Antifungal activity of eicosanoic acids isolated from the endophytic fungus Mycosphaerella sp. against Cryptococcus neoformans and C. gattii.

The antifungal effects of two eicosanoic acids, 2-amino-3,4-dihydroxy-2-25-(hydroxymethyl)-14-oxo-6,12-eicosenoic acid (compound 1) and myriocin (compound 2), isolated from Mycosphaerella sp. were evaluated against Cryptococcus neoformans and C. gattii. The compounds displayed antifungal activities against several isolates of C. neoformans and C. gattii, with minimal inhibitory concentration (MIC) values ranging from 0.49 to 7.82 µM for compound 1 and 0.48-1.95 µM for compound 2. In the checkerboard microtiter test, both compounds exhibited synergistic activity with amphotericin B against C. gattii. Ultrastructural analysis revealed several signs of damage in C. gattii and C. neoformans cells treated with compounds 1 and 2, including deformities in cell shape, depressions on the surface, and withered cells. The cells of C. gattii treated with compounds 1 and 2 showed less loss of cellular material in comparison to those treated with amphotericin B. The difference in cellular material loss increased in a test compound concentration-dependent manner. Consistent with this observation, compounds 1 and 2 were able to internalize propidium iodide (PI) in C. gattii cells. In addition, compound 2 induced the formation of several pseudohyphae, suggesting that it could reduce virulence in C. gattii cells. The study results show that these natural products led to membrane damage; however, this may not be the main target of action. These compounds have potential antifungal activity and could be useful in further studies for developing more effective combination therapies with amphotericin B and reducing side effects in patients.

Inhibition of DNA topoisomerases I and II and cytotoxicity of compounds from Ulmus davidiana var. japonica.

Twenty five compounds including ten triterpenes (1-3, 5-11), six flavonoids (12-15, 24, 25), five lignans (17, 18, 21-23), two butenyl clohexnone glycosides (19-20), one fructofuranoside (16) and one fatty acid (4) were isolated from the roots of Ulmus davidiana var. japonica. The structures of those compounds were identified by comparing their physicochemical and spectral data with those of published in literatures. All the compounds were evaluated for DNA topoisomerase inhibitory activities and cytotoxicities. Among the purified compounds, 4 and 19 showed more potent inhibitory acitivities (IC(50): 39 and 19 µM, respectively) than camptothecin, as the positive control (IC(50): 46 µM) against topoisomerase I. Compounds, 4, 10, 12, 19, 24 and 25 showed strong inhibitory activities toward DNA topoisomerase II (IC(50): 0.1, 0.52, 0.47, 0.42, 0.17 µM and 17 nM, respectively), which were more potent than that of etoposide as positive control (IC(50): 20 µM). In A549 cell line, 5 and 6 showed cytotoxicities (IC(50): 4 µM and 3 µM, respectively, with IC(50) of camptothecin as positive control: 10.3 µM). In the HepG2 cell line, 3, 5 and 7 showed cytotoxicity (IC(50): 4, 3 and 4 µM, respectively, with IC(50) of camptothecin: 0.3 µM). Compounds 6, 12 and 23 showed cytotoxicities in the HT-29 cell line (IC(50): 19, 19 and 15 µM, respectively, with IC(50) of camptothecin: 2 µM).

[A new chemical component of Pueraria lobata (Willd.) Ohwi].

Pueraria lobata (Willd.) Ohwi. was extracted for two times with 70% ethanol and the 70% ethanol-extracts was condensed. Various column chromatography with AB-8 macroreticular resin, Toyopearl HW-40, pharmadex LH-20, and silica gel were employed for the isolation and purification of the 70% ethanol-extracts from Pueraria lobata (Willd.) Ohwi. Five compounds were isolated and their structures were identified by physiochemical properties and spectral analysis (UV, IR, MS, 1H NMR, 13C NMR, HMQC, HMBC, etc.): (4R)-3-[ 2-hydroxy-4-methoxyphenyl]-4-(4-beta-D-glucopyranosyloxybenzyl) but-2-en-4-olide (1), 4', 8-dimethoxyl-7-O-beta-D-glucopyranosyl isoflavone (2), eicosanoic acid (3), hexadecanoic acid (4), tetracosanoid acid-2,3-dihydroxypropyl ester (5). Compound 1 was a new compound, and compounds 2, 3, 4 were isolated from this plant for the first time.

Cytotoxic compounds of Schizolaena hystrix from the Madagascar rainforest.

Bioassay-guided fractionation of a crude ethanol extract from a Madagascar collection of Schizolaena hystrix afforded the two new long-chain compounds, 3 S-acetoxyeicosanoic acid ethyl ester ( 1) and 3 S-acetoxydoeicosanoic acid ( 2), and the known long-chain compound 3 S-acetoxyeicosanoic acid ( 3). In addition, the long-chain alcohol 1-hydroxydodecan-2-one ( 7), as well as the new flavonoid schizolaenone C ( 4) and the two known flavonoids diplacol ( 5) and 3'-prenylnaringenin ( 6) were isolated from a methanol extract of the same plant. Isolation and structure elucidation of the novel compounds and the cytotoxicities of all the isolates are reported.

Sesquiterpene and long chain ester from Tanacetum longifolium.

A new naturally occurring linear sesquiterpene (tanacetene) (1). having irregular non-head- to- tail attachment of isoprene units, a new long chain ester (2). and 10 known compounds have been isolated from hexane and chloroform extracts of the roots and aerial parts of Tanacetum longifolium wall. The structure of (tanacetene) (1). was established as (2E,6E,10E)-2,6,11-trimethyl-dodeca-2,6,10-triene and that of (2). as heptatriacontanyl eicosanoate by spectroscopic methods along with 10 known compounds. From the oily fraction twelve volatile compounds were identified by GC-MS. The roots of this plant were found to be a new major source of Z-spiroketalenol ether-6,7-epoxy-diyne in 3.2% yield on dry weight basis.

[The research on the chemical components of Schizonepeta tenuifolia Briq].

Eicosane acid, beta-sitosterol, oleanolic acid, ursolic acid and daucosterol have been firstly isolated and identified from Schizonepeta tenuifolia Briq..

[Study of supercritical-CO2 fluid extraction in extracting essential oils of Amomun tsao-ko].

Essential oils from Amomum tsao-ko are extracted by supercritical-CO2 fluid and the oils are analyzed by GC-MS. 1, 8-cineole, trans-geraniol eicosatrienoic acid methylester and d-nerolidol of them are the major constitutents, 12 constitutents of them are first obtained from the plant.

Fungal metabolites. Part 14. Novel potent immunosuppressants, mycestericins, produced by Mycelia sterilia.

Mycestericins A, B, C, D and E were isolated from the culture broth of Mycelia sterilia ATCC 20349 along with thermozymocidin (= myriocin). Their structures were elucidated on the basis of spectroscopic studies and chemical evidence. The acetate of mycestericin C was identical with the acetate of 6,7-dihydromyriocin. Mycestericins suppressed the proliferation of lymphocytes in the mouse allogeneic mixed lymphocyte reaction, with a potency similar to that of myriocin.

[Chemical constituents of Pedicularis muscicola Maxim].

It is one of the main components in an effective secret recipe for the treatment of snakebite in Mabian, an autonomaus county of the Yi nationality in Sichuan province. Four compounds have been isolated from the herb of Pedicularis muscicola. On the basis of spectrometric analysis and physico-chemical constants, they were identified as pediculariside, D-mannitol, arachidic acid and hentriacontane.

Characterization of a novel arachidonic acid-derived neutrophil chemoattractant.

The oxidation of arachidonic acid in a superoxide-generating environment results in the formation of a potent chemoattractant that appears to be identical to a chemotactic material generated by hepatocytes when they metabolize alcohol. The product was extracted, chromatographed and characterized by physical methods including GC/MS. The physical properties are consistant with the parent structure: 19-hydroperoxy, 20-hydroxyarachidic acid. This novel saturated 20 carbon product, derived from arachidonic acid by free radical-generating reactions, may play a role in the neutrophilic infiltration observed during the course of acute alcoholic hepatitis.

Modern high-performance liquid chromatographic-radioimmunoassay strategies for the study of eicosanoids in biological samples.

An evaluation of the most recent literature on the determination of eicosanoids by immunoassay methods confirms that owing to the inherent lack of specificity of many of the antibodies used for this purpose, immunological assays (radioimmunoassay or enzyme immunoassay) are often preceded by solid-phase extraction followed by further purification of the antigens of interest by routine reversed-phase high-performance liquid chromatographic methods. In this way the analytical potential of radioimmunoassay is remarkably enhanced and accuracy and precision of the assay are ensured.

Gamma-interferon induces prostacyclin and prostaglandin but not lipoxygenase product synthesis in rat endothelial cells.

The major arachidonic acid metabolites excreted by untreated rat heart endothelial cells were prostacyclin (PGI2), 5-hydroxyeicosatetraenoic acid (5-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). Trace amounts of both prostaglandin E (PGE) and thromboxane B2 (TXB2) were also produced. The activity of leukotriene B4 (LTB4) was the same as baseline activity. Gamma-interferon (gamma-IFN) treatment increased the production of PGI2 and PGE more than three-fold compared to control values. Thromboxane production increased two-fold while the excretion of 5-HETE and 15-HETE was unaffected. No LTB4 excretion was seen after gamma-IFN stimulus. Methylprednisolone downregulated the 5-HETE, 15-HETE and prostacyclin production both in normal and gamma-IFN treated cells. Indomethacin decreased prostacyclin excretion in untreated cells and also decreased gamma-IFN-induced prostacyclin and PGE excretion. Taken together these results indicate that rat microvascular endothelial cells excrete prostaglandins and prostacyclin, but not leukotrienes.

Psoriatic skin lesions contain a novel lipid neutrophil chemokinetic compound which is distinct from known chemoattractant eicosanoids.

1. Lipid extracts of scale from the lesions of the skin disease psoriasis were purified by high performance liquid chromatography (h.p.l.c.). Assay of fractions by an agarose microdroplet method showed the presence of a novel neutrophil chemokinetic compound which possessed the chromatographic properties of a monohydroxy fatty acid, yet was distinct from the chemoattractant eicosanoid, 12-hydroxyeicosatetraenoic acid, previously isolated in psoriasis. 2. The novel, material, termed compound X, was also detected in fractions collected on h.p.l.c. of extracts of chamber fluid samples obtained from abraded psoriatic lesions, but was not detectable in samples from clinically normal skin. 3. Comparison of the straight and reversed phase h.p.l.c. retention times of compound X with those of a range of standard monohydroxy fatty acids, together with further analysis by gas chromatography-mass spectrometry and assay of selected standards for neutrophil chemokinetic activity, failed to reveal the structural identity of compound X. 4. The finding of a further compound in psoriatic lesions, which stimulates neutrophil movement, highlights the complexity of inflammatory mediator production in this disease.

Location of double bonds in two unsaturated forms of phytanic acid from Refsum disease as determined by mass spectrometry.

A monounsaturated and a triunsaturated form of phytanic acid (3,7,11,15-tetramethylhexacosanoate) were isolated from plasma lipids of a patient with Refsum disease. Both were converted to their methyl esters, oxidized to polyhydroxy acids by treatment with OsO4 and converted to their vicinal trimethylsilyl ethers. These derivatives were analyzed by gas chromatography-mass spectrometry using both electron impact ionization (at 21 and 70 eV) and chemical ionization conditions to obtain clear evidence to establish the structure of the monounsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-15-monoenoic acid and that of the triunsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-6,10,14-trienoic acid. The possible metabolic and dietary sources for these novel fatty acids are discussed.

Neutrophil-aggregating activity of monohydroxyeicosatetraenoic acids.

The following oxidative derivatives of arachidonic acid were prepared and assayed for their ability to aggregate cytochalasin-B-pretreated human neutrophils: 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids. The compounds were prepared by oxidation of arachidonic acid and purified by direct and reverse phase high performance liquid chromatography. Each lipid was racemic at the hydroxy residue and had a cistrans conjugated double bond adjacent to the hydroxy residue. Except for racemization, therefore, they were identical to hydroxyeicosatetraenoic acids generated by neutrophils exposed to diverse aggregating stimuli. In addition, 15-L-hydroxyeicosatetraenoic acid was prepared from soybean lipoxygenase. Of these 7 fatty acid preparations, only 5- and 12-hydroxyeicosatetraenoic acid aggregated the cells. Thus, the bioactions of these lipids are crucially dependent upon the position of the hydroxy residue. The 5- and 12-hydroxy derivatives were potent aggregating agents, inducing half-maximal responses at 200 and 40 nM, respectively. Their bioactions required extracellular calcium and magnesium. And the response to both fatty acids was effectively blocked by three inhibitors of cellular arachidonic acid metabolism: nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, and indomethacin. The 5- and 12- hydroxyeicosatetraenoic acids, therefore, may induce neutrophils to metabolize their endogenous arachidonate. Alternatively, the two hydroxy acids themselves may be further metabolized through pathways inhibited by arachidonate antimetabolites into a final mediator(s) of aggregate formation.

Identification of leukotriene C-1 as a major component of slow-reacting substance from rat mononuclear cells.

Slow-reacting substance (SRS) was produced by rat peritoneal mononuclear cells after stimulation with the ionophore A23187. The SRS consisted of two main components as judged by high-pressure liquid chromatography (HPLC) on Florisil. The larger and more polar component consisted mainly of leukotriene (LT) C-1 as judged by ultraviolet spectroscopy, mass spectrometry (after desulfurization), amino acid analysis, and conversion by soybean lipoxygenase. Comparisons with authentic LTC-1 showed identity on reverse phase HPLC and guinea pig ileum bioassay. The latter methods are known to distinguish between LTC-1 and stereoisomers of LTC-1.