Fatty Acid Synthase Inhibitors Isolated from Punica granatum L

Este trabalho tem por objetivo o isolamento de inibidores da enzima ácido graxo sintase (FAS) a partir de acetato de etila proveniente de extratos de cascas de frutas da Punica granatum L. A investigação química guiada por bioensaios das cascas das frutas resultou no isolamento de dezessete compostos incluindo principalmente triternóides e compostos fenólicos, dos quais um novo triterpeno do tipo oleanano (punicaone) juntamente com quatorze compostos conhecidos foram isolados pela primeira vez a partir desta planta. Sete dos componentes isolados foram avaliados para atividades inibitórias de FAS e dois deles apresentaram-se ativos. Em particular, o ácido flavogalônico que exibiu forte atividade inibitória de FAS com valor de IC50 de 10,3 μmol L .


Introduction
The animal fatty acid synthase (FAS; EC 2.3.1.85)][4][5] At the same time, many FAS inhibitors, such as cerulenin, C75, orlistat and EGCG (epigallocatechin gallate), have joint effects of weight-loss and anti-tumor. 6,7These prove that FAS may be a clinical and experimental target for scientific research and drug discovery.In other words, natural products inhibiting FAS are appearing as potential therapeutic agents to treat cancer and obesity.Therefore, it is important to find novel compounds especially from natural sources that reduce FAS activity or expressing levels that may be useful for the treatment of obesity and cancer.

General procedures
Optical rotations were measured on a Horiba SEPA-300 polarimeter.UV spectra were obtained on a Shimadzu double-beam 210A spectrometer.Infrared (IR) spectra were obtained on a Tensor 27 spectrometer with KBr pellets.NMR spectra were recorded on Bruker AV-400 or DRX-500 spectrometer with TMS (tetramethylsilane) as an internal standard using CDCl 3 as solvent.Electrospray ionization mass spectrometry (ESI-MS) and high-resolution-ESI-MS were recorded with an API QSTAR Pulsar 1 spectrometer.Silica gel (200-300 mesh, Qingdao Marine Chemical Inc., People's Republic of China), RP-18 (40-70 µm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences, Sweden) were used for column chromatography.Fractions were monitored by TLC (thin layer chromatography) and spots were visualized by heating after spraying with 10% H 2 SO 4 in ethanol.

Plant material
The peels of Punica granatum were purchased from a market in Kunming (People's Republic of China) and identified by Prof. Li Gong Lei from Kunming Institute of Botany, Chinese Academy of Sciences.The specimen (JHZ20081201) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China from Kunming Institute of Botany, Chinese Academy of Sciences.

Results and Discussion
Compound 1 was obtained as a yellow oil and had the molecular formula C 30 H 44 O 4 determined by HR-EI-MS (m/z 468.3250, calcd.for C 30 H 44 O 4 : 468.3240).The IR spectrum showed absorption bands at 3432, 2931, 1722, 1688, 1640, 1461 and 1382 cm -1 , which were indicative of hydroxyl groups, carbonyls and double bonds.The 1 H and 13 C NMR spectra (Table 1) displayed 30 carbon resonances comprising six methyl groups, nine methylenes (including one oxygenated at d C 69.4), six methines (including three olefinic at d C 150.7, 128.1 and 123.7), nine quaternary carbons (one carbonyl at 206.0, one carboxyl at 183.1 and one olefinic at 141.9), suggesting that compound 1 owns a typical oleanane skeleton. 11,12The NMR data of 1 were similar to that of amooranin. 13,14However, different carbon and proton chemical shifts for C-1, C-2 and C-3 indicated that the structure of 1 differed from amooranin.In the 13  The FAS inhibitory activity of compounds 2, 3, 7, 12-14 and 17 was determined by previous described method. 15The results are presented in Table 2.The known FAS inhibitor EGCG (IC 50 of 51.97 µmol L -1 ) was used as positive control in this assay.Compounds 2 (oleane-type triterpene) and 17 (gallogen-type phenols) exhibited strong inhibitory activity with the lowest IC 50 values (68.4 and 10.3 µmol L -1 , respectively), and the other five compounds showed weak activity.Oleanetype triterpene and its oligoglycosides from the hulls of Nephelium lappaceum L. were found to have effective FAS inhibitions. 16The gallogen-type phenols could also be considered as another type of natural FAS inhibitors based on this test.

Table 1 .
1H and 13 C NMR data of 1 in CD 3 OD