Minor Prenylated Flavonoids from the Twigs of Macarangaadenantha and Their Cytotoxic Activity

Three new minor prenylated flavonoids, named macadenanthins A–C (1–3), together with three known ones (4–6), were isolated from the twigs of Macaranga adenantha. Their structures were elucidated on the basis of extensive spectroscopic analysis including NMR, UV and MS. The prenyl moieties in compounds 1–3 were further modified by cyclization and hydroxylation. The new compounds were tested for their cytotoxicity against four cancer cell lines (MCF-7, Hep G2, Hela and P388) and showed IC50 values in the range of 13.76–22.27 μM.


Introduction
Prenylated flavonoids are attracting great attention from the scientific community due to their structural uniqueness and remarkable biological activities [1]. The different prenylation position, various lengths of prenyl chain and further modifications on the prenyl moiety such as cyclization and hydroxylation resulted in the chemical diversity of the prenylated products, and which also made them exhibit promising biological activities. Prenylated flavonoids have a relatively narrow distribution in the plant kingdom and an overview of the literature indicated that prenylated flavonoids are the typical secondary metabolites of the genus Macaranga [2].
Macaranga adenantha Gagnep (Euphorbiaceae) is an arbor distributed in the tropical rainforests, previous studies showed that it contained triterpenoids, steroids and phenolic compounds [3][4][5]. As part of our program to discover new anticancer prenylated aromatic products from the genus Macaranga [6][7][8], a phytochemical investigation on this plant led to the isolation and characterization of three minor new prenylated flavonoids, named macadenanthins A-C (1-3), along with three known ones, including glyasperin A (4) [9], broussoflavonol F (5) [10], and macarangin (6) [11]. The new compounds 1-3 were evaluated for their cytotoxicity against a small panel of cancer cell lines. Described herein are the isolation, structure elucidation and cytotoxicity of these compounds (Fig. 1).

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter. CD spectra were obtained on an automated circular dichroism spectrometer (Applied Photophysics). UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained on a Bruker Tenor 27 spectrometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AM-400, DRX-500 or AV III-600 spectrometers with TMS as an internal standard. ESIMS were recorded using a Finnigan MAT 90 instrument and HRESIMS was performed on an API QSTAR time-of-flight spectrometer, HREIMS were recorded on a Waters AutoSpec Premier P776 instrument.

Plant Material
The twigs of M. adenantha were collected from Malipo County of Yunnan province, P. R. China, in June 2013. A voucher specimen (Yangyp-20130619) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, which was identified by one of the authors (Prof. Yong-Ping Yang).

Extraction and Isolation
The dried and powdered twigs of M. adenantha (9.5 kg) were extracted three times with 90 % EtOH (25 L) for 24 h at room temperature and filtrated. The filtrate was concentrated and partitioned between H 2 O and EtOAc and then the EtOAc portion was decolorized on MCI gel (eluting with 95 % EtOH). The residue (380 g) was chromatographed on silica gel (CHCl 3 -MeOH 1:0 to 1:1) to afford fractions A-F. Fraction B was purified over a Sephadex LH-20 CC (CHCl 3 -MeOH 1:1) and then

Cytotoxicity Bioassays
Compounds 1-3 were tested for their cytotoxicity against human breast adenocarcinoma (MCF-7), human hepatocellular (Hep G2), human cervical carcinoma (Hela), mouse leukemia (P388) by the MTT method, and 5-FU was used as a positive control. Briefly, 100 lL cell suspension (1 9 10 5 cells/mL) was seeded into 96-well microteter plates and cultured for 24 h before the compound was added. Then, different concentrations of the compounds were added to the plates, the cells were cultivated for 48 h, and 10 lL of MTT (5 mg/mL) was added to each well. After 4 h, the culture medium was removed and the formazan crystal was completely dissolved with 150 lL DMSO to each well by vigorously shaking the plate. Finally, formazan absorbance was assessed by a BioRad microplate reader at 570 nm.