Hairy roots of Ruta graveolens L. – a rich source of coumarins and furanocoumarins

Abstract

Ruta graveolens L. (Rutaceae) is one of the richest natural sources of coumarins (i.a.: umbelliferone) and linear furanocoumarins (i.a.: psolaren, bergapten, xanthotoxin). Ruta has been used in the treatment of leucoderma, vitiligo and psoriasis diseases. The aim of our work was to obtain hairy root cultures of R. graveolens after transformation of explants of in vitro grown plant with Agrobacterium rhizogenes. Hypocotyls were transformed with 2 strains of A. rhizogenes (A4, LBA9402) using 2 methods. Axenic cultures derived from a single root tip were established after 3–5 weeks of subculture in an Erlenmeyer flask containing B5 medium, 3% sucrose, claforan and carbenicillin (500mg l-1). Hairy roots were maintained on a liquid B5 medium without antibiotics (16/8h photoperiod or in the darkness), on a rotary shaker at 110 rpm. Subcultures were made every 4 weeks. The transformation of Ruta tissue was confirmed on molecular level using PCR tests with primers based on the sequence of rolB and rolC genes of A. rhizogenes. Quantitative and qualitative determination of coumarins and furanocoumarins in chloroform and methanol extracts was performed by using a Hewlett- Packard Model 5890 gas chromatograph coupled with mass spectrometer TRIO 3000. Separations were performed on a capillary column BP-1 (30m x 0,25 i.d., 0, 25µm film thickness). The growth index (t 30 /t 1) for control shoots grown in vitro amounted to 5.5 and for hairy roots 4.8. In comparison to control shoots of R. graveolens in hairy roots growing in the dark we detected 2 times more bergapten; 16.5 times more izopimpinellin and 4 times more rutacultin isomer. In addition new compounds were identified, like osthol, 3-(1,1-dimetylallylo)scopoletin, 2,5-dimetyl-7-hydroxychromon, 5-(3-metyl-2-butenyl)-8-metoxypsolaren and chalepin. The level of these compounds in hairy roots growing under a 16h photoperiod was on average 2–3 times lower than in roots growing in the dark. The obtained results indicate that hairy roots of R. graveolens grown in the dark are a rich source of coumarins and furanocoumarins.