The study aimed to elucidate the effects of benzothiadiazole (BTH) and saccharin on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, and furoquinolone alkaloids in shoots of R. graVeolens cultivated in vitro. The biosynthesized metabolites were analyzed and identified by GC-MS and by comparison of Kovats indices. Eight coumarin metabolites were identified: bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin, and also four alkaloids: dictamnine, γ-fagarine, skimmianine, and kokusaginine. Each of the tested BTH concentrations induced a significant production of furanocoumarins and furoquinolone alkaloids. The use of saccharin also increased the production of bergapten, isopimpinelin, pinnarin, psoralen, and xanthotoxin several times. IntroductionThe in vitro culture of R. graVeolens is a useful biotechnological source of biologically active linear furanocoumarins and furoquinolone alkaloids. Linear furanocoumarins, particularly xanthotoxin, bergapten, and isopimpinelin, have been applied in the treatment of skin diseases characterized by excessive cell proliferation (e.g., psoriasis, mycosis fungoides) or in pigmentation disorders (e.g., vitiligo) (1) and also in neurology (the symptomatic treatment of demyelinating diseases, particularly multiple sclerosis) (2). In addition, alkaloids like dictamnine and methoxydictamnine from R. graVeolens tissues are wellknown antimicrobial factors (3). Apart from their antibiotic effect, furoquinolone alkaloids appear to have a spasmolytic effect (4). The biological effects of furanocoumarins and furoquinolone alkaloids makes them attractive for pharmaceutical uses, hence the considerable interest shown in their availability and sources. However, the commercial production of secondary metabolites is usually limited by their low yield. Elicitation has therefore become an extensively used tool for enhancing secondary metabolites: it is an integral part of any large-scale process for secondary metabolite production.The mechanisms of action of biotic and abiotic elicitors are thought to be different; they are complex, and many hypotheses have been set up in this regard. Moreover, since little is known about the biosynthetic pathways of secondary metabolites, the effect of an elicitor on a plant cell or tissue culture is not easily predictable; the majority of elicitation approaches are therefore empirical (5). The search for elicitors is of the utmost importance in order to obtain the most effective biotechnological system for producing the required metabolites. The objective of our study was to elucidate the effects of saccharin, as well as benzothiadiazole (BTH, benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester) on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, simple coumarins, and furoquinolone alkaloids in shoots of R. graVeolens cultivated in vitro.Benzothiadiazole is the main component of the BION preparation (6, 7). Chemically, it is a functional...

Background:Paris quadrifolia L. is a medicinal plant which contains steroidal saponins. The present study reports isolation and structural identification of six pennogenyl saponins obtained from P. quadrifolia rhizomes. The four spirostan saponins were obtained from P. quadrifolia for the first time. The cytotoxic effects of the sub-fractions and six compounds isolated from the plant extract were evaluated on tumour cells.Materials and Methods:Ethanol extract from the rhizomes of P. quadrifolia were partinioned using column chromatography. The saponins were isolated from the obtained sub-fractions by isocratic RP HPLC and their structures were determined by means of 1D and 2D NMR spectroscopy and MALDI TOF MS. The cytotoxic effects of the sub-fractions and the isolated compounds were tested against human promyelocytic leukaemia cells (HL-60), human cervical adenocarcinoma cells (HeLa) and human breast cancer cells (MCF-7) using the [(3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay.Results:Six pennogenyl saponins were isolated from P. quadrifolia rhizomes: pennogenin 3-O-β-D-glucopyranoside (1), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside (2), pennogenin 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (3), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside (4), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (5), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (6). Pennogenyl saponins 5 and 6 exhibited cytotoxic activity against HL-60, HeLa and MCF-7 tumour cells with IC50 values of 1.0 ± 0.04 μg/ml, 1.8 ± 0.072 μg/ml and 2.4 ± 0.096 μg/ml respectively, and 2.0 ± 0.08 μg/ml, 2.5 ± 0.125 μg/ml and 3.2 ± 0.128 μg/ml respectively.Conclusion:Compounds 1-4 were isolated from this species for the first time.

Steroidal saponins isolated from many plant species belonging to Monocotyledones display potent cytotoxic activity towards many human tumor cells. We examined the cytotoxic effects of crude Paris quadrifolia extract for the first time, testing isolated saponin-rich fractions against four different human cell lines using the [(3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxic activity was tested against human promyelocytic leukemia (HL-60) cells, human cervical adenocarcinoma (HeLa) cells and human breast cancer (MDA-MB-468) cells. Human skin fibroblasts were used as non-neoplastic control cells. Our results show significant activity of the weakly water-soluble solid residue and butanolic fraction against HL-60 and HeLa cells. The solid residue exerted cytotoxicity against all tested cell lines.K Ke ey y w wo or rd ds s: : In vitro cytotoxicity, MTT assay, Paris quadrifolia extract, steroidal saponins, human tumor cell lines.

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