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The cytotoxic effects of the natural photosensitizing agent hypericin were evaluated. A dramatic difference in the sensitivity of several different human and mouse cell lines towards photoactivated hypericin (4 J cm-2) was demonstrated using a neutral red assay (e.g. A431, IC50 = 0.14 +/- 0.02 microM; HeLa, IC50 = 0.32 +/- 0.05 microM, MCF7, IC50 = 1.84 +/- 0.22 microM). Dark cytotoxicity was absent, even at high hypericin concentration (25 microM). The differential phototoxicity of hypericin did not correlate with the expression of the epidermal growth factor (EGF) receptor nor with the expression of the P170 glycoprotein in the cell. The reduction of the intracellular glutathione content did not enhance further the cytotoxic effects of photoactivated hypericin, as investigated with the A431, HeLa and MCF7 cells. Conversely, using confocal laser microscopy, it was shown that the phototoxicity correlated well with the hypericin cellular uptake.

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The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. Athymic nude mice xenografted with A431 cells were intraperitoneally administered with different hypericin doses and the tumours were locally irradiated 2 h later with white light (180 J/cm2) using a cold light source. When treatment was started one day after tumour inoculation, a dose-dependent antitumour effect was observed in light-treated animals. Complete inhibition of the tumour growth was achieved with 2.5 mg/kg hypericin. When the efficacy of a single hypericin dose (5 mg/kg) followed by a single light treatment on established tumours (60 mm3) was investigated, an 80% reduction in tumour mass was seen. Furthermore, an accumulation of the photosensitizer in A431 xenografts was observed after local light irradiation. Our results strongly suggest that hypericin holds promise as a new, safe, efficient and selective PDT photosensitizer.

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A method has been developed for the analysis of phosphoserine, phosphothreonine and phosphotyrosine in 32P-phosphoprotein hydrolysates. The hydrolysates are treated with dabsyl reagent (28.8 mM) for 10 min at 70 degrees C. After a clean-up using a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC aluminum sheets using a one-dimensional solvent system. The method is straightforward and permits the simultaneous analysis of numerous samples. Very clean chromatograms are obtained enabling the unambiguous identification of the well separated dabsylated phosphoamino acids with autoradiography. The phosphoamino acids can be quantified by simply cutting out the relevant spots from the aluminum sheets followed by 32P-quantification using liquid scintillation spectrometry.

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The photodynamic inhibitory effect of hypericin and a number of hypericin-derivatives were investigated in vitro using numerous growth-factor regulated protein kinases including receptor-bound (Insulin-R, EGF-R) and non-receptor (Lyn, c-Fgr, CSK, Syk) protein tyrosine kinases as well as Ser/Thr (PK-C, protein kinase CK-2, CK-1) protein kinases. Modification of the hypericin structure altered significantly the specificity of the protein kinase inhibition. In particular, methylation or attachment of long lipophilic chains to both methyl groups of the hypericin molecule strongly enhanced the specificity toward PK-C.

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We have developed a new method for the quantification of phosphoserine, phosphothreonine, and phosphotyrosine as dabsyl derivatives in acid-hydrolyzed extracts of 32P-labeled A431 cells. In the first step the phosphamino acids are concentrated using a disposable anion-exchange column. Subsequently, the phosphoamino acid fraction is treated with dabsyl reagent (28.8 mM) for 10 min at 70 degrees C. After cleanup with a second anion-exchange column followed by separation on a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC sheets using a one-dimensional solvent system. The major advantages of this method are the complete separation of dabsylated P-Ser, P-Thr, and P-Tyr on silica aluminum sheets in a very reproducible way without the interference of 32P contaminants originating from hydrolyzed cell extracts. Very clean chromatograms are obtained, enabling the fast and unambiguous quantification of the phosphoamino acids by simply cutting out the relevant spots from the aluminum sheets. A high sensitivity is achieved by the removal of the amino acids before derivatization of the sample. This allows the use of relatively low amounts of [32P]orthophosphate to load up the cells. Most important, the method allows the simultaneous analysis of dozens of samples within 1 day, making it a very convenient technique for routine analysis of the phosphorylation state of cultured cells. Consequently the method is well suited to implementation in large screenings for inhibitors of protein kinases, e.g., PTK inhibitors, in whole-cell studies.

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The influence of the intestinal microbial reduction of rhein anthraquinone on the formation of deterioration products was studied. Therefore [14C]rhein and [14C]rhein anthrone were mixed with sterilized or non-sterilized cecal mass of rats and incubated for 20 hours at 37 degrees C. Extractions with a methanol-water (50:50) mixture or 4-nitroso-N,N-dimethylaniline (0.1%) in pyridine revealed several radioactive derivatives after TLC and autoradiography, except in the case where the anthraquinone was mixed with sterilized cecal content. Gel permeation on a styrene-divinylbenzene copolymer column of an methanol/water extract of non-sterilized cecal content incubated with [14C]rhein, showed radioactive deterioration products with a molecular weight higher than rhein anthraquinone. The high molecular weight of some deterioration products was confirmed by an ultrafiltration study where the methanol/water extract was centrifuged on a Centricon-3 microconcentrator (nominal cutoff: 3000 MW). Aqueous extracts of non-sterilized cecal content incubated with rhein were extracted with chloroform to remove rhein anthraquinone, rhein anthrone and sennidins before being intracecally injected in rats. No laxative activity was found. Furthermore it was shown that the deterioration products which are probably formed through radical reactions, no longer develop a color with a solution of KOH. Therefore it is concluded that the reduction process of dihydroxy-anthraquinones in the gut microflora followed by an extraction, accounts for the loss of anthranoid equivalents in in vivo circumstances, as several times reported in the past.

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Bianthraquinone glycosides are formed during the preparation and conservation of fluid extracts of cascara. Analysis of these preparations has shown that up to 20% of total amount of anthracene glycosides can be dimerized. These dimers are devoid of laxative activity. They are absorbed in the intestine and very slowly excreted.

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Differentiation of CASSIA SENNA and C. ANGUSTIFOLIA is possible by chromatographic identification of the naphthalene glycosides.

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From leaves and pods of Cassia senna L. and C. angustifolia Vahl. were isolated the naphthalene glycosides 6-hydroxymusizin glycoside and the new tinnevellin glycoside. The structures were established mainly by spectroscopic methods ( (1)H NMR, (13)C NMR, MS).

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