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. 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All issues are dispatched by airmail throughout the world, excluding the USA and Canada. NPC 2013 Vol. 8 No. 9 1213 - 1216 Natural Product Communications Isolation of Cycloeucalenol from Boophone disticha and Evaluation of its Cytotoxicity Emmanuel Adekanmi Adewusia*, Paul Steenkampb,c, Gerda Foucheb and Vanessa Steenkampa a Department of Pharmacology, Faculty of Health Sciences, University of Pretoria, Private Bag X323, Arcadia 0007, South Africa b Natural Product Chemistry Group, Biosciences, Council for Scientific and Industrial Research, PO Box 395, Pretoria 0001, South Africa c Department of Biochemistry, University of Johannesburg, Auckland Park 2006, South Africa adewusiadekanmi@gmail.com Received: November 21st, 2012; Accepted: June 6th, 2013 Boophone disticha (Amaryllidaceae) is widely used in traditional medicine in southern Africa. Several alkaloids, volatile oils and fatty acids have been isolated from the plant. However, there has been no literature report of a triterpene from B. disticha. Cycloeucalenol, a cycloartane triterpene, together with its regioisomer, was isolated from the ethyl acetate extract of the bulbs using column chromatography and preparative thin layer chromatography. Structural elucidation was carried out using 1D and 2D NMR and mass spectroscopy. The MTT and neutral red assays were used to assess the cytotoxicity of the compound in human neuroblastoma (SH-SY5Y) cells. The compound was obtained as a mixture of two regio-isomers, which were separated for the first time by chromatographic optimization. Integration of the 1H NMR spectrum showed that cycloeucalenol and its regio-isomer were present in a ratio of 1.04:1. A dose-dependent decrease in cell viability was observed using both cytotoxicity assays. IC50 values of 173.0 ± 5.1 µM and 223.0 ± 6.4 µM were obtained for the MTT and neutral red assays, respectively, indicative of the low toxicity of the compound. This work describes for the first time, the presence of triterpene compounds from the genus Boophone. Keywords: Amaryllidaceae, Boophone disticha, Cycloeucalenol, Cytotoxicity, SH-SY5Y cells, Regio-isomer. Boophone disticha (L.f.) Herb, a member of the Amaryllidaceae family, is an attractive, bulbous plant with a thick covering of dry scales [1]. The large, round heads occur on short stems so that they appear to grow directly from the bulb, almost at ground level. The flowers vary from shades of pink to red and are sweetly scented [2]. The pedicels (flower stalks) elongate after flowering to form a large seed-head. This breaks off at the top of the scape (stalk) and tumbles across the veld dispersing the seed. The greyish green leaves are erect, arranged in a conspicuous fan and are usually produced after flowering [2]. B. disticha is used traditionally to treat several diseases. Fresh scales are applied to burns and used to treat rashes and skin disorders including eczema. It is also used to relieve rheumatic pains, arthritic swelling, sprains, muscular strains, painful wounds, eye conditions, headaches, anxiety, the pain of abrasions and inflammatory conditions [3,4]. Bulb decoctions are administered either orally or as enemas to adults suffering from headaches, abdominal pain, weakness, sharp chest pains and persistent bladder pains [3]. The bulb is also used in the treatment of varicose ulcers and for the relief of urticaria, as well as a treatment for cancer [3]. The Amaryllidaceae alkaloids, a group of isoquinoline alkaloids are found in various Boophone species [3]. Alkaloids isolated to date include crinine, buphanisine, buphanamine, distichamine, buphacetine, crinamidine, lycorine, nerbowdine, undulatine, 3-O-acetylnerbowdine, buphanidrine and 6-hydroxycrinamine [5,6]. Buphanidrine, buphanamine and distichamine have been reported to have affinity to the serotonin transporter indicating their potential in treatment of depression and anxiety [7,8]. Also, 6hydroxycrinamine has been shown to contain acetylcholinesterase inhibitory activity [6]. Several other compounds have been isolated from the plant and these include; a volatile oil containing furfuraldehyde, acetovanillone, chelidonic acid, copper, laevulose, 18 CH3 R 19 2 3 H HO 1 4 10 5 11 9 6 12 8 7 13 17 16 14 15 CH 29 3 H CH3 28 Side chain (R) 30 30 21 20 21 22 23 24 26 20 22 26 25 27 27 Cycloeucalenol (1) 24 23 25 Regio-isomer of cycloeucalenol (2) Figure 1: Structure of cycloeucalenol and its regio-isomer. petatriacontane, ipuranol and a mixture of free and combined fatty acids [3,9]. However, there has been no literature report of the detection of a triterpene from B. disticha. This paper describes the isolation and structural elucidation of a cycloartane triterpene from B. disticha. Toxicity of the isolated compound was determined using both the 3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide (MTT) and neutral red uptake assays. In addition, as the compound was obtained as a mixture of two regio-isomers, the separation of the regio-isomers was achieved by chromatographic optimization. The triterpene was isolated from the ethyl acetate extracts of the bulbs of B. disticha as white crystals. MS data showed the pseudo 1214 Natural Product Communications Vol. 8 (9) 2013 molecular ion [M + H]+ peak as the base peak at m/z 427 which corresponds to the molecular formula, C30H50O (MW = 426.3942 Da; iFit = 0; DBE = 6). The compound was observed to be nonpolar and was dissolved in deuterated chloroform for NMR analysis (1H, 13C and 2D experiments). The signals obtained from both the 1 H and 13C NMR spectra were complex suggesting that the isolated compound was a mixture of two regio-isomers. Analyses of both the NMR and MS data revealed that the structure of the isolated compound was cycloeucalenol (1), together with its regio-isomer (2) (Figure 1). The NMR data obtained was compared with that of the published data on cycloeucalenol [10,11], and our extensive literature search revealed that cycloeucalenol and its regio-isomer have not previously been isolated from any species of Boophone. However, this class of compound, the cycloartanes, including cycloeucalenol, have previously been reported from Ammocharis coranica, a member of the Amaryllidaceae family [12]. The first literature report of a cycloartane from this family was from the plant Crinum asiaticum var. japonicum [13]. The 1H NMR spectra of cycloeucalenol and its regio-isomer are very similar, with the only difference observed in the position of the double bond on the side chain. The methyl protons of the regioisomer (2) (Figure 1), Me-28 and Me-21, appeared as broad singlets (0.95 and 0.86); Me-26 appeared as a multiplet ( H 1.64), while Me-29 was observed as a singlet ( H 0.88). A hextet was observed at H 2.22 (J = 7.0 Hz), while an olefinic proton, which appeared as a doublet, was observed at H 1.00 (J = 6.6 Hz). The 1H NMR data compares well with that of Akihisa et al. [10]. The 13C NMR spectra of cycloeucalenol and its regio-isomer are very similar for C-1 to C-21, with the only difference observed in the side chain from C-22, because of the difference in position of the double bond. C-25 is an olefinic quaternary carbon at C 150.5, while C-27 is an exomethylene carbon at C 109.6. Cycloeucalenol and its regio-isomer co-chromatographed. To date there has been no literature report in which the separation of these regio-isomers was accomplished. This study is the first to separate these isomers into two distinct compounds, as evident from the chromatographic profile. Integration of the 1H NMR spectrum showed that cycloeucalenol and its regio-isomer are present in a ratio of 1.04:1. The continuous use and growing demand for herbal therapies have invigorated the quest for validating the efficacy and safety or toxic implications of medicinal plants. This is very important, as it helps in developing safe and cheap alternative medicines. One of the fundamental in vitro toxicological assays performed is the direct assessment of the effects of a plant extract or compound on the viability of a cell line. Data obtained in these assays are very useful in selecting the most promising candidate for further development and obtaining data for future studies [14]. The human neuroblastoma (SH-SY5Y) cell line, which is widely used in experimental neurological studies, analysis of neuronal differentiation, metabolism and function related to neurodegenerative and neuroadaptive processes, neurotoxicity and neuroprotection [15], was selected to assess the cytotoxicity of cycloeucalenol and its regio-isomer. The MTT and neutral red uptake assays were selected to determine cell viability. Both assays were run in parallel in order to improve the reliability of the cytotoxicity data thereby providing a more comprehensive picture of the potential cellular toxicity through different mechanisms. Cytotoxicity tests were carried out to assess the effect of cycloeucalenol and its regio-isomer on the viability of the cells. A dose-dependent effect on cell viability was observed and results Adewusi et al. obtained from both cytotoxicity assays were comparable (Figure 2). IC50 values of 173.0 ± 5.1 µM and 223.0 ± 6.4 µM were obtained for the MTT and neutral red assays, respectively. Cycloeucalenol and its regio-isomer were observed to have high IC50 values for both assays, which is indicative of their low toxicity. Two cycloartane triterpenes; 25-O-acetylcimigenol-3-O- -Dglucopyranosyl (1 →2 )- -D-xylopyranoside and 25-O-acetylcimigenol-3-O- -D-galactopyranoside showed low toxicity when tested against mouse hepatocytes, with IC50 values >100 µM [16]. This result supports the findings of the present study. Cycloeucalenol has been reported to show anti-inflammatory, cardiotonic and spasmolytic effects [17,18], and its low toxicity indicates that it could be studied further as a potential lead in developing drugs useful in treating inflammation and with cardioprotective properties. In conclusion, we have described the isolation of cycloeucalenol, a cycloartane triterpene, together with its regio-isomer, from the bulbs of Boophone disticha. The separation of both regio-isomers into two distinct compounds is also reported for the first time. The low toxicity of cycloeucalenol and its regio-isomer make it a suitable agent for further testing for pharmacological activity. Experimental General experimental procedures: NMR spectroscopy was performed using a 600 MHz Varian NMR spectrometer. Structural characterizations were carried out using a combination of 1D (1H, 13 C) and various 2D experiments. The 2D experiments carried out included DEPT, COSY, HSQC and HMBC. Chemical shifts are reported in units of (ppm) and coupling constants (J) are expressed in Hz. UV-VIS detection was achieved on a WATERS PDA scanning from 200 – 600 nm. All chemicals for UPLC-MS were of ultra-pure LC-MS grade and purchased from Fluka (Steinheim, Germany), while ultra-pure solvents were purchased from Honeywell (Burdick & Jackson, Muskegon, USA). Ultra-pure water was generated from a Millipore Elix 5 RO system and Millipore Advantage A10 Milli-Q system (Millipore SAS, Molsheim, France). Silica gel 60 (0.063-0.2 mm) was used for CC, while pre-coated glass plates (Merck, SIL G-25 UV254, 20 cm x 20 cm) were used for TLC and preparative TLC. Compounds on the TLC plates were detected under UV light at short wave (250 nm) and long wave (365 nm) lengths, and by spraying with vanillinH2SO4 reagent. MTT and neutral red dye, purchased from Sigma were used for the cytotoxicity assays. Plant material: Bulbs of Boophone disticha (L.f.) Herb. (Amaryllidaceae) were a gift from the South African National Biodiversity Institute, Pretoria. Extraction and isolation of cycloeucalenol and its regio-isomer: Plant material was cut into small pieces and air-dried at room temperature. Dried material was ground to a fine powder using an Ika Analytical Mill (Staufen, Germany), and stored at ambient temperature in the dark till use. Powdered plant material (250 g) was extracted with 2.5 L of ethyl acetate for 24 h while shaking. The extracts were filtered, concentrated using a rotary vacuum evaporator and further dried under reduced pressure. The ethyl acetate extract (1.4 g) was subjected to silica gel CC (65 g; particle size 0.063 - 0.2 mm). The separation and purification was carried out using a stepwise gradient mixture of n-hexane: ethyl acetate starting from 100:0 until 0:100 as eluent to give 70 fractions. Fractions were collected every Cycloeucalenol from Boophone disticha Natural Product Communications Vol. 8 (9) 2013 1215 5 min at a rate of 1 mL/min. The fractions were pooled together based on the similarity in their Rf values on a TLC plate to give 4 sub-fractions. Sub-fraction 2, which contained cycloeucalenol, was further purified by CC. This sub-fraction was subjected to further silica gel column chromatographic purification and subsequently eluted using a stepwise gradient mixture of n-hexane: ethyl acetate, starting from 90:10 until 0:100, to give another set of 18 fractions. These fractions were pooled together based on the similarity in their Rf values on a TLC plate. Cycloeucalenol and its regio-isomer (0.3 g) were obtained as white crystals. These were further analyzed using UPLC-QTOF (mass spectrometric determination) and NMR spectroscopy (1D and 2D experiments). The separation of the 2 regio-isomers into 2 distinct compounds was evident from the chromatographic profile (data not shown). Instrumental: A Waters UPLC coupled in tandem to a Waters photodiode array (PDA) detector and a SYNAPT G1 HDMS mass spectrometer was used to generate accurate mass data. Chromatographic separation of the purified sample utilized a Waters HSS C18 column (150 mm x 2.1 mm, 1.8 µm) with temperature controlled at 60ºC. A binary solvent mixture was used consisting of water (Eluent A) containing 10 mM formic acid (natural pH of 2.3) and methanol (Eluent B). The initial conditions were 40% A at a flow rate of 0.4 mL/min, which was maintained for 1 min, followed by a linear gradient to 5% A at 12 min. The conditions were kept constant for 3 min and then changed to the initial conditions. The runtime was 20 min and the injection volume was 5 µL. The PDA detector was scanned between 200 and 500 nm (1.2 nm resolution), which collected 20 spectra per second. The SYNAPT G1 mass spectrometer was used in V-optics and operated in electrospray ionization mode to enable detection of terpenes. Leucine enkephalin (50 pg/mL) was used as reference calibrant to obtain typical mass accuracies between 1 and 3 mDa. The mass spectrometer was operated in positive mode with a capillary voltage of 3.0 kV, the sampling cone at 25 V and the extraction cone at 4 V. The scan time was 0.1 sec covering the 100 to 1000 Da mass range. The source temperature was 120ºC and the desolvation temperature was set at 400ºC. Nitrogen gas was used as the nebulization gas at a flow rate of 800 L/h. The software used to control the hyphenated system and for data manipulation was MassLynx 4.1 (SCN 704). Cells and cell culture: Human neuroblastoma (SH-SY5Y) cells (ATCC CRL-2266) were used for the cytotoxicity studies. Cells were cultured in Ham’s F-12 supplemented with 2% heatinactivated fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37oC in a humidified incubator at 95% air and 5% CO2. For use in the assay, the cells were trypsin-treated for 10 min, decanted from culture flasks and centrifuged (200 g, 10 min). The pellet was re-suspended in 1 mL Ham’s F-12 medium supplemented with fetal calf serum, and enumerated by staining with trypan blue. The cells were diluted to a concentration of 1 × 105 cells/well in Ham’s F-12 medium and 100 µL of the cell suspension plated into each of the wells of a 96-well microtiter plate. Ham’s F-12 medium (80 µL) was added and plates were then incubated for 1 h at 37oC in a humidified incubator with 95% air and 5% CO2 to allow for cellular re-attachment. MTT assay: The MTT assay as described by Mossmann [19] was used to measure cell viability. The principle of the assay is based on generation of formazan (a blue product) in the mitochondria of active cells, which is measured by photometric techniques [20]. The compound was dissolved in 0.3%, v/v, DMSO in distilled water. The vehicle was used as control. Figure 2: Effect of cycloeucalenol and its regio-isomer on the viability of SH-SY5Y cell lines as determined by the MTT and neutral red uptake assays after 72 h of incubation. The cells were plated into 96-well culture plates, as described above, and treated with various concentrations of the compound ranging from 3.125 μM to 400 μM for 72 h. Thereafter, 20 μL of MTT solution (5 mg/mL) was added to the wells and further incubated for 3 h. A solution (50 μL) containing 30%, w/v, N,Ndimethylformamide and 20% sodium dodecyl sulfate in water was then added to breach the cells and dissolve the formazan crystals. The plates were incubated overnight at 37oC, after which absorbance was measured at 570-630 nm using a microtiter plate reader (Labsystems Multiscan EX type 355). Wells without cells were used as blanks and were subtracted as background from each sample. Cytotoxicity results are expressed as the percentage cell survival compared with the untreated control using a dose response curve and extract concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage versus extract concentration. Neutral red assay: The neutral red uptake assay, as described by Borenfreund and Puerner [21], was also used to assess cell viability. This method is based on the determination of the accumulation of the neutral red dye in the lysosomes of viable, uninjured cells. The compound was dissolved in 0.3%, v/v, DMSO in distilled water. The vehicle was used as control. The cells were plated into 96-well culture plates, as described above, and treated with various concentrations of the compound ranging from 3.125 μM to 400 μM for 72 h. Thereafter, 150 µL of neutral red dye (100 µg/mL) dissolved in serum free medium (pH 6.4) was added to the culture medium for 3 h at 37°C. Cells were washed with Phosphate Buffered Saline (PBS), and 150 μL of elution medium (EtOH/AcCOOH/H2O, 50%/1%/49%) was added, followed by gentle shaking for 60 min, so that complete dissolution could be achieved. Absorbance was recorded at 540-630 nm using a microtiter plate reader (Labsystems Multiscan EX type 355). Cytotoxicity results are expressed as the percentage cell survival compared with the untreated control using a dose response curve and extract concentration providing 50% inhibition (IC50) of cell death was calculated from the graph. Statistical analysis: Tests were carried out where possible at least in triplicate and on 3 different occasions. The results are reported as mean ± standard deviation (S.D.). Standard curves were generated and calculation of the 50% inhibitory concentration (IC50) values was made using GraphPad Prism Version 4.00 for Windows 1216 Natural Product Communications Vol. 8 (9) 2013 (GraphPad Software Inc.). Cytotoxicity results are expressed as the percentage cell survival compared with the untreated control using a dose response curve. Data obtained from mass spectroscopy were analyzed using MassLynx 4.1 (SCN 704) software. Adewusi et al. 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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