Hypoglycaemic constituents of Stachytarpheta cayennensis leaf.

The aqueous infusion (tea) of Stachytarpheta cayennensis leaves is used ethnomedically in Peru, Nigeria and other tropical countries for the management of diabetes. Oral administration (p. o.) of aqueous (125 mg/kg) and methanolic (2000 mg/kg) extracts of the leaves to alloxan-diabetic rats showed significant blood glucose reductions by 43 and 53%, respectively, at the end of a 4 hour period similar to the strong effect of glibenclamide (5 mg/kg, P. O.). The methanolic extract was successively partitioned into ethyl acetate, butanol and water fractions, and the same test showed that the butanol fraction (2000 mg/kg) had the highest (50%) hypoglycaemic activity at 4 hours after oral administration. It was also the most active fraction when tested in vitro [insulin release from an insulin secreting cell line (INS-1)] and was also active in normal rats and rats made hyperglycaemic by a glucose load. Its activity was comparable to that of glibenclamide (positive control) in these models. This active butanol fraction was subjected to chromatographic subfractionation; some subfractions reduced hyperglycaemia in alloxan-diabetic rats to 60 and 78% and induced insulin release from the INS-1 cells; other subfractions, however, gave hyperglycaemic activities IN VIVO and inhibition of insulin release from the INS-1 cells. Three major compounds of the butanol fraction were isolated and characterised as 6beta-hydroxyipolamide, ipolamide and isoverbascoside; they increased insulin secretion from INS-1 cells to 125, 128 and 127%, respectively, whereas glibenclamide increased insulin secretion to 157%. The results justify the ethnomedical use of the plant in the management of diabetes and suggest that the butanol fraction and some of its isolated constituents mediate their actions primarily by stimulating insulin release directly.

The metabolite products of chlorocholine chloride (CCC) in eggs and meat of laying hens fed 15N-CCC containing diets.

An experiment was conducted to evaluate the metabolic products of chlorocholine chloride (CCC) in eggs and meat of laying hens fed a diet containing (15)N-CCC. Ten brown laying hens were randomly divided into two groups of five each. One group was offered (15)N-CCC free diet while the other group received a diet with 100 ppm (15)N-CCC for 11 days. Samples of eggs and meat from the laying hens were collected. Egg yolks and albumen were separated. Meat was collected from the breast and femur. The metabolic products of CCC were measured using ion trap electrospray ionisation mass spectrometry (ion trap-ESI-MS/MS). Determination of CCC or its metabolites in eggs and meat showed that CCC was metabolised to choline. Corresponding MS/MS spectra were obtained for m/z 104 (choline) or 105 ((15)N-choline), whereas nothing was detected at m/z 122 (CCC) or 123 ((15)N-CCC). The results from this study indicate that CCC will be metabolised in tissues of laying hens.

Bioactive natural products from marine invertebrates and associated fungi.

Marine natural products with their unique structural features and pronounced biological activities continue to provide lead structures in the search for new drugs from nature. Invertebrates such as sponges, tunicates, mollusks and others that are either sessile or slow moving and mostly lack morphological defense structures have so far provided the largest number of marine-derived secondary constituents including some of the most interesting drug candidates. This review highlights recent research findings of our group related to natural products from marine invertebrates. Areas that are covered include ecological functions of secondary constituents from sponges against predatory fish, the search for new pharmacologically active constituents from sponges and tunicates, and sponge-associated fungi as an evolving source for new bioactive natural products.

Drugs from the seas - current status and microbiological implications.

The oceans are the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds (especially the tunicate metabolite ET-743) show pronounced pharmacological activities and are interesting candidates for new drugs primarily in the area of cancer treatment. Other compounds are currently being developed as an analgesic (ziconotide from the mollusc Conus magus) or to treat inflammation. Numerous natural products from marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (bacteria, microalgae) are at least involved in their biosynthesis or are in fact the true sources of these respective metabolites. This assumption is corroborated by several studies on natural products from sponges that proved these compounds to be localized in symbiotic bacteria or cyanobacteria. Recently, molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the bryozoan Bugula neritina.

Rocaglamides, glycosides, and putrescine bisamides from Aglaia dasyclada.

A phytochemical analysis of the leaves of Aglaia dasyclada collected in Yunnan Province (People's Republic of China) yielded five cyclopentabenzofurans (1-5) of the rocaglamide family that are common secondary metabolites of Aglaia species as well as four biogenetically related compounds of the aglain (7), aglaforbesin (8) and forbaglin (9, 10) types. In addition, the cinnamic acid amide dasyclamide (6), which is a putative biogenetic precursor of these compounds (7-10), was isolated. The structures of the new compounds (6-10) were assigned unambiguously from the combined use of 1D and 2D NMR spectroscopy and mass spectrometry.

Hortein, a new natural product from the fungus Hortaea werneckii associated with the sponge Aplysina aerophoba.

The fungus Hortaea werneckii isolated from the Mediterranean sponge Aplysina aerophoba, collected at Banyuls-sur-Mer in southern France, yielded a new compound named hortein (1), which possesses a unique ring system hitherto unknown for natural products. The structure of 1 was established on the basis of 1D and 2D NMR spectroscopic and mass spectrometric (ESI, CI, FAB, EI) data.

Biological screening of rain forest plot trees from Palawan Island (Philippines).

Study plots totaling 0.2 Ha were established in primary forest in the highlands of central Palawan Island, Philippines. Samples of various anatomical parts [typically leaf + twig (If/tw), stem bark (sb), and root (rt)] were collected from all tree species represented within the plots by individuals having a diameter at breast height > or = 10 cm. In all, 211 distinct samples were obtained from 68 tree species, representing 35 families (not including samples from 4 indeterminate species). Methanol extracts of these samples were tested in in vitro antiplasmodial, brine shrimp toxicity, and cytotoxicity assays. The following samples showed an IC50 < or = 10 microg/mL against either chloroquine-sensitive or chloroquine-resistant clones of Plasmodium falciparum: Acronychia laurifolia (sb), Agathis celebica (lf/tw), Aglaia sp. 1 (sb), Aglaia sp. 2 (lf/tw, rt), Bhesa sp. 1 (rt), Cinnamomum griffithii (lf/tw), Croton leiophyllus (rt), Dysoxylum cauliflorum (rt), Garcinia macgregorii (sb), Lithocarpus sp. 1 (rt, sb), Meliosma pinnata ssp. macrophylla (lf/tw, rt), Myristica guatteriifolia (lf/tw), Ochrosia glomerata (rt, sb), Swintonia foxworthyi (lf/tw), Syzygium sp. 1 (rt), Turpinia pomifera (rt), and Xanthophyllum flavescens (sb). Secondly, those samples which displayed > or = 50% immobilization of brine shrimp at 100 microg/mL were: Acronychia laurifolia (lf/tw/fruit, rt, sb), Agathis celebica (lf/tw, sb), Aglaia sp. 1 (lf/tw), Alphonsea sp. 1 (rt), Ardisia iwahigensis (lf/tw), Arthrophyllum ahernianum (lf/tw, rt, sb), Castanopsis cf. evansii (rt), Cinnamomum griffithii (lf/tw, rt), Croton argyratus (lf/tw), C. leiophyllus (lf/tw, rt), Dysoxylum cauliflorum (fruit, lf/tw, rt), Euonymus javanicus (rt), Glochidion sp. 1 (rt), Polyosma sp. 1 (rt), Symplocos polyandra (rt), Timonius gammillii (sb), and Xanthophyllum flavescens (rt). Lastly, samples which exhibited an IC50 < or = 20 microg/mL against one or more of the cancer cell lines employed (LU1, KB, KB-V1, P-388, LNCaP, or ZR-75-1) include: Acronychia laurifolia (lf/tw/fruit, rt, sb), Aglaia sp. 1 (sb), Aglaia sp. 2 (rt), Alphonsea sp. 1 (rt), Ardisia iwahigensis (lf/tw, rt, sb), Astronia cumingiana (sb), Croton argyratus (lf/tw, rt, sb), C. leiophyllus (lf/tw, rt), Dimorphocalyx murina (lf/tw, rt, sb), Lithocarpus caudatifolius (rt, sb), Litsea cf. sibuyanensis (rt), Syzygium cf. attenuatum (rt, sb), S. confertum (sb), Ternstroemia gitingensis (rt), and Ternstroemia sp. 1 (rt, sb).

Anthraquinones and betaenone derivatives from the sponge-associated fungus Microsphaeropsis species: novel inhibitors of protein kinases.

An undescribed fungus of the genus Microsphaeropsis, isolated from the Mediterranean sponge Aplysina aerophoba, produces two new betaenone derivatives (1, 2) and three new 1,3,6, 8-tetrahydroxyanthraquinone congeners (5-7). The structures of the compounds were established on the basis of NMR spectroscopic and mass spectrometric data and by CD spectroscopy. This is the first report wherein the (1)H and (13)C NMR data of the betaenone congeners are fully and unambiguously assigned on the basis of two-dimensional NMR spectroscopy. Furthermore, we describe the first elucidation of the absolute configuration of 1-(2'-anthraquinonyl)ethanols such as 5 and 6, by quantum chemical calculation of their circular dichroism (CD) and comparison with experimentally measured spectra. Moreover, it was shown that compounds 1, 5, 6, and 7 are inhibitors of PKC-epsilon, CDK4, and EGF receptor tyrosine kinases.

Novel spiciferone derivatives from the fungus Drechslera hawaiiensis isolated from the marine sponge Callyspongia aerizusa.

From the marine sponge Callyspongia aerizusa collected from the Sea of Bali, Indonesia, fungal isolates of Drechslera hawaiiensis were obtained. Culture filtrates of the fungus yielded four spiciferone derivatives which include spiciferone A (1) and B (2), and two other novel derivatives including spiciferol A (3) which is an alcohol congener of spiciferone A (1) and compound 4 which is an monocyclic spiciferone congener featuring a butoxyl side chain. The structures of the novel compounds were established on the basis of NMR spectroscopic (1H, 13C, COSY) and mass spectrometric (EIMS) data.

Bioactive terpenes from the soft coral Heteroxenia sp. from Mindoro, Philippines.

A marine soft coral species of the genus Heteroxenia collected from Mindoro Island, Philippines yielded two cadinene sesquiterpenes, (+)-alpha-muurolene (1) and a novel derivative (+)-6-hydroxy-alpha-muurolene (2), as well as the biologically active polyhydroxysterol, sarcoaldosterol A (3). The structure of the novel compound was unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, 1H-detected direct and long range 13C-1H correlations) and mass spectrometric (EIMS) data. All compounds were active against the phytopathogenic fungus Cladosporium cucumerinum. The isolated terpenes were also active in the brine shrimp lethality test.

A new meroditerpenoid dimer from an undescribed Philippine marine sponge of the genus Strongylophora.

An undescribed Philippine marine sponge of the genus Strongylophora yielded a new meroditerpenoid-strongylophorine dimer (1) and the known meroditerpenoids, strongylophorine-2 (2), strongylophorine-3 (3), and strongylophorine-4 (4). The structures of the compounds were established on the basis of NMR spectroscopic and mass spectrometric data. The position of the inter-unit linkage in the new compound was elucidated after methylation and 1D 1H NOE difference experiments. This is the first report wherein the1H and 13C NMR data of the strongylophorine congeners are fully and unambiguously assigned on the basis of 2D NMR spectroscopy. Compounds 2 and 3 exhibited slight activity against Micrococcus luteus and Salmonella typhii, respectively. Compound 3 was active against the phytopathogenic fungusCladosporium cucumerinum and also against the neonate larvae of the polyphagous pest insectSpodoptera littoralis (EC50 of 69 [+/-0.48 (S.E.)] ppm) when incorporated into artificial diet. Compound 1 was found to be the most active in the brine shrimp lethality test with a LC50 of 10.5 [+/-0.43 (S.E.)] microg/mL.

Four new bioactive lobane diterpenes of the soft coral Lobophytum pauciflorum from Mindoro, Philippines.

The marine soft coral Lobophytum pauciflorum collected from Mindoro Island, Philippines, yielded four new lobane diterpene derivatives: the acetate congeners of epoxylobatrienol and lobatrienediol (2 and 7, respectively), a methoxyl congener of lobatetraene (10), and an oxepin congener of lobatrienetriol (11), and six known derivatives (1, 3-6, and 8). The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, 1H-detected direct, and long-range 13C-1H correlations) and mass spectrometric (EIMS) data. All of the compounds were active against the phytopathogenic fungus Cladosporium cucumerinum. Compound 1 was found to be active against the Gram-positive bacteria Bacillus subtilis and the yeast Saccharomyces cerevisiae. The isolated lobane diterpenes were also active in the brine shrimp lethality test. In the latter bioassay, compounds 8 and 10 were the most active congeners with LC50's of 0.64 and 4.18 micrograms/mL, respectively.

Isolation and structure elucidation of ardisenone: a new, cytotoxic alkenylphenol from Ardisia iwahigensis.

A methanol extract of leaves and twigs from Ardisia iwahigensis demonstrated toxicity toward brine shrimp as well as LNCaP, ZR-75-1, and Lu1 human cancer cells in culture. A novel alkenylphenol, (Z)-1,16-bis(3-hydroxy-5-methoxyphenyl)-10-hexadecene-1,15-dione (ardisenone) (1), was isolated from the extract by bioassay-directed fractionation. This compound demonstrated moderate cytotoxicity against BC1, Lu1, Col2, KB, KB-V1, and LNCaP cell lines.

Four new bioactive polybrominated diphenyl ethers of the sponge Dysidea herbacea from West Sumatra, Indonesia.

The marine sponge Dysidea herbacea collected from Indonesia yielded four new polybrominated diphenyl ether congeners 2-5 and the known derivatives 1, 6, and 7. The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, 1H-detected direct and long-range 13C-1H correlations) and mass spectrometric (EIMS) data. All of the compounds were active against the Gram-positive bacteria Bacillus subtilis and the phytopathogenic fungus Cladosporium cucumerinum. The isolated polybrominated compounds were also active in the brine shrimp lethality test. In the latter bioassay, compounds 1 and 6 were the most active with LC50's of 0.96 [SE +/- 0.19] and 0.94 [SE +/- 0.70] microg/mL, respectively.

Four new bioactive manzamine-type alkaloids from the Philippine marine sponge Xestospongia ashmorica.

Analysis of the Philippine marine sponge Xestospongia ashmorica afforded four new manzamine congeners 1-4 and four known compounds 5 and 7-9. Compound 1 is the 6-deoxy derivative of manzamine X, while 2-4 are the N-oxides of manzamine J (5), 3,4-dihydromanzamine A (6), and manzamine A (7), respectively. The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, 1H-detected direct, and long-range 13C-1H correlations) and mass spectrometric (EI, FAB-MS, and electrospray ionization) data. Alkaloid N-oxide structures were confirmed by conversion to the corresponding tertiary bases by reduction with Zn/HCl. This is the first report of the occurrence of bioactive manzamine N-oxides in marine sponges. Compound 7 exhibited insecticidal activity toward neonate larvae of the polyphagous pest insect Spodoptera littoralis (with an ED50 of 35 ppm) when incorporated in artificial diet and offered to larvae in a chronic feeding bioassay. Compound 7 was also active against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. Cytotoxicity was studied in vitro using L1578y mouse lymphoma cells. From the alkaloids studied, the N-oxides 3 and 4 were the most active (ED50 = 1.6 micrograms/mL) followed by compound 7 (ED50 = 1.8 micrograms/mL).