Esperanza Maribel G. Agoo

Chemical constituents of Cinnamomum cebuense

AIM To investigate the chemical constituents of Cinnamomum cebuense, an endemic and critically endangered tree found only in Cebu, Philippines. METHODS The compounds were isolated by silica gel chromatography. The structures of the isolates were elucidated by NMR spectroscopy. RESULTS The dichloromethane (DCM) extract of the bark of C. cebuense afforded a new monoterpene natural product 1 and a new sesquiterpene 2, along with the known compounds, 4-hydroxy-3-methoxycinnamaldehyde (3), 4-allyl-2-methoxyphenol (4), α-terpineol (5) and humulene (6). The DCM extract of the leaves of C. cebuense yielded 6, β-caryophyllene (7), squalene (8), and a mixture of α-amyrin (9), β-amyrin (10) and bauerenol (11). The structures of 1-7 were elucidated by extensive 1D and 2D NMR spectroscopy, while the structures of 8-11 were identified by comparison of their (13)C NMR data with those reported in the literature. CONCLUSION The bark of C. cebuense afforded monoterpenes, sesquiterpenes and phenolics, while the leaves yielded sesquiterpenes and triterpenes.

Chemical Constituents of Cinnamomum rupestre and Cinnamomum nanophyllum

Cinnamomum rupestre Kosterm. (Lauraceae) is an endemic species in the Philippines originally discovered in Palawan [1], while Cinnamomum nanophyllum Kosterm. is a widespread endemic species in the country. In Cebu, the local people collect and prepare the leaves and the bark as a decoction for relief of stomach and abdominal ailments. There are no reported chemical and biological studies on C. rupestre and C. nanophyllum. These are two of the 21 species of Cinnamomum found in the Philippines, of which 16 are known to be endemic [1]. This study was conducted as part of our research on the chemical constituents of Cinnamomum species found in the Philippines. We earlier reported the isolation of eugenol and safrole from the bark, polyprenol from the leaves, and trilinolein from the roots of Cinnamomum cebuense [2]. Silica gel chromatography of the dichloromethane (DCM) extract of the bark of C. rupestre afforded 4 -hydroxy5,7,3 -trimethoxyflavan-3-ol (1), 4-hydroxy-3-methoxycinnamaldehyde (2), and 4-allyl-2-methoxyphenol or eugenol (3), while the leaves yielded -sitosterol (4). The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy and confirmed by comparison of its 13C NMR data with those reported in the literature for 4 -hydroxy-5,7,3 -trimethoxyflavan-3-ol (1) [3]. The structures of 4-hydroxy-3-methoxycinnamaldehyde (2) [4], 4-allyl-2-methoxyphenol or eugenol (3) [5], and -sitosterol (4) [6] were confirmed by comparison of their 13C NMR data with those reported in the literature. The DCM extract of the bark of C. nanophyllum afforded 1, 2, 4, and trilinolein (5), while the leaves yielded 4. The structure of trilinolein (5) [7] was confirmed by comparison of its 13C NMR data with those reported in the literature. To the best of our knowledge, this is the first report on the chemical constituents of C. rupestre and C. nanophyllum. The bark and leaves of Cinnamomum rupestre and Cinnamomum nanophyllum were collected from Hagnaya, Carmen, Cebu, Philippines in June 2010. Voucher specimens of Cinnamomum rupestre and Cinnamomum nanophyllum were authenticated by one of the authors (EMGA) and deposited in the De La Salle University-Manila Herbarium (DLSU-3101 and DLSU-3102, respectively). The air-dried bark and leaves of C. rupestre were chopped into small pieces and then air dried. The air-dried bark (371.8 g) and leaves (89.1 g) were soaked in DCM for 3 days and then filtered. The filtrates were concentrated under vacuum to afford the crude extracts of bark (16.6 g) and leaves (7.0 g), which were fractionated by silica gel chromatography using increasing proportions of acetone in DCM (10% increment by volume) as eluents. The DCM fraction from the bark was rechromatographed in petroleum ether (7 ) to afford 3 (28 mg). The 30% acetone in DCM fraction was rechromatographed with 12.5% ethyl acetate in petroleum ether as eluent to afford 2 (18 mg). The 40% acetone in DCM fraction was rechromatographed (5 ) in DCM, then (8 ) in diethyl ether–acetonitrile–DCM (0.5:0.5:9) to afford 1 (12 mg). The DCM fraction from the leaves was rechromatographed (3 ) in 5% ethyl acetate in petroleum ether to afford 4 (10 mg). The air-dried bark and leaves of C. nanophyllum were chopped into small pieces and then air dried. The air-dried bark (493.5 g) and leaves (49.5 g) were soaked in DCM for three days and then filtered. The filtrates were concentrated under vacuum to afford the crude extracts of bark (8.0 g) and leaves (9.5 g), which were fractionated by silica gel chromatography using increasing proportions of acetone in DCM (10% increment by volume) as eluents. The crude DCM extract of the bark of C. nanophyllum was chromatographed in increasing proportions of acetone in DCM at 10% increment as eluents. The 10%

Chemical constituents of Cinnamomum griffithii

1) Chemistry Department and Center for Natural Sciences and Ecological Research, De La Salle University, 2401 Taft Avenue, 1004, Manila, Philippines, fax: (+0632) 536 02 30, e-mail: consolacion.ragasa@dlsu.edu.ph; 2) Biology Department and Center for Natural Sciences and Ecological Research, De La Salle University-Manila; 3) National Research Institute of Chinese Medicine, 155-1, Li-Nong St., Sec. 2, Taipei 112, Taiwan. Published in Khimiya Prirodnykh Soedinenii, No. 1, January–February, 2013, p. 111. Original article submitted November 22, 2011. Chemistry of Natural Compounds, Vol. 49, No. 1, March, 2013, 127-128.

Chemical Constituents of Cinnamomum utile

1) Chemistry Department and Center for Natural Sciences and Ecological Research, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines, fax: (+0632) 536 02 30, e-mail: consolacion.ragasa@dlsu.edu.ph; 2) Biology Department and Center for Natural Sciences and Ecological Research, De La Salle UniversityManila; 3) National Research Institute of Chinese Medicine, 155-1, Li-Nong St., Sec. 2, Taipei 112, Taiwan.

Methyl esters (biodiesel) from Pachyrhizus erosus seed oil

ABSTRACT The search for additional or alternative feedstocks is one of the major areas of interest regarding biodiesel. In this paper the fuel properties of Pachyrhizus erosus (commonly known as yam bean or Mexican potato or jicama) seed oil methyl esters were investigated by methods prescribed in biodiesel standards and comprehensively reported. As a result of the elevated content of saturated fatty acid methyl esters, the cloud point is high, the fatty acid methyl esters obtained from P. erosus seed oil generally meet fuel property specifications in the American and European biodiesel standards ASTM (American Society for Testing and Materials) D6751 and EN 14214, respectively, including a high cetane number.

Chemical Constituents of Cinnamomum trichophyllum

Cinnamomum trichophyllum Quis. & Merr. (Lauraceae) grows in mid-elevation mountains in the Philippines and Indonesia [1]. To date, there are no reported chemical studies and biological activities of C. trichophyllum. There are 21 species of Cinnamomum founded in the Philippines, of which 16 are known to be endemic. This study was conducted as part of our research on the chemical constituents of Cinnamomum species founded in the Philippines. We reported herein the isolation and identification of the chemical constituents of the dichloromethane extracts of the bark and leaves of C. trichophyllum from Jamildan, Capiz, Philippines. The leaves of C. trichophyllum yielded eugenol (1) [2], -sitosterol (2) [3], and polyprenol (3) [4], while the bark afforded trilinolein (4) [5] and a mixture of -sitosterol (2) and stigmasterol (5) [3]. To the best of our knowledge, this is the first report on the isolation of these compounds from C. trichophyllum. Eugenol (1) is a constituent common to five Philippine Cinnamomum species, namely, C. cebuense [6], C. iners [7], C. utile [8], C. trichophyllum, and C. rupestre [9]. It was reported to be cytotoxic against HL-60 leukemia cells [10], human osteoblastic cell line U2OS [11], human HFF fibroblasts, and human HepG2 hepatoma cells [12]. It also possesses significant antioxidant, anti-inflammatory, analgesic, local anesthetic, and cardiovascular activities [13]. Polyprenols (3) are constituents found in five Philippine Cinnamomum species, namely, C. cebuense [6], C. griffithii [14], C. utile [8], C. trichophyllum, and C. rupestre [9]. They exhibited hepatoprotective effects [15], significant triglyceride and cholesterol lowering effects [16], and chemotherapeutic properties on human breast cancer cells [17]. -Sitosterol (2) is a constituent of five Philippine Cinnamomum species, namely, C. iners [7], C. utile [8], C. trichophyllum, C. rupestre, and C. nanophyllum [9]. It inhibited the proliferation and induced apoptosis in human solid tumors such as colon and breast cancers [18]. Trilinolein (4) is a constituent of C. cebuense [6], C. nanophyllum [9], and C. trichophyllum. It exhibited myocardial protective effects [19] and inhibited endothelin-1-induced hypertension [20]. Stigmasterol (5) is a constituent of C. utile [8] and C. trichophyllum. It decreases plasma cholesterol levels, inhibits intestinal cholesterol and plant sterol absorption, and suppresses hepatic cholesterol and classic bile acid synthesis [21]. The bark and leaves of Cinnamomum trichophyllum were collected from Jamildan, Capiz, Philippines in May 2011. Voucher specimens were authenticated by one of the authors (EMGA) and deposited in the De La Salle University-Manila Herbarium (DLSU3103). The air-dried leaves of C. trichophyllum (559.3 g) were ground in an osterizer, soaked in CH2Cl2 for three days, and then filtered. The filtrate was concentrated under vacuum to afford the crude extract (21.8 g), which was chromatographed in increasing proportions of acetone in CH2Cl2 at 10% increments as eluents. The combined 10–20% acetone in the CH2Cl2 fractions from the chromatography of the crude extract was rechromatographed (8 ) in petroleum ether and (5 ) in 1% EtOAc in petroleum ether. The less polar eluents provided 1 (25.0 mg), while the more polar eluents gave 3 (10.0 mg). The 30% acetone in the CH2Cl2 fraction was rechromatographed (2 ) each in 7.5% EtOAc in petroleum ether and 10% EtOAc in petroleum ether to afford 2 (15.0 mg). The bark of C. trichophyllum was chopped into small pieces and then air dried. The air-dried bark (124.7 g) was soaked in CH2Cl2 for three days and then filtered. The filtrate was concentrated under vacuum to afford the crude extract (1.4 g), which was chromatographed in increasing proportions of acetone in CH2Cl2 at 10% increments as eluents. The CH2Cl2 fraction from

Chemical Constituents of Cinnamomum iners

Cinnamomum iners, a species closely similar to C. subcuneatum Miq., is an evergreen medium height tree with reddish brown and smooth branchlets. Cinnamomum iners is widely distributed in Southeast Asia from Indochina, Sumatra, Pensular Malaysia, Java, and the Philippines [1]. Previous studies reported the presence of -caryophyllene, stigmasterol, cardiac glycoside, flavonoid, polyphenol, saponin, sugar, tannin and terpenoid in the tree [2]. This study was conducted as part of our research on the chemical constituents of Cinnamomum species found in the Philippines. We earlier reported the isolation of a new monoterpene natural product and a new sesquiterpene, along with the known compounds 4-hydroxy-3-methoxycinnamaldehyde, 4-allyl-2-methoxyphenol, -terpineol, and humulene from the bark of C. cebuense, while the leaves afforded humulene, -caryophyllene, squalene, and a mixture of -amyrin, -amyrin, and bauerenol [3]. We also reported the presence of 1 ,4 ,7 -trihydroxyeudesmane, 4 -hydroxy-5,7,3 -trimethoxyflavan-3-ol, squalene, polyprenol, and a mixture of -amyrin, -amyrin, and bauerenol from the dichloromethane (DCM) extract of the bark of C. griffithii, and squalene and benzyl benzoate from the leaves [4]. Silica gel chromatography of the DCM extract of the bark of C. iners from Guimaras Island, Philippines afforded 5,7-dimethoxy-3 ,4 -methylenedioxyflavan-3-ol (1) and -sitosterol (2), which were also obtained from the DCM extract of the twigs of C. iners together with 4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one (3), cinnamaldehyde (4), linoleic acid (5), and vanillin (6). The leaves of C. iners afforded eugenol (7), linoleic acid (5), and -sitosterol (2). The structures of 1, 3, and 4 were elucidated by extensive 1D and 2D NMR spectroscopy and confirmed by comparison of their 13C NMR data with those reported in the literature for 5,7-dimethoxy-3 ,4 -methylenedioxyflavan-3-ol [5], 4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one [6], and cinnamaldehyde [7], respectively. The structures of -sitosterol (2) [8], linoleic acid (5) [9], vanillin (6) [10], and eugenol (7) [11] were confirmed by comparison of their 13C NMR data with those reported in the literature. The bark and leaves of Cinnamomum iners were collected from Guimaras, Philippines in May 2011. Voucher specimens were authenticated by one of the authors (EMGA) and deposited in the De La Salle University-Manila Herbarium (DLSU3103). The bark of C. iners was chopped into small pieces and then air dried. The air-dried bark (313.3 g) was soaked in DCM for 3 days and then filtered. The filtrate was concentrated under vacuum to afford the crude extract (7.1 g), which was chromatographed in increasing proportions of acetone in DCM at 10% increments as eluents. The 10% acetone in DCM fraction from the chromatography of the crude extract was rechromatographed (3 ) in 1% ethyl acetate in petroleum ether, then (2 ) in 2.5% ethyl acetate in petroleum ether to afford 2 (25.0 mg). The 20% acetone in DCM fraction was rechromatographed (5 ) in 5% ethyl acetate in petroleum ether, then (3 ) in 7.5% ethyl acetate in petroleum ether as eluents to afford 1 (12.0 mg). The air-dried twigs of C. iners (112.9 g) were ground in an osterizer, soaked in DCM for 3 days, and then filtered. The filtrate was concentrated under vacuum to afford the crude extract (1.6 g), which was chromatographed in increasing proportions of acetone in DCM at 10% increments as eluents. The DCM fraction from the chromatography of the crude extract was rechromatographed (6 ) in petroleum ether to afford 4 (10.0 mg). The 20% acetone in DCM fraction was rechromatographed (3 ) in 15% ethyl acetate in petroleum ether, then (3 ) in DCM as eluents. The less polar fractions yielded 2 (15.0 mg), while the more polar eluents afforded 3 (12.0 mg). The 30% acetone in DCM and 40% acetone in DCM

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.