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The rhamnose biosynthetic pathway, which is highly conserved across nematode species, was characterized in the nematode Caenorhabditis elegans. The pathway is up-regulated before each larval molt, suggesting that rhamnose biosynthesis plays a role in cuticle or surface coat synthesis., L-Rhamnose is a common component of cell-wall polysaccharides, glycoproteins and some natural products in bacteria and plants, but is rare in fungi and animals. In the present study, we identify and characterize a biosynthetic pathway for dTDP-rhamnose in Caenorhabditis elegans that is highly conserved across nematode species. We show that RML-1 activates glucose 1-phosphate (Glc-1-P) in the presence of either dTTP or UTP to yield dTDP-glucose or UDP-glucose, respectively. RML-2 is a dTDP-glucose 4,6-dehydratase, converting dTDP-glucose into dTDP-4-keto-6-deoxyglucose. Using mass spectrometry and NMR spectroscopy, we demonstrate that coincubation of dTDP-4-keto-6-deoxyglucose with RML-3 (3,5-epimerase) and RML-4 (4-keto-reductase) produces dTDP-rhamnose. RML-4 could only be expressed and purified in an active form through co-expression with a co-regulated protein, RML-5, which forms a complex with RML-4. Analysis of the sugar nucleotide pool in C. elegans established the presence of dTDP-rhamnose in vivo. Targeting the expression of the rhamnose biosynthetic genes by RNAi resulted in significant reductions in dTDP-rhamnose, but had no effect on the biosynthesis of a closely related sugar, ascarylose, found in the ascaroside pheromones. Therefore, the rhamnose and ascarylose biosynthetic pathways are distinct. We also show that transcriptional reporters for the rhamnose biosynthetic genes are expressed highly in the embryo, in the hypodermis during molting cycles and in the hypodermal seam cells specifically before the molt to the stress-resistant dauer larval stage. These expression patterns suggest that rhamnose biosynthesis may play an important role in hypodermal development or the production of the cuticle or surface coat during molting.

Australian native plants have a long history of therapeutic use in indigenous cultures, however, they have been poorly studied scientifically. We analysed the effects of 14 plant derived compounds from the species Pilidiostigma glabrum, Myoporum montanum, Geijera parviflora, and Rhodomyrtus psidioides for their potential wound healing properties by assessing their ability to induce or suppress Collagen I and Collagen III expression in human skin fibroblasts in culture. The compound 7-geranyloxycoumarin was able to significantly increase Collagen I (23.7%, p < 0.0002) expression in comparison to control. Significant suppression of Collagen III was observed for the compounds flindersine (11.1%, p < 0.02), and (N-acetoxymethyl) flindersine (27%, p < 0.00005). The implications of these finding is that these compounds could potentially alter the expression of different collagens in the skin allowing for the potential development of new wound healing therapies and new approaches for treating various skin diseases as well as photo (sun) damaged, and aged skin. Refereed/Peer-reviewed

Ethnopharmacological relevance Australian Aboriginal people used crushed leaves of Geijera parviflora Lindl. both internally and externally for pain relief, including for toothache ( Cribb and Cribb, 1981 ). This study tested the hypothesis that this traditional use might be at least in part explained by the presence of compounds with anti-inflammatory activity. Materials and methods A crude extract (95% EtOH) was prepared from powdered dried leaves. From the CH 3 Cl fraction of this extract compounds were isolated by bioassay-guided fractionation and tested for: (1) cytotoxicity in RAW 264.7 murine leukemic monocyte–macrophages, (2) prostaglandin E 2 (PGE 2 ) inhibitory activity in 3T3 Swiss albino mouse embryonic fibroblast cells, as well as (3) nitric oxide (NO) and (4) tumour necrosis factor alpha (TNFα) inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Isolated compounds were also tested for (5) antibacterial activity against a panel of Gram-positive ( Staphylococcus aureus ATCC 29213 and ATCC 25923, Staphylococcu s epidermidis ATCC 35984, biofilm-forming) and Gram-negative ( Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) strains by broth microdilution. Results Eleven compounds were isolated, including one new flavone and one new natural product, with a further four compounds reported from this species for the first time. Some of the compounds showed good anti-inflammatory activity in vitro . In particular, flindersine ( 1 ) and N-(acetoxymethyl) flindersine ( 3 ) inhibited PGE 2 release with IC 50 values of 5.0 μM and 4.9 μM, respectively, without any significant cytotoxicity. Several other compounds showed moderate inhibition of NO ( 5 , 6 , 7 ) and TNF-α ( 6) , with IC 50 in the low micromolar range; however much of this apparent activity could be accounted for by the cytotoxicity of these compounds. None of the compounds showed anti-bacterial activity. Conclusions The inhibition of PGE 2 , an important mediator of inflammation and pain, by flindersine and a derivative thereof, along with the moderate anti-inflammatory activity shown by several other compounds isolated from Geijera parviflora leaf extract, support the traditional use of this plant for pain relief by Australian Aboriginal people.

Polyketides and nonribosomal peptides are two important types of natural products that are produced by many species of bacteria and fungi but are exceedingly rare in metazoans. Here, we elucidate the structure of a hybrid polyketide-nonribosomal peptide from Caenorhabditis elegans that is produced in the canal-associated neurons (CANs) and promotes survival during starvation-induced larval arrest. Our results uncover a novel mechanism by which animals respond to nutrient fluctuations to extend survival.

In an effort to identify natural compounds with immunosuppressive activity, nine new flavonoids, including one isoflav-3-ene derivative ( 1 ), one coumaronochromone ( 2 ), two isoflavanones ( 3 , 4 ), one isoflavone derivative ( 6 ), one isoflavone ( 7 ), three flavonols ( 8 , 9 , 10 ), as well as one known compound, hydroisoflavone C ( 5 ), were isolated from the roots of Campylotropis hirtella . The structures of these compounds were elucidated by extensive spectroscopic measurements. All of the compounds were assessed for immunosuppressive activity. Among the isolates, compound 2 showed good inhibitory activity against mitogen-induced splenocyte proliferation with an IC 50 of 0.28 μM and relatively low cytotoxicity.

In an effort to identify new anti-inflammatory and antibacterial agents with potential application in wound healing, five new dibenzofurans, 1,3,7,9-tetrahydroxy-2,8- dimethyl-4,6-di(2-methylbutanoyl)dibenzofuran (1), 1,3,7,9- tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2- methylpropionyl)dibenzofuran (2), 1,3,7,9-tetrahydroxy-2,8- dimethyl-4,6-di(2-methylpropionyl)dibenzofuran (3), 1,3,7,9- tetrahydroxy-4,6-dimethyl-2-(2-methylbutanoyl)-8-(2- methylpropionyl)dibenzofuran (4), and 1,3,7,9-tetrahydroxy- 4,6-dimethyl-2,8-di(2-methylpropionyl)dibenzofuran (5), were isolated from the leaves of Pilidiostigma glabrum together with one previously described dibenzofuran. Structure elucidation was achieved by way of spectroscopic measurements including 2D-NMR spectroscopy. Compounds with 2,8-acyl substitutions had potent antibacterial activity against several Gram-positive strains (MIC in the low micromolar range), while compounds with 4,6- acyl substitutions were less active. All compounds except 3 inhibited the synthesis of nitric oxide in RAW264 macrophages with IC50 values in the low micromolar range. Compounds with 2,8-acyl substitutions also inhibited the synthesis of PGE2 in 3T3 cells, whereas 4,6-acyl-substituted compounds were inactive. None of the compounds inhibited the synthesis of TNF-α in RAW264 cells. The compounds showed variable but modest antioxidant activity in the oxygen radical absorbance capacity assay. These findings highlight that much of the Australian flora remains unexplored and may yet yield many new compounds of interest. Initial clues are provided on structure/activity relationships for this class of bioactives, which may enable the design and synthesis of compounds with higher activity and/or selectivity. Refereed/Peer-reviewed

Seven new oleanane-type triterpenoid saponins (glabasaponin A-G) were isolated from the roots of Glycyrrhiza glabra together with a known compound macedonoside A. The structures of compounds 1-7 were determined on the basis of 1D and 2D NMR as well as the MS data analyses. The sugar moieties of compound 5 were confirmed by gas chromatography after hydrolysis.

Phytochemical investigation of the fresh bulbs of Fritillaria anhuiensis S. C. Chen et S. E. Yin, resulted in the isolation of a known steroidal alkaloid solanidine of (22S,25S)-solanid-5-en-3beta-ol (1), which has never yet been found as a natural substance, and of a new steroidal alkaloid (22S,25S)-solanid-5,20(21)-dien-3beta-ol. Compounds 1 and 2 were the first solanidine-type alkaloids with 22-S configuration discovered from nature. Their structures were elucidated based on spectroscopic analysis, including 1D and 2D NMR experiments.

From the dried roots of Campylotropis hirtella (Franch.) Schindl., five novel isoflavonoids, 3( S)-7,2',4'-trihydroxy-5,5'-dimethoxy-6-(3-methylbut-2-enyl)-isoflavan ( 1), 3( S)-2',4'-dihydroxy-5,5'-dimethoxy-(6'',6''-dimethylpyrano)-(2'',3'':7,6)-isoflavan ( 2), 3( R)-5,4'-dihydroxy-2'-methoxy-3'-(3-methylbut-2-enyl)-(6'',6''-dimethylpyrano)-(7,6 : 2'',3'')-isoflavanone ( 3), 3( R)-5,4'-dihydroxy-2'-methoxy-(6'',6''-dimethylpyrano)-(7,6: 2'',3'')-isoflavanone ( 4), and 3( R)-5,4'-dihydroxy-7,2'-dimethoxy-6-geranylisoflavanone ( 5) were isolated. The structures of the compounds were elucidated on the basis of spectroscopic analysis. All isolates exhibited good immunosuppressive activities on mitogen-induced splenocyte proliferation, and their cytotoxicity on splenic lymphocytes was also tested.

Four novel compounds, rhodomyrtals A–D (1–4), with two unprecendented carbon frameworks of phloroglucinol coupled eudesmane, together with the known compound eucalyptin A (5) have been isolated from the leaves of the Australian plant Rhodomyrtus psidioides. The structures of compounds 1–4 were elucidated by spectroscopic analysis and ECD calculations. Some of the compounds showed good antibacterial activity against selected Gram-positive strains.

Two novel diterpenoids were isolated from the bulbs of Fritillaria anhuiensis S. C. Chen and S. E. Yin. Compound 1 was the first diterpenoid containing a sulfonyl group isolated from nature. Compound 2 was a novel kaurane-type diterpenoid. Their structures were determined by extensive spectroscopic analysis (IR, MS, NMR, and X-ray diffraction). Compound 1 significantly attenuated nitric oxide (NO) production of a macrophage cell line of Raw 264.7 cells stimulated with IFN-γ.

Four novel compounds, rhodomyrtals A–D (1–4), with two unprecendented carbon frameworks of phloroglucinol coupled eudesmane, together with the known compound eucalyptin A (5) have been isolated from the leaves of the Australian plant Rhodomyrtus psidioides. The structures of compounds 1–4 were elucidated by spectroscopic analysis and ECD calculations. Some of the compounds showed good antibacterial activity against selected Gram-positive strains. Refereed/Peer-reviewed

Five anthranilic acid derivatives, a mixture I of three new compounds 11′-hexadecenoylanthranilic acid ( 1 ), 9′-hexadecenoylanthranilic acid ( 2 ), and 7′-hexadecenoylanthranilic acid ( 3 ), as well as a new compound 9,12,15-octadecatrienoylanthranilic acid ( 4 ) together with a new natural product, hexadecanoylanthranilic acid ( 5 ), were isolated from Geijera parviflora Lindl. (Rutaceae). Their structures were elucidated by extensive spectroscopic measurements, and the positions of the double bonds in compounds 1 – 3 of the mixture I were determined by tandem mass spectrometry employing ozone-induced dissociation. The mixture I and compound 5 showed good antibacterial activity against several Gram-positive strains.

Two novel alkaloids (parvifloranines A and B), possessing an unusual 11-carbon skeleton linked with amino acids, were isolated from Geijera parviflora , an endemic Australian Rutaceae. Their structures were elucidated by extensive spectroscopic measurements including 2D NMR analyses. Parvifloranine A was found to be a mixture of two enantiomers, (S)-1 and (R)-1 , in a ratio of 1:4, based on their separation using a chiral column. Parvifloranine B is also believed to be a mixture of enantiomers. Proposed biosynthetic pathways are discussed. Parvifloranine A inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages with an IC 50 value of 23.4 μM. Refereed/Peer-reviewed

Pilidiostigmin, a novel dimeric acylphloroglucinol derivative, was isolated from the leaves of the Australian plant species Pilidiostigma glabrum (Myrtaceae). Pilidiostigmin exists in the plant in the form of a sodium salt, which is rare in natural products. The elucidation of the structure and relative configuration was achieved by spectroscopic measurements with special emphasis on 1D and 2D NMR techniques. Pilidiostigmin was found to display biological activity; it significantly and dose-dependently inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages at low micromolar concentrations. (C) 2013 Elsevier Ltd. All rights reserved.

A method of reversed-phase high performance liquid chromatography (RP-HPLC) using diode array detection (DAD) was developed for the quantitative determination of 3'-geranyl-5,7,4'-trihydroxyisoflavone (compound 1) and 8,9-dihydroxy-1-methoxy-[6',6'-dimethylpyrano(2',3': 2,3)]pterocarpene (compound 2) in Campylotropis hirtella. The separation and quantification were achieved using an Agilent Zorbax SB-C18 column (250 mm x 4.6 mm, 5 microm), and mobile phases of acetonitrile and 0. 1% formic acid with gradient elution at a flow rate of 1.0 mL/min and 30 degrees C. The calibration curves for compounds 1 and 2 were linear in the ranges of 4.4-13.2 microg and 0.428-1.284 microg, respectively. The recoveries were 99.65% and 99.11% with the relative standard deviations of 1.83% and 2.59% (n=5), respectively. This RP-HPLC-DAD method is rather simple, accurate and convenient. It can be used for the quantitative determination of the active flavonoids in Campylotropis hirtella (Franch.) Schindl.

A pair of isomeric isoflavonoid derivatives, Hirtellanines A ( 1 ) and B ( 2 ), has been isolated from the roots of Campylotropis hirtella (Franch.) Schindl. and their structures were elucidated on the basis of spectroscopic methods, with special emphasis on 1D and 2D NMR techniques. The in vitro assay showed that Hirtellanine A had strong B lymphocyte suppression activity (IC 50 : 0.06 μM) and T lymphocyte suppression activity (IC 50 : 0.92 μM). Hirtellanine B showed moderate B lymphocyte suppression activity (IC 50 : 3.00 μM) and T lymphocyte suppression activity (IC 50 : 9.55 μM). Due to the potent immunosuppressive activities and lower cytotoxicity, Hirtellanine A could be a promising lead towards novel immunosuppressive agents.

In an effort to identify new immunosuppressive agents from natural sources, 12 new geranylated flavonoids, 5,7,4'-trihydroxy-3'-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]isoflavone (1), a racemate of 5,7,2',4'-tetrahydroxy-3'-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]isoflavanone (2), 2''(S)-5,7-dihydroxy-[2''-methyl-2''-(4-methyl-3-pentenyl)pyrano]-5'',6'':3',4'-isoflavone (3), (2''S,3''R,4''S)-5,7,3'',4''-tetrahydroxy[2''-methyl-2''-(4-methyl-3-pentenyl)pyrano]-5'',6'':3',4'-isoflavone (4), a racemate of 3'-geranyl-5,7,2',4'-tetrahydroxyisoflavanone (5), a racemate of 3'-geranyl-4'-methoxy-5,7,2'-trihydroxyisoflavanone (6), 3'-geranyl-5,7,4',5'-tetrahydroxyisoflavone (8), 3'-geranyl-5,7,2',5'-tetrahydroxyisoflavone (9), 3'-geranyl-4'-methoxy-5,7,2'-trihydroxyisoflavone (10), 2(R),3(R)-3'-geranyl-2,3-trans-5,7,4'-trihydroxyflavonol (12), (2R,3R)-6-methyl-3'-geranyl-2,3-trans-5,7,4'-trihydroxyflavonol (13), and 5,7-dihydroxy-4'-O-geranylisoflavone (14), were isolated from the roots of Campylotropis hirtella (Franch.) Schindl. together with three previously described flavonoids. Their structures were elucidated by spectroscopic measurements, including two-dimensional nuclear magnetic resonance (NMR) techniques. The immunosuppressive effects of these compounds were assessed using mitogen-induced splenocyte proliferation, and the cytotoxicity of the compounds was also examined. The IC50 values of the compounds were found to be in the range of 1.49-61.23 microM for T lymphocyte suppression and 1.16-73.07 microM for B lymphocyte suppression. An analysis of their structure-activity relationships revealed that an isoflavonoid carbon skeleton with a C10 substituent at the C3' position was necessary for the activity. As many of the compounds exhibited good immunosuppressive activities, they may be promising as novel immunosuppressive agents.

A pair of isomeric isoflavonoid derivatives, Hirtellanines A ( 1 ) and B ( 2 ), has been isolated from the roots of Campylotropis hirtella (Franch.) Schindl. and their structures were elucidated on the basis of spectroscopic methods, with special emphasis on 1D and 2D NMR techniques. The in vitro assay showed that Hirtellanine A had strong B lymphocyte suppression activity (IC 50 : 0.06 μM) and T lymphocyte suppression activity (IC 50 : 0.92 μM). Hirtellanine B showed moderate B lymphocyte suppression activity (IC 50 : 3.00 μM) and T lymphocyte suppression activity (IC 50 : 9.55 μM). Due to the potent immunosuppressive activities and lower cytotoxicity, Hirtellanine A could be a promising lead towards novel immunosuppressive agents.

A method of reversed-phase high performance liquid chromatography (HPLC) coupled with evaporative light scattering detection (ELSD) was developed for the determination of jervine and veratramine in veratrum plants. The extraction method of total active alkaloids from the raw material was also established. The separation and quantification were achieved using a Kromasil C8column (250 mm x 4.6 mm, 5 microm), and a mobile phase of acetonitrile and 0.1% trifluoroacetic acid with the following gradient elution: 20% acetonitrile at the first 5 mm, 20%-40% acetonitrile at the 5-30 mm, 40%-20% acetonitrile at the 30-40 mm, 20% acetonitrile at the 40-45 mm with a flow rate of 0.8 mL/min; column temperature of 35 t and monitored by an ELSD detector with the drift tube of 98 t and the nitrogen flow rate of 2.2 L/min. The calibration curves for jervine and veratramine were linear over the ranges of 42.05-980 mg/L and 43.52-1020 mg/L, respectively. The recoveries were 99.2% and 101.4% with relative standard deviations of 1.7% and 2.1% (n=6), respectively. The limits of detection for jervine and veratramine in raw material were 18.37 mg/kg and 21.50 mg/kg, respectively, with 3 times of the signal to noise ratio. This HPLC-ELSD method is rapid, simple, accurate and convenient. It can be used as one of the direct and reliable means for quantitative determination of the active alkaloids in veratrum plants.

In an effort to identify hemostatic components from Liparis nervosa (Thunb.) Lindl. using a bioactivity-guided fractionation approach, the n-BuOH extract was found to promote ADP-induced platelet aggregation and two compounds were isolated from the active extract. Compound 1 was a new nervogenic acid glycoside and the structure was elucidated as 3,5-bis(3-methyl-but-2-enyl)-4- O-[β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl]-benzoic acid by extensive spectroscopic measurements. Adenosine (2) was isolated from this plant for the first time. Compound 1 also showed good pro-coagulant activity in vitro.