Furoquinoline Alkaloids and Methoxyflavones from the Stem Bark of Melicope madagascariensis (Baker) T.G. Hartley

Melicope madagascariensis (Rutaceae) is an endemic plant species of Madagascar that was first classified as a member of the genus Euodia J. R. & G. Forst (Rutaceae) under the scientific name Euodia madagascariensis Baker. Based on morphological characteristics, Thomas Gordon Hartley taxonomically revised E. madagascariensis Baker to be M. madagascariensis (Baker) T.G. Hartley. Chemotaxonomical studies have long been used to help the identification and confirmation of taxonomical classification of plant species and botanicals. Aiming to find more evidences to support the taxonomical revision performed on E. madagascariensis, we carried out phytochemical investigation of two samples of the plant. Fractionation of the ethanol extracts prepared from two stem bark samples of M. madagascariensis (Baker) T.G. Hartley led to the isolation of seven known furoquinoline alkaloids 1–7 and two known methoxyflavones 8 and 9. The presence of furoquinoline alkaloids and methoxyflavones in the title species is in agreement with its taxonomic transfer from Euodia to Melicope. Antiprotozoal evaluation of the isolated compounds showed that 6-methoxy-7-hydroxydictamnine (heliparvifoline, 3) showed weak antimalarial activity (IC50 = 35 µM) against the chloroquine-resistant strain Dd2 of Plasmodium falciparum. Skimmianine (4) displayed moderate cytotoxicity with IC50 value of 1.5 µM against HT-29 colon cancer cell line whereas 3,5-dihydroxy-3′,4′,7-trimethoxyflavone (9) was weakly active in the same assay (IC50 = 13.9 µM).

M. madagascariensis is a tree up to 10-20 m tall which is encountered in the rainforest of Madagascar and is traditionally employed as exhilarating agent, purgative, and in formulations for the treatment of liver, kidney and stomach disorders, bronchitis and mumps [2]. Members of the genus Melicope are rich sources of furoquinoline alkaloids, methoxyflavones and acetophenones, some of which have elicited antiplatelet aggregation [3], cytotoxic [4] and antimalarial activities [5]. A previous phytochemical study on M. madagascariensis reported the isolation of methoxyflavones [6], a class of compounds that have also been found in Euodia species already transferred into Melicope. As part of our ongoing projects aiming to characterize secondary metabolites of plants originated from Madagascar rainforest for biological and chemotaxonomical studies, we carried out phytochemical investigation of M. madagascariensis, which is one of the eleven Melicope species endemic to Madagascar. This paper deals with the isolation of nine known compounds including seven furoquinoline alkaloids 1-7 and two methoxyflavones 8 and 9 from two samples of the stem bark of this species. Herein, we discuss the chemotaxonomic significance as well as the antimalarial and cytotoxic activities of the isolated compounds.
With the exception of few species including those of the genus Euodia, furoquinoline alkaloids are widespread in the Rutaceae family [14]. In particular, a number of furoquinoline alkaloids and derivatives have been reported from species of the genus Melicope [3,15,16]. The isolation of furoquinoline alkaloids from M. madagascariensis chemotaxonomically confirmed the classification of the present plant species in the genus Melicope of the Rutaceae family. Furthermore, polyoxygenated flavones (polymethoxy and methylenedioxy) have been considered to be characteristic chemical components of plant species of the genus Melicope inside the Rutaceae family [17]. The isolation of furoquinoline alkaloids and methoxylated flavones from the present plant species is in good agreement with its taxonomic reassignment from Euodia into Melicope. This study added one more species to the Melicopeproducing methoxyflavones. Whether all of the Euodia species containing furoquinoline alkaloids and methoxyflavones should be moved to the genus Melicope or not is one of the major questions that can be unfolded from the present investigation. Apart from the morphological and phytochemical contributions, full genome sequencing of all species of the genus to be transferred would complete this taxonomical classification.
The sample MG250 was collected for the International Cooperative Biodiversity Group (ICBG)/Madagascar program that was mainly aimed at searching for antimalarial and antiproliferative agents from natural resources from Madagascar. Evaluation of compounds 1-3 for their antimalarial activity against the multidrug-resistant strain Dd2 of Plasmodium falciparum showed that only heliparvifoline (3) exhibited growth inhibitory effect at relatively high concentration (IC 50 = 35 lM). Positive control consisted of fosmidomycin (IC 50 = 0.31 lM). Moreover, in vitro antimalarial activity against the chloroquine-susceptible HB3 (Honduras) and the chloroquine resistant W-2 (Indochina) strains of P. falciparum has been previously performed on some Rutaceous furoquinoline alkaloids such as skimmianine (4), haplopine (10), kokusaginine (11), acronycidine (12) and acronydine (13) [18]. Although the compounds isolated in the present study did not exhibit strong antimalarial activity against the multidrug-resistant strain Dd2 of Plasmodium falciparum, the present paper added more data on the antimalarial activity of furoquinoline alkaloids of Rutaceous plants. In addition, compounds 4-9 isolated from the sample ST1375 were assayed against the human colorectal adenocarcinoma (HT-29) cell line. As results, compounds 5-8 were inactive in this assay while skimmianine (4) and 3,5-dihydroxy-3 0 ,4 0 ,7-trimethoxyflavone (9) exhibited moderate and weak activity with IC 50 values of 1.5 lM and 13.9 lM, respectively.

Antimalarial Assay
The antimalarial assay was carried out at Virginia Tech against the multidrug-resistant strain Dd2 of P. falciparum by using the SYBR Green I-based plate assay as previously reported [19].

Cytotoxicity Assay
Cytotoxicity assay against HT-29 human colon cancer cell line was performed at the College of Pharmacy, The Ohio State University, USA by using a sulforhodamine B reduction assay procedure as described in a previous paper [20].

Plant Material
The first sample coded MG250 of M. madagascariensis

Extraction and Isolation
The dried and powdered sample MG250 (300 g) was macerated in EtOH for 48 h to yield 11.5 g of a crude EtOH extract. A portion (1.5 g) was suspended in 90 % aqueous MeOH (150 mL) and extracted with n-hexane ( The dried and powdered sample ST1375 (1.3 kg) was extracted as above to afford a crude EtOH extract (65.3 g). A portion (25 g) was liquid-liquid partitioned using same procedures described above. The resulting CHCl 3 fraction was subjected to a conventional acid-base extraction to afford a basic CHCl 3 -soluble fraction (Fraction A, 343.7 mg) and a neutral CHCl 3 -soluble fraction (Fraction B, 11.2 g). Fraction A was first gel filtered on a Sephadex LH-20 column eluted with n-hexane/CH 2 Cl 2 /MeOH, 4:3:3 to give four pooled fractions (A1-A4). Fraction A2 was subjected to a combination of silica gel open column chromatography and HPLC on a semi-preparative C18 column (Purospher column, 5 lm, 25 9 1 cm) or silica gel preparative TLC to furnish 4 (1.4 mg) and 7 (0.9 mg). Fraction A4 (191.4 mg) was rechromatographed over silica gel open column eluted with n-hexane/EtOAc mixtures to give 5 (3.1 mg). A part of the fraction B (4 g) was flash chromatographed on silica gel column to furnish six subfractions (B1-B6). Subfraction B2 (171.5 mg) was separated by C18 open column chromatography and then by silica gel preparative TLC with CHCl 3 /MeOH, 50:1 as eluent to furnish 8 (3.4 mg), 9 (2.7 mg) and 6 (1.4 mg).
Structures of isolated compounds were established by the interpretation of their 1 H NMR spectra and comparison of data obtained with those published in the literature. The 1 H NMR spectroscopic data of the isolated compounds can be obtained free of charge from the corresponding author. study. Thanks are also due to the College of Pharmacy at The Ohio State University, USA and the Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, USA for facilitating the acquisition of the NMR spectra.

Compliance with Ethical Standards
Conflict of Interest The authors declare no conflict interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.