Cytotoxic and Anti-infective Phenolic Compounds Isolated from Mikania decora and Cremastosperma microcarpum

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1597

Cytotoxic and Anti-infective Phenolic Compounds Isolated from Mikania decora and Cremastosperma microcarpum

Abstract !

An anticancer-bioassay guided isolation of the ethanol extract and fractions of two plants from the Peruvian rainforest, Mikania decora and Cremastosperma microcarpum, led to the characterization of one abundant diterpene, ent-pimara-8(14),15-dien19-oic acid (1), three thymol derivatives, 10-acetoxy-8,9-dehydro-6-methoxythymol butyrate (2), 10-acetoxy-8,9-epoxy-6methoxythymol isobutyrate (3), and acetylschizoginol (4), as well as one neolignan, (±)-trans-dehydrodiisoeugenol (5). Only the latter was isolated from C. microcarpum. These compounds exhibited significant cytotoxic activity against a panel of human tumor cell lines. Compounds 3 and 4 were also investigated for their in vitro antileishmanial and trypanocidal activity against Leishmania amazonensis axenic amastigotes and Trypanosoma cruzi trypomastigotes.

Key words Mikania decora · Compositae · Cremastosperma microcarpum – Annonaceae · anticancer · antileishmania · trypanoside Supporting information available online at http://www.thieme-connect.de/ejournals/toc/plantamedica

The importance of plant-based drug discovery research has diminished in recent years despite the fact that most cancer chemotherapy agents currently in clinical use have originated from plants or are analogs of plant-derived compounds [1, 2]. Reasons for this decline include concerns about resupply, legal barriers to foreign access in developing countries, and the increased popularity of combinatorial chemical libraries, even though the latter contains only a fraction of the chemical diversity present in natural product extracts. Indeed, reliance on synthetic libraries has been cited as one of the factors responsible for the relative lack of success in developing new chemotherapy agents in recent years [3]. Another argument used against plant-based drug discovery programs is that the chance of reisolating a known compound is high, but what is less known is that only

Fig. 1 Chemical structures of compounds 1–5 isolated from M. decora and C. microcarpum.

41.4 % of the compounds reported in natural product databases, such as Napralert, has been studied from a biological perspective [4]. Our group has had access to a large repository of extracts from plants collected in the Amazonian region of Peru – one of the most biodiverse, but relatively under-explored, regions of the world. This collection contains some very rare families of plants and was gathered after lengthy negotiations with the Peruvian government and the Aguaruna tribe, under the auspices of the International Cooperative Biodiversity Group Program [5, 6]. Close to 1600 crude plant extracts, belonging to families that are either rare (i.e., unlikely to have been previously studied) or that contain a high proportion of species with medicinal properties, have been screened by our group for potential anticancer activity [7–9]. Herein, we report the results of our investigations of the ethanol extracts of Mikania decora (Compositae) and Cremastosperma microcarpum (Annonaceae). Mikania is one of 1600 genera belonging to the Asteraceae (Compositae) family, the second largest family of flowering plants [10, 11]. Originally from Central and South America, and with over 450 species, Mikania constitutes the largest genus in the Eupatorieae tribe. M. decora occurrence is widespread in the tropical and subtropical areas of Ecuador, Peru, and Bolivia. Its roots, stems, and leaves are known to be toxic and poisonous and are not used by the Aguaruna community of northwestern Peru. To the best of our knowledge, there have been no previous phytochemical or pharmacological studies of M. decora, and the only phytochemical report on one of the forty-three known species of Cremastosperma describes the isolation of bisbenzylisoquinoline alkaloids from the bark of C. polyphlebum [12]. There are no investigations reported for C. microcarpum itself. We found that the ethanolic extracts of M. decora and C. microcarpum showed significant cytotoxic activity. Bioactivity-guided

Aponte JC et al. Cytotoxic and Anti-infective …

Planta Med 2011; 77: 1597–1599

This is a copy of the authorʼs personal reprint

This is a copy of the authorʼs personal reprint

José C. Aponte 1, 2, Zhuang Jin 1, Abraham J. Vaisberg 3, Denis Castillo 3, Edith Málaga 3, Walter H. Lewis 4, Michel Sauvain 5, Robert H. Gilman 6, Gerald B. Hammond 1 1 Department of Chemistry, University of Louisville, Louisville, KY, USA 2 Current address: Department of Geological Sciences, Brown University, Providence, RI, USA 3 Departamento de Microbiología y Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Perú 4 Department of Biology, Washington University, St. Louis, MO, USA 5 Universidad de Toulouse III – IRD, UMR 152 (Pharma-Dev), France, and UPCH, Lima, Peru 6 Johns Hopkins School of Public Health, Baltimore, MD, USA

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Letters

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Table 1 Cytotoxicity of compounds 1–5 isolated from M. decora and C. microcarpum (GI50 values µM). Cell linea

a

Compound

3T3

1 2 3 4 5 Doxorubicin

> 200 > 200 2.5 4.7 63.8 0.029

H460

> 200 > 200 6.5 24.1 60.4 0.044

DU145

MCF-7

> 200 > 200 2.5 10.0 85.2 0.055

> 200 > 200 1.9 8.5 38.9 0.073

M-14

> 200 > 200 5.9 15.6 68.7 0.092

HT-29

K562

> 200 > 200 5.9 19.4 93.5 0.129

VERO

> 200 > 200 2.5 5.9 48.8 0.044

> 200 > 200 3.1 10.3 67.8 0.020

3T3, BALB/3T3 clone A31 embryonic mouse fibroblast cells; H460, human large lung cancer cell; DU145, human prostate carcinoma; MCF-7, human breast adenocarcinoma;

fractionation of M. decora using silica gel, C18 column chromatography, and HPLC normal phase, led to the isolation and characterization of one abundant diterpene, ent-pimara-8(14),15-dien19-oic acid (1) [13, 14], and three thymol derivatives: 10-acetoxy-8,9-dehydro-6-methoxythymol butyrate (2) [15], 10-acetoxy-8,9-epoxy-6-methoxythymol isobutyrate (3) [16], and ace" Fig. 1). Only the anti-inflammatory tylschizoginol (4) [17] (l activity of 4 has been reported [18], and there has been no bioactivity reported for the remaining thymol derivatives. Our antitumor-bioassay guided fractionation showed that 3 and 4 were the major antiproliferative compounds present in M. decora " Table 1). In the case of C. microcarpum, we found that its anti(l cancer activity was due to the presence of the neolignan " Table 1) (l " Fig. 1) [19, 20]. (±)-trans-dehydrodiisoeugenol (5, l This phenolic compound has been studied previously for its antioxidant and anticancer properties [21–23], and it has been recently included on a skin formulation patent [24]. A comparison of the structures of compounds 2, 3, and 4 would appear to indicate that the antitumor activity is due to the presence of the 8,9epoxy-10-acetoxy groups and the 9,10-diester glycerol moieties in 3 and 4, respectively, and that the allylacetate moiety in 3 would reduce its antiproliferative activity. Cytotoxic compounds 3 and 4 were further investigated for their trypanocidal and antileishmanial activities using Leishmania amazonensis axenic amastigotes and Trypanosoma cruzi trypomasti" Table 2). These parasites are responsible for two of the gotes (l so-called neglected diseases, leishmaniasis and Chagas disease. Compound 3 was only moderately active against L. amazonensis with a selectivity index (SI = IC50 macrophages/IC50 L. amazonensis) of 3.0, while compound 4 exhibited a higher antileishmanial activity and an SI of 2.4. Only compound 3 was active below the minimum concentration tested (30 µM) against trypomastigotes.

Materials and Methods !

Mikania decora was collected at Bajo Naranjillo, province of Rioja, San Martin Department, Perú, in May 1999 (voucher accession number, Lewis et al. 20 222). C. microcarpum leaflets and stems of a tree (13 m tall) were collected at Copallin, province of Bagua, Amazonas Department, Perú, in May 1999 (voucher accession number, R. Castro et al. 19 759). All voucher specimens are kept at the Museo de Historia Natural, Universidad Nacional Mayor de San Marcos (UNMSM), Lima, Perú, and at the Missouri Botanical Garden (MBG), St. Louis, MO, USA. All plant material was identified by the taxonomic staff at the MBG (W. L.). Air-dried and ground roots of M. decora (50 g) were extracted with ethanol 95 % at room temperature for 10 days. The ethanolic solution was concentrated to dryness under reduced pressure to

Aponte JC et al. Cytotoxic and Anti-infective …

Planta Med 2011; 77: 1597–1599

Table 2 Antiprotozoal (IC50 µM) activity of compounds 3 and 4.

a

Microorganism

3

L. amazonensis Macrophages T. cruzi

29.5 89.7 10.9

4

Anfa

Nifb

3.2 7.6 > 30

0.14 5.84 –

– – 0.7

Amphotericin B; b Nifurtimox

yield 5.57 g of extract. An aliquot (3 g) was partitioned between diethyl ether and water. The diethyl ether-soluble extract (337.1 g), in which the cytotoxic activity was concentrated, was subjected to column chromatography using 120 g of silica gel (22 × 4 cm) and a hexanes/EtOAc step gradient (100 : 1, 20 : 1, 10 : 1, 8 : 1, 4 : 1, 2 : 1, 1 : 1, 0 : 1, MeOH), obtaining 8 fractions (500–200 mL each), which were analyzed by TLC and pooled into 7 subfractions: A (48.3 mg), B (44.5 mg), 10-acetoxy-8,9-dehydro-6-methoxythymol butyrate (2) [15] (9.1 mg), D (47.1 mg), E (17.1 mg), F (53.6 mg), and G (148.3 mg). Fractions D, E, and F were the most cytotoxic, with GI50 values lower than 15 µg/mL for all cancer cell lines. Fraction B was subjected to flash column chromatography using hexanes/CHCl3 (4 : 1) to afford ent-pimara-8(14),15-dien-19-oic acid (1) [13, 14] (34.6 mg, approximately 10 % of total diethyl ether fraction). Active fraction D was first subjected to reversed-phase column chromatography using 4.2 g of C18 silica gel (1 × 3 cm) and H2O/MeOH (4 : 6, 2 : 8, 1 : 9, MeOH, CHCl3) as mobile phase, obtaining four subfractions (D1– D4, 50 mL each). Fraction D2 was successively chromatographed using reversed-phase and normal-phase HPLC to finally yield 10acetoxy-8,9-epoxy-6-methoxythymol isobutyrate (3) [16] (3.8 mg, Rt = 13 min) using hexanes/EtOAc (9 : 1) as mobile phase. Fraction F was subjected to reversed-phase column chromatography using 4.1 g of C18 silica gel (1 × 3 cm) and H2O/MeOH (5 : 5, 4 : 6, 3 : 7, 2 : 8, 0 : 10, Et2O) as mobile phase, obtaining six subfractions (F1–F3, 25 mL each). Normal-phase HPLC of fraction F1 (33 mg) using hexane/EtOAc (85 : 15) afforded acetylschizoginol (4) [17] (16.0 mg, Rt = 13 min). Air-dried and ground leaflets and stems of C. microcarpum (50 g) were extracted with ethanol 95% at room temperature for 7 days. The ethanolic solution was concentrated to dryness under reduced pressure to yield 3.66 g of extract. An aliquot (2.0 g) was partitioned between dichloromethane (45 mL) and water (45 mL), which afforded an organic fraction (ACM1, 1240 mg), insoluble residue (ACM2, 100 mg), and an aqueous fraction (ACM3, 108 mg). In vitro cytotoxic studies indicated that ACM1 was the only active fraction. Hence, it was purified on silica gel chromatography and eluted with hexanes, ethyl acetate, and methanol

This is a copy of the authorʼs personal reprint

This is a copy of the authorʼs personal reprint

M-14, human melanoma; HT-29, human colon adenocarcinoma; K562, human chronic myelogenous leukemia cells; VERO, normal African green monkey kidney epithelial cells

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in gradient mode to yield five subfractions: ACM11 (163 mg), ACM12 (165 mg), ACM13 (650 mg), ACM14 (123 mg), and ACM15 (110 mg). ACM13, the most cytotoxic fraction, was further chromatographed using a reverse-phase C18 column in a solvent gradient of methanol-water (80%, V/V) to pure methanol, yielding (±)-trans-dehydrodiisoeugenol (5) (7.0 mg). Physical and spectroscopic data matched those in the literature [19, 20]. The purity of the tested compounds was ≥ 80 % as determined by analytical HPLC with PDA detection.

Supporting information

Detailed description of the materials and methods used and 1H and 13C spectra of compounds 1–5 are available as Supporting Information.

Acknowledgements

This is a copy of the authorʼs personal reprint

!

The authors are grateful to the U. S. Department of Defense Prostate Cancer Research Program (PCRP) of the Office of the Congressionally Directed Medical Research Programs (CDMRP) (Grant #W81XWH‑07-1-0299). A. J. V. and W. H. L. are grateful to the NIH and NSF (ICBG grant U01TW00331). Our gratitude is extended to the Aguaruna people for generously sharing their traditional medicinal information. In particular, we acknowledge the assistance of organizational/clan leaders, Apus, and members of OCCAAM, FAD, FECONARIN, and OAAM. We also acknowledge the support provided by the CREAM Mass Spectrometry Facility (University of Louisville) funded by NSF/EPSCoR grant # EPS0447479. The authors of this work state that no conflict of interest exists in this work.

11 Krautmann M, de Riscala EC, Burgueno-Tapia E, Mora-Perez Catalán CAN, Joseph-Nathan P. C‑15-Functionalized eudesmanolides from Mikania campanulata. J Nat Prod 2007; 70: 1173–1179 12 Cava MP, Wakisaka K, Noguchi I, Edie DL. Phlebicine, a new biphenylbisbenzylisoquinoline alkaloid from Cremastosperma polyphlebum. J Org Chem 1974; 39: 3588–3591 13 Mihashi S, Yanagisawa I, Tanaka O, Shibata S. Further study on the diterpenes of Aralia spp. Tetrahedron Lett 1969; 21: 1683–1686 14 Matsuo A, Uto S, Nakayama M, Hayashi S, Yamasaki K, Kasai R, Tanaka O. (−)-Thermarol, a new ent-pimarane-class diterpene diol from Jungermannia thermarum (Liverwort). Tetrahedron Lett 1976; 28: 2451– 2454 15 González AG, Bermejo Barrera J, Estévez Rosas F, Yanes Hernandez AC, Espiñeira J, Josep-Nathan P. Thymol derivatives from Schizogyne glaberrima. Phytochemistry 1986; 25: 2889–2891 16 Bohlmann F, Jakupovic J, Lonitz M. Über Inhaltsstoffe der EupatoriumGruppe. Chem Ber 1977; 110: 301–314 17 González AG, Bermejo J, Estévez F, Yanes AC, Joseph-Nathan P. Derivados fenolicos del genero Schizogyne. Rev Latinoam Quím 1986; 17: 54–56 18 González AG, Bermejo Barrera J, Castaneda Acosta J, Estévez Rosas F. Anti-inflammatory activity of Schizogyne species. Fitoterapia 1988; 59: 476–478 19 Aiba CJ, Correa RGC, Gottlieb OR. Natural occurrence of Erdtmanʼs dehydrodiisoeugenol. Phytochemistry 1973; 12: 1163–1164 20 Enriquez RG, Chaves MA. Phytochemical investigations of plant of the genus Aristolochia, 1. Isolation and NMR spectral characterization of eupomatenoid derivatives. J Nat Prod 1984; 47: 896–899 21 Bortolomeazzi R, Verardo G, Liessi A, Callea A. Formation of dehydrodiisoeugenol and dehydrodieugenol from the reaction of isoeugenol and eugenol with DPPH radical and their role in the radical scavenging activity. Food Chem 2010; 118: 256–265 22 Atsumi T, Fujisawa S, Satoh K, Sakagami H, Iwakura I, Uehai T, Sugita Y, Yokoe I. Cytotoxicity and radical intensity of eugenol, isoeugenol or related dimers. Anticancer Res 2000; 20: 2519–2524 23 Le Quesne PW, Larrahondo JE, Raffauf RF. Antitumor plants. X. Constituents of Nectandra rigida. J Nat Prod 1980; 43: 353–359 24 Meyer I, Koch O, Krutmann J. Use of dihydrodehydrodiisoeugenol as skin care preparations. PCT Int Appl 2010; WO 2010070152 A2 20100624

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received revised accepted

December 16, 2010 March 10, 2011 March 12, 2011

Bibliography DOI http://dx.doi.org/10.1055/s-0030-1270960 Published online April 6, 2011 Planta Med 2011; 77: 1597–1599 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943

Correspondence Univ.-Prof. Dr. Gerald B. Hammond Department of Chemistry University of Louisville 2308 S. Brook St Louisville, KY 40292 USA Phone: + 1 50 28 52 59 98 Fax: + 1 50 28 52 38 99 [email protected]

Aponte JC et al. Cytotoxic and Anti-infective …

Planta Med 2011; 77: 1597–1599

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This is a copy of the authorʼs personal reprint

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