Triterpenes and Acylglycerols from Canarium ovatum

Consolacion Y. Ragasa, Oscar B. Torres, Jessa Mae P. Gutierrez, Hannah Patricia Beatriz C. Kristiansen, and Chien-Chang Shen Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna 4024, Philippines Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines. National Research Institute of Chinese Medicine, 155-1, LiNong St., Sec. 2, Taipei 112, Taiwan.


INTRODUCTION
Canarium ovatum Engl. of the family Burseraceae and locally known as pili is indigenous to the Philippines (Brown, 1954).It is the most important nut producing tree in the Philippines (Coronel, 1996) where the roasted or candied nuts are sold commercially (Pili, 2014).The young shoots are edible, while the green pulp can be pickled (Pili, 2014).The oil from the pulp is used for cooking and lighting (Brown, 1954).The tree is also used as lumber and fuel (Coronel, 1996).The C. ovatum resin is employed as an ointment for healing wounds and as a plaster, while raw nuts are used as purgative (Canarium ovatum, 2014).The roasted and unroasted pili nut oil scavenged DPPH radicals by 24.66% and 9.52%, respectively at a concentration of 140μg/mL (Zarinah et al., 2014).A recent study reported that the This study is part of our research on the chemical constituents of Canarium species found in the hilippines.We earlier reported the isolation of a mixture of new triterpene diastereomers, asperol a and asperol b, and the major triterpene constituents, β-amyrin and α-amyrin from Canarium asperum resin (Ragasa et al., 2014a).We report herein the isolation from the dichloromethane extracts of the leaves of C. ovatum of a mixture of β-amyrin (1a) and α-amyrin (1b) in a 1:3 ratio, another.mixture of epi-β-amyrin (2a), epi-α-amyrin (2b) and epi-lupeol (2c) in a 2:1:0.5 ratio, β-carotene (3) and lutein (4), while the twigs yielded a mixture of 1a and 1b in a 1:2 ratio.The dichloromethane extracts of the nuts and pulp of C. ovatum yielded triacylglycerols (5); the mesocarp also afforded 1a, 1b, 1,2-dioleylglycerol (6) (Fig. 1) and a mixture of monounsaturated and saturated fatty acids in a 3:2 ratio; the nutshell also provided 6; and the kernel also yielded a mixture of monounsaturated and saturated fatty acid in a 2:1 ratio.To the best of our knowledge this is the first report on the chemical constituents of the leaves, twigs and fruits of C. ovatum.Previous studies were conducted on the chemical constituents of the oil (Kakuda et al., 2000) and methanol extract of the resin (Kikuchi et al., 2012) of C. ovatum.

General Experimental Procedure
NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl 3 at 500 MHz for 1 H NMR; and 125 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.

General Isolation Procedure
A glass column 20 inches in height and 2.0 inches internal diameter was packed with silica gel.The crude extracts were fractionated by silica gel chromatography using increasing proportions of acetone in dichloromethane (10% increment) as eluents.
One hundred 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.Five milliliter fractions were collected.Final purifications were conducted using Pasteur pipettes as columns.One milliliter fractions were collected.

Sample collection and preparation
The leaves and stems of Canarium ovatum were collected from the De La Salle University-Manila campus in July 2012.The fruit sample was collected from the province of Camarines Norte, Philippines in September 2012.It was identified as Canarium ovatum Engl.at the Bureau of Plant Industry in San Andres, Malate, Manila, Philippines.
The leaves and stems of C. ovatum were air-dried for about two weeks.The whole fruit of C. ovatum was separated into mesocarp (thick flesh), nutshell and kernel.The mesocarp and kernel were separately ground in a blender and freeze-dried.The nutshell was ground in a mortar and pestle and air-dried.

Isolation of the chemical constituents of the leaves
The air-dried leaves (250 g) was soaked in CH 2 Cl 2 for three days and then filtered.The filtrate was concentrated under vacuum to afford a crude extract (16 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 petroleum ether to afford 3 (3 mg).The 20% and 30% acetone in CH 2 Cl 2 fractions were combined and rechromatographed (4 ×) in 15% EtOAc in petroleum ether to afford a mixture of 1a-1b (7 mg) and another mixture of 2a-2c (5 mg) after washing with petroleum ether.The 60% acetone in CH 2 Cl 2 fraction was rechromatographed (5×) in Et 2 O:CH 3 CN:CH 2 Cl 2 (0.5:0.5:9, v/v) to yield 4 (7 mg).

Isolation of the chemical constituents of the twigs
The air-dried twigs (105 g) was soaked in CH 2 Cl 2 for three days and then filtered.The filtrate was concentrated 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 20% CH 2 Cl 2 fraction was rechromatographed (4×) using 5% EtOAc in petroleum ether to afford a mixture of 1a and 1b (4 mg) after washing with petroleum ether.

Isolation of the chemical constituents of the kernel
The freeze-dried kernel (190 g) was soaked in CH 2 Cl 2 for three days and then filtered.The filtrate was concentrated under vacuum to afford a crude extract (48 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.The 10% and 20% acetone in CH 2 Cl 2 fractions were combined and rechromatographed (3×) using 5% EtOAc in petroleum ether to afford 5 (25 mg).The 30% acetone in CH 2 Cl 2 fraction was rechromatographed (2×) using 10% EtOAc in petroleum ether to afford a mixture of monounsaturated and saturated fatty acids (10 mg).

Isolation of the chemical constituents of the nutshell
The air-dried nutshell (953 g) was soaked in CH 2 Cl 2 for three days and then filtered.The filtrate was concentrated under vacuum to afford a crude extract (9 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.

Isolation of the chemical constituents of the mesocarp
The freeze-dried mesocarp (707 g) was soaked in CH 2 Cl 2 for three days and then filtered.The filtrate was concentrated under vacuum to afford a crude extract (27.3 g) which was chromatographed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment.
Silica gel chromatography of the dichloromethane extracts of the kernel, nutshell, mesocarp and endocarp of Canarium ovatum Engl.yielded triacylglycerols (5) (Ragasa et al., 2013).Based on the integrations of the triacylglycerol (5) protons in the nutshell, the fatty acids attached to the glycerol are oleic acid (Human Metabolome, 2014b), linoleic acid (Human Metabolome, 2014a) and saturated fatty acid, possibly palmitic acid (Human Metabolome, 2014c).Oleic acid, linoleic acid and palmitic acid were reported as major constituents of C. ovatum oil (Kakuda et al., 2000).Based on the integrations of the triacylglycerol (5) protons in the mesocarp and kernel, the fatty acids attached to the glycerol are oleic acid (2 ×) and palmitic acid.The mesocarp also yielded 1,2-diacylglycerol (6) (Vlahov, 1999;Ragasa et al., 2005) and a mixture of monounsaturated and saturated fatty acid.The fatty acid esterified to the 1,2diacylglycerol ( 5) is oleic acid (Human Metabolome, 2014b) as deduced from the integrations of the 1 H NMR resonances of the olefinic protons at δ 5.33, the allylic protons at δ 1.97-2.04,the αmethylene protons at δ 2.32 and the terminal methyl protons at δ 0.86.Thus, 6 is 1,2-dioleylglycerol which was confirmed by comparison of its 13 C NMR data with literature data (Vlahov, 1999).Compound 6 was also obtained from the nutshell.The 3:2 ratio of the monounsaturated and saturated fatty acids obtained from the mesocarp was deduced from the integrations of the olefinic acid proton resonances at δ 5.33, the allylic protons at δ 2.0, the α-methylene protons at δ 2.30 and the terminal methyl protons at δ 0.87 for the monounsaturated fatty acid and the αmethylene protons at δ 2.30 and the terminal methyl protons at δ 0.87 for the saturated fatty acid.The monounsaturated fatty acid is possibly oleic acid, while the saturated fatty acid is possibly palmitic acid.Both fatty acids were reported as major constituents of C. ovatum oil (Kakuda et al., 2000).The kernel also afforded oleic acid and palmitic acid in a 2:1 ratio.
Although no biological activity tests were conducted on the isolated compounds (1-6 and fatty acids), literature search revealed that these have diverse biological activities as follows.
β-Carotene (3) dose-dependently induced apoptosis and cell differentiation in cultured leukemia cells, but not in normal cells (Upadhyaya et al., 2007).Another study reported that βcarotene could reduce damage caused by radiation therapy and decrease local cancer recurrence (Meyer et al., 2007).It also inhibited angiogenesis by altering the cytokine profile and the activation and nuclear translocation of transcription factors (Guruvayoorappan and Kuttan, 2007).
Dietary lutein (4), especially at 0.002%, inhibited tumor growth by selectively modulating apoptosis, and by inhibiting angiogenesis (Chew et al., 2003).Another study reported that the chemopreventive properties of all-trans retinoic acid and lutein may be attributed to their differential effects on apoptosis pathways in normal versus transformed mammary cells (Sumatran et al., 2000).Moreover, very low amounts of dietary lutein (0.002%) can efficiently decrease mammary tumor development and growth in mice (Park et al., 1998).Another study reported that lutein and zeaxanthine reduces the risk of age related macular degeneration (SanGiovanni, 2007).Triacylglycerols (5) 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).sn-1,2-Diacylglycerols (6) modulate vital biochemical mechanisms since they function as second messengers in many cellular processes (Christie, 2013).Another study reported that the directed migration of leukocytes can be stimulated by 1,2-diacylglycerol (Wright et al., 1988).
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).A recent study reported that oleic acid, a monounsaturated fatty acid inhibited cancer cell growth and survival in gastric carcinoma SGC7901 and breast carcinoma MCF-7 cell lines (Li et al., 2014).Another study demonstrated that oleic acid promotes apoptotic cell death of breast cancer cells (Menendez et al., 2005).It was also shown to be effective at depressing lipogenesis and cholesterologenesis (Natali et al., 2007).Furthermore, it may contribute to the prevention of atherogenesis (Carluccio et al., 1999).Monounsaturated fatty acids were reported to lower total and LDL cholesterol levels, increase HDL cholesterol levels and decrease plasma triglyceride levels (Kris-Etherton, 1999).Palmitic acid, a saturated fatty acid showed selective cytotoxicity to human leukemic cells, induced apoptosis in the human leukemic cell line MOLT-4 and exhibited in vivo antitumor activity in mice (Harada et al., 2002).

CONCLUSION
The leaves of C. ovatum afforded triterpenes and carotenoids; the twigs yielded triterpenes; and the fruits gave diacylglycerol, fatty acids and triacylglycerols with varying fatty acid compositions.The compounds obtained from the leaves, twigs and fruits of C. ovatum were reported to exhibit diverse biological activities.