Phytochemistry (v.71, #2-3)

Plant NADPH-cytochrome P450 oxidoreductases by Kenneth Jensen; Birger Lindberg Møller (132-141).
NADPH-cytochrome P450 oxidoreductase (CPR) serves as the electron donor to almost all eukaryotic cytochromes P450. It belongs to a small family of diflavin proteins. Dependent on the species, plants contain one, two or three CPR paralogs of which one is inducible. CPRs from different species or even from different kingdoms at least partially complement each other functionally and show that the nature of P450-CPR interacting domains is well conserved. This makes CPR an ideal bio-brick in synthetic biology.NADPH-cytochrome P450 oxidoreductase (CPR) serves as the electron donor to almost all eukaryotic cytochromes P450. It belongs to a small family of diflavin proteins and is built of cofactor binding domains with high structural homology to those of bacterial flavodoxins and to ferredoxin-NADP+ oxidoreductases. CPR shuttles electrons from NADPH through the FAD and FMN-cofactors into the central heme-group of the P450s. Mobile domains in CPR are essential for electron transfer between FAD and FMN and for P450 interaction. Blast searches identified 54 full-length gene sequences encoding CPR derived from a total of 35 different plant species. CPRs from vascular plants cluster into two major phylogenetic groups. Depending on the species, plants contain one, two or three paralogs of which one is inducible. The nature of the CPR–P450 interacting domains is well conserved as demonstrated by the ability of CPRs from different species or even from different kingdoms to at least partially complement each other functionally. This makes CPR an ideal bio-brick in synthetic biology approaches to re-design or develop entirely different combinations of existing biological systems to gain improved or completely altered functionalities based on the “share your parts” principle.
Keywords: CPR; P450; Evolution; Structural domains; Electron transfer; Physiology; Homologs; Subcellular localization;

The expression of γ-conglutin during seed germination can be elicited by treatment with chitosan. This strengthen the hypothesis of an actual role of the proteins in plant defence mechanisms. Despite structurally belonging to the XEGIP protein family, this lupin protein does not display any inhibitory activity against the representative endo-glucanase belonging to GH12 family and the other enzymes tested.γ-Conglutin, a glycoprotein from Lupinus albus seed, has been characterized at molecular level but its physiological function is still unknown. γ-Conglutin shares a high structural similarity with xyloglucan-specific endo-β-1,4-glucanase inhibitor proteins (XEGIPs) and Triticum aestivum xylanase inhibitor (TAXI-I), which act specifically against fungal glycosyl hydrolase belonging to families 12 and 11, respectively. To assess the possible involvement of γ-conglutin in plant defense, germinating lupin seeds were incubated with chitosan. The relative quantification of γ-conglutin mRNA extracted from cotyledons was then carried out by RT-qPCR and indicated that chitosan strongly elicited the expression of γ-conglutin. Moreover, biochemical trials aimed to test the inhibitory capacity of the protein have been also carried out. γ-Conglutin failed to inhibit representative fungal endo-glucanases and other cell wall-degrading enzymes. To explain the lack of inhibitory capacity we investigated the possible structural differences between γ-conglutin and XEGIPs and TAXI-I, including the construction of a predictive 3D model of the protein. Bioinformatic analysis suggests that the lack of inhibitory activity of γ-conglutin can be attributed to sequence differences in the inhibitor interaction domains, and in particular to a sequence deletion in one of the functional loops.
Keywords: Lupinus albus; Seed proteins; Enzyme inhibitors; Plant defence; XEGIP;

Acetylcholinesterase inhibitory activity of lycopodane-type alkaloids from the Icelandic Lycopodium annotinum ssp. alpestre by Elsa Steinunn Halldorsdottir; Jerzy W. Jaroszewski; Elin Soffia Olafsdottir (149-157).
Docking studies using 3D model of EeAChE provided rationale for low inhibitory activity of lycopodane-type alkaloids from Lycopodium annotinum, as well as hints for future structure-based design. Along with isolation of a new alkaloid, NMR and conformational data for several classical Lycopodium alkaloids are reported.The aim of this study was to investigate structures and acetylcholinesterase inhibitory activities of lycopodane-type alkaloids isolated from an Icelandic collection of Lycopodium annotinum ssp. alpestre. Ten alkaloids were isolated, including annotinine, annotine, lycodoline, lycoposerramine M, anhydrolycodoline, gnidioidine, lycofoline, lannotinidine D, and acrifoline, as well as a previously unknown N-oxide of annotine. 1H and 13C NMR data of several of the alkaloids were provided for the first time. Solvent-dependent equilibrium constants between ketone and hemiketal form of acrifoline were determined. Conformation of acrifoline was characterized using NOESY spectroscopy and molecular modelling. The isolated alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Ligand docking studies based on mutated 3D structure of Torpedo californica acetylcholinesterase provided rationale for low inhibitory activity of the isolated alkaloids as compared to huperzine A or B, which are potent acetylcholinesterase inhibitors belonging to the lycodine class. Based on the modelling studies the lycopodane-type alkaloids seem to fit well into the active site gorge of the enzyme but the position of their functional groups is not compatible with establishing strong hydrogen bonding interactions with the amino acid residues that line the binding site. The docking studies indicate possibilities of additional functionalization of the lycopodane skeleton to render potentially more active analogues.
Keywords: Lycopodium annotinum; Lycopodiaceae; Alkaloids; NMR spectroscopy; Acetylcholinesterase; Butyrylcholinesterase; Ligand docking;

Petunia floral volatile benzenoid/phenylpropanoid genes are regulated in a similar manner by Thomas A. Colquhoun; Julian C. Verdonk; Bernardus C.J. Schimmel; Denise M. Tieman; Beverly A. Underwood; David G. Clark (158-167).
Petunia flowers emit volatile compounds as a group. Likewise, seven floral volatile gene transcripts accumulate as a group spatially, developmentally, and after ethylene treatment.Petunia (Petunia x hybrida cv ‘Mitchell Diploid’ [MD]) flowers emit high levels of multiple floral volatile benzenoid/phenylpropanoid (FVBP) compounds from anthesis to senescence in a concerted manner. Here we show seven genes responsible for the production of emitted FVBPs share similar transcript accumulation profiles through an analysis of four expression criteria. As a group, the FVBP gene transcripts accumulate to high levels in petal limb tissue of MD flowers from anthesis to senescence. Two to four hours of exogenous ethylene exposure reduces transcript levels of all FVBP genes examined, but 2 h of treatment will not accelerate senescence or reduce volatile emissions in MD flowers. The FVBP genes show two obvious rhythmic patterns of transcript accumulation; however, corresponding enzyme activities of a subset of FVBP gene products do not. Together, these results depict floral volatile benzenoid/phenylpropanoid biosynthesis as a specific system with multiple regulatory features. One such feature is the highly regulated transcript accumulation of the FVBP genes. Additionally, ethylene may have a regulatory role in the FVBP system prior to a floral senescence program.
Keywords: Petunia x hybrida; Solanaceae; Petunia; Benzenoid/phenylpropanoid; Ethylene; Flower; Volatiles;

Hydroxylation of carotenoid β- and ε-rings is critical for their modification to generate a diversity of carotenoids, including ketocarotenoids. Assay of carotenoid profiles in multiple Arabidopsis thaliana lines overexpressing four carotenoid hydroxylases alone or in combination with an algal β-carotenoid ketolase provides insight into in vivo enzyme functions and pathway cross-talk.Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of α- and β-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo β-carotene hydroxylations of β,β-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the ε-ring of α-carotene or the β-ring of β,ε-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the β- and ε-rings of α-carotene and β-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) β-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 β-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of α-carotene derivatives, accompanied by a reduction of α-carotene, were observed. These data suggest possible interactions between the β-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways.
Keywords: Arabidopsis thaliana; Cruciferae; Thale CMSS; Ketocarotenoid; P450 hydroxylase; Ketolase; β-Carotene hydroxylase; bkt1; CrtO; Cytochrome P450;

Artemisinin biosynthesis in growing plants of Artemisia annua. A 13CO2 study by Nicholas Schramek; Huahong Wang; Werner Römisch-Margl; Birgit Keil; Tanja Radykewicz; Bernhard Winzenhörlein; Ludger Beerhues; Adelbert Bacher; Felix Rohdich; Jonathan Gershenzon; Benye Liu; Wolfgang Eisenreich (179-187).
A plant of Artemisia annua was labelled with 13CO2. The isotopologue profile of artemisinin indicated its formation from (E,E)-farnesyl diphosphate with a prenyl moiety (marked in red) predominantly derived from the non-mevalonate pathway.Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate pathway. In order to address that question, a plant of A. annua was grown in an atmosphere containing 700 ppm of 13CO2 for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by 13C NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosynthesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic conversion of FPP to amorphadiene and show that either of the two methyl groups can undergo deprotonation.
Keywords: Terpene; Biosynthesis; Mevalonate; 1-Deoxyxylulose phosphate; Artemisinin;

A detailed spectroscopic and microscopic investigation of the mechanisms involved in metal delivery and storage in the Ni hyperaccumulator Alyssum murale showed that Ni is transported in the stem and leaf xylem sap complexed with malate, tartrate, possibly histidine and low molecular weight organic acids (LMWOA) and/or counter-ions and stored in leaf epidermal vacuoles complexed primarily with malate.The Kotodesh genotype of the nickel (Ni) hyperaccumulator Alyssum murale was examined to determine the compartmentalization and internal speciation of Ni, and other elements, in an effort to ascertain the mechanism used by this plant to tolerate extremely high shoot (stem and leaf) Ni concentrations. Plants were grown either hydroponically or in Ni enriched soils from an area surrounding an historic Ni refinery in Port Colborne, Ontario, Canada. Electron probe micro-analysis (EPMA) and synchrotron based micro X-ray fluorescence (μ-SXRF) spectroscopy were used to determine the metal distribution and co-localization and synchrotron X-ray and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopies were used to determine the Ni speciation in plant parts and extracted sap. Nickel is concentrated in the dermal leaf and stem tissues of A. murale bound primarily to malate along with other low molecular weight organic ligands and possibly counter anions (e.g., sulfate). Ni is present in the plant sap and vasculature bound to histidine, malate and other low molecular weight compounds. The data presented herein supports a model in which Ni is transported from the roots to the shoots complexed with histidine and stored within the plant leaf dermal tissues complexed with malate, and other low molecular weight organic acids or counter-ions.
Keywords: Alyssum murale; Hyperaccumulation; Nickel (Ni); Extended X-ray absorption fine structure (EXAFS) spectroscopy; Synchrotron X-ray fluorescence (SXRF) spectroscopy; Tolerance; Malate; Histidine;

Biotransformation of naringin and naringenin by cultured Eucalyptus perriniana cells by Kei Shimoda; Naoji Kubota; Koji Taniuchi; Daisuke Sato; Nobuyoshi Nakajima; Hatsuyuki Hamada; Hiroki Hamada (201-205).
Naringin (1) was converted into biotransformation products 28 by cultured cells of Eucalyptus perriniana. On the other hand, 2, 47, and 911 were isolated from E. perriniana treated with naringenin (3).The biotransformation of naringin and naringenin was investigated using cultured cells of Eucalyptus perriniana. Naringin (1) was converted into naringenin 7-O-β-d-glucopyranoside (2, 15%), naringenin (3, 1%), naringenin 5,7-O-β-d-diglucopyranoside (4, 15%), naringenin 4′,7-O-β-d-diglucopyranoside (5, 26%), naringenin 7-O-[6-O-(β-d-glucopyranosyl)]-β-d-glucopyranoside (6, β-gentiobioside, 5%), naringenin 7-O-[6-O-(α-l-rhamnopyranosyl)]-β-d-glucopyranoside (7, β-rutinoside, 3%), and 7-O-β-d-gentiobiosyl-4′-O-β-d-glucopyranosylnaringenin (8, 1%) by cultured cells of E. perriniana. On the other hand, 2 (14%), 4 (7%), 5 (13%), 6 (2%), 7 (1%), naringenin 4′-O-β-d-glucopyranoside (9, 4%), naringenin 5-O-β-d-glucopyranoside (10, 2%), and naringenin 4′,5-O-β-d-diglucopyranoside (11, 5%) were isolated from cultured E. perriniana cells, that had been treated with naringenin (3). Products, 7-O-β-d-gentiobiosyl-4′-O-β-d-glucopyranosylnaringenin (8) and naringenin 4′,5-O-β-d-diglucopyranoside (11), were hitherto unknown.
Keywords: Eucalyptus perriniana; Mystaceae; Eucalyptus; Cultured plant cells; Biotransformation; Glycosylation; Naringin; Naringenin; β-Glycosides;

O-Methylation of phenylphenalenone phytoalexins in Musa acuminata and Wachendorfia thyrsiflora by Felipe Otálvaro; Kusuma Jitsaeng; Tobias Munde; Fernando Echeverri; Winston Quiñones; Bernd Schneider (206-213).
Labelled precursors were used to study biosynthetic O-methylation in various positions of the backbone of phenylphenalenones by NMR spectroscopic methods. The inducible formation of (methoxy)phenylphenalenones after treatment with jasmonic acid and copper chloride has been shown.Biosynthetic O -methylation at various sites along the backbone of inducible phenylphenalenones in Musa acuminata var. “Williams” (Musaceae) and Wachendorfia thyrsiflora (Haemodoraceae) was investigated using 13C-labelled precursors. The inducibility of O-methylated metabolites was demonstrated in both species and the origin of methoxyl group from [methyl-13C]l-methionine was confirmed. In addition to known phenylphenalenones, a methoxylated metabolite, 4-(4-hydroxy-3-methoxy-phenyl)-benzo[de]isochromene-1,3-dione, was detected and its structure elucidated mainly by NMR spectroscopic techniques. The experiments were used to discriminate methionine-derived and artificial methoxy groups formed during methanolic extraction. Finally, demethylation of 4′-methoxycinnamic acid and subsequent conversion to 3′,4′-methylenedioxycinnamic acid was demonstrated in M. acuminata.
Keywords: Musa acuminata; Wachendorfia thyrsiflora; Haemodoraceae; Musaceae; Elicitor; Labelling; Methylation; NMR; Phenylphenalenones; Phytoalexins;

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition by Joana Tomomi Sumikawa; Marlon Vilela de Brito; Maria Ligia Rodrigues Macedo; Adriana F. Uchoa; Antonio Miranda; Ana Paula U. Araujo; Rosemeire A. Silva-Lucca; Misako Uemura Sampaio; Maria Luiza Vilela Oliva (214-220).
The study addressed the defensive role of plant protease inhibitors, including presence of the functional domain. A very short functional domain with less than 10 amino acids, which is not directly involved in proteinase inhibitory activity, was found to be crucial for the toxic effect of PI.Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH2 (RGE) and IVYYPDRGETGL-NH2 (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED50 0.16% and LD50 0.09%), this being even more effective than the native protein.
Keywords: Bauhinia sp.; Fabaceae; Caesalpinodae; Callosobruchus maculatus; Vigna unguiculata; Leguminosae; Cowpea; Defense protein; Insect attack; Kunitz inhibitors; Pesticide; Plant peptidase inhibitors; RGD; Trypsin inhibitor;

In vitro chemopreventive potential of fucophlorethols from the brown alga Fucus vesiculosus L. by anti-oxidant activity and inhibition of selected cytochrome P450 enzymes by Sabine Parys; Stefan Kehraus; Anja Krick; Karl-Werner Glombitza; Shmuel Carmeli; Karin Klimo; Clarissa Gerhäuser; Gabriele M. König (221-229).
From Fucus vesiculosus three fucophlorethols with potential chemopreventive activities have been isolated. All three compounds were identified as potent radical scavengers, and may contribute to the prevention of carcinogenesis by inhibition of cytochrome P450 enzymes like CYP1A, involved in carcinogen activation, and aromatase, essential for estrogen biosynthesis.Within a project focusing on the chemopreventive potential of algal phenols, two phloroglucinol derivatives, belonging to the class of fucophlorethols, and the known fucotriphlorethol A were obtained from the ethanolic extract of the brown alga Fucus vesiculosus L. The compounds trifucodiphlorethol A and trifucotriphlorethol A are composed of six and seven units of phloroglucinol, respectively.The compounds were examined for their cancer chemopreventive potential, in comparison with the monomer phloroglucinol. Trifucodiphlorethol A, trifucotriphlorethol A as well as fucotriphlorethol A were identified as strong radical scavengers, with IC50 values for scavenging of 1,1-diphenyl-2 picrylhydrazyl radicals (DPPH) in the range of 10.0–14.4 μg/ml. All three compounds potently scavenged peroxyl radicals in the oxygen radical absorbance capacity (ORAC) assay. In addition, the compounds were shown to inhibit cytochrome P450 1A activity with IC50 values in the range of 17.9–33 μg/ml, and aromatase (Cyp19) activity with IC50 values in the range of 1.2–5.6 μg/ml.
Keywords: Fucus vesiculosus; Fucaceae; Fucophlorethols; Phlorotannin; Anti-oxidant; Radical-scavenging; Phloroglucinol; Cytochrome P450 (Cyp); Aromatase; Chemoprevention;

Antifungal constituents isolated from the seeds of Aegle marmelos by Bhuwan B. Mishra; Desh D. Singh; Navneet Kishore; Vinod K. Tiwari; Vyasji Tripathi (230-234).
Fungal infections are a challenge, particularly in the growing number of immunosuppressed patients seen in modern medical facilities. In present communication isolation, structure elucidation and antifungal activity of furo-anthraquinone (5) and coumarin (9) has been described.Antifungal constituents, 2-isopropenyl-4-methyl-1-oxa-cyclopenta[b]anthracene-5,10-dione and (+)-4-(2′-hydroxy-3′-methylbut-3′-enyloxy)-8H-[1,3]dioxolo[4,5-h]chromen-8-one in addition to known compounds imperatorin, β-sitosterol, plumbagin, 1-methyl-2-(3′-methyl-but-2′-enyloxy)-anthraquinone, β-sitosterol glucoside, stigmasterol, vanillin and salicin were isolated during phytochemical investigation on seeds of Aegle marmelos Correa.
Keywords: Rutaceae; Aegle marmelos Correa; Anthraquinone; Coumarin; Antifungal activity;

Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and anti-viral activity by Sharmistha Sinha; Akram Astani; Tuhin Ghosh; Paul Schnitzler; Bimalendu Ray (235-242).
A guluronic acid-rich alginate (B) and a fucoidan (C) extracted from Sargassum tenerrimum are inhibitor of herpes simplex virus type 1, whereas their sulfated derivatives (BS and CS) show higher activity.Herpes simplex viruses (HSVs) display affinity for cell-surface heparan sulfate proteoglycans with biological relevance in virus entry. Here, we exploit an approach to inhibiting HSV infection by using a sulfated fucoidan, and a guluronic acid-rich alginate derived from Sargassum tenerrimum, mimicking the active domain of the entry receptor. These macromolecules have apparent molecular masses of 30 ± 5 and 26 ± 5 kDa, respectively. They and their chemically sulfated derivatives showed activity against herpes simplex virus type 1 (HSV-1). Their inhibitory concentration 50% (IC50) values were in the range 0.5–15 μg/ml and they lacked cytotoxicity at concentrations up to 1000 μg/ml. The anti-HSV activity increased with increasing sulfate ester content. Our results suggest the feasibility of inhibiting HSV infection by blocking viral entry with polysaccharide having specific structure.
Keywords: Sargassum tenerrimum; Sargassaceae; Herpes simplex virus; Anti-viral activity; Poly-guluronate; Fucoidan;

Effects of diterpenes from latex of Euphorbia lactea and Euphorbia laurifolia on human immunodeficiency virus type 1 reactivation by Liliana Avila; Moises Perez; Gonzalo Sanchez-Duffhues; Rosario Hernández-Galán; Eduardo Muñoz; Fabio Cabezas; Winston Quiñones; Fernando Torres; Fernando Echeverri (243-248).
Through bioassays, three diterpenes were isolated and characterized by spectroscopic analysis from Euphorbia laurifolia and Euphorbia lactea; differential and specific activity on HIV-1 reactivation was observed.The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication in patients treated with highly active antiretroviral therapy, referred to as HAART. HIV-1 reservoirs are long-lived resting CD4+ memory cells containing the virus latently integrated. Since the HIV-1 reservoirs are not targeted by HAART, reactivation therapy has been suggested to purge viral latency. Bioassay-guided study of an ethyl acetate extract of Euphorbia laurifolia afforded two isomeric diterpenes that showed differential activity over HIV-1 reactivation. A previously reported compound was isolated too from Euphorbia lactea. This compound showed a potent HIV-1 reactivating effect. Bioassays results showed that HIV-1 reactivation activity is influenced by distinct structural characteristics.
Keywords: Euphorbiaceae; E. laurifolia; E. lactea; HIV; Latency; Reactivation therapy;

Bioactive jatrophane diterpenes from Euphorbia guyoniana by Mohamed-Elamir F. Hegazy; Abou El-Hamd H. Mohamed; Nobuwa Aoki; Toshitaka Ikeuchi; Emi Ohta; Shinji Ohta (249-253).
Chromatographic investigation of the methylenechloride/methanol extract of the aerial parts of Euphorbia guyoniana afforded two jatrophane diterpenes designated guyonianins E (1) and F (2), in addition to a known jatrophane diterpene (3). The structures of the compounds were determined by comprehensive NMR analyses, including DEPT, COSY, HMQC, HMBC, NOESY and HRMS. These compounds exhibited cytotoxicity against human embryonic kidney 293 (HEK293) cells with IC50 values of 35–100 μM.Chromatographic investigation of the methylenechloride/methanol extract of the aerial parts of Euphorbia guyoniana afforded two jatrophane diterpenes, designated guyonianins E and F, in addition to a known jatrophane diterpene. The structures of the compounds were determined by comprehensive NMR analyses, including DEPT, COSY, HMQC, HMBC, NOESY and HRMS. These compounds exhibited cytotoxicity against human embryonic kidney 293 (HEK293) cells with IC50 values of 35–100 μM.
Keywords: Euphorbia guyoniana; Euphorbiaceae; Jatrophane diterpenes; Cytotoxicity;

Rheediinosides A and B, two antiproliferative and antioxidant triterpene saponins from Entada rheedii by Laurence Kegah Nzowa; Luciano Barboni; Remy Bertrand Teponno; Massimo Ricciutelli; Giulio Lupidi; Luana Quassinti; Massimo Bramucci; Léon Azefack Tapondjou (254-261).
Two triterpenoid saponins have been isolated from the seed kernels of Entada rheedii. Their structures have been established using 1D- and 2D-NMR and MS as 3-O-β-d-xylopyranosyl-(1 → 3)-O-α-l-arabinopyranosyl-(1 → 6)-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside A, 1) and 3-O-β-d-glucopyranosyl-(1 → 3)-O-[β-d-xylopyranosyl-(1 → 3)-α-l-arabinopyranosyl-(1 → 6)]-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside B, 2). Moderate cytotoxic potency and antioxidant properties were found for these compounds.Two triterpenoid saponins have been isolated from the seed kernels of Entada rheedii. Their structures have been established using 1D- and 2D-NMR and mass spectrometry as 3-O-β-d-xylopyranosyl-(1 → 3)-O-α-l-arabinopyranosyl-(1 → 6)-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside A, 1) and 3-O-β-d-glucopyranosyl-(1 → 3)-O-[β-d-xylopyranosyl-(1 → 3)-α-l-arabinopyranosyl-(1 → 6)]-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside B, 2). Compounds 1 and 2 were tested for their antiproliferative activity against T98G, A431, PC3 and B16-F1 cell lines, and further for their antioxidant properties. Moderate cytotoxic potency and antioxidant properties were found for these compounds whereas Rheediinoside B was in all assays more active than Rheediinoside A.
Keywords: Entada rheedii; Mimosaceae; Triterpene saponins; Rheediinosides A–B; Antioxidant; Antiproliferative;

Bioactive microbial metabolites from glycyrrhetinic acid by Galal T. Maatooq; Amani M. Marzouk; Alexander I. Gray; John P. Rosazza (262-270).
Isolation and structural assignment of seven microbial metabolites of 18β-glycyrrhetinic acid are reported. Two major metabolites displayed significant hepatoprotective activity.Biotransformation of 18β-glycyrrhetinic acid, using Absidia pseudocylinderospora ATCC 24169, Gliocladium viride ATCC 10097 and Cunninghamella echinulata ATCC 8688a afforded seven metabolites, which were identified by different spectroscopic techniques (1H, 13C NMR, DEPT, 1H-1H COSY, HMBC and HMQC). Three of these metabolites, viz. 15α-hydroxy-18α-glycyrrhetinic acid, 13β-hydroxy-7α,27-oxy-12-dihydro-18β-glycyrrhetinic acid and 1α-hydroxy-18β-glycyrrhetinic acid are new. The 13C NMR data and full assignment for the known metabolite 7β, 15α-dihydroxy-18β-glycyrrhetinic acid are described here for the first time. The major metabolites were evaluated for their hepatoprotective activity using different in vitro and in vivo models. These included protection against FeCl3/ascorbic acid-induced lipid peroxidation of normal mice liver homogenate, induction of nitric oxide (NO) production in rat macrophages and in vivo hepatoprotection against CCl4-induced hepatotoxicity in albino mice.
Keywords: 18β-Glycyrrhetinic acid; Microbial transformation; Hepatoprotection; Lipid peroxidation; Nitric oxide;

Cardiovascular protective flavonolignans and flavonoids from Calamus quiquesetinervius by Chao-Lin Chang; Guei-Jane Wang; Li-Jie Zhang; Wen-Jen Tsai; Ru-Yin Chen; Yang-Chang Wu; Yao-Haur Kuo (271-279).
Tricin-type flavonolignans (16), along with six known phenolic compounds (712), were isolated from the EtOAc extract of Calamus quiquesetinervius.Tricin-type flavonolignans, (2S)-dihydrotricin 4′-O-(erythro-β-guaiacylglyceryl) ether, (2S)-dihydrotricin 4′-O-(threo-β-guaiacylglyceryl) ether, (2S)-dihydrotricin 4′-O-(threo-β-4-hydroxyphenylglyceryl) ether, tricin 4′-O-(erythro-β-4-hydroxyphenylglyceryl) ether, tricin 4′-O-(threo-β-4-hydroxylphenylglyceryl) ether, and (2S)-dihydrotricin 4′-O-(β-6′′-methoxy-4′′-oxo-chroman-3′′-yloxy) ether namely calquiquelignan A–F, respectively, were isolated and characterized from the EtOAc extract of Calamus quiquesetinervius. Additionally, six known phenolic compounds, including dihydrotricin, tricin, salcolin A, p-hydroxybenzoic acid, (2S, 3S)-trans-dihydrokapempferol and (2S)-naringenin, were also obtained and identified from the extract. Structures of the flavonolignans were assigned based on spectroscopic analyses that included 1D and 2D NMR spectroscopic techniques, such as HMQC, HMBC, and NOESY. Bioassay results showed that calquiquelignan A, dihydrotricin and (2S)-naringenin exhibited significant vasodilatory potencies, as indicated by 60.3%, 80.3% and 60.9% relaxations, respectively, at 100 μM. Salcolin A showed potent platelet aggregation inhibition, compared with aspirin. Most of the tricin-type derivatives (calquiquelignan A–B, dihydrotricin and tricin) also exhibited more potent hydroxyl radical (OH) scavenging activity than trolox as characterized by the ultraweak chemiluminescence assay.
Keywords: Calamus quiquesetinervius; Arecaceae; Vasodilatory potencies; Platelet aggregation; Hydroxyl radical; Flavonolignan; Calquiquelignan A–F;

Different contributions of side-chains in β-d-(1 → 3,6)-galactans on intestinal Peyer’s patch-immunomodulation by polysaccharides from Astragalus mongholics Bunge by Hiroaki Kiyohara; Taichi Uchida; Manami Takakiwa; Toshiake Matsuzaki; Noriyasu Hada; Tadahiro Takeda; Toshiro Shibata; Haruki Yamada (280-293).
β-d-(1 → 3)-Galactan and galactooligosaccharide glyco-cluster bearing β-d-(1 → 6)-oligogalactosyl side-chains were found as immunomodulator for intestinal Peyer’s patch immunocompetent cells from aerial parts of Astragalus mongholics.Thirteen polysaccharides isolated from an extract of the aerial portions of Astragalus mongholics Bunge demonstrated immunomodulating activity against Peyer’s patch immunocompetent cells. Nine of the active polysaccharide fractions were composed of either arabinogalactans, pectic arabinogalactans or pectins. The activities of the arabinogalactans and pectic arabinogalactans were associated with β-d-(1 → 3)-galactan moieties branched with β-d-(1 → 6)-galactooligosaccharide side-chains having degrees of polymerization of 8 or less. Degradation of the β-d-(1 → 3)-galactan or β-d-(1 → 6)-galactosyl side-chains in the arabinogalactans significantly decreased immunomodulating activity. Rhamnogalacturonan I (RG-I) with β-d-(1 → 3,6)-galactosyl side-chains having terminal β-d-GlcA showed activity in the pectin-enriched fractions. Interestingly, the terminal GlcA was not required for activity of the arabinogalactan-enriched fractions, suggesting at least two different immunomodulating structures.
Keywords: Astragalus mongholics Bunge; Leguminosae; Polysaccharides; Oligosaccharide side-chain; Enzymatic digestion; Exo-β-d-(1 → 3)-galactanase; High performance anion-exchange chromatography; Immunomodulating activity; Intestinal immune system; Peyer’s patches;

Flavones and phenylpropanoids from a sedative extract of Lantana trifolia L. by Lisieux de Santana Julião; Suzana Guimarães Leitão; Cinzia Lotti; Anna Lisa Picinelli; Luca Rastrelli; Patricia D. Fernandes; François Noël; Jean-Pierre Barros Thibaut; Gilda Guimarães Leitão (294-300).
The flavones scutellarein-7-O-β-d-apiofuranoside (1), celtidifoline (5,6,4′,5′-tetrahydroxy-7,3′-dimethoxyflavone) (2), and apigenin-7-O-β-d-apiofuranosyl-(1 → 2)-β-d-apiofuranoside (3), were isolated along with other 11 known compounds from leaves of the sedative ethyl acetate extract of Lantana trifolia L. by combined High Speed Countercurrent Chromatography (HSCCC) and HPLC. Their structures were elucidated using 2D NMR spectroscopic and mass spectrometry (ESI-MS) techniques. The sedative properties of L. trifolia extracts were evaluated by the open field method in mice as well as by the affinity for the [3H] flunitrazepam binding sites of the isolated compounds 15.The flavone glycosides, named scutellarein-7-O-β-d-apiofuranoside and apigenin-7-O-β-d-apiofuranosyl-(1 → 2)-β-d-apiofuranoside, and the flavone celtidifoline (5,6,4′,5′-tetrahydroxy-7,3′-dimethoxyflavone), along with other 11 known compounds, were isolated from leaves of the ethyl acetate extract of Lantana trifolia L. using step gradient High Speed Countercurrent Chromatography (HSCCC) and High Performance Liquid Chromatography (HPLC), respectively. Their structures were elucidated by spectroscopic methods, including 2D NMR and mass spectrometry (ESI-MS) techniques. The ethanolic and ethyl acetate extracts produced an intense sedative effect in mice, one hour after oral administration of 1 mg/kg. This effect was neither due to a benzodiazepine-like effect of the three flavone derivatives neither of the phenylpropanoids, betonyoside F and verbascoside, that were tested for their affinity for the [3H] flunitrazepam binding sites.
Keywords: Lantana trifolia; Verbenaceae; HSCCC; Scutellarein-7-O-β-d-apiofuranoside; Apigenin-7-O-β-d-apiofuranosyl-(1 → 2)-β-d-apiofuranoside; Celtidifoline; 1D and 2D NMR spectroscopy; Sedative activity; Benzodiazepine receptor;

Glycosides of benzodioxole-indole alkaloids from Narcissus having axial chirality by Tomáš Řezanka; Pavel Řezanka; Karel Sigler (301-306).
Glycosides made up of glucose, Rha–Glc, and Xyl–Rha–Glc of the benzodioxole-indole alkaloid 6-hydroxy-galanthindole, having axial chirality were isolated from Narcissus cultivar ‘Dutch Master’. The structure, including absolute configuration, was determined by means of extensive spectroscopic data such as UV, IR, CD, MS, 1D and 2D NMR spectra, and computational chiroptical methods.Glycosides of benzodioxole-indole alkaloid 6-hydroxy-galanthindole (7-(6′-(hydroxymethyl)benzo[d][1′,3′]dioxol-5′-yl)-1-methyl-1H-indol-6-ol) having axial chirality were isolated from Narcissus cultivar ‘Dutch Master’. The structure, including absolute configuration, was determined by means of extensive spectroscopic data such as UV, IR, CD, MS, 1D and 2D NMR spectra, and computational chiroptical methods. The aglycone has a structure containing two aromatic moieties with substituents hindering rotation about the biaryl axis and is connected to a saccharide moiety linked at C-6 and made up of one, two, or three sugars (glucose, α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranose, and trisaccharide ([β-d-xylopyranosyl(1 → 2)]-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranose).
Keywords: Narcissus cultivar ‘Dutch Master’; Amaryllidaceae; 6-Hydroxy-galanthindole; Benzodioxole-indol alkaloids; Axial chirality; Glycosides;

Hantupeptins B and C, cytotoxic cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula by Ashootosh Tripathi; Jonathan Puddick; Michele R. Prinsep; Peter Peng Foo Lee; Lik Tong Tan (307-311).
Members belonging to the marine cyanobacterial genus Lyngbya are an important source of bioactive compounds. This article details the isolation and structural determination of a cytotoxic cyclic depsipeptides, hantupeptins B and C, from a collection of Lyngbya majuscula from Singapore waters.Hantupeptins B (2) and C (3) were isolated, along with the previously reported hantupeptin A (1), from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu Besar, Singapore. Their structures were elucidated by interpretation of extensive 1D and 2D NMR spectroscopic data. Compounds 2 and 3 are cyclic depsipeptides consisting of five α-amino/hydroxy acid residues, including phenyllactic acid, proline, N-methyl-valine, valine, N-methyl-isoleucine, and a β-hydroxy acid unit with different degrees of unsaturation at the terminal end of each molecule. The absolute configurations of the common amino acids and phenyllactic acid were determined by the advanced Marfey’s and chiral HPLC analyses, respectively. The complete stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid moiety in hantupeptin A was elucidated by a combination of homonuclear J-resolved 2D NMR experiments and by Mosher’s method. Hantupeptins B and C showed moderate in vitro cytotoxicity when tested against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines.
Keywords: Lyngbya majuscula; Oscillatoriaceae; Marine cyanobacteria; Brine shrimp toxicity; Cytotoxicity; Cyclic depsipeptide;

The antibacterial properties of 6-tuliposide B. Synthesis of 6-tuliposide B analogues and structure–activity relationship by Kengo Shigetomi; Kazuaki Shoji; Shinya Mitsuhashi; Makoto Ubukata (312-324).
6-Tuliposide B and tulipalin B analogues were synthesized and structure–activity relationship (SAR) was studied against a broad panel of bacterial strains.6-Tuliposide B is a secondary metabolite occurring specifically in tulip anthers. Recently, a potent antibacterial activity of 6-tuliposide B has been reported. However, its molecular target has not yet been established, nor its action mechanism. To shed light on such issues, 6-tuliposide B and tulipalin B analogues were synthesized and a structure–activity relationship (SAR) was examined using a broad panel of bacterial strains. As the results of SAR among a total of 25 compounds, only tulipalin B and the compounds having 3′,4′-dihydroxy-2′-methylenebutanoate (DHMB) moieties showed any significant antibacterial activity. Moreover, the 3′R analogues of these compounds displayed essentially the same activities as 6-tuliposide B and the structure of the 3′R-DMBA moiety was the same as that of the proposed active moiety of cnicin. These results suggest that 6-tuliposide B has the same action mechanism as proposed for cnicin and bacterial MurA is one of the major molecular targets of 6-tuliposide B.
Keywords: Tuliposide; Tulipalin; SAR; MurA;

Glycosides from Breynia fruticosa and Breynia rostrata by Dahai Meng; Jian Wu; Weimin Zhao (325-331).
Eight glycosides, along with 30 known glycosides were isolated from Breynia fruticosa and Breynia rostrata (Euphorbiaceae). Their structures were determined on the basis of spectroscopic analysis and chemical methods.Glycosides, 3-acetyl-(−)-epicatechin 7-O-β-glucopyranoside (1), 3-acetyl-(−)-epicatechin 7-O-(6-isobutanoyloxyl)-β-glucopyranoside (2), 3-acetyl-(−)-epicatechin 7-O-[6-(2-methyl-butanoyloxyl)]-β-glucopyranoside (3), (5Z)-6-[5-(2-hydroxypropan-2-yl)-2-methyl-tetrahydrofuran-2-yl]-3-methylhexa-1,5-dien-3-O-β-glucopyranoside (4), hydroquinone O-[6-(3-hydroxyisobutanoyl)]-β-galactopyranoside (5), 4-(4-O-β-glucopyranosyl-phenoxy)-1-O-β-glucopyranosyl-1,3-benzenediol (6), 7,8-erythro-dihydroxy-3,4,5-trimethoxy-phenyl-propane8-O-β-glucopyranoside (7), 6,7-dimethylbenzofuranol 5-O-β-xylopyranosyl-(1 → 6)-β-glucopyranoside (8), along with 30 known glycosides, were isolated from Breynia fruticosa and Breynia rostrata (Euphorbiaceae). Their structures were determined on the basis of spectroscopic analysis and chemical methods.
Keywords: Breynia fruticosa; Breynia rostrata; Euphorbiaceae; Glycosides;