Chemical Constituents of Cycas sancti-lasallei

Vincent Antonio S. Ng, Esperanza Maribel G. Agoo, Chien-Chang Shen, Consolacion Y. Ragasa Chemistry Department, De La Salle University 2401 Taft Avenue, Manila 1004, Philippines. Biology Department, De La Salle University 2401 Taft Avenue, Manila 1004, Philippines. National Research Institute of Chinese Medicine, 155-1, Li-Nong St., Sec. 2, Taipei 112, Taiwan. Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Binan City, Laguna 4024, Philippines.


INTRODUCTION
Cycas, the only currently known genus of the Family Cycadaceae, are considered as fossil plants though they may have evolved only about 12 million years ago (Nagalingum et al., 2011).The cycads resemble palms in morphology and are commonly called sago palm.These are widely distributed in the Tropics, with species found in Asia, Africa, Southeast Asia, Pacific, and Australia (Donaldson, 2013).They also grow on volcanic, limestone, ultramafic, sandy, or even water-logged soils in grassland and forest habitats (Madulid and Agoo, 2009) Agoo (Madulid and Agoo, 2009;Lindstrom et al., 2008;Agoo and Madulid, 2012).All species, . .except for C. edentata, are endemic to the Philippines (Lindstrom et al., 2008).C. revoluta, a widely cultivated species, is an introduced species from Japan and Taiwan.The demand of Cycas species for domestic and international horticultural trade, grassland and forest fires of anthropogenic origin, and conversion of their natural habitats to settlements and other land uses have threatened to varying degrees the wild populations of the genus ( IUCN 2010).Some of these species which have been assessed and evaluated in the 2010 IUCN Red List of Threatened Species are C. curranii (Agoo et al., 2010), C. wadei (Hill, 2010) and C. zambalensis as Critically Endangered (CR) (Agoo et al., 2010), C. riuminiana as Endangered (E) (Agoo et al., 2010), and C. saxatilis as Vulnerable (V) (Bosenberg, 2010).Cycas sancti-lasallei (Cycadaceae) is a new species from Mindanao, Philippines (Agoo and Madulid, 2012).There are no reported chemical and biological activity studies on C. sancti-lasallei.However, some Cycas species have been studied for their chemical constituents and biological activities.The most studied Cycas are Cycas revoluta Thunb.and C. circinalis which contain the carcinogenic toxin cycasin (Nishida et al., 1956;Laqueur et al., 1963).The methanolic extract of the leaflets of C. circinalis L. and the chloroform extract of C. revoluta Thunb.yielded biflavonoids, lignans, flavan-3-ols, flavone-C-glucosides, nor-isoprenoids, and a flavanone.Three of the biflavonoids exhibited moderate activity against S. aureus and methicillin-resistant S. aureus (Moawad et al., 2010).Further studies on the chemical constituents of the leaves of C. revoluta Thunb.and C. circinalis L. afforded lariciresinol, naringenin and biflavonoids which are derivatives of amentoflavone and hinokiflavone (Ferreira et al., 2009).Studies on other Cycas species have also been conducted.The seeds of C. micronesica K. D. Hill yielded β-sitosterol β-D-glucoside, stigmasterol β-Dglucoside, β-sitosterol, and stigmasterol (Marler et al., 2006).C. beddomei afforded a new biflavonoid, along with pinoresinol, hinokiflavone, and amento flavones (Das et al., 2006(Das et al., , 2005)).The leaves of C. panzhihuaensis afforded a new flavone, along with .2,3-dihydrohinokiflavone, a biflavone, vanillic acid, sitosterol and daucosterol (Zhou et al., 2002).The methanolic extracts of the stems, flowers and seeds of C. panzhihuaensis L. yielded chavicol β-rutinoside, amentoflavone, podocarpusflavone A, a biflavone, βsitosterol, daucosterol and palmitic acid (Zhou et al., 2009).

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.

Sample Collection
Cycas sancti-lasallei endotesta, sarcotesta, and bark were collected from Malasag, Cugman, Cagayan de Oro, Misamis Oriental, Philippines on April 25, 2014.Voucher specimens were collected and authenticated by one of the authors (EMGA) and deposited in the De La Salle University-Manila Herbarium (DLSUH 3116).

General Isolation Procedure
A glass column 18 inches in height and 1.0 inch internal diameter was packed with silica gel.The crude extracts were fractionated by silica gel chromatography using increasing proportions of acetone in CH 2 Cl 2 (10% increment) as eluents.Fifty 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.Two milliliter fractions were collected.Final purifications were conducted using Pasteur pipettes as columns.One milliliter fractions were collected.

Isolation
The freeze-dried sarcotesta of C. sancti-lasallei (218 g) was 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 CH 2 Cl 2 fraction was rechromatographed (3 ×) using 1% EtOAc in petroleum ether to afford 1 (2 mg).The 20% acetone in CH 2 Cl 2 fraction was rechromatographed (4 ×) in 7.5% EtOAc using petroleum ether to afford 3 (7 mg).The 40% acetone in CH 2 Cl 2 fraction was rechromatographed (2 ×) using 20% EtOAc in petroleum ether to afford a mixture of 2a and 2b after washing with petroleum ether (5 mg).
The freeze-dried endotesta of C. sancti-lasallei (273.5 g) was 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 (11.3 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.The CH 2 Cl 2 fraction was rechromatographed (4 ×) using 2.5% EtOAc in petroleum ether to afford 4 (2mg).The 20% acetone in CH 2 Cl 2 fraction was rechromatographed (3 ×) using 7.5% EtOAc in petroleum ether to afford 3 (9mg).The 40% acetone in CH 2 Cl 2 fraction was rechromatographed (2 ×) using 20% EtOAc in petroleum ether to afford a mixture of 2a and 2b after washing with petroleum ether (6 mg).
The air-dried bark of C. sancti-lasallei (98 g) was 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 (1.0 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.The CH 2 Cl 2 fraction was rechromatographed (3 ×) using 2.5% EtOAc in petroleum ether to afford 5 (5 mg).The 20% acetone in CH 2 Cl 2 fraction was rechromatographed (4 ×) using 7.5% EtOAc in petroleum ether to afford 3 (8 mg).
The fatty acids esterified to the glycerol in the triglycerides of endotesta are oleic acid, linoleic acid and saturated fatty acid.These were deduced from the integrations of the resonances at δ 0.87 (t, J = 6.6 Hz, CH 3 ), 1.97-2.06(allylic CH 2 ), 2.74 (double allylic CH 2 ), and 5.30-5.37(CH=) for the linoleic acid; δ 0.87 (t, J = 6.6 Hz, CH 3 ), 1.97-2.06(allylic CH 2 ), and 5.30-5.37(CH=) for the oleic acid; and δ 0.87 (t, J = 6.6 Hz, CH 3 ) for the saturated fatty acid.A small amount of linolenic acid was detected from the low intensity resonance at δ 0.95 (t, J = 7.2 Hz) for the terminal methyl and 2.76 (double allylic CH 2 ' s) of this fatty acid (Human Metabolome, 2013).For the sarcotesta and sclerotesta triglycerides, the same fatty acids esterified to the glycerol were deduced from the integrations in the 1 H NMR spectra.However, larger amounts of linolenic acid were detected from the more intense resonances of the terminal methyl at δ 0.95 (t, J = 7.2 Hz) and the double allylic methylenes at δ 2.76.
Although bioassays were not conducted on the isolated compounds (1-3), there were previous studies that reported on their biological activities.
Squalene (1) was reported to significantly suppress colonic ACF formation and crypt multiplicity which strengthened the hypothesis that it possesses chemopreventive activity against colon carcinogenesis (Rao et al., 1998).It showed cardioprotective effect which is related to inhibition of lipid accumulation by its hypolipidemic properties and/or its antioxidant properties (Farvin et al., 2006).A recent study reported that tocotrienols, carotenoids, squalene and coenzyme Q10 haveanti-proliferative effects on breast cancer cells (Loganathan et al., 2013).The preventive and therapeutic potential of squalene containing compounds on tumor promotion and regression have been reported (Desai et al., 1996).A recent review on the bioactivities of squalene has been provided (Ronco and De Stéfani, 2013).
β-Sitosterol (2a) 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).It was also reported to attenuate βcatenin 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 downregulation of Akt in MCA-102 murine fibrosarcoma cells (Moon et al., 2007).Stigmasterol (2b) shows therapeutic efficacy against Ehrlich ascites carcinoma bearing mice while conferring protection against cancer induced altered physiological conditions (Ghosh et al., 2011).It 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 stigmasterol 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), exhibited antimutagenic (Lim et al., 2005), topical anti-inflammatory (Garcia et al., 1999), antiosteoarthritic (Gabay et al., 2010) and antioxidant (Panda et al., 2009) activities.Triacylglycerides (3) exhibited antimicrobial activity against S. aureus, P. aeruginosa, B. subtilis, C. albicans, and T. mentagrophytes (Ragasa et al., 2013).Another study reported that triglycerides showed a direct relationship between toxicity and increasing unsaturation, which in turn correlated with increasing susceptibility to oxidation (Ferruzzi and Blakeslee, 2007).The fatty acids esterified to the triglycerides were deduced to be oleic acid, linoleic acid and linolenic acid.Oleic acid has been reported to be responsible for the reduction of blood pressure induced by olive oil (Teres et al., 2008).It may hinder the progression of adrenoleukodystrophy, a fatal disease that affects the brain and adrenal glands (Rizzo et al., 1986).Oleic acid inhibited cancer cell growth and survival in low metastatic carcinoma cells, such as gastric carcinoma SGC7901 and breast carcinoma MCF-7 cell lines (Li et al., 2014).Linoleic acid belongs to the omega-6 fatty acids.It was reported to be a strong anticarcinogen in a number of animal models.It reduces risk of colon and breast cancer (Chan et al., 2002) and lowers cardiovascular disease risk and inflammations (Whelan, 2008).Linolenic acid belongs to omega-3 fatty acid.A previous study reported that α-linolenic acid (ALA) inhibited the human renal cell carcinoma (RCC) cell proliferation (Yang et al., 2013).Another study reported that apoptosis of hepatoma cells was induced by the α-linolenic acid enriched diet which correlated with a decrease in arachidonate content in hepatoma cells and decreased cyclooxygenase-2 expression (Vecchini et al., 2004).γ-Linolenic acid (GLA) and α-linolenic acid (ALA) exhibited greater than 90% cytotoxicity between 500 μM and 1 mM against all but two malignant microorgan cultures tested in 5-10% serum.GLA and ALA killed tumor at concentrations of 2 mM and above in tests using 30-40% serum (Scheim, 2009).