ISSN: 0975-8585 Research Journal of Pharmaceutical, Biological and Chemical Sciences Chemical Constituents of Polyscias nodosa. Consolacion Y Ragasa1,2,*, Virgilio D Ebajo Jr2, Mariquit M De Los Reyes3,4, Robert Brkljača5, and Sylvia Urban5. 1 Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna 4024, Philippines. 2 Biology Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines. 3 Biology Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna 4024, Philippines. 4 Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines. 5 School of Applied Sciences (Discipline of Chemistry), RMIT University (City Campus), Melbourne 3001, Victoria, Australia. ABSTRACT Chemical investigation of the dichloromethane extracts of Polyscias nodosa (Bl.) Seem. yielded squalene (1), phytyl fatty acid esters (2), lutein (3), a d β-sitosteryl- β-glucopyranoside-6'-O-palmitate (4) from the leaves; and 1, triacylglycerols (5), and a mixture of stigmasterol (6a) and β-sitosterol (6b) in a 5:1 ratio from the twigs. The structures of 1-6b were identified by comparison of their NMR data with those reported in the literature. Keywords: Polyscias nodosa, Araliaceae, squalene, phytyl fatty acid esters, lutein, β-sitosteryl- βglucopyranoside-6'-O-palmitate, stigmasterol, β-sitosterol *Corresponding author September - October 2015 RJPBCS 6(5) Page No. 1210 ISSN: 0975-8585 INTRODUCTION Polyscias nodosa locally known as malapapaya is native to Tropical Asia – Indonesia, Papua New Guinea and the Philippines and the Southwestern Pacific – Solomon Islands [1]. This tree occurs throughout the Philippines where it is commercially used for making woodworks, boxes, pencil slats, chopsticks, matchsticks, ice cream spoons, plywood, native wooden shoes, lollipops, popsicle sticks, toothpicks, and similar articles [2]. Traditionally, the leaves are powdered and applied as fish poison and used medicinally against purpuric fever and as a contraceptive [2]. The only studies conducted on the chemical constituents of P. nodosa were the isolation of saponins from the leaves of the plant [3-5]. We report herein the isolation of squalene (1), phytyl fatty acid esters (2), lutein (3), and β-sitosterylβ-glucopyranoside-6'-O-palmitate (4) from the leaves; and 1, triacylglycerols (5) and a mixture of stigmasterol (6a), β-sitosterol (6b) from the twigs of P. nodosa. To the best of our knowledge this is the first report on the isolation of these compounds from P. nodosa. 17 20 O 1 7 11 3 CH2OCR 1' 16 2 R = long chain fatty acid 1 OH HO 3 O CH2OCR O CHOCR' OH O O HO HO CH2OCR" 4 O 5 O R, R', R" = long chain fatty acids (CH2)14CH3 O HO HO 6b 6a Chemical structures of squalene (1), phytyl fatty acid esters (2), lutein (3), β-sitosteryl- β-glucopyranoside-6'-Opal itate , triacylglycerols , stig asterol a a d β-sitosterol (6b) from P. nodosa. MATERIALS AND METHODS General Experimental Procedure 1 NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for H NMR and 13 150 MHz for C NMR spectra. Column chromatography was performed with silica gel 60 (70-230 mesh). Thin September - October 2015 RJPBCS 6(5) Page No. 1211 ISSN: 0975-8585 layer chromatography was performed with plastic backed plates coated with silica gel F 254 and the plates were visualized by spraying with vanillin/H2SO4 solution followed by warming. Sample Collection Polyscias nodosa (Bl.) Seem. was collected from the De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna, Philippines in April 2014. The sample was authenticated at the Botany Division of the Philippine National Museum with control no. 830. General Isolation Procedure A glass column 18 inches in height and 1.0 inch internal diameter was packed with silica gel. The crude extract from the twigs were fractionated by silica gel chromatography using increasing proportions of acetone in CH2Cl2 (10% increment) as eluents. Fifty milliliter fractions were collected. All fractions were monitored by thin layer chromatography. Fractions with spots of the same Rf 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 of the Chemical Constituents of the Leaves The air-dried leaves of P. nodosa (149.3 g) was ground in a blender, soaked in CH2Cl2 for 3 days and then filtered. The solvent was evaporated under vacuum to afford a crude extract (3.5 g) which was chromatographed using increasing proportions of acetone in CH 2Cl2 at 10% increment by volume. The CH2Cl2 fraction was rechromatographed (3 ×) in petroleum ether to afford 1 (5 mg). The 10% acetone in CH2Cl2 fraction was rechromatographed using 5% EtOAc in petroleum ether (2 ×) to afford 2 (2 mg). The 50% acetone in CH2Cl2 fraction was rechromatographed (4 ×) using CH 3CN:Et2O:CH2Cl2 (0.5:0.5:1 by volume ratio) to afford 3 (3 mg) after washing with petroleum ether, followed by Et 2O. The 60% acetone in CH2Cl2 fraction was rechromatographed (4 ×) using CH3CN:Et2O:CH2Cl2 (2:2:6 by volume ratio) to afford 4 (2 mg) after trituration with petroleum ether. Isolation of the Chemical Constituents of the Twigs The air-dried twigs of P. nodosa (123 g) were ground in a blender, soaked in CH2Cl2 for 3 days and then filtered. The solvent was evaporated under vacuum to afford a crude extract (1.5 g) which was chromatographed using increasing proportions of acetone in CH 2Cl2 at 10% increment by volume. The CH2Cl2 fraction was rechromatographed (3 ×) in petroleum ether to afford 1 (3 mg). The 20% acetone in CH2Cl2 fraction was rechromatographed (2 ×) using 5% EtOAc in petroleum ether (2 ×) to afford 5 (4 mg). The 40% acetone in CH2Cl2 fraction was rechromatographed using 20% EtOAc in petroleum ether (3 ×) to afford a mixture of 6a and 6b (5 mg) after washing with petroleum ether. RESULTS AND DISCUSSION Silica gel chromatography of the dichloromethane extract of P. nodosa afforded squalene (1) [6], phytyl fatty acid esters (2) [7], lutein (3) [8], and β-sitosteryl- β-glucopyranoside-6'-O-palmitate (4) [9] from the leaves; and 1, triacylglycerols (5) [6], and a mixture of stigmasterol (6a) [10] and β-sitosterol (6b) [10] in a 1 5:1 ratio from the twigs. The structures of 1-6b were identified by comparison of their H NMR data with those reported in the literature [6-10]. Although no biological activity tests were conducted on the isolated compounds, a literature search of 1, 3, 4, 6a and 6b revealed that these have a range bioactivities. 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 [11]. It showed cardioprotective effect which is related to inhibition of lipid accumulation by its hypolipidemic properties and/or its antioxidant properties [12]. A recent study reported that tocotrienols, carotenoids, squalene and coenzyme Q10 have anti-proliferative effects on breast cancer cells [13]. The preventive and September - October 2015 RJPBCS 6(5) Page No. 1212 ISSN: 0975-8585 therapeutic potential of squalene containing compounds on tumour promotion and regression have been reported [14]. A recent review on the bioactivities of squalene has been provided [15]. Dietary lutein (3), especially at 0.002%, inhibited tumor growth by selectively modulating apoptosis, and by inhibiting angiogenesis [16]. 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 [17]. Moreover, very low amounts of dietary lutein (0.002%) can efficiently decrease mammary tumor development and growth in mice [18]. Another study reported that lutein and zeaxanthine reduces the risk of age related macular degeneration [19]. β-Sitosteryl- β-glucopyranoside-6'-O-palmitate (4) was reported to exhibit cytotoxicity against Bowes (melanoma) and MCF7 (breast) cancer cell lines with IC50 values of 152 mM and 113 mM, respectively [9]. Furthermore, 4 exhibited cytotoxicity against human stomach adenocarcinoma (AGS) cell line with 60.28% growth inhibition [20]. In search of substances that inhibit the hemolytic activity of human serum against erythrocytes, 4 was evaluated on its anti-complement activity. Compound 4 was found to exhibit potent anticomplement activity (IC50 = . ± . μM) o the classical pathway of the co ple e t, as co pared to the positive control, tiliroside (IC50 = . ± . μM) [ ]. Stigmasterol (6a) shows therapeutic efficacy against Ehrlich ascites carcinoma bearing mice while conferring protection against cancer induced altered physiological conditions [22]. 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 [23]. Other studies reported that 6a showed cytostatic activity against Hep-2 and McCoy cells [24], markedly inhibited tumour promotion in two stage carcinogenesis experiments [25], exhibited antimutagenic [26], topical anti-inflammatory [27], anti-osteoarthritic [28] and antioxidant [29] activities. β-Sitosterol (6b) was reported to exhibit growth inhibitory effects on human breast MCF-7 and MDAMB-231 adenocarcinoma cells [30]. It was shown to be effective for the treatment of benign prostatic hyperplasia [ ]. It atte uated β-catenin and PCNA expression, as well as quenched radical in-vitro, making it a potential anticancer drug for colon carcinogenesis [32]. It was reported to induce apoptosis mediated by the activation of ERK and the downregulation of Akt in MCA-102 murine fibrosarcoma cells [33]. It can inhibit the expression of NPC1L1 in the enterocytes to reduce intestinal cholesterol uptake [34]. ACKNOWLEDGEMENT A research grant from the De La Salle University Science Foundation through the University Research Coordination Office is gratefully acknowledged. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?29323. Downloaded on July 19, 2014. rafi.org.ph/greenin-philippines/green-almanac/malapapaya. Downloded on July 19, 2015. van der Haar AW. Archiv der Pharmazie 1913; 250: 424-35. van der Haar AW. Archiv der Pharmazie 1909; 247: 213-20. Van Der Haar AW. Pharmaceutisch Weekblad 1909; 45: 1184-91. Ragasa CY, Ng VAS, De Los Reyes MM, Mandia EH, Oyong GG, Shen C-C. Der Pharma Chemica 2014; 6(5): 182-187. Ng VAS, Agoo EM, Shen C-C, Ragasa CY. J Appl Pharm Sci 2015; 5(Suppl. 1): 12-17. Ragasa CY, Torres OB, Gutierrez JMP, Kristiansen HPBC, Shen C-C. J Appl Pharm Sci 2015; 5(4): 94-100. Nguyen AT, Malonne H, Duez P, Vanhaelen-Fastre R, Vanhaelen M, Fontaine J. Fitoter 2004; 75:500 504. Ng VAS, Agoo EM, Shen C-C, Ragasa CY. Int J Pharmacog Phytochem Res 2015; 7(4): Rao CV, Mark HLN, Reddy BS. Carcinogenesis 1998; 19: 287-290. Farvin KHS, Anandan R, Hari S, Kumar S, Shing KS, Mathew S, Sankar TV, Nair PGV. J Med Food 2006; 9(4): 531-536. Loganathan R, Selvaduray KR, Nesaretnam K, Radhakrisnan A. J Oil Palm Res 2013; 25: 208-215. September - October 2015 RJPBCS 6(5) Page No. 1213 ISSN: 0975-8585 [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] Desai KN, Wei H, Lamartiniere CA. Cancer Lett 1996; 101: 93-96. Ronco AL, De Stéfani E. Functional Foods in Health and Disease 2013; 3: 462-476. Chew BP, Brown CM, Park JS, Mixter PF. Anticancer Res 2003; 23(4): 3333-3339. Sumantran VN, Zhang R, Lee DS, Wicha MS. Cancer Epidemiol Biomarkers Prev 2000; 9: 257-263. Park JS, Chew BP, Wong TS. J Nutr 1998; 128(10): 1650–1656. SanGiovanni JP, Chew EY, Clemons TE. Arch Ophthalmol 2007; 125(9): 1225–1232. Tsai P-W, de Castro-Cruz K, Shen C-C, Ragasa CY. Pharm Chem J 2012 ; 46(4):225-227. Yoon NY, Min BS, Lee HK, Park JC, Choi JS. Arch Pharmacal Res 2005; 28(8):892-896. Ghosh T, Maity TK, Singh J. Orient Pharm Exp Med 2011; 11: 41–49. Batta AK, Xu G, Honda A, Miyazaki T, Salen G. Metabolism 2006; 55(3): 292–299. Gómez MA, García MD, Sáenz MT. Phytother Res 2001; 15(7): 633–634. Kasahara Y, Kumaki K, Katagiri S, Yasukawa K, Yamanouchi S, Takido M. Phytother Res 1994; 8(6): 327–331. Chu LJ, Hee PJ, Milos B, Alexander K, Hwan HY, Byung-Soo K. Chem Pharm Bull 2005; 53(5): 561–564. García MD, Sáenz MT, Gómez MA, Fernández MA. Phytother Res 1999; 13(1): 78–80. Gabay O, Sanchez C, Salvat C, Chevy F, Breton M, Nourissat G. Osteoarthritis Cartilage 2010; 18(1): 106–116. Panda S, Jafri M, Kar A, Meheta BK. Fitoter 2009; 80(2): 123–126. Awad AB, Chinnman M, Fink CS, Bradford PG. Phytomed 2007; 14: 747–754. Jayaprakasha GK, Mandadi KK, Poulose SM, Jadegoud Y, Gowda GA, Patil BS. Bioorg Med Chem 2007; 15; 4923–4932. Baskar AA, Ignacimuthu S, Paulraj G, Numair K. BMC Comp Alt Med 2010; 10: 24. Moon DO, Kyeong-Jun L, Yung HC, Gi-Young K. Int Immunopharmacol 2007; 7: 1044–1053. ED Jesch, JM. Seo, TP. Carr, J. Y. Lee. Nutr Res 2009; 29(12): 859–66. September - October 2015 RJPBCS 6(5) Page No. 1214