Chemical constituents of Cycas aenigma

1 Chemistry Department, De La Salle University 2401 Taft Avenue, Manila 1004, Philippines. 2 Biology Department, De La Salle University 2401 Taft Avenue, Manila 1004, Philippines. 3 National Research Institute of Chinese Medicine, 155-1, Li-Nong St., Sec. 2, Taipei 112, Taiwan. 4 Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Binan City, Laguna 4024, Philippines.


General experimental procedure
NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl 3 at 600 MHz for 1 H NMR and 150 MHz for 13 C NMR spectra.Column chromatography was performed with silica gel 60 (70-230 mesh).Thin layer chromatography was performed with plastic backed plates coated with silica gel F 254 and the plates were visualized by spraying with vanillin/H 2 SO 4 solution followed by warming.

Plant material
Cycas aenigma leaflets and petiole and rachis were collected in 2013.Voucher specimens were collected and authenticated by one of the authors (EMGA) and deposited in the De La Salle University-Manila Herbarium (DLSUH).

General isolation procedure
A glass column 18 inches in height and 1.0 inch internal diameter was used for the chromatography of the crude extracts.Twenty milliliter fractions were collected.All fractions were monitored by thin layer chromatography.Fractions with spots of the same R f values were combined and rechromatographed in appropriate solvent systems until TLC pure isolates were obtained.A glass column 12 inches in height and 0.5 inch internal diameter was used for the rechromatography of smaller fractions from the first column.Five milliliter fractions were collected.Final purifications were conducted using Pasteur pipettes as columns.One milliliter fractions were collected.

Isolation of the chemical constituents of the leaflets
The air-dried bark of C. aenigma (105 g) were ground in a blender, soaked in CH 2 Cl 2 for 3 days and then filtered.The solvent was evaporated under vacuum to afford a crude extract (2.3 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 20% increment.The 20% acetone in CH 2 Cl 2 fraction was rechromatographed (2 ×) using 15% EtOAc in petroleum ether to afford 3 (3 mg) after washing with petroleum ether.

Isolation of the chemical constituents of the petiole and rachis
The air-dried petiole and rachis of C. aenigma (47 g) were ground in a blender, soaked in CH 2 Cl 2 for 3 days and then filtered.The solvent was evaporated under vacuum to afford a crude extract (0.7 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.The 20% acetone in CH 2 Cl 2 fraction was rechromatographed (4 ×) using 7.5% EtOAc in petroleum ether to yield 4 (8 mg).The 30% acetone in CH 2 Cl 2 fraction was rechromatographed using 15% EtOAc in petroleum ether to afford a mixture of 5a and 5b in a 1:1 ratio (4 mg) after washing with petroleum ether.
Results showed that 1 did not exhibit any antiallergic activity at a 1.5 μg/ml concentration (Koyama et al., 2008).On the other hand, pinoresinol (2) was found to have antioxidant and Ca 2+ antagonist properties (Páska et al., 2002).It was reported to exhibit strong antiinflammatory properties by acting on the NF-κB signaling pathway (During et al, 2012).Furthermore, 2 attenuates inflammatory responses of microglia and could be useful in modulation of inflammatory status in brain disorders (Jung et al., 2010).Lignan 2 was shown to possess fungicidal activities and therapeutic potential as an antifungal agent for the treatment of fungal infectious diseases in humans (Hwang et al., 2010).It exhibited inhibitory activity against rat intestinal maltase with an IC 50 value of 34.3 μM (Wikul et al., 2012).Long-chain fatty alcohols (3) were reported to exhibit a protective effect on some mediators involved in the inflammatory damage development (Fernandez-Arche et al., 2015).On the other hand, triacylglycerides (4) exhibited antimicrobial activity against S. aureus, P. aeruginosa, B. subtilis, C. albicans, and T. mentagrophytes (Ragasa et al., 2013).Another study reported that 4 showed a direct relationship between toxicity and increasing unsaturation, which in turn correlated with increasing susceptibility to oxidation (Ferruzzi and Blakeslee, 2007).β-Sitosterol (5a) was observed to have growth inhibitory effects on human breast MCF-7 and MDA-MB-231 adenocarcinoma cells (Awad et al., 2007).It was shown to be effective for the treatment of benign prostatic hyperplasia (Jayaprakasha et al., 2007).Compound 5a was also reported to attenuate β-catenin and PCNA expression, as well as quench radical in vitro, making it a potential anticancer drug for colon carcinogenesis (Baskar et al., 2010).It can inhibit the expression of NPC1L1 in the enterocytes to reduce intestinal cholesterol uptake (Jesch et al., 2009).It was reported to induce apoptosis mediated by the activation of ERK and the down-regulation of Akt in MCA-102 murine fibrosarcoma cells (Moon et al., 2007).On the other hand, stigmasterol (5b) shows therapeutic efficacy against Ehrlich ascites carcinoma bearing mice while conferring protection against cancer induced altered physiological conditions (Ghosh et al., 2011).Compound 5b lowers plasma cholesterol levels, inhibits intestinal cholesterol and plant sterol absorption, and suppresses hepatic cholesterol and classic bile acid synthesis in Winstar as well as WKY rats (Batta et al., 2006).Other studies reported that 5b showed cytostatic activity against Hep-2 and McCoy cells (Gómez et al., 2001), markedly inhibited tumour promotion in two stage carcinogenesis experiments (Kasahara et al., 1994), and exhibited antimutagenic (Lim et al., 2005), topical anti-inflammatory (García et al., 1999), antiosteoarthritic (Gabay et al., 2010) and antioxidant (Panda et al., 2009) activities.