Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils.

Owing to their multiple side effects, the use of steroidal drugs is becoming more and more controversial, resulting in an increasing need for new and safer anti-inflammatory agents. In the inflammatory process, reactive oxygen species produced by phagocytic cells are considered to play an important role. We showed that apocynin (4'-hydroxy-3'-methoxy-acetophenone or acetovanillone), a non-toxic compound isolated from the medicinal plant Picrorhiza kurroa, selectively inhibits reactive oxygen species production by activated human neutrophils. Apocynin proved to be effective in the experimental treatment of several inflammatory diseases such as arthritis, colitis and atherosclerosis. These features suggest that apocynin could be a prototype of a novel series of non-steroidal anti-inflammatory drugs (NSAIDs). So far, apocynin is mainly used in vitro to block NADPH oxidase-dependent reactive oxygen species generation by neutrophils. In order to get a better insight in what chemical features play a role in the anti-inflammatory effects of apocynin, a structure-activity relationship study with apocynin analogs was performed. We show here that especially substances with an additional methoxy group at position C-5 display enhanced anti-inflammatory activity in vitro. Our approach may lead to the development of more effective anti-inflammatory agents which are safe and which lack the side effects of steroids.

Immunomodulatory and anti-inflammatory activity of Picrorhiza scrophulariiflora.

Extracts of the rhizomes of Picrorhiza scrophulariiflora Pennell (Scrophulariaceae) were investigated for their in vitro and in vivo immunomodulatory properties. Diethyl ether extracts showed potent inhibitory activity towards the classical pathway of the complement system, the respiratory burst of activated polymorphonuclear leukocytes, and mitogen-induced proliferation of T-lymphocytes. Furthermore, such extracts showed anti-inflammatory activity towards carrageenan-induced paw edema. No effects were observed in experimentally induced arthritis in mice.

Inhibition of T-lymphocyte proliferation by cucurbitacins from Picrorhiza scrophulariaeflora.

Two cucurbitacin aglycons were isolated from the dried rhizomes of Picrorhiza scrophulariaeflora and were identified as 25-acetoxy-2,3, 16,20-tetrahydroxy-9-methyl-19-norlanosta-5,23-dien-22-one (picracin, 1) and 2,3,16,20,25-pentahydroxy-9-methyl-19-norlanosta-5, 23-dien-22-one (deacetylpicracin, 2). Both compounds inhibit mitogen-induced T-lymphocyte proliferation at an IC(50) value of 1 microM.

Inhibition of human complement by beta-glycyrrhetinic acid.

Licorice, the root extract of Glycyrrhiza glabra I., is used as a medicine for various diseases. Anti-inflammatory as well as anti-allergic activities have been attributed to one of its main constituents, glycyrrhizin. These activities are mainly ascribed to the action of the aglycone, beta-glycyrrhetinic acid. beta-Glycyrrhetinic acid has a steroid-like structure and is believed to have immunomodulatory properties. To determine whether interference with complement functions may contribute to the immunomodulatory activity of beta-glycyrrhetinic acid, its effects on the classical and alternative activation pathways of human complement were investigated. We found that beta-glycyrrhetinic acid is a potent inhibitor of the classical complement pathway (IC50 = 35 microM), whereas no inhibitory activity was observed towards the alternative pathway (IC50 > 2500 microM). The anticomplementary activity of beta-glycyrrhetinic acid was dependent on its conformation, since the alpha-form was not active. It was also established that naturally occurring steroids, e.g. hydrocortisone and cortisone, did not inhibit human complement activity under similar conditions. Detailed mechanistic studies revealed that beta-glycyrrhetinic acid acts at the level of complement component C2.

Podacycline A and B, two cyclic peptides in the latex of Jatropha podagrica.

Two novel cyclic peptides were isolated from the latex of Jatropha podagrica, which we named podacycline A and B. Podacycline A is a cyclic nonapeptide with the sequence Gly1-Leu2-Leu3-Gly4-Ala5-Val6-Trp7-Ala8-Gly9+ ++-Gly1. The sequence of podacycline B, a cyclic heptapeptide, was determined to be Phe1-Ala2-Gly3-Thr4-Ile5-Phe6-Gly7-Phe1. The amino acid residues of both compounds were found to have the L-configuration.

Cyclogossine A: a novel cyclic heptapeptide isolated from the latex of Jatropha gossypifolia.

From the latex of Jatropha gossypifolia L. (Euphorbiaceae) a novel cyclic heptapeptide was isolated, which we named cyclogossine A. A combination of amino acid analysis, FAB mass spectrometry, and two dimensional 1H-NMR spectroscopy (TOCSY and ROESY) was used to determine the primary structure. The compound was found to contain one glycine, one alanine, one valine, two leucine, one threonine, and one tryptophan residue; its amino acid sequence is: Leu 1 - Ala 2 - Thr 3 - Trp 4 - Leu 5 - Gly 6 - Val 7. The absolute configurations of the amino acids were determined by chiral gas chromatography; all have the L-configuration.

Ayurvedic herbal drugs with possible cytostatic activity.

Ayurveda is considered to be the traditional science of health in India and is based on the principle of subjectivity. All matter is composed of five basic elements, which can be perceived by the five sense organs. All food and drugs are classified according to their pharmacological properties, which are derived from these five elements. To investigate which Ayurvedic plants might have cytostatic activity, an Ayurvedic model for the pathogenesis of cancer was made. Based on this, selection criteria were formed, that were used to select plants from a list of Ayurvedic herbal drugs. Some of the selected species could be collected in India and Nepal. The dried material of 14 species was submitted to ethanol (70% v/v) extraction and the extracts were tested for cytotoxicity on COLO 320 tumour cells, using the microculture tetrazolium (MTT) assay. The IC50-value, the concentration causing 50% growth inhibition of the tumour cells, was used as a parameter for cytotoxicity. Extracts of the flowers of Calotropis procera (Ait.) R. Br. (Asclepiadaceae) and of the nuts of Semecarpus anacardium L.f. (Anacardiaceae) displayed the strongest cytotoxic effect with IC50-values of 1.4 micrograms/ml and 1.6 micrograms/ml, respectively. The extracts of several other plants did not show a cytotoxic effect up to 100 micrograms/ml, the highest concentration tested.

Curcacycline A--a novel cyclic octapeptide isolated from the latex of Jatropha curcas L.

From the latex of Jatropha curcas L. (Euphorbiaceae) a novel cyclic octapeptide was isolated, which we named curcacycline A. The compound was found to contain one threonine, one valine, two glycine, and four leucine residues. By two-dimensional 1H-NMR spectroscopy (HOHAHA and ROESY), its sequence was determined to be Gly1-Leu2-Leu3-Gly4-Thr5-Val6-Leu7-Leu8-Gly1+ ++. Curcacycline A displays a moderate inhibition of (i) classical pathway activity of human complement and (ii) proliferation of human T-cells.

Fermentation in traditional medicine: the impact of Woodfordia fruticosa flowers on the immunomodulatory activity, and the alcohol and sugar contents of Nimba arishta.

The impact of Woodfordia fruticosa flowers on the immunomodulatory activity, and alcohol and sugar contents of the ayurvedic drug 'Nimba arishta' was investigated by means of model preparations. The use of Woodfordia flowers in model preparations resulted in a substantial increase of the inhibition of both human complement activity and chemiluminescence generated by zymosan-stimulated human polymorphonuclear leukocytes. It was established that the increased biological activity was not due to microbial interference, but to immuno-active constituents released from the Woodfordia flowers. It was also found that the flowers themselves are not the source of alcohol-producing microorganisms. Experiments performed with yeasts isolated from commercial Nimba arishtas showed, in agreement with empirical findings, significantly raised alcohol content upon addition of Woodfordia. An invertase activity exhibited by Woodfordia flowers may be causative of this effect.

Anti-inflammatory activity of gallic acid.

Gallic acid was found to possess antiinflammatory activity towards zymosan-induced acute food pad swelling in mice. In vitro studies on the mode of action of gallic acid revealed that this compound interferes with the functioning of polymorphonuclear leukocytes (PMNs). Scavenging of superoxide anions, inhibition of myeloperoxidase release and activity as well as a possible interference with the assembly of active NADPH-oxidase may account for the inhibition of inflammatory process by gallic acid. Structure-activity relationship analysis showed that the o-dihydroxy group of gallic acid is important for the inhibitory activity in vitro.

Effects of apocynin, a drug isolated from the roots of Picrorhiza kurroa, on arachidonic acid metabolism.

Apocynin is a constituent of root extracts of the medicinal herb Picrorhiza kurroa and has been shown to possess anti-inflammatory properties. We investigated the effects of apocynin on the production of arachidonic acid-derived inflammatory mediators by guinea pig pulmonary macrophages. Apocynin concentration-dependently inhibited the formation of thromboxane A2, whereas the release of prostaglandins E2 and F2 alpha was stimulated. Apocynin potently inhibited arachidonic acid-induced aggregation of bovine platelets, possibly through inhibition of thromboxane formation. The present results suggest that apocynin might, beside its therapeutic effects in inflammatory conditions when given in a root extract of P. kurroa, also be a valuable tool in the development of new anti-inflammatory or anti-thrombic drugs.

Ethnopharmacognostical survey of Azadirachta indica A. Juss (Meliaceae).

Literature data on respectively botany, chemistry, ethnopharmacology, pharmacology and toxicology of Azadirachta indica A. Juss. (Meliaceae) are reviewed and evaluated. In traditional literature, preparations of the tree are claimed to be vulnerable in wide spectrum of diseases. Especially for inflammation-related diseases a good correlation is found with the results of recent experimental investigations. In addition, a variety of other biological activities are reported. Most frequently the effects can be attributed to compounds representing the structural classes of the limonoids, phenolics and macromolecules. Reported toxicity of preparations and isolated compounds are low, except for the seed oil. In conclusion, A. indica can be regarded as a valuable plant source for the rationalisation of its use in traditional medicine and for modern drug development.

Immunomodulatory activity of three Sri-Lankan medicinal plants used in hepatic disorders.

The effects of aqueous extracts of Osbeckia octandra whole plant, Melothria maderaspatana whole plant and Phyllanthus debelis leaves on the human immune system were investigated. The extracts showed strong anticomplement effects on both the classical and alternate pathways of the human complement system in vitro. The effects were dose-dependent and most pronounced in the classical complement pathway assay. The extracts also exhibited a direct dose-dependent inhibition of luminol-induced chemiluminescence of human polymorphonuclear leukocytes upon stimulation with zymosan.

Activity-guided isolation and identification of Azadirachta indica bark extract constituents which specifically inhibit chemiluminescence production by activated human polymorphonuclear leukocytes.

The A. indica crude aqueous bark extract inhibits the generation of chemiluminescence by activated human polymorphonuclear leukocytes (PMN). Guided by this activity the responsible compounds were purified by extraction with different organic solvents and HPLC. Gallic acid, (+)-gallocatechin, (-)-epicatechin, and (as a 2:1 mixture) (+)-catechin and epigallocatechin were isolated and identified by means of HPLC, TLC, MS, 1H-NMR, UV, and CD data. Commercial samples of gallic acid, (+)-catechin and (-)-epicatechin showed the same effects. To our knowledge the identified catechins have never been described as constituents of A. indica.

The newly developed neutrophil oxidative burst antagonist apocynin inhibits joint-swelling in rat collagen arthritis.

Methylated catechols like apocynin selectively inhibits neutrophil superoxide production without significant side-effects. When rats were treated orally with low doses of apocynin the severity of collagen-induced arthritis was significantly reduced, pointing at a role of oxyradicals in the induction of this disease.

Reaction products of 1-naphthol with reactive oxygen species prevent NADPH oxidase activation in activated human neutrophils, but leave phagocytosis intact.

Activation of human neutrophils with opsonized particles in the presence of a nontoxic dose of 1-naphthol resulted in inhibition of superoxide anion production but not of the phagocytotic activity of the cells. In this study we have investigated the mechanism of action of 1-naphthol. The inhibition is not at the level of cellular activation since the FMLP-induced rise of intracellular free calcium was unaffected. Our results show that the (metabolic) activation of 1-naphthol to 1,4-naphthoquinone by reaction with H2O2 from the oxidative burst is a necessary event for the inhibition to occur. The study provides evidence that by its reactivity with essential thiol groups 1,4-naphthoquinone (1,4-NQ) prevents the assembly of a functional NADPH-oxidase in the neutrophil membrane.

How flavonoids inhibit the generation of luminol-dependent chemiluminescence by activated human neutrophils.

The mechanism by which (a panel of) flanonoids inhibit the production of luminol-dependent chemiluminescence (CLlum) by activated human neutrophils is subject to this study. CLlum is frequently used as a bio-assay to quantify the effect of xenobiotics on the production of reactive oxygen species (ROS). Most of the flavonoids decreased CLlum by inhibition of ROS production by the cells. Four selected flavonoids (Taxifolin, Eriodictyol, Hesperetin and Luteolin), inhibited myeloperoxidase (MPO) release, while two of these (Taxifolin and Eriodictyol) strongly inhibited MPO activity. Because CLlum is a MPO-dependent process these activities might mask effects of the flavonoids on ROS production. Finally, our results provide evidence that essential determinants for inhibition of O2(-)-release are the OH-groups located in the B-ring of the flavonoid molecule. Flavonoids methylated at a single OH-group in the B-ring are only inhibitory when they react with activated neutrophils in the presence of myeloperoxidase.

Metabolic activation of natural phenols into selective oxidative burst agonists by activated human neutrophils.

Phenols isolated from the traditional medicinal plant Picrorhiza kurroa inhibit the release of superoxide anion (O2-) by activated human neutrophils, but leave the phagocytotic capacity intact. Resting neutrophils and resting or activated human lymphocytes are insensitive to these agents. The underlying mechanism of this highly selective activity is investigated. A critical event is the reaction of the phenols with secretory products from the activated neutrophils. The reaction products interfere with the assembly of a functional NADPH-oxidase in the membrane. Analysis of the mode of activation of the phenols reveals two possible pathways. Catechols react directly with reactive oxygen species (ROS) from the oxidative burst. For the activation of the orthomethoxy-substituted catechols the combined activity of ROS and myeloperoxidase (MPO) is obligatory. Catechols with a dimethoxy substitution cannot be activated metabolically by neutrophil-derived ROS.