Chemical Constituents of Strongylodon Macrobotrys
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Available online a t www.derpharmachemica.com Scholars Research Library Der Pharma Chemica, 2014, 6(6):366-373 (http://derpharmachemica.com/archive.html) ISSN 0975-413X CODEN (USA): PCHHAX Chemical constituents of Strongylodon macrobotrys Consolacion Y. Ragasa 1,2* , Virgilio D. Ebajo Jr. 2, Vincent Antonio S. Ng 2, Mariquit M. De Los Reyes 3,4 and Chien-Chang Shen 5 1Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna, Philippines 2Chemistry Department, De La Salle University, Taft Avenue, Manila, Philippines 3Biology Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna, Philippines 4Biology Department, De La Salle University, Taft Avenue, Manila, Philippines 5National Research Institute of Chinese Medicine, 155-1, Li-Nong St., Sec. 2, Taipei, Taiwan _____________________________________________________________________________________________ ABSTRACT Chemical investigation of the dichloromethane extracts of Strongylodon macrobotrys led to the isolation of taraxerone ( 1), stigmasterol ( 2), β-sitosterol (3), and triglycerides (4) from the stems; 2, 3, and β-stigmasteryl 3-O- β-D-glucopyranoside (5) from the flowers; and polyprenol (6), lutein (7), squalene ( 8) and chlorophyll a (9) from the leaves. The structures of these compounds were identified by comparison of their 13 C NMR data with those reported in the literature. Keywords: Strongylodon macrobotrys, Leguminosae, taraxerone, stigmasterol, β-sitosterol, β-stigmasteryl 3-O-β- D-glucopyranoside, polyprenol, lutein, squalene, chlorophyll a _____________________________________________________________________________________________ INTRODUCTION Strongylodon macrobotrys A. Gray, of the family Leguminosae, commonly known as jade vine or emerald creeper and locally known as “tayabak”, is a leguminous perennial woody vine that can reach up to 18 m in length. It is native to the Philippines, thriving best in tropical forests, along streams or in ravines from 700 to 1,000 m asl [1, 2]. It is considered as one of the most beautiful of all tropical climbers because of its elegant and striking turquoise flowers that dangle in mid-air when in full bloom. In the Philippines, jade vine is cultivated as an ornamental plant. Naturally pollinated by bats, the destruction of rainforests in the Philippines threatens S. macrobotrys in the wild resulting in the plant being listed as vulnerable in the IUCN list of threatened species [3]. There are few studies on the chemical constituents of S. macrobotrys . An earlier study reported that the major visible pigment in the jade vine flower is the anthocyanin, malvidin 3,5-di-O-glucoside, accompanied with C- glycosylflavones, isovitexin 7-O-glucoside and isovitexin [4]. Recently, the flower color of S. macrobotrys which is luminous blue green was attributed to a mixture of an anthocyanin, malvin, and a flavone, saponarin, in approximately 1:9 molar ratio [5]. The isolation and identification of two saponins, the dimethyl esters of pseudoginsenoside-RP1 and zingibroside-R1 from the seeds of S. macrobotrys were also reported [6]. There is no reported study on the biological activity of jade vine. 366 www.scholarsresearchlibrary.com Consolacion Y. Ragasa et al Der Pharma Chemica, 2014, 6 (6):366-373 ___________________ __________________________________________________________ This study is part of our research on the chemical constituents of endemic and native Philippine ornamental plants. Our chemical investigation of Hoya mindorensis , an endemic ornamental plant of the Philippines, led to the isolation of lupenone and lupeol from the roots; lupeol, squalene and β-sitosterol from the leaves; and betulin from the stems of the plant. Except for lupenone, all the isolated secondary metabolites from H. mindorensis are known anticancer compounds [7]. We report herein the isolation and identification of taraxerone ( 1), a mixture of stigmasterol ( 2) and β-sitosterol ( 3) in a 2:1 ratio, and triglycerides ( 4) from the stems; a mixture of 2 and 3 in a 3:1 ratio and stigmasterol-β-D- glucoside ( 5) from the flowers; and polyprenol ( 6), lutein ( 7), squalene ( 8) and chlorophyll a ( 9) from the leaves (Fig. 1) of S. macrobotrys . To the best of our knowledge this is the first report on the isolation of these compounds from the plant. 30 20 27 18 18 25 11 14 28 1 9 3 5 O HO 2 HO 3 23 1 O 28 21 CH2OCR O 6' 20 23 25 HOH2C 19 11 13 CHOCR' HO 16 O O HO 3' 1 9 1' O 3 5 CH2OCR" OH 5 4 R, R', R" = long chain fatty acids OH [ ]n [ ]3 6 18' 17 16 OH 20 5' 3' 15 13' 1' 1 13 9' 6 15' 3 5 16' 17' HO 18 7 2b 2a CH3 alpha 2 3 4a 1a 13 4b H3C 1 12 4 N 14 N beta 11 delta Mg N 15 12 13 N 5 14 18 16 5a H 8 17 5 gamma CH3 10 7 6 10 9 3 7 H3C 8a H H 10a C O 7b H3C O P3a P7a P11a P15a 15 1 8 10b O 7c O C O P1 P3 P7 P11 P15 P16 9 Fig. 1. Chemical constituents of Strongylodon macrobotrys : taraxerone (1), stigmasterol (2), β-sitosterol (3), triglycerides (4), β- stigmasteryl 3-O-β-D-glucopyranoside (5), polyprenol (6), lutein (7), squalene (8) and chlorophyll a (9). 367 www.scholarsresearchlibrary.com Consolacion Y. Ragasa et al Der Pharma Chemica, 2014, 6 (6):366-373 ___________________ __________________________________________________________ MATERIALS AND METHODS Sample Collection Samples of leaves, twigs and flowers of Strongylodon macrobotrys A. Gray were a generous gift collected from the Center for Ecozoic Living and Learning (CELL), Silang, Cavite in May 2014. The samples were authenticated at the Botany Division of the National Museum, Manila and deposited with voucher # 268-2014. General Experimental Procedure 1 NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl 3 at 600 MHz for 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 2SO 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 extract from the leaves 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 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. Five milliliter fractions were collected. Final purifications were conducted using Pasteur pipettes as columns. One milliliter fractions were collected. Isolation The air-dried stems of S. macrobotrys (40.8 g) was ground in a blender, soaked in CH 2Cl 2 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 2Cl 2 at 10% increment. The CH 2Cl 2 fraction was rechromatographed (3 ×) using 5% EtOAc in petroleum ether to afford 1 (5 mg) after washing with petroleum ether. The 30% acetone in CH 2Cl 2 fraction was rechromatographed (2 ×) in 5% EtOAc using petroleum ether to afford 4 (7 mg). The 40% acetone in CH 2Cl 2 fraction was rechromatographed (4 ×) using CH 3CN:Et 2O:CH 2Cl 2 (0.5:0.5:9, v/v) to afford a mixture of 2 and 3 (9 mg) after washing with petroleum ether. The air-dried flowers of S. macrobotrys (28.5 g) were ground in a blender, soaked in CH 2Cl 2 for 3 days and then filtered. The solvent was evaporated under vacuum to afford a crude extract (0.15 g) which was chromatographed using increasing proportions of acetone in CH 2Cl 2 at 10% increment. The 40% acetone in CH 2Cl 2 fraction was rechromatographed (3 ×) using CH 3CN:Et 2O:CH 2Cl 2 (0.5:0.5:9, v/v) to afford a mixture of 2 and 3 (5 mg) after washing with petroleum ether. The 60% acetone in CH 2Cl 2 was rechromatographed (5 ×) using CH 3CN:Et 2O:CH 2Cl 2 (2:2:6, v/v) to afford 5 (4 mg) after trituration with petroleum ether. The air-dried leaves of S. macrobotrys (69.2 g) was ground in a blender, soaked in CH 2Cl 2 for 3 days and then filtered. The solvent was evaporated under vacuum to afford a crude extract (3.0 g) which was chromatographed using increasing proportions of acetone in CH 2Cl 2 at 10% increment. The CH 2Cl 2 fraction was rechromatographed (2 ×) using 1% EtOAc in petroleum to afford 8 (5 mg). The 20% acetone in CH 2Cl 2 fraction was rechromatographed (3 ×) using 12.5 % EtOAc in petroleum ether to afford 6 (6 mg). The 40% acetone in CH 2Cl 2 fraction was rechromatographed (4 ×) using 20% EtOAc in petroleum ether to afford 9 (8 mg) after washing with petroleum ether, followed by Et 2O. The 60% acetone in CH 2Cl 2 fraction was rechromatographed (5 ×) using CH 3CN:Et 2O:CH 2Cl 2 (1:1:8, v/v) to afford 7 (9 mg) after washing with petroleum ether, followed by Et 2O. 1 Taraxerone (1): H NMR (600MHz, CDCl 3) δ: 5.54 (dd, J = 3.6, 8.4 Hz, H-15), 1.06 (s, H 3-23), 1.05 (s, H 3-24), 13 1.07 (H 3-25), 0.888, 0.894 (s, H 3-26, H 3-30), 1.12 (s, H 3-27), 0.81 (s, H 3-28), 0.93 (s, H 3-29); C NMR (CDCl 3) δ: 38.33 (C-1), 34.14 (C-2), 217.60 (C-3), 47.58 (C-4), 55.76 (C-5), 19.94 (C-6), 35.08 (C-7), 38.86 (C-8), 48.68 (C-9), 35.77 (C-10), 17.43 (C-11), 37.73, 37.67 (C-12, C-13), 157.58 (C-14), 117.18 (C-15), 36.65 (C-16), 37.52 (C-17), 48.76 (C-18), 40.61 (C-19), 28.79 (C-20), 33.55 (C-21), 33.06 (C-22), 26.08 (C-23), 21.33 (C-24), 14.80 (C-25), 29.84 (C-26), 25.56 (C-27), 29.91 (C-28), 33.34 (C-29), 21.48 (C-30).