Phytochemistry (v.69, #2)

Plant bioactives for ruminant health and productivity by Simone Rochfort; Anthony J Parker; Frank R. Dunshea (299-322).
Plants can be used to enhance animal health. Research on ruminant specific treatments reinforces the importance of understanding phytochemistry. For example, researchers have recently demonstrated that saponins with different core structures (1 and 2) have disparate effects on methane production. Plant use for animal health is analysed in this review.Plants have been used throughout history for their medicinal properties. This use has often focused on human health but plants have also been, and still are, applied in ethnoveterinary practice and animal health management.In recent times, the use of synthetic chemicals has become prevalent. Public awareness of the potential environmental and health risks associated with heavy chemical use has also increased. This has put pressure on regulatory bodies to reduce the use of chemicals in agriculture. The most striking example is the 2006 banning of antibiotics in animal feed by the European Union. Moves such as this have increased the drive to find alternatives to synthetic chemicals and research has again turned to the use of plant bioactives as a means of improving animal health.Current scientific evidence suggests there is significant potential to use plants to enhance animal health in general and that of ruminants (cattle, deer, sheep, etc.) in particular. Active areas of research for plant bioactives (particularly saponin and tannin containing plants) include reproductive efficiency, milk and meat quality improvement, foam production/bloat control and methane production. Nematode control is also a significant area of research and the evidence suggests a much broader range of phytochemicals may be effective. This review presents a summary of the literature and examines international research efforts towards the development of plant bioactives for animal health.
Keywords: Ruminant; Anthelmintic; Methanogensis; Bloat control; Saponin; Tannin; Animal health;

Critical view on the monochlorodimedone assay utilized to detect haloperoxidase activity by Claudia Wagner; Ilka M. Molitor; Gabriele M. König (323-332).
Haloperoxidase activity guided protein purification from the cyanobacterium Fischerella ambigua revealed the isolation of a protein positive in the monochlorodimedone-assay, but an involvement in halogenating processes could not be verified. Our data indicate that the reaction of MCD with proteins of the cytochrome c – family leads to unspecific products giving false positive results.The current study aimed to identify the halogenating enzymes involved in the biosynthesis of the ambigols A, B, C and tjipanazole D, isolated from the cyanobacterium Fischerella ambigua. Haloperoxidase (HPO) activity within F. ambigua was therefore assayed spectrophotometrically by using monochlorodimedone (MCD) during protein purification. This strategy revealed the isolation of a protein positive in the MCD-assay, but an involvement in halogenating processes could not be verified. N-terminal sequencing rather demonstrated homology to cytochrome c 6 from other cyanobacteria and green algae. From our findings it thus has to be concluded that the spectrophotometrical MCD-assay routinely used to detect HPO activity may yield false positive results, mainly since the assay focuses on the decline of the educt and not on the formation of the product. Our data indicate that the reaction of MCD with proteins of the cytochrome c– family leads to unspecific products.
Keywords: Biohalogenation; Biosynthesis; Cyanobacteria; Cytochrome c 6; Haloperoxidase; Monochlorodimedone; Organohalogens; Protein purification;

Suppression of reactive oxygen species by glyceraldehyde-3-phosphate dehydrogenase by Dongwon Baek; Yinhua Jin; Jae Cheol Jeong; Hyo-Jung Lee; Haejeong Moon; Jiyoung Lee; Dongjin Shin; Chang Ho Kang; Doh Hoon Kim; Jaesung Nam; Sang Yeol Lee; Dae-Jin Yun (333-338).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a classical glycolytic enzyme, is involved in cellular energy production and has important housekeeping functions. In this report, we show that GAPDH controls generation of H2O2 by Bax and heat shock, which in turn suppresses cell death in yeast and plants.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a classical glycolytic enzyme, is involved in cellular energy production and has important housekeeping functions. In this report, we show that a GAPDH from Arabidopsis, GAPDHa, has a novel function involved in H2O2-mediated cell death in yeast and Arabidopsis protoplasts. GAPDHa was cloned along with other plant genes that suppress Bax-induced cell death in yeast. Flow cytometry analyses with dihydrorhodamine 123 indicated that H2O2 production mediated by Bax expression in yeast cells was greatly reduced when Bax was coexpressed with GAPDHa. In plants, GAPDHa transcript levels were greatly increased by H2O2 treatment. Furthermore, transformation of GAPDHa into Arabidopsis protoplasts strongly suppressed heat shock-induced H2O2 production and cell death. Together, our results indicate that GAPDH controls generation of H2O2 by Bax and heat shock, which in turn suppresses cell death in yeast and plant cells.
Keywords: Arabidopsis thaliana; Cruciferae; Saccharomyces cerevisiae; Glyceraldehyde-3-phosphate dehydrogenase; Bax-induced cell death; Reactive oxygen species; Multifunctional protein;

A role for a Hevea latex lectin-like protein in mediating rubber particle aggregation and latex coagulation by Rapepun Wititsuwannakul; Piyaporn Pasitkul; Kamonwan Kanokwiroon; Dhirayos Wititsuwannakul (339-347).
A lectin-like protein was isolated and purified from the bottom membrane fraction of centrifuged fresh Hevea brasiliensis latex. Its general biochemical properties and physiological involvement in rubber latex coagulation is reported.An in vitro aggregation of washed lutoid membrane and rubber particles, respectively, prepared from the bottom (lutoid) fraction and rubber layer of centrifuged fresh latex, leading to the formation of rubber coagulum necessary for a latex coagulation was demonstrated. A Triton X-100 extract of washed lutoid membrane proteins, isolated and prepared from the bottom fraction of centrifuged fresh latex was examined for its role in the latex coagulation process. It induced agglutination of rabbit erythrocytes, indicating the presence of a lectin-like protein. Hevea latex lectin-like protein (HLL) was purified to homogeneity by active chitin binding separation, followed by DEAE-Sepharose chromatography. Its M r analyzed by SDS–PAGE was 17 kDa, whereas that determined by gel filtration was 267 kDa. The HLL had a pI value of 7.2. Several glycoproteins were shown to inhibit the HLL-induced hemagglutination. The hemagglutinin activity of HLL was enhanced by Ca2+. Of most interest was the finding that HLL strongly induced aggregation of the Hevea latex rubber particles (RP). This strong RP aggregation leads to latex coagulation, indicating the possibility that it is involved in the formation of the coagulum that plugs the latex vessel ends and stops the flow of latex upon tapping. In addition, the purified HLL also induced aggregation of RP taken from several other non-Hevea latex producing plants. This might indicate either a common or universal role of this lectin-like protein in RP aggregation and hence latex coagulation. This paper, for the first time, provides clear and unequivocal evidence for either a key biological role or physiological function of an endogeneous latex lectin-like protein in the sequential process of latex coagulation.
Keywords: Hevea brasiliensis; Euphorbiaceae; Rubber latex; Lectin; Agglutinin; Rubber particle; Latex coagulation;

Tyr residues located on the protein surface of CWPO-C were considered to be important for oxidation of a wide range of substrates including sinapyl alcohol and polymeric lignin. The Tyr is considered to be an unique oxidation site found in the plant peroxidase family.It was previously reported that an unique peroxidase isoenzyme, cationic cell-wall-bound peroxidase (CWPO-C), from poplar callus oxidizes sinapyl alcohol, ferrocytochrome c and synthetic lignin polymers, unlike other plant peroxidases. Here, the catalytic mechanism of CWPO-C was investigated using chemical modification and homology modeling. The simulated CWPO-C structure predicts that the entrance to the heme pocket of CWPO-C is the same size as those of other plant peroxidases, suggesting that ferrocytochrome c and synthetic lignin polymers cannot interact with the heme of CWPO-C. Since Trp and Tyr residues are redox-active, such residues located on the protein surface were predicted to be active sites for CWPO-C. Modification of CWPO-C Trp residues did not suppress its oxidation activities toward guaiacol and syringaldazine. On the other hand, modification of CWPO-C Tyr residues using tetranitromethane strongly suppressed its oxidation activities toward syringaldazine and 2,6-dimethoxyphenol by 90%, respectively, and also suppressed its guaiacol oxidation activity to a lesser extent. Ferrocytochrome c was not oxidized by Tyr-modified CWPO-C. These results indicate that the Tyr residues in CWPO-C mediate its oxidation of syringyl compounds and high-molecular-weight substrates. Homology modeling indicates that Tyr-177 and Tyr-74 are located near the heme and exposed on the protein surface of CWPO-C. These results suggest that Tyr residues on the protein surface are considered to be important for the oxidation activities of CWPO-C with a wide range of substrates, and potentially unique oxidation sites for the plant peroxidase family.
Keywords: Poplar; Poplus alba; Salicaceae; Homology modeling; Lignin biosynthesis; Long-range electron transfer; Plant peroxidase; Tetranitromethane;

5’-Adenylylsulfate reductase from soybean (Glycine max) was cloned and the recombinant protein expressed, purified, and biochemically characterized. The tissue distribution and changes in expression as a response to environmental stresses indicate that the sulfur assimilation pathway in soybean plays a key role in early seed development and in responding to cold stress.Soybean seeds are a major source of protein, but contain low levels of sulfur-containing amino acids. With the objective of studying the sulfur assimilation pathway of soybean, a full-length cDNA clone for 5′-adenylylsulfate reductase (APS reductase) was isolated and characterized. The cDNA clone contained an open reading frame of 1414 bp encoding a 52 kDa protein with a N-terminal chloroplast/plastid transit peptide. Southern blot analysis of genomic DNA indicated that the APS reductase in soybean is encoded by a small multigene family. Biochemical characterization of the heterologously expressed and purified protein shows that the clone encoded a functional APS reductase. Although expressed in tissues throughout the plant, these analyses established an abundant expression of the gene and activity of the encoded protein in the early developmental stages of soybean seed, which declined with seed maturity. Sulfur and phosphorus deprivation increased this expression level, while nitrogen starvation repressed APS reductase mRNA transcript and protein levels. Cold-treatment increased expression and the total activity of APS reductase in root tissues. This study provides insight into the sulfur assimilation pathway of this nutritionally important legume.
Keywords: Glycine max; Leguminosae; Legume; Soybean; Cloning; Kinetic analysis; Sulfur; 5′-Adenylyl reductase; APS reductase; Seed development; Nutrient stress; Cold stress;

Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase by Shiu-Cheung Lung; Andy Leung; Rainbow Kuang; Yu Wang; Priscilla Leung; Boon-Leong Lim (365-373).
Elevated phytase activity was detected in tobacco root exudates in response to phosphorus starvation. The enzyme was purified to homogeneity and its identity was confirmed to be a purple acid phosphatase by molybdate-inhibition assay and cDNA cloning. Its catalytic properties implicate a specific role in mobilizing organic phosphorus in soil.Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco (Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg−1 and an overall yield of 3%. SDS–PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0–5.5 and 45 °C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate (pNPP; K m  = 13.9 μM) and phytate (K m  = 14.7 μM), but a higher kcat for pNPP (2,056 s−1) than phytate (908 s−1). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.
Keywords: Nicotiana tabacum; Solanaceae; Phytase; Purple acid phosphatase; Phytate; Phosphorus; Root exudates; Tobacco;

UDP-glucose:(6-methoxy)podophyllotoxin 7-O-glucosyltransferase from suspension cultures of Linum nodiflorum by Anna Berim; Rainer Ebel; Bernd Schneider; Maike Petersen (374-381).
Cell-free protein extracts from suspension cultures of Linum nodiflorum L. (Linaceae) catalyse the 7-O-β-glucosidation of podophyllotoxin and its 6-methoxy derivative with high specificity. The enzyme activity correlates with lignan accumulation by the plant cells and is competitively inhibited by β-peltatin.Cell cultures of Linum species store 6-methoxypodophyllotoxin (MPTOX), podophyllotoxin (PTOX) and related lignans as O-glucosides. UDP-glucose:(M)PTOX 7-O-glucosyltransferase has been detected and characterised in protein preparations of suspension-cultured cells of Linum nodiflorum L. (Linaceae). The maximal lignan glucoside contents in the cells are preceded by a rapid increase of the specific glucosyltransferase activity on day six of the culture period. MPTOX glucoside is the major lignan with up to 1.18 mg g−1 of the cell dry wt which is more than 30-fold of the PTOX glucoside content. Of the three aryltetralin lignans tested as substrates, PTOX and MPTOX display comparable apparent K m values of 4.7 and 5.4 μM, respectively. 5′-Demethoxy-6-methoxypodophyllotoxin is converted with the highest velocity of 25.2 pkat mg−1 while also possessing a higher K m of 14.7 μM. Two-substrate test series indicate that all three compounds compete for the active site of a single protein. The structurally similar lignan β-peltatin acts as competitive inhibitor as well. However, the 6-O-glucosidation is most likely catalysed by a separate enzyme. The (M)PTOX 7-O-glucosyltransferase works best at a pH around 9 and a temperature around 35 °C. A 15–30% increase of the reaction rate is effected by the addition of 0.9 mM Mn2+.
Keywords: Linum nodiflorum L.; Linaceae; Lignans; Glucosyltransferase; 6-Methoxypodophyllotoxin; Podophyllotoxin; β-Peltatin;

Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin by María Belén Pascual; Zhong Ping Jing; Edward G. Kirby; Francisco M. Cánovas; Fernando Gallardo (382-389).
Glutamine synthetase (GS) is a key enzyme in nitrogen metabolism, and the target of the herbicide phosphinothricin (PPT). Overexpression of cytosolic GS enhances the tolerance of poplar to PPT. Increases in GS, large subunit of Rubisco (LSU), and NADP+-isocitrate dehydrogenase (IDH) levels could be considered as mechanisms operating in young poplar leaves to overcome the toxic effect of PPT.Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula ×  P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19–26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411–418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137–145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine syntetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729–736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31–39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha−1, growth of GS transgenic poplar plantlets was 5-fold greater than controls. The response of young leaves to PPT treatment depends on physiological state as indicated by GS and Rubisco (LSU) levels. Young leaves from control plants, typically in a low differentiation state, respond to the herbicide showing up-regulation of GS and LSU. In contrast, young leaves from transgenic lines, with higher initial GS and LSU levels compared to control, display up-regulation of NADP+-isocitrate dehydrogenase. Differences between control and GS transgenics in their response to PPT are discussed in relation to their differences in photosynthetic and photorespiratory capacities ().
Keywords: Populus tremula ×  P. alba; Salicaceae; Poplar; Phosphinothricin; Herbicide; Glutamine synthetase; Isocitrate dehydrogenase;

Biosynthesis of trigonelline from nicotinate mononucleotide in mungbean seedlings by Xin-Qiang Zheng; Ayu Matsui; Hiroshi Ashihara (390-395).
Enzyme activities and in situ tracer experiments suggest that part of nicotinate mononucleotide synthesised by de novo pyridine nucleotide synthesis is converted to nicotinate via nicotinate riboside, and is used for trigonelline synthesis in mungbean seedlings.To determine the biosynthetic pathway to trigonelline, the metabolism of [carboxyl-14C]nicotinate mononucleotide (NaMN) and [carboxyl-14C]nicotinate riboside (NaR) in protein extracts and tissues of embryonic axes from germinating mungbeans (Phaseolus aureus) was investigated. In crude cell-free protein extracts, in the presence of S-adenosyl-l-methionine, radioactivity from [14C]NaMN was incorporated into NaR, nicotinate and trigonelline. Activities of NaMN nucleotidase, NaR nucleosidase and trigonelline synthase were also observed in the extracts. Exogenously supplied [14C]NaR, taken up by embryonic axes segments, was readily converted to nicotinate and trigonelline. It is concluded that the NaMN → NaR → nicotinate → trigonelline pathway is operative in the embryonic axes of mungbean seedlings. This result suggests that trigonelline is synthesised not only from NAD but also via the de novo biosynthetic pathway of pyridine nucleotides.
Keywords: Mungbean; Phaseolus aureus; Leguminosae; Biosynthesis; Pyridine alkaloid; Trigonelline;

Cuticular wax composition of Salix varieties in relation to biomass productivity by Mark A. Teece; Thomas Zengeya; Timothy A. Volk; Lawrence B. Smart (396-402).
The leaf cuticular wax composition (fatty acids, n-alcohols, n-aldehydes, and n-alkanes) and biomass productivities of six Salix clones grown under similar environmental conditions were determined. Contrary to previous studies, no relationship was found between wax composition and biomass productivity.The leaf cuticular waxes of six Salix clones (one Salix miyabeana, one Salix dasyclados, one Salix eriocephala, two Salix purpurea, and one interspecific hybrid of Salix eriocephala × interior) with different biomass productivities were characterized by gas chromatography–mass spectrometry. Total wax content ranged from 6.3 to 16.8 μg cm−2, and two distinct patterns of wax were measured. The wax from leaves of S. dasyclados ‘SV1’ differed from all other clones and was dominated by fatty acids (42%), high concentrations of n-alkanes (25%) and n-alcohols (28%), with low n-aldehyde content (4%). All other clones produced cuticular wax dominated by n-alcohols (32–51%), particularly 1-hexacosanol, with fatty acids (14–37%) and n-aldehydes (19–26%) present in lower abundances. Clones of Salix grown under identical environmental conditions produce noticeably different amounts of cuticular wax. In contrast to previous studies of Salix, total wax content was independent of biomass productivity, measured as basal area, suggesting that wax production is not directly linked with woody biomass production by shrub willows under these site conditions.
Keywords: Salix eriocephala; Salix dasyclados; Salix miyabeana; Salix purpurea; Salicaceae; Willow; Biomass; Cuticular waxes; Ecological biochemistry section;

Seasonal variation in glucosinolate content in Brassica oleracea crops grown in northwestern Spain by María Elena Cartea; Pablo Velasco; Sara Obregón; Guillermo Padilla; Antonio de Haro (403-410).
Aliphatic, indolyl and aromatic glucosinolates were identified from leaves of kale, cabbage, and Tronchuda cabbage varieties (Brassica oleracea sp.) grown in northwestern Spain. Sinigrin makes the major contribution of glucosinolates to kales, while glucobrassicin or glucoiberin do so to cabbage leaves. The influence of sowing date on glucosinolate content and the nutritional effects of these compounds glucosinolates are discussed. Brassica oleracea L. crops including kales, cabbages, and Tronchuda cabbages are widely grown in northwestern Spain and Portugal but little information is available on leaf glucosinolate content of these crops. The objectives were to determine the diversity for the total glucosinolate content and profile on leaves in a collection of 153 kales, 26 cabbages, and three Tronchuda cabbages varieties grown at two growing seasons and to determine the seasonal variation of glucosinolates in cabbages and Tronchuda cabbage varieties. Sinigrin, glucoiberin, and glucobrassicin were the major glucosinolates found in kales. Glucoiberin was the most common glucosinolate in Tronchuda cabbages in both planting seasons and in cabbages sown in fall season whereas glucobrassicin and glucoiberin were the most common glucosinolates in cabbages in spring season. In kales the total glucosinolate content ranged from 11.0 to 53 μmol g−1  dw, with a mean value of 26.3 μmol g−1  dw. Four kale varieties (MBG-BRS0468, MBG-BRS0476, MBG-BRS0060 and MBG-BRS0223) showed the highest total sinigrin or glucobrassicin contents. So, they could be good candidates for future breeding programs. In cabbages, the total glucosinolate content ranged from 10.9 to 27 g−1  dw. Total glucosinolate concentration during spring sowing (22 μm g−1  dw) was higher than those in fall sowing (13 μm g−1  dw). Regarding both high glucosinolate content and the agronomic value, MBG-BRS0057 and MBG-BRS0074 could be good sources of beneficial glucosinolates. The presence of high concentrations of sinigrin, glucoiberin, and glucobrassicin warrant further search into their potential use to enhance the level of these important phytochemicals in these edible crops.
Keywords: Brassica oleracea; Cabbage; Glucosinolates; Kale; Sinigrin; Tronchuda cabbage;

cis-Vaccenic acid (C18:1 (n-7)), an isomer of oleic acid (C18:1 (n-9)) is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Δ9 desaturase and elongation by an elongase giving C18:1 (n-7). The fatty acid composition of 48 different varieties from 12 Brassica species was analyzed. Various amount of the C18:1(n-7), C20:1 (n-7) and C22:1(n-7) were found in all the tested species suggesting the elongation of the C18:1 (n-7) to longer chain fatty acids in the twelve Brassica species. These Brassica species could be grouped into three broad categories based on similarities in C18:1 (n-7)/(n-9) ratios. C20:1 (n-7) had a statistically significant positive effect on the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. cis-Vaccenic acid or cis-11-octadecenoic acid, a C18:1 (n-7) isomer of oleic acid (C18:1 (n-9)) has been found in several oilseeds. It is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Δ9 desaturase and elongation by an elongase giving C18:1 (n-7). In this study, the fatty acid composition of 12 Brassica species was analyzed by GC-FID and confirmed by GC–MS. All species contained C18:1 (n-7), C20:1 (n-7) and C22:1 (n-7) fatty acid isomers, suggesting that C18:1 (n-7) was elongated. The levels of these fatty acids varied according to the species. C18:1(n-7)) represented from 0.4% to 3.3% of the total relative fatty acid contents of the seeds. The contents of C20:1(n-7) and C22:1(n-7) levels were lower than C18:1(n-7) contents; the relative fatty acid composition varied from 0.02% to 1.3% and from below the limit of detection to 1.3% for C20:1 (n-7) and C22:1 (n-7), respectively. The ratios of (n-7)/(n-9) ranged from 2.8% to 16.7%, 0.6% to 29.5% and 0% to 2.6% for C18:1, C20:1 and C22:2, respectively.Using statistical similarities or differences of the C18:1 (n-7)/(n-9) ratios for chemotaxonomy, the surveyed species could be arranged into three groups. The first group would include Brassica napus, B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The second group would include B. tournefortii, Raphanus sativus and Sinapis alba. The last group would include B. juncea, B. carinata and B. nigra with no similarity/relationship between them and between the other species.Results suggested that the level of C20:1 (n-7) influenced the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. On the other hand, palmitoleic acid (C16:1) levels, C16:1 being the parent of all (n-7) fatty acids, had no statistically significant correlation with the content of any of the fatty acids of the (n-7) or (n-9) family.
Keywords: Brassicaceae; Brassica carinata; B. juncea; B. napus; B. nigris; B. rapa; B. tournefortii; Camelina sativa; Crambe abyssinica; Eruca sativa; Raphinus sativa; Sinapis alba; (n-7) Fatty acids; cis-Vaccenic acid; Chemotaxonomy;

Anticancer and antiproliferative activity of natural brassinosteroids by Jana Malíková; Jana Swaczynová; Zdeněk Kolář; Miroslav Strnad (418-426).
The influence of natural types of brassinosteroids on viability and proliferation on different cancer cell lines is reported. 28-Homocastasterone (1) and 24-epibrassinolide (2) were shown to be effective in a micromolar range, despite having minimal effects on normal cells.Brassinosteroids (BRs) are steroid plant hormones that are essential for many plant growth and developmental processes, including cell expansion, vascular differentiation and stress responses. Up to now the inhibitory effects of BRs on cell division of mammalian cells are unknown. To determine basic anticancer structure–activity relationships of natural BRs on human cells, several normal and cancer cell lines have been used. Several of the tested BRs were found to have high cytotoxic activity. Therefore, in our next series of experiments, we tested the effects of the most promising and readily available BR analogues with interesting anticancer properties, 28-homocastasterone (1) and 24-epibrassinolide (2), on the viability, proliferation, and cycling of hormone-sensitive/insensitive (MCF-7/MDA-MB-468) breast and (LNCaP/DU-145) prostate cancer cell lines to determine whether the discovered cytotoxic activity of BRs could be, at least partially, related to brassinosteroid-nuclear receptor interactions. Both BRs inhibited cell growth in a dose-dependent manner in the cancer cell lines. Flow cytometry analysis showed that BR treatment arrested MCF-7, MDA-MB-468 and LNCaP cells in G1 phase of the cell cycle and induced apoptosis in MDA-MB-468, LNCaP, and slightly in the DU-145 cells. Our results provide the first evidence that natural BRs can inhibit the growth, at micromolar concentrations, of several human cancer cell lines without affecting the growth of normal cells. Therefore, these plant hormones are promising leads for potential anticancer drugs.
Keywords: Brassinosteroids; Anticancer activity; Cell cycle; Hormone-dependent/independent cancers;

Isolation and identification of alectrol as (+)-orobanchyl acetate, a germination stimulant for root parasitic plants by Xiaonan Xie; Kaori Yoneyama; Dai Kusumoto; Yoichi Yamada; Takao Yokota; Yasutomo Takeuchi; Koichi Yoneyama (427-431).
Alectrol, a germination stimulant for root parasitic plants, was purified from root exudates of red clover (Trifolium pratense L.) and identified as a novel strigolactone, (+)-orobanchyl acetate, by 1D and 2D NMR spectroscopy and ESI- and EI-MS spectrometry. Orobanchyl acetate afforded an [M–42]+ ion in EI-MS and thus had been recognized as an isomer of strigol.Alectrol, a germination stimulant for root parasitic plants, was purified from root exudates of red clover (Trifolium pratense L.) and identified as a strigolactone, (+)-orobanchyl acetate [(3aS,4S,8bS,E)-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-octahydro-2H-indeno[1,2-b]furan-4-yl acetate], by 1D and 2D NMR spectroscopy and ESI- and EI-MS spectrometry. Orobanchyl acetate afforded an [M–42]+ ion in EI-MS and thus had been recognized as an isomer of strigol. Orobanchyl acetate was detected in root exudates of soybean (Glycine max L.) and cowpea (Vigina unguiculata L.) along with orobanchol.
Keywords: Trifolium pratense; Leguminosae; Orobanche minor; Orobanchaceae; Alectrol; Orobanchyl acetate; Strigolactone;

Cytotoxic and apoptosis-inducing effect of ent-15-oxo-kaur-16-en-19-oic acid, a derivative of grandiflorolic acid from Espeletia schultzii by Yarimar Ruiz; Juan Rodrígues; Francisco Arvelo; Alfredo Usubillaga; Mariugenia Monsalve; Nardy Diez; Iván Galindo-Castro (432-438).
ent-Kaurenic acid and many natural derivatives of this diterpene are known to have interesting biological properties. ent-15-oxo-kaur-16-en-19-oic acid (EOKA), obtained from grandiflorolic acid from Espeletia schultzii, induces changes in the expression level of proteins associated with the execution and regulation of apoptosis on the human prostate carcinoma epithelial cell line PC-3. ent-Kaurenic acid and many natural derivatives of this diterpene are known to have interesting biological properties. ent-15-Oxo-kaur-16-en-19-oic acid can be easily obtained from grandiflorolic acid which was first isolated from Espeletia grandiflora. The present work describes the proapoptotic effect of ent-15-oxo-kaur-16-en-19-oic acid on the human prostate carcinoma epithelial cell line PC-3 as evidenced by the changes in the expression level of proteins associated with the execution and regulation of apoptosis. Cell viability was affected upon exposure to the compound, the IC50 were determined as 3.7 μg/ml, which is 4 times lower than that corresponding to a primary cell culture of fibroblasts (14.8 μg/mL). Through Western blot analysis, active forms of caspace-3 associated with the specific proteolysis of Poly(ADP-ribose) polymerase (PARP) were detected. Reduced levels of the antiapoptotic protein Bcl-2, as well as the appearance of internucleosomal DNA fragmentation, were also demonstrated. Thus, ent-15-oxo-kaur-16-en-19-oic acid may be a promising lead compound for new chemopreventive strategies, alone or in combination with traditional chemotherapy agents to overcome drug resistance in tumoral cells.
Keywords: Espeletia schultzii; Espeletiinae; Frailejón; ent-Kaurenoids; ent-5-Oxo-kaur-16-en-19-oic acid; Apoptosis; PC-3 cell line;

Dihydroisocoumarin from Xyris pterygoblephara active against dermatophyte fungi by Keller Guilherme Guimarães; José Dias de Souza Filho; Thiago Rennó dos Mares-Guia; Fernão Castro Braga (439-444).
(3R,4R)-(−)-6-methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one (1), isolated from the Brazilian species Xyris pterygoblephara, showed potent in vitro antifungal activity against clinical isolates of the dermatophytes Epidermophyton floccosum, Trichophyton mentagrophytes and Trichophyton rubrum.The ethanol extract from Xyris pteygoblephara aerial parts was evaluated against five microorganism strains, by the microdilution and agar diffusion methods. Extract fractionation led to the isolation of three compounds, whose structures were assigned by spectrometric data (1D and 2D NMR, IR, MS and UV) as (3R,4R)-(−)-6-methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one (1), moronic acid and quercetin. The absolute configuration of 1 was defined by circular dichroism spectroscopy and comparison with data reported for other dihydroisocoumarins. Assay of 1 (100 μg/disc) by the agar diffusion method against clinical isolates of the dermatophytes Epidermophyton floccosum (inhibition zone, mm ± s.d.: 4.5 ± 0.8), Trichophyton mentagrophytes (4.8 ± 0.4) and Trichophyton rubrum (10.2 ± 0.8) revealed similar inhibition zones to the positive control amphotericin B (32 μg/disc; 5.0 ± 0.2; 5.0 ± 0.6 and 8.8 ± 1.2, respectively). The result corroborates the ethnomedical use of Xyris species to treat dermatitis.
Keywords: Xyris pertygoblephara; Xyridaceae; (3R,4R)-(−)-6-methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one; Circular dichroism; Moronic acid; Quercetin; Dermatophyte fungi;

Trypanocidal tetrahydrofuran lignans from Peperomia blanda by Lidiane Gaspareto Felippe; Debora Cristina Baldoqui; Massuo Jorge Kato; Vanderlan da Silva Bolzani; Elsie Franklin Guimarães; Regina Maria Barreto Cicarelli; Maysa Furlan (445-450).
Five tetrahydrofuran lignans were isolated from the aerial parts of Peperomia blanda, of which compounds 14 are diastereomeric lignans while 5 is a novel compound. The trypanocidal activities of 15 were evaluated against epimastigotes of Trypanosoma cruzi strain Y.Five tetrahydrofuran lignans and two known flavones were isolated from the aerial parts of Peperomia blanda. The structures of the isolated lignans were elucidated by interpretation of their spectroscopic data, including by gHMQC and gHMBC. The relative and absolute configurations of the isolates were determined from NOESY interactions and optical properties, respectively. Four of the lignans were diastereomeric whilst one was of mixed biosynthetic origin. All but one of the lignans exhibited high in vitro trypanocidal activity when assayed against epimastigotes of Trypanosoma cruzi strain Y.
Keywords: Peperomia blanda; Piperaceae; Tetrahydrofuran lignans; Structural elucidation; Trypanocidal activity;

Antioxidant aryl-prenylcoumarin, flavan-3-ols and flavonoids from Eysenhardtia subcoriacea by José M. Narváez-Mastache; Fernando Novillo; Guillermo Delgado (451-456).
The antioxidant activity assay-guided chemical analysis of Eysenhardtia subcoriacea allowed the isolation of the new compound named subcoriacin together with known flavan-3-ols as bioactive constituents. Subcoriacin, (+)-catechin, (−)-epicatechin and afzelechin improved the reduced glutathione levels with rat pancreatic homogenate.Antioxidant activity (AOA) assay-guided chemical analysis, using a rat pancreas homogenate model, of aerial parts from Eysenhardtia subcoriacea, led to isolation of the new compound subcoriacin (3-(2′-hydroxy-4′,5′-methylendioxyphenyl)-6-(3″-hydroxymethyl-4″-hydroxybut-2″-enyl)-7-hydroxycoumarin) together with the known substances: (+)-catechin, (−)-epicatechin, (+)-afzelechin, eriodictyol, (+)-catechin 3-O-β-d-galactopyranoside and quercetin 3-O-β-d-galactopyranoside as bioactive constituents. The structure of the compound was determined from 1D and 2D NMR spectroscopic analyses. Additional known constituents were characterized. The bioactive compounds showed also moderate to strong radical scavenging properties against diphenylpicrylhydrazyl radical (DPPH). In addition, subcoriacin, (+)-catechin, (−)-epicatechin and (+)-afzelechin improved the reduced glutathione levels in rat pancreatic homogenate.
Keywords: Eysenhardtia subcoriacea; Fabaceae; Coumarins; Flavan-3-ols; 3-(2′-hydroxy-4′,5′-methylendioxyphenyl)-6-(3″-hydroxymethyl-4″-hydroxybut-2″-enyl)-7-hydroxycoumarin; Subcoriacin; Antioxidant activity; Reduced glutathione;

Antibacterial stilbenoids from the roots of Stemona tuberosa by Li-Gen Lin; Xin-Zhou Yang; Chun-Ping Tang; Chang-Qiang Ke; Ji-Bao Zhang; Yang Ye (457-463).
Twelve dihydrostilbenes and one phenanthraquinone were isolated from roots of Stemona tuberosa. Their structures were established by 1D and 2D NMR and other spectroscopic analyses. Dihydrostilbene 8 exhibited strong activity against the hospital pathogenic bacterium Bacillus pumilus.Twelve dihydrostilbenes, stilbostemins NY (112), and a phenanthraquinone, stemanthraquinone (13), were isolated and identified from roots of Stemona tuberosa, along with five known dihydrostilbenes. Their structures were established on the basis of 1D and 2D NMR and other spectroscopic analyses. Dihydrostilbene 8 exhibited strong activity against Bacillus pumilus (MIT 12.5–25 μg/mL). Many tested compounds exhibited moderate antibacterial activities.
Keywords: Stemona tuberosa; Stemonaceae; Dihydrostilbenes; Stilbostemins NY; Phenanthraquinone; Stemanthraquinone; Antibacterial;

Effect of storage xyloglucans on peritoneal macrophages by M.M.T. Rosário; G.R. Noleto; J.F. Bento; F. Reicher; M.B.M. Oliveira; C.L.O. Petkowicz (464-472).
Xyloglucans isolated from seeds of Copaifera langsdorffii, Hymenaea courbaril and Mucuna sloanei showed immunomodulatory activity.Xyloglucans from seeds of Copaifera langsdorffii (XGC), Hymenaea courbaril (XGJ) and Mucuna sloanei (XGM) were obtained from milled and defatted cotyledons by aqueous extraction at 25 °C. The resulting fractions contained Glc, Xyl and Gal in molar ratios of 2.5: 1.5: 1.0 (XGC), 3.8: 2.6: 1.0 (XGJ) and 2.5: 1.6: 1.0 (XGM). HPSEC-MALLS/RI analysis showed that each polysaccharide fraction was homogeneous; M w values were 1.6 × 105, 2.0 × 105 and 1.5 × 105  g/mol, respectively. The effect of the xyloglucans on the production of O 2 · - and NO· and on the recruitment of macrophages to the mouse peritoneum was evaluated. All polysaccharides promoted an increase in the number of peritoneal macrophages in a dose-dependent manner. The largest increase, of 576% in comparison to the control group, was elicited by XGJ at 200 mg/kg. The effect of XGC, XGJ and XGM on O 2 · - production, in the presence or absence of phorbol 12-myristate 13-acetate (PMA), was not statistically significant. For NO· production, the lowest concentration of XGC (10 μg/ml) gave rise to an increase of 262% when compared to the control group; the effect was dose-dependent, reaching 307% at 50 μg/ml. On the other hand, XGJ at a concentration of 50 μg/ml enhanced NO· production by 92%. XGM did not affect NO· production significantly. The results indicate that xyloglucans from C. langsdorffii, H. courbaril and M. sloanei have immunomodulatory activity.
Keywords: Copaifera langsdorffii; Hymenaea courbaril; Mucuna sloanei; Leguminosae; Macrophages; O 2 · - ; NO·; Cell-eliciting activity; Xyloglucans;

Polar secondary metabolites of Ferula persica roots by Mehrdad Iranshahi; Mehdi Mojarab; Hamid Sadeghian; Mohammad Yahya Hanafi-Bojd; Bernd Schneider (473-478).
Phytochemical investigation of the methanolic extract of the dried roots of Ferula persica resulted in four sesquiterpene coumarin glycosides, persicaosides A–D, and two known phytosterol glucosides, sitosterol 3-O-β-glucoside and stigmasterol 3-O-β-glucoside.Phytochemical investigation of the methanolic extract of the dried roots of Ferula persica resulted in four sesquiterpene coumarin glycosides, persicaosides A–D, and two known phytosterol glucosides, sitosterol 3-O-β-glucoside and stigmasterol 3-O-β-glucoside. The structures of these compounds were elucidated by extensive spectroscopic methods including 1D-(1H and 13C) and 2D NMR experiments (DQF-COSY, HSQC, HMBC, and ROESY) as well as ESIMS and TOFMS analyses.
Keywords: Ferula persica; Apiaceae; Persicaosides; Roots; Sesquiterpene coumarin glycosides;

Astaxanthin glucoside esters from snow alga Chlamydomonas nivalis were identified by means of liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC–MS/APCI) based on preparative HPLC and subsequent identification by microbore LC–MS/APCI. The combination of these two techniques was used to identify more than 100 molecular species. The astaxanthin diglucoside diester was also synthesized to unambiguously confirm its structure.A method is described for the identification of astaxanthin glucoside esters from snow alga Chlamydomonas nivalis by means of liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization (LC–MS/APCI). The method is based on the use of preparative HPLC and subsequent identification of astaxanthin diglucoside diesters by microbore LC–MS/APCI. The combination of these two techniques was used to identify more than 100 molecular species. The astaxanthin diglucoside diester, i.e. (all-E)-[di-(6-O-oleoyl-β-d-glucopyranosyloxy)]-astaxanthin, was also synthesized to unambiguously confirm its structure.
Keywords: Chlamydomonas nivalis; Snow alga; Astaxanthin diglucoside diesters; Liquid chromatography–mass spectrometry–atmospheric pressure chemical ionization;

Characterization of short-chain poly3-hydroxybutyrate in baker’s yeast by Yoshikatsu Suzuki; Yasuaki Esumi; Hiroyuki Koshino; Masashi Ueki; Yoshiharu Doi (491-497).
A calcium-polyphosphate complex-derived short-chain poly3-hydroxybutyrate (Yeast cPHA-1) was isolated from baker’s yeast (Saccharomyces serevisiae) and characterized.A short-chain poly3-hydroxybutyrate including four comonomers, originating from a complex with calcium polyphosphate, was isolated from commercial baker’s yeast cells (Saccharomyces cerevisiae) and characterized as the second complexed poly(3-hydroxyalkanoate) (cPHA) in eukaryotes. The number-average molecular weight of 4982.5 Da with a polydispersity index of 1.11 was much lower than that of beet cPHA previously isolated. End-group analysis suggested that at least 60% of the molecules form the cyclic structures. Here, the organism-dependent structural diversity of cPHAs was completely established. It was also found that a change of culture medium influences the molecular weight but not the polydispersity of baker’s yeast cPHA.
Keywords: Saccharomycs cerevisiae; Baker’s yeast; Time-of-flight MALDI MS; Number-average molecular weight; Weight-average molecular weight; Polydispersity; Short-chain poly3-hydroxyalkanoate; cPHA;

Diterpene constituents of leaves from Juniperus brevifolia by Ana M.L. Seca; Artur M.S. Silva; Isabel L. Bazzocchi; Ignacio A. Jimenez (498-505).
Six abietane and sandaracopimarane derivatives and 15 other known compounds, mainly diterpenes, were isolated from leaves of Juniperus brevifolia. Their structures were established on the basis of 1D and 2D NMR and MS evidences.The dichloromethane extract from leaves of Juniperus brevifolia, through chromatographic fractionations yield six compounds: 3β-hydroxy-abieta-8,11,13-trien-7-one, 18-hydroxy-sandaracopimara-8(14),15-dien-7-one, sandaracopimara-8(14),15-dien-18-yl formate; and the first examples of sandaracopimaranes and abieta-8,11,13-triene diterpenoids with a large aliphatic chain on C-18, abieta-8,11,13-trien-18-yl hexadecanoate, 7-oxoabieta-8,11,13-trien-18-yl hexadecanoate, sandaracopimara-8(14),15-dien-18-yl hexadecanoate. Moreover fifteen known compounds were also isolated, some of them for the first time identified on Juniperus genus. The compound abieta-8,11,13-trien-18-yl formate is reported for the first time as a natural product. All the structures were established by spectroscopic methods. 2D NMR techniques have allowed the revision of certain previously reported 13C NMR assignments. Studies on the isolated new compounds showed those possessing a diterpenol ester of a long-chain fatty acid present lipophilicity very distinct from other diterpenoid compounds.
Keywords: Juniperus brevifolia; Cupressaceae; Cedro do mato; Abietanes; Sadaracopimaranes; Fatty acid diterpenol ester;

Polyhydroxyserratane triterpenoids from Diphasiastrum complanatum by Jian Yan; Ping Yi; Baohui Chen; Lu Lu; Zhongrong Li; Xianmin Zhang; Lin Zhou; Minghua Qiu (506-510).
Five polyhydroxyserratane triterpenoids was isolated from Diphasiastrum complanatum including serratane-3α,14α,15α,20β,21β,24, 29-heptol (1), given the trivial name diphasiastrol.Serratane triterpenoids were identified from Diphasiastrum complanatum (L.) Holub, including serratane-3α,14α,15α,20β,21β,24,29-heptol (1), 3α,20β,21β-trihydroxyserrat-14-en-24-oic acid (2), 3β,20β,21β-trihydroxyserrat-14-en-24-oic acid (3), 3α,20β,21β-trihydroxy-16-oxoserrat-14-en-24-oic acid (4), and 16-oxolyclanitin-29-yl E-4′-hydroxyl-3′-methoxycinnamate (5) on the basis of their spectroscopic data as well as nine known analogs.
Keywords: Diphasiastrum complanatum; Lycopodiaceae; Serratane triterpenoid; Polyhydroxylated derivatives;

A-seco-oleane-type triterpenes from Phomopsis sp. (strain HKI0458) isolated from the mangrove plant Hibiscus tiliaceus by Liya Li; Isabel Sattler; Zhiwei Deng; Ingrid Groth; Grit Walther; Klaus-Dieter Menzel; Gudrun Peschel; Susanne Grabley; Wenhan Lin (511-517).
A-seco-oleane-type triterpenes (1–4) were isolated from the fermentation broth of a fungus Phomopsis sp. (HKI0458), which was isolated from the mangrove plant Hibiscus tiliaceus (L.). Their structures were elucidated by extensive spectroscopic data analyses.From the fermentation broth of an unidentified Phomopsis sp. (strain HKI0458) isolated from the mangrove plant Hibiscus tiliaceus, four A-seco-oleane-type triterpenes, namely 3,4-seco-olean-11,13-dien-4,15α, 22β,24-tetraol-3-oic acid (1), 3,4-seco-olean-11,13-dien-4,7β,22β,24-tetraol-3-oic acid (2), 3,4-seco- olean-13-en-4,7,15,22,24-pentaol-3-oic acid (3), and 3,4-seco-olean-13-en-4,15,22,24-tetraol-3-oic acid (4) were obtained. Their structures were elucidated by extensive spectroscopic (UV, IR, FABMS, and 2D NMR) data analyses.
Keywords: Fungus; Phomopsis sp.; Mangrove plant; Hibiscus tiliaceus; Malvaceae; A-seco-oleane-type triterpenes; Spectroscopic analyses;

Diterpenoids from the pericarp of Platycladus orientalis by Ya-Zhou Wang; Chun-Ping Tang; Chang-Qiang Ke; Hans-Christoph Weiss; Ernst-Rudolf Gesing; Yang Ye (518-526).
Eight labdane-type diterpenes along with 20 known diterpenoid compounds were isolated from the pericarp of Platycladus orientalis.Eight labdane-type diterpenes, 7β,13S-dihydroxylabda-8(17),14-dien-19-oic acid (1), 12R,15-dihydroxylabda-8(17),13E-dien-19-oic acid (3c), 12R,15-dihydroxylabda-8(17),13Z-dien-19-oic acid (3d), 12R,13R,14S-trihydroxylabda-12,15-epoxy-8(17)-en-19-oic acid (4a), 12S,13S,14R-trihydroxylabda-12,15-epoxy-8(17)-en-19-oic acid (4b), 15-hydroxy-12-oxolabda-8(17),13E-dien-19-oic acid (5), 14R,15-dihydroxylabda-8(17),12Z-dien-19-oic acid (7a) and 14S,15-dihydroxylabda-8(17),12Z-dien-19-oic acid (7b), along with 20 known diterpenoids, were isolated from the pericarp of Platycladus orientalis. Their structures were unambiguously elucidated by NMR spectroscopic and single crystal X-ray diffraction analyses, as well as via chemical correlation conversion. NMR spectroscopic data of known isomers 8c and 8d were reported as a supplement to existing data.
Keywords: Platycladus orientalis; Cupressaceae; Labdane-type; Diterpenes;

Alisolide, alisols O and P from the rhizome of Alisma orientale by Ming Zhao; Li-Jia Xu; Chun-Tao Che (527-532).
A nor-protostane, alisolide (1), a rearranged protostane, alisol O (2), and a 2,3-seco-protostane triterpene, alisol P (3), were isolated from the rhizomes of Alisma orientale.A nor-protostane, alisolide (1), a rearranged protostane, alisol O (2), and a 2,3-seco-protostane triterpene, alisol P (3), were isolated from the rhizomes of Alisma orientale, along with eight known protostane triterpenes. The structures were elucidated to be (17S)-3,11-dioxo-23-nor-protost-12-en-23(17)-olide, 3-oxo-11β,23-dihydroxy-24,24-dimethyl-26,27-dinorprotost-13(17)-en-25-oic acid, and (20R,23S,24R)-23,24,25-trihydroxy-2,3-seco-protost-13(17)-en-3-oic acid 2,11β-lactone, respectively, by interpretation of spectroscopic data.
Keywords: Alisma orientale; Alismataceae; Protostane triterpenes; Alisolide; Alisol O; Alisol P;

Cytotoxic turrianes of Kermadecia elliptica from the New Caledonian rainforest by Claire Jolly; Odile Thoison; Marie-Thérèse Martin; Vincent Dumontet; Aline Gilbert; Bruno Pfeiffer; Stéphane Léonce; Thierry Sévenet; Françoise Guéritte; Marc Litaudon (533-540).
Eight new cyclophanes (1–8), named kermadecins A-H, were isolated from the bark of Kermadecia elliptica (PROTEACEAE). A LC/APCI-MS study provided a reliable method of detection for most of the compounds.In the course of an automated screening for small molecules presenting cytotoxic activity, eight new cyclophanes named kermadecins A–H (1–8), have been isolated from the bark of a New Caledonian plant, Kermadecia elliptica, Proteaceae. A LC/APCI-MS study performed on kermadecins A, B and C, provided a reliable method for the detection of other analogues existing in small quantities in the extract. This led to the isolation of five other members of this chemical series. The structures were elucidated by extensive mono- and bi-dimensional spectroscopy and mass spectrometry. The cytotoxic activity of four of them was evaluated on various cell lines.
Keywords: Kermadecia elliptica; Proteaceae; Kermadecin; Turriane; Cyclophane; Cytotoxicity; LC/APCI-MS;

Flavones from Struthiola argentea with anthelmintic activity in vitro by Sloan Ayers; Deborah L. Zink; Kenneth Mohn; Joanne S. Powell; Christine M. Brown; Terry Murphy; Robert Brand; Seef Pretorius; Dennis Stevenson; Donald Thompson; Sheo B. Singh (541-545).
Bioassay-guided fractionation of Struthiola argentea (Thymelaeaceae) led to the isolation of three anthelmintic flavones 13, including the flavone 5,6,2′,5′,6′-pentamethoxy-3′,4′-methylenedioxyflavone (3). The flavone 3 exhibited the most potent in vitro activity against Haemonchus contortus with 90% inhibition of larval motility (EC90) at 3.1 μg/mL.Parasitic diseases caused by helminthes lead to significant health hazards to animals resulting in enormous economic impact. While a number of anthelmintics are currently available, all are encountering resistance and ones with a mode of action are needed. We report herein bioassay-guided isolation of three anthelmintic flavones 13, including the flavone, 5,6,2′,5′,6′-pentamethoxy-3′,4′-methylenedioxyflavone (3) from the methanol extract of Struthiola argentea (Thymelaeaceae). The structure of 3 was elucidated by analysis of its 1D and 2D NMR and MS data. The two major flavones produced by this plant were also isolated and identified as yuankanin (4) and amentoflavone (5). A number of flavones related to the compounds isolated from S. argentea were acquired and tested to ascertain structure activity relationships. The isolation, structure, anthelmintic activity and structure activity relationships of the flavones are described. Compound 3 exhibited the most potent in vitro activity with 90% inhibition of larval motility at 3.1 μg/mL and compound 15 showed modest in vivo activity.
Keywords: Flavones; Struthiola argentea; Anthelmintics; Haemonchus contortus; Heligmosomoides polygyrus; Thymelaeaceae;

Estrogenic constituents of the heartwood of Dalbergia parviflora by Kaoru Umehara; Kiyomitsu Nemoto; Kyoko Kimijima; Ayako Matsushita; Eri Terada; Orawan Monthakantirat; Wanchai De-Eknamkul; Toshio Miyase; Tsutomu Warashina; Masakuni Degawa; Hiroshi Noguchi (546-552).
From the heartwood of Dalbergia parviflora, five compounds, dalparvin A and B, isodalparvinol A, dalparvinol C, and neokhriol A, along with 11 known compounds, were isolated and characterized. By evaluating their activity in human breast cancer cells, dalparvin B and isodalparvinol A stimulated both MCF-7 and T-47D cell proliferation, but no isolates showed significant effects on BT20.From the heartwood of Dalbergia parviflora, five compounds, dalparvin A (1), B (2), C (3), dalparvinol C (4), and neokhriol A (5), along with 11 known compounds, kenusanone G (6), cajanin (7), sophorol (8), alpinetin (9), hesperetin (10), 3′-O-methylorobol, odoratin, (2R)(3R)-2,3-trans 7-hydroxy-5-methoxydihydroflavonol, (6aR, 11aR)-3,8-dihydroxy-9-methoxypterocarpan, (6aR, 11aR)- vesticarpan, and methyl-3,4-dihydroxy-2-methoxybenzoate were isolated and characterized. Isolates were evaluated for their cell proliferation stimulatory activity against MCF-7, T-47D, and BT20 human breast cancer cell lines. Along with 710, two compounds 2 and 3 stimulated not only MCF-7, but also T-47D human breast cancer cell proliferation. Compound 6 had activity only against MCF-7 cells, and the activity of 7 was more than equivalent to that of daidzein. On the other hand, none of the isolates had any significant effects on BT20 cell proliferation, and these results indicated that the stimulative activity of these compounds was not general to any cell proliferations. Furthermore, these compounds were tested in the estrogen-responsive transient luciferase reporter assay.
Keywords: Dalbergia parviflora; Leguminosae; Heartwood; Estrogenic activity; Luciferase; Flavonoid; Dalparvin A; Dalparvin B; Isodalparvinol A; Dalparvinol C; Neokhriol A;

Furoquinoline alkaloids of Ertela (Monnieria) trifolia (L.) Kuntze from the Suriname rainforest by Shugeng Cao; Adnan J. Al-Rehaily; Peggy Brodie; Jan H. Wisse; Etienne Moniz; Stan Malone; David G.I. Kingston (553-557).
7-(2’-Hydroxy-3’-chloroprenyloxy)-4,8-dimethoxyfuroquinoline (1) and 6-(2’-hydroxy-3’-chloroprenyloxy)-4,7-dimethoxyfuroquinoline (2), together with ten known compounds, have been isolated from the aerial parts of Ertela (Monnieria) trifolia (L.) Kuntze. All the isolates were tested for antiproliferative activity against the A2780 human ovarian cancer cell line.
Keywords: Ertela trifolia; Monnieria trifolia; Rutaceae; Furoquinoline alkaloids; Acridone alkaloids; Furofuran lignans; NMR; Cytotoxicity;

Methyl chanofruticosinate alkaloids from Kopsia arborea by Kuan-Hon Lim; Toh-Seok Kam (558-561).
Six alkaloids belonging to the methyl chanofruticosinate group, prunifolines A–F, in addition to six other known methyl chanofruticosinate alkaloids, were isolated from the leaf extract of Kopsia arborea.Six alkaloids belonging to the methyl chanofruticosinate group, viz., prunifolines A–F, in addition to six other known methyl chanofruticosinate alkaloids, were isolated from the leaf extract of Kopsia arborea. The structures were determined using NMR and MS analysis and comparison with known related compounds.
Keywords: Kopsia species; Apocynaceae; Indole alkaloids;

Direct NMR analysis of cannabis water extracts and tinctures and semi-quantitative data on Δ9-THC and Δ9-THC-acid by M. Politi; W. Peschel; N. Wilson; M. Zloh; J.M. Prieto; M. Heinrich (562-570).
Diffusion-edited 1H NMR (1D DOSY) and 1H NMR with suppression of the ethanol and water signals were used for the analyses of different tinctures and infusions of cannabis. Cannabis sativa L. is the source for a whole series of chemically diverse bioactive compounds that are currently under intensive pharmaceutical investigation. In this work, hot and cold water extracts as well as ethanol/water mixtures (tinctures) of cannabis were compared in order to better understand how these extracts differ in their overall composition. NMR analysis and in vitro cell assays of crude extracts and fractions were performed. Manufacturing procedures to produce natural remedies can strongly affect the final composition of the herbal medicines. Temperature and polarity of the solvents used for the extraction resulted to be two factors that affect the total amount of Δ9-THC in the extracts and its relative quantity with respect to Δ9-THC-acid and other metabolites. Diffusion-edited 1H NMR (1D DOSY) and 1H NMR with suppression of the ethanol and water signals were used. With this method it was possible, without any evaporation or separation step, to distinguish between tinctures from different cannabis cultivars. This approach is proposed as a direct analysis of plant tinctures.
Keywords: Cannabis; Cannabis water extract; Tinctures; Nuclear magnetic resonance; Solvent signal suppression; Principal component analysis; NFκB;

Penicidones A–C, three cytotoxic alkaloidal metabolites of an endophytic Penicillium sp. by Hui Ming Ge; Yao Shen; Chun Hua Zhu; Shu Hua Tan; Hui Ding; Yong Chun Song; Ren Xiang Tan (571-576).
Penicidones A–C, three cytotoxic alkaloids, were characterized from the culture of Penicillium sp. IFB-E022, an endophytic fungal strain on Quercus variabilis.Along with the known secondary metabolites lumichrome, physcion, and emodin-1,6-dimethyl ether, three alkaloids named penicidones A–C (1–3) were isolated from the culture of Penicillium sp. IFB-E022, an endophytic fungal strain residing in the stem of Quercus variabilis (Fagaceae). The structures of penicidones A–C were established by a correlative interpretation of spectroscopic data including IR, UV and HR-ESI-MS, as well as by analysis of a set of 1D and 2D NMR experiments. The stereochemistry of compounds 1 and 2 was obtained by comparison of the optical rotation with those of vermistatin and its analogues. Penicidones A–C were the first group of natural products possessing a penicidone framework. Compounds 1–3 exhibited moderate cytotoxicity against four cancer cell lines.
Keywords: Penicillium sp.; Quercus variabilis; Fagaceae; Endophyte; Alkaloid; Penicidone A; Penicidone B; Penicidone C; Cytotoxicity;