(+)-Altholactone exhibits broad spectrum immune modulating activity by inhibiting the activation of pro-inflammatory cytokines in RAW 264.7 cell lines.

An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflammatory activity in a nuclear factor-kappa B (NF-κB) luciferase and MTT assay using transfected macrophage immune (Raw264.7) cells. A high-throughput LC/MS-ELSD based library approach of the extract in combination with the NF-κB and MTT assays revealed the styryl lactone (+)-altholactone (2) was responsible for the activity. Compound 2, its acetylated derivate (+)-3-O-acetylaltholactone (3), and the major compound of this class, (+)-goniothalmin (1), were further evaluated to determine their anti-inflammatory potential in the NF-κB assay. Concentration-response studies of 1-3 indicated that only 2 possessed NF-κB based anti-inflammatory activity. Compound 2 reduced the LPS-induced NO production, phosphorylation of IκBα, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) using Western blot analysis. Further studies using qPCR indicated 2 reduced the expression of eight pro-inflammatory cytokines/enzymes (0.8-5.0µM) which included: COX-2, iNOS, IP-10, IL-1ß, MCP-1, GCS-F, IL-6 and IFN-ß. These results indicated that 2 displays broad spectrum immune modulating activity by functioning as an anti-inflammatory agent against LPS-induced NF-κB signaling. Conversely the selective cytotoxicity and in vivo anti-tumor and anti-inflammatory activity previously reported for 1 do not appear to arise from a mechanism that is linked to the NF-κB immune mediated pathway.

Natural product libraries to accelerate the high-throughput discovery of therapeutic leads.

A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.

Cytotoxic and NF-kappaB inhibitory constituents of Artocarpus rigida.

Four new prenylated flavonoids (1-4), a new stilbenoid (5), and nine known compounds were isolated from the twigs of Artocarpus rigida, collected in Indonesia. The structures of the new compounds were determined by analysis of their spectroscopic data, and the absolute configuration at C-12 of 1 and 2 and the known compounds artonin O (6), artobiloxanthone (7), and cycloartobiloxanthone (8) was determined from their CD and NMR spectroscopic data. Several of the compounds obtained were cytotoxic toward HT-29 human colon cancer cells, with the most potent being compound 2 and the known compounds 6 and 8. Of the substances obtained, compounds 1 and 7 were the most active in the NF-kappaB p50 and p65 assay, respectively.

Isolation and characterization of minor analogues of silvestrol and other constituents from a large-scale re-collection of Aglaia foveolata.

Two new minor silvestrol analogues [2'''-episilvestrol (1) and 2''',5'''-diepisilvestrol (2)], together with a new 21-norbaccharane-type triterpene (3), two new 3,4-secodammarane triterpenes (4 and 5), and a new eudesmane sesquiterpene (6), as well as nine known compounds, were isolated from a large-scale re-collection of the CHCl(3)-soluble extract of the stem bark of Aglaia foveolata obtained in Kalimantan, Indonesia. The structures of the new compounds were established by interpretation of their spectroscopic data. All of the isolates were tested for cytotoxicity against HT-29 cells. The new silvestrol analogues, 1 and 2, were considerably less active as cytotoxic agents than silvestrol (7) and episilvestrol (5'''-episilvestrol) (8) against this cell line, showing the importance of the configuration at C-2''' in mediating such activity within this compound class. Several of the compounds isolated were also evaluated in a NF-κB (p65) inhibition assay.

Proteasome-inhibitory and cytotoxic constituents of Garcinia lateriflora: absolute configuration of caged xanthones.

A new biflavonoid (1), a new xanthone enantiomer (2), five new caged xanthones (3-7), and several known compounds were isolated from the stem bark of Garcinia lateriflora, collected in Indonesia. The structures of the new compounds were determined by analysis of spectroscopic data, and the absolute configuration of the caged xanthones was shown for the first time at carbons 5, 7, 8, 8a, 10a, and 27, by analysis of COSY and NOESY NMR and ECD spectra. The biflavonoids exhibited proteasome inhibitory activity, and the known compound, morelloflavone (8) was found to have the greatest potency (IC(50) = 1.3 muM). The caged xanthones were cytotoxic towards HT-29 cells, with the known compound, morellic acid (10) being the most active (ED(50) = 0.36 muM). However, when tested in an in vivo hollow fiber assay, it was inactive at the highest dose tested (20 mg/kg).

Cytotoxic xanthone constituents of the stem bark of Garcinia mangostana (mangosteen).

Bioassay-guided fractionation of a chloroform-soluble extract of Garcinia mangostana stem bark, using the HT-29 human colon cancer cell line and an enzyme-based ELISA NF-kappaB assay, led to the isolation of a new xanthone, 11-hydroxy-3-O-methyl-1-isomangostin (1). The structure of 1 was elucidated by spectroscopic data analysis. In addition, 10 other known compounds, 11-hydroxy-1-isomangostin (2), 11alpha-mangostanin (3), 3-isomangostin (4), alpha-mangostin (5), beta-mangostin (6), garcinone D (7), 9-hydroxycalabaxanthone (8), 8-deoxygartanin (9), gartanin (10), and cratoxyxanthone (11), were isolated. Compounds 4-8 exhibited cytotoxicity against the HT-29 cell line with ED50 values of 4.9, 1.7, 1.7, 2.3, and 9.1 microM, respectively. In an ELISA NF-kappaB assay, compounds 5-7, 9, and 10 inhibited p65 activation with IC50 values of 15.9, 12.1, 3.2, 11.3, and 19.0 microM, respectively, and 6 showed p50 inhibitory activity with an IC50 value of 7.5 microM. Alpha-mangostin (5) was further tested in an in vivo hollow fiber assay, using HT-29, LNCaP, and MCF-7 cells, but it was found to be inactive at the highest dose tested (20 mg/kg).

Bioactive 5,6-dihydro-alpha-pyrone derivatives from Hyptis brevipes.

Six new 5,6-dihydro-alpha-pyrone derivatives (1-6), namely, brevipolides A-F, together with seven known compounds, including a 5,6-dihydro-alpha-pyrone derivative (7), three flavonoids, a steroid glycoside, and two triterpenoids, were isolated from the entire plant of Hyptis brevipes. Compounds 1-7 were assigned with the absolute configuration 5R, 6S, 7S, and 9S, as elucidated by analysis of data obtained from their CD spectra and by Mosher ester reactions. Compounds 2, 6, and 7 exhibited ED(50) values of 6.1, 6.7, and 3.6 microM against MCF-7 cells, and compounds 1, 2, 6, and 8 (the known 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavone) gave ED(50) values of 5.8, 6.1, 7.5, and 3.6 microM against HT-29 cells, respectively. However, no significant cytotoxicity was found against Lu1 cells for any of the compounds isolated. When these compounds were subjected to evaluation in a panel of mechanism-based in vitro assays, compound 7 was found to be active in an enzyme-based ELISA NF-kappaB assay, with an ED(50) value of 15.3 microM. In a mitochondrial transmembrane potential assay, compounds 3, 7, and 8 showed ED(50) values of 8.5, 75, and 310 nM, respectively. No potent activity was found in a proteasome inhibition assay for any of the isolated compounds.

Constituents of the Leaves and Stem Bark of Aglaia foveolata.

The previously known potent cytotoxic agent silvestrol (1) (0.002% w/w yield) and five new flavagline derivatives (2-6) were isolated from the leaves of Aglaia foveolata collected in Indonesia. The new compound 5 has an unprecedented cyclic amide moiety in its cyclopenta[b]benzopyran skeleton, while compound 6 is a novel benzo[b]oxepine derivative in which the oxepine ring is cleaved. Pyramidatine (7), a biogenetic precursor of the new flavaglines 2-6, was isolated from the leaf extract investigated. Silvestrol was also isolated from the stem bark of A. foveolata (yield of 0.02% w/w) along with a new baccharane-type triterpenoid (8). The structures of the new compounds were elucidated on the basis of their NMR and mass spectrometric data. All new compounds isolated were tested against a panel of cancer cell lines, but only compound 2 was cytotoxic (IC(50) range = 1.4-1.8 muM), and is the first member of the cyclopenta[b]benzopyran class found to exhibit this type of activity. Compound 2 also showed significant NF-kappaB inhibitory activity in an Elisa assay (IC(50) = 0.37 muM).

Dioxadispiroketal Compounds and a Potential Acyclic Precursor from Amomum aculeatum.

Four new compounds having an unusual 1,7-dioxadispiro[5.1.5.2]-12-ene-11-one tricyclic ring system (1-4), their potential precursor, 5R-hydroxy-1-(4-hydroxyl-phenyl)-eicosan-3-one (5), and two known compounds, aculeatins A (6) and B (7), have been isolated from Amomum aculeatum. All compounds were characterized by spectroscopic methods and the configurations were established by 2D NOE correlations. Compounds 1-4, 6 and 7 showed cytotoxic activity against several human cancer cell lines.

Cytotoxic clerodane diterpenoids from the leaves of Premna tomentosa.

Three clerodane diterpenoids, premnones A-C (1-3), were isolated from a chloroform-soluble fraction of Premna tomentosa along with four known flavonoids and three known triterpenoids. Among these isolates, premnones A-C exhibited cytotoxic activity when evaluated against a small panel of tumor cell lines. However, premnone A was found to be inactive when evaluated in a follow-up in vivo hollow fiber assay at the highest dose tested (50mg/kg), using LNCaP, Lu1, and MCF-7 cells.

Beccaridiol, an unusual 28-nortriterpenoid from the leaves of Diplectria beccariana.

A C29-triterpene, beccaridiol (1), a dihydrochalcone natural product, 2',4'-dihydroxy-3-(4-methoxyphenyl)-propiophenone (2), as well as three known compounds, 4'-hydroxy-1',2'-dihydro-beta-ionone, 4'-O-methyldavidigenin (3), and ursolic acid, have been isolated from an EtOAc-soluble extract of the leaves of Diplectria beccariana. Beccaridiol (1) was characterized as an ursane-type 28-nortriterpene possessing an unusual aromatic E-ring by spectroscopic data interpretation. The relative configuration of this unusual isolate was established by analyzing the observed NOESY NMR correlations, and the absolute stereochemistry of 1 was then determined based on the circular dichroism (CD) spectrum of its 2,3-di-p-bromobenzoate (1b) derivative. All isolates were evaluated for their potential cancer chemopreventive properties utilizing a cell culture assay to determine quinone reductase induction.

Ellagic acid derivatives and cytotoxic cucurbitacins from Elaeocarpus mastersii.

Bioassay-guided investigation of the bark of Elaeocarpus mastersii using KB (human oral epidermoid carcinoma) cells as a monitor led to the isolation of two cucurbitacins, cucurbitacin D and cucurbitacin F as cytotoxic principles, together with two ellagic acid derivatives, 4'-O-methylellagic acid 3-(2",3"-di-O-acetyl)-alpha-L-rhamnoside (1) and 4,4'-O-dimethylellagic acid 3-(2",3"-di-O-acetyl)-alpha-L-rhamnoside (2). These compounds were evaluated against a panel of human tumor cell lines.

Cytotoxic triterpenes from the twigs of Celtis philippinensis.

Two triterpene esters, 3beta-trans-sinapoyloxylup-20(29)-en-28-ol (1) and 3beta-trans-feruloyloxy-16 beta-hydroxylup-20(29)-ene (2), were isolated as cytotoxic constituents from the chloroform-soluble extract of the twigs of Celtis philippinensis, along with five known triterpenes, 3beta-O-(E)-feruloylbetulin (3), 3beta-O-(E)-coumaroylbetulin (4), betulin (5), 20-epibryonolic acid (6), and ursolic acid (7). The structures of 1 and 2 were assigned from their 1D and 2D NMR spectroscopic data. All isolates were evaluated for cytotoxicity against several human cancer cell lines.

Constituents of the stems of Macrococculus pomiferus and their inhibitory activities against cyclooxygenases-1 and -2.

As part of our program directed towards the discovery of new cancer chemopreventive agents from plants, the EtOAc-soluble extract of the stems of M. pomiferus was found to inhibit the enzyme cyclooxygenase-2 (COX-2). Bioassay-directed fractionation of this extract led to the isolation of two dibenzylbutyrolactone lignans, (8R,8'R)-3'-O-demethyl-5-hydroxymatairesinol (1) and (8R,8'R)-3'-O-demethyl-5-methoxymatairesinol (2), as well as seven known compounds, (-)-5'-methoxyyatein (3), blumenol A, (-)-deoxypodophyllotoxin (anthricin), (-)-deoxypodorhizone, 2,6-dimethoxyhydroquinone, 4-hydroxybenzaldehyde, and beta-sitosterol glucoside. The structures of compounds 1 and 2 were determined using spectroscopic data (1D and 2D NMR, and HREIMS), and the 8R and 8'R absolute stereochemistry was established for both 1 and 2 on the basis of their CD spectra. All isolates obtained in the present study were evaluated for their inhibitory effects with both COX-1 and -2. Of these, only 5'-methoxyyatein (3) showed weak activity against COX-2, while all other compounds isolated were inactive. The COX-2 inhibitory activity of the EtOAc extract was also traced to the presence of several common fatty acids by LC-MS.

Prenylated flavonoids of the leaves of Macaranga conifera with inhibitory activity against cyclooxygenase-2.

Two prenylated flavonoid derivatives, 5-hydroxy-4'-methoxy-2",2"-dimethylpyrano-(7,8:6",5")flavanone (1) and 5,4'-dihydroxy-[2"-(1-hydroxy-1-methylethyl)dihydrofurano]-(7,8:5",4")flavanone (2), were isolated from an ethyl acetate-soluble extract of the leaves of Macaranga conifera using an in vitro activity-guided fractionation procedure based on the inhibition of cyclooxygenase-2. Also obtained were eight known compounds, 5,7-dihydroxy-4'-methoxy-8-(3-methylbut-2-enyl)flavanone (3), lonchocarpol A (4), sophoraflavanone B (5), 5,7-dihydroxy-4'-methoxy-8-(2-hydroxy-3-methylbut-3-enyl)flavanone (6), tomentosanol D (7), lupinifolinol (8), isolicoflavonol (9), and 20-epibryonolic acid (10). The structures of compounds 1 and 2 were determined using spectroscopic methods. All isolates were tested for their inhibitory effects against both cyclooxygenases-1 and -2, and selected compounds were evaluated in a mouse mammary organ culture assay.

Potential cancer chemopreventive constituents of the leaves of Macaranga triloba.

Activity-guided fractionation of the leaves of Macaranga triloba, using an in vitro bioassay based on the inhibition of cyclooxygenase-2, resulted in the isolation of a rotenoid, 4,5-dihydro-5'alpha-hydroxy-4'alpha-methoxy-6a,12a-dehydro-alpha-toxicarol (1), as well as 12 known compounds, (+)-clovan-2beta,9alpha-diol, ferulic acid, 3,7,3',4'-tetramethylquercetin, 3,7,3'-trimethylquercetin, 3,7-dimethylquercetin, abscisic acid, 1beta,6alpha-dihydroxy-4(15)-eudesmene, 3beta-hydroxy-24-ethylcholest-5-en-7-one, loliolide, scopoletin, taraxerol, and 3-epi-taraxerol. The structure of compound 1 was determined using spectroscopic methods. All isolates were evaluated for their potential to inhibit cyclooxygenases-1 and -2 by measuring PGE(2) production, and to induce quinone reductase in cultured Hepa 1c1c7 mouse hepatoma cells.

Bioactive prenylated flavonoids from the stem bark of Artocarpus kemando.

Four known prenylated flavonoids, artonins E (1) and O (2), artobiloxanthone (3), and cycloartobiloxanthone (4), were isolated from the stem bark of Artocarpus kemando by bioassay-guided fractionation using the DNA strand-scission and the KB cytotoxicity assays as monitors. Compounds 1 and 3 exhibited strong DNA strand-scission activity, and all four compounds were found to be cytotoxic.

Effect of media compositions on α-glucosidase inhibitory activity, growth and fatty acid content in mycelium extracts of Colletotrichum sp. TSC13 from Taxus Sumatrana (Miq.) de Laub.

The active α-glucosidase inhibitor compounds in the endophytic fungus Colletotrichum sp. TSC13 were found to be the unsaturated fatty acids (oleic, linoleic and linolenic acids). These compounds have potential as antidiabetic agents. The aim of the present study is to investigate the effects of various media composition on growth (mycelium dry weight) and the fatty acids content (µg mg(-1) mycelium DW) of Colletotrichum sp. TSC13 in relation to its α-glucosidase inhibitory activity. For that purpose, the experiments were set up by varying the carbon and nitrogen sources, metal ions and desaturase and fatty acid synthase inhibitors in the media. Colletotrichum sp. TSC13 grown on potato dextrose broth (PDB) was used as control. The α-glucosidase inhibitory activities were (range from 43.9 ± 2.5 to 88.6 ± 5.2%) at 10 µg mL(-1). This activity seemed to correlate with the unsaturated fatty acids content of the samples. Different sugars as carbon source experiment showed that xylose gave the highest growth (938.7 ± 141.6 mg). However, the highest fatty acids content was obtained from fructose medium which containing linoleic acid (38.8 ± 4.9 µ g mg(-1) DW). Soluble starch gave better growth (672.5 ± 62.3 mg) but very low fatty acids content (2.8 ± 0.1 µg mg(-1) DW) was obtained. Yeast extract was the best nitrogen source. Fatty acids production was better as compared to beef extract and soytone. This is the first report of various media compositions on fatty acids content in Colletotrichum sp. TSC13 in relation to its α-glucosidase inhibitory activity.

Phytochemical and Bioactivity Studies on Constituents of the Leaves of Vitex Quinata.

A phytochemical investigation of the leaves of Vitex quinata (Lour.) F.N. Williams (Verbenaceae), guided by the MCF-7 human breast cancer cell line, led to the isolation of a new δ-truxinate derivative (1) and a new phytonoic acid derivative (2), together with 12 known compounds. The structures of the new compounds were determined by spectroscopic methods as dimethyl 3,4,3',4'-tetrahydroxy-δ-truxinate (1) and methyl 10R-methoxy-12-oxo-9(13),16E-phytodienoate (2), respectively. In a cytotoxicity assay, (S)-5-hydroxy-7,4'-dimethoxyflavanone (3) was found to be the sole active principle, with ED(50) values of 1.1-6.7 µM, respectively, when tested against a panel of three human cancer cells. Methyl 3,4,5-O-tricaffeoyl quinate (4) showed activity in an enzyme-based ELISA NF-κB p65 assay, with an ED(50) value of 10.3 µM.

Bioactivity-guided isolation of cytotoxic sesquiterpenes of Rolandra fruticosa.

Cytotoxicity-guided fractionation of a methanol extract of the leaves and twigs of Rolandra fruticosa using the HT-29 human colon cancer cell line led to the isolation of seven sesquiterpene lactones, including the hitherto unknown isorolandrolide, 13-methoxyisorolandrolide (1), and bourbonenolide, 2alpha,13-diacetoxy-4alpha-hydroxy-8alpha-isobutyroyloxybourbonen-12,6alpha-olide (2), as well as five known compounds, 13-acetoxyrolandrolide (3), 8-desacyl-13-acetoxyrolandrolide-8-O-tiglate (4), 2-epi-glaucolide E (5), 2alpha,13-diacetoxy-4alpha-hydroxy-8alpha-methacryloyloxybourbonen-12,6alpha-olide (6), and 2alpha,13-diacetoxy-4alpha-hydroxy-8alpha-tigloyloxybourbonen-12,6alpha-olide (7). The structures of the two sesquiterpenes were elucidated on the basis of spectroscopic methods. All isolates were evaluated for their cytotoxicity using the HT-29 cell line, and only 13-acetoxyrolandrolide (3) was found to possess a potent inhibitory effect against this cell line. Compounds 3, 5 and 6 were also tested in a NF-kappaB (p65) inhibition assay, and 3 was assessed in an in vivo hollow fiber assay.