Jiří Slanina

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC50 values for individual alkaloids were: sanguinarine IC50 = 0.8 μg/ml, sanguilutine IC50 = 8.3 μg/ml, chelerythrine IC50 = 6.2 μg/ml, chelilutine IC50 = 5.2 μg/ml, coptisine IC50 = 2.6 μg/ml and berberine IC50 >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity.

New and facile method of preparation of the anti-HIV-1 agent,1,3-dicaffeoylquinic acid

A facile and inexpensive preparation of 1,3-dicaffeoylquinic acid (cynarin) from the leaves Cynara cardunculus L. (Asteraceae) without the use any chromatographic steps is described. The procedure was based on separation of the fraction rich in 1,5-dicaffeoylquinic acid, isomerisation of 1,5-dicaffeoylquinic acid to cynarin and, owing to its higher polarity, the simple isolation of cynarin from the reaction mixture. Cynarin inhibited HIV-1 replication in MT-2 cell culture at nontoxic concentrations similar to other previously tested dicaffeoylquinic acids, which have been recently established as a potent and highly selective class of HIV-1 integrase inhibitors.

Determination of lignans in Schisandra chinensis using micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MEKC) has been developed as a promising method for the determination of lignans in plant samples. The separation conditions have been optimized with respect to the different parameters including sodium dodecyl sulfate (SDS) and acetonitrile concentration, pH of the background electrolyte, separation voltage, and capillary temperature. The background electrolyte consisting of 40 mM SDS and 35% acetonitrile in 10 mM tetraborate buffer (pH 9.3) was found to be the most suitable electrolyte for this analysis. The applied voltage of 28 kV (positive polarity) and the capillary temperature 25°C gave the best separation of lignans. The interday reproducibility of the peak areas and the migration times was below 2.0%. The results of MEKC analyses were compared with those obtained by capillary electrochromatography (CEC) and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The possibilities of using this method for the determination of lignans in drug and in serum samples were also tested.

Quaternary benzo[c]phenanthridine alkaloids--novel cell permeant and red fluorescing DNA probes.

Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs–macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells.

Dibenzocyclooctadiene lignans overcome drug resistance in lung cancer cells - Study of structure-activity relationship

A panel of nine dibenzo[a,c]cyclooctadiene lignans, schizandrin, gomisin A, gomisin N, gomisin J, angeloylgomisin H, tigloylgomisin P, deoxyschizandrin, gamma-schizandrin and wuweizisu C was examined for their effect on multidrug resistance, as well as their anti-proliferative activities. COR-L23/R, a multidrug resistant sub-line, which has been reported to over-express multidrug resistance-associated protein (MRP1), was used for the experiments together with its parent cell line COR-L23 (human lung cell carcinoma). We found that lignans deoxyschizandrin and gamma-schizandrin at relatively non-toxic concentrations restored the cytotoxic action of doxorubicin to COR-L23/R cells. Deoxyschizandrin and gamma-schizandrin also significantly enhanced the accumulation of doxorubicin in drug resistant cells. Both lignans alone had no effect on the cell cycle; however, when combined with sub-toxic doses of doxorubicin, they induced cell cycle arrest in the G2/M phase, which is typical for toxic doses of doxorubicin. Our results suggest that deoxyschizandrin and gamma-schizandrin potentiate the cytotoxic effect of doxorubicin in doxorubicin resistant lung cancer cells COR-L23/R by increasing the accumulation of doxorubicin inside the cells. The common structural feature of both active lignans is the R-biaryl configuration and the absence of a hydroxy group at C-8. Unlike the reversal effect, the cytotoxicity of lignans with the R-biaryl configuration was similar to that observed for lignans with the S-biaryl configuration.

Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography.

The main constituents of artichoke extract were separated by micellar electrokinetic chromatography (MEKC), using a buffer consisting of 100 mM sodium dodecyl sulfate (SDS) in 20 mM sodium dihydrogen phosphate, 20 mM disodium tetraborate (pH 8.6) as background electrolyte. Optimum separation voltage of 28 kV (positive polarity) and a capillary temperature of 25°C gave the best analysis. The UV detection was performed at 200 nm. The method was successfully used to analyze plant and drug samples as well as for the study of artichoke antioxidant activity. The quantitative MEKC results were in good agreement to those obtained previously by reversed‐phase high‐performance liquid chromatography (RP‐HPLC).

Supercritical fluid extraction of lignans from seeds and leaves of Schizandra chinensis

Supercritical carbon dioxide was used to extract lignans from seeds and leaves of Schizandra chinensis (Turcz.) Baill. Kinetics of the extraction was found to be controlled by intra-particle diffusion of lignans. The extraction yields were approximately 96% of lignans from seeds and about 26% from leaves, both yields being based on the total lignan content determined from two successive Soxhlet extractions with petroleum ether and methanol.

Evaluation of cytotoxic activity of Schisandra chinensis lignans

Using exhaustive chromatographic separation we have isolated (-)-tigloyl-deangeloyl-gomisin F as a novel dibenzocyclooctadiene lignan from schisandra chinensis. With the help of HPLC, we further isolated (+)-schisandrin, (+)-deoxyschisandrin, (+)-γ-schisandrin, (-)-gomisin J, (+)-gomisin A, (-)-gomisin N, (-)-tigloyl-gomisin P, (-)-wuweizisu C, (-)-gomisin D, rubrisandrin A, (-)-gomisin G, (+)-gomisin K (3) and (-)-schisantherin C. A full NMR description of (-)-schisantherin C was carried out with the aim to confirm previous reports of its structure. Compounds isolated were identified on the basis of UV, IR, (1)H- and (13)C-NMR and MS. The cytotoxicity of lignans was tested for the BY-2 cell line alone and as a synergistic effect with the cytotoxic agent camptothecin. Lignans showed various toxicity and synergistic and antagonistic effects on camptothecin-induced cytotoxicity. Cytotoxicity against colon cancer cell line LoVo was also tested.

Evaluation of the Antiradical Activity of Schisandra Chinensis Lignans Using Different Experimental Models

The in vitro antiradical activity of Schisandra chinensis lignans was investigated using DPPH, ABTS+, Fenton reaction inhibition and tyrosine-nitration inhibition assays, as were the in vivo antidiabetic activities of selected lignans in an animal model of alloxan-induced diabetes. Different degrees of antiradical activity were found, depending upon the structural parameters of the tested compounds. Unfortunately, the compounds showed no antidiabetic activity in concentration range tested.

Formation of lignans in plant cell culture of Schisandra chinensis

The fruit of Schisandra chinensis, a woody liana, has been used for centuries in traditional Chinese medicine. The fruit is prescribed for the treatment of hepatitis in China. The active principles are lignans with unusual structures derived from dibenzo[a,c]cyclooctadiene. These lignans have been shown to possess a broad range of biological activities. Recently, it has been found that lignan gamma-schizandin strongly inhibits P-glycoprotein, the overexpression of which is the most frequent cause for cancer multidrug resistance. S. chinensis cultures derived from immature zygotic embryos were developed on Murashige and Skoog and VW5 medium. Embryogenic cultures were established on the medium containing thidiazuron, 2,4-dichlorophenoxyacetic acid and benzylaminopurine. An analytical method based on HPLC-UV was used for the determination of six lignans in plant cell cultures. Our previous results showed that the concentration of lignans in plant cell cultures was mostly lower than that in fruit and leaves. We also detected the lignans in liquid culture media, but the amount of lignans inside the cells was substantially higher. In order to release the lignans stored within the cells, we explored the cultivation of five cell lines with neutral polymeric resin, Amberlite XAD2. We found that the addition of Amberlite XAD2 not only notably enlarged portions of extracellular lignans, but also greatly enhanced the production of lignan deoxyschizandrin, whereas the formation of other lignans was increased only moderately (less than 5fold). Importantly, Amberlite XAD-2 did not reduce the growth of cell cultures. Deoxyschizandrin adsorbed on the polymeric resin can be easily recovered and purified without destruction of the cells.