Chien-Chang Shen

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 Ficus linearifolia and Ficus triangularis

Ficus linearifolia Elmer is an endemic Philippine fig tree distributed in the forests of Luzon, Negros, and Leyte at altitudes up to 900 m above sea level [1]. It was earlier considered as a mere variety of Ficus botryocarpa Miq. [2]. This fig tree was first described in 1907 as an erect tree that reaches a height of 20 m and bears linear lanceolate leaves that are more or less scattered but usually confined towards ends of the branchlets. It grows in low damp wooded ground or along streams in deep ravines at the base of Mt. Banahao (Lucban, Luzon Island) at 750 m above sea level [3]. This fig tree is locally known in the Philippines as daing-daing [4]. Ficus triangularis, commonly known as triangular fig and sweetheart tree, is a native to tropical Africa. It was recently introduced in the Philippines as an ornamental tree [5]. This study was conducted as part of our research on the chemical constituents of the genus Ficus. We earlier reported the isolation of a new neohopane [6] from Ficus pumila. We also reported the isolation of terpenoids and sterols from the endemic Philippine trees, Ficus pseudopalma and Ficus ulmifolia [7]. Recently, we reported the isolation of terpenoids and sterols from another endemic Philippine tree, Ficus odorata. -Sitosteryl-3 -glucopyranoside-6-O-palmitate from the leaves of F. odorata exhibited cytotoxicity against human stomach adenocarcinoma cell line (AGS) with 60.28% growth inhibition [8]. There are no reported chemical constituents and biological activities of F. linearifolia and F. triangularis. We report herein the isolation and structure elucidation of 1–9 from the dichloromethane extract of the air-dried leaves of Ficus linearifolia and 4–13 from the leaves of F. triangularis. To the best of our knowledge, this is the first report on the isolation of these compounds from these trees. Silica gel chromatography of the dichloromethane extract of the leaves of F. linearifolia afforded 1–9. The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy and confirmed by comparison of their 13C NMR data with those of 11 ,12 -epoxyurs-14-en-3 -yl acetate [9]. The structures of 2–9 were identified by comparison of their 13C NMR data with those reported in the literature for -amyrin (2) [10], -amyrin (3) [10], squalene (4) [11], -sitosterol (5) [12], -stigmasterol (6) [12], polyprenol (7) [13], linoleic acid (8) [14], lutein (9) [15]. The leaves of Ficus linearifolia were collected from the foot of Mt. Banahaw, at around 600 m above sea level (masl), at Avila s Farm, Brgy Palola, Lucban, Quezon, Philippines in March 2012. Voucher specimens were authenticated by Danilo N. Tandang of Philippine National Museum. The air-dried leaves (310 g) of F. linearifolia were soaked in CH2Cl2 for 3 days and then filtered. The filtrate was concentrated under vacuum to afford the crude extract (6 g), which was chromatographed in increasing proportions of acetone in CH2Cl2 at 10% increments by volume as eluents. The CH2Cl2 fraction was rechromatographed (4 ) in petroleum ether to afford 4 (8 mg). The 10% acetone in CH2Cl2 fraction from the chromatography of the crude extract was rechromatographed (3 ) in 7.5% EtOAc in petroleum ether to afford 1 (12 mg). The 20% acetone in CH2Cl2 fraction from the chromatography of the crude extract was rechromatographed (4 ) in 10% EtOAc in petroleum ether to afford 7 (10 mg). The 30% acetone in CH2Cl2 fraction was rechromatographed (3 ) in 15% EtOAc in petroleum ether to afford 8 (15 mg) and a mixture of 2 and 3 (9 mg).

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 Amaranthus viridis

Amaranthus viridis L., known in the Philippines as Spinach Tagalog and Kolitis, is sold in the market as vegetable. It is reported to exhibit antiviral and anticancer properties [1] and antinociceptive activity [2]. Another study reported that after treatment with A. viridis methanol extract, normal and streptozotocin induced diabetic rats exhibited a significant increase in body weight and a decrease in blood glucose, total cholesterol, and serum triglycerides [3]. A recent study reported that methanol extracts of A. viridis showed considerable antimicrobial activity against selected bacterial and fungal strains with MIC ranging from 179–645 g/mL. The seed extracts exhibited superior antioxidant and antimicrobial activity [4]. Earlier studies reported the isolation of quercetin and lutein [5] and rutin and -carotene [6] from A. viridis. Another study reported the isolation of amasterol, an ecdysone precursor and a growth inhibitor, from the roots of A. viridis [7]. We report herein the isolation of squalene (1) from the stems and leaves; spinasterol (2) from the stems and roots; and trilinolein (3), polyprenol (4), and phytol (5) from the leaves of A. viridis L. To the best of our knowledge, this is the first report on the isolation of these compounds from the plant. 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), while TLC was performed with plastic backed plates coated with silica gel F254. The plates were visualized with vanillin–H2SO4 and warming. Approximately 10 kg of the plant was obtained from Divisoria Market, Manila, Philippines. It was identified as Amaranthus viridis L. by the Jose Vera Santos Herbarium, Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City. The air-dried stems (233.26 g), leaves (293.92), and roots (128.9) of A. viridis were ground in a blender, soaked in CH2Cl2 for 3 days, and then filtered. The filtrates were concentrated in vacuo to afford the crude extracts (4.47, 5.80, and 2.05 g, respectively), which were fractionated by silica gel chromatography using increasing proportions of acetone in CH2Cl2 (10% by volume increment) as eluents. The CH2Cl2 fraction from the chromatography of the stem extract was rechromatographed in petroleum ether. The less polar fraction was rechromatographed (4 ) in petroleum ether to yield squalene (1, 6 mg). The 60% and 80% acetone in CH2Cl2 fractions were rechromatographed in a step-grade elution from 7.5–15% EtOAc in petroleum ether to afford spinasterol (2, 16 mg) after washing with petroleum ether. The CH2Cl2 fraction from the chromatography of the leaf extract was rechromatographed via step-grade elution from 2.5% to 7.5% EtOAc in petroleum ether. The fractions eluted with 5% EtOAc in petroleum ether yielded squalene (1, 15 mg). The more polar fractions eluted with 7.5% EtOAc in petroleum ether were rechromatographed with petroleum ether (3 ) to afford trilinolein (3, 18 mg). The 30% acetone in CH2Cl2 fraction was eluted in a step-gradient process from 5–20% EtOAc in petroleum ether. Fractions eluted with 20% EtOAc in petroleum ether were combined and rechromatographed with 5% EtOAc in petroleum ether (2 ) to afford polyprenol (4, 10 mg). The 40% acetone in CH2Cl2 fraction was rechromatographed (3 ) in 15% EtOAc in petroleum ether to afford phytol (5, 14 mg). The 40% acetone in CH2Cl2 fraction from the chromatography of the root extract was rechromatographed in 5% EtOAc in petroleum ether. The more polar fractions were combined and rechromatographed in 15% EtOAc in petroleum ether (3 ) to afford spinasterol (2, 17 mg) after washing with petroleum ether.

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.

Chemical Constituents of Hoya madulidii

The genus Hoya of the family Apocynaceae is represented in the Philippines by at least 109 species. Among the 88 species considered endemic to the country is Hoya madulidii Kloppenb. found in Ifugao, Ilocos Norte, Quezon, and Rizal Provinces in Luzon Island, Davao Oriental, and Zamboanga Provinces in Mindanao island, and in Palawan and Mindoro islands. It is a terrestrial plant that climbs up trees as a strong, fleshy vine, so it appears epiphytic [1]. With flowers varying in color from yellowish to purple to almost black red depending on the clone, hanging plants are attractive even with just the lush green foliage and are suitable as ornamental plants or landscaping materials. The closest relative of H. madulidii is H. coronaria Blume, which is found in northern Australia, Papua New Guinea, throughout the Indonesian archipelago, and in Peninsular Malaysia. H. coronaria has yellowish white or reddish yellow often violet-dotted flowers [2]. In Java, its latex is mixed with leaves of capsicum and ingested to promote digestion, while in the Moluccas, the latex is applied to stings from poisonous fish [3]. The latex of H. coronaria is composed of cis-polyisoprene (rubber), free triterpenols, and triterpene esters such as triterpene acetates, triterpene aromates (mostly cinnamic acid), and long-chain fatty acids [4]. For H. madulidii, however, no chemical analysis or biological activities have been reported. In this study, the dichloromethane extracts of H. madulidii yielded a mixture of lupeol cinnamate (1a), -amyrin cinnamate (1b), and -amyrin cinnamate (1c) in a 2:2:1 ratio, lupeol (2), a mixture of -sitosterol (3a) and stigmasterol (3b) in a 5:1 ratio and unsaturated hydrocarbons from the stems; and a mixture of 3a and 3b in a 3:1 ratio, squalene (4), chlorophyll a (5), and saturated hydrocarbons from the leaves. The air-dried stems (122.5 g) and leaves (153.5 g) of H. madulidii were ground in a blender, soaked in CH2Cl2 for three days, and then filtered. The filtrates were concentrated under vacuum to afford crude extracts of stems (6.15 g) and leaves (11 g), which were each chromatographed by gradient elution with CH2Cl2, followed by increasing amounts of acetone at 10% increment by volume as eluents. The crude extracts were fractionated by silica gel chromatography using increasing proportions of acetone in dichloromethane (10% increment) as eluents. Fifty 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. All fractions were monitored by thin layer chromatography. A glass column 12 inches in height and 0.5 inch internal diameter was used for further purification. Five milliliter fractions were collected. Rechromatography and final purifications were conducted using Pasteur pipettes as columns. One milliliter fractions were collected. The CH2Cl2 fraction from the chromatography of the crude extract of the stems was rechromatographed using petroleum ether to yield unsaturated hydrocarbons after washing with petroleum ether (12 mg). The 10% acetone in CH2Cl2 fraction was rechromatographed (2 ) using 5% EtOAc in petroleum ether to afford 1 (4 mg) after washing with petroleum ether. The 30% acetone in CH2Cl2 fraction was rechromatographed using 15% EtOAc in petroleum ether. The less polar fractions were rechromatographed using15% EtOAc in petroleum ether to yield a mixture of 3a and 3b (5 mg) after washing with petroleum ether. The more polar fractions were rechromatographed using CH2Cl2 to yield 2 (3 mg) after washing with petroleum ether.

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.

Chemical constituents and bioactivities of Glinus oppositifolius.

Objectives: To isolate the secondary metabolites from the dichloromethane (DCM) extracts of Glinus oppositifolius; to test for the cytotoxicity of a new triterpene, oppositifolone (1); and to test for the hypoglycemic, analgesic, and antimicrobial potentials of 1, DCM and aqueous leaf extracts of G. oppositifolius. Methods: The compounds were isolated by silica gel chromatography and identified by nuclear magnetic resonance spectroscopy. The cytotoxicity potential of 1 was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Triterpene 1, DCM, and aqueous leaf extracts were tested for hypoglycemic potential using the oral glucose tolerance test; analgesic potential using the tail-flick assay, and antimicrobial potential using the disc diffusion method. Results: The DCM extracts of G. oppositifolius afforded 1, squalene, spinasterol, oleanolic acid, phytol, and lutein from the leaves; squalene and spergulagenin A from the stems; and spinasterol from the roots. Triterpene 1 was cytotoxic against human colon carcinoma 116 with an IC 50 value of 28.7 but did not exhibit cytotoxicity against A549. The aqueous leaf extract at 200 mg/kg body weight (BW) exhibited hypoglycemic activity with a pronounced % blood glucose reduction of 70.76% ±17.4% within 0.5 h after introduction. The DCM leaf extract showed a lower % blood glucose reduction of 18.52% ±13.5% at 200 mg/kg BW within 1.5 h after introduction, while 1 did not exhibit hypoglycemic activity. The samples did not exhibit analgesic property and were inactive against multiple drug resistant bacterial pathogens. Conclusion: The compounds responsible for the hypoglycemic activity of G. oppositifolius which are fast acting (0.5 h) are found in the aqueous leaf extract.

Phenolics from Knema stellata subsp. cryptocaryoides

Ten out of the 75 known Malesian species of Knema (Family Myristicaceae) occur in the Philippines [1, 2]. Of these, seven taxa are endemic to the country, mostly occurring in the island of Luzon [1]. Among these taxa is Knema stellata subsp. cryptocaryoides (Elmer) W. J. de Wilde, locally known as durogo. It is a medium-size dioecious tree growing in the lowland forests of Luzon, Sibuyan, and Mindanao. Its wood is locally used for light construction, plywood production, wall paneling, making matchboxes, splints, crates, and wooden patterns, and in the manufacture of wrapping and writing papers [3]. Interestingly, its seeds contain oil that locals use for illumination purposes [3]. Like all other endemic Philippine Knema, this particular subspecies has not been the subject of any chemical study. This study is part of our research on the chemical constituents of trees endemic to the Philippines. 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], F. pseudopalma and F. ulmifolia [10], and F. odorata [11]. We report herein the isolation and structure elucidation of mixtures of 2-[(Z)-heptadec-8-enyl]-6-hydroxybenzoic acid (1a) and 2-[(Z)-pentadec-8-enyl]-6-hydroxybenzoic acid (1b); 3-(heptadec-8-enyl)phenol (2a), 3-(pentadec-8-enyl)phenol (2b), and 3-pentadecylphenol (2c); and saturated long-chain 4-hydroxycinnamate fatty acid esters (3) and -stigmasterol (4) from Knema stellata subsp. cryptocaryoides. This is the first report on the isolation of these compounds from Knema stellata subsp. cryptocaryoides. Samples of Knema stellata subsp. cryptocaryoides were collected from a private farm in Sitio Usiwan, Barangay Palola, Lucban, Quezon in March 2012 by Maryl V. Arceta and Leah D. J. Madrazo of the Southern Luzon State University (SLSU) in Lucban, Quezon. Within this private farm is a patch of secondary forest at 620 m asl, which interestingly harbors a number of endemic species. Voucher specimens were collected and identified by one of the authors (EHM) with collection #903 and deposited at De La Salle University, Manila. The air-dried leaves (330 g) of Knema stellata subsp. cryptocaryoides were ground in a blender, soaked in CH2Cl2 for 3 days, and then filtered. The air-dried stems (255 g) were chopped into small pieces, soaked in CH2Cl2 for 3 days, and then filtered. The filtrates were concentrated under vacuum to afford the crude extracts: 12 g (leaves) and 8 g (stems). The crude extracts were fractionated by silica gel chromatography using increasing proportions of acetone in CH2Cl2 (10% increment) as eluents.