Extraction, purification and determination of Solasodine in cultures of Solanum mauritianum Scop.
Solasodine, a steroidal alkaloid, is used by the pharmaceutical industry in certain parts of the world, as a raw material in the synthesis of steroid drugs. The compound is contained in many members of the genus Solanum, including S. mauritianum Scop., a common weed in South Africa. The levels of solasodine in three culture systems of S. mauritianum under various cultural conditions were examined. A high performance liquid chromatographic (HPLC) method was developed for the detection of solasodine. In order that low-cost, fixed wavelength ultra-violet detectors could be used, which would make the technique more widely applicable, a derivatization step, namely benzoylation, was included in the sample preparation. An extraction and purification protocol was then established, that complemented the HPLC technique and allowed successful detection of solasodine levels in a whole range of different sample types, including callus, suspension cultured cells, roots, stems and leaves. The three culture systems examined were callus, suspension and hairy root cultures. The callus system was used to establish which cultural parameters affected solasodine content in vitro to the greatest extent. A control culture was grown on a MURASHIGE and SKOOG (1962) medium (excluding glycine) supplemented with 3 % sucrose, 0.1 g I ¯¹ myo-inositol and lacking hormones. This culture contained an average of 9.2 μg g ¯¹ DW of solasodine. Many factors, including alteration of the carbon : nitrogen ratio and substitution of Gelrite for agar as the gelling agent, had no significant effect on the solasodine content of the callus or its growth. Greatly increased solasodine productivity of the callus was recorded when glucose was substituted for sucrose, the medium strength was reduced by half, or certain combinations of the hormones benzyladenine and naphthaleneacetic acid were added to the medium. The maximum levels of solasodine recorded in these cultures, on a per gram dry weight basis, equalled those of the vegetative parts of an intact S. mauritianum plant, but were approximately three times lower than those of the green berries. Suspension cultures could not be grown on the same medium as the callus cultures. Substitution of the vitamin complement of MURASHIGE and SKOOG (1962) with the so-called RT vitamin complement of KHANNA and STAB A (1968), resulted in successful growth and maintenance of S. mauritianum suspension cultures. The auxin, 2,4-dichlorophenoxyacetic acid (1 mg I ¯¹) was included in the medium. None of the suspension cultures grown on this medium, or slight modifications thereof, contained any trace of solasodine. This system could therefore not be used for the synthesis of solasodine in vitro. Hairy root cultures were initiated by inoculation of an excised hypocotyl of an in vitro-grown seedling of S. mauritianum with a 48 hour culture of Agrobacterium rhizogenes LBA 9402. Transformation frequency was extremely low. The transformed roots could be excised and grown successfully on a phytohormone-free medium, either in the solid or liquid form. Solasodine was extracted from hairy roots grown in a full-strength liquid MURASHIGE and SKOOG (1962) medium (excluding glycine) supplemented with 3 % sucrose and 0.1 g I ¯¹ myo-inositol, a half-strength such medium and a full-strength medium with 3 % glucose substituting for 3 % sucrose. In the latter medium, growth was very poor, whereas in the other two media, growth was very rapid . Both solasodine content (126 μg g¯¹DW) and root growth were greatest in the full-strength medium supplemented with 3 % sucrose. This level of solasodine was greater than that found in any of the callus cultures or vegetative parts of the plant and approached that of the green berries of S. mauritianum. Overall, of the culture types of S. mauritianum tested, the hairy root culture system appears to be most favourable for the in vitro production of solasodine.