Vincent Antonio S. Ng

Cytotoxic Triterpenes and Sterols from Pipturus arborescens (Link) C.B. Rob.

The triterpenes, squalene (1), friedelin (2) and a mixture of ursolic acid (3a) and oleanolic acid (3b) in a 2:3 ratio, and a mixture of β-sitosterol (4a) and stigmasterol (4b) in a 2:1 ratio, obtained from the dichloromethane extract of Pipturus arborescens (Link) C.B. Rob., were evaluated for their anti-proliferative activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal fibroblast- neonatal (HDFn) using the in vitro PrestoBlue� cell viability assay. The HCT-116 cell line was most susceptible to the compounds and mixtures tested. Triterpene 1 was most cytotoxic against HCT-116 and MCF-7 with IC50 values of 4.21 and 5.92 μg/mL, respectively. Triterpene 2 and the mixture of 3a and 3b were highly anti-proliferative against HCT-116 cells (IC50 of 1.22 and 1.66 μg/mL, respectively) and moderately inhibitory against MCF-7 cells (IC50 of 16.51 and 23.97 μg/mL, respectively). The mixture of 4a and 4b exhibited high cytotoxicity against HCT-116 cells (IC50 of 1.14 μg/mL). Compounds 1-4b showed the least activity against HT-29 cells (IC50 of 11.97 to 52.52 μg/mL). Cytotoxic effect was not observed against HDFn cells (>100 μg/mL). Comparing the effects of 1-4b on the two colon cancer cell lines, the IC50 values of 1-4b against HCT-116 were lower than those of HT-29.

Cytotoxic Compounds from Wrightia pubescens (R.Br.)

Background: Mixtures of ursolic acid (1) and oleanolic acid (2) (1:1 and 1:2), oleanolic acid (2), squalene (3), chlorophyll a (4), wrightiadione (5), and α-amyrin acetate (6) were isolated from the dichloromethane (CH2Cl2) extracts of the leaves and twigs of Wrightia pubescens (R.Br.). Objectives: To test for the cytotoxicity potentials of 1–6. Materials and Methods: The antiproliferative activities of 1–6 against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal fibroblast neonatal (HDFn), were evaluated using the PrestoBlue® cell viability assay. Results: Compounds 4, 1 and 2 (1:2), 2, 1 and 2 (1:1), and 5 exhibited the most cytotoxic effects against HT-29 with half maximal inhibitory concentration (IC50) values of 0.68, 0.74, 0.89, 1.70, and 4.07 μg/mL, respectively. Comparing 2 with its 1:1 mixture with 1 (IC50 = 1.70 and 7.18 μg/mL for HT-29 and HCT-116, respectively) and 1:2 mixture with 1 (0.74 and 3.46 μg/mL for HT-29 and HCT-116, respectively), 2 also showed strong cytotoxic potential against HT-29 and HCT-116 (0.89 and 2.33 μg/mL, respectively). Unlike the mixtures which exhibited low effects on MCF-7 (IC50 = 20.75 and 30.06 μg/mL for 1:1 and 1:2, respectively), 2 showed moderate activity against MCF-7 (10.99 μg/mL). Compound 6 showed the highest cytotoxicity against HCT-116 (IC50 = 4.07 μg/mL). Conclusion: Mixtures of 1 and 2 (1:1 and 1:2), 2, 3, 4, 5, and 6 from the CH2Cl2extracts of the leaves and twigs of W. pubescens (R.Br.) exhibited varying cytotoxic activities. All the compounds except 6 exhibited the strongest cytotoxic effects against HT-29. On the other hand, 6 was most cytotoxic against HCT-116. Overall, the toxicities of 1–6 were highest against HT-29, followed by HCT-116 and MCF-7. All the compounds showed varying activities against HDFn (IC50 < 30 μg/mL). Abbreviation Used: IC50: Half maximal inhibitory concentration.

Cytotoxic Compounds from Kibatalia gitingensis (Elm.) Woodson

Ursolic acid (1), squalene (2), a mixture of α-amyrin acetate (3a) and lupeol acetate (3b), and isoscopoletin (4), isolated from the dichloromethane extracts of the leaves and twigs of Kibatalia gitingensis, were evaluated for their cytotoxic activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal fibroblast-neonatal (HDFn), using the in vitro PrestoBlue® cell viability assay. Compounds 1-4 exhibited strong cytotoxic activities against HT-29 cells with IC50 values ranging from 0.6931 to 1.083 μg/mL. Furthermore, 1-4 were moderately cytotoxic against HCT-116 cells with IC50 values ranging from 4.065 to 11.09 μg/mL. These compounds were least cytotoxic against MCF-7 cells with IC50 values ranging from 8.642 to 25.87 μg/mL. The most cytotoxic against HT-29 cells, HCT-116 cells and MCF-7 cells are 2, 4 and 1, respectively.

Chemical Constituents of Cycas Zambalensis

Cycas, the only currently known genus of the family Cycadaceae, are considered fossil plants though they may have evolved only about 12 million years ago [1]. Their long existence and persistence through time have sparked special interest in their biology and evolution. The cycads resemble palms in morphology and thus are commonly called sago palm. They bear naked seeds and are dioecious (male and female as separate individuals). They are widely distributed in the Tropics, with species found in Asia, Africa, Southeast Asia, the Pacific, and Australia [2]. They also grow on volcanic, limestone, ultramafic, sandy, or even water-logged soils in grassland and forest habitats [3]. This study was conducted as part of our research on the chemical constituents of endemic Philippine plants. We earlier reported the chemical constituents of Cinnamomum utile [4], C. griffithii [5], C. rupestre, C. nanophyllum [6], C. trichophyllum [7], Ardisia squamulosa [8], Ficus linearifolia, and F. triangularis [9]. Chemical investigation of Cycas zambalensis Madulid & Agoo, a plant endemic to the Philippines, led to the isolation of dihydrodehydrodiconiferyl alcohol (1), squalene (2), -carotene (3), chlorophyllide a (4), and lutein (5) from the leaflets; 2, 5, balanophonin (6), and -sitosterol (7) from the petiole and rachis; 7, isopimaran-19-ol (8), and 3-oxoisopimara-7,15-diene (9) from the bark; 1, 2, 7, and stigmasterol (10) from the roots; 2, 3, 5, and 7 from the sarcotesta; and 7 from the endotesta. The structures of 1, 6, 8, and 9 were elucidated by extensive 1D and 2D NMR spectroscopy. This is the first report on the occurrence of 6, 8, and 9 in the genus Cycas and the family Cycadaceae. This is also the first study on the chemical constituents of C. zambalensis. NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for 1H NMR and 150 MHz for 13C 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 F254, and the plates were visualized by spraying with vanillin– H2SO4 solution followed by warming. Cycas zambalensis leaflets, petiole and rachis, bark, roots, sarcotesta, and endotesta were collected from Pundaquit, San Antonio, Zambales, Philippines on April 9, 2013. Voucher specimens were collected and authenticated by one of the authors (EMGA) and deposited in the De La Salle University-Manila Herbarium (DLSU-M 3111). The air-dried leaflets of C. zambalensis (600.0 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 (14.8 g) which was chromatographed using increasing proportions of acetone in CH2Cl2 at 10% increment. The CH2Cl2 fraction was rechromatographed in petroleum ether to afford 2 (9 mg) and 3 (6 mg) after washing with petroleum ether. The 40–50% acetone in CH2Cl2 fractions were rechromatographed (5 ) in 20% EtOAc in petroleum ether to afford 4 after washing with petroleum ether, followed by Et2O. The 60% acetone in CH2Cl2 fraction was rechromatographed (3 ) in CH3CN–Et2O–CH2Cl2 (1:1:8 by volume) to afford 5 (10 mg) after washing with petroleum ether, followed by Et2O. The 90% acetone in CH2Cl2 fraction was rechromatographed (4 ) in CH3CN–Et2O–CH2Cl2 (2:2:6 by volume) to afford 1 (7 mg) after washing with petroleum ether, followed by Et2O.

Terpenoids and Sterols from Cycas vespertilio

Cycas vespertilio A. Lindstr. & K. D. Hill of the family Cycadaceae is one of the 12 cycad species found in the Philippines. Ten of these species are endemic to the Philippines, namely C. aenigma K. D. Hill & Lindstrom, C. curranii (J. Schust.) K. D. Hill, C. lacrimans Lindstrom & K. D. Hill, C. nitida K. D. Hill & Lindstrom, C. riuminiana Porte ex Regel, C. saxatilis K. D. Hill & Lindstrom, C. sancti-lasallei Agoo & Madulid, C. wadei Merr., C. vespertilio Lindstrom & K. D. Hill, and C. zambalensis Madulid & Agoo [1–3]. C. edentata de Laub. is not Philippine endemic, while C. revoluta is an introduced species from Japan and Taiwan. We earlier reported the isolation of pinoresinol, lariciresinol, mixtures of -amyrin acetate and lupeol acetate, -sitosterol and stigmasterol, triglycerides, and fatty alcohols from the male cone of C. vespertilio [4]. We also reported the isolation of pinoresinol, sesamin, paulownin, a mixture of -sitosterol and stigmasterol, triacylglycerols, and lariciresinol from the cataphylls; -sitosterol from the megasporophyll lamina; -sitosterol and a mixture of trans-4-hydroxycinnamate fatty acid esters and cis-4-hydroxycinnamate fatty acid esters from unripe sarcotesta, and -sitosterol and triacylglycerols from the ripe sarcotesta of C. vespertilio [5]. Chemical investigation of Cycas vespertilio, a plant endemic to the Philippines, led to the isolation of 9 H-isopimara-7,15-diene (1), squalene (2), -sitosterol (3), and stigmasterol (4) from the bark; 2, 3 and phytyl fatty acid ester (5) from the petiole and rachis; 3–5 and triglycerides (6) from the endotesta; 1–3, 6 and adianenone (7) from the roots; 3, 4, 6 and chlorophyll a from the leaflets; and 3 and 6 from the sclerotesta. The structures of 1, 5, and 7 were elucidated by extensive 1D and 2D NMR spectroscopy. This is the first report on the occurrence of 7 in the genus Cycas and the family Cycadaceae. NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for 1H NMR and 150 MHz for 13C 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 F254, and the plates were visualized by spraying with vanillin/H2SO4 solution followed by warming. Cycas vespertilio A. Lindstr. & K. D. Hill bark, petiole and rachis, endotesta, roots, leaves, and sclerotesta were collected from Iloilo, Panay Island, Philippines in April 2013. Voucher specimens were collected and authenticated by one of the authors (EMGA) and deposited in the De La Salle University-Manila Herbarium (DLSUH 3112). The crude extracts were fractionated by silica gel chromatography eluted with increasing proportions of acetone in CH2Cl2 at 10% increment. A glass column 18 inches in height and 1.0 inch internal diameter was used for the fractionation of the crude extracts. Ten-milliliter fractions were collected. 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.

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.