Phytochemistry (v.69, #11)

Fujenal, a diterpenoid saga of neighbouring group participation by James R. Hanson; Braulio M. Fraga (2104-2109).
The chemistry, the biosynthesis and the biotransformations related to fujenal ant its analogues have been reviewed. Despite the opportunity for free rotation about the C-9:C-10 bond, the chemistry of the ring B of this diterpenoid is dominated by neighbouring group participation between C-6, C-7 and C-19.The chemistry, the biosynthesis and the biotransformations related to fujenal and its analogues are reviewed. Despite the opportunity for free rotation about the C-9:C-10 bond, the chemistry of the ring B of this diterpenoid is dominated by neighbouring group participation between C-6, C-7 and C-19.
Keywords: Gibberella fujikuroi; Diterpenoids; Fujenal; Fujenoic acid; Fujenoic triacid; Neighbouring group; Review;

Indicain, a dimeric serine protease from Morus indica cv. K2 by Vijay Kumar Singh; Ashok Kumar Patel; A.J. Moir; Medicherla V. Jagannadham (2110-2119).
▪A high molecular mass serine protease has been purified to homogeneity from the latex of Morus indica cv. K2 by the combination of techniques of ammonium sulfate precipitation, hydrophobic interaction chromatography, and size-exclusion chromatography. The protein is a dimer with a molecular mass of 134.5 kDa and with two monomeric subunits of 67.2 kDa and 67.3 (MALDI-TOF), held by weak bonds susceptible to disruption on exposure to heat and very low pH. Isoelectric point of the enzyme is pH 4.8. The pH and temperature optima for caseinolytic activity were 8.5 and 80 °C, respectively. The extinction coefficient ( ε 280 1 % ) of the enzyme was estimated as 41.24 and the molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residues. The enzyme activity was inhibited by phenylmethylsulfonylflouride, chymostatin and mercuric chloride indicating the enzyme to be a serine protease. The enzyme is fairly stable and similar to subtilases in its stability toward pH, strong denaturants, temperature, and organic solvents. Polyclonal antibodies specific to enzyme and immunodiffusion studies reveal that the enzyme has unique antigenic determinants. The enzyme has activity towards broad range of substrates comparable to those of subtilisin like proteases. The N-terminal residues of indicain (T-T-N-S-W-D-F-I-G-F-P) exhibited considerable similarity to those of other known plant subtilases, especially with cucumisin, a well-characterized plant subtilase. This is the first report of purification and characterization of a subtilisin like dimeric serine protease from the latex of M. indica cv. K2. Owing to these unique properties the reported enzyme would find applications in food and pharma industry.
Keywords: Morus indica cv. K2; Moraceae; Protease purification; Serine protease; Indicain;

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds by Navneet S. Chaudhary; Chandan Shee; Asimul Islam; Faizan Ahmad; Dinesh Yernool; Pravindra Kumar; Ashwani K. Sharma (2120-2126).
Putranjiva roxburghii is an ornamental tree of tropical India known as child life tree that belongs to the Euphorbiaceae family. A highly stable and potent trypsin inhibitor (PRTI) of approximately 34 kDa was purified and characterized from its seeds.A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS–PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4 × 10−11  M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2–12), temperature (20–80 °C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 °C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 °C. The CD spectra of inhibitor at room temperature exhibited an α, β pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.
Keywords: Putranjiva roxburghii; Euphorbiaceae; Seeds; Trypsin inhibitor; Kinetic studies; CD studies; Stability studies;

Fungicidal activities of C6-aldehydes, such as (Z)-3-hexenal, (E)-2-hexenal, and n-hexanal, formed by hydroperoxide lyase are found to be one of most important components for the defense against necrtrophic fungal pathogen, Botrytis cinerea, in Arabidopsis.C6-aldehydes, such as (Z)-3-hexenal, (E)-2-hexenal, and n-hexanal, are volatile compounds formed by hydroperoxide lyase (HPL) and found in most terrestrial plants. They are fungicidal and bactericidal compounds, and are also signaling compounds to induce defense responses in plants. Transgenic plants having overexpressed or suppressed HPL activity (SH or ASH, respectively) showed lower or higher susceptibility against a necrotrophic fungal pathogen, Botrytis cinerea. In this study, we examined whether the modulated susceptibility was accountable to the direct fungicidal activity or to the signaling potency of C6-aldehydes. When wild-type Arabidopsis leaves were inoculated with B. cinerea, HPL expression was upregulated, and concomitantly, the amounts of C6-aldehydes increased. Higher amounts of C6-aldehydes found in inoculated SH plants inhibited growth of B. cinerea in vitro, while lower amounts found in ASH plants caused no inhibitory effect on the fungi. Thus, it was suggested that direct fungicidal activity of C6-aldehydes accounted for the modulated susceptibility. With SH plants higher amounts of camalexin could be found, but with the ASH plants no difference from wild-type plants could be found. Surplus amounts of C6-aldehydes could induce formation of camalexin as signaling compounds; however, this was not the case with wild-type and ASH plants. Accordingly, it could be assumed that direct fungicidal activity of C6-aldehydes were prominently responsible to the defense against B. cinerea but their signaling roles could be little responsible if any.
Keywords: Arabidopsis thaliana; Brassicaceae; Defense response; Botrytis cinerea; C6-aldehydes; Camalexin;

Overexpression of apple spermidine synthase gene (MdSPDS1), one of the polyamine biosynthetic genes, conferred tolerance to NaCl and mannitol stress in European pear (Pyrus communis L.) in vitro shoots through enhancing enzymatic and non-enzymatic antioxidant capacity (photo, 15 days after stress treatment; #32, transgenic pear; WT, wild type).In our previous work, an apple spermidine synthase (SPDS)-overexpressing transgenic European pear (Pyrus communis L. ‘Ballad’), line no. 32 (#32), demonstrated attenuated susceptibility to stress treatment. In the current paper, changes in enzymatic and non-enzymatic antioxidant capacity of the transgenic pear (line #32) were investigated in response to NaCl or mannitol stress. Under non-stressed conditions (before stress treatment), spermidine (Spd) contents and SPDS activity of line #32 were higher than those of the non-transformant (wild type). However, no significant differences were detected between line #32 and the wild type as regards contents of malondialdehyde (MDA) and H2O2, and activities of antioxidant enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). When exposed to NaCl or mannitol stress, both the wild type and line #32 exhibited accumulation of Spd with the latter accumulating more. The transgenic line contained higher antioxidant enzyme activities, less MDA and H2O2 than the wild, implying it suffered from less injury. These results suggested that increase of Spd content in the transgenic line could, at least in part, lead to enhancing enzymatic and non-enzymatic antioxidant capacity.
Keywords: ADC; APX; AsA; DHA; DCIP; DTPA; DW; FW; GR; IBA; MDA; MDHAR; ODC, ornithine decarboxylase; Put; ROS; SAMDC; SH; SOD; Spd; SPDS; Spm; Pyrus communis; Rosaceae; Antioxidant enzymes; Polyamines; Stress tolerance; Transgenic plant;

It has been shown that young leaves of runner bean plants grown under natural conditions showed an unusually high content of γ-tocopherol, accounting for up to 90% of all tocopherol isomers. The level of γ-tocopherol gradually decreased during the first two weeks of leaves development. During simultaneous drought and light stress, γ-tocopherolquinone, an oxidation product of γ-tocopherol, was preferentially accumulated. This indicates specific function of γ-tocopherol in the protection against drought stress in young leaves.It has been shown that young leaves of runner bean (Phaseolus coccineus) plants grown under natural conditions have an unusually high content of γ-tocopherol, accounting for up to 90% of all tocopherols and 50% of the chlorophyll content. The level of γ-tocopherol gradually decreased during the first two weeks of leaf development. The high content of γ-tocopherol in young leaves was not significantly influenced by growth conditions. In contrast to seeds, γ-tocopherol was also the main tocopherol found in light-grown and etiolated primary leaves of runner bean. The obtained results suggest that γ-tocopherol decline during leaf development is not only due to conversion of γ- to α-tocopherol but probably also due to degradation of γ-tocopherol to non-tocochromanol compounds. We have also shown that γ-tocopherol found in young leaves is mainly localized in thylakoid membranes within chloroplast. In the primary leaves subjected to different abiotic stresses, only during simultaneous drought and light stress, γ-tocopherolquinone, an oxidation product of γ-tocopherol, was preferentially accumulated. Since one of the other possible functions of γ-tocopherol could be its action as a nitric oxide scavenger, young leaves were analyzed for the presence of nitro-γ-tocopherol. However, despite the use of a sensitive detection method, it was not found. The possible physiological function of the increased level of γ-tocopherol in the young leaves was discussed.
Keywords: Phaseolus coccineus; γ-Tocopherol; α-Tocopherol; Etiolated leaves; Chloroplast; Nitric oxide; Drought stress;

Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum; consequences for phenolic accumulation and UV-tolerance by Carla Clé; Lionel M. Hill; Ricarda Niggeweg; Cathie R. Martin; Yves Guisez; Els Prinsen; Marcel A.K. Jansen (2149-2156).
Hydroxycinnamoyl CoA quinate transferase (HQT) mediates chlorogenic acid biosynthesis in tomato (Solanum lycopersicum). Our study revealed that increased chlorogenic acid accumulation was associated with UV-protection in transgenics with altered HQT activity. However, manipulation of HQT activity also resulted in more complex alterations in the profiles of phenolics. Our data suggests the existence of regulatory mechanisms that direct the flow of phenolic precursors in response to both metabolic parameters and environmental conditions.Chlorogenic acid (CGA) is one of the most abundant phenolic compounds in tomato (Solanum lycopersicum). Hydroxycinnamoyl CoA quinate transferase (HQT) is the key enzyme catalysing CGA biosynthesis in tomato. We have studied the relationship between phenolic accumulation and UV-susceptibility in transgenic tomato plants with altered HQT expression. Overall, increased CGA accumulation was associated with increased UV-protection. However, the genetic manipulation of HQT expression also resulted in more complex alterations in the profiles of phenolics. Levels of rutin were relatively high in both HQT gene-silenced and HQT-overexpressing plants raised in plant growth tunnels. This suggests plasticity in the flux along different branches of phenylpropanoid metabolism and the existence of regulatory mechanisms that direct the flow of phenolic precursors in response to both metabolic parameters and environmental conditions. These changes in composition of the phenolic pool affected the relative levels of UV-tolerance. We conclude that the capability of the phenolic compounds to protect against potentially harmful UV radiation is determined both by the total levels of phenolics that accumulate in leaves as well as by the specific composition of the phenolic profile.
Keywords: Chlorogenic acid; Rutin; UV-B radiation; Phenylpropanoid pathway; Tomato; Solanum lycopersicum;

Metabolism of (−)-grandisin from Piper solmsianum in Coleoptera and Lepidoptera species by Clécio S. Ramos; Sérgio A. Vanin; Massuo J. Kato (2157-2161).
The major tetrahydrofuran lignan grandisin from leaves of Piper solmsianum was mono- and di-O-demethylated during the digestive process of the beetle Naupactus bipes as well as in the caterpillars Heraclides hectorides and Quadrus u-lucida. Additionally, 3-hydroxy-4,5-dimethoxycinnamyl and 3,4,5-trimethoxycinnamyl alcohols were identified in the fecal extracts of N. bipes.The biotransformation of the major Piper solmsianum leaf phenylpropanoids, such as the tetrahydrofuran lignan grandisin and derivatives was investigated in the beetle Naupactus bipes as well as in the caterpillars Heraclides hectorides and Quadrus u-lucida. Analysis of fecal material indicated that metabolism occurred mainly through mono- and di-O-demethylation at para positions of 3,4,5-trimethoxyphenyl rings of tetrahydrofuran lignans during digestion by these insects. Additionally, 3-hydroxy-4,5-dimethoxycinnamyl and 3,4,5-trimethoxycinnamyl alcohols were identified in fecal extracts of N. bipes.
Keywords: Piperaceae; Piper solmsianum; Lignans; Biotransformation; Naupactus bipes; Heraclides hectorides; Quadrus u-lucida;

Phytochemistry and molecular systematics of Triaenophora rupestris and Oreosolen wattii (Scrophulariaceae) by Søren Rosendal Jensen; Hong-Qing Li; Dirk C. Albach; Charlotte H. Gotfredsen (2162-2166).
The relationships between the genera Triaenophora, Oreosolen and Rehmannia have been investigated. The content of iridoid glucosides in Triaenophora and Rehmannia were almost identical while Oreosolen wattii was different. These results are consistent with a phylogenetic tree based on DNA sequencing.The relationships between the genera Triaenophora, Oreosolen and Rehmannia were investigated. All three genera were previously included in tribe Veroniceae which was part of Scrophulariaceae but which is now included in Plantaginaceae. With regard to the content of iridoid glucosides, Triaenophora rupestris and the much-investigated Rehmannia were almost identical in containing catalpol, ajugol and 6-feruloylajugol. Oreosolen wattii was rather different in having compounds typical for the tribe Scrophularieae (Scrophulariaceae), namely aucubin, harpagide, harpagoside as well as two diesters of rhamnopyranosylcatalpol, one of which, here named oreosolenoside, had not previously been described in the literature. These results are consistent with recent analyses based on DNA sequencing and a phylogenetic tree illustrating the taxonomic relationships is presented.
Keywords: Triaenophora; Oreosolen; Scrophulariaceae; Chemotaxonomy; Iridoid glucosides; Scorodioside; Oreosolenoside; Phylogeny;

Sterols and fatty acids of three harmful algae previously assigned as Chattonella by José-Luis Giner; Hui Zhao; Carmelo Tomas (2167-2171).
The sterol and fatty acids were investigated of three harmful marine algae previously regarded as Chattonella species. The lipids of Chloromorum toxicum are consistent with its inclusion in the Raphidophyceae. Both species of the new genus Verrucophora (Dictyochophyceae) contained the rare 27-norsterol occelasterol.Sterol and fatty acid compositions were determined for three harmful algal species previously classified in the genus Chattonella (Raphidophyceae): the new genus Chloromorum toxicum (ex Chattonella cf. verruculosa), Verrucophora farcimen (Dictyochophyceae), previously Chattonella aff. verruculosa, and Verrucophora verruculosa (=Pseudochattonella verruculosa) previously Chattonella verruculosa. The major fatty acids of C. toxicum were 14:0, 16:0, 18:1n−9, 18:4n−3 and 20:5n−3, and those of the Verrucophora strains were. 14:0, 16:0, 18:0, 18:4n−3, 18:5n−3 and 22:6n−3. C. toxicum contained the 24β-ethyl sterols, poriferasterol and clionasterol, as its major sterols. For comparison, the stereochemistry of the 24-ethyl sterols of two raphidophytes, Chattonella marina and Heterosigma akashiwo, was determined to be 24α and 24β, respectively. Both Verrucophora strains contained the 27-nor sterol occelasterol as the only detected sterol. This was the first time occelasterol has been found in algae.
Keywords: Chloromorum toxicum; Pseudochattonella verruculosa; Verrucophora farcimen; Dictyochophyceae; Raphidophyceae; Harmful algae; Chemotaxonomy; Marine lipids; Fatty acids; Sterols;

The natural compound benzoxazolin-2(3H)-one selectively retards cell cycle in lettuce root meristems by Adela M. Sánchez-Moreiras; Teodoro Coba de la Peña; Manuel J. Reigosa (2172-2179).
Previous experiments showed an effect on germination and radicle growth of 2-benzoxazolinone (BOA), a compound released mainly by grasses to the environment. Our group investigated this mode of action in the cell cycle progression related with seedling growth inhibition in lettuce seedlings.Benzoxazolin-2(3H)-one (BOA) is a natural plant product that is phytotoxic to target plant species, inhibiting germination and growth and causing oxidative damage. We investigated its effects on the root meristems of seedlings of lettuce (Lactuca sativa) by means of light and transmission electron microscopy, flow cytometry, and conventional determination of mitotic index. Flow cytometry analyses and mitotic index showed a retard of cell cycle in BOA-treated meristems with selective activity at G2/M checkpoint.
Keywords: BOA phytotoxicity; Cyclic hydroxamic acid; G2/M checkpoint; Flow cytometry; Mitotic activity; Natural plant product;

Acylated pregnane glycosides from Caralluma tuberculata and their antiparasitic activity by Essam Abdel-Sattar; Fathalla M. Harraz; Soliman Mohammed Abdullah Al-ansari; Sahar El-Mekkawy; Chikara Ichino; Hiroaki Kiyohara; Aki Ishiyama; Kazuhiko Otoguro; Satoshi Omura; Haruki Yamada (2180-2186).
Six pregnane glycosides were isolated from Caralluma tuberculata, including five ones (15). The structures of the isolated compounds were elucidated by the analysis of NMR data and FAB–MS experiments. All the isolated compounds were tested for their antimalarial and antitrypanosomal activities as well as their cytotoxicity against human diploid embryonic cell line (MRC-5).Five pregnane glycosides were isolated from Caralluma tuberculata (15), in addition to a known one (russelioside E, 6). The structures of the isolated compounds were elucidated by the analysis of NMR data and FAB–MS experiments. All the isolated compounds were tested for their antimalarial and antitrypanosomal activities as well as their cytotoxicity against human diploid embryonic cell line (MRC5).
Keywords: Caralluma tuberculata; Asclepiadaceae; Pregnane glycosides; Antimalarial activity; Antitrypanosomal activity; Cytotoxicity;

Xanthones with growth inhibition against HeLa cells from Garcinia xipshuanbannaensis by Quan-Bin Han; Nian-Yun Yang; Hong-Lei Tian; Chun-Feng Qiao; Jing-Zheng Song; Donald C. Chang; Shi-Lin Chen; Kathy Q. Luo; Hong-Xi Xu (2187-2192).
Polyprenylated xanthones, bannaxanthones A–H (18), together with seven known compounds, were isolated from the acetone extract of the twigs of Garcinia xipshuanbannaensis. Their structures were elucidated by spectroscopic data interpretation. Some of these compounds showed growth inhibition effects against HeLa cells.Eight prenylated xanthones, bannaxanthones A–H (18), together with seven known compounds, were isolated from the acetone extract of the twigs of Garcinia xipshuanbannaensis. Their structures were elucidated by spectroscopic data interpretation. The cytotoxic activities of these compounds were evaluated using the MTT method. The results showed that xanthones with an unsaturated prenyl group had stronger cytotoxic activity against cancer cells, whereas those with hydroxylated prenyl groups had none.
Keywords: Garcinia xipshuanbannaensis; Clusiaceae; Xanthone; Growth inhibition; HeLa cells; Bannaxanthones;

Anti-herpetic activity of a sulfated xylomannan from Scinaia hatei by Pinaki Mandal; Carlos A. Pujol; María J. Carlucci; Kausik Chattopadhyay; Elsa B. Damonte; Bimalendu Ray (2193-2199).
The sulfated xylomannan (F1) of Scinaia hatei is selective inhibitors of herpes simplex virus types 1 and 2, whereas its desulfated derivative (F1D) shows little activity. The anti-herpetic activity of this polymer is higher than heparin.Many viruses display affinity for cell surface heparan sulfate proteoglycans with biological relevance in virus entry. This raises the possibility of the application of sulfated polysaccharides in antiviral therapy. In this study we have analyzed polysaccharide fractions isolated from Scinaia hatei. The crude water extract (ShWE) as well as one fraction (F1) obtained by size exclusion chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC50) values ranging from 0.5 to 4.6 μg/ml were much lower than the cytotoxic concentration 50% (CC50) values (⩾1000 μg/ml). These fractions had very low anticoagulant activity. Furthermore, they had a weak inactivating effect on virions in a virucidal assay at concentrations in the range of 60–100 μg/ml. Chemical, chromatographic and spectroscopic methods showed that the major polysaccharide, which had 0.4 sulfate group per monomer unit and an apparent molecular mass of 160 kDa, contained a backbone of α-(1 → 3)-linked d-mannopyranosyl residues substituted at C-6, C-4 and C-2 with single stub of β-d-xylopyranosyl residues. Sulfate groups, when present, are located at C-4 of α-(1 → 3)-linked d-mannopyranosyl units, and appeared to be very important for the anti-herpetic activity of this polymer.
Keywords: Scinaia hatei; Liagoraceae; Herpes simplex virus; Antiviral activity; Xylomannan sulfate;

Chemical constituents of Aeschynanthus bracteatus and their weak anti-inflammatory activities by Su-Mei Li; Xian-Wen Yang; Yun-Heng Shen; Lin Feng; Yue-Hu Wang; Hua-Wu Zeng; Xiao-Hua Liu; Jun-Mian Tian; Ya-Na Shi; Chun-Lin Long; Wei-Dong Zhang (2200-2204).
Phenylethanol glycosides, aeschynanthosides A–D (14), and 55 known constituents were isolated from the aerial parts of Aeschynanthus bracteatus. Aeschynanthoside D (4) and naringenin (41) showed weak inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages within the concentration range tested (50–100 μg/mL).Chemical examination of the EtOAc extract from the aerial parts of Aeschynanthus bracteatus led to isolation of four phenylethanol glycosides, aeschynanthosides A–D (14), and 55 known constituents, including 8 phenylethanoids, 23 phenols, 5 lignans, 7 flavonoids, 9 terpenoids, and 4 others. Their structures were elucidated mainly by detailed spectroscopic studies and comparison with published data. All 59 compounds were isolated for the first time from an Aeschynanthus species. The isolates were also tested for inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages. Aeschynanthoside D (4) and naringenin (41), within the concentration arrange tested (50–100 μg/mL), showed very weak dose-dependent effects with the inhibition rate of 24.2%, 35.4%, 66.0%, and 9.5%, 40.1%, 65.0%, respectively, relative to positive controls.
Keywords: Aeschynanthus bracteatus; Gesneriaceae; Phenylethanoids; Aeschynanthosides A–D; RAW 264.7 macrophages; Anti-inflammatory; NO;

Ascorbic acid potentiates the cytotoxicity of the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin by Rodrigo R. Kitagawa; Luiz M. da Fonseca; Valdecir F. Ximenes; Najeh M. Khalil; Wagner Vilegas; Maria Stella G. Raddi (2205-2208).
The synergic cytotoxic effect of adding ascorbic acid on 5-methoxy-3,4-dehydroxanthomegnin, was investigated. Results indicated that ascorbic acid reduced 5-methoxy-3,4-dehydroxanthomegnin via a semiquinone free radical intermediate, which generate reactive oxygen species. This association resulted in the cytotoxic index being 7 times lower than when 5-methoxy-3,4-dehydroxanthomegnin was added alone to McCoy cell line.The interaction of ascorbic acid with 5-methoxy-3,4-dehydroxanthomegnin, an 1,4-naphthoquinone, was investigated using the cytotoxic index for McCoy cells by neutral red assay. The synergstic effect was observed when such compounds were added simultaneously, most probably due to hydrogen peroxide being generated by ascorbate – driven 5-methoxy-3,4-dehydroxanthomegnin redox cycling. Incubation of cells in the presence of 5-methoxy-3,4-dehydroxanthomegnin/ascorbic acid/catalase, an enzyme that destroys H2O2, resulted in an increase of cell survival, reinforcing the involvement of hydrogen peroxide generated as an important oxidizing agent that kills McCoy cells.
Keywords: Paepalanthus latipes; Eriocaulaceae; Naphthoquinone; Ascorbic acid; Cytotoxic activity; Antitumor drug;

Nitrile glucosides and serotobenine from Campylospermum glaucum and Ouratea turnarea by Auguste Abouem à Zintchem; Dominique Ngono Bikobo; Alex de Théodore Atchadé; Joséphine Ngo Mbing; Joseph Gangoue-Pieboji; Raphael Ghogomu Tih; Alain Blond; Dieudonné Emmanuel Pegnyemb; Bernard Bodo (2209-2213).
Two new nitrile glucosides, campyloside A (1) and campyloside B (2) were isolated from the stem roots of Campylospermum glaucum, whereas serotobenine was isolated from Ouratea turnarea. The structure elucidations were based on spectroscopic evidence. The biological assays of compounds and crude extract of plant species showed good antimicrobial activity of crude extracts against Gram-positive cocci.Two nitrile glucosides (1S,3S,4S,5R)-4-benzoyloxy-2-cyanomethylene-3,5-dihydroxycyclohexyl-1-O-β-glucopyranoside (campyloside A) and (1S,3S,4S,5R)-5-benzoyloxy-2-cyanomethylene-3-hydroxy-4-(2-pyrrolcarboxyloxy)cyclohexyl-1-O-β-glucopyranoside (campyloside B) were isolated from the stem roots of Campylospermum glaucum, whereas serotobenine was isolated from Ouratea turnarea. The structure elucidations were based on spectroscopic evidence.The biological assays of compounds and crude extract of plant species showed good antimicrobial activity of crude extracts against Gram-positive cocci.
Keywords: Campylospermum glaucum; Ouratea turnarea; Ouratea; Ochnaceae; Nitrile glucosides; Lanceolin C; Campyloside A; Campyloside B; Serotobenine; Flavonoids; Antimicrobial activity;

Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry by Ondřej Novák; Eva Hauserová; Petra Amakorová; Karel Doležal; Miroslav Strnad (2214-2224).
A simple, high-throughput batch immunoextraction (IAE) micropurification procedure for extracting a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) from plant tissues in solutions that are compatible with ultra-performance liquid chromatography (UPLC), thereby facilitating sensitive subsequent analysis was developed.We have developed a simple, high-throughput batch immunoextraction (IAE) micropurification procedure for extracting a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) from plant tissues in solutions that are compatible with ultra-performance liquid chromatography (UPLC), thereby facilitating sensitive subsequent analysis. The UPLC system was coupled to a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). Small (mg) amounts of tissues were purified by solid-phase extraction (SPE) followed by an immunoaffinity clean-up step and two fast chromatographic separations of most cytokinin metabolites (bases, ribosides, and 9-glucosides in the first, O-glucosides and nucleotides in the second). Using UPLC, the runs were up to 4-fold faster than in standard cytokinin analyses, and both retention times and injection volumes were less variable (RSDs, 0.15–0.3% and 1.0–5.5%, respectively). In multiple reaction monitoring (MRM) mode, the detection limit for most of the cytokinins analyzed was close to 1 fmol (5–25 fmol for O-glucosides and nucleotides) and the linear range spanned at least five orders of magnitude. The extraction and purification method was optimized using poplar (Populus  ×  canadensis Moench, cv Robusta) leaf samples, and the analytical accuracy was further validated using IAE-purified 10-day-old Arabidopsis thaliana plants spiked with 1 and 10 pmol of cytokinin derivatives. This approach can be used for rapid, sensitive qualitative and/or quantitative analysis of more than 50 natural cytokinins in minute amounts of plant tissues with high performance, robustness, and accuracy.
Keywords: Ultra-performance liquid chromatography (UPLC); Tandem mass spectrometry (MS/MS); Microextraction; Immunoaffinity purification; Cytokinins; A. thaliana;

Tomoeones A–H, cytotoxic phloroglucinol derivatives from Hypericum ascyron by Waka Hashida; Naonobu Tanaka; Yoshiki Kashiwada; Michiko Sekiya; Yasumasa Ikeshiro; Yoshihisa Takaishi (2225-2230).
Eight phloroglucinols, tomoeones A–H were isolated from the leaves of Hypericum ascyron. Their structures were established based on spectroscopic analyses. Cytotoxicities of tomoeones (A–H) against human tumor cell lines were evaluated. Tomoeone F (6) demonstrated significant cytotoxicity against KB cells with an IC50 value of 6.2 μg/mL.Phloroglucinol derivatives tomoeones A–H (18) and three known compounds were isolated from leaves of Hypericum ascyron. Their structures were established based on spectroscopic analyses. They are all acylphloroglucinol derivatives possessing a spiro skeleton with geminal isoprenyl groups and a monoterpene moiety, and they are stereoisomers to each other at C-4 and C-13. They appear to be a class of phloroglucinol derivatives. Cytotoxicities of the isolated phloroglucinol derivatives against human tumor cell lines, including multidrug-resistant (MDR) cancer cell lines, were evaluated. Tomoeone F (6) demonstrated significant cytotoxicity against KB cells with an IC50 value of 6.2 μM. Compound 6 was also cytotoxic against MDR cancer cell lines (KB-C2 and K562/Adr), which was more potent than doxorubicin.
Keywords: Hypericum ascyron; Clusiaceae; Phloroglucinol; Cytotoxicity;

Cytotoxic sesquiterpenes from Ligularia platyglossa by Jian-Qun Liu; Mian Zhang; Chao-Feng Zhang; Huan-Yang Qi; Alan Bashall; S.W. Annie Bligh; Zheng-Tao Wang (2231-2236).
Four sesquiterpenes (14) along with two known ones were isolated from Ligularia platyglossa. A dimer of eremophilenolides (1) linked at C-2 β/C-8′α was obtained for the first time. Compound 3 showed cytotoxic activities against HL-60, B16, BEL7402 and Hela cancer cells.Four sesquiterpene lactones including an eremophilenolide dimer, named as biligulaplenolide, 1, 8β-hydroxy-1-oxo-(14α,15α eremophil-7(11),9(10)-dien-12,8α-olide, 2, 1-hydroxy-2-oxo-(14α,15α eremophil-1(10),7(11),8(9)-trien-12,8-olide, 3, 4α,8β,9α-trihydroxy- 5αΗ-7(11)-eudesmen-12,8α-olide, 4, along with two known ones, 10α-hydroxy-1-oxo-eremophil-7(11),8(9)-dien-12,8-olide, 5, and furanoeremophil-1(10)-ene-2,9-dione, 6, were isolated from the underground organs of Ligularia platyglossa (Franch.) Hand.-Mazz. Their structures were elucidated by spectroscopic methods including single-crystal X-ray diffraction analysis (2 and 3). Their in vitro cytotoxicities against seven cancer cell lines (BGC-823, A549, HL-60, B16, SMMC-7721, BEL7402, Hela) were evaluated. Compounds 2, 3, 5 showed cytotoxic activities on HL-60 cancer cells with IC50 in the range of 24.0 to 51.1 μM, whereas compound 3 exhibited only weak cytotoxic activity against the B16, BEL7402 and Hela cancer cells. Flow cytometric analysis indicated that compound 3 induces Hela cells to apoptotic death after 48 h treatment with 0.38 mM of this compound.
Keywords: Ligularia platyglossa; Asteraceae; Bioactive sesquiterpene lactones; Eremophilenolide dimer; Biligulaplenolide; Cytotoxicity;

Metabolite profiling of Panax notoginseng using UPLC–ESI-MS by Mo Dan; Mingming Su; Xianfu Gao; Tie Zhao; Aihua Zhao; Guoxiang Xie; Yunping Qiu; Mingmei Zhou; Zhong Liu; Wei Jia (2237-2244).
UPLC–ESI-MS/MS coupled with multivariate statistical analysis was used for metabolic profiling and identification of multiple saponins in Panax notoginseng revealing saponin differences and critical markers among different parts of P. notoginseng.The metabolite profiling of different parts of Panax notoginseng was carried out using rapid ultra-performance liquid chromatography–electrospray ionization mass spectrometry (UPLC–ESI-MS) and multivariate statistical analysis. Principal component analysis (PCA) of the UPLC–ESI-MS data showed a clear separation of compositions among the flower buds, roots and rhizomes of P. notoginseng. The saponins accounting for such variations were identified through the corresponding loadings weights and were further verified by accurate mass, tandem mass and retention times of available standard saponins using UPLC quadrupole time-of-flight mass spectrometer (UPLC–QtofMS). Finally, the influential factors of different metabolic phenotypes of P. notoginseng was elucidated. The currently proposed UPLC–ESI-MS/MS analytical method coupled with multivariate statistical analysis can be further utilized to evaluate chemical components obtained from different parts of the plant and/or the plant of different geographical locations, thereby classifying the medicinal plant resources and potentially elucidating the mechanism of inherent phytochemical diversity.
Keywords: Panax notoginseng; Metabolite profiling; Saponin; Ultra-performance liquid chromatography (UPLC); Electrospray ionization (ESI); Quadrupole time-of-flight mass spectrometer (QtofMS); Multivariate statistical analysis;

Flavonols and an oxychromonol from Piliostigma reticulatum by Olalekan J. Babajide; Omotola O. Babajide; Abimbola O. Daramola; Wilfred T. Mabusela (2245-2250).
Chemical investigation of Piliostigma reticulatum leaves lead to the isolation of a chromone, three new and three known flavonoids, namely, 1, C-methyl-p-phenoxychromonol (Piliostigmol), 2, 6,8-di-C-methylquercetin-3,3′,7-trimethyl ether, 3, 6,8-di-C-methylquercetin-3,3′-dimethyl ether, 4, 3′,6,8-tri-C-methylquercetin-3,7-dimethyl ether, 5, 6-C-methylquercetin-3-methyl ether, 6, 6,8-di-C-methylkaempferol-3-methyl ether and 7, 6-C-methylquercetin-3,3′,7-trimethyl ether, respectively. The compounds showed variable cytotoxic and antimicrobial activities.The leaf extract from the plant Piliostigma reticulatum was found to exhibit antimicrobial activity against some bacteria and fungi such as Staphylococcus aureus (NCTC 6571), Escherichia coli (NCTC 10418), Bacillus subtilis (NCTC 8236), Proteus vulgaris (NCTC 4175), Aspergillus niger (ATCC 10578) and Candida albicans (ATCC 10231). Upon investigation of the chemical constituents present in the leaf extract, a total of seven compounds were isolated and their structures were unambiguously established by spectroscopic methods including HR-MS and NMR spectrometry. Four of the isolated compounds were novel, namely 6- C-methyl-2-p-hydroxyphenyloxychromonol (piliostigmol), 1, 6,8-di-C-methylquercetin-3,3′,7-trimethyl ether, 2, 6,8-di-C-methylquercetin-3,3′-dimethyl ether, 3 and 3′,6,8,-tri-C-methylquercetin-3,7-dimethyl ether, 4. The other three were known C-methylated flavonols and they were isolated from P. reticulatum for the first time. These were 6-C-methylquercetin-3-methyl ether, 5, 6,8-di-C-methylkaempferol-3-methyl ether, 6 and 6-C-methylquercetin-3,3′,7-trimethyl ether 7. All the isolated compounds were tested for cytotoxicity using the brine shrimp toxicity assay and all of them were active albeit at different levels. With respect to antibacterial activity piliostigmol, 1 showed the highest activity against E. coli (MIC = 2.57 μg/ml, 0.006 μmol), which is three times more that of Amoxicillin, where as 4 and 7 showed the least activity.
Keywords: Piliostigma reticulatum; Caesalpiniaceae; Flavonols; Piliostigmol; Phenoxychromonol; C-methylated quercetins; Cytotoxicity;

Absolute configuration of eremophilanoids by vibrational circular dichroism by Eleuterio Burgueño-Tapia; Pedro Joseph-Nathan (2251-2256).
The absolute configurations of natural occurring 6-hydroxyeuryopsin (1), of its acetyl derivative 2, and of eremophilanolide 8 were confirmed by comparison of the experimental vibrational circular dichroism spectra with their respective theoretical curves generated from density functional theory calculations.The absolute configurations (AC) of natural occurring 6-hydroxyeuryopsin (1), of its acetyl derivative 2, and of eremophilanolide 8 were confirmed by comparison of the experimental vibrational circular dichroism (VCD) spectra with theoretical curves generated from density functional theory (DFT) calculations. Initial analyses were carried out using a Monte Carlo searching with the MMFF94 molecular mechanics force field. All MMFF94 conformers were further optimized using DFT at the B3LYP/6-31G(d) level of theory, followed by calculations of their vibrational frequencies at the B3LYP/6-31G(d,p); the VCD spectra of 2 and 8 were also calculated at the B3PW91/DGDZVP level of theory. Good agreement between theoretical and experimental VCD curves unambiguously verified the 4S,5R,6S absolute configuration for 1 and 2, and the 1S,4S,5R,6S,8S,10S configuration for 8.
Keywords: 6-Hydroxyeuryopsin; 6-Acetyloxyeuryopsin; 1α-Angeloyloxy-8β,10β-dihydroxyeremophil-7(11)-en-8α,12-olide; Vibrational circular dichroism; Absolute configuration;