Extraction, purification and determination of Solasodine in cultures of Solanum mauritianum Scop.
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Date
1993
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Abstract
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.
Description
Thesis (Ph.D)-University of Natal, Pietermaritzburg, 1993.
Keywords
Steroids., Bugweed., Solanum mauritianum., Theses--Botany.