Dinah L. Espineli

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.

A new triterpene from Barringtonia asiatica

The freeze-dried bark of Barringtonia asiatica afforded a new triterpene: (3β,11α)-11-hydroxyolean-12-en-3-yl palmitate (1). The bark also yielded mixtures of (3β)-olean-12-en-3-yl palmitate (2a), (3β)-urs-12-en-3-yl palmitate (2b) and (3β)-olean-18-en-3-yl palmitate (2c) in a 2 : 1 : 4 ratio; β-amyrin (3a), α-amyrin (3b) and germanicol (3c) in a 3 : 1 : 4 ratio; 22-O-tigloylcamelliagenin A (4a) and betulinic acid (4b) in a 2 : 1 ratio; olean-12-en-3β,16β,22α-triol (5), β-sitosterol, spinasterol, squalene and trilinolein. The roots yielded 2a–c and 3a–c as well as trilinolein, spinasterol and squalene, while the flowers afforded verimol k (6), linoleic acid, spinasterol, squalene, phytyl fatty acid ester and trilinolein. Compounds 1–4 and 6 were tested for antimicrobial property against seven microorganisms. All compounds tested exhibited slight activity against Candida albicans and were found inactive against Escherichia coli, Bacillus subtilis, Trichophyton mentagrophytes and Aspergillus niger. Except for the mixture of 4a and 4b that proved to be inactive, all the compounds were slightly active against the bacterium Staphylococcus aureus, while 3a–c were slightly active against Pseudomonas aeruginosa.

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.

TERPENOIDS FROM Ardisia squamulosa

The genus Ardisia Swartz belongs to the family Myrsinaceae. Ardisia has 68 recorded species in the Philippines [1], 60 of which are endemic. The hexane extract from A. squamulosa was reported to have a significant effect on sperm count but has a negligible effect on sperm morphology and viability [2]. No chemical study of A. squamulosa has been reported. However, we earlier studied a congener of this plant, Ardisia pyramidalis, which afforded spinasterol, spinasteryl acetate, a mixture of -amyrin, -amyrin and bauerenol, squalene, lutein, and triglycerides from the dichloromethane (DCM) extract of the leaves. Spinasterol was found to exhibit anti-angiogenic potential [3]. This study was conducted as part of our research on the chemical constituents of Ardisia species found in the Philippines. We report herein the chemical constituents of the DCM extract of the leaves of A. squamulosa. Fresh leaves of Ardisia squamulosa were collected from Kanawan, Morong Bataan, Philippines in 2010 by Ramon Bandong. The sample was authenticated by the Jose Vera Herbarium Collection at the Institute of Biology of the University of the Philippines-Diliman and the Philippine National Museum, Manila. The DCM extracts of the air-dried leaves of A. squamulosa afforded terpenoids by silica gel chromatography. These compounds were identified by comparison of their 13C NMR data with those of -caryophyllene (1) [4], squalene (2) [5], bauerenone (3a) [6], ursenone (3b) [7], oleanone (3c) [7], bauerenol (4a) [6], -amyrin (4b) [8], -amyrin (4c) [8], and lutein (5) [9] reported in the literature. Air-dried leaves of Ardisia squamulosa (1 kg) were ground in an Osterizer and then soaked in DCM for three days and filtered. The filtrate was concentrated in vacuo to afford a crude extract (55 g), which was chromatographed in increasing proportions of acetone in dichloromethane at 10% increment. The DCM fraction was rechromatographed (6 ) in petroleum ether to afford 1 (5 mg) and 2 (20 mg). The 10% acetone in DCM and 20% acetone in DCM fractions were combined and rechromatographed (4 ) with 2.5% ethyl acetate in petroleum ether as eluent to afford a mixture of 3a–3c (12 mg). The 30% acetone in DCM fraction was rechromatographed (5 ) using 5% ethyl acetate in petroleum ether to afford a mixture of 4a–4c (15 mg). The 40% acetone in DCM and 50% acetone in DCM fractions were combined and rechromatographed (3 ) with DCM–diethyl ether–acetonitrile (9:0.5:0.5) as eluent to afford 5 (18 mg) after washing with diethyl ether.

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

A new triterpene from Glinus oppositifolius

Abstract Aim To investigate the chemical constituents of Glinus oppositifolius. Methods The compounds were isolated by silica gel chromatography. The structure of the new triterpene was elucidated by extensive 1D and 2D NMR spectroscopy. Results The dichloromethane extract of the air-dried leaves of Glinus oppositifolius afforded a new triterpene, oppositifolone ( 1 ), spinasterol ( 2 ), squalene ( 3 ) and lutein ( 4 ). The structure of 1 was elucidated by NMR spectroscopy, while 2 - 4 were identified by comparison of their 13 C NMR data with those reported in the literature. Conclusion A new triterpene was isolated from G. oppositifolius .

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

Cytotoxic Triterpene from Barringtonia asiatica

Triterpenes isolated from the dichloromethane extract of Barringtonia asiatica, namely, a mixture of betulinic acid (1) and 22-O-tigloylcamelliagenin A (2) in a 1 : 2 ratio and a mixture of 3β-olean-18-en-3-yl palmitate (7), 3β-urs-12-en-3-yl palmitate (8) and 3β-olean-12-en-3-yl palmitate (9) in a 4 : 1 : 2 ratio (isolated from the bark), as well as germanicol caffeoyl ester (3), germanicol trans-coumaroyl ester (4), germanicol cis-coumaroyl ester (5) and germanicol (6) (from the leaves), and a phenolic compound, verimol K (10) from the flowers, were assessed for cytotoxicity against a human cancer cell line, colon carcinoma (HCT 116), using the MTT assay. The mixture of 1 and 2 exhibited IC50 =8.0 μg · mL–1 against this cell line, while 6 exhibited an IC50 value of 29.6 μg · mL–1. The other compounds tested (3 – 5, 7 – 10) were inactive against the HCT 116 cell line. The mixture of 1 and 2 and compound 6 were further tested for cytotoxicity against the non-small cell lung adenocarcinoma (A549) cell line. The mixture of 1 and 2 and compound 6 exhibited IC50 values of 6.0 and 35.6 μg · mL–1, respectively. The cytotoxicity for the mixture of 1 and 2 may be attributed to betulinic acid (1), a known cytotoxic compound.