Biotransformation of Flavonoid Conjugates with Fatty Acids and Evaluations of Their Functionalities.

Enzymatic conjugation with fatty acids including omega-3 polyunsaturated fatty acids (ω-3 PUFAs) derived from fish oil to three citrus fruit-derived flavonoids: grapefruit extract, naringin, and neohesperidin dihydrochalcone were investigated. The conversions were achieved over 85% under the catalysis of lipase Novozyme 435 in acetone at 45°C at semi-preparative scale. The conjugates were purified via solvent partition and silica gel chromatography and achieved 90-98% in purity. The NMR analysis of the conjugates confirmed that the fatty acid carbon chain was linked onto the primary -OH group on the glucose moiety of the flavonoids. The purified flavonoid conjugates alongside their original flavonoids were analyzed for antioxidant activities via 2,2-diphenyl-1-picrylhydrazyl scavenging assay, and anti-peroxidation test via peroxide values measured during a 1-week fish oil storage trial. Vascular endothelial growth factor (VEGF) assay was conducted with 1, 10, and 100 µM of naringin and grapefruits and their conjugates, respectively, and total VEGF levels were measured at 24 and 48 h, respectively, using ELISA and dot blot analysis. The results from these functionality experiments demonstrated that flavonoid FA conjugates have at least comparable (if not higher) antioxidant activity, anti-peroxidation activity, and anti-angiogenic activity.

Antileishmanial activity of hydrolyzable tannins and their modulatory effects on nitric oxide and tumour necrosis factor-alpha release in macrophages in vitro.

A series of 27 hydrolyzable tannins and related compounds was tested for antiparasitic effects against both extracellular promastigote and intracellular amastigote Leishmania donovani organisms. In parallel, the compounds were evaluated for their immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and interferon (IFN)-like properties using several functional assays. Of the series of polyphenols tested, only gallic acid (54 microM NO) and its methyl ester (32 microM NO) induced murine macrophage-like RAW 264.7 cells to release NO in appreciable amounts (IFN-gamma/LPS 119 microM NO). The in vitro TNF-inducing potential of the polyphenols examined increased in the order of oligomeric ellagitannins (EC(50) > 25 microg/ml) < monomeric ellagitannins, gallotannins (EC(50) 8.5 to > 25 microg/ml) < C-glucosidic ellagitannins, dehydroellagitannins (EC(50) 0.6 - 2.8 microg/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, promastigotes of Leishmania donovani were assayed in the presence of these polyphenols and the results showed that none of the compounds was significantly toxic (EC(50) > 25 microg/ml) to the extracellular forms. In contrast, all polyphenols showed pronounced antileishmanial activities (EC(50) < 0.4 - 12.5 versus 7.9 microg/ml for Pentostam) against intracellular amastigotes of L. donovani residing within RAW cells. Noteworthy, most compounds exhibited low cytotoxicity against the murine host cells (EC(50) >25 microg/ml). Furthermore, some ellagitannins and the majority of dehydroellagitannins induced potent interferon-like activities as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L929 cells. This is the first report on hydrolyzable tannins as a new class of natural products with leishmanicidal activity including their potential for inducing the release of NO, TNF and IFN-like activity in macrophage-like RAW cells.

Proanthocyanidins and related compounds: antileishmanial activity and modulatory effects on nitric oxide and tumor necrosis factor-alpha-release in the murine macrophage-like cell line RAW 264.7.

A series of 17 proanthocyanidins and structurally related compounds was tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Most of the polyphenols significantly inhibited the intracellular survival of L. donovani amastigotes (EC50 0.8-10.6 nM) when compared with the antileishmanial drug Pentostam (EC50 10.6 nM), but all were inactive against the extracellular form (EC50 7.8 to >86 nM). Noteworthy is that all compounds exhibited only moderate or no cytotoxicity against the murine host cells (EC50 7.8 to >56 nM; >25 microg/ml). These polyphenols were further evaluated for immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumor necrosis factor-alpha (TNF) and interferon (IFN)-like properties using several functional assays. The results showed that all compounds induced murine RAW 264.7 cells only moderately to release NO (7-26 microM) relative to the reference stimulus IFN-gamma/LPS (119 microM). The TNF-inducing potential of the polyphenols producing 50% lysis in murine L929 cells ranged from absent to 138 U/ml at the host cell subtoxic concentration of 50 microg/ml. The highest TNF-inducing activity was associated with those flavan-3-ols with galloyl groups (98-127 U/ml). For proanthocyanidins, it appeared that an increase in the flavanyl chain length did not enhance the induction of TNF-release (32-86 U/ml and below detection limits for oligomers and polymers, respectively). With interferon-like activities, phylloflavan and a prodelphinidin polymer showed appreciable cytoprotective effects, as reflected by the inhibition of the cytopathic effect of encephalomyocarditis virus on L929 fibroblast cells (38 and 36 U/ml, respectively). All remaining compounds displayed only negligible or moderate protective effects at subtoxic concentrations up to 25 microg/ml (<5 to 12 U/ml). These results indicate that proanthocyanidins and related compounds have favorable antileishmanial activity in vitro and might be considered as beneficial immunological response modifiers provided there are no bioavailability problems.

Chemical studies of proanthocyanidins and hydrolyzable tannins.

We investigated a number of natural polyphenols representing flavan-3-ols, gallotannins, and ellagitannins with regard to their antioxidant potential. For this purpose we used pulse radiolysis to determine scavenging rate constants with hydroxyl radicals and decay rates of the respective aroxyl radicals and EPR spectroscopy to identify the radicals after in situ oxidation. Using NMR spectroscopy, we could confirm phenolic coupling reactions of epigallocatechin gallate and pentagalloyl glucose after radical-induced oxidation.

Phenolic glycosides of forage legume Onobrychis viciifolia.

A chemical examination of the extractives of the leaves of sainfoin was undertaken as part of a programme directed at understanding the factors which may contribute to its nutritive value as animal feed. Among the low molecular weight phenolic compounds characterized were seven cinnamic acid derivatives and nine flavonoid glycosides all of which were identified by NMR spectroscopy. Included among these compounds were two new natural hydroxycinnamic esters namely methyl 6-O-p-trans-coumaroyl-beta-D-glucopyranoside and methyl 6-O-p-cis-coumaroyl-beta-D-glucopyranoside and a novel flavonoid chrysoeriol-4'-O-(6''-O-acetyl)-beta-D-glucopyranoside.

Inhibitory activity of flavonoids and tannins against HIV-1 protease.

Twenty-nine flavonoids and six hydrolyzable tannins were studied for their inhibitory activity against human immunodeficiency virus (HIV)-1 protease using fluorescence and HPLC assays. Among the flavonoids, flavones, flavanones, flavonols, catechols and chalcones, the flavonols were the most active category while flavanones and catechols displayed low activity. Quercetin was the most potent inhibitor of the target enzyme with an IC50 value of 58.8 microM, while butein and luteolin showed moderate activity. Of the hydrolyzable tannins tested, three ellagitannins which contain a hexahydroxvdiphenoyl (HHDP) unit linked to the O-3 and 0-6 positions of the sugar, were found to strongly inhibit HIV-1 protease. The IC50 values of corilagin and repandusinic acid on HIV-1 protease were 20.7 and 12.5 microM, respectively.

A-Type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic P-fimbriated Escherichia coli.

Three proanthocyanidin trimers possessing A-type interflavanoid linkages, epicatechin-(4beta-->6)-epicatechin-(4beta-->8, 2beta-->O-->7)-epicatechin (4), epicatechin-(4beta-->8, 2beta-->O-->7)-epicatechin-(4beta-->8)-epicatechin (5), and epicatechin-(4beta-->8)-epicatechin-(4beta-->8, 2beta-->O-->7)-epicatechin (6), were isolated from the ripe fruits of Vaccinium macrocarpon (cranberry) and prevented adherence of P-fimbriated Escherichia coli isolates from the urinary tract to cellular surfaces containing alpha-Gal(1-->4)beta-Gal receptor sequences similar to those on uroepithelial cells. The structure of 4 was elucidated by a combination of spectroscopic methods and acid-catalyzed degradation with phloroglucinol. Also isolated were the weakly active epicatechin-(4beta-->8, 2beta-->O-->7)-epicatechin (procyanidin A2) (3) and the inactive monomer epicatechin (1) and the inactive dimer epicatechin-(4beta-->8)-epicatechin (procyanidin B2) (2).

The phenols and prodelphinidins of white clover flowers.

White clover flowers (Trifolium repens L.) contain an abundance of phenolics, namely cis- and trans-p-coumaric acid 4-O-beta-D-glucopyranoside, the 3-O-beta-D-galactopyranosides of myricetin, quercetin and kaempferol together with two new derivatives namely myricetin 3-O-(6"-acetyl)-beta-D-galactopyranoside and kaempferol 3-O-(6"-acetyl)-beta-D-galactopyranoside. Gallocatechin, epigallocatechin, gallocatechin-(4alpha-8)-epigallocatechin and their corresponding prodelphinidin polymers were also present. The 13C-NMR spectra showed that the polymers consisted of only gallocatechin and epigallocatechin monomeric units with the latter being about twice as abundant in the extenders but only slightly more than that in the terminating units. The average degree of polymerization was estimated by 13C-NMR and ES-MS, which gave a remarkably consistent result of about 5.8 flavanol units.

The structure of cranberry proanthocyanidins which inhibit adherence of uropathogenic P-fimbriated Escherichia coli in vitro.

Ethyl acetate extracts of Sephadex LH20-purified proanthocyanidins of American cranberry (Vaccinium macrocarpon Ait.) exhibited potent biological activity by inhibiting adherence of uropathogenic isolates of P-fimbriated Escherichia coli bacteria to cellular surfaces containing alpha-Gal(1-->4)beta-Gal receptor sequences similar to those on epithelial cells in the urinary tract. The chemical structures of the proanthocyanidins were determined by 13C NMR, electrospray mass spectrometry, matrix-assisted laser absorption time-of-flight mass spectrometry and by acid catalyzed degradation with phloroglucinol. The proanthocyanidin molecules consisted predominantly of epicatechin units with mainly DP of 4 and 5 containing at least one A-type linkage. The procyanidin A2 was the most common terminating unit occurring about four times as frequently as the epicatechin monomer.

Flavonoid and phenolic glycosides from Salvia officinalis.

Two novel phenolic glycosides cis-p-coumaric acid 4-O-(2'-O-beta-D-apiofuranosyl)-beta-D-glucopyranoside and trans-p-coumaric acid 4-O-(2'-O-beta-D-apiofuranosyl)-beta-D-glucopyranoside were isolated and identified from Salvia officinalis together with 4-hydroxyacetophenone 4-O-(6'-O-beta-D-apiofuranosyl)-beta-D-glucopyranoside, luteolin 7-O-beta-D-glucoside, 7- and 3'-O-beta-D-glucuronide, 6-hydroxyluteolin 7-O-beta-D-glucoside and 7-O-glucuronide, and 6,8-di-C-beta-D-glucosylapigenin (vicenin-2). The luteolin glucuronides and vicenin-2 were identified as new sage constituents.

Differential inhibition of eukaryote protein kinases by condensed tannins.

Condensed tannins, isolated from a variety of plant sources, were characterized according to the constituent flavans, being based on procyanidin and/or prodelphinidin and having a cis or trans stereochemistry at positions 2 and 3. All the tannin preparations are potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) with IC50 values (concentrations for 50% inhibition) ranging from 0.009 to 0.2 microM. The tannin preparations are very good inhibitors of rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC) (IC50 values in the range 0.3-7 microM), wheat embryo Ca(2+)-dependent protein kinase (CDPK) (IC50 values in the range 0.8-7 microM) and of calmodulin (CaM)-dependent myosin light chain kinase (MLCK) (IC50 values in the range 7-24 microM). One of the most effective preparations, that from the leaves of Ribes nigrum, has IC50 values with respect to cAK, PKC, CDPK and MLCK of 0.009, 0.6, 2.0 and 16 microM, respectively. In general, the order with respect to sensitivity to inhibition by these condensed tannins is cAK > PKC > CDPK > MLCK. The Ribes nigrum preparation is a competitive inhibitor of cAK with respect to both ATP and synthetic peptide substrate. These condensed tannin preparations are the most potent plant-derived inhibitors of cAK yet found.

Inhibition of signal-regulated protein kinases by plant-derived hydrolysable tannins.

A variety of hydrolysable tannins purified from Phyllanthus amarus are potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) with IC50 values (concentrations for 50% inhibition) in the range 0.2-1.7 microM. The three most effective compounds of this series of hydrolysable tannins have five phenolic substituents. These three compounds are also the most effective inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK), rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC) and Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK). The order of sensitivity for protein kinase inhibition by the hydrolysable tannins studied is cAK > CDPK > PKC > MLCK. Thus the IC50 values for protein kinase inhibition by the most potent compound are 0.2 microM (for cAK), 1.8 microM (for CDPK), 26 microM (for PKC) and 56 microM (for MLCK) when protein kinase affinity is measured using synthetic peptide substrates. These hydrolysable tannin inhibitors found are the most specific and potent plant-derived inhibitors of cAK yet found.

Inhibition of eukaryote signal-regulated protein kinases by plant-derived catechin-related compounds.

The cladodes of Phyllocladus trichomanoides, the bark of Pseudotsuga menziesii and the heartwood of Acacia melanoxylon contain catechin derivatives that are potent inhibitors of rat brain protein kinase C. Most of these compounds are also inhibitors of bovine heart cyclic AMP-dependent protein kinase catalytic subunit and wheat Ca(2+)-dependent protein kinase. However, these compounds are either not inhibitors or are relatively poor inhibitors of avian myosin light chain kinase. The most potent protein kinase inhibitors are the procyanidin dimer, trimer and tetramer from the bark of Pseudotsuga menziesii. These compounds are among the most potent plant-derived protein kinase inhibitors yet found.

Scombroid poisoning--recapitulation on the role of histamine.

The histamine content and the total vagal stimulating activities in various fish samples involved in an outbreak of food poisoning were compared. There was a close correlation between the two levels and there was lack of potentiation between histamine and other non-active fractions. The role of histamine in scombroid poisoning is discussed in the light of these results.

Simple and rapid paper chromatographic method for the simultaneous determination of histidine and histamine in fish samples.

A simple and rapid method for the simultaneous determination of histamine and histidine in fish samples is described. The method entails paper chromatography and is sensitive from 0.5 mug to at least 30 mug. The method is reproducible to within +/- 5% and compares favorably with a biological method for histamine determination.

The content of histamine and fish food poisoning.

The histamine content of various fish samples that were involved in food poisoning incidents was determined. There was an elevated level of histamine and this is discussed in terms of fish spoilage. The histamine level could be helpful in gauging the degree of spoilage of commercially canned fish.

Scombroid-type poisoning induced by the ingestion of smoked kahawai.

Three incidents of presumptive scombroid poisoning were reported in the Wellington area in 1973. Two of these cases were attributed to canned mackerel and the third to smoked kahawai. The marked similarity of the symptoms of the kahawai poisoning to those of the other two outbreaks of scombroid fish poisoning and the relief following anti-histamine therapy suggests that the poisoning arose from a similar source. Analysis of the extracts from the smoked kahawai revealed an abnormally high level of histamine and the presence of an unknown toxin. The unknown substance resembled saurine, the toxin that has been incriminated in scromboid poisoning, thus suggesting that scombroid intoxication may not be confined to fish belonging to the sub-order Scombroidei.