Isolation and characterization of irritant components of Euphorbia pilulifera L.

Euphorbia pilulifera is commonly found weed along road sides and loamy soils. This weed is commonly used as treatment of female disorders and respiratory problems. The latex of this weed causes irritation on hand on contact. To evaluate its irritant potentials, the dermatological investigation of irritant principles from locally occurring Euphorbia pilulifera was carried out. For this purpose, after collection and drying, a series of solvents with increasing polarity were used for the successive extraction of non-polar compounds (petroleum ether extract), constituents of intermediate polarities (chloroform extract) and polar constituents (methanol extract) from the whole herb of Euphorbia pilulifera. The chloroform extract of this weed was found most irritant to rabbit ' s skin. Chloroform extract was further subjected to column chromatography; four fractions Ep 1 to Ep 4 were isolated from active chloroform extract by column and thin layer chromatography. The irritant potentials of these isolated fractions were evaluated on rabbit 's skin. Two fractions out of the four, Ep 1 and Ep 3 appeared to be the most irritant than others. A possible structure activity relationship of these active compounds was discussed in order to establish their activity.

Irritant and co-carcinogenic diterpene esters from the latex of Euphorbia cauducifolia L.

Ten (1-10) irritant and mild co-carcinogenic diterpene esters were isolated from the latex of Euphorbia cauducifolia L. using bioassay-guided countercurrent distribution and other chromatographic techniques. The isolated compounds were characterized on the basis of spectroscopic results and mass measurements. As an outcome, the ingenane-type esters were established with the following structures: 3-O-angeloyl-17-O-palmatoylingenol (1), 3-O-palmatoyl-5-O-angeloylingenol (2), 5-O-angeloyl-17-O-palmatoylingenol (3), 3-O-angeloyl-5-O-palmatoylingenol (4), 17-O-(2Z,4E,6Z)-2,4,6-tetradecatrienoyl-20-O-palmatoylingenol (5), 5-O-angeloyl-17-O-benzoylingenol (6), 5-O-angeloyl-17,20-diacetoxyingenol (7), 3-O-angeloyl-17-O-benzoyl-20-acetoxyingenol (8), 3-acetoxy-5-O-angeloyl-17-O-benzoylingenol (9), and 5-O-angeloyl-3,17,20-triacetoxyingenol (10). Their biological screening revealed that they are moderate irritants, and low to moderate tumor promoters compared to TPA, but hardly showed any solitary carcinogenic activity. The isolated esters represent new compounds and were not reported before from any source.

Composition of the fresh leaves and stems of Melissa officinalis and evaluation of skin irritation in a reconstituted human epidermis model.

The composition of a centrifuged product obtained from the fresh leaves and stems of Melissa officinalis and skin irritation in the reconstituted human epidermis (Episkin model) have been investigated in comparison to the EtOH-H(2)O (1:1) extract obtained by Soxhlet from the dried plant. Two new sulfated triterpenes (1 and 2) and two ionol derivatives have been isolated for the first time from Melissa officinalis together with caffeic and rosmarinic acids. The structures of compounds 1 and 2 were established by analysis of their spectroscopic data. Both the centrifuged material and its major constituents neither affected cell viability nor caused the release of pro-inflammatory mediators or the decrease of trans-epithelial electrical resistance (TEER) in the reconstituted human epidermis.

Anti-tumor 12-deoxyphorbol esters from Euphorbia cornigera.

Nine (1-8 and 10) new and two (9 and 11) known compounds have been isolated from roots of Euphorbia cornigera Boiss. Their structure and relative stereochemistry were acquired through NMR ((1)H, (13)C, COSY-45, HOHAHA, HMQC, HMBC, NOE and HMBC) spectroscopic measurements. Compounds 1-10 were identified as diesters of 13,20-O-diacyl and 11 as 13-O-acetyl of 12-deoxyphorbol. Cytotoxic activity of the compounds was investigated on human KB cells by reduction of MTT. Compounds 8-10 displayed IC(50) of 0.8, 0.5, and 1.0 microg mL(-1), respectively, whereas the activity of rest of the compounds (1-7) was either very low or (11) zero even up to 1000 microg mL(-1). The inhibition of DNA synthesis through Trypan blue exclusion and Brd-U assay was investigation to figure out the role of compounds 8-10 and concluded that these were responsible for the death of KB cells. Significant correlation has been found between the cytotoxicity and DNA cross-link and DNA strand-break formation.

Chemistry and biological activity of secondary metabolites in Euphorbia from Iran.

The chemical constituents of some species of Euphorbia, which grow mostly in semi-desert areas in Iran and on the Alborz Mountains in the north of Tehran, have been found to include chemotaxonomically important myrsinane diterpenoids and cycloartane triterpenoids. The Euphorbia plants collected in province of Azarbaijan in the northwestern part of Iran contained mostly derivatives of skin-irritating ingenol esters. Some of the diterpenoids with myrsinane carbon skeleton inhibited enzymes such as alpha-glycosidase, urease, HIV-1 reverse transcriptase, and prolyl endopeptidase. They also showed analgesic and DNA-damaging activities. The present review describes the chemistry and biological activity of several compounds isolated from different species of Iranian Euphorbia: diterpenoids with myrsinane skeletons, flavonoids, tannins, alkanes, sterols, mono-, sesqui- and triterpenoids, skin-irritating and tumor-promoting latexes and their active ingenol diterpenoids.

(R)-(+)-palasonin, a cantharidin-related plant toxin, also occurs in insect hemolymph and tissues.

Gas chromatographic and mass spectroscopic analyses of extracts of cantharidin-containing meloid, clerid, and staphylinid beetles revealed the presence of minor to significant amounts of palasonin, previously only known from seeds and fruits of the Indian shrub Buteafrondosa (Leguminaceae). Unlike (S)-(-)-palasonin (> 99% ee) from B. frondosa, the insects produce palasonin of low ee with the (R)-(+)-enantiomer (0-50% ee) prevailing. The ee of palasonin from individual specimens of predatory insects (Trichodes apiarius), which acquire their chemical protection from cantharidin-producing insects, may vary considerably. The absolute configuration of (S)-(-)-palasonin, previously deduced from indirect chemical and spectroscopic methods, was confirmed by X-ray crystal structure analysis of a cyclic imide derived from (S)-(-)-palasonin and (S)-(-)- 1 -(4-nitrophenyl)-ethylamine.

Irritant potential of triterpenoids from Ficus carica leaves.

The irritant potential of total methanolic extract and five triterpenoids newly isolated from the leaves of Ficus carica investigated by open mouse ear assay. Total methanolic extract, calotropenyl acetate, methyl maslinate and lupeol acetate showed potent and persistent irritant effects.

Cytotoxic effects of cantharidin on the growth of normal and carcinoma cells.

Cantharidin is isolated from Mylabris phalerata Pallas and is a potent inhibitor of hepatocellular carcinoma cells (Hep 3B cells). In the present study, the IC(50) values of cantharidin on Hep 3B cells and normal Chang liver cells were found to be 2.2 and 30.2 microM for 36 h, respectively. Furthermore, cantharidin-treated Hep 3B cells induced cell death within 1 h (IC(50)=52.8 microM), suggesting that cantharidin is an acute cytotoxic agent. We found that although cantharidin could induce cell death, it could not directly inhibit the activity of nucleic acid biosynthesis by the cellular incorporation of 3H-thymidine, 3H-uridine or 3H-leucine. Cantharidin-treated Hep 3B cells showed no evidence of major alterations in the cell cycle distribution within 1 h. However, examination of cells after treatment for 36 h showed that cantharidin regulated the cell cycle at the G(2)/M phase. Moreover, the treated Hep 3B cells had a rounded and shrunken appearance. The microvilli of treated Hep 3B cells were reduced in number and replaced by numerous blebs. Other ultrastructural changes following cantharidin treatment included the presence of lipid droplets, swelling of the mitochondria and accumulation of glycogen particles. The findings of damaged mitochondria in the cantharidin treated Hep 3B cells in this study suggest that cantharidin can induce acute and lethal toxic effects on Hep 3B cells by inhibiting the mitochondria energy system. In conclusion, this study had demonstrated that cantharidin could inhibit progression of all phases of the Hep 3B cell cycle.

Jatrophane diterpenoids from Euphorbia peplus.

From the pro-inflammatory active extract of Euphorbia peplus, a new diterpene polyester (1) based on the jatrophane skeleton was isolated together with the known compounds 2-5. The irritant activities of some jatrophane diterpenes (2, 3 and 6-9) were also investigated: only compound 2 was found to exert a weak pro-inflammatory activity on mouse ear.

Development of a flow cytometry assay for the identification and differentiation of chemicals with the potential to elicit irritation, IgE-mediated, or T cell-mediated hypersensitivity responses.

These studies were conducted to investigate the potential use of a flow cytometric analysis method for the identification and differentiation of chemicals with the capacity to induce irritation, IgE- or T cell-mediated hypersensitivity responses. An initial study investigated the ability of equally sensitizing concentrations (determined by local lymph node assay) of IgE-mediated (Toluene Diisocyanate-TDI) and T cell-mediated (Dinitrofluorobenzene-DNFB) allergens to differentially modulate the IgE+B220+ population in the lymph nodes draining the dermal exposure site. Sodium lauryl sulfate (SLS) was also tested as a nonsensitizing irritant control. Female B6C3F1 mice were dermally exposed once daily for 4 consecutive days, with the optimum time point for analysis determined by examining the IgE+B220+ population 8, 10, and 12 days post-initial chemical exposure. At the peak time point, day 10, the IgE+B220+ population was significantly elevated in TDI (41%), while moderately elevated in DNFB (18%) exposed animals when compared to the vehicle (0.8%), and remained unchanged in SLS (2.2%) exposed animals when compared to the ethanol control (2.5%). Experiments in our laboratory and others have demonstrated that the draining lymph node B220+ population becomes significantly elevated following exposure to allergens (IgE- and T cell-mediated), not irritants, allowing for their differentiation. An existing mouse ear swelling assay was used to identify chemical irritants. Therefore, using the endpoints of percent ear swelling, percent B220+ cells, and percent IgE+B220+ cells, a combined irritancy/phenotypic analysis assay was developed and tested with tetradecane (irritant), toluene diisocyanate, trimellitic anhydride (IgE-mediated allergens), benzalkonium chloride, dinitrofluorobenzene, oxazolone, and dinitrochlorobenzene (T cell-mediated allergens) over a range of concentrations. Based upon the pattern of response observed, a paradigm was developed for continued evaluation: Irritant exposure will result in significant ear swelling without altering the B220+ or IgE+B220+ populations. Exposure to sensitizers (IgE-mediated or T cell-mediated) will increase the B220+ population and the percent ear swelling will remain unchanged or will significantly increase, depending on the irritancy capacity of the chemical. Both the IgE+B220+ and B220+ populations will become elevated at the same test concentration following exposure to IgE-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will be equal to the percent of B220+ cells. The B220+ population will increase at a lower test concentration than the IgE+B220+ population, following exposure to T cell-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will reach less than half that of the percent of B220+ cells. The irritancy/phenotypic analysis method may represent a single murine assay able to identify and differentiate chemicals with the capacity to induce irritation, or IgE-mediated or T cell-mediated responses.

Formaldehyde allergy: A follow-up study.

BACKGROUND: Formaldehyde is a commonly used preservative in cosmetic products, cleaning agents and industrial products, and sensitization to formaldehyde frequently occurs. OBJECTIVE: The aim of the study was to evaluate the effect of extensive information and exposure assessment of formaldehyde on the prognosis of eczema. METHODS: The present study was designed as a clinical follow-up study of formaldehyde-sensitive patients. 105 patients diagnosed as formaldehyde allergic in the period January 1 1990 to December 31 1994 were thoroughly informed about their allergy, and exposure assessment was performed as a formaldehyde analysis on the patients' products. RESULTS: At follow-up 1 to 5 years later, 57 patients accepted the invitation to attend the outpatient clinic for an interview and a skin examination by a dermatologist, and were also at this time asked to bring products to which they were currently exposed for formaldehyde analysis. Evaluated by reported number of eczema eruptions and from objective skin examination patients had generally improved from their first visit to the department. At follow-up, 38 of 49 patients bringing products for analysis were still exposed to formaldehyde, as assessed by formaldehyde analysis on their products. There was a trend that severe eczema was found more often in patients still exposed to formaldehyde, but this finding was not statistically significant. Thirty-seven patients reported that they "paid attention to their allergy" when buying and using cosmetics or consumer products. CONCLUSION: Patients who paid attention to their allergy had statistically significantly fewer eruptions than those who did not.

Demonstration of the contact sensitizer p-tert-butylcatechol in p-tert-butylphenol formaldehyde resin.

BACKGROUND: In regard to patients hypersensitive to p-tert-butylphenol formaldehyde resin (PTBP-F-R), it is for diagnostic, therapeutic, and preventive reasons necessary to know the identity of the primary sensitizing substances and their sensitizing capacities, as well as their cross-reaction patterns. In patients hypersensitive to PTBP-F-R, we have demonstrated a statistically significant overrepresentation of simultaneous reactions to p-tert-butylcatechol (PTBC). In the guinea pig, we have shown that PTBC is a strong sensitizer, giving cross reactions to p-tert-butylphenol. Furthermore, PTBC cross reacts to the PTBP-F-R monomer 2,6-methylol p-tert-butylphenol, which is a strong sensitizer in the guinea pig. OBJECTIVE: The aim of this study was to investigate the background to the observed simultaneous reactions to PTBP-F-R and PTBC and to see if these could be explained by the presence of PTBC in PTBP-F-R. METHODS: High pressure liquid chromatography (HPLC), nuclear magnetic resonance spectrometry (NMR), mass spectrometry (MS). RESULTS: PTBC was isolated and identified; the concentrations in two resins were determined. HPLC analyses showed the presence of 0.099% and 0.020% wt/wt PTBC, respectively. CONCLUSION: This study shows that PTBC can be present in at least in some brands of PTBP-F-R. The study also indicates that a positive patch test reaction to PTBP-F-R could be an indication of allergy to PTBC, at least in patients with high sensitivity to PTBC. When detecting contact allergies to PTBP-F-R in a patient for whom no clinically relevant exposure to this resin can be found, the possibility of PTBC as the eliciting factor should be considered.

Improved access to highly unsaturated skin irritants of the daphnane type from latex of Excoecaria oppositifolia.

A new and more efficient procedure was developed to obtain 5 beta-hydroxyresiniferonol-6 alpha,7 alpha-epoxide- 9,13,14-ortho(2,4,6-decatrienoate) [Excoecaria factor O1] by alkaline transesterification of corresponding 20-ester(s) genuinely present in latex of Excoecaria oppositifolia. It was obtained in a 10-fold better yield than previously, probably due to speeding up the separation procedures of the sensitive compound. O1 is an appropriate starting material for the preparation of the daphnane prototype irritant and tumor-promoting orthoester simplexin (SIM) especially of a multiple tritium-labeled form thereof. Moreover, a new Excoecaria factor O3, i.e. the 9,13,14-ortho(2,4,6,8-hexadecate-traenoate) of 5 beta,12 beta-dihydroxyresiniferonol-6 alpha,7 alpha-epoxide was isolated after alkaline transesterification of a TLC-uniform fraction, composed of corresponding 20-ester(s).

A skin irritant principle from Euphorbia matabelensis Pax.

A diterpene of the ingenane-type parent alcohol with tetradecanoic acid as the acid substituent was isolated by chromatographic methods from the latex of Euphorbia matabelensis. The ingenol ester exhibited irritant activity on the mouse ear.

Irritant principles of the mezereon family (Thymelaeaceae), V. New skin irritants and tumor promoters of the daphnane and 1 alpha-alkyldaphnane type from Synaptolepis kirkii and Synaptolepis retusa.

Seventeen mostly new, skin irritant diterpene esters (DTE) of the daphnane and 1 alpha-alkyldaphnane types were isolated from roots of Synaptolepis kirkii and Synaptolepis retusa. The parent alcohols of the daphnane types are shown to be 5 beta-hydroxyresiniferonol-6 alpha,7 alpha-oxide [1] and 5 beta, 12 beta-dihydroxyresiniferonol-6 alpha,7 alpha-oxide [2]. Ten of the daphnane types are 9,13,14-orthoesters and three are conventional esters involving tertiary or secondary hydroxyl groups at C-13 or C-14, respectively. The latter may be considered immediate precursors of corresponding orthoesters. The four 1 alpha-alkyldaphnane types are intramolecular 9,13,14-ortho-(2-hexadecenoic acid)-esters in which, formally, the second to last C atom of the orthoester moiety is linked covalently to C-1 alpha of the diterpene parent alcohols 1 or 2. Thus, in the new structure, a macrocyclic ring bridges the alpha side of the diterpene moiety in an "ansa" type manner. The irritancies on the mouse ear of the DTE obtained cover a wide range (I24 = 0.05-670 nmole-1). Some of them are considerably more irritant than the daphnane type standard simplexin. Structure/activity investigations reveal that an ester group instead of a free hydroxyl group at C-20 ("cryptic types"), or presence of a hydroxy or an acetoxy group in position 12 diminishes the irritancies of the daphnane types isolated, similar to what is known in corresponding tigliane types. In the standardized initiation/promotion protocol on the back skin of mice, some of the irritant DTE exhibit tumor-promoting activities higher than that of simplexin.

On the active principles of the Euphorbiaceae, XII. Highly unsaturated irritant diterpene esters from Euphorbia tirucalli originating from Madagascar.

The latex of Euphorbia tirucalli originating from Madagascar contains as irritant constituents ingenane- and tigliane-type diterpene esters derived from the parent alcohols ingenol and phorbol. The main irritant constituents are isomeric 12,13-acetates, acylates of phorbol as well as 3-acylates of ingenol. As acyl groups, they carry homologous, highly unsaturated aliphatic acids of the general structure CH3-(CH2)m-(CH = CH)n-COOH (m = 2,4; n = 2,3,4,5; total number N of C-atoms = 2n + m + 2). The lack of 4-deoxyphorbol esters in this latex as compared to latex of South African origin is probably indicative of the existence of chemical races of E. tirucalli. In the acyl moiety of phorbol esters investigated in detail, an increasing number of C-atoms or an increasing number of double bonds at a fixed number of C-atoms leads to an increase of irritant activity. As compared to their saturated analogs, corresponding unsaturated phorbol esters exhibit similar irritant activities. On the other hand, by an increasing number of conjugated double bonds in the acyl moieties of phorbol esters, the promoting activity is decreased, thus indicating that irritant activity is a necessary, but insufficient, requirement for promoting activity of phorbol esters. An assessment of a potential carcinogenic risk involved in mass production and handling of the plant should point to the very weak tumor-promoting activity and the chemical instability demonstrated for the diterpene constituents in the latex and hence in all plant parts.