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Fractionation of the aqueous extract of Derris scandens stems extract using tests for eicosanoid inhibition resulted in the isolation of three isoflavonoids, genistein, its 7- O-alpha-rhamno(1-->6)-beta-glucosyl glycoside, a new compound, and two known isoprenyl derivatives 3'-gamma,gamma-dimethylallylweighteone and scandenin. The isoprenylated compounds showed a high inhibitory effect on eicosanoid production in vitro but HPLC analysis showed that the genistein accounted for most of the activity of the total extract. Antioxidant studies showed that genistein and the isoprenylated compounds showed activity comparable to standard antioxidants. Genistein and its glycoside demonstrated no cytotoxicity in the MTT test but the prenylated compounds showed some toxicity and also increased LDH release from polymorphonucleocytes, at concentrations much greater than would be encountered in an aqueous extract of D. scandens.

Salvia lavandulaefolia Vahl. (Spanish sage) essential oil and individual monoterpenoid constituents have been shown to inhibit the enzyme acetylcholinesterase in-vitro and in-vivo. This activity is relevant to the treatment of Alzheimer's disease, since anticholinesterase drugs are currently the only drugs available to treat Alzheimer's disease. Other activities relevant to Alzheimer's disease include antioxidant, anti-inflammatory and estrogenic effects. Results of in-vitro tests for these activities are reported here for S. lavandulaefolia extracts, the essential oil and its major constituents. Antioxidant activity (inhibition of bovine brain liposome peroxidation) was found in the EtOH extract of the dried herb (5 mg mL−1) and the monoterpenoids (0.1 M) α- and β-pinene and 1,8-cineole. Thujone and geraniol had lower antioxidant effects, while camphor had no antioxidant effects. Possible anti-inflammatory activity (eicosanoid inhibition in rat leucocytes) was found in the EtOH extract (50 μg mL−1) and was shown by the monoterpenoids α-pinene and geraniol (0.2 mM), but not 1,8-cineole, thujone or camphor. Possible estrogenic activity (via induction of β-galactosidase activity in yeast cells) was found in the essential oil (0.01 mg mL−1) and the monoterpenoid geraniol (0.1–2 mM). 1,8-Cineole, α- and β-pinene and thujone did not exhibit estrogenic activity in this analysis. These results demonstrate that S. lavandulaefolia, its essential oil and some chemical constituents have properties relevant to the treatment of Alzheimer's disease and provide further data supporting the value of carrying out clinical studies in patients with Alzheimer's disease using this plant species.

We have used tachykinin neurokinin-1 receptor (NK1 receptor) knockout mice to learn of the link between NK1 receptors and neutrophil accumulation in normal naive skin, as compared with inflamed skin. Intradermal substance P (300 pmol) induced edema formation in wild-type mice, but not in NK1 knockout mice, as expected. However, in contrast to IL-1β (0.3 pmol), substance P did not induce neutrophil accumulation in wild-type mice. IL-1β-induced neutrophil accumulation was similar in wild-type and knockout mice, but a significant (p < 0.05) contributory effect of added NK1 agonists, which by themselves have no effect on neutrophil accumulation in normal skin, was observed. The results support the concept that NK1 agonists such as substance P cannot act on their own to mediate neutrophil accumulation in naive skin and provide direct evidence that in inflamed skin, under certain circumstances, the NK1 receptor can play a pivotal role in modulating neutrophil accumulation during the ongoing inflammatory process. We investigated responses to two inflammatory stimuli (carrageenin and zymosan). Neutrophil accumulation was significantly attenuated (p < 0.001) in carrageenin- but not zymosan-induced inflammation in NK1 knockout mice. The carrageenin (500 μg)-induced response was inhibited (p < 0.05) by a NK1 receptor antagonist, SR140333 (480 nmol/kg i.v. at −5 min), in the wild-type group. The bradykinin B1 and B2 receptor antagonists (desArg9[Leu8]bradykinin and HOE 140) each reduced neutrophil accumulation to carrageenin in wild-type animals (p < 0.05), but did not cause further reduction of the suppressed response of knockout mice. The results provide evidence that kinin receptors participate in NK1 receptor-dependent neutrophil accumulation in inflamed mouse skin.

Low density lipoproteins (LDL) can be oxidatively modified in vitro by macrophages and certain other cell types so that macrophages will take them up much faster. This process may be important in the formation of cholesterol-laden foam cells derived from macrophages in atherosclerotic lesions. In this study, we have shown that certain flavonoids, plant constituents found in the diet, are potent inhibitors of the modification of 125I-labelled LDL by macrophages, with IC50 values in the micromolar range (e.g. morin and fisetin 1 microM; quercetin and gossypetin 2 microM). The potencies of individual flavonoids in inhibiting LDL modification did not correlate with their previously determined potencies as inhibitors of 5-lipoxygenase and cyclo-oxygenase. The modification of LDL by macrophages exhibits a lag period of about 4-6 hr before enhanced uptake is detected. During this time, there is a rapid depletion in its content of alpha-tocopherol (an endogenous antioxidant found in lipoproteins) followed by a large increase in the level of hydroperoxides. The flavonoids conserved the alpha-tocopherol content of LDL and delayed the onset of detectable lipid peroxidation. Flavonoids also inhibited the cell-free oxidation of LDL mediated by CuSO4. These findings raise the possibility that flavonoids may protect LDL against oxidation in atherosclerotic lesions and may therefore be natural anti-atherosclerotic components of the diet, although this will depend to a large extent on their pharmacokinetics.

The major phenolics from the polar fraction of virgin olive oil (caffeic acid, oleuropein, tyrosol and hydroxytyrosol) have well-established antioxidant activities but their effects on reactive nitrogen species and nitrergic neurotransmission have not been fully investigated. The three catechol compounds were active as scavengers of nitric oxide generated spontaneously from the decomposition of sodium nitroprusside (approximately 50% inhibition achieved at 75 microM), and had similar ability to scavenge chemically generated peroxynitrite, as determined by an alpha1-antiproteinase inactivation assay (67.2%-92.4% reduction when added at 1 mM). Tyrosol was less active in these tests, but does not possess the catechol functionality. Despite their ability to interact with chemically prepared nitric oxide, neither oleuropein nor hydroxytyrosol at 5 microM altered NO*-mediated relaxations of the nerve-stimulated rat anococcygeus preparation, but this may be because the nitrergic transmitter is protected from the effects of externally applied scavengers. In conclusion, the phenolics found in virgin olive oil possess ability to scavenge reactive oxygen and nitrogen species that are implicated in human pathologies, but their impact may be restricted to those species present in the extracellular environment., We thank the “Comisión Interministerial de Ciencia y Tecnología” (CICYT) and the “Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria” (INIA) for partial financial support of this work ( Projects ALI99-0863 and CAO99-006 respectively).

Interest in the health-promoting effects of virgin olive oil, an important part of the ‘Mediterranean diet’, prompted us to determine the anti-eicosanoid and antioxidant effects in leukocytes of the principal phenolic compounds from the ‘polar fraction’: oleuropein, tyrosol, hydroxytyrosol, and caffeic acid. In intact rat peritoneal leukocytes stimulated with calcium ionophore, all four phenolics inhibited leukotriene B4 generation at the 5-lipoxygenase level with effectiveness hydroxytyrosol > oleuropein > caffeic acid > tyrosol (approximate ec50 values: 15, 80, 200, and 500 μM, respectively). In contrast, none of these compounds caused substantial inhibition of thromboxane generation via the cyclo-oxygenase pathway. Hydroxytyrosol, caffeic acid, oleuropein, and tyrosol (decreasing order of effectiveness) also quenched the chemiluminescence signal due to reactive oxygen species generated by phorbol myristate acetate-stimulated rat leukocytes. None of these compounds were toxic to leukocytes at the concentrations tested. We conclude that the phenolics found in virgin olive oil possess an array of potentially beneficial lipoxygenase-inhibitory, prostaglandin-sparing, and antioxidant properties., We thank the Spanish Ministry of Education for a Research Fellowship awarded to R. de la Puerta and the Spanish Interministerial Commission of Science and Technology (CICYT) for partial financial support of this work (Projects ALI95-0073 and ALI96-0456).

The involvement of prostaglandins in feedback modulation of the lipopolysaccharide-inducible isoforms of cyclooxygenase and nitric oxide was studied. This was done by testing the effects of arachidonic acid and indomethacin in the murine macrophage cell line J774. When added before lipopolysaccharide, arachidonic acid (10−6–10−4 M) dose-dependently reduced inducible nitric oxide synthase and cyclooxygenase-2 activity as well as reducing the expression of both enzyme proteins. Indomethacin (10−7–10−4 M) suppressed prostaglandin E2 release into the medium and reduced cyclooxygenase-2 activity irreversibly, as expected, but the amount of cyclooxygenase-2 protein was increased (because indomethacin removes the inhibitory effect of prostaglandins on cyclooxygenase-2 expression). However, the same doses of indomethacin also inhibited the release of nitric oxide into the medium. This was accompanied by a reduction in the amount of inducible nitric oxide synthase protein. The drug was not cytotoxic. Thus these results show that pharmacological treatments can have opposing actions on inducible nitric oxide synthase and cyclooxygenase-2 induction, with indomethacin showing an additional unexpected action in macrophages which may diminish their cytotoxic potential.

Myricetin and gossypetin, two hexahydroxylated flavonoids, are capable of modifying low density lipoprotein (LDL) to increase greatly its uptake by macrophages. When human 125I-labelled LDL was incubated with 100-1000 microM myricetin or gossypetin, it was subsequently endocytosed much faster by mouse peritoneal macrophages. This modification did not occur at a concentration of 10 microM. Nine other flavonoids containing up to five hydroxyl substituents did not modify LDL to any great extent at 100 microM. The modification of LDL by 100 microM myricetin was time-dependent and complete by 6 hr. Flavonoids can sometimes act as pro-oxidants but myricetin did not act by oxidizing the LDL, as the LDL lipid hydroperoxide content was not increased by myricetin, nor did it promote the depletion of the endogenous antioxidant alpha-tocopherol in the LDL. High concentrations of myricetin caused the aggregation of LDL particles, as judged by light microscopy, agarose gel electrophoresis, retention by a membrane filter and sedimentability by centrifugation. SDS-PAGE indicated that the apolipoprotein B-100 molecules of LDL particles were covalently crosslinked. The uptake and degradation by macrophages of myricetin-modified 125I-labelled LDL reached saturation at about 10 micrograms protein/mL, suggesting the existence of a high affinity uptake process for the modified LDL. The uptake of myricetin-modified 125I-labelled LDL was not competed for by a large excess of non-labelled native LDL or acetylated LDL. We conclude that myricetin and gossypetin at high concentrations are capable of modifying LDL by a novel non-oxidative mechanism to a form taken up by macrophages by a high affinity process.