Shehadeh, Mayadah Bashir; (1997) Phytochemical and biological investigations of plants from the families Euphorbiaceae and Thymelaeaceae. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Extracts of forty samples from the plant families Euphorbiaceae and Thymelaeaceae were screened for the presence of biologically active diterpene esters. Plant extracts from Gnidia kraussianus, Lasiosiphone kraussianus, Thymelaea hirsuta and Daphne blagayana (Thymelaeaceae) as well as extracts from Sapium inclicum (Euphorbiaceae) induced intense inflammation. Crude extracts and methanol fractions from the Thymelaeaceae species inhibited TPA-induced differentiation of HL-60 cells. Ether fractions induced platelet aggregation of the human blood at concentrations of 10-50 μg/ml. Biologically active diterpene esters were isolated and identified. These compounds were purified by multistage fractionation procedures. These involved liquid-liquid partition, centrifugal liquid chromatography, column chromatography, preparative adsorption and partition liquid chromatography. Final purification was carried out using HPLC. A series of one and two dimension NMR experiments were carried out to facilitate the structure elucidation of the isolated diterpenes. Mass spectrometry and infrared were used to confirm the chemical structures. Both thin layer and normal phase HPLC proved to be valuable in analysing and purifying small quantities of daphnanes and tiglianes. Thymelaeatoxin-A and resiniferonol-9,13,14-orthophenyl acetate were isolated from the twigs and leaves of Thymelaea hirsuta. The intense irritant effect of Daphne blagayana was partially due to the presence of mezerein. The novel sapintoxin-E, 12-O-(2-methylanminobenzoyl)-4,20-dideoxy phorbol-13-acetate, in addition to sapintoxin-A and sapintoxin-D were isolated from the latex of Sapium indicum. The twigs and leaves of Sapium insigne yielded the semi-synthetic 12-O-(2-methyl aminobenzoyl)-4-deoxy-5-hydroxyphorbol-5,13,20-triacetate. The established technique of irritancy testing was used to assess the potency of selected diterpenes. Compared to tiglianes irritancy induced by daphnanes was rapid and short lasting. Epidermal hyperplasia induced by mezerein was comparable to that induced by TPA. In vitro, the isolated compounds were evaluated for their platelet aggregating activity and for their ability to induce differentiation of HL-60 cells. At doses of 1nM and greater, TPA was the most potent diterpene ester in both in vivo and in vitro assays. For the detection of differentiation of HL-60 cells, a rapid and effective microdilution in vitro assay was developed. This technique was based on quantitation of absorbence of protein-bound eosin dye using a single beam minireader. Irritant daphnanes and tiglianes induced the HL-60 cells to differentiate at doses of 20 nM and greater. Differentiation effect of TPA obtained from the new assay was closely related to the established methods.

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