Antiarthritic activity of the newly developed neutrophil oxidative burst antagonist apocynin.

The plant-phenol 4-hydroxy-3-methoxyacetophenone (trivial name apocynin) is a strong inhibitor of neutrophil superoxide anion (O2-) release in vitro. In vitro the inhibitory effect of apocynin is restricted to cells with the capacity to release peroxidase and reactive oxygen species (ROS). Peroxidase deficient cells are insensitive to apocynin. In the present study the antiinflammatory activity of apocynin was tested in collagen-induced arthritis in rats. Collagen-immunized rats were treated with different doses of apocynin in the drinking water starting at the onset of joint-swelling and terminating 14 days later, at the time when joint swelling in the control group was maximal. Apocynin-treated animals had a normal plasma level of collagen-specific antibodies, but showed a significant reduction of the joint swelling. Also the plasma IL-6 level in apocynin-treated animals was substantially lower than in control animals. No flare-up of joint swelling after termination of the treatment was observed in the apocynin-treated groups.

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.

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.

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.

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.

Evidence that superoxide-anion, produced by PMA-activated human polymorphonuclear leukocytes, is the cytolytic agent for rabbit, but not for sheep red blood cells.

The lysis of sheep and rabbit red blood cells (SRBC and RRBC, respectively) upon exposure to PMA-activated human polymorphonuclear leukocytes (PMN) was investigated. The lysis of these target cells, which was measured by the release of 51Cr, showed different kinetics and scavenger-sensitivity. The lysis of RRBC, which was already detectable within 45 min of incubation, was sensitive to superoxide dismutase (SOD), but was only poorly influenced by scavengers of hydroxyl radical formation, such as desferal or thiourea. In contrast, lysis of SRBC was first detectable after 90 to 135 min of incubation and sensitive to desferal and thiourea, but not to SOD. Finally, only RRBC were sensitive to the artificial superoxide-generating system hypoxanthine/xanthine oxidase. Taken together, these data point at a cytolytic activity of superoxide anion O2- towards RRBC. SRBC are relatively resistant to O2-, but are lysed by an H2O2- and hydroxyl radical-dependent process.

Suppression of migration inhibition factor (MIF) production by mitomycins in vitro.

The effects of a series of mitomycins in a modified version of the Migration Inhibition Factor (MIF) test were investigated. This modified assay, which uses monocytes of the mouse cell line WEHI3 as target cells for MIF, discriminates between effects on the migrating monocytes, the production of MIF by lymphocytes and the activity of the produced MIF. The 7-methoxy mitomycins were found to be more active than the 7-amino and 7-hydroxy derivatives in suppressing both the spontaneous migration of target monocytes and the MIF production by lymphocytes. Neither of the compounds had a significant effect on the activity of produced MIF. A relationship between the activities of the mitomycins and their lipophilicity and polarographic half-wave potential is discussed. Further attention is paid to the stability of the mitomycins under the test conditions and additional advantages of the modified MIF assay over related in vitro tests for cell-mediated immunity.

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.

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.

Decomposition of mitomycin and anthracycline cytostatics in cell culture media.

The chemical stability of members of two groups of cytostatics, mitomycins and anthracyclines, has been studied in four different cell culture media enriched with serum. Stability was determined with the use of high performance liquid chromatography. In the group of mitomycins, the 7-aminomitosanes appeared to be relatively stable during a seven days incubation period at 37 degrees C when compared to the 7-methoxy congeners. The anthracycline derivatives, 4-demethoxy-daunorubicin, doxorubicin and its 4'-analogues showed half-lives of about 10-20 hours. Doxorubicinol and daunorubicin were found to be more stable. Anthracycline degradation products could be traced with the use of thin layer chromatography. All main degradation products originate from hydrolytic reactions. No enzymatic conversions could be observed. These observations may be of importance for the correct interpretation of the effects of mitomycins or anthracyclines on cells incubated in a cell culture medium.

In vitro immunosuppressive activities of recently developed anthracycline cytostatics.

The effects of a series of seven anthracycline cytostatics on various human leukocyte functions, namely the production of Migration Inhibition Factor (MIF) by lymphocytes and the production of chemiluminescence by activated polymorphonuclear leukocytes (PMNs), as well as on classical and alternative pathways of activation of human complement were investigated. In addition, lipophilic and electrochemical properties of the compounds were determined, by measuring their High Performance Liquid Chromatography (HPLC) capacity ratio (k') and half-wave reduction potential (E1/2). The antitumor agents under investigation suppressed, in most cases, lymphocyte and PMN functions, whereas complement activity remained unaffected. The extent of suppressive effects showed a good correlation with the lipophilicity of the compounds, while no correlation with reduction potentials was found.

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.

A rapid and sensitive micro-assay to determine the capacity of quinones to undergo redox cycling.

Using a series of aziridinyl-benzoquinones it is shown that the conversion of oxyhemoglobin to methemoglobin in sheep erythrocytes is correlated with the capacity of each quinone to undergo redox cycling. Based on these findings a semiquantitative assay is developed for the rapid screening of redox cycling quinones.

Problems and possibilities in the use of traditional drugs.

Research and development of traditional drugs, as tools in health care, need a special approach, preferably in a multidisciplinary setting. With special reference to the situation of the indigenous medicine in Sri Lanka, an ethnopharmacognostic approach which has been practised there during the last 4 years is outlined. Besides the study of literature data the significance and relevancy of a field inquiry into the nature, the position and the use of traditional drugs is stressed. In such field inquiries a questionnaire which covers the problems of an ethnobotanical, ethnopharmaceutical, ethnopharmacological and ethnomedical nature is used. The problems encountered are discussed according to four categories: the non-professional users, the professionals who prescribe the drugs and direct the medications, the professional producers and the non-professional preparation of drugs. The possibilities for advancement of traditional medicine is outlined as a programmed development aiming at standardization of the quality of ingredients and manufacturing processes and schemes of quality control. However, simultaneous clinical research along with experimentally based standardized production is of vital importance. Within the framework of such developmental endeavours serious scientific attention should be given to conceptual aspects of the traditional system and the context in which their drugs are used. Finally it is argued that major advancements of traditional medicine will only be achieved if research programmes are focused on clear-cut topics.

Evaluation of Asoka Aristha, an indigenous medicine in Sri Lanka.

PIP: This paper presents the results of an investigation of Asoka Aristha, an indigenous medicine used in rural Sri Lanka for menstrual disorders. This investigation was part of a broader evaluation of ayurvedic drugs aimed at establishing the botanic identity of drug ingredients, establishing possible modes of action, and identifying the chemical constituents responsible for the drug effects. It is projected that the results of this research can be used to standardize ayurvedic drugs and optimize the quality and production procedures. Asoka Aristha, a fermented potion composed largely of Asoka bark, is produced on a large scale by the state-owned Ayurvedic Drugs Corporation. It has been indicated for women in cases of menorrhagia, metrorrhagia, leukorrhea, primary amenorrhea, general menstrual disorders, and skin disorders. 3 types of experiments are projected to evaluate the biologic activity of Asoka Aristha: measurement of the occurrence of cornification of the uterus epithelium in hypophysectomized rats after administration of the drug to determine the estrogenic mode of action, in vitro measurement of the direct uterine activity of the drug to determinme the oxytocic effect, and testing of the inhibiting properties of the drug in prostaglandin synthetase enzyme reactions in vitro. Experiments carried out on rat uteri so far seem to rule out the mechanism based on oxytocic action. It is urged that clinical trials on the therapeutic effect of Asoka Aristha be carried out in Sri Lanka.^ieng

Inhibitory activity of Jatropha multifida latex on classical complement pathway activity in human serum mediated by a calcium-binding proanthocyanidin.

This study isolates and characterizes the anti-complement constituent(s) present in the latex of Jatropha multifida, in an attempt to explain the traditional application of the latex in the treatment of infected wounds. Guided by the inhibition of classical pathway (CP) complement activity in human serum, a polymer was isolated which could be characterized as a proanthocyanidin. The polymer inhibits CP activation of the complement cascade, while alternative pathway (AP) activation is relatively insensitive to the polymer. This is due to the selective depletion of Ca2+, but not Mg2+, from the incubation medium.

Two functionally and chemically distinct immunomodulatory compounds in the gel of Aloe vera.

An aqueous extract of Aloe vera gel was analyzed guided by modulatory activity with regard to the in vitro activation of human complement and of human polymorphnuclear leucocytes (PMN). Upon ultrafiltration a high (h-Mr) and a low (l-Mr) molecular mass fraction were obtained. Pre-incubation of human pooled serum with the h-Mr fraction resulted in a depletion of classical and alternative pathway complement activity. In contrast, only the l-Mr fraction could inhibit the production of free oxygen radicals by activated PMNs. The latter activity cannot be attributed to non-specific effects like toxicity, interference with stimulant binding or scavenger activity.

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.

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 an aqueous extract of Azadirachta indica stem bark.

The interference of an aqueous extract of the stem bark of Azadirachta indica with different parts of the human immune system was investigated. The extract showed strong anticomplementary effects which were dose-and time-dependent and most pronounced in the classical complement pathway assay. Moreover, a dose-dependent decrease in the chemiluminescence of polymorphonuclear leukocytes was observed and a dose-dependent increase in the production of migration inhibition factor by lymphocytes.