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We report here an analysis of plants used by traditional healers of ancient Persia to induce abortions. Six herbal formulations that contain 39 different species from 21 plant families with their vernacular names, English names, amounts used, and their methods of preparation are reported. Our initial objective of this ethnobotanical analysis was to evaluate the validity and the efficacy of the plants used by (1) comparing other reported uses of these plants in traditional medicine, (2) investigating the medical and pharmacological literature on the medicinal properties of the plant species used, and (3) investigating the reported cytotoxic effects of compounds prevalent in these plants. Previous phytochemical analyses have shown that a number of plant species are rich in coumarin compounds that have potential antineoplastic or cytotoxic activities. Our results indicate a correlation between the reported use of these plants as abortifacients and their cytotoxic (antineoplastic) effects. In addition, we discuss the process in which this ethnobotanical investigation led to the discovery of dicoumarol (a coumarin anticoagulant) as a potential chemotherapeutic agent.

The marine natural produce manoalide has been reported to inactivate venom phospholipase A2 from several sources and phospholipase A2 from polymorphonuclear leukocytes. In this investigation, the regulation of eicosanoid production was studied both in an in vitro and in an in vivo model. The release of arachidonic acid and prostaglandin E2 was inhibited by manoalide when cultured mouse peritoneal macrophages were stimulated with phorbol myristate acetate (apparent IC50 = 0.23 microM), calcium ionophore A23187 (apparent IC50 = 0.23 microM) and zymosan (apparent IC50 = 0.18 microM). Leukotriene C4 production was inhibited by manoalide when macrophages were stimulated by A23187 (IC50 = 0.35 microM) but was enhanced when the cells were stimulated with zymosan. In an in vivo model, manoalide antagonized zymosan-induced peritoneal writhing in the mouse (ED50 = 0.71 mg/kg) and inhibited the i.p. release of 6-keto-prostaglandin F1 alpha (ED50 = 0.2 mg/kg) and leukotriene C4 (ED50 = 0.24 mg/kg). Our results suggest that: 1) manoalide modifies arachidonic acid release and metabolism to prostaglandins and leukotrienes in mouse cultured peritoneal macrophages stimulated by phorbol myristate acetate, calcium ionophore A23187 and zymosan and 2) the analgesic properties of manoalide seem to be partially correlated with reduced eicosanoid production in zymosan-stimulated mouse peritoneal exudates.

Stypolidione, an orthoquinone derived from the brown seaweed Stypopodium zonale, inhibited the polymerization of three-cycle-purified bovine brain microtubule protein in vitro in a concentration-dependent manner. Fifty per cent inhibition of the extent of polymerization beginning under initiating conditions occurred at a stypoldione concentration of approximately 25 microM, and 50% inhibition of tubulin addition to the assembly ends of microtubules at steady state occurred at a concentration of approximately 8 microM. Only slight structural abnormalities could be detected by negative stain electron microscopy in some of the microtubules that did assemble in the presence of the drug, and no aberrant structural forms of microtubule protein were detected. Stypoldione inhibited the binding of [3H]colchicine to tubulin, with 50% inhibition of colchicine binding activity occurring at a stypoldione concentration of 12-15 microM. Inhibition of colchicine binding activity appeared noncompetitive and was at least partially reversible, suggesting that stypoldione and colchicine bind at separate sites. By assuming that the inhibition constant for the ability of stypoldione to prevent the binding of colchicine to tubulin was equivalent to the dissociation constant for the binding of stypoldione to tubulin, we calculated that approximately 62% of the tubulin present free in solution under initiating conditions and 35-37% of the soluble tubulin under steady-state conditions was complexed with stypoldione when polymerization was inhibited by 50%. These data are consistent with a mechanism in which stypoldione interacts with soluble tubulin and inactivates the tubulin so that it is unable to add to microtubule ends, although a colchicine-like mechanism involving an action of stypoldione at microtubule ends has not been eliminated.

We have found that stypoldione, a bright red o-quinone isolated from the brown alga Stypopodium zonale, inhibits the division of sea urchin embryos in a concentration-dependent manner (IC50 approximately 2.5 X 10(-6) M). Although previous studies have shown this marine natural product to inhibit beef brain microtubule assembly in vitro [Fed. Proc. 39:26-29 (1980); Mol. Pharmacol. 24:493-499 (1983)], we have found that stypoldione does not accumulate sea urchin embryos in mitosis and hence does not act like a mitotic spindle poison. We have also shown this marine natural product to inhibit both amino acid and nucleoside uptake. By preloading sea urchin embryos with nucleoside (i.e., [3H]thymidine) in order to dissociate effects on uptake from those on incorporation, we found that stypoldione in fact produces no significant inhibition of the M phase-independent S1 period of DNA synthesis, a result which suggests that stypoldione has no direct effect on DNA synthesis. In contrast, stypoldione did reduce the extent of amino acid incorporation in embryos preloaded with [3H]leucine. An inhibition of incorporation was apparent as early as 20 min after fertilization, and incorporation was reduced to 50% of control by 40 min postfertilization. This result suggests that stypoldione might inhibit cleavage via an inhibition of translation, although the existence of other inhibitory mechanisms cannot yet be ruled out. Cytological examination revealed that sea urchin embryos did not progress beyond-interphase or very early prophase when incubated in the presence of 1.0 X 10(-5) M stypoldione. The nuclear membranes remained intact, and chromatin did not condense into chromosomes in these arrested embryos. These results indicate that embryos exposed to stypoldione early in the cell cycle initiate and complete the M phase-independent S1 period of DNA synthesis, but stop cell cycle progression prior to the start of prophase of mitosis. The period between S phase and mitosis is referred to, by definition, as the "G2" phase of the cell cycle. The result therefore suggest that stypoldione blocks cell cycle progression (and, ultimately, cell division) by inhibiting progression through G2. This compound may represent a new class of G2-accumulating agents.

The efficiency of 35 nitroaromatic and nitroheterocyclic compounds in radiosensitizing hypoxic Chinese Hamster cells in vitro was determined. The concentration C of the compound required to achieve an enhancement ratio of 1·6 was measured, and the redox and partition properties were quantified as the one-electron reduction potential at pH 7, E, and the octanol: water partition coefficient, P, respectively. Most of the compounds studied were 2-nitroimidazoles, but some 4- and 5-nitroimidazoles, 5-nitrofurans and nitrobenzenes were investigated for comparison. Together with data for nine nitroimidazoles previously reported, the results were fitted to a structure-activity relationship of the form [image omitted] using multiple linear regression analysis. Statistical tests showed that the coefficients b2 and b3 were not significantly different from zero and the simpler equation, obtained by omitting the terms in log P, explained 85 per cent of the variance in log C. Earlier reports that the radiosensitization efficiency of nitro compounds in vitro largely depends on the reduction potential were confirmed. The conclusive demonstration that P is unimportant in vitro is valuable in interpreting the results of experiments in vivo, where P is expected to have a much greater influence on biological response.

Manoalide is a potent antiinflammatory marine natural product and a direct inactivator of venom phospholipase A2 (PLA2; EC 3.1.1.4). Manoalide has been shown to irreversibly inhibit PLA2, with the corresponding modification of a selective number of lysine residues. The mechanism of inactivation has not yet been elucidated and structure-activity relationship studies were, therefore, performed in order to determine the contributions of the various functional groups incorporated in the gamma-hydroxybutenolide, alpha-hydroxydihydropyran, and trimethylcyclohexenyl ring systems to the efficacy (irreversibility) and potency of this series of inhibitors. These studies indicate that 1) the presence of the hemiacetal in the alpha-hydroxydihydropyran ring is required for irreversible binding of manoalide, 2) the gamma-hydroxybutenolide ring is involved in the initial interaction of manoalide with PLA2, and 3) the hydrophobic nature of the trimethylcyclohexenyl ring system allows nonbonded interactions between manoalide and PLA2 that enhance the potency of these analogs. These structure-activity relationship studies suggest that the closed ring form of manoalide is the predominant molecular species that accounts for the selective and potent inhibition of PLA2 by manoalide. Elucidation of the mechanism awaits further detailed physicochemical studies on the structure of the manoalide (analog)-protein adducts in model systems and using PLA2.

Stypoldione is a marine natural product that inhibits cells division in marine embryos and in mammalian cell cultures. The mechanism responsible for the ability of the compound to inhibit cell division is not known. The compound was found in early studies to inhibit polymerization of tubulin into microtubules in vitro, which lead to the suggestion that inhibition of microtubule polymerization in cells might be responsible for the ability of the compound to inhibit cell division. More recently, stypoldione was found to react covalently with the sulfhydryl groups of a number of proteins including tubulin and with sulfhydryl groups of peptides and small molecules. Thus, stypoldione could potentially react with a large number of cellular targets. In the present study, we have examined the effects of stypoldione on the organization of microtubules and chromatin in cells, in relation to the ability of the compound to inhibit cell division. We used indirect immunofluorescence light microscopy of fixed and stained sea urchin embryos during the first and second divisions after fertilization, with stains specific for tubulin and DNA. We found that stypoldione exerted qualitatively different effects on cell division and microtubule organization and function at different concentrations. At the lowest effective concentrations, 5-10 microM, stypoldione selectively inhibited cytokinesis. Mitotic division occurred normally, usually with no discernible perturbation of microtubule organization or function, and cells became multinuclear. At somewhat higher concentrations, 20-40 microM, stypoldione blocked embryos before streak stage of the first division and, although microtubules were present, their organization was perturbed and they often formed unusual "spiral aster" arrays. At 80 microM and above, microtubules in blocked cells were largely absent. Thus, stypoldione uncouples cytokinesis from mitosis at the lowest effective concentrations and, although it can disrupt microtubules at relatively higher concentrations, it inhibits cell division at the lowest effective concentrations by a selective action on cytokinesis through a mechanism that does not appear to involve disassembly of microtubules.

Fuscoside (FSD) is a potent and long-lasting anti-inflammatory drug that selectively inhibits leukotriene production in murine models of inflammation. In the present study, the effects of FSD on the lipoxygenase pathways in human polymorphonuclear leukocytes are explored in order to better understand the mechanism of action of this novel drug. In adherent and suspended polymorphonuclear leukocytes, FSD irreversibly inhibits leukotriene B4 (LTB4) synthesis (IC50 = 10 microM) and the release of 14C-labeled LTB4 from neutrophils prelabeled with [14C]arachidonic acid. Unlike the reversible 5-lipoxygenase inhibitor L-651,896, FSD has no observable effect on LTB4 biosynthesis in whole blood, but does express activity as blood is successively diluted. In 10,000 x g supernatants of human platelets and polymorphonuclear leukocytes, FSD does not inhibit platelet 12-lipoxygenase, but is extremely effective in inhibiting the metabolism of arachidonic acid and 5-hydroperoxyeicosatetraenoic acid to LTB4 via neutrophil 5-lipoxygenase. FSD has no effect on the conversion of leukotriene A4 to LTB4 in this system. Interestingly, concurrent with FSD inhibition of leukotriene synthesis is a concentration-dependent increase in 5-hydroxyeicosatetraenoic acid, suggesting that FSD may selectively inhibit the leukotriene A4 synthase activity associated with human 5-lipoxygenase. FSD is therefore representative of a new class of nonantioxidant 5-lipoxygenase inhibitors that may be effective local therapeutic agents in the management of diseases such as psoriasis, arthritis and inflammatory bowel and lung diseases.

The biological and biochemical pharmacology of fuscoside, a novel anti-inflammatory marine natural product isolated from the Caribbean gorgonian Eunicea fusca, has recently been characterized using murine (part I) and human (part II) models of inflammation. Topically applied fuscoside (FSD) effectively inhibits phorbol myristate acetate (PMA)-induced edema in mouse ears at levels comparable with indomethacin over a 3.3-hr exposure period, and is significantly more efficacious than indomethacin over 24 hr in the PMA model. Histological preparations and quantification of the neutrophil-specific marker, myeloperoxidase, demonstrate that FSD inhibits neutrophil infiltration into PMA-induced regions of edema and inflammation. In systemic studies, where FSD is injected i.p. before the topical application of PMA, negligible effects on ear inflammation are observed. FSD does not inhibit bee venom or human synovial fluid phospholipase A2 up to concentrations of 500 microM. In calcium ionophore-activated cultures of mouse peritoneal macrophages, FSD selectively and irreversibly inhibits leukotriene C4 biosynthesis (IC50 = 8 microM), yet has negligible effects on prostaglandin E2 production. FSD is also without effect on the conversion of arachidonic acid to prostaglandin E2 by ram seminal vesicle cyclooxygenase. Chromatographic and spectroscopic studies suggest that FSD is not metabolized, and that drug uptake/binding by macrophages is time dependent, saturable and independent of active transport mechanisms. These studies represent the first report of an anti-inflammatory marine natural product that selectively inhibits leukotriene biosynthesis.

The subsynaptic actions of theophylline were studied by employing intracellular recording techniques in the isolated cat tenuissimus muscle. Theophylline produced a dose-dependent increase in miniature end-plate potential (MEPP) amplitude. The maximal increase (30%) in MEPP amplitude occurred at the 1.8 mM concentration of theophylline with a concomitant depolarization of the resting membrane potential. Depolarizations induced by acetylcholine were potentiated by 1.8 mM theophylline. This effect was less evident with a carbachol-induced depolarization, which indicates a possible anticholinesterase action. Input resistance decreased significantly; this effect correlated with the decline in resting membrane potential produced by theophylline. The equilibrium potential of the EPP was shifted to a more positive value in a reversible manner. In addition, the conductance change due to MEPPs, acetylcholine and carbachol was increased by theophylline treatment. These results suggest that the shift in equilibrium potential of the EPP coupled with a slight anticholinesterase action may account for the increase in MEPP amplitude observed with theophylline treatment.

The effects of 4-aminopyridine (4AP) on the extracellularly recorded nerve terminal action potential (NTAP) and end-plate potential were studied at the frog neuromuscular junction. An in-depth analysis of the time course of the NTAP was performed in the presence and absence of extracellular Ca++. Low concentrations (5 X 10(-6) M) of 4AP produced no significant alterations in the time course of the NTAP, yet increased quantal content of the end-plate potential 2-fold. In contrast, high concentrations (5 X 10(-4) M) of 4AP prolonged the duration of the NTAP by selectively flattening the K+ slope of the NTAP and increased the quantal content of the end-plate potential. It is concluded that both potassium channel blockade and facilitation of transmitter release by 4AP can be demonstrated in this preparation, and that it is possible to separate these actions by varying the concentration of 4AP. Interpretation of these data suggests that there is a second site or mechanism of action by which 4AP potentials transmitter release. Possible mechanisms of action for 4AP are discussed.

Low concentrations (1-10 microM) of the marine natural product pseudopterolide inhibited cytokinesis and induced formation of multinucleate cells in fertilized Strongylocentrotus purpuratus embryos. As determined by immunofluorescence microscopy using fluorescent stains for actin filaments, microtubules, and chromosomes, pseudopterolide inhibited cytokinesis selectively by disrupting the contractile ring, whereas spindle microtubule organization and mitotic chromosome segregation to opposite spindle poles were unimpaired. At somewhat higher concentrations (16-20 microM), pseudopterolide induced formation of microtubule spiral asters, which are believed to be caused by rotation of the cytoplasm relative to the cell cortex. The effects of pseudopterolide on cytokinesis were cell-cycle dependent. The actions of pseudopterolide in fertilized sea urchin embryos were strikingly similar to the actions of another marine natural product, stypoldione, a structurally unrelated orthoquinone that reacts covalently with the sulfhydryl groups of glutathione, beta-mercaptoethanol, cysteine, and a number of proteins [Mol. Pharmacol. 35:635-642 (1989)]. In the present study, pseudopterolide was also found to react with sulfhydryl groups of glutathione, beta-mercaptoethanol, and cysteine. The results indicate that the cellular target for pseudopterolide, like the target for stypoldione, may be an especially sensitive sulfhydryl-containing protein involved in the formation or function of the contractile ring.

Pseudopterosin A is a diterpene riboside isolated from Pseudopterogorgia bipinata, a soft coral of the order Gorgonacea. Pseudopterosin A inhibits the first cleavage in fertilized sea urchin eggs (Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, and Lytechinus pictus) with an IC50 of 25 microM. In time of addition studies, Pseudopterosin A (4 X 10(-5) M) progressively blocked the first cleavage when added within the first 50 min post fertilization; when added after 50 min the first division occurred normally. Cell cycle studies show that Pseudopterosin A had to be in continuous contact with the sea urchin eggs during the initial 45 min post fertilization to significantly inhibit the first cleavage. Shorter drug contact time progressively reduced the degree of inhibition, suggesting that inhibition of cytokinesis by Pseudopterosin A was correlated with mitosis. Pseudopterosin A (4 X 10(-5) M) inhibited the uptake and incorporation of [3H]thymidine during the S2 phase. This concentration of Pseudopterosin A also inhibits protein synthesis as measured by the uptake and incorporation of [3H]phenylalanine. In this case the inhibition started before the S1 phase. Cytological examination revealed that sea urchin embryos did not progress beyond early prophase. Notably, the nuclear envelope remained intact and chromatin was condensed into chromosomes in the arrested embryos. These synchronously dividing embryos did not show any abnormalities such as lysis, swelling, or morphological changes different from control embryos.

Facilitatory effects of SQ 20009 [1-ethyl-4-(isopropylidenehydrazine)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl] were investigated at the frog sartorious neuromuscular junction. A dose-dependent increase in miniature end-plate potential frequency occurred without changes in miniature end-plate potential amplitude. Significant increases in the amplitude and the rate of rise of the end-plate potential and reduction in the incidence of end-plate potential failures were observed in magnesium blocked muscle. The increase in end-plate potential amplitude and rate of rise appeared calcium dependent. No significant changes in passive membrane resistance or muscle membrane sensitivity to acetylcholine were observed. Twitch studies employing direct and indirect stimulation of the rat diaphragm preparation demonstrated a preferential facilitation of the indirect response rather than a direct action on the muscle fiber. The facilitatory effect of SQ 20009 on evoked and spontaneous release were of approximately equal orders of magnitude, suggesting that the drug may affect a common mechanism in the two release processes.

A specimen of Plakortis lita from Palau yielded the new carboxylic acids, homo-plakotenin (2a), the sodium salt of homo-plakotenin (2b), the sodium salt of nor-plakotenin (3), the sodium salt of plakotenin (1b), and the known compound plakotenin (1a). The structures of the new acids were elucidated by interpretation of spectral data and by comparison with the known compound. Compounds 1a, 1b, and 2a were found to significantly reduce proliferation of rheumatoid synovial fibroblasts.