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Cytotoxic Compounds from the Leaves of Garcinia polyantha by Alain Meli Lannang* a ) b ), Simplice J. N. Tatsimo a ), Hugues Fouotsa c ), Jean Paul Dzoyem d ), Ajit Kumar Saxena e ), and Norbert Sewald b ) a
) Department of Organic Chemistry, Higher Teachers Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon (phone: þ 237-77-534830; fax: þ 237-22-229116; e-mail: [email protected]) b ) Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, D-33501 Bielefeld c ) Department of Organic Chemistry, Faculty of Science, University of Yaounde´ I, P.O. Box 812, Yaounde´, Cameroon d ) Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon e ) Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu, India
A new compound, named banganxanthone C ( ¼ 12-(1,1-dimethylprop-2-en-1-yl)-5,10-dihydroxy-9methoxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one; 4), together with five known compounds, were isolated from the leaves of Garcinia polyantha. The structures of the compounds were elucidated on the basis of 1D- and 2D-NMR spectroscopy. Among the known compounds, two were xanthones, one was a pentacyclic triterpene, one sterol, and one benzophenone derivative. Isoxanthochymol (2) and 4-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-1,5,8-trihydroxy-3-methoxy-9H-xanthen-9-one (3) exhibited significant antiproliferative activity against the leukemia cell line TPH-1 with IC50 inhibition values of 1.5 and 2.8 mg/ml, respectively. The cytotoxic activity was found to be related to apoptosis induction.
Introduction. – Plants of the genus Garcinia (Guttiferae), widely distributed in tropical Africa, Asia, New Caledonia, and Polynesia, have yielded a plethora of biologically active and structurally intriguing natural products [1]. Garcinia species are known to contain a wide variety of O-bearing and prenylated xanthones, as well as polyisoprenylated benzophenones such as the guttiferones [2]. Xanthones possess a wide range of biological and pharmacological properties including antioxidant, antiinflammatory, antimicrobial, and cytotoxic activities, as well as cholinesterase and aglycosidase inhibition [2 – 4]. Guttiferones have been reported as anti-HIV, trypanocidal, and cytotoxic agents [5 – 7]. These two groups of compounds are probably responsible for the therapeutic potential of the Garcinia species, justifying their various uses in traditional medicine. For example, in West and Central Africa, the stem bark of Garcinia polyantha is used against female infertility, skin diseases, and as antidote and purgative. Its resinous sap is used for wound healing [8]. This wide range of biological and traditional uses prompted structureactivity relationship and cytotoxic studies. As part of our ongoing research program on the identification of bioactive constituents from the plants of the genus Garcinia, we investigated the petroleum ether extract of the leaves of Garcinia polyantha. Herein, we describe the isolation and characterization of six compounds, 1 – 6, i.e., of one new xanthone 4, together with two known xanthones 2014 Verlag Helvetica Chimica Acta AG, Zrich
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Fig. 1. Structures of compounds 1 – 6
3 and 5, one pentacyclic triterpene 1, one sterol 6, and one benzophenone derivative 2 (Fig. 1). We also report on their activities against five human tumor cell lines (A549, MCF-7, PC-3, HeLa, and THP-1) evaluated by a sulforhodamine B (SRB) assay. Results and Discussion. – The petroleum ether extract of the leaves of G. polyantha was separated by column chromatography on silica gel to give a new xanthone derivative named banganxanthone C (4), along with the five known compounds, echinocystic acid (1) [9], isoxanthochymol (2) [7], 4-[(2E)-3,7-dimethylocta-2,6-dien1-yl]-1,5,8-trihydroxy-3-methoxy-9H-xanthen-9-one (3) [1], 2-hydroxy-1,7-dimethoxyxanthone (5) [1], and b-sitosterol (6) [10]. The molecular formula of compound 4, isolated as optically active yellow powder, was deduced as C29H32O6 from the HR-MS data. Both the 1H- and 13C-NMR spectra of 4 (Table 1) revealed the presence of a chromene ring system indicated by an AB Hatom system (d(H) 6.76 (AB, J ¼ 10.1, HC(4)) and 5.69 (AB, J ¼ 10.1, HC(3))) further supported by the signals at d(C) 117.0 (C(4)), 127.0 (C(3)), 82.0 (C(2)), and 24.1 (C(7’)). The lack of a Me signal corresponding to the 2,2-dimethylchromene system, and the appearance of the CH2 signals at d(C) 42.3 (C(1’)) and 25.9 (C(2’)), two olefinic signals at d(C) 132.2 (C(4’)) and 124.9 (C(3’)), and two Me signals at 17.8 (C(5’)) and 25.9 (C(6’)) suggested that one Me group was replaced by a 4-methylpent-3-en-1-yl group. This was supported by the base peak at m/z 393 (100, [M 83] þ ) resulting from
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Table 1. 1H- and 13C-NMR Data for Compound 4 in ( D6 )Acetone (500 and 125 MHz; respectively, d in ppm, J in Hz) Position 1 2 3 4 4a 5 5a 6 6a 7 8 9 10 10a 11a 12
d( H )
5.69 ( AB, J ¼ 10.1) 6.76 ( AB, J ¼ 10.1)
7.83 (d, J ¼ 8.8) 7.05 (d, J ¼ 8.8)
d(C ) 82.0 127.0 117.0 105.6 157.5 114.8 181.8 103.7 122.1 114.8 160.2 136.0 151.5 156.2 114.6
Position 12a 1’ 2’ 3’ 4’ 5’ 6’ 7’ 1’’ 2’’ 3’’ 4’’ 5’’ 5-OH 10-MeO
d( H ) 1.75 – 1.90, 1.70 – 1.76 (2m) 1.98 – 2.17 (m) 5.14 (br. t) 1.65 (s) 1.44 (s) 1.59 (s) 6.40 (dd, J ¼ 10.6, 17.6) 4.86 (dd, J ¼ 1.1, 10.6), 4.96 (dd, J ¼ 1.1, 17.6) 1.76 (s) 1.78 (s) 13.89 (s) 3.95 (s)
d(C ) 157.4 42.3 25.9 124.9 132.2 17.8 25.9 24.1 41.9 151.8 108.4 27.6 27.2 62.0
the loss of the 4-methylpent-3-en-1-yl group through scission of the C(2)C(1’) bond. The substitution pattern of the chromene ring was confirmed by HMBC experiment (Fig. 2), in which the HC(4) signal at d(H) 6.76 correlated with those of the C-atoms at d(C) 82.0 (C(2)), 157.4 (C(12a)), and 157.5 (C(5)), while the HC(3) signal at d(H) 5.69 correlated with those at d(C) 105.6 (C(4a)), 24.1 (C(7’)), 82.0 (C(2)), and 42.3 (C(1’)). Further analyses indicated the presence of a conjugated C¼O and a chelated OH group with the signal at d(C) 181.9 (C(6)) and d(H) 13.89 (HOC(5)), respectively. The long-range correlations between the H-atom signal at 13.89 (HOC(5) with those at d(C) 157.5 (C(5)), 105.6 (C(4a)), and 114.8 (C(5a)) allowed an unequivocal assignment of ring A as in pruniflorones G and H [11]. Signals attributed to ring B were also similar to those found previously in pruniflorones A and B, except that signals of a MeO group and O-bearing C-atoms were observed at d(C) 62.0, 136.0, and 160.2, respectively. The MeO group was attached to C(10) (d(C) 136.0) according to the HMBC experiment, implying that both O-bearing C-atoms were in ortho-position within ring A. The ortho-coupled H-atom signals at d(H) 7.83 and 7.05 were located at C(7) and C(8), respectively, according to the HMBCs (Fig. 2). Thus, compound 4 was characterized as 12-(1,1-dimethylprop-2-en-1-yl)-5,10-dihydroxy-9-methoxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen6-one, named banganxanthone C. The cytotoxic activities of compounds 1 – 4 were evaluated with a panel of five human cancer cell lines including A549, MCF-7, PC-3, HeLa, and THP-1 (Table 2). Compounds 2 and 3 had a strong inhibitory effect on THP-1 cell proliferation with IC50 inhibition values of 1.5 and 2.8 mg/ml, respectively. On the other hand, only compound 4 showed a weak inhibitory effect on the MCF-7 cell line. Unlike compounds 2 and 3, 1 and 4 displayed weak growth inhibition effects. Our findings that isoxanthochymol (2) and 4-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-1,5,8-trihydroxy-3-methoxy-9H-xanthen-
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Fig. 2. Selected HMBCs of 4
Table 2. IC50 Values of Compounds 1 – 4 on Human Cancer Cell Lines [mg/ml] Compound
Cell lines HeLa (Cervix) A549 ( Lung) PC-3 ( Prostate) THP-1 ( Leukemia) MCF-7 ( Breast)
1 nd a ) 2 10.09 0.76 3 8.10 1.41 4 – Paclitaxel c ) 12.00 1.10 a
– b) 21.04 1.83 – 40.06 2.79 1.03 0.05
– – 22.01 2.56 – –
40.02 3.38 1.52 0.28 2.80 0.65 31.04 2.37 4.02 0.04
– – – 24.77 1.18 3.07 0.77
) nd, Not determined. b ) –, > 50 mg/ml. c ) Positive control.
9-one (3) have an interesting antiproliferative effect on cancer cells are in agreement with previous studies demonstrating that benzophenone derivatives and xanthones inhibit the proliferation of tumor cell lines [12]. Furthermore, Garcinia species have been reported as sources of xanthones with useful biological properties including cytotoxic activity [13]. DNA Content assays were performed to assess whether the cytotoxic effects of compounds 2 and 3 on THP-1 might be mediated by apoptosis. THP-1 Cells were treated with compounds 2 and 3 for 24 h at concentrations of 10 and 20 mg/ml, respectively, the DNA content of cells was measured by propidium iodide (PI) staining, and flow cytometry analysis was performed to detect apoptotic cells. The apoptotic cells can be observed on a DNA histogram as subdiploid or sub-G1 peak (Fig. 3). The appearance of a sub-diploid DNA peak is a specific marker of apoptosis; necrosis induced by metabolic poisons, or lysis produced by complement did not induce any subG1 peak in the DNA fluorescence histogram [14]. Thus, compounds 2 and 3 induced apoptosis in the leukemia cell line THP-1. Matsumoto et al. [15] previously reported the cytotoxic effect of benzophenone derivatives from Garcinia species with a strong apoptosis-inducing effect against the human leukemia cell line HL60. In this study, we have demonstrated that isoxanthochymol (2) and 4-[(2E)-3,7-dimethylocta-2,6-dien-1yl]-1,5,8-trihydroxy-3-methoxy-9H-xanthen-9-one (3) exhibited cytotoxic effects on different human cancer cells to an extent comparable with that of paclitaxel. Flow cytometry analysis clearly revealed that cytotoxic activity might be related to apoptosis induction. These results indicate that compounds 2 and 3 are potential candidates for the development as therapeutic agents against blood cancer.
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Fig. 3. Effects of compounds 2 and 3 on apoptosis in THP-1 cells. Cells were treated with compounds at the indicated concentrations for 24 h. Plots show Sub-G1 peaks with the percentages of apoptotic population cell.
Conclusions. – In this work, we elucidated the structures of the compounds 1 – 6 isolated from G. polyantha. The isolation and identification of one of them was accomplished for the first time. The cytotoxicity results provided baseline information for the possible use of compounds 1 – 4 for the control of cancer diseases and, especially, leukemia. We are grateful to Alexander von Humboldt-Stiftung for having provided a Georg Forster Fellowship for Experienced Researchers (ID Nr: 3.4-KAM/1137675) to A. M. L.
Experimental Part General. Column chromatography (CC): silica gel 60 (SiO2 ; 70 – 230 and 240 – 300 mesh; E. Merck). TLC: Precoated SiO2 plates (E. Merck, F254 ); visualization by spraying with Ce(SO4 )2 reagent. Optical
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rotations: in MeOH soln.; JASCO digital polarimeter (model DIP-3600). IR Spectra: JASCO A-302 IR spectrophotometer, in CHCl3 ; ˜n in cm 1. 1H- and 13C-, and 2D-NMR spectra: Bruker AMX-500 spectrometer with (D6 )acetone as solvent; d [ppm] relative to the residual (D6 )acetone signal at d(H) 2.05, and d(C) 29.84 and 206.26; J in Hz. Homonuclear 1H,1H connectivities were determined by the COSY 458 experiment. One-bond 1H,13C connectivities were determined by HMQC. Two- and threebond 1H,13C connectivities were determined by HMBC. EI-MS: Varian MAT 311A double-focusing mass spectrometer; in m/z (rel. %). HR-EI-MS: JEOL HX 110 mass spectrometer; in m/z. Plant Material. The leaves of G. polyantha were collected in Yaounde´, central region, Cameroon, in February 2009, and identified by Mr. Victor Nana from Cameroon National Herbarium where a voucher specimen (21337/SRF/Cam/Mt Kala) has been deposited. Extraction and Isolation. The dried and powdered leaves of G. polyantha (2.0 kg) were extracted at r.t. with MeOH (3 20 l) for 3 d. The extract was concentrated and suspended in H2O, followed by successive partition with petroleum ether (PE), CH2Cl2 , AcOEt, and BuOH, resp. The PE extract (10.8 g) was subjected to flash column chromatography (FC; SiO2 (10 40 cm, 200.0 g); PE/AcOEt 20 : 1 – 3 : 1 (3 l each)) to afford six fractions, Frs. 1 – 6. Fr. 3 (2.2 g) was subjected to FC (SiO2 (4 30 cm, 20.0 g); PE/AcOEt 10 : 1 to 3 : 1 (100 ml each)) to give six subfractions, Subfrs. 4a – 4f, and compound 1 (20 mg) was obtained from Subfr. 4a. Subfr. 4d (150.0 mg) was subjected to CC (SiO2 (5 40 cm, 50.0 g); PE/AcOEt 20 : 1 to 3 : 1 (50 ml each)) to afford compounds 2 (10 mg), 3 (6 mg), and 4 (25 mg). Fr. 5 (1.3 g) was subjected to CC (SiO2 (5.5 30 cm, 70.0 g); PE/AcOEt 4 : 1 to 1 : 1 (100 ml each)) to give five subfractions, Subfrs. 5a – 5e. Subfr. 5a (70.0 mg) was repeatedly purified by CC (SiO2 (2 45 cm, 20.0 g); PE/AcOEt 7 : 1) to yield compound 3 (11.0 mg). Subfr. 5b (74 mg) was separated by CC (SiO2 (3 30 cm, 20.0 g); PE/AcOEt 4 : 1) to yield compound 4 (10.6 mg). Subfr. 5c (55.2 mg) was purified by CC (SiO2 (1.2 34 cm, 9.0 g); PE/AcOEt 5 : 1) to yield compound 5 (3.8 mg). Fr. 6 (1.7 g) was separated by CC (SiO2 (4 30 cm, 32 g); PE/AcOEt 4 : 1 to 1 : 1 (2 l each)) to afford two subfractions, Subfrs. 6a and 6b. Subfr. 6b (75.7 mg) was gel-filtered over Sephadex LH-20 (2 100 cm; CHCl3/MeOH 1 : 1) to yield compounds 5 (4.9 mg) and 6 (4.1 mg). All isolated compounds were more than 97% pure. Banganxanthone C ( ¼ 12-(1,1-Dimethylprop-2-en-1-yl)-5,10-dihydroxy-9-methoxy-2-methyl-2-(4methylpent-3-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one; 4). Yellow amorphous powder. [a] 25 D ¼ þ 56 (c ¼ 3.5 10 4, CHCl3 ). M.p. 106.5 – 107.58. UV (CHCl3 ): 250 (3.96), 275 (4.35), 320 (4.00). IR (CHCl3 ): 3400, 1603, 1583. 1H- and 13C-NMR: see Table 1. EI-MS: 476 (18, M þ ), 461 (11), 394 (25), 393 (100), 339 (20), 325 (4). HR-EI-MS: 476.2181 (M þ , C29H32O þ6 ; calc. 476.2193). Bioassay. The cytotoxic activities of compounds 1 – 4 were determined against five human tumor cells including lung A549 adenocarcinoma, breast carcinoma MCF-7, prostate carcinoma PC-3, cervical carcinoma HeLa, and acute monocytic leukemia THP-1 cell lines, using sulforhodamine B (SRB) assay as described in [14]. Briefly, cells were harvested in log phase using trypsin (0.05% trypsin, 0.02% EDTA, in phosphate buffer saline (PBS)). The cell suspensions were diluted with appropriate growth medium to obtain the cell density of 104 cells/well. Aliquots of 100 ml of each suspension were seeded in 96-wells cell culture plates. The cells were incubated at 378 in an atmosphere of 5% CO2 and 95% relative humidity in a CO2 incubator. After 24 h of incubation, test materials (100 ml/well) at different concentrations (1, 10, 30, and 50 or 100 mg/ml) were added to the wells containing cells. Paclitaxel (Sigma Chem. Co., USA, purity 95%; 0.1, 1.0, and 10 mm) was used as positive reference. Suitable controls with equivalent concentration of DMSO were also included. The plates were further incubated for 48 h in a CO2 incubator after addition of test material. After incubation, cells were fixed by gently layering CCl3COOH (50 ml/well, 50% w/v) on top of the medium in all the wells and incubated at 48 for 1 h. The plates were washed five times with dist. H2O and air-dried. Cell growth was measured by staining with SRB dye (0.4% (w/v) in 1% AcOH, 100 ml/well). The unbound dye was washed 3 – 5 times with 1% AcOH, and plates were air-dried. The adsorbed dye was dissolved in Tris buffer (100 ml/well, 0.01m, pH 10.4), and the plates were gently shaken for 10 min on a mechanical shaker. The optical density (OD) was recorded using a 96-well plate reader. Growth inhibition was calculated by subtracting mean OD values of respective blank from the mean OD value of experimental set. Percentage growth in presence of test material was calculated considering the growth in absence of any test material as 100%, and in turn percentage growth inhibition in presence of test material was calculated. The viability and growth in the presence of test material was calculated by the following formula:
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Growth inhibition [%] ¼ 100 [OD(test sample) OD(blank) /OD(control) OD(blank) ] 100 IC50 Value is the concentration of sample required to inhibit 50% of the cell proliferation and was calculated by plotting the percentage survival vs. the concentrations, using Microsoft Excel. For all samples, each compound concentration was tested thrice in triplicates. Compounds 5 and 6 were not tested due to their small quantity. DNA Content Analysis. Sub-G1 DNA Content analysis was performed as described in [16]. Briefly, THP-1 cells (106/ml) in exponential phase of growth were seeded in 24-well tissue culture plates and allowed to adhere for 24 h. On day 2, the old medium was changed, and the cells were treated with extract/compounds at different concentrations and further incubated for 24 h. Cells were trypsinized, centrifuged, and washed with PBS, then fixed gently by adding 70% EtOH. They were fixed in 70% EtOH, washed with PBS, and then successively treated with RNase and with propidium iodide (PI; 25 mg/ ml) and incubated at 378 for 30 min. The percentages of DNA content in sub-G1 cells population were measured using BD-LSR flow cytometer equipped with electronic doublet discrimination capability using blue (488 nm) excitation from Ar laser. Data were collected in list mode on 10,000 events for FL2A vs. FL2-W. Apoptotic nuclei appear as a broad hypodiploid DNA peak at lower fluorescence intensity compared to nuclei in G0/G1 phase.
REFERENCES [1] A. Meli Lannang, G. N. Louh, B. M. Biloa, J. Komguem, C. D. Mbazoa, B. L. Sondengam, L. Naesens, C. Pannecouque, E. De Clercq, E. S. H. El Ashry, Planta Med. 2010, 76, 708. [2] H. Fouotsa, A. Meli Lannang, C. D. Mbazoa, S. Rasheed, B. P. Marasini, Z. Ali, K. P. Devkota, A. E. Kengfack, F. Shaheen, M. I. Choudhary, S. Sewald, Phytochemistry Lett. 2012, 5, 236. [3] A. Meli Lannang, B. S. Noudou, S. Sewald, Phytochemistry Lett. 2013, 6, 157. [4] A. S. Ampofo, G. P. Waterman, Phytochemistry 1986, 25, 2351. [5] Nilar, L. H. D. Nguyen, G. Venkatraman, K. Y. Sim, L. J. Harrison, Phytochemistry 2005, 66, 1718. [6] N. G. Louh, A. Meli Lannang, D. C. Mbazoa, J. G. Tangmouo, J. Komguem, P. Castillo, N. F. Mofo, Q. Naz, D. Lontsi, M. C. Iqbal, B. L. Sondengam, Phytochemistry 2008, 69, 1013. [7] K. R. Gustafson, J. W. Blunt, M. H. G. Munro, R. W. Fuller, T. C. McKee, J. H. Cardellina II, J. B. McMahon, G. M. Cragg, M. R. Boyd, Tetrahedron 1992, 48, 10093. [8] A. Bouquet, Feticheurs et Medecine traditionnelles du Congo (Brazzaville), O.R.ST.O.M., Paris, 1969. [9] Y. Shao, O. Poobrasert, C. T. Ho, C. K. Chin, G. A. Cordell, Phytochemistry 1996, 43, 195. [10] A. F. Kamdem Waffo, D. Mulholland, J. D. Wansi, L. M. Mbaze, R. Powo, T. N. Mpondo, Z. T. Fomum, W. Kçnig, A. E. Nkengfack, Chem. Pharm. Bull. 2006, 54, 448. [11] N. Boonnak, C. Karalai, S. Chantrapromma, C. Ponglimanont, H. K. Fun, A. Kanjana-Opas, Laphookhieo, Tetrahedron 2006, 62, 8850. [12] A. J. Hou, F. Toshio, S. Manabu, S. Hiroshi, H. D. Sun, N. Taro, J. Nat. Prod. 2001, 64, 65. [13] K. Matsumoto, Y. Akao, A. Kobayashi, T. Ito, K. Ohguchi, T. Tanaka, M. Iinuma, Y. Nozawa, Biol. Pharm. Bull. 2003, 26, 569. [14] I. Nicoletti, G. Migliorati, M. C. Pagliacci, F. Grignani, C. Riccardi, J. Immunol. Methods 1991, 139, 271. [15] P. Skehan, R. Storeng, D. Scudiero, J. Natl. Cancer Inst. 1990, 82, 1107. [16] W. H. Park, Y. W. Han, S. W. Kim, S. H. Kim, K. W. Cho, S. Z. Kim, Cancer Lett. 2007, 251, 68. Received September 18, 2013
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Cytotoxic Compounds from the Leaves of Garcinia polyantha by Alain Meli Lannang* a ) b ), Simplice J. N. Tatsimo a ), Hugues Fouotsa c ), Jean Paul Dzoyem d ), Ajit Kumar Saxena e ), and Norbert Sewald b ) a
) Department of Organic Chemistry, Higher Teachers Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon (phone: þ 237-77-534830; fax: þ 237-22-229116; e-mail: [email protected]) b ) Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, D-33501 Bielefeld c ) Department of Organic Chemistry, Faculty of Science, University of Yaounde´ I, P.O. Box 812, Yaounde´, Cameroon d ) Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon e ) Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu, India
A new compound, named banganxanthone C ( ¼ 12-(1,1-dimethylprop-2-en-1-yl)-5,10-dihydroxy-9methoxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one; 4), together with five known compounds, were isolated from the leaves of Garcinia polyantha. The structures of the compounds were elucidated on the basis of 1D- and 2D-NMR spectroscopy. Among the known compounds, two were xanthones, one was a pentacyclic triterpene, one sterol, and one benzophenone derivative. Isoxanthochymol (2) and 4-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-1,5,8-trihydroxy-3-methoxy-9H-xanthen-9-one (3) exhibited significant antiproliferative activity against the leukemia cell line TPH-1 with IC50 inhibition values of 1.5 and 2.8 mg/ml, respectively. The cytotoxic activity was found to be related to apoptosis induction.
Introduction. – Plants of the genus Garcinia (Guttiferae), widely distributed in tropical Africa, Asia, New Caledonia, and Polynesia, have yielded a plethora of biologically active and structurally intriguing natural products [1]. Garcinia species are known to contain a wide variety of O-bearing and prenylated xanthones, as well as polyisoprenylated benzophenones such as the guttiferones [2]. Xanthones possess a wide range of biological and pharmacological properties including antioxidant, antiinflammatory, antimicrobial, and cytotoxic activities, as well as cholinesterase and aglycosidase inhibition [2 – 4]. Guttiferones have been reported as anti-HIV, trypanocidal, and cytotoxic agents [5 – 7]. These two groups of compounds are probably responsible for the therapeutic potential of the Garcinia species, justifying their various uses in traditional medicine. For example, in West and Central Africa, the stem bark of Garcinia polyantha is used against female infertility, skin diseases, and as antidote and purgative. Its resinous sap is used for wound healing [8]. This wide range of biological and traditional uses prompted structureactivity relationship and cytotoxic studies. As part of our ongoing research program on the identification of bioactive constituents from the plants of the genus Garcinia, we investigated the petroleum ether extract of the leaves of Garcinia polyantha. Herein, we describe the isolation and characterization of six compounds, 1 – 6, i.e., of one new xanthone 4, together with two known xanthones 2014 Verlag Helvetica Chimica Acta AG, Zrich
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Fig. 1. Structures of compounds 1 – 6
3 and 5, one pentacyclic triterpene 1, one sterol 6, and one benzophenone derivative 2 (Fig. 1). We also report on their activities against five human tumor cell lines (A549, MCF-7, PC-3, HeLa, and THP-1) evaluated by a sulforhodamine B (SRB) assay. Results and Discussion. – The petroleum ether extract of the leaves of G. polyantha was separated by column chromatography on silica gel to give a new xanthone derivative named banganxanthone C (4), along with the five known compounds, echinocystic acid (1) [9], isoxanthochymol (2) [7], 4-[(2E)-3,7-dimethylocta-2,6-dien1-yl]-1,5,8-trihydroxy-3-methoxy-9H-xanthen-9-one (3) [1], 2-hydroxy-1,7-dimethoxyxanthone (5) [1], and b-sitosterol (6) [10]. The molecular formula of compound 4, isolated as optically active yellow powder, was deduced as C29H32O6 from the HR-MS data. Both the 1H- and 13C-NMR spectra of 4 (Table 1) revealed the presence of a chromene ring system indicated by an AB Hatom system (d(H) 6.76 (AB, J ¼ 10.1, HC(4)) and 5.69 (AB, J ¼ 10.1, HC(3))) further supported by the signals at d(C) 117.0 (C(4)), 127.0 (C(3)), 82.0 (C(2)), and 24.1 (C(7’)). The lack of a Me signal corresponding to the 2,2-dimethylchromene system, and the appearance of the CH2 signals at d(C) 42.3 (C(1’)) and 25.9 (C(2’)), two olefinic signals at d(C) 132.2 (C(4’)) and 124.9 (C(3’)), and two Me signals at 17.8 (C(5’)) and 25.9 (C(6’)) suggested that one Me group was replaced by a 4-methylpent-3-en-1-yl group. This was supported by the base peak at m/z 393 (100, [M 83] þ ) resulting from
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Table 1. 1H- and 13C-NMR Data for Compound 4 in ( D6 )Acetone (500 and 125 MHz; respectively, d in ppm, J in Hz) Position 1 2 3 4 4a 5 5a 6 6a 7 8 9 10 10a 11a 12
d( H )
5.69 ( AB, J ¼ 10.1) 6.76 ( AB, J ¼ 10.1)
7.83 (d, J ¼ 8.8) 7.05 (d, J ¼ 8.8)
d(C ) 82.0 127.0 117.0 105.6 157.5 114.8 181.8 103.7 122.1 114.8 160.2 136.0 151.5 156.2 114.6
Position 12a 1’ 2’ 3’ 4’ 5’ 6’ 7’ 1’’ 2’’ 3’’ 4’’ 5’’ 5-OH 10-MeO
d( H ) 1.75 – 1.90, 1.70 – 1.76 (2m) 1.98 – 2.17 (m) 5.14 (br. t) 1.65 (s) 1.44 (s) 1.59 (s) 6.40 (dd, J ¼ 10.6, 17.6) 4.86 (dd, J ¼ 1.1, 10.6), 4.96 (dd, J ¼ 1.1, 17.6) 1.76 (s) 1.78 (s) 13.89 (s) 3.95 (s)
d(C ) 157.4 42.3 25.9 124.9 132.2 17.8 25.9 24.1 41.9 151.8 108.4 27.6 27.2 62.0
the loss of the 4-methylpent-3-en-1-yl group through scission of the C(2)C(1’) bond. The substitution pattern of the chromene ring was confirmed by HMBC experiment (Fig. 2), in which the HC(4) signal at d(H) 6.76 correlated with those of the C-atoms at d(C) 82.0 (C(2)), 157.4 (C(12a)), and 157.5 (C(5)), while the HC(3) signal at d(H) 5.69 correlated with those at d(C) 105.6 (C(4a)), 24.1 (C(7’)), 82.0 (C(2)), and 42.3 (C(1’)). Further analyses indicated the presence of a conjugated C¼O and a chelated OH group with the signal at d(C) 181.9 (C(6)) and d(H) 13.89 (HOC(5)), respectively. The long-range correlations between the H-atom signal at 13.89 (HOC(5) with those at d(C) 157.5 (C(5)), 105.6 (C(4a)), and 114.8 (C(5a)) allowed an unequivocal assignment of ring A as in pruniflorones G and H [11]. Signals attributed to ring B were also similar to those found previously in pruniflorones A and B, except that signals of a MeO group and O-bearing C-atoms were observed at d(C) 62.0, 136.0, and 160.2, respectively. The MeO group was attached to C(10) (d(C) 136.0) according to the HMBC experiment, implying that both O-bearing C-atoms were in ortho-position within ring A. The ortho-coupled H-atom signals at d(H) 7.83 and 7.05 were located at C(7) and C(8), respectively, according to the HMBCs (Fig. 2). Thus, compound 4 was characterized as 12-(1,1-dimethylprop-2-en-1-yl)-5,10-dihydroxy-9-methoxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen6-one, named banganxanthone C. The cytotoxic activities of compounds 1 – 4 were evaluated with a panel of five human cancer cell lines including A549, MCF-7, PC-3, HeLa, and THP-1 (Table 2). Compounds 2 and 3 had a strong inhibitory effect on THP-1 cell proliferation with IC50 inhibition values of 1.5 and 2.8 mg/ml, respectively. On the other hand, only compound 4 showed a weak inhibitory effect on the MCF-7 cell line. Unlike compounds 2 and 3, 1 and 4 displayed weak growth inhibition effects. Our findings that isoxanthochymol (2) and 4-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-1,5,8-trihydroxy-3-methoxy-9H-xanthen-
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Fig. 2. Selected HMBCs of 4
Table 2. IC50 Values of Compounds 1 – 4 on Human Cancer Cell Lines [mg/ml] Compound
Cell lines HeLa (Cervix) A549 ( Lung) PC-3 ( Prostate) THP-1 ( Leukemia) MCF-7 ( Breast)
1 nd a ) 2 10.09 0.76 3 8.10 1.41 4 – Paclitaxel c ) 12.00 1.10 a
– b) 21.04 1.83 – 40.06 2.79 1.03 0.05
– – 22.01 2.56 – –
40.02 3.38 1.52 0.28 2.80 0.65 31.04 2.37 4.02 0.04
– – – 24.77 1.18 3.07 0.77
) nd, Not determined. b ) –, > 50 mg/ml. c ) Positive control.
9-one (3) have an interesting antiproliferative effect on cancer cells are in agreement with previous studies demonstrating that benzophenone derivatives and xanthones inhibit the proliferation of tumor cell lines [12]. Furthermore, Garcinia species have been reported as sources of xanthones with useful biological properties including cytotoxic activity [13]. DNA Content assays were performed to assess whether the cytotoxic effects of compounds 2 and 3 on THP-1 might be mediated by apoptosis. THP-1 Cells were treated with compounds 2 and 3 for 24 h at concentrations of 10 and 20 mg/ml, respectively, the DNA content of cells was measured by propidium iodide (PI) staining, and flow cytometry analysis was performed to detect apoptotic cells. The apoptotic cells can be observed on a DNA histogram as subdiploid or sub-G1 peak (Fig. 3). The appearance of a sub-diploid DNA peak is a specific marker of apoptosis; necrosis induced by metabolic poisons, or lysis produced by complement did not induce any subG1 peak in the DNA fluorescence histogram [14]. Thus, compounds 2 and 3 induced apoptosis in the leukemia cell line THP-1. Matsumoto et al. [15] previously reported the cytotoxic effect of benzophenone derivatives from Garcinia species with a strong apoptosis-inducing effect against the human leukemia cell line HL60. In this study, we have demonstrated that isoxanthochymol (2) and 4-[(2E)-3,7-dimethylocta-2,6-dien-1yl]-1,5,8-trihydroxy-3-methoxy-9H-xanthen-9-one (3) exhibited cytotoxic effects on different human cancer cells to an extent comparable with that of paclitaxel. Flow cytometry analysis clearly revealed that cytotoxic activity might be related to apoptosis induction. These results indicate that compounds 2 and 3 are potential candidates for the development as therapeutic agents against blood cancer.
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Fig. 3. Effects of compounds 2 and 3 on apoptosis in THP-1 cells. Cells were treated with compounds at the indicated concentrations for 24 h. Plots show Sub-G1 peaks with the percentages of apoptotic population cell.
Conclusions. – In this work, we elucidated the structures of the compounds 1 – 6 isolated from G. polyantha. The isolation and identification of one of them was accomplished for the first time. The cytotoxicity results provided baseline information for the possible use of compounds 1 – 4 for the control of cancer diseases and, especially, leukemia. We are grateful to Alexander von Humboldt-Stiftung for having provided a Georg Forster Fellowship for Experienced Researchers (ID Nr: 3.4-KAM/1137675) to A. M. L.
Experimental Part General. Column chromatography (CC): silica gel 60 (SiO2 ; 70 – 230 and 240 – 300 mesh; E. Merck). TLC: Precoated SiO2 plates (E. Merck, F254 ); visualization by spraying with Ce(SO4 )2 reagent. Optical
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rotations: in MeOH soln.; JASCO digital polarimeter (model DIP-3600). IR Spectra: JASCO A-302 IR spectrophotometer, in CHCl3 ; ˜n in cm 1. 1H- and 13C-, and 2D-NMR spectra: Bruker AMX-500 spectrometer with (D6 )acetone as solvent; d [ppm] relative to the residual (D6 )acetone signal at d(H) 2.05, and d(C) 29.84 and 206.26; J in Hz. Homonuclear 1H,1H connectivities were determined by the COSY 458 experiment. One-bond 1H,13C connectivities were determined by HMQC. Two- and threebond 1H,13C connectivities were determined by HMBC. EI-MS: Varian MAT 311A double-focusing mass spectrometer; in m/z (rel. %). HR-EI-MS: JEOL HX 110 mass spectrometer; in m/z. Plant Material. The leaves of G. polyantha were collected in Yaounde´, central region, Cameroon, in February 2009, and identified by Mr. Victor Nana from Cameroon National Herbarium where a voucher specimen (21337/SRF/Cam/Mt Kala) has been deposited. Extraction and Isolation. The dried and powdered leaves of G. polyantha (2.0 kg) were extracted at r.t. with MeOH (3 20 l) for 3 d. The extract was concentrated and suspended in H2O, followed by successive partition with petroleum ether (PE), CH2Cl2 , AcOEt, and BuOH, resp. The PE extract (10.8 g) was subjected to flash column chromatography (FC; SiO2 (10 40 cm, 200.0 g); PE/AcOEt 20 : 1 – 3 : 1 (3 l each)) to afford six fractions, Frs. 1 – 6. Fr. 3 (2.2 g) was subjected to FC (SiO2 (4 30 cm, 20.0 g); PE/AcOEt 10 : 1 to 3 : 1 (100 ml each)) to give six subfractions, Subfrs. 4a – 4f, and compound 1 (20 mg) was obtained from Subfr. 4a. Subfr. 4d (150.0 mg) was subjected to CC (SiO2 (5 40 cm, 50.0 g); PE/AcOEt 20 : 1 to 3 : 1 (50 ml each)) to afford compounds 2 (10 mg), 3 (6 mg), and 4 (25 mg). Fr. 5 (1.3 g) was subjected to CC (SiO2 (5.5 30 cm, 70.0 g); PE/AcOEt 4 : 1 to 1 : 1 (100 ml each)) to give five subfractions, Subfrs. 5a – 5e. Subfr. 5a (70.0 mg) was repeatedly purified by CC (SiO2 (2 45 cm, 20.0 g); PE/AcOEt 7 : 1) to yield compound 3 (11.0 mg). Subfr. 5b (74 mg) was separated by CC (SiO2 (3 30 cm, 20.0 g); PE/AcOEt 4 : 1) to yield compound 4 (10.6 mg). Subfr. 5c (55.2 mg) was purified by CC (SiO2 (1.2 34 cm, 9.0 g); PE/AcOEt 5 : 1) to yield compound 5 (3.8 mg). Fr. 6 (1.7 g) was separated by CC (SiO2 (4 30 cm, 32 g); PE/AcOEt 4 : 1 to 1 : 1 (2 l each)) to afford two subfractions, Subfrs. 6a and 6b. Subfr. 6b (75.7 mg) was gel-filtered over Sephadex LH-20 (2 100 cm; CHCl3/MeOH 1 : 1) to yield compounds 5 (4.9 mg) and 6 (4.1 mg). All isolated compounds were more than 97% pure. Banganxanthone C ( ¼ 12-(1,1-Dimethylprop-2-en-1-yl)-5,10-dihydroxy-9-methoxy-2-methyl-2-(4methylpent-3-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one; 4). Yellow amorphous powder. [a] 25 D ¼ þ 56 (c ¼ 3.5 10 4, CHCl3 ). M.p. 106.5 – 107.58. UV (CHCl3 ): 250 (3.96), 275 (4.35), 320 (4.00). IR (CHCl3 ): 3400, 1603, 1583. 1H- and 13C-NMR: see Table 1. EI-MS: 476 (18, M þ ), 461 (11), 394 (25), 393 (100), 339 (20), 325 (4). HR-EI-MS: 476.2181 (M þ , C29H32O þ6 ; calc. 476.2193). Bioassay. The cytotoxic activities of compounds 1 – 4 were determined against five human tumor cells including lung A549 adenocarcinoma, breast carcinoma MCF-7, prostate carcinoma PC-3, cervical carcinoma HeLa, and acute monocytic leukemia THP-1 cell lines, using sulforhodamine B (SRB) assay as described in [14]. Briefly, cells were harvested in log phase using trypsin (0.05% trypsin, 0.02% EDTA, in phosphate buffer saline (PBS)). The cell suspensions were diluted with appropriate growth medium to obtain the cell density of 104 cells/well. Aliquots of 100 ml of each suspension were seeded in 96-wells cell culture plates. The cells were incubated at 378 in an atmosphere of 5% CO2 and 95% relative humidity in a CO2 incubator. After 24 h of incubation, test materials (100 ml/well) at different concentrations (1, 10, 30, and 50 or 100 mg/ml) were added to the wells containing cells. Paclitaxel (Sigma Chem. Co., USA, purity 95%; 0.1, 1.0, and 10 mm) was used as positive reference. Suitable controls with equivalent concentration of DMSO were also included. The plates were further incubated for 48 h in a CO2 incubator after addition of test material. After incubation, cells were fixed by gently layering CCl3COOH (50 ml/well, 50% w/v) on top of the medium in all the wells and incubated at 48 for 1 h. The plates were washed five times with dist. H2O and air-dried. Cell growth was measured by staining with SRB dye (0.4% (w/v) in 1% AcOH, 100 ml/well). The unbound dye was washed 3 – 5 times with 1% AcOH, and plates were air-dried. The adsorbed dye was dissolved in Tris buffer (100 ml/well, 0.01m, pH 10.4), and the plates were gently shaken for 10 min on a mechanical shaker. The optical density (OD) was recorded using a 96-well plate reader. Growth inhibition was calculated by subtracting mean OD values of respective blank from the mean OD value of experimental set. Percentage growth in presence of test material was calculated considering the growth in absence of any test material as 100%, and in turn percentage growth inhibition in presence of test material was calculated. The viability and growth in the presence of test material was calculated by the following formula:
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Growth inhibition [%] ¼ 100 [OD(test sample) OD(blank) /OD(control) OD(blank) ] 100 IC50 Value is the concentration of sample required to inhibit 50% of the cell proliferation and was calculated by plotting the percentage survival vs. the concentrations, using Microsoft Excel. For all samples, each compound concentration was tested thrice in triplicates. Compounds 5 and 6 were not tested due to their small quantity. DNA Content Analysis. Sub-G1 DNA Content analysis was performed as described in [16]. Briefly, THP-1 cells (106/ml) in exponential phase of growth were seeded in 24-well tissue culture plates and allowed to adhere for 24 h. On day 2, the old medium was changed, and the cells were treated with extract/compounds at different concentrations and further incubated for 24 h. Cells were trypsinized, centrifuged, and washed with PBS, then fixed gently by adding 70% EtOH. They were fixed in 70% EtOH, washed with PBS, and then successively treated with RNase and with propidium iodide (PI; 25 mg/ ml) and incubated at 378 for 30 min. The percentages of DNA content in sub-G1 cells population were measured using BD-LSR flow cytometer equipped with electronic doublet discrimination capability using blue (488 nm) excitation from Ar laser. Data were collected in list mode on 10,000 events for FL2A vs. FL2-W. Apoptotic nuclei appear as a broad hypodiploid DNA peak at lower fluorescence intensity compared to nuclei in G0/G1 phase.
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