The cytotoxic effect of palytoxin on Caco-2 cells hinders their use for in vitro absorption studies.

Palytoxin (PLTX), found in Palythoa zoanthids and Ostreopsis dinoflagellates, has also been detected in crabs and fish, through which it can enter into the food chain. Indeed, PLTX is considered the causative agent of several cases of human seafood poisoning resulting in systemic symptoms. Available epidemiological data on PLTX human toxicity suggest that the intestinal tract may be one of its in vivo targets and its potential site of access into the bloodstream. Hence, the purpose of this study was to investigate the suitability of the human intestinal Caco-2 cell line for evaluating PLTX oral absorption. A detailed analysis of PLTX cytotoxicity revealed a high sensitivity of Caco-2 cells: 4h toxin exposure reduced mitochondrial activity (MTT assay, EC(50) of 8.9±3.7×10(-12)M), cell density (SRB assay, EC(50) of 2.0±0.6×10(-11)M) and membrane integrity (LDH release, EC(50) of 4.5±1.4×10(-9)M and PI uptake, EC(50) of 1.0±0.8×10(-8)M). After low PLTX concentration (1.0×10(-11)M) exposure for 1-8h, followed by 24h recovery time in toxin-free medium, cell density reduction was only partially reversible. These results indicate that, due to the high susceptibility to PLTX cytotoxic effects, Caco-2 cells do not represent an appropriate and reliable model for investigating intestinal barrier permeation by this toxin.

Screening of the topical anti-inflammatory activity of the bark of Acacia cornigera Willdenow, Byrsonima crassifolia Kunth, Sweetia panamensis Yakovlev and the leaves of Sphagneticola trilobata Hitchcock.

ETHNOPHARMACOLOGICAL RELEVANCE: An investigation of topical anti-inflammatory activity was undertaken on plants used in Central America traditional medicine. AIM OF STUDY: Four herbal drugs used in the folk medicine of Central America to treat inflammatory skin affections (Acacia cornigera bark, Byrsonima crassifolia bark, Sphagneticola trilobata leaves and Sweetia panamensis bark) were evaluated for their topical anti-inflammatory activity. MATERIALS AND METHODS: Petroleum ether, chloroform and methanol extracts were obtained for herbal medicines and then extracts were tested on Croton oil-induced ear dermatitis model in mice. RESULTS: Almost all the extracts reduced the Croton oil-induced ear dermatitis in mice and the chloroform ones showed the highest activity, with ID(50) (dose giving 50% oedema inhibition) values ranging from 112 microg/cm(2) (Byrsonima crassifolia) to 183 microg/cm(2) (Sphagneticola trilobata). As reference, ID(50) of the non-steroidal anti-inflammatory drug indomethacin was 93 microg/cm(2). CONCLUSIONS: Lipophilic extracts from these species can be regarded as potential sources of anti-inflammatory principles.

An extract of Apium graveolens var. dulce leaves: structure of the major constituent, apiin, and its anti-inflammatory properties.

Flavonoids, natural compounds widely distributed in the plant kingdom, are reported to affect the inflammatory process and to possess anti-inflammatory as well as immunomodulatory activity in-vitro and in-vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of the ethanol/water (1:1) extract of the leaves of Apium graveolens var. dulce (celery) on iNOS expression and NO production in the J774.A1 macrophage cell line stimulated for 24 h with Escherichia coli lipopolysaccharide (LPS) were evaluated. The extract of A. graveolens var. dulce contained apiin as the major constituent (1.12%, w/w, of the extract). The extract and apiin showed significant inhibitory activity on nitrite (NO) production in-vitro (IC50 0.073 and 0.08 mg mL(-1) for the extract and apiin, respectively) and iNOS expression (IC50 0.095 and 0.049 mg mL(-1) for the extract and apiin, respectively) in LPS-activated J774.A1 cells. The croton-oil ear test on mice showed that the extract exerted anti-inflammatory activity in-vivo (ID50 730 microg cm(-2)), with a potency seven-times lower than that of indometacin (ID50 93 microg cm(-2)), the non-steroidal anti-inflammatory drug used as reference. Our results clearly indicated the inhibitory activity of the extract and apiin in-vitro on iNOS expression and nitrite production when added before LPS stimulation in the medium of J774.A1 cells. The anti-inflammatory properties of the extract demonstrated in-vivo might have been due to reduction of iNOS enzyme expression.

An extract of Lannea microcarpa: composition, activity and evaluation of cutaneous irritation in cell cultures and reconstituted human epidermis.

Lannea microcarpa (Anacardiaceae) is a tropical tree used in African folk medicine and commercial dermopharmaceutical formulations. Fractionation and analysis of its polar extract allowed the identification of 4'-methoxy-myricetin 3-O-alpha-L-rhamnopyranoside, myricetin 3-O-alpha-L-rhamnopyranoside, myricetin 3-O-beta-D-glucopyranoside, vitexin, isovitexin, gallic acid and epi-catechin, as the major constituents. In-vivo assay (the croton oil ear test in mice) showed that the extract had significant anti-inflammatory effect (ID50 = 900 microg cm(-2)) but ten times lower than that of indometacin (ID50 = 93 microg cm(-2)), the non-steroidal anti-inflammatory drug used as reference. Cytotoxicity and cutaneous irritation of the extract and its constituents were investigated. The crude extract and its major components did not affect cell viability in-vitro either in three different cultures (J774. A1, WEHI-164 and HEK-293) of cells grown in monolayers or in the reconstituted human epidermis (RHE, 3D model), nor did they cause release of pro-inflammatory mediators (IL-1alpha) or histomorphological modification of RHE.

Topical anti-inflammatory activity of Solenostemma argel leaves.

The topical anti-inflammatory activity of Solenostemma argel Hayne leaves was evaluated using the Croton oil ear test in mice. A bioassay-guided fractionation procedure led to a highly active chloroform extract: at 300 microg/cm(2), it induced 73% oedema reduction, while the reference drug indomethacin (100 microg/cm(2)) induced 56% reduction. The extract contained a new pregnene glycoside (1, solenoside A) and the known 14 beta,15 alpha-dihydroxy-Delta(4)pregnene-3,20 dione (2), kaempferol-3-O-glucoside (3) and kaempferol-3-O-rutinoside (4). Their structures were determined by ID and 2D NMR experiments as well as HRMS. Compounds 2 and 4 showed anti-inflammatory activity comparable to that of indomethacin.

Extracts and constituents of Lavandula multifida with topical anti-inflammatory activity.

The topical anti-inflammatory activity of the ethanol and aqueous extracts from the aerial parts of Lavandula multifida L. (Lamiaceae), used in the Moroccan traditional medicine, was investigated by inhibition of the Croton oil-induced ear edema in mice. The biological assay revealed a dose-dependent anti-inflammatory activity for the ethanol extract, while the aqueous one was less active. Bioassay-guided fractionation of the ethanol extract led to identify four triterpenic acids of oleanane series, four pimarane and one iso-pimarane diterpenes, as well as the phenolic monoterpene carvacrol and its glucoside. Some of these compounds revealed an anti-inflammatory activity comparable to that of indomethacin.

In vivo topical anti-inflammatory and in vitro antioxidant activities of two extracts of Thymus satureioides leaves.

Four extracts at increasing polarity were prepared from the leaves of Thymus satureioides Coss. (Labiatae) and assayed for the in vivo topical anti-inflammatory effect using the croton oil ear test in mice, and for in vitro both antioxidant (DPPH degrees test) and anti-bacterial (broth microdilution method) activities. The chloroform extract showed a topical anti-inflammatory activity (ID50=282 microg cm(-2)), only three times lower than that of the reference drug indomethacin (ID50=93 microg cm(-2)) and its active components were identified as ursolic and oleanolic acids. The methanol extract, showing a significant radical-scavenging effect (SC50=14.54 microg), was characterized by the isolation and identification of some flavonoids. On the contrary, the extracts did not show any anti-bacterial effect against four standard aerobial bacteria strains.

Short-term oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice.

A short-term toxicity study after 7 days oral daily administration of yessotoxin (YTX; 2 mg/kg/day), homoYTX (1 mg/kg/day), 45-hydroxy-homoYTX (1 mg/kg/day) and of the main diarrhoetic shellfish toxin okadaic acid (OA; 1 mg/kg/day) was carried out in mice. Symptoms, lethality, food consumption, body and organ weights, gross pathology and histopathology of the main organs and tissues, leukocytes formula as well as plasmatic levels of transaminases, lactate dehydrogenase and creatinine phosphokinase were evaluated. Heart tissue was studied also hystochemically for the presence of apoptotic nuclei and by transmission electron microscopy. No mortality, signs of toxicity or cumulative effects were induced by the repeated oral exposure to YTXs. Only ultrastructural changes in the cardiac muscle cells near the capillaries, such as package of rounded mitochondria and alteration of the cells boundary were observed, without any increase of lactate dehydrogenase, an index of cardiac damage. OA induced diarrhoea, body weight loss, reduced food consumption, and the death of 2/5 mice after 5 days. Necroscopy and/or light microscopy analysis revealed toxic effects mainly at forestomach (ulceration and hyperplasia), liver and, indirectly to body weight loss of mice, atrophic signs in the lymphoid organs and exocrine pancreas. Electron microscopy of heart tissue showed alterations of mitochondria and fibers in myocardiocytes, although no apoptotic change was recorded.

Topical anti-inflammatory activity of Bauhinia tarapotensis leaves.

The topical anti-inflammatory properties of Bauhinia tarapotensis Benth. (Leguminosae) leaves have been studied by the inhibition of the croton oil-induced ear edema in mice. A bioassay-guided fractionation showed an interesting anti-inflammatory activity of the chloroform extract, that justifies the activity of the whole herbal drug. The main anti-inflammatory principles of B. tarapotensis leaves are triterpenic acids of ursane and oleanane series. The antiphlogistic activity of mixtures constituted of two ursane and oleanane isomers with different hydroxylation pattern, in the ratio 2:1, is comparable to that of indomethacin (ID50 ranging from 95 to 147 microg/cm2 and 93 microg/cm2, respectively).

Topical anti-inflammatory activity of a new germacrane derivative from Achillea pannonica.

The topical anti-inflammatory activity of a germacrane derivative [1,4-dihydroxy-germacra-5E-10(14)-diene; DHGD] isolated from Achillea pannonica Scheele (Asteraceae) was investigated employing the Croton oil-induced dermatitis in the mouse ear. Its effects on the oedematous response and on leukocytes infiltration are described. The germacrane derivative significantly inhibited ear oedema in a dose-dependent manner, with an ID(50) of 0.40 micromol/cm(2). DHGD (0.75 micromol/cm(2)) provoked a global inhibition of the oedematous response (61 %) higher than that induced by an equimolar dose of indomethacin (43 %) within 24 hours; the reduction induced by hydrocortisone (0.10 micromol/cm(2)) was 68 %. The effect of DHGD (61 % inhibition) was higher than that of the equimolar dose of indomethacin (51 % inhibition) also on granulocytes recruitment at the site of inflammation. Hydrocortisone (0.10 micromol/cm(2)) reduced the cellular infiltrate by 44 %.

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.

Topical anti-inflammatory activity of Thymus willdenowii.

The topical anti-inflammatory activity of Thymus willdenowii Boiss (Labiatae) leaves, a herbal drug used in Moroccan folk medicine, has been studied using the croton oil ear test in mice. A bioassay-oriented fractionation procedure showed that the activity concentrates in the chloroform extract, which has a potency similar to that of indometacin, the non-steroidal anti-inflammatory drug used as reference (ID50 (dose giving 50% oedema inhibition) = 83 microg cm(-2) and 93 microg cm(-2), respectively). The main compounds responsible for the anti-inflammatory activity of T. willdenowii are ursolic acid and oleanolic acid. The flavonoids luteolin-3'-O-glucuronide and eriodictyol-7-O-glucoside were found for the first time in the genus Thymus.

The antiinflammatory activity of Icacina trichantha tuber.

Five fractions (hexane, chloroform, ethylacetate, methanol and water) of Icacina trichantha tuber were obtained by gradient solvent extraction and tested for their ability to inhibit the Croton oil-induced ear edema in mice. The most active fraction was the chloroform one which significantly inhibited ear edema in a dose-dependent manner, showing an ID50 (dose giving 50% edema inhibition) of 107 micrograms/cm2. The ID50 of the reference drug indomethacin was 93 micrograms/cm2. The chloroform fraction significantly reduced also the carrageenin-induced paw edema in rats, after oral adiminstration: 50, 100 or 200 mg/kg of the fraction reduced the global edematous response by 15, 20 or 34%, whereas 10 mg/kg of indomethacin induced 40% inhibition.

Methodological improvement of the protein phosphatase inhibition assay for the detection of okadaic acid in mussels.

A simplified procedure for the enzyme inhibition assay to measure okadaic acid and DTX-1 in mussels, based on the use of a commercially available enzyme preparation, is presented. The detection limit is 10 ng of toxin per g of digestive glands. Using Certified Reference Material (MUS-2), high accuracy and good precision is demonstrated for contamination levels higher than 32 ng g(-1). Twenty samples can be processed in about 9 h by one operator, at the cost of US$ 10 per sample. Some possibilities for further enhancing the sensitivity and reducing the processing time are discussed and a monitoring example is presented.

Anti-inflammatory activities of hypocretenolides from Leontodon hispidus.

Hypocretenolides, a small group of sesquiterpene lactones with an unusual ring structure, are constituents of a small number of species from the Lactuceae tribe (Asteraceae). Three biogenetically closely related 14-hypocretenolides from Leontodon hispidus L. were investigated for a putative anti-inflammatory activity. 14-Hydroxyhypocretenolide-beta-D-glucoside-4'-14"-hydroxyhypocr etenoate significantly exhibited in vivo anti-inflammatory activity in the croton oil-induced mouse ear edema. To obtain first information regarding the molecular targets which might be affected by this constituent, two in vitro bioassays were performed: (i) DNA binding activity of the transcription factor NF-kappa B was evaluated by electrophoretic mobility shift assay (EMSA) using TNF-alpha-activated Jurkat T cells and (ii) nitrite accumulation in cell culture supernatants of LPS-activated RAW 264.7 macrophages was determined as a parameter for inducible nitric oxide synthase (iNOS)-dependent nitric oxide release. In order to gain information about structure-activity relationships, additionally the aglycone 14-hydroxyhypocretenolide and its D-glycoside were investigated in these in vitro systems. 14-Hydroxyhypocretenolide-beta-D-glucoside-4'-14"-hydroxyhypocr etenoate as well as its aglycone exhibited activity in both test systems, whereas the D-glucoside was not or only weakly active.

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.

Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels.

The suitability of a cytotoxicity assay based on the MTT colorimetric method has been evaluated for the detection of okadaic acid in mussels. On KB cells, okadaic acid exhibited a dose-dependent cytotoxic effect, the IC50 being inversely related to the exposure time (IC50 = 6.3 ng/ml, 4.0 ng/ml and 1.1 ng/ml after 24, 48 and 72 hr of contact, respectively). Using a contact time of 24 hr, the MTT cytotoxicity assay is suitable for revealing okadaic acid concentrations in mussel samples as low as 50 ng/g of digestive glands, with a sensitivity higher than that of the commercially available kits for enzyme-linked immunosorbent assay (ELISA). In the okadaic acid concentration range from 50 to 1500 ng/g of digestive glands the MTT cytotoxicity assay showed satisfactory accuracy and reproducibility. A high degree of correlation was found between the okadaic acid content of 16 naturally contaminated samples measured by the MTT cytotoxicity assay and by an ELISA.

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.

The role of triterpenoids in the topical anti-inflammatory activity of Calendula officinalis flowers.

By means of a bioassay-oriented fractionation of the CO2 extract of Calendula flowers, the triterpenoids are shown as the most important anti-inflammatory principles of the drug. Among them, the faradiol monoester appears to be the most relevant principle for the activity of the drug, due to its quantitative prevalence. The unesterified faradiol, not present in the extract, is the most active of the tested compounds and equals indomethacin in activity, whereas the monools psi-taraxasterol, lupeol, taraxasterol, and beta-amyrin are less active than the free diol. The anti-inflammatory activity of different CO2 extracts is proportional to their content of faradiol monoester, which can be taken as a suitable parameter for the quality control of Calendula preparations.

A comparison of methods for diarrhoeic shellfish poison detection.

Samples of diarrhoeic shellfish poison (DSP) mussels from several parts of the Italian Adriatic coastline were extracted and tested according to a number of different methods presently available, i.e. Yasumoto's mouse biotest, Kat's biotest, the ELISA test and the HPLC method. Results were compared for toxic levels detected in each sample. While a common qualitative result (toxic/non-toxic) was given by all the methods, no clear quantitative agreement was found. The differences between methods and consequent lack of agreement in results are discussed.