Silvio Sosa

Topical anti-inflammatory activity of Salvia officinalis L. leaves: the relevance of ursolic acid.

Salvia officinalis L. leaves, obtained from four plant populations of different origin, were investigated for their topical anti-inflammatory properties. The n-hexane and the chloroform extracts dose-dependently inhibited the Croton oil-induced ear oedema in mice, the chloroform extracts being the most active. By contrast, the methanol extracts showed a very low effect and the essential oil was inactive. Chemical and pharmacological investigation of the most potent chloroform extract, issued from an autochthonous sage population grown in the submediterranean climatic region of Slovenia, revealed ursolic acid as the main component involved in its anti-inflammatory activity. The anti-inflammatory effect of ursolic acid (ID50 = 0.14 microMoles/cm2) was two fold more potent than that of indomethacin (ID50 = 0.26 microMoles/cm2), which was used as a reference non-steroidal anti-inflammatory drug (NSAID). The content of ursolic acid in sage and sage-based remedies for the topical treatment of inflammatory diseases is proposed as a parameter for quality control purposes.

In vivo anti-inflammatory and in vitro antioxidant activities of Mediterranean dietary plants.

Five hydroalcoholic extracts of edible plants from Calabria region (Italy) used in local traditional medicine for the treatment of inflammatory diseases were evaluated for their in vivo topical anti-inflammatory activity (inhibition of croton oil-induced ear oedema in mice) and in vitro antioxidant and antiradical properties (inhibition of linoleic acid oxidation and bovine brain liposomes peroxidation, DPPH radical scavenging). All the extracts showed an anti-inflammatory effect: 300 microg/cm(2) provoked oedema reductions ranging from 21 to 27%. All the extracts exerted also radical scavenging and/or antioxidant properties, the most active plant being Mentha aquatica L. (Lamiaceae) which contained the highest amount of phenolics (337 mg/g) and of flavonoids (15.75 mg/g). Moreover, the content and the composition of sterols were assessed by GC-MS in the examined plants Borago officinalis L. (Boraginaceae) contained the highest number of sterols.

Screening of the topical anti-inflammatory activity of some Central American plants.

Hexane, chloroform and methanol extracts of seven herbal drugs used in the folk medicine of Central America against skin disorders (Aristolochia trilobata leaves and bark, Bursera simaruba bark, Hamelia patens leaves, Piper amalago leaves, and Syngonium podophyllum leaves and bark) were evaluated for their topical anti-inflammatory activity against the Croton oil-induced ear oedema in mice. Most of the extracts induced a dose-dependent oedema reduction. The chloroform extract of almost all the drugs exhibited interesting activities with ID(50) values ranging between 108 and 498 micro g/cm(2), comparable to that of indomethacin (93 micro g/cm(2)). Therefore, the tested plants are promising sources of principles with high anti-inflammatory activity.

A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels

The specific inhibitory activity exerted by okadaic acid on protein phosphatase 2A was used to assess the presence of okadaic acid in mussels, using a commercially available protein phosphatase 2A preparation. Under the conditions used, okadaic acid inhibits the enzymatic activity dose-dependently, with an IC50 = 0.26 ng/ml (0.32 nM). The assay is accurate and reproducible. Okadaic acid was detected in concentrations as low as 0.063 ng/ml in aqueous solutions and 2 ng/g in mussel digestive glands. Thirty naturally contaminated mussel samples were submitted to the protein phosphatase 2A inhibition assay as well as to an ELISA assay and to a MTT cytotoxicity assay, with similar results. The proposed assay is sensitive, rapid and does not require expensive equipment. These characteristics make it a good candidate for employment in the routine assessment of okadaic acid shellfish contamination.

Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice

The acute toxicity of yessotoxin (YTX), homoyessotoxin (homoYTX) and 45-hydroxy-homoyessotoxin (45-OH-homoYTX) has been studied in comparison to that of okadaic acid (OA), the main diarrhogenic toxin, both after intraperitoneal (i.p.) and oral administration. After i.p. administration, homoYTX and YTX showed similar lethality (LD(50)=444 microg/kg and 512 microg/kg), higher than that of OA (LD(50)=225 microg/kg), while 750 microg/kg of 45-OH-homoYTX did not cause death. OA induced the already known toxic signs: before death, mice were motionless and cyanotic; small intestine and liver damage were shown at post-mortem. Mice treated with YTX and homoYTX were restless and jumped before death; necroscopy did not show major changes. After oral treatment, 2 mg/kg of OA induced diarrhoea and body weight loss, causing 4/5 deaths; necroscopy and/or histology revealed degenerative lesions to small intestine, forestomach and liver (confirmed by increased plasma transaminase), but no myocardium alterations. On the contrary, the oral treatment with YTX (1 and 2 mg/kg) and its derivatives (1 mg/kg) did not cause any death or signs of toxicity, except some ultrastructural myocardiocyte alterations, adjacent to capillaries, such as cytoplasmic protrusions (YTX, 1 and 2 mg/kg), fibrillar alteration (YTX, 1 mg/kg) or mitochondria assemblage (45-OH-homoYTX). Altogether, our data show that YTX and its derivatives are less toxic than OA after acute oral and i.p. treatments, at doses which may represent up to 100 times of the possible human daily intake.

Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.)

Separation and isolation of the genuine faradiol esters (1, 2) from flower heads of Marigold (Calendula (officinalis L., Asteraceae) could be achieved by means of repeated column chromatography (CC) and HPLC for the first time. Structure elucidation of faradiol-3-myristic acid ester 1, faradiol-3-palmitic acid ester 2 and psi-taraxasterol 3 has been also performed, without any previous degradation by means of MS, 1H-NMR, 13C-NMR and 2D-NMR experiments. The anti-oedematous activities of these three compounds were tested by means of inhibition of Croton oil-induced oedema of the mouse ear. Both faradiol esters showed nearly the same dose dependent anti-oedematous activity and no significant synergism appeared with their mixture. The free monol, psi-taraxasterol, had a slightly lower effect. Furthermore, faradiol was more active than its esters and than psi-taraxasterol and showed the same effect as an equimolar dose of indomethacin.

Characterization of Topical Antiinflammatory Compounds in Rosmarinus officinalis L.

The topical antiinflammatory activity of three extracts at increasing polarity (n-hexane, chloroform, and methanol) from the leaves of Rosmarinus officinalis L. (Labiatae) has been tested using the croton oil ear test in mice. Both the n-hexane and the chloroform (CE-1) extracts from the leaves showed a dose-dependent activity, the last one possessing an antiinflammatory potency similar to that of indomethacin, the nonsteroidal antiinflammatory drug used as a reference drug (ID50 = 83 and 93 microg/cm2, respectively). The bioassay-oriented fractionation of CE-1 led to the identification of tritepenes, ursolic acid, oleanolic acid, and micromeric acid as the main antiinflammatory principles. Furthermore, the CE-1 extract obtained from the residue of the steam distillation of the leaves (extract A) showed the same antiinflammatory potency of CE-1, suggesting this waste product as a source of antiinflammatory products.

Yessotoxins: A toxicological overview

Yessotoxins (YTXs) are polyciclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter feeding shellfish. These toxins can be ingested by humans through contaminated seafood consumption. Initially, YTXs were classified as Diarrhetic Shellfish (DS) toxins but the biological activity of these compounds, which lack of diarrheogenic effects, differs from that of diarrheic toxins. Thus, YTXs have been recently classified as a separate group of algal toxins. Yessotoxin (YTX), homoyessotoxin and 45-hydroxy-homoyessotoxin are lethal after intraperitoneal injection to mice but not after single or repeated oral administration. The target organ seems to be the cardiac muscle cells, where these toxins induce light and electron microscopy ultrastructural changes not only after intraperitoneal injection, but also after oral exposure. On the other hand, di-desulfo-yessotoxin affects liver and pancreas, where it induces fatty degeneration. The mechanisms at the basis of the cardiac effects of YTX and homoyessotoxins are still not completely understood. No short term and chronic toxicity data are available as well as pharmacokinetic studies are lacking. Nevertheless, YTX is known to exert different in vitro activities, such as changes of intracellular calcium and cyclic AMP levels, alteration of cytoskeletal and adhesion molecules, caspases activation and opening of the permeability transition pore of mitochondria. This review reports the current knowledge on the in vivo toxicity and in vitro effects of these toxins.

Topical anti-inflammatory activity of extracts and compounds from Thymus broussonettii

The topical anti‐inflammatory activity of four extracts from Thymus broussonetii Boiss (Labiatae) leaves, a herbal drug used in Moroccan traditional medicine, has been studied using the croton oil ear test in mice. A bioassay‐oriented fractionation revealed that the pharmacological activity is mainly in the chloroform extract. Fractionation and analysis of this extract allowed the identification of ursolic acid and oleanolic acid as the main anti‐inflammatory principles. Some flavonoids (luteolin, eriodictyol, thymonin) and glycosides (luteolin‐7‐O‐glucoside, luteolin‐3′‐O‐glucuronide, eriodictyol‐7‐O‐glucoside) were also isolated from the methanol extract.

Topical anti-inflammatory activity of extracts and compounds from Hypericum perforatum L.

Three preparations of Hypericum perforatum L. (a hydroalcoholic extract, a lipophilic extract and an ethylacetic fraction) and the pure compounds hypericin, adhyperforin, amentoflavone, hyperoside, isoquercitrin, hyperforin dicyclohexylammonium (DHCA) salt and dicyclohexylamine were evaluated for their topical anti‐inflammatory activity. H. perforatum preparations provoked a dose‐dependent reduction of Croton‐oil‐induced ear oedema in mice, showing the following rank order of activity: lipophilic extract > ethylacetic fraction > hydroalcoholic extract (ID50 (dose that inhibited oedema by 50%) 220, 267 and >1000 μg cm−2, respectively). Amentoflavone (ID50 0.16μmol cm−2), hypericin (ID50 0.25μmol cm−2), hyperforin DHCA salt (ID50 0.25μmol cm−2) and adhyperofrin (ID50 0.30μmol cm−2) had anti‐inflammatory activity that was more potent or comparable to that of indometacin (ID50 0.26 μmol cm−2), whereas isoquercitrin and hyperoside were less active (ID50 about 1μmol cm−2). As dicyclohexylamine alone was inactive, the effect of hyperforin DHCA salt can be attributed completely to the phloroglucinol moiety. The pharmacological activity and phytochemical profile of the tested extracts and fraction suggest that different constituents are involved in the topical antiphlogistic property of H. perforatum in‐vivo.