NPC Natural Product Communications EDITOR-IN-CHIEF DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA agrawal@naturalproduct.us EDITORS PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain afbarre@ugr.es PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy braca@farm.unipi.it PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China gda5958@163.com PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan mimakiy@ps.toyaku.ac.jp PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia spyne@uow.edu.au PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA m.reinecke@tcu.edu PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA wsetzer@chemistry.uah.edu PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan tezuka@inm.u-toyama.ac.jp PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK david.thurston@pharmacy.ac.uk HONORARY EDITOR PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT U.K. axuf64@dsl.pipex.com ADVISORY BOARD Prof. Berhanu M. Abegaz Gaborone, Botswana Prof. Viqar Uddin Ahmad Karachi, Pakistan Prof. Øyvind M. 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Pynea*, Malai Taweechotipatrc and Sumalee Kamchonwongpaisand a School of Chemistry, University of Wollongong, Wollongong, NSW, 2522, Australia Manjong Sorig Pharmaceuticals, Ministry of Health, Thimphu, Bhutan c Department of Microbiology, Faculty of Medicine, Srinakharinwirot University, Sukhumvit 23, Bangkok, 10110, Thailand d Medical Molecular Biology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Pathumthani, 12120, Thailand b spyne@uow.edu.au Received: July 8th, 2013; Accepted: July 19th, 2013 We have hydrodistilled the essential oil (EO) from the aerial parts of the Bhutanese medicinal plant, Pleurospermum amabile using a Clevenger apparatus and evaluated this EO by GC/GC-MS and NMR analysis followed by testing for bioactivity. The GC-MS analysis identified 52 compounds with (E)-isomyristicin as a major component (32.2%). Repeated purification yielded four compounds; (E)-isomyristicin (1), (E)-isoapiol (2), methyl eugenol (3) and (E)-isoelemicin (4). Compound 2 and the mother EO showed the best antiplasmodial activity against the Plasmodium falciparum strains, TM4/8.2 (chloroquine and antifolate sensitive) and K1CB1 (multidrug resistant). They exhibited mild antibacterial activity against Bacillus subtilis. None of the test samples showed cytotoxicity. Keywords: Pleurospermum amabile, Medicinal plant, Essential oil, (E)-isomyristicin, Antibacterial, Antimalarial, Multidrug resistant strain, Cytotoxicity. Essential oils (EOs) are secondary metabolites of aromatic plants with strong odors and volatile constituents composed of terpenoids (mono-, sesqui- and di-terpenes), alcohols, ketones, aldehydes, alkanes, and phenylpropanoids. They are responsible for the protection of plants against microbes, insects, and herbivores [1]. Since antiquity, mankind has used EOs and the EO-containing plants as medicinals (ethnomedicine), perfumeries, incense, fragrances and embalmments and in culinary and the preservation of foods [2]. While many plants from various genera have been studied and some even commercially explored for EOs, the genus Pleurospermum (family Apiaceae) has been rarely investigated. Out of 30-50 species of Pleurospermum reported from eastern Europe, north Asia and the Himalayan region [3], only five species; P. lindleyanum [4], P. austriacum [5], P. hookeri [6], P. wrightianum [7], and P. giraldii [8] have been investigated for EOs. From the EO of P. lindleyani, 73 compounds were identified with 1-propoxy-2propanol, myristicine, cis-asarone, n-hexane, apiol, dimethyl ether, acetic acid, ethyl ester, spathulenol, 4-trimethylbenzene methanol, (E)-methyl isoeugenol and β-phellandrene as the main constituents [4]. Apparently, 1-propoxy-2-propanol, dimethyl ether, and n-hexane appear to be solvent contaminants or artefacts. From the EO of P. austriacum, 205 compounds were identified with germacrene D, β-caryophyllene, β-farnesene, β-phellandrene, -cadinene, epi-cubebol, bicyclogermacrene, humulene, α-cadinol and hexadecanoic acid as some of the major components [5]. Out of 72 GC peaks detected, 51 compounds were identified from the EO of P. hookeri and the major ones were palmitic acid, decanoic acid, ligustilide, piperitenal, (Z)-2-decenaldehyde and 2,4,5-trimethyl benzaldehyde [6]. From the EO of P. wrightianum, about 49 compounds were identified with (E)-9-octadecenoic acid and 1,3,5,7-cyclooctatetraene as the major components of the oil [7]. Out of 45 compounds identified from the EO of P. giraldii, Lcarvone and limonene were found to be the major components [8]. In this study, we have analyzed a Bhutanese Himalayan medicinal plant, P. amabile (synonym Hymenidium amabile) for its essential oil (EO) components and biological activities for the first time. The aerial components of this plant are used in Bhutanese traditional medicine (BTM) for treating dyspepsia, poisoning (antidote) and fever (febrifuge) that correlates to the symptoms of microbial infections and malaria [9]. The hydrodistillation of the dried powdered aerial plant material (250 g dry weight) yielded 0.7 % EO. Fifty two component peaks were detected and identified by GC/GC-MS analysis including MS library matching and Kovats retention indices (KI) comparison techniques (Table 1). The percent contents of the EO were determined on the basis of their FID responses upon GC (Figure 1). (E)-isomyristicin (1) was the major component (32.5%) of the EO (Figure 1, Table 1) followed by limonene (17.0%), (E)-isoapiol (7.6%), β-sesquiphellandrene (3.9%), methyl eugenol (3.8%), geranyl pentanoate (2.4%), (Z)-isomyristicin (2.3%), myrcene (2.3%), β-caryophyllene (2.2%), geraniol (1.6%), (E)-isoelemicin (1.3%), geranyl isobutyrate (1.3%), myristicin (1.3%), valeranone (1.2%), β-citronellol (1.2%), β-selinene (1.2%), myrtenyl acetate (1.1%) and citronellyl acetate (1%). Although slightly different column and protocol conditions were used in the EOs analysis, many EO compounds of P. amabile including limonene, β-caryophyllene, germacrene D, α-cadinol, α-bisabolene, α-pinene, myrcene, α-phellandrene, β-selinene, geranyl acetate, β-ocimene, and few others (see Table 1 and literature for details) were found common to the EO of P. austriacum [5]. 1306 Natural Product Communications Vol. 8 (9) 2013 Wangchuk et al. Table 1: Chemical compositions of EO from P. amabile. GC Peak No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 % oil 0.7 0.3 2.3 0.4 0.2 17.0 0.5 0.6 0.6 0.6 0.4 0.2 0.2 0.2 0.2 0.2 0.8 0.6 1.2 0.2 1.6 0.6 0.3 1.1 1.0 0.5 0.2 0.2 3.8 2.2 0.8 0.3 0.2 0.3 1.2 0.5 1.3 1.3 3.9 2.3 0.2 0.7 0.7 2.4 32.5 1.4 0.3 1.3 0.5 1.2 7.6 0.2 Compound name α-Pinene β-Pinene Myrcene α-Phellandrene Isoamyl isobutyrate Limonene (Z)-β-Ocimene (E)-β-Ocimene Terpinolene Linalool Isoamyl valerate 2-Methylbutyl isovalerate Fenchol Valeric acid, 3-methylbut-2-enyl-ester Terpinen-4-ol α-Terpineol Myrtenol Fenchyl acetate β-Citronellol 2,4-Dimethoxytolouene Geraniol Citronellyl formate Pinocarvyl acetate Myrtenyl acetate Citronellyl acetate Geranyl acetate 2-methyl-5,7-dimethylene-1,8-nonadiene Benzyl 2-methylbutanoate Methyl eugenol β-Caryophyllene Aromadendrene Geranyl n-propionate Citronellyl isobutyrate Germacrene D β-Selinene α-Bisabolene Geranyl isobutyrate Myristicin β-Sesquiphellandrene (Z)-Isomyristicin Germacrene D-4-ol Caryophyllene oxide Geranyl isovalerate Geranyl pentanoate (E)-Isomyristicin Dillapiole α-Cadinol (E)-Isoelemicin Apiole Valeranone (E)-Isoapiol Hexadecanal Table 2: Antibacterial, antimalarial and cytotoxicity activities of EO and compounds 1, 2 and 4 isolated from P. amabile. KI 934a 977a 991a 1005a 1013b 1030a 1038a 1048c 1090c 1100c 1106a 1109c 1115a 1149b 1179a 1189a 1193a 1223c 1229a 1240b 1256a 1276a 1293a 1329b 1354a 1384c 1388b 1396b 1406d 1427c 1462a 1475a 1484a 1489a 1500c 1507a 1514a 1526a 1529b 1575e 1584a 1587a 1605b 1611c 1624f 1630i 1640a 1654g 1665i 1684a 1722h 1840c Samples CH2Cl2 extract CHCl3 extract Essential oil (EO) (E)-Isomyristicin (1) (E)-Isoapiol (2) (E)-Isoelemicin (4) Amoxicillinb Vancomycinb Chloroquinec Cycloguanilc Pyrimethaminec Ellipticined Doxorubicind Antibacterial (MIZ in mm) B. subtilis MRSA 14a 12a 6a NT 5 NA 5 NA 6 NA NA NA 8 13.5 Antiplasmodial (IC50 in g/mL) TM4/8.2 K1CB1 12.1±0.4a 10.9±2.3a 7.8±1.8a 7.3±2.6a 79.0±4.6 72.3±2.1 >100 >100 52.9± 2.9 69.9±2.0 >20 >20 0.010 0.009 0.020 Cytotoxicity (IC50 in g/mL) Vero KB >25a >25a >25a >25a >100 >100 >100 >100 >100 >100 >20 >20 0.089 0.810 7.700 0.093 0.56 NT: Not Tested, NA: Not Active. a Original activity taken from [9]. b Positive controls for antibacterial activity. c Positive controls for antiplasmodial activity. d Positive controls for cytotoxicity activity. minimum inhibition zones (MIZ) ranging from 5-6 mm (Table 2). However, unlike the CH2Cl2 and CHCl3 extracts (from previous study, Table 2), the EO and its compounds (1, 2 and 4) did not show any activity against MRSA and other microbial strains tested here. The mother EO also exhibited moderate in vitro antiplasmodial activity against the P. falciparum strains: TM4/8.2 (a wild type chloroquine and antifolate sensitive strain) and K1CB1 (multidrug resistant strain) with IC50 values of 79.0 µg/mL and 72.3 µg/mL, respectively. These activities were lower than those exhibited by the CH2Cl2 and CHCl3 extracts (Table 2). Such correlations, including the antimicrobial activities, suggested that the more potent chemical components are present in the CH2Cl2 and CHCl3 extracts rather than in EO. In comparison to the antiplasmodial activity of the mother EO, compound 2 showed improved activity with IC50 values of 52.9 µg/mL and 69.9 µg/mL against TM4/8.2 and K1CB1, respectively. Compounds 1 did not show any antiplasmodial activity and compound 4 had poor solubility which restricted the determination of its accurate IC50 values for its highest test concentrations at 20 µg/mL. None of the test samples in this study showed any major cytotoxicity which thereby supports the assumption that the use of P. amabile in BTM is safe. * Retention time of the compounds based on GC-FID peaks (see Figure 1) and the isolated compounds were highlighted in bold face. a Identified by NIST and NISTREP mass spectra library and agrees with [16]. b Identified tentatively by NIST and NISTREP mass spectra library. c Identified by NIST and NISTREP mass spectra library and agrees with [5]. d Identified by its KI, 1H and 13C-NMR spectra comparisons with literature [13-14]. e Identified by NIST and NISTREP mass spectra library and agrees with [10]. f Identified by NIST and NISTREP mass spectra library and [10] and the comparison of its NMR spectra with [11]. g Identified by 1H and 13C-NMR spectra comparison with [15]. h Identified by 1H and 13C-NMR spectra comparison with [12]. I Identified by comparing the calculated KI with [17]. Subsequent purification of the EO of P. amabile using column chromatography and preparative TLC resulted in the isolation of four compounds which were identified by KI and NMR spectroscopy data analysis as (E)-isomyristicin (1) [10-11], (E)isoapiol (2) [12], methyl eugenol (3) [13-14], and (E)-isoelemicin (4) [15] (Figure 1). Given the traditional use of P. amabile to treat fever and various disorders bearing relevance to microbial infections and malaria and the evidence that its CH2Cl2 and CHCl3 extracts exhibited significant antibacterial and antiplasmodial activity (Table 2) [9], we investigated the mother EO and three major compounds (1, 2 and 4) isolated above for their antimicrobial, antimalarial and cytotoxicity activities (Table 2). The mother EO and compounds 1 and 2 showed moderate antibacterial activity against B. subtilis with In conclusion, this study found that P. amabile, which is one of the important medicinal ingredients of the Bhutanese traditional medicine, has an EO (0.7% oil w/w) with (E)-isomyristicin (32.5% oil) as the major component. The EO and compounds 1 and 2 inhibited the growth of only B. subtilis and no other strains. The EO and compound 2 also demonstrated moderate in vitro antiplasmodial activity without mammalian cell toxicity. This in vitro bioactivity of the EO supports the reported biological activities [9] of the crude extracts of the plant which further verifies the safety record of P. amabile used in BTM for treating the various aforementioned disorders. However, in vivo experiments will be required to further justify the use of the EO for treating humans. Experimental Plant material and essential oil: P. amabile is an endangered species inhabiting the open scrub, alpine turf and the semi-stable screes of high altitude Himalayan mountains (3950 to 4700 meters above sea level) in Bhutan [18-19]. It grows to 15-50 cm tall with stout root, solitary stem, sheathed broad leaves, white to dark purple flower and ovoid-oblong fruit [3,18]. For this study, the aerial components of P. amabile were collected from Lingzhi (Altitude: 4200 m) in Thimphu district in July 2009. A herbarium specimen (voucher number 29) was deposited at the herbarium of the Manjong Sorig Pharmaceuticals, Ministry of Health, Thimphu, Natural Product Communications Vol. 8 (9) 2013 1307 Relative abundance (%) Pleurospermum amabile Essential oil Retention time (min) Figure 1: GC-FID peaks for % oil and the structures of the isolated compounds (1-4) from the EO of P. amabile Bhutan. The air-dried plant material (250 g) was powdered and hydro-distilled using a Clevenger apparatus for 3 hours to obtain the pale green pleasantly aromatic EO (1.8 mL). The EO collected was dried over MgSO4. The EO was stored at 0–5°C until analysed. Analysis of EO using GC and GC-MS: The EO was analysed for its chemical composition using GC and GC-MS systems and equipment as described by us previously [20]. The GC analysis was performed on a Shimadzu GC-2010 Plus gas chromatograph. Hydrogen was used as carrier gas and the separation was achieved using a Restek fused silica capillary column (Rxi-5MS: 30 m × 0.25 mm i.d., 0.25 m film thickness). Injector and detector temperature were set at 260 °C and 300 °C, respectively. The starting oven temperature was programmed at 40 °C with an increasing temperature of 6 °C/min until it reached 290 °C. KI were obtained by GC-FID analysis of an aliquot of the EOs spiked with a commercially available n-alkane mixture (C9 to C21). The GC-MS analysis was performed using Shimadzu QP5050A GC-MS system (electron impact (EI) mode at 70 eV). The column and the GC-MS chromatographic conditions were same as that for GC with the exception that the He was used as carrier gas. The EO constituents were identified by comparing mass spectra with NIST and NISTREP mass spectra library of GC-MS data system and further confirmed by comparing their KI with those reported [5,10,16-17]. Isolation of compounds from EO: The equipment, general protocols and isolation techniques were carried out as described by us previously [21]. The known compounds were identified through MS library matching techniques (NIST and NISTREP mass spectra library) and then confirmed through comparison of their MS and NMR spectra (500 MHz, CDCl3) with those reported (see footnote of Table 1 for references). The EO (383 mg) was column chromatographed on a silica gel (120 g, 200-300 mesh) eluting with a gradient solvent system of CHCl3petroleum spirit (v/v ratio of 0:100, 5:95, 10:90, 15:85, 20:80, 30:70, 50:50, 70:30, 100:0) to obtain nine fractions, PAoil.1-9. Further separation of PAoil.1 using reversed phase preparative silica gel plates in highly polar solvent system (10% H2O:90% MeOH) yielded compound 1 (185.3 mg) (major constituent of oil, Figure 1) which was identified as (E)-isomyristicin through NMR spectral data analysis. Eluting fraction PAoil.5 with isocratic or fixed ratio solvent system (10% petroleum spirit:90% CHCl3) on a normal phase silica gel column furnished (E)-isoapiol (2) (30.7 mg, Figure 1) and methyl eugenol (3) (44.7 mg, Fig. 1). Separation of fraction PAoil.8 on a preparative silica plate using the solvent system of 30% diethyl ether:96% petroleum spirit, yielded (E)-isoelemicin (4) (3.4 mg, Figure 1). Bioassay methods: Antimicrobial, antiplasmodial and cytotoxicity bioassays were carried out using the standard protocols reported by us previously [9]. The test strains of Plasmodium falciparum used for the antiplasmodial bioassay were K1CB1, a multidrug resistant strain; and TM4/8.2, a wild type chloroquine and antifolate sensitive strain. Chloroquine (Sigma-Aldrich), cycloguanil (Sigma-Aldrich) and pyrimethamine (Sigma-Aldrich) were used as positive controls in the antiplasmodial assays. For the antimicrobial assay, the test organisms including Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), methicillin resistant S. aureus (MRSA), (DMST 20651), S. epidermidis (ATCC 12228), Vibrio cholerae (DMST 2873) and Candida albicans (ATCC 10231) were used. Amphotericin B (Sigma-Aldrich, USA) was used as a positive control for antifungal testing against Candida albicans (not shown in Table 2 as the samples were found to be inactive). Vancomycin (Edicin, Slovenia) and amoxicillin (GPO, Thailand) were used as the positive controls for antibacterial assays. For the cytotoxicity assay, normal vero cells from kidney of African green monkey, Cecopithecus aethiops and the human oral carcinoma KB cells were used. Ellipticine (SigmaAldrich, USA) and doxorubicin (Sigma-Aldrich, USA) were used as reference drugs for cytotoxicity activities. All the experiments were performed three times in duplicate (3x2) and DMSO (0.1%) and distilled water were used as controls to rule out the solvent effects on the bioassay results of the test samples. 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Pharmaceutical and Research Unit, Ministry of Health, Thimphu, Bhutan. Wangchuk P, Keller PA, Pyne SG, Korth J, Samten, Taweechotipatr M, Rattanajak R, Kamchonwongpaisan S. (2013) Antimicrobial, antimalarial and cytoxicity activities of constituents of a Bhutanese variety of Ajania nubigena. Natural Product Communications, 8, 733-736. Wangchuk P, Keller PA, Pyne SG, Willis AC, Kamchonwongpaisan S. (2012) Antimalarial alkaloids from a Bhutanese traditional medicinal plant Corydalis dubia. Journal of Ethnopharmacology, 143, 310-313. Natural Product Communications Vol. 8 (9) 2013 Published online (www.naturalproduct.us) In vitro Anti-diabetic Activity of Sclerocarya birrea and Ziziphus mucronata Nuno M.H. Da Costa Mousinho, Jacob J. van Tonder and Vanessa Steenkamp Secondary Metabolites from the Fungus Emericella nidulans Amer H. Tarawneh, Francisco Leόn, Mohamed M. Radwan, Luiz H. Rosa and Stephen J. Cutler A New Glucuronolactone Glycoside Phoenixoside B from the Seeds of Phoenix dactylifera Sumbul Azmat, Rehana Ifzal, Faryal Vali Mohammad, Viqar Uddin Ahmad and Aqib Zahoor Cancer-Suppressive Potential of Extracts of Endemic Plant Helichrysum zivojinii: Effects on Cell Migration, Invasion and Angiogenesis Ivana Z. Matić, Ivana Aljančić, Vlatka Vajs, Milka Jadranin, Nevenka Gligorijević, Slobodan Milosavljević and Zorica D. Juranić Analysis of Volatile Components, Fatty Acids, and Phytosterols of Abies koreana growing in Poland Anna Wajs-Bonikowska, Karol Olejnik, Radosław Bonikowski and Piotr Banaszczak Cytotoxic Effects of Air Freshener Biocides in Lung Epithelial Cells Jung-Taek Kwon, Mimi Lee, Gun-Baek Seo, Hyun-Mi Kim, Ilseob Shim, Doo-Hee Lee, Taksoo Kim, Jung Kwan Seo, Pilje Kim and Kyunghee Choi GC/GC-MS Analysis, Isolation and Identification of Bioactive Essential Oil Components from the Bhutanese Medicinal Plant, Pleurospermum amabile Phurpa Wangchuk, Paul A. Keller, Stephen G. Pyne, Malai Taweechotipatr and Sumalee Kamchonwongpaisan Antibacterial Activity of the Essential Oil of Heracleum sibiricum Dragoljub L. Miladinović, Budimir S. Ilić, Tatjana M. Mihajilov-Krstev, Dejan M. Nikolić, Olga G. Cvetković, Marija S. Marković and Ljiljana C. Miladinović Assessment of the Chemical Composition and in vitro Antimicrobial Potential of Extracts of the Liverwort Scapania aspera Danka R. Bukvicki, Amit K. Tyagi, Davide G. Gottardi, Milan M. Veljic, Snezana M. Jankovic, Maria E. Guerzoni and Petar D. Marin Essential Oils of Alpinia rafflesiana and Their Antimicrobial Activities Shariha Jusoh, Hasnah Mohd. Sirat and Farediah Ahmad Chemical Composition and Synergistic Antioxidant Activities of Essential Oils from Atractylodes macrocephala and Astragalus membranaceus Jinkui Li, Feng Li, Yan Xu, Wenjian Yang, Lili Qu, Qian Xiang, Cong Liu and Dapeng Li Chemical Analysis and Antioxidant Activity of the Essential Oils of Three Piperaceae Species Growing in the Central Region of Cuba Elisa Jorge Rodríguez, Yanelis Saucedo-Hernández, Yvan Vander Heyden, Ernesto F. Simó-Alfonso, Guillermo Ramis-Ramos, María Jesús Lerma-García, Urbano Monteagudo, Luis Bravo, Mildred Medinilla, Yuriam de Armas and José Manuel Herrero-Martínez The Composition, Anti-mildew and Anti-wood-decay Fungal Activities of the Leaf and Fruit Oils of Juniperus formosana from Taiwan Yu-Chang Su, Kuan-Ping Hsu, Eugene I-Chen Wang and Chen-Lung Ho 1279 1285 1289 1291 1297 1301 1305 1309 1313 1317 1321 1325 1329 Meeting/Report Meeting Report: First National Meeting on Aloe, April 20-21, 2013, Isernia, Italy New Perspectives in Aloe Research: from Basic Science to Clinical Application Raffaele Capasso, Massimiliano Laudato and Francesca Borrelli 1333 Review/Account Alkaloids of the South African Amaryllidaceae: a Review Jerald J. Nair, Jaume Bastida, Carles Codina, Francesc Viladomat and Johannes van Staden 1335 Natural Product Communications 2013 Volume 8, Number 9 Contents Original Paper Page Alternate Biosynthesis of Valerenadiene and Related Sesquiterpenes Shashikumar K. Paknikar, Shahuraj H. Kadam, April L. Ehrlich and Robert B. Bates A Facile Synthesis of (±)-Heliannuol-D Tao Zhang, Liang-Zhu Huang, You-Qiang Li, Yimg-Meng Xu and Zhen-Ting Du A New Bioactive Diterpene Glycoside from Molinaea retusa from the Madagascar Dry Forest Alexander L. Eaton, Liva Harinantenaina, Peggy J. Brodie, Maria B. Cassera, Jessica D. Bowman, Martin W. Callmander, Richard Randrianaivo, Roland Rakotondrajaona, Etienne Rakotobe, Vincent E. Rasamison and David G. I. Kingston Nitric Oxide and Tumor Necrosis factor-alpha Inhibitory Substances from the Rhizomes of Kaempferia marginata Kanidta Kaewkroek, Chatchai Wattanapiromsakul, Palangpon Kongsaeree and Supinya Tewtrakul Biscembranoids from the Marine Sponge Petrosia nigricans Nguyen Xuan Nhiem, Ngo Van Quang, Chau Van Minh, Dan Thi Thuy Hang, Hoang Le Tuan Anh, Bui Huu Tai, Pham Hai Yen, Nguyen Thi Hoai, Do Cong Thung and Phan Van Kiem Isolation of Cycloeucalenol from Boophone disticha and Evaluation of its Cytotoxicity Emmanuel Adekanmi Adewusi, Paul Steenkamp, Gerda Fouche and Vanessa Steenkamp Chemical Constituents from an Endophytic Fungus Chaetomium globosum Z1 Chun-Yan Zhang, Xiao Ji, Xuan Gui and Bao-Kang Huang Determination of C-23 Configuration in (20R)-23-Hydroxycholestane Side Chain of Steroid Compounds by 1H and 13 C NMR Spectroscopy Alla A. Kicha, Anatoly I. Kalinovsky, Alexander S. Antonov, Oleg S. Radchenko, Natalia V. Ivanchina, Timofey V. Malyarenko, Alexander M. Savchenko and Valentin A. Stonik Oxasetin from Lophiostoma sp. of the Baltic Sea: Identification, in silico Binding Mode Prediction and Antibacterial Evaluation against Fish Pathogenic Bacteria Muftah Ali M. Shushni, Faizul Azam and Ulrike Lindequist Chemical Constituents from the Fruit Body of Chlorophyllum molybdites Zushang Su, Ping Wang, Wei Yuan, and Shiyou Li Pulchranins B and C, New Acyclic Guanidine Alkaloids from the Far-Eastern Marine Sponge Monanchora pulchra Tatyana N. Makarieva, Ekaterina K. Ogurtsova, Yuliya V. Korolkova, Yaroslav A. Andreev, Irina V. Mosharova, Ksenya M. Tabakmakher, Alla G. Guzii, Vladimir A. Denisenko, Pavel S. Dmitrenok, Hyi-Seung Lee, Eugene V. Grishin and Valentin A. Stonik Cloning and Characterization of a cDNA Encoding Calcium/Calmodulin-dependent Glutamate Decarboxylase from Scutellaria baicalensis Yeon Bok Kim, Md Romij Uddin, Do Yeon Kwon, Min-Ki Lee, Sun-Ju Kim, Chanhui Lee and Sang Un Park Biflavonoids, Main Constituents from Garcinia bakeriana Leaves Ahmed Al-Shagdari, Adonis Bello Alarcón, Osmany Cuesta-Rubio, Anna Lisa Piccinelli and Luca Rastrelli Analysis of Flavonoids and Iridoids in Vitex negundo by HPLC-PDA and Method Validation Somendu K. Roy, Khemraj Bairwa, Jagdeep Grover, Amit Srivastava and Sanjay M. Jachak Chemical Constituents of the Leaves of Triumfetta semitriloba Alejandra Barraza-Morales, Deisy Medrano-Nahuat, Sergio R. Peraza-Sánchez Phytochemical Evaluation of Lythrum salicaria Extracts and Their Effects on Guinea-pig Ileum Tímea Bencsik, Loránd Barthó, Viktor Sándor, Nóra Papp, Rita Benkó, Attila Felinger, Ferenc Kilár and Györgyi Horváth New Flavonol Glycosides from the Leaves of Triantha japonica and Tofieldia nuda Tsukasa Iwashina, Minoru N. Tamura, Yoshinori Murai and Junichi Kitajima Cytotoxic Activity of Dihydrochalcones Isolated from Corema album Leaves against HT-29 Colon Cancer Cells Antonio J. León-González, Miguel López-Lázaro, José L. Espartero and Carmen Martín-Cordero Immunomodulatory Activities of α-Mangostin on Peripheral Blood Mononuclear Cells Pimolkan Kasemwattanaroj, Primchanien Moongkarndi, Kovit Pattanapanyasat, Supachoke Mangmool, Ekkarat Rodpai, Jutima Samer, Julaporn Konlata and Kasama Sukapirom Antiplasmodial Quinones from the Rhizomes of Kniphofia foliosa Martha Induli, Meron Gebru, Negera Abdissa, Hosea Akala, Ingrid Wekesa, Robert Byamukama, Matthias Heydenreich, Sylvia Murunga, Ermias Dagne and Abiy Yenesew Biphenyl Derivatives from Garcinia schomburgkiana and the Cytotoxicity of the Isolated Compounds Chihiro Ito, Takuya Matsui, Eri Noda, Nijsiri Ruangrungsi and Masataka Itoigawa Anticarcinogenic Effect and Carcinogenic Potential of the Dietary Phenolic Acid: o-Coumaric Acid Alaattin Sen, Pelin Atmaca, Gulsum Terzioglu and Sevki Arslan Bioproduction and Optimization of Rosmarinic Acid Production in Solenostemon scutellarioides through Media Manipulation and Conservation of High Yielding Clone via Encapsulation Ranabir Sahu, Saikat Dewanjee and Moumita Gangopadhyay Continued inside backcover 1195 1197 1201 1205 1209 1213 1217 1219 1223 1227 1229 1233 1237 1241 1245 1247 1251 1255 1257 1261 1265 1269 1275