Triterpenoids from Gentiana scabra

Five triterpenoids, (20S)-dammara-13(17),24-dien-3-one, (20R)-dammara-13(17),24-dien-3-one, chirat-16-en-3-one, chirat-17(22)-en-3-one and 17beta,21beta-epoxyhopan-3-one, were isolated from the rhizomes and roots of Gentiana scabra together with five known ones, chiratenol, hop-17(21)-en-3-one, hop-17(21)-en-3beta-ol, lupeol and alpha-amyrin. The structures of new compounds were elucidated on the basis of spectroscopic studies.     

Leishmanicidal cycloartane-type triterpene glycosides from Astragalus oleifolius

Two new cycloartane-type glycosides oleifoliosides A (1) and B (2) were isolated from the lower stem parts of Astragalus oleifolius. Their structures were identified as 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-xylopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane and 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-glucopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane, respectively, by means of spectroscopic methods (IR, 1D and 2D NMR, ESI-MS). Three known cycloartane glycosides cyclocanthoside E (3), astragaloside II (4) and astragaloside IV (5) were also isolated and characterized. All five compounds were evaluated for in vitro trypanocidal, leishmanicidal and antiplasmodial activities as well as their cytotoxic potential on primary mammalian (L6) cells. Except for the compound 5, all compounds showed notable growth inhibitory activity against Leishmania donovani with IC50 values ranging from 13.2 to 21.3 microg/ml. Only weak activity against Trypanosoma brucei rhodesiense was observed with the known compounds astragaloside II (4, IC50 66.6 microg/ml) and cyclocanthoside E (3, IC50 85.2 microg/ml), while all compounds were inactive against Trypanosoma cruzi and Plasmodium falciparum. None of the compounds were toxic to mammalian cells (IC50's > 90 microg/ml). This is the first report of leishmanicidal and trypanocidal activity of cycloartane-type triterpene glycosides.     

Ent-3,4-seco-labdane and ent-labdane diterpenoids from Croton stipuliformis (Euphorbiaceae)

From a methanolic extract of the leaves of Croton stipuliformis, three ent-3,4-seco-labdanes (1-3) and an ent-labdane (4) together with the known compounds 6-hydroxynidorellol (5), maravuic acid, and sitosterol were isolated and identified from their spectroscopic data. The absolute stereochemistry of compound 4 was determined by application of Mosher's method in the NMR tube.     

Echinostome Infections in the Striped-Field Mouse, Apodemus agrarius, and the Ussuri White-Toothed Shrew, Crocidura lasiura, Caught Near the Demilitarized Zone, Gyeonggi-do (Province), Republic of Korea

A total of 1,498 small mammals (rodents and insectivores), including Apodemus agrarius (n = 1,366), Crocidura lasiura (54), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutes (6), and Cricetulus triton (3), were live-trapped in Gyeonggi-do (Province) (Paju-si, Pocheon-gun, and Yeoncheon-gun) near the demilitarized zone (DMZ) from December 2004 to September 2005. A. agrarius was found to be infected with 3 species of echinostomes (Echinostoma hortense, Echinostoma cinetorchis, and Euparyphium murinum), while C. lasiura was infected with 1 species (Echinochasmus japonicas) of echinostome. Other mammals were free from echinostome infections. Total 16 E. hortense were detected in 7 (0.5%) mice, 9 E. cinetorchis from 5 (0.4%), and 3 E. murinum from 2 (0.1%) out of 1.366 A. agrarius examined. E. japonicus was found only in 1 (1.9%; total 3 specimens) C. lasiura. These results demonstrate that A. agrarius and C. lasiura, inhabiting near the DMZ of Gyeonggi-do serve as the natural definitive hosts for several species of echinostomes, although their infection rates are low. This is the first record of natural infections of A. agrarius with E. cinetorchis and C. lasiura with E. japonicus in the Republic of Korea.     

Gene amplification profiling of esophageal squamous cell carcinomas by DNA array CGH

Gene amplification is one of the basic mechanisms that lead to overexpression of oncogenes. DNA array comparative genomic hybridization (CGH) has great potential for comprehensive analysis of both a relative gene-copy number and altered chromosomal regions in cancers, which enables us to identify new amplified genes and unstable chromosomal loci. We examined the amplification status in 32 esophageal squamous cell carcinomas (ESCCs) and 13 ESCC cell lines on 51 frequently amplified loci in a variety of cancers by both DNA array CGH and Southern blot analyses. The 1p34 locus containing MYCL1, 2p24 (MYCN), 7p12 (EGFR), and 12q14 (MDM2) were amplified in one of the 32 cases (3%), and the 17q12 locus (ERBB2) and 8p11 (FGFR1) in two of the 32 cases (6%), while only the 11q13 locus (Cyclin D1, FGF4, and EMS1) was frequently amplified (28%, 9/32), demonstrating this locus to be a major target in ESCCs. One locus, 8q24 (c-MYC) was found to be amplified only in the cell lines. Eight out of 51 loci (15.7%) were found to be amplified in at least one of the 32 primary ESCCs or the 13 ESCC cell lines, suggesting that chromosomal loci frequently amplified in a type of human cancer may also be amplified in other types of cancers. This paper is the first report of an application of DNA array CGH to ESCCs.     

Antimalarial xanthones from Calophyllum caledonicum and Garcinia vieillardii

The antimalarial activity of 22 xanthones against chloroquino-resistant strains of Plasmodium falciparum was evaluated. Natural caloxanthone C (1), demethylcalabaxanthone (2), calothwaitesixanthone (3), calozeyloxanthone (4), dombakinaxanthone (5), macluraxanthone (6), and 6-deoxy-gamma-mangostin (7) were isolated from Calophyllum caledonicum. 1,6-dihydroxyxanthone (8), pancixanthone A (9), isocudraniaxanthone B (10), isocudraniaxanthone A (11), 2-deprenylrheediaxanthone B (12) and 1,4,5-trihydroxyxanthone (13) were isolated from Garcinia vieillardii. Moreover, synthetic compounds (14-22) are analogues or intermediates of xanthones purified from Calophyllum caledonicum (Oger J.M., Morel C., Helesbeux J.J., Litaudon M., Seraphin D., Dartiguelongue C., Larcher G., Richomme P., Duval O. 2003. First 2-Hydroxy-3-Methylbut-3-Enyl substituted xanthones isolated from Plants: structure elucidation, synthesis and antifungal activity. Natural Product Research 17(3), 195-199; Helesbeux J.J., Duval O., Dartiguelongue C., Seraphin D., Oger J.M., Richomme P., 2004. Synthesis of 2-hydroxy-3-methylbut-3-enyl substituted coumarins and xanthones as natural products. Application of the Schenck ene reaction of singlet oxygen with ortho-prenylphenol precursors. Tetrahedron 60(10), 2293-2300). The relationship between antimalarial activity and molecular structure of xanthones has also been explored. The most potent xanthones (2), (3) and (7) (IC50 = c.a. 1.0 microg/mL) are 1,3,7 trioxygenated and prenylated on the positions 2 and 8.     

Intestinal helminthiases and schistosomiasis among school children in an urban center and some rural communities in southwest Nigeria

Intestinal helminths and schistosomiasis among school children were investigated in an urban and some rural communities of Ogun State, southwest Nigeria. Fecal samples of 1,059 subjects (524 males, 535 females) aged 3-18 years were examined using direct smear and brine concentration methods between June 2005 and November 2006. The pooled prevalence of infection was 66.2%. Ascaris lumbricoides showed the highest prevalence (53.4%) (P < 0.001) followed by hookworms (17.8%), Trichuris trichiura (10.4%), Taenia sp. (9.6%), Schistosoma mansoni (2.3%), Strongyloides stercoralis (0.7%), Schistosoma haematobium (0.6%), and Enterobius vermicularis (0.3%). The prevalences of A. lumbricoides, hookworms, Taenia sp., S. mansoni, and S. stercoralis in the urban centre were similar (P > 0.05) to those in the rural communities. The fertile and infertile egg ratios of A. lumbricoides in the urban centre and the rural communities were 13: 1 and 3.7: 1, respectively. Each helminth had similar prevalences among both genders (P > 0.05). The prevalence of A. lumbricoides increased significantly with age (P < 0.001). The commonest double infections were Ascaris and hookworms, while the commonest triple infections were Ascaris, hookworms, and Trichuris. The study demonstrates the need for urgent intervention programmes against intestinal helminthiases and schistosomiasis in the study area.     

Characterization of aerobic bacterial and fungal microbiota on surfaces of historic Scottish monuments

Twenty samples were taken from the inner or outer surfaces of stone monuments of six historic Scottish buildings and ruins. Biofilms developing on mineral substrates were analysed by in situ scanning electron microscopy and cultivation. Various methods were used to characterize the isolates including automated ribotyping, RAPD and sequencing of the 16S rRNA gene for bacteria, and stereomicroscopy and sequencing of the Internal Transcribed Spacers (ITS) for fungi. Most samples contained microbes between 10(5) and 10(7)cfug(-1) substrate. Actinobacteria belonging to the genus Streptomyces (17 samples/5 monuments) or Arthrobacter (12/3) and Pseudomonas (9/3) were frequently detected. Most streptomycetes were in terms of their 16S rRNA gene sequence most closely related to S. microflavus (10/3) or to the undescribed species S. "vulgaris" (8/3). Indoor and outdoor biofilms exhibited significant differences in their microbiota, as shown by both microscopy and isolation studies. Pigmented coccoid Arthrobacter species were typical for the outdoor samples, whereas Pseudomonas species were common in the indoor samples. Based on the low phylogenetic relationship to a known species (type strain), potential novel pigmented bacterial species belonging to the genera Arthrobacter, Brevundimonas, Cryseobacterium, Deinococcus and Dyadobacter were detected from the outdoor samples and to Pseudomonas from the indoor samples. Hyaline fungal species of Acremonium (10/4) mainly occurred in indoor samples, whereas pigmented species of Cladosporium (8/3), Penicillium (6/3) and Phialophora (6/2) were found outdoors. Using in situ microscopy diatom algae were also detected.     

21beta-Hydroxy-oleanane-type triterpenes from Hippocratea excelsa

Stem bark of Hippocratea excelsa afforded six pentacyclic triterpenes, five oleanane and one ursane types. They were identified as 11beta,21beta-dihydroxy-olean-12-ene-3-one (2), 3alpha,11alpha,21beta-trihydroxy-olean-12-ene (3), 3alpha,21beta-dihydroxy-11alpha-methoxy-olean-12-ene (4), 3alpha,21beta-dihydroxy-olean-9(11),12-diene (5), 3alpha,21beta-dihydroxy-olean-12-ene (6) and 3alpha,21beta-dihydroxy-11alpha-methoxy-urs-12-ene, isolated as its diacetate derivative (7), as well as 3alpha,21beta-dihydroxy-olean-12-ene (1) previously isolated from the root bark. The known alpha- and beta-amyrin, oleanoic and ursolic acids, trans-polyisoprene, and the ubiquitous beta-sitosterol were also isolated. Structures were elucidated on the basis of spectroscopic analyses, including homo- and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC and HMBC) and comparison with literature data. The antigiardial activity of compounds 2-5 was not significant.     

Up-regulation of ras-GAP genes is reversed by a MEK inhibitor and doxorubicin in v-Ki-ras-transformed NIH/3T3 fibroblasts

Ras-GTPase-activating proteins (Ras-GAPs) have been implicated both as suppressors of Ras and as effectors in regulating cellular activities. To study whether Ras-GAPs have roles in tumor cell survival or not, mRNA levels of ras-related genes were measured in v-Ki-ras-transformed (DT) and the parental NIH/3T3 cells, using real-time PCR. mRNA levels of p120-Gap, Gap1(m), and PIK3CA were increased in DT cells compared with NIH/3T3 cells. p120-Gap and PIK3CA genes were induced by addition of serum or epidermal growth factor to serum-starved DT cells. Three anti-cancer drugs, an ERK kinase (MEK) inhibitor PD98059, a topoisomerase II poison doxorubicin (adriamycin), and a histone deacetylase inhibitor trichostatin A, selectively blocked the overexpression of p120-Gap and Gap1(m) genes in DT cells. These drugs also caused reversion of DT cells to the adherent shape associated with growth arrest. Our results suggest that p120-Gap and Gap1(m) genes provide important biomarkers for cancer therapies.     

Biotransformation of betulinic and betulonic acids by fungi

Betulinic acid (1), a triterpenoid found in many plant species, has attracted attention due to its important pharmacological properties, such as anti-cancer and anti-HIV activities. The closely related, betulonic acid (2) also has similar properties. In order to obtain derivatives potentially useful for detailed pharmacological studies, both compounds were submitted to incubations with selected microorganisms. In this work, both were individually metabolized by the fungi Arthrobotrys, Chaetophoma and Dematium, isolated from the bark of Platanus orientalis as well as with Colletotrichum, obtained from corn leaves; such fungal transformations are quite rare in the scientific literature. Biotransformations with Arthrobotrys converted betulonic acid (2) into 3-oxo-7beta-hydroxylup-20(29)-en-28-oic acid (3), 3-oxo-7beta,15alpha-dihydroxylup-20(29)-en-28-oic acid (4) and 3-oxo-7beta,30-dihydroxylup-20(29)-en-28-oic acid (5); Colletotrichum converted betulinic acid (1) into 3-oxo-15alpha-hydroxylup-20(29)-en-28-oic (6) acid whereas betulonic acid (2) was converted into the same product and 3-oxo-7beta,15alpha-dihydroxylup-20(29)-en-28-oic acid (4); Chaetophoma converted betulonic acid (2) into 3-oxo-25-hydroxylup-20(29)-en-28-oic acid (7) and both Chaetophoma and Dematium converted betulinic acid (1) into betulonic acid (2). Those fungi, therefore, are useful for mild, selective oxidations of lupane substrates at positions C-3, C-7, C-15, C-25 and C-30.     

Polyketides from Penicillium sp. JP-1, an endophytic fungus associated with the mangrove plant Aegiceras corniculatum

Four polyketides, leptosphaerone C (1), penicillenone (2), arugosin I (3) and 9-demethyl FR-901235 (4), as well as five known compounds, bacillosporin A (5), bacillosporin C (6), sequoiamonascin D (7), sequoiatone A (8), and sequoiatone B (9) were isolated from the Penicillium sp. JP-1, an endophytic fungus isolated from Aegiceras corniculatum. Their structures were determined by spectroscopic methods, mainly by 2D NMR spectroscopic analyses. Compound 1 showed cytotoxicity against A-549 cells with an IC50 value of 1.45 microM, while compound 2 showed cytotoxicity against P388 cells with an IC50 value of 1.38 microM.     

Potential anti-allergic ent-kaurene diterpenes from the bark of Suregada multiflora

Two ent-kaurene diterpenes, ent-16-kaurene-3beta,15beta,18-triol (1) and ent-3-oxo-16-kaurene-15beta,18-diol (2), were isolated from a dichloromethane extract of the bark of Suregada multiflora along with five known diterpenes:ent-16-kaurene-3beta,15beta-diol (3), abbeokutone (4), helioscopinolide A (5), helioscopinolide C (6) and helioscopinolide I (7). Their structures were elucidated on the basis of spectroscopic analysis. Compounds 1-7 possessed appreciable anti-allergic activities in RBL-2H3 cells model with IC50 values ranging from 22.5 to 42.2 microM.     

Fatty acid composition of individual polar lipid classes from marine macrophytes

Major glycolipids [monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)) and phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG)] as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from Anfeltia tobuchiensis (Rhodophyta), Laminaria japonica, Sargassum pallidum (Phaeophyta), Ulva fenestrata (Chlorophyta) and Zostera marina (Embriophyta), harvested in the Sea of Japan. GC analysis of their fatty acid (FA) composition revealed that the n-6 polyunsaturated FAs (PUFAs) shared the most part of the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG. In algae, it was related to the prevalence of 20:4n-6 over 20:5n-3 in non-photosynthetic lipids. Percentage of n-6 PUFAs as well as the sum of n-3 and n-6 PUFAs decreased in the following sequence: PC-->PE-->PG. The saturation increased in the lines of MGDG-->DGDG-->SQDG and PC-->PE-->PG. PG was close to SQDG by the level of saturation. Distribution of C(18) and C(20) PUFAs in polar lipids depended on taxonomic position of macrophytes. Balance between C(18) and C(20) PUFAs was preferably shifted to the side of C(20) PUFAs in PC and PE that was observed in contrast to glycolipids and PG from L. japonica containing both series of FAs. The set of major FAs of polar lipid classes can essentially differ from each other and from total lipids of macrophytes. For example, MGDG was found to accumulate characteristic fatty acids 16:4n-3, 16:3n-3, 18:3n-6 and 18:4n-3, 20:3n-6 in U. fenestrata, Z. marina, L. japonica and S. pallidum, respectively.     

Antimicrobial constituents of Scrophularia deserti

A study of the chemistry and antibacterial activity of Scrophularia deserti led to the isolation of eight compounds, including the metabolite 3(zeta)-hydroxy-octadeca-4(E),6(Z)-dienoic acid (1). The known compounds ajugoside (2), scropolioside B (3), 6-O-alpha-L-rhamnopyranosylcatalpol (4), buddlejoside A(8) (5), scrospioside A (6), laterioside (7) and 3R-1-octan-3-yl-3-O-beta-D-glucopyranoside (8) were also isolated. Compounds 1-3 exhibited moderate antibacterial activity against strains of multidrug and methicillin-resistant Staphylococcus aureus (MRSA) and a panel of rapidly growing mycobacteria with minimum inhibitory concentration (MIC) values ranging from 32 to 128 microg/ml.     

5-Hydroxy-seco-carotenoids from Pittosporum tobira

Three 5-hydroxy-seco-carotenoids were isolated from seeds of Pittosporum tobira. These structures were determined to be (3S,3'S,5'?)-3,3'-di(tetradecanoyloxy)-5'-hydroxy-5,6,5',6'-diseco-beta,beta-carotene-5,6,6'-trione (1), (3S,5?,3'S,5'R,6'S,9'Z)-3-tetradecanoyloxy-5',6'-epoxy-5,3'-dihydroxy-5',6'-dihydro-5,6-seco-beta,beta-caroten-6-one (2), and (3S,5?,3'S,5'R,6'R)-3-tetradecanoyloxy-5,3',5'-trihydroxy-6',7'-didehydro-5',6'-dihydro-5,6-seco-beta,beta-caroten-6-one (3) based on analysis of UV-vis, IR, FAB MS, and NMR spectroscopic data.     

Antioxidant constituents of Nymphaea caerulea flowers

As part of an ongoing search for antioxidants from medicinal plants, 20 constituents were isolated from the Nymphaea caerulea flowers, including two 2S,3S,4S-trihydroxypentanoic acid (1), and myricetin 3-O-(3'-O-acetyl)-alpha-L-rhamnoside (2), along with the known myricetin 3-O-alpha-L-rhamnoside (3), myricetin 3-O-beta-D-glucoside (4), quercetin 3-O-(3'-O-acetyl)-alpha-L-rhamnoside (5), quercetin 3-O-alpha-L-rhamnoside (6), quercetin 3-O-beta-D-glucoside (7), kaempferol 3-O-(3'-O-acetyl)-alpha-L-rhamnoside (8), kaempferol 3-O-beta-D-glucoside (9), naringenin (10), (S)-naringenin 5-O-beta-D-glucoside (11), isosalipurposide (12), beta-sitosterol (13), beta-sitosterol palmitate (14), 24-methylenecholesterol palmitate (15), 4alpha-methyl-5alpha-ergosta-7,24(28)-diene-3beta,4beta-diol (16), ethyl gallate (17), gallic acid (18), p-coumaric acid (19), and 4-methoxybenzoic acid (20). The structures were determined by spectroscopic means. Compounds were tested for antioxidant activity and nine compounds 2-7, 11, 12 and 18 were considered active with IC(50) of 1.16, 4.1, 0.75, 1.7, 1.0, 0.34, 11.0, 1.7 and 0.95 microg/ml, respectively, while 1 was marginally active (IC(50)>31.25 microg/ml). The most promising activity was found in the EtOAc fraction (IC(50) 0.2 microg/ml). This can be attributed to the synergistic effect of the compounds present in it.     

Bioactive constituents of Artemisia monosperma

During a study on the chemistry and biological activity of Kuwaiti plants, new metabolites including 4,6-dihydroxy-3-[3'-methyl-2'-butenyl]-5-[4'-hydroxy-3'-methyl-2'-butenyl]-cinnamic acid (1), the 3R,8R stereoisomer of the C17 polyacetylene dehydrofalcarindiol (2) and a C10 polyacetylene glucoside (3) were characterised by spectroscopic means. Additionally, the previously characterised natural products 1,3R,8R-trihydroxydec-9-en-4,6-yne (4), spathulenol (5) and eriodyctiol-7-methyl ether (6) were also isolated. Compounds 2, 3, and 4 were evaluated for their ability to inhibit the enzyme 12-lipoxygenase and 3 and 4 showed moderate activity at 30 microg/ml. Compound 2 was evaluated against a panel of colorectal and breast cancer cell lines and IC50 values ranged from 5.8 to 37.6 microg/ml. Against a panel of fast-growing mycobacteria and a standard ATCC strain of Staphylococcus aureus, compound 6 exhibited minimum inhibitory concentrations in the range of 64-128 microg/ml.     

Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR

Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.     

Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples

The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.