dichloromethane-soluble material. Chromatography of the dichloromethane fraction on Sephadex LH-20 (30 × 400 mm) using 50% MeOH/CH2Cl2 gave six fractions (A–F). Fraction D (19.8 mg) was separated by solid phase extraction with Hypersep C18 eluting with 50% MeOH/H2O to give three subfractions (D1–D3). Subfraction D1 (13.3 mg) was purified by HPLC on a C18 column with 50% MeOH/H2O to provide 1 (1.5 mg). HPLC of subfraction D2 (5.3 mg) on a C18 column with a solvent gradient from 80% MeOH/H2O to 100% MeOH gave 7 (1.4 mg). Fraction E (4.7 mg) was separated by the same procedure as fraction D to furnish three subfractions (E1–E3). Separation of subfraction E2 (4.3 mg) by HPLC on a C18 column with the solvent gradient from 80% MeOH/H2O to 100% MeOH gave 11 (1.3 mg) and 12 (1.7 mg). Fraction F (5.2 mg) was separated by the same procedure as fraction D2 to furnish 8 (2.4 mg).
A 10.00 g portion of the barks extract was suspended in H2O and partitioned with dichloromethane to afford 232.8 mg of dichloromethane-soluble fraction. Chromatography of the dichloromethane-soluble fraction (232.8 mg) on Sephadex LH-20 (30 × 400 mm) using 50% MeOH/CH2Cl2 as a mobile phase gave eight fractions (BD1–BD8). Fraction BD5 (37.7 mg) was separated by Hypersep C18 eluting with 50% MeOH/H2O to give three subfractions (BD51–BD53). HPLC separation of subfraction BD51 (29.7 mg) on a C18 column with a solvent gradient from 50–90% MeOH/H2O gave 10 (1.4 mg) and 6 (0.5 mg). Separation of subfraction BD52 (5.4 mg) by HPLC on a C18 column with 80% MeOH/H2O gave 5 (1.8 mg). Fraction BD6 (21.0 mg) was separated by the same procedure as fraction BD5 to furnish three subfractions (BD61–BD63). The first subfraction (16.9 mg) was purified by HPLC on a C18 column with 50% MeOH/H2O to afford 3 (1.6 mg) and 9 (0.5 mg). Separation of subfraction BD62 (4.2 mg) by HPLC by the same procedure as subfraction BD52 gave 2 (0.9 mg) and 4 (2.0 mg).
Lakoochanone (1): yellow gum; [α]21D −2.3 (c 0.45, MeOH); UV λmax (MeOH) nm (log ε): 225 (4.31), 292 (3.95), 311 (3.24); IR (neat) νmax cm−1: 3365, 1690, 1605; 1H-NMR (500 MHz, methanol-d4) data (Table 1); 13C-NMR (125 MHz, methanol-d4) data (Table 1). HRLC-TOF-MS m/z: [M + H]+ found: 503.1691 (calcd for C29H27O8 503.1691).
Acknowledgments
S. Boonyaketgoson thanks the Science Achievement Scholarship of Thailand and the Graduate School, Chiang Mai University for a scholarship and financial support. K. Trisuwan thanks the Office of the Higher Education Commissions and the Thailand Research Fund for the TRF Grant for New Researcher (Grant No. MRG5980004), the Department of Chemistry, Faculty of Science, Chiang Mai University. This research work was partially supported by Chiang Mai University.
Conflict of Interest
The authors declare no conflict of interest.
Supplementary Materials
The online version of this article contains supplementary materials.
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