Cytotoxic Coumarins from Toddalia asiatica Ratchanee Phatchana, Chavi Yenjai Natural Products Research Unit, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand
Abstract !
Three new coumarins and 13 known compounds were isolated from the stem bark of Toddalia asiatica. Compounds 1, 3, 8, and 9 showed cytotoxicity against the NCI-H187 cell line with IC50 values ranging from 6 to 9 µg/mL. Compounds 4 and 9 exhibited cytotoxicity against the MCF-7 cell line with IC50 values of 3.17 and 9.79 µg/mL, respectively. Compound 9 also displayed cytotoxic activity against KB cells with an IC50 value of 8.63 µg/mL. In addition, compound 14 showed antimalarial activity against Plasmodium falciparum with an IC50 value of 3.66 µg/mL. Compounds 5, 9, and 16 exhibited antituberculosis activity against Mycobacterium tuberculosis with MIC values of 50, 50, and 25 µg/mL, respectively.
Key words Toddalia asiatica · Rutaceae · coumarin · cytotoxicity · antimalarial Supporting information available online at http://www.thieme-connect.de/products
As part of our research on the phytochemistry of Thai medicinal plants, we have intensively evaluated biological activities of extracts and pure compounds [1, 2]. Our interest focuses on cyto-
toxicity, antimalarial, antifungal, and antituberculosis (anti-TB) properties [3]. We report herein the isolation and structural identification of 3 new coumarins and 12 known coumarins as well as an alkaloid from Toddalia asiatica (L) Lam. (Rutaceae). All compounds were investigated for cytotoxicity against KB, MCF-7, and NCI-H187 cell lines. Antimalarial activity against Plasmodium falciparum and anti-TB activity against Mycobacterium tuberculosis H37Ra were also evaluated. T. asiatica is a medicinal plant widely distributed in Thailand, India, and China as well as in south and tropical Africa. All parts of this plant show some therapeutic activities such as to treat chronic lumbago, scelalgia, colds, stomachache, and injuries from falls [4]. The fruits are used to treat malaria and cough, the roots to treat indigestion, and the leaves to treat lung diseases [5]. T. asiatica contains mainly coumarins, especially prenylated and geranylated, triterpenes, phenanthridine alkaloids, and also volatile oils [6–9]. Many compounds from this plant have shown anticancer activity against the U-937 cell line [10], as well as antidiabetic, antioxidant [11], and antibacterial activities [5].
Results and Discussion !
In this paper, three new coumarins 1–3 together with 13 known compounds were isolated from the crude EtOAc extract of T. asiatica bark. Thirteen known compounds included 4-O-geranylconiferyl aldehyde (4) [12], nelumol A (5) [13], phellopterin (6) [14], 5-methoxy-8-geranyloxylpsoralen (7) [15], artanin (8) [16], 8geranyloxy-5,7-dimethyloxycoumarin (9) [16], 5,7,8-trimethoxycoumarin (10) [16], leptodactylone (11) [17], toddalolactone (12) [18], toddalolactone methyl ether (13) [19], 1,2-secodihydromethylumbelliferone methyl ester (14) [20], toddalosin (15) [16], and norchelerythrine (16) [21] were discovered " Fig. 1). Compounds 4, 5, 7, 11 and 14 were found for the first (l time in this plant. Compound 1 was found as a pale yellow oil and assigned the molecular formula C21H26O6 by HRESIMS. The 1H and 13C NMR spec-
Fig. 1
Structure of compounds 1–16.
Phatchana R, Yenjai C. Cytotoxic Coumarins from …
Planta Med 2014; 80: 719–722
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Letters
Letters
Table 1
1
H NMR spectral data of compounds 1–3 (δ in ppm and J in Hz).
Table 2
13
C NMR spectral data of compounds 1–3 (δ in ppm and J in Hz).
Position
1
2
3
Position
1
2
3
3 4 6 8 1′ 2′ 4′ 5′ 6′ 8′ 9′ 10′ OCH3-5 OCH3-7 OH
6.14, d (9.6) 7.96 d (9.6) 6.32, s
6.13, d (9.6) 7.95, d (9.6) 6.32, s
6.22, d (9.6) 7.88, d (9.6)
4.59, d (7.2) 5.60, m 2.71, (6.0) 5.52, d (15.6) 5.61, d (15.6) 1.31, s 1.31, s 1.61, s 3.90 3.95 1.71
4.58, d (7.2) 5.57, m 2.68, d (6.0) 5.55, d (12.0) 5.60, d (12.0) 1.29, s 1.29, s 1.63, s 3.89 3.94 1.73
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ OCH3-5 OCH3-7
161.1 111.4 138.9 152.5 91.6 156.7 128.9 149.3 104.1 70.0 121.1 140.7 42.4 128.8 135.7 82.2 24.4 24.4 16.7 56.2 56.7
161.0 111.3 138.9 152.5 91.5 156.7 128.9 149.3 104.0 70.1 120.8 141.1 42.2 124.2 140.3 70.7 29.9 29.9 16.6 56.1 56.6
161.0 112.8 138.9 155.7 116.6 161.6 95.6 154.9 107.6 118.3 136.9 143.0 117.8 18.3
6.59, s 6.62, d (16.8) 7.23, d (16.8) 5.09, 5.08, s 1.97, s
3.76, s 3.89, s
" Table 1 and 2) showed a 5,7-dimethoxy-8-substituted tra of 1 (l coumarin unit as in co-occurring compound 9. The methylene protons at δ 4.59 (2H, d, J = 7.2 Hz, H-1′) correlated with C-8 (δ 128.9), C-2′ (δ 121.1), and C-3′ (δ 140.7) in the HMBC experiment. In this experiment, the methylene protons at δ 2.71 (2H, d, J = 6.0 Hz, H-4′) correlated with C-2′ (δ 121.1), C-3′ (δ 140.7), C-5′ (δ 128.8), and C-6′ (δ 135.7). Two methyl groups (CH3-8′ and 9′) showed singlet signals at δ 1.31 and correlated with C-6′ (δ 135.7) and C-7′ (δ 82.2) in the HMBC spectrum. In the 1H NMR spectrum, the coupling constant of H-5′ (δ 5.52) and 6′ (δ 5.61) exhibited 15.6 Hz indicating 5′,6′-trans geometry. In addition, the carbon at δ 82.2 was assigned as C-7′ which connected with the hydroxyl group. Furthermore, the NOESY experiment revealed a correlation between methylene protons at H-1′ and methyl protons at H-10′ (δ 1.61, s, 3H) which indicated the E geometry of a 2′,3′ double bond. Therefore, compound 1 was identified as 8-(3′,7′-dimethyl-7′-hydroxy-2′E,5′E-octadienyl)oxy-5,7dimethoxycoumarin. Compound 2 was a pale yellow oil, assigned the molecular formula C21H26O6 by HRESIMS. The 1H NMR spectrum of this com" Table 2) closely resembled that of 1, except for the coupound (l pling constant of H-5′ and 6′ (J = 12.8 Hz), which indicated cis ge" Table 2) was also similar to ometry. The 13C NMR spectrum (l that of 1, but the signals of C-5′, C-6′, C-7′, and 8′/9′ were shifted compared to those of 1. The HMBC correlations of this compound were nearly the same as 1. Two protons at δ 5.55 (d, J = 12 Hz) and δ 5.60 (d, J = 12 Hz) were assigned as H-5′ and H-6′, respectively, which indicated the cis geometry of the 5′,6′ double bond. Therefore, compound 2 was identified as 8-(3′,7′-dimethyl-7′-hydroxy2′E,5′Z-octadienyl)oxy-5,7-dimethoxycoumarin. Compound 3 was a white solid, assigned the molecular formula " Table 1 C16H16O4 by HRESIMS. The 1H and 13C NMR spectra (l and 2) indicated that this compound had a coumarin core structure similar to co-occurring compound 12. The coupling constants of two doublet protons at δ 6.62 (J = 16.8, H-1′) and 7.23 (J = 16.8, H-2′) indicated a trans geometry double bond and connected with carbons at δ 118.3 (C-1′) and 136.9 (C-2′), respectively, in the HMQC experiment. Correlations between H-1′ with C-5 (δ 155.7), C-7 (δ 161.6), and C-3′ (δ 143.0) in the HMBC spectrum confirmed the connection of an isoprene unit at the C-6 (δ 116.6) position. Terminal olefinic protons H-4′ (δ 5.09 and 5.08, s) correlated with methylenecarbon at δ 117.8 in the HMQC experiment.
Phatchana R, Yenjai C. Cytotoxic Coumarins from … Planta Med 2014; 80: 719–722
62.0 56.3
These protons displayed correlation with C-2′ (δ 136.9) and C-5′ (δ 18.3) in the HMBC spectrum. Furthermore, the NOESY experiment revealed a correlation between H-1′ and H-5′ which indicated that these conjugated double bonds prefer a transoid conformation. From these data, compound 3 was identified as 6-(3′methyl-1′,3′-butadienyl)-5,7-dimethoxycoumarin. Compounds 1 and 2 showed cytotoxicity against the MCF-7 cell line with IC50 values of 28.23 and 24.80 µg/mL, respectively " Table 3). These two compounds showed cytotoxicity against (l the NCI-H187 cell line with IC50 values of 6.42 and 24.46 µg/mL, respectively. The results suggest that a trans double bond at C5′/6′ is required for activity against the NCI-H187 cell line. Compounds 4 and 5 showed cytotoxic activity against all cell lines tested. Compound 4 exhibited the strongest cytotoxic activity against the MCF-7 cell line with an IC50 value of 3.17 µg/mL. The results show convincingly that an aldehyde group seems to have an important role against the MCF-7 cell line. From the literature search, 4 and 5 showed cytotoxicity against six human cancer cell lines including U373, OE21, A549, PC-3, SKMEL−28, and LoVo with IC50 mean values of 16 and 47 µg/mL, respectively [22]. Furanocoumarin 6 demonstrated cytotoxicity against the MCF-7 cell line (IC50 = 23.20 µg/mL), while furanocoumarin 7 displayed cytotoxicity against the other cell lines (KB and NCI-H187) with IC50 values around 10 µg/mL. The results suggest that the isopentenyl group is required for activity against the MCF-7 cell line but the geranyl group is essential against KB and NCI-H187 cell lines. Kimura and coworkers reported that 6 exhibited cytotoxicity against B16F10 melanoma cells with an IC50 value of 0.52 µg/mL [23]. In the cases of 8 and 9, compound 9 exhibited stronger cytotoxicity against all cell lines (IC50 ≈ 8 µg/mL), while 8 showed good activity only against the NCI-H187 (IC50 = 7.41 µg/mL). This suggests that the geranyl group at the C8 position is favorable against all cell lines. It was reported that 8 showed cytotoxicity against leukemic cells (U-937) with an IC50 value of 177.3 µM [24]. In addition, all compounds were evaluated for anti-TB activity against M. tuberculosis H37Ra and antimalarial activity against P. falciparum (K1, multidrug resistant strain). Compounds 5, 9, and
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720
Letters
1 2 3 4 5 6 7 8 9 11 14 16 10, 12–13, 15 Ellipticine Doxorubicin
Cytotoxicity (IC50, µg/mL)* KB
MCF-7
NCI-H187
28.12 inactivea inactivea 23.68 15.50 inactivea 12.34 31.02 8.63 inactivea inactivea inactivea inactivea 1.27 –
28.23 24.80 inactivea 3.17 17.82 23.20 inactivea 25.44 9.79 inactivea inactivea 23.20 inactivea 0.44 2.84
6.42 24.46 9.58 10.61 23.09 inactivea 10.53 7.41 7.97 29.68 19.45 inactivea inactivea 0.684 –
Table 3 Cytotoxicity of all compounds.
* Data shown are from triplicate experiments; a inactive at > 50 µg/mL
16 showed anti-TB activity with MIC values of 50, 50, and 25 µg/ mL, respectively. Fortunately, only compound 14 displayed antimalarial activity by showing an IC50 value of 3.66 µg/mL. It was reported that 5,7-dimethoxy-8-(3′-hydroxy-3′-methyl-1′-butene)-coumarin isolated from this plant displayed moderate antimalarial activity against P. falciparum K39 and V1 S strains with IC50 values of 16.2 and 8.8 µg/mL, respectively [25].
Materials and Methods !
General experimental procedures: Melting points were determined on a Sanyo Gallenkamp melting point apparatus and are uncorrected. UV spectra were measured on an Agilent 8453 UVVisible spectrophotometer. IR spectra were recorded as KBr disks or thin films, using Perkin Elmer Spectrum One FT‑IR spectrophotometer. The NMR spectra were recorded on a Varian Mercury plus spectrometer operating at 400 MHz (1H) and at 100 MHz (13C). Mass spectra were determined on a Micromass Q‑TOF 2 hybrid quadrupole time-of-flight (Q‑TOF) mass spectrometer with a Z-spray ES source (Micromass). Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 TLC aluminium sheet. Column chromatography was done with silica gel 0.063–0.200 mm or less than 0.063 mm. Preparative layer chromatography (PLC) was carried out on glass supported silica gel plates using silica gel 60 PF254 for preparative layer chromatography. All solvents were routinely distilled prior to use. Plant material: The bark of T. asiatica was collected in April 2011 from Phetchabun Province. The plant was identified by Dr. Pranom Chantaranothai, Faculty of Science, Khon Kaen University. A botanically identified voucher specimen (KKU0042011) was deposited at the herbarium of the Department of Chemistry, Faculty of Science, Khon Kaen University. Extraction and isolation: Air-dried and finely powdered stem bark (2.9 kg) of T. asiatica was sequentially extracted at room temperature for three days with hexane (2 × 3 L), EtOAc (2 × 3 L), and MeOH (2 × 3 L). The extracts were evaporated in vacuo to obtain three dry extracts, crude hexane (69 g), EtOAc (89 g), and crude MeOH (121 g). The crude EtOAc extract (89.7 g) was subjected to column chromatography on silica gel 60 and subsequently eluted with a gradient of three solvents (hexanes, EtOAc,
and MeOH) by gradually increasing the polarity of the elution solvents system to isolate pure compounds 1–16 (purity ≥ 95%, see Supporting Information). Bioassays: Cytotoxicity assay: Cytotoxicity assays against human epidermoid carcinoma (KB, ATCC CCL−17), breast adenocarcinoma (MCF-7, ATCC HTB−22) and human small cell lung cancer (NCI-H187, ATCC CRL−5804) cell lines were performed employing resazurin microplate assay (REMA; Sigma-Aldrich, dye content 75 %) [26], while cytotoxicity assay against Vero cells (African green monkey kidney, ATCC CCL−81) was performed by green fluorescent protein (GFP)-based assay [27]. Ellipticine (Fluka, purity ≥ 99 %) and doxorubicin (Fluka, purity ≥ 98%) were included as the reference substances. Antimalarial assay: Antimalarial activity was performed against P. falciparum (K1, multidrug resistant strain) which was cultured continuously according to the method of Trager and Jensen [28]. Quantitative assessment of antimalarial activity in vitro was determined by means of the microdilution radioisotope technique based on the method described by Desjardins [29]. The inhibitory concentration was that which caused 50% reduction in parasite growth as indicated by the in vitro uptake of [3H] hypoxanthine by P. falciparum. The standard compound was dihydroartemisinin (Sigma, purity ≥ 97 %). Antimycobacterial assay: The antimycobacterial activity was performed against M. tuberculosis H37Ra using the green fluorescent protein microplate assay (GFPMA) [30]. The standard drugs isoniazid (Fluka, purity ≥ 99%) and rifampicin (Fluka, purity ≥ 97 %) were used as the reference compounds. Spectroscopic data of compounds: Compound 1: Pale yellow oil; UV (CH3CN) λmax (log ε) 262 (4.20), 318 (4.20) nm; IR (KBr) νmax 3436, 2944, 1724, 1604, 1505, 1438, 1344, 1150, 1120, 992, 754 cm−1; HRESIMS m/z 397.1620 [M + Na]+ (calcd. 397.1619); 1 " Table 1 and 2. H and 13C NMR spectroscopic data, see l Compound 2: Pale yellow oil; UV (CH3CN) λmax (log ε) 262 (4.20), 320 (4.24) nm; IR (KBr) νmax 3404, 3017, 2945, 1720, 1604, 1505, 1344, 1216, 1150, 1121, 752 cm−1; HRESIMS m/z 397.1629 [M + Na]+ (calcd. 397.1619); 1H and 13C NMR spectroscopic data, see " Table 1 and 2. l Compound 3: White solid; mp. 103–104 °C, UV (CH3CN) λmax (log ε) 287 (4.54), 335 (4.04) nm; IR (KBr) νmax 2959, 1727, 1611,
Phatchana R, Yenjai C. Cytotoxic Coumarins from …
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Compound
721
Letters 1386, 1203, 1141, 1088, 973, 827 cm−1; HRESIMS m/z 273.1132 [M + H]+ (calcd. 273.1128); 1H and 13C NMR spectroscopic data, " Table 1 and 2. see l
Supporting information Detailed protocols for extraction and isolation as well as 1D and 2D NMR spectra of compounds 1–3 are available as Supporting Information.
Acknowledgements !
We thank the Rajamangala University of Technology Isan, Khon Kaen Campus, for financial support to R. Phatchana and the Bioassay Laboratory of the National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand, for biological activity assays. The National Research University Project of Thailand through the Advanced Functional Materials Cluster of Khon Kaen University is also gratefully acknowledged.
Conflict of Interest !
There are no conflicts of interest of all authors with respect to this work.
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received revised accepted
June 12, 2013 April 29, 2014 May 11, 2014
Bibliography DOI http://dx.doi.org/10.1055/s-0034-1368568 Published online June 17, 2014 Planta Med 2014; 80: 719–722 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Chavi Yenjai Natural Products Research Unit Department of Chemistry and Center of Excellence for Innovation in Chemistry Faculty of Science, Khon Kaen University Khon Kaen 40002 Thailand Phone: + 66 43 20 22 22 41 ext. 1 22 43 Fax: + 66 43 20 23 73 [email protected]
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Abstract !
Three new coumarins and 13 known compounds were isolated from the stem bark of Toddalia asiatica. Compounds 1, 3, 8, and 9 showed cytotoxicity against the NCI-H187 cell line with IC50 values ranging from 6 to 9 µg/mL. Compounds 4 and 9 exhibited cytotoxicity against the MCF-7 cell line with IC50 values of 3.17 and 9.79 µg/mL, respectively. Compound 9 also displayed cytotoxic activity against KB cells with an IC50 value of 8.63 µg/mL. In addition, compound 14 showed antimalarial activity against Plasmodium falciparum with an IC50 value of 3.66 µg/mL. Compounds 5, 9, and 16 exhibited antituberculosis activity against Mycobacterium tuberculosis with MIC values of 50, 50, and 25 µg/mL, respectively.
Key words Toddalia asiatica · Rutaceae · coumarin · cytotoxicity · antimalarial Supporting information available online at http://www.thieme-connect.de/products
As part of our research on the phytochemistry of Thai medicinal plants, we have intensively evaluated biological activities of extracts and pure compounds [1, 2]. Our interest focuses on cyto-
toxicity, antimalarial, antifungal, and antituberculosis (anti-TB) properties [3]. We report herein the isolation and structural identification of 3 new coumarins and 12 known coumarins as well as an alkaloid from Toddalia asiatica (L) Lam. (Rutaceae). All compounds were investigated for cytotoxicity against KB, MCF-7, and NCI-H187 cell lines. Antimalarial activity against Plasmodium falciparum and anti-TB activity against Mycobacterium tuberculosis H37Ra were also evaluated. T. asiatica is a medicinal plant widely distributed in Thailand, India, and China as well as in south and tropical Africa. All parts of this plant show some therapeutic activities such as to treat chronic lumbago, scelalgia, colds, stomachache, and injuries from falls [4]. The fruits are used to treat malaria and cough, the roots to treat indigestion, and the leaves to treat lung diseases [5]. T. asiatica contains mainly coumarins, especially prenylated and geranylated, triterpenes, phenanthridine alkaloids, and also volatile oils [6–9]. Many compounds from this plant have shown anticancer activity against the U-937 cell line [10], as well as antidiabetic, antioxidant [11], and antibacterial activities [5].
Results and Discussion !
In this paper, three new coumarins 1–3 together with 13 known compounds were isolated from the crude EtOAc extract of T. asiatica bark. Thirteen known compounds included 4-O-geranylconiferyl aldehyde (4) [12], nelumol A (5) [13], phellopterin (6) [14], 5-methoxy-8-geranyloxylpsoralen (7) [15], artanin (8) [16], 8geranyloxy-5,7-dimethyloxycoumarin (9) [16], 5,7,8-trimethoxycoumarin (10) [16], leptodactylone (11) [17], toddalolactone (12) [18], toddalolactone methyl ether (13) [19], 1,2-secodihydromethylumbelliferone methyl ester (14) [20], toddalosin (15) [16], and norchelerythrine (16) [21] were discovered " Fig. 1). Compounds 4, 5, 7, 11 and 14 were found for the first (l time in this plant. Compound 1 was found as a pale yellow oil and assigned the molecular formula C21H26O6 by HRESIMS. The 1H and 13C NMR spec-
Fig. 1
Structure of compounds 1–16.
Phatchana R, Yenjai C. Cytotoxic Coumarins from …
Planta Med 2014; 80: 719–722
719
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Letters
Letters
Table 1
1
H NMR spectral data of compounds 1–3 (δ in ppm and J in Hz).
Table 2
13
C NMR spectral data of compounds 1–3 (δ in ppm and J in Hz).
Position
1
2
3
Position
1
2
3
3 4 6 8 1′ 2′ 4′ 5′ 6′ 8′ 9′ 10′ OCH3-5 OCH3-7 OH
6.14, d (9.6) 7.96 d (9.6) 6.32, s
6.13, d (9.6) 7.95, d (9.6) 6.32, s
6.22, d (9.6) 7.88, d (9.6)
4.59, d (7.2) 5.60, m 2.71, (6.0) 5.52, d (15.6) 5.61, d (15.6) 1.31, s 1.31, s 1.61, s 3.90 3.95 1.71
4.58, d (7.2) 5.57, m 2.68, d (6.0) 5.55, d (12.0) 5.60, d (12.0) 1.29, s 1.29, s 1.63, s 3.89 3.94 1.73
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ OCH3-5 OCH3-7
161.1 111.4 138.9 152.5 91.6 156.7 128.9 149.3 104.1 70.0 121.1 140.7 42.4 128.8 135.7 82.2 24.4 24.4 16.7 56.2 56.7
161.0 111.3 138.9 152.5 91.5 156.7 128.9 149.3 104.0 70.1 120.8 141.1 42.2 124.2 140.3 70.7 29.9 29.9 16.6 56.1 56.6
161.0 112.8 138.9 155.7 116.6 161.6 95.6 154.9 107.6 118.3 136.9 143.0 117.8 18.3
6.59, s 6.62, d (16.8) 7.23, d (16.8) 5.09, 5.08, s 1.97, s
3.76, s 3.89, s
" Table 1 and 2) showed a 5,7-dimethoxy-8-substituted tra of 1 (l coumarin unit as in co-occurring compound 9. The methylene protons at δ 4.59 (2H, d, J = 7.2 Hz, H-1′) correlated with C-8 (δ 128.9), C-2′ (δ 121.1), and C-3′ (δ 140.7) in the HMBC experiment. In this experiment, the methylene protons at δ 2.71 (2H, d, J = 6.0 Hz, H-4′) correlated with C-2′ (δ 121.1), C-3′ (δ 140.7), C-5′ (δ 128.8), and C-6′ (δ 135.7). Two methyl groups (CH3-8′ and 9′) showed singlet signals at δ 1.31 and correlated with C-6′ (δ 135.7) and C-7′ (δ 82.2) in the HMBC spectrum. In the 1H NMR spectrum, the coupling constant of H-5′ (δ 5.52) and 6′ (δ 5.61) exhibited 15.6 Hz indicating 5′,6′-trans geometry. In addition, the carbon at δ 82.2 was assigned as C-7′ which connected with the hydroxyl group. Furthermore, the NOESY experiment revealed a correlation between methylene protons at H-1′ and methyl protons at H-10′ (δ 1.61, s, 3H) which indicated the E geometry of a 2′,3′ double bond. Therefore, compound 1 was identified as 8-(3′,7′-dimethyl-7′-hydroxy-2′E,5′E-octadienyl)oxy-5,7dimethoxycoumarin. Compound 2 was a pale yellow oil, assigned the molecular formula C21H26O6 by HRESIMS. The 1H NMR spectrum of this com" Table 2) closely resembled that of 1, except for the coupound (l pling constant of H-5′ and 6′ (J = 12.8 Hz), which indicated cis ge" Table 2) was also similar to ometry. The 13C NMR spectrum (l that of 1, but the signals of C-5′, C-6′, C-7′, and 8′/9′ were shifted compared to those of 1. The HMBC correlations of this compound were nearly the same as 1. Two protons at δ 5.55 (d, J = 12 Hz) and δ 5.60 (d, J = 12 Hz) were assigned as H-5′ and H-6′, respectively, which indicated the cis geometry of the 5′,6′ double bond. Therefore, compound 2 was identified as 8-(3′,7′-dimethyl-7′-hydroxy2′E,5′Z-octadienyl)oxy-5,7-dimethoxycoumarin. Compound 3 was a white solid, assigned the molecular formula " Table 1 C16H16O4 by HRESIMS. The 1H and 13C NMR spectra (l and 2) indicated that this compound had a coumarin core structure similar to co-occurring compound 12. The coupling constants of two doublet protons at δ 6.62 (J = 16.8, H-1′) and 7.23 (J = 16.8, H-2′) indicated a trans geometry double bond and connected with carbons at δ 118.3 (C-1′) and 136.9 (C-2′), respectively, in the HMQC experiment. Correlations between H-1′ with C-5 (δ 155.7), C-7 (δ 161.6), and C-3′ (δ 143.0) in the HMBC spectrum confirmed the connection of an isoprene unit at the C-6 (δ 116.6) position. Terminal olefinic protons H-4′ (δ 5.09 and 5.08, s) correlated with methylenecarbon at δ 117.8 in the HMQC experiment.
Phatchana R, Yenjai C. Cytotoxic Coumarins from … Planta Med 2014; 80: 719–722
62.0 56.3
These protons displayed correlation with C-2′ (δ 136.9) and C-5′ (δ 18.3) in the HMBC spectrum. Furthermore, the NOESY experiment revealed a correlation between H-1′ and H-5′ which indicated that these conjugated double bonds prefer a transoid conformation. From these data, compound 3 was identified as 6-(3′methyl-1′,3′-butadienyl)-5,7-dimethoxycoumarin. Compounds 1 and 2 showed cytotoxicity against the MCF-7 cell line with IC50 values of 28.23 and 24.80 µg/mL, respectively " Table 3). These two compounds showed cytotoxicity against (l the NCI-H187 cell line with IC50 values of 6.42 and 24.46 µg/mL, respectively. The results suggest that a trans double bond at C5′/6′ is required for activity against the NCI-H187 cell line. Compounds 4 and 5 showed cytotoxic activity against all cell lines tested. Compound 4 exhibited the strongest cytotoxic activity against the MCF-7 cell line with an IC50 value of 3.17 µg/mL. The results show convincingly that an aldehyde group seems to have an important role against the MCF-7 cell line. From the literature search, 4 and 5 showed cytotoxicity against six human cancer cell lines including U373, OE21, A549, PC-3, SKMEL−28, and LoVo with IC50 mean values of 16 and 47 µg/mL, respectively [22]. Furanocoumarin 6 demonstrated cytotoxicity against the MCF-7 cell line (IC50 = 23.20 µg/mL), while furanocoumarin 7 displayed cytotoxicity against the other cell lines (KB and NCI-H187) with IC50 values around 10 µg/mL. The results suggest that the isopentenyl group is required for activity against the MCF-7 cell line but the geranyl group is essential against KB and NCI-H187 cell lines. Kimura and coworkers reported that 6 exhibited cytotoxicity against B16F10 melanoma cells with an IC50 value of 0.52 µg/mL [23]. In the cases of 8 and 9, compound 9 exhibited stronger cytotoxicity against all cell lines (IC50 ≈ 8 µg/mL), while 8 showed good activity only against the NCI-H187 (IC50 = 7.41 µg/mL). This suggests that the geranyl group at the C8 position is favorable against all cell lines. It was reported that 8 showed cytotoxicity against leukemic cells (U-937) with an IC50 value of 177.3 µM [24]. In addition, all compounds were evaluated for anti-TB activity against M. tuberculosis H37Ra and antimalarial activity against P. falciparum (K1, multidrug resistant strain). Compounds 5, 9, and
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720
Letters
1 2 3 4 5 6 7 8 9 11 14 16 10, 12–13, 15 Ellipticine Doxorubicin
Cytotoxicity (IC50, µg/mL)* KB
MCF-7
NCI-H187
28.12 inactivea inactivea 23.68 15.50 inactivea 12.34 31.02 8.63 inactivea inactivea inactivea inactivea 1.27 –
28.23 24.80 inactivea 3.17 17.82 23.20 inactivea 25.44 9.79 inactivea inactivea 23.20 inactivea 0.44 2.84
6.42 24.46 9.58 10.61 23.09 inactivea 10.53 7.41 7.97 29.68 19.45 inactivea inactivea 0.684 –
Table 3 Cytotoxicity of all compounds.
* Data shown are from triplicate experiments; a inactive at > 50 µg/mL
16 showed anti-TB activity with MIC values of 50, 50, and 25 µg/ mL, respectively. Fortunately, only compound 14 displayed antimalarial activity by showing an IC50 value of 3.66 µg/mL. It was reported that 5,7-dimethoxy-8-(3′-hydroxy-3′-methyl-1′-butene)-coumarin isolated from this plant displayed moderate antimalarial activity against P. falciparum K39 and V1 S strains with IC50 values of 16.2 and 8.8 µg/mL, respectively [25].
Materials and Methods !
General experimental procedures: Melting points were determined on a Sanyo Gallenkamp melting point apparatus and are uncorrected. UV spectra were measured on an Agilent 8453 UVVisible spectrophotometer. IR spectra were recorded as KBr disks or thin films, using Perkin Elmer Spectrum One FT‑IR spectrophotometer. The NMR spectra were recorded on a Varian Mercury plus spectrometer operating at 400 MHz (1H) and at 100 MHz (13C). Mass spectra were determined on a Micromass Q‑TOF 2 hybrid quadrupole time-of-flight (Q‑TOF) mass spectrometer with a Z-spray ES source (Micromass). Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 TLC aluminium sheet. Column chromatography was done with silica gel 0.063–0.200 mm or less than 0.063 mm. Preparative layer chromatography (PLC) was carried out on glass supported silica gel plates using silica gel 60 PF254 for preparative layer chromatography. All solvents were routinely distilled prior to use. Plant material: The bark of T. asiatica was collected in April 2011 from Phetchabun Province. The plant was identified by Dr. Pranom Chantaranothai, Faculty of Science, Khon Kaen University. A botanically identified voucher specimen (KKU0042011) was deposited at the herbarium of the Department of Chemistry, Faculty of Science, Khon Kaen University. Extraction and isolation: Air-dried and finely powdered stem bark (2.9 kg) of T. asiatica was sequentially extracted at room temperature for three days with hexane (2 × 3 L), EtOAc (2 × 3 L), and MeOH (2 × 3 L). The extracts were evaporated in vacuo to obtain three dry extracts, crude hexane (69 g), EtOAc (89 g), and crude MeOH (121 g). The crude EtOAc extract (89.7 g) was subjected to column chromatography on silica gel 60 and subsequently eluted with a gradient of three solvents (hexanes, EtOAc,
and MeOH) by gradually increasing the polarity of the elution solvents system to isolate pure compounds 1–16 (purity ≥ 95%, see Supporting Information). Bioassays: Cytotoxicity assay: Cytotoxicity assays against human epidermoid carcinoma (KB, ATCC CCL−17), breast adenocarcinoma (MCF-7, ATCC HTB−22) and human small cell lung cancer (NCI-H187, ATCC CRL−5804) cell lines were performed employing resazurin microplate assay (REMA; Sigma-Aldrich, dye content 75 %) [26], while cytotoxicity assay against Vero cells (African green monkey kidney, ATCC CCL−81) was performed by green fluorescent protein (GFP)-based assay [27]. Ellipticine (Fluka, purity ≥ 99 %) and doxorubicin (Fluka, purity ≥ 98%) were included as the reference substances. Antimalarial assay: Antimalarial activity was performed against P. falciparum (K1, multidrug resistant strain) which was cultured continuously according to the method of Trager and Jensen [28]. Quantitative assessment of antimalarial activity in vitro was determined by means of the microdilution radioisotope technique based on the method described by Desjardins [29]. The inhibitory concentration was that which caused 50% reduction in parasite growth as indicated by the in vitro uptake of [3H] hypoxanthine by P. falciparum. The standard compound was dihydroartemisinin (Sigma, purity ≥ 97 %). Antimycobacterial assay: The antimycobacterial activity was performed against M. tuberculosis H37Ra using the green fluorescent protein microplate assay (GFPMA) [30]. The standard drugs isoniazid (Fluka, purity ≥ 99%) and rifampicin (Fluka, purity ≥ 97 %) were used as the reference compounds. Spectroscopic data of compounds: Compound 1: Pale yellow oil; UV (CH3CN) λmax (log ε) 262 (4.20), 318 (4.20) nm; IR (KBr) νmax 3436, 2944, 1724, 1604, 1505, 1438, 1344, 1150, 1120, 992, 754 cm−1; HRESIMS m/z 397.1620 [M + Na]+ (calcd. 397.1619); 1 " Table 1 and 2. H and 13C NMR spectroscopic data, see l Compound 2: Pale yellow oil; UV (CH3CN) λmax (log ε) 262 (4.20), 320 (4.24) nm; IR (KBr) νmax 3404, 3017, 2945, 1720, 1604, 1505, 1344, 1216, 1150, 1121, 752 cm−1; HRESIMS m/z 397.1629 [M + Na]+ (calcd. 397.1619); 1H and 13C NMR spectroscopic data, see " Table 1 and 2. l Compound 3: White solid; mp. 103–104 °C, UV (CH3CN) λmax (log ε) 287 (4.54), 335 (4.04) nm; IR (KBr) νmax 2959, 1727, 1611,
Phatchana R, Yenjai C. Cytotoxic Coumarins from …
Planta Med 2014; 80: 719–722
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Compound
721
Letters 1386, 1203, 1141, 1088, 973, 827 cm−1; HRESIMS m/z 273.1132 [M + H]+ (calcd. 273.1128); 1H and 13C NMR spectroscopic data, " Table 1 and 2. see l
Supporting information Detailed protocols for extraction and isolation as well as 1D and 2D NMR spectra of compounds 1–3 are available as Supporting Information.
Acknowledgements !
We thank the Rajamangala University of Technology Isan, Khon Kaen Campus, for financial support to R. Phatchana and the Bioassay Laboratory of the National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand, for biological activity assays. The National Research University Project of Thailand through the Advanced Functional Materials Cluster of Khon Kaen University is also gratefully acknowledged.
Conflict of Interest !
There are no conflicts of interest of all authors with respect to this work.
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received revised accepted
June 12, 2013 April 29, 2014 May 11, 2014
Bibliography DOI http://dx.doi.org/10.1055/s-0034-1368568 Published online June 17, 2014 Planta Med 2014; 80: 719–722 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Chavi Yenjai Natural Products Research Unit Department of Chemistry and Center of Excellence for Innovation in Chemistry Faculty of Science, Khon Kaen University Khon Kaen 40002 Thailand Phone: + 66 43 20 22 22 41 ext. 1 22 43 Fax: + 66 43 20 23 73 [email protected]
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