The effect of extracts from ginger rhizome on inflammatory mediator production.

Compounds from rhizomes of Zingiber officinale, commonly called ginger, have been purported to have anti-inflammatory actions. We have used an in vitro test system to test the anti-inflammatory activity of compounds isolated from ginger rhizome. U937 cells were differentiated and exposed to lipopolysaccharide (LPS) from Escherichia coli (1 microg/ml) in the presence or absence of organic extracts or standard compounds found in ginger (6-, 8-, 10-gingerol or 6-shogaol) for 24 h. Supernatants were collected and analyzed for the production of prostaglandin E(2) (PGE(2)) and tumor necrosis factor alpha (TNF-alpha) by standard ELISA assays. Predominant compounds in the organic extracts were identified as 6-, 8- 10-gingerols and 6-, 8-, 10-shogaols. Organic extracts or standards containing gingerols were not cytotoxic, while extracts or standards containing predominantly shogaols were cytotoxic at concentrations above 20 microg/ml. Crude organic extracts of ginger were capable of inhibiting LPS induced PGE(2) (IC(50)<0.1 microg/ml) production. However, extracts were not nearly as effective at inhibiting TNF-alpha (IC(50)>30 microg/ml). Thirty three fractions and subfractions, prepared by column chromatography, were analyzed for bioactivity. Extracts containing either predominantly gingerols or shogaols (identified by HPLC) were both highly active at inhibiting LPS-induced PGE(2) production (IC(50)<0.1 microg/ml), while extracts that contained unknown compounds were less effective (IC(50)<3.2 microg/ml). Extracts or standards containing predominantly gingerols were capable of inhibiting LPS-induced COX-2 expression while shogaol containing extracts had no effect on COX-2 expression. These data demonstrate that compounds found in ginger are capable of inhibiting PGE(2) production and that the compounds may act at several sites.

The effect of turmeric extracts on inflammatory mediator production.

Major compounds of several commonly used botanicals, including turmeric, have been purported to have anti-inflammatory actions. In order to test the anti-inflammatory activity of compounds isolated from rhizomes of Curcuma longa L. (Zingiberaceae), we have established an in vitro test system. HL-60 cells were differentiated and exposed to lipopolysaccharide (LPS) from Escherichia coli (1 microg/ml) in the presence or absence of botanical compounds for 24 h. Supernatants were collected and analyzed for the production of tumor necrosis factor alpha (TNF-alpha) and prostaglandin E2 (PGE2) using standard ELISA assays. Water-soluble extracts were not cytotoxic and did not exhibit biological activity. Organic extracts of turmeric were cytotoxic only at concentrations above 50 microg/ml. Crude organic extracts of turmeric were capable of inhibiting LPS-induced TNF-alpha (IC50 value = 15.2 microg/ml) and PGE2 (IC50 value = 0.92 microg/ml) production. Purified curcumin was more active than either demethoxy- or bisdemethoxycurcumin. Fractions and subfractions of turmeric extracts collected via preparative HPLC had differing biological activity, ranging from no activity to IC50 values of < 1 microg/ml. For some fractions, subfractionation resulted in a loss of activity, indicating interaction of the compounds within the fraction to produce an anti-inflammatory effect. A combination of several of the fractions that contain the turmeric oils was more effective than the curcuminoids at inhibiting PGE2. While curcumin inhibited COX-2 expression, turmeric oils had no effect on levels of COX-2 mRNA.

Inhibition of Mycobacterium tuberculosis growth by saringosterol from Lessonia nigrescens.

Assay-guided fractionation of an antitubercular extract obtained from Lessonia nigrescens yielded the phytosterol saringosterol as its active component. No appreciable toxicity against Vero cells was observed for this compound. Saringosterol was also synthesized by oxidation of fucosterol. The MIC values for antitubercular activity of saringosterol and its 24S and 24R epimers were determined as 0.25, 1, and 0.125 microg/mL.

tetra-O-methylnordihydroguaiaretic acid inhibits melanoma in vivo.

tetra-O-methylnordihydroguaiaretic acid is a derivative of a naturally-occurring lignan, nordihydroguaiaretic acid, that has previously been shown to inhibit various cancer types in vitro and in vivo. Additionally, nordihydroguaiaretic acid has been shown to have nephrotoxic effects in the rat. Here we show that tetra-O-methylnordihydroguaiaretic acid inhibits the growth of a number of tumor cell lines in vitro by inducing apoptosis in a non-schedule-dependent manner. Further, this compound inhibits the synthesis of DNA by melanoma cells and causes cell cycle arrest in G0/G1 and G2/M phases of the cell cycle. tetra-O-Methylnordihydroguaiaretic acid also inhibits the growth of both murine and human melanomas and human colon cancer in vivo without apparent hepatic or renal toxicity.

Propolis from Chilean matorral hives.

Viscidone (0.5%), vanillin, 3',4'-(methylendioxy)acetophenone, 3-ethoxy-4-methoxybenzaldehyde, cinnamic acid, 3-methoxy-4-hydroxymethyl ester were isolated from propolis of hives from Cuncumen. This is the first report on propolis composition of an arid and a Mediterranean type climate area.

Phenolic compounds of propolis from Central Chilean matorral.

Pinocembrin, acacetin, galanguin, izalpin, kaempferide, prenyletin and diarytheptane were isolated from propolis from Central Chile.

Pharmacokinetic analysis by high-performance liquid chromatography of intravenous nordihydroguaiaretic acid in the mouse.

Nordihydroguaiaretic acid (NDGA) has been shown to inhibit both 5-lipoxygenase and ornithine decarboxylase and is active against several cancer cell lines and at least one mouse tumor model. Despite these findings, there have been no reports on the pharmacokinetics of NDGA. A reverse-phase high-performance liquid chromatography (HPLC) method was developed to detect NDGA in mouse plasma. The limit of detection of this method was 0.5 microg/ml. Administration of NDGA (50 mg/kg, i.v.) to mice resulted in a peak plasma concentration of 14.7 microg/ml. The terminal half-life of NDGA was 135.0 min with a clearance of 201.9 ml/min x kg.

Antitubercular activity of triterpenoids from Lippia turbinata.

Assay-guided fractionation of the antitubercular MeOH-CH(2)Cl(2) extract obtained from Lippia turbinata led to the isolation of four novel triterpenoids-3beta,25-epoxy-3alpha,21alpha-dihydroxy-22beta-(3-methylbut-2-en-1-oyloxy)olean-12-ene-28-oic acid (1); 3beta,25-epoxy-3alpha,21alpha-dihydroxy-22beta-angeloyloxyolean-12-ene-28-oic acid (2); 3beta,25-epoxy-3alpha,21alpha-dihydroxy-22beta-tigloyloxyolean-12-ene-28-oic acid (3); and 3beta,25-epoxy-3alpha-hydroxy-22beta-(2-methylbutan-1-oyloxy)olean-12-ene-28-oic acid (4)-together with the known triterpenoids lantanilic acid (5), camaric acid (6), lantanolic acid (7), and rehmannic acid (8). The MIC values of 1-8 for growth inhibition of Mycobacterium tuberculosis were determined in the radiorespirometric BACTEC system.

Antioxidant chemistry of green tea catechins. New oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin from their reactions with peroxyl radicals.

The green tea catechins (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) react with peroxyl radicals generated by thermolysis of the azo initiator 2,2'-azobis(2, 4-dimethylvaleronitrile) (AMVN) to produce several oxidation products. Structure elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. We isolated and identified a previously unreported reaction product of EGCG and three reaction products of EGC. In the EGCG product, the B-ring was transformed into a ring-opened unsaturated dicarboxylic acid moiety. The EGC products include a seven-membered B-ring anhydride and a symmetrical EGC dimer, both analogues of previously described EGCG oxidation products. The third EGC product was an unsymmetrical dimer. In all identified products, changes occurred solely in the B-ring of EGCG or EGC. This confirmed our previous observation that the principal site of antioxidant reactions in EGCG and EGC is the trihydroxyphenyl B-ring, regardless of the presence of a 3-galloyl moiety. A stoichiometric factor n of 4.16 +/- 0.51 was measured for EGCG, whereas factors of 2.20 +/- 0.26 was found for EGC and 2.33 +/- 0.18 measured for methyl gallate. These values represent the net peroxyl radical trapping per catechin molecule by several competing reactions. EGCG and EGC oxidation involves addition of oxygen, which is not derived from water, but most likely from atmospheric oxygen via peroxyl radicals. Characteristic oxidation products may be useful markers for antioxidant actions in living systems.

Tandem mass spectrometry studies of green tea catechins. Identification of three minor components in the polyphenolic extract of green tea.

Liquid chromatography/electrospray ionization mass and tandem mass spectrometry (MS/MS) techniques were used to identify two minor components and one new compound in the polyphenolic extract of green tea (Camellia sinensis). Identification and structure assignments were based on previously reported sub-structural features in the MS/MS product, precursor and neutral loss scans of reference samples. The structures of two minor components, related to the known green tea components epicatechin gallate (ECG, 5) and epigallocatechin gallate (EGCG, 6), are formed by methylation at the 3"-O-position of the gallic acid moiety. The new compound contained a gallic acid ester group, but had only one phenolic group in either the A- or B-ring, relative to the structure of 5. High-resolution mass measurements supported the empirical formula assigned to the new compounds. An important fragmentation for defining the position of methylation of the ester function involves ionization of the phenolic group at the 4"-position of the gallic acid, followed by elimination of the ester function as a neutral with concomitant formation of the m/z 169 ion. If the 4"-position is blocked by methylation, the formation of m/z 169 incorporating the gallic acid group would be blocked. Thus, the presence of an ion representing the ester group indicates a free 4"-phenol and the absence of this ion would signify the 4"-position as a site of methylation. The operation of this mechanism should be general and useful in assigning the site of methylation of any polyphenolic ester group in natural products. A similar conclusion can be drawn concerning alkylation or esterification of the 4'-position of the catechins, i. e. blocking the 4'-phenol would prevent formation of the m/z 125 ion common to all of the catechin compounds thus far examined. Therefore, mass spectral data are of considerable importance in deducing the sites of alkylation or esterification in the structures of the components of green tea.

Reactions of Genistein with alkylperoxyl radicals.

Antioxidant actions of the soy isoflavone genistein are believed to contribute to its overall chemopreventive activity. However, the mechanisms of its antioxidant reactions remain unknown. The objective of this study was to characterize the reaction products of genistein (5,7,4'-trihydroxyisoflavone) with peroxyl radicals generated by thermolysis of 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Genistein oxidations with AMVN-derived peroxyl radicals yielded orobol (5,7,3',4'-tetrahydroxyisoflavone), a hydroxylated derivative of genistein, and several stable adducts of 4'-oxogenistein with AMVN-derived radicals. Some of these adducts include novel structures resulting from secondary oxidations of the AMVN-derived moiety. For all the observed oxidation products, the modifications occurred on the B-ring of the molecule. Genistein oxidation product structures provide potentially useful markers of genistein antioxidant chemistry.

New phenylethanoids from Buddleja cordata subsp. cordata.

Bioassay-guided fractionation of a crude extract of the stem bark of Buddleja cordata subsp. cordata with significant antimycobacterial activity led to the isolation of a mixture composed by ten new long-chain esters of 2[4'-hydroxyphenyl]-ethanol (1-10), along with the lichen metabolites methyl beta-orcinolcarboxylate (11) and beta-orcinolcarboxylate (12). Extensive HPLC allowed the separation of the major components of the mixture, which were characterized by spectral means as 2[4'-hydroxyphenyl]-ethyl stearate (3), 2[4'-hydroxyphenyl]-ethyl behenate (6), and 2[4'-hydroxyphenyl]-ethyl lignocerate (8). The minor esters were identified as 2[4'-hydroxyphenyl]-ethyl palmitate (1), 2[4'-hydroxyphenyl]-ethyl heptadecanoate (2), 2[4'-hydroxyphenyl]-ethyl nonadecanoate (4), 2[4'-hydroxyphenyl]-ethyl arachidate (5), 2[4'-hydroxyphenyl]-ethyl tricosanoate (7), 2[4'-hydroxyphenyl]-ethyl pentacosanoate (9), and 2[4'-hydroxyphenyl]-ethyl hexacosanoate (10) by GC-MS analysis of the methyl esters derivatives of the fatty acids obtained by alkaline hydrolysis of the mixture. Compound 8 exhibited moderate antibacterial activity against Mycobacterium tuberculosis (MIC = 64 micrograms/ml).

Oleanane triterpenes from Junellia tridens.

Three novel triterpenes, 3,4-seco-olean-12-ene-3,28-dioic acid (4), 3alpha-hydroxyolean-11-en-28,13beta-olide (5), and 3alpha-hydroxyoleane-11:13(18)-dien-28-oic acid (6), were isolated from the aerial parts of the Argentinean shrub, Junellia tridens. Another five compounds-oleanolic (1), oleanonic (2), and epioleanolic acids (3), all biosynthetically related to the three new oleananes, and epibetulinic acid (7) and sitosterol (8)-were also isolated. Structures were elucidated primarily by 1D and 2D NMR and mass spectrometry, and all protons and carbons of the three novel compounds were fully assigned by NMR. We report the minimum inhibitory concentrations of these compounds against Mycobacterium tuberculosis and conclude that they are responsible for antitubercular activity originally observed in the crude plant extract. LC-MS data is provided on the occurrence of triterpenes 1-6 in six other species of Junellia.

Two novel flavanones from Greigia sphacelata.

As part of our continuing phytochemical investigations of plants from arid environments in Chile, the aerial parts of Greigia sphacelata were examined. Two novel flavanones, 5,7,3'-trihydroxy-6, 4',5'-trimethoxyflavanone (1) and 5,3'-dihydroxy-6,7,4', 5'-tetramethoxyflavanone (2), as well as eight known compounds-1, 3-O-di-trans-p-coumaroylglycerol (3), 1-O-trans-p-coumaroylglycerol (4), a mixture of 1-(omega-feruloyldocosanoyl)glycerol (5) and 1-(omega-feruloyltetracosanoyl)glycerol (6), trans-ferulic acid 22-hydroxydocosanoic acid ester (7), arborinone (8), arborinol (9), and isoarborinol (10)-were isolated.

Antibacterial diterpenoid acids from Azorella compacta.

The two novel diterpenoid acids mulin-12,14-dien-11-on-20-oic acid (1) and mulin-12-ene-11,14-dion-20-oic acid (2) have been isolated from Azorella compacta. Their structures have been elucidated by 1D and 2D NMR methods. In contrast to the closely related known mulinolic acid (3) and its dehydration product (4) these new natural products have been shown to exhibit antimicrobial activity.

Phytochemical, morphological, and biological investigations of propolis from Central Chile.

Propolis from central Chile was investigated for its plant origin by microscopical analysis of pollen grains and leaf fragments found in the sample. The pollen grains that appear with significant higher frequency in the sample corresponded to four native and two introduced species, whereas leaf fragments corresponded to four native species. Seventeen phenolic compounds that belong to the phenylpropane, benzaldehyde, dihydrobenzofuran, or benzopyran classes, were isolated from an organic extract that was found to have a moderate growth inhibitory activity against Mycobacterium avium, M. tuberculosis, and two strains of Staphylococcus aureus. The components responsible for activity were determined.

Antioxidant chemistry of green tea catechins. Identification of products of the reaction of (-)-epigallocatechin gallate with peroxyl radicals.

(-)-Epigallocatechin gallate (EGCG), isolated from green tea, displays antioxidant properties and is thought to act as an antioxidant in biological systems. However, the specific mechanisms of its antioxidant actions remain unclear. In this study, we have isolated and identified for the first time two reaction products of EGCG derived from its reaction with peroxyl radicals generated by thermolysis of the initiator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). The products include a seven-membered B-ring anhydride and a novel dimer. The identification of these products provides the first unambiguous proof that the principal site of antioxidant reactions on the EGCG molecule is the trihydroxyphenyl B ring, rather than the 3-galloyl moiety. In contrast to phenoxyl radicals from simple phenolic antioxidants, an initially formed EGCG phenoxyl radical apparently does not form stable addition products with AMVN-derived peroxyl radicals. Characteristic reaction products may provide novel markers for EGCG antioxidant reactions in living systems.

Inhibition of ultraviolet B--induced c-fos gene expression and p38 mitogen-activated protein kinase activation by (-)-epigallocatechin gallate in a human keratinocyte cell line.

(-)-Epigallocatechin gallate (EGCG), the major polyphenol isolated from green tea, is an active chemoprevention agent against cancer. However, the molecular mechanisms that underlie the inhibitory effects of EGCG are not well understood. In this study, we tested the effects of EGCG on ultraviolet (UV) B radiation-induced c-fos gene expression in a human keratinocyte cell line, HaCaT. EGCG inhibited UVB-induced steady-state message and transcriptional activation of the c-fos gene in a dose-dependent manner. Western analyses further indicated that EGCG had an inhibitory effect on UVB-induced accumulation of the c-fos protein within the same dose range. To further examine the mechanism by which EGCG inhibits UVB-induced c-fos expression, we tested the effect of EGCG on upstream activators of the c-fos gene. We found that EGCG significantly inhibited activation of p38 mitogen-activated protein kinase but not c-jun NH2-terminal kinase or extracellular signal-regulated protein kinase activation. Our previous studies have indicated that UVB-induced c-fos expression may play a key role in UVB-induced activation of the activator protein-1 transcription factor and EGCG-inhibited, UVB-induced activation of AP-1 in HaCaT cells. Because AP-1 is important for tumor promotion and c-fos is a major component of AP-1, the inhibitory effects of EGCG on c-fos expression may further explain the anti-tumor-promoting effects of EGCG.

Diterpenoids from Baccharis pingraea.

From the aerial parts of Baccharis pingraea the known furolabdane, angeloyl-gutierrezianolic acid (1); two novel diterpenoids, furolabda-6,8-dien-17-oic acid (2) and furolabd-7-en-17-oic acid (3); and the known linear diterpenoid, (10E)-centipedic acid (4), were isolated. LC/MS suggested the presence of gutierrezianolic acid (5). The structures of the new compounds were elucidated by 1D and 2D NMR methods.

Antitubercular activity of pentacyclic triterpenoids from plants of Argentina and Chile.

Screening of plants from South America for antitubercular activity and subsequent assay-guided fractionation resulted in the isolation and characterization of several pentacyclic triterpenoids. The MIC values of 22 triterpenoids were determined using the radiorespiratory BACTEC assay and range from 8 microM to above 128 microM. The structure-activity relationships are discussed.