Symphytum officinale L. and Symphytum asperum L. (Boraginaceae) are allopatric taxa, which are able to intercross and to form interspecific hybrids with different chromosome numbers. The species differ not only in a number of morphological characters but also ecologically, Symphytum asperum being a species of higher elevations (upper montane zone), Symphytum officinale of lowland and the lower montane zone. Symphytum asperum is a Caucasian species, which has the sporophytic chromosome number 2n = 32. It was introduced from the Caucasus into Europe as a fodder plant. Symphytum officinale is variable, containing cytotypes with 2n = 24, 48, 56, 40, and occurs throughout Europe.
The most common chromosome number of Symphytum officinale is 2n = 48. Scattered diploid populations of 2n = 24 occur in Western, Central, and Eastern Europe; they are white-flowered throughout. The tetraploids are white- or purple-flowered in western Europe and purple in eastern Europe. Populations in which purple- and white-flowered individuals occur intermingled are very common in western Europe. In eastern Europe, mixed populations are very rare and consist of white-flowered diploid plants with purple-flowered tetraploid plants.
The cytotype 2n = 56 has been found at only one location in the high Tatra mountains of Czechoslovakia and the cytotype 2n = 40 is very common in low-lying peat regions in The Netherlands. There exists, however, some controversy whether the latter represents a true subspecies, Symphytum uplandicum Nym., or a hybrid between Symphytum asperum (2n = 32) and Symphytum officinale ().
There has been renewed interest in Symphytum officinale regarding its use in the treatment of several diseases, as well as for treatment of neuralgia and paradontosis. Also for the treatment of burns, a preparation containing an extract of Symphytum officinale has been used recently. In the DAC (Deutscher Arzneimittel Codex) Symphytum officinale has been included in the monograph “Symphyti radix”. Also a number of other described physiological activities of alcoholic extracts of Symphytum officinale led to a strongly increased interest for this plant in phy-totherapeutics.
It is not only for phytotherapeutic reasons that Symphytum (comfrey) taxa have regained interest. Hills reported that comfrey is a plant which should be able to produce vegetable protein at the highest yield, per unit of time and culturing area, known for plants. He also claimed that comfrey should be well suited not only for fodder, but also human consumption was considered. Furthermore, Farnsworth has pointed out that comfrey is used in the most common herbal teas sold to the American public, which points to its widespread use. However, application should be considered with caution for its possible hepatotoxic and carginogenic properties.
In 1900, Greiner demonstrated the presence of small amounts of alkaloids in roots of Symphytum officinale, which had been purchased under the name “Radix consolidae”. It was in 1968 that Furuya and Araki isolated symphytine and echimidine from Symphytum officinale. These alkaloids belong to the diester pyrrolizidine alkaloids with an allylic-ester structure. This latter configuration determines the hepatotoxic action of pyrrolizidine alkaloids (); it also has been shown that symphytine is carcinogenic to rats.
Indicine-N-oxide (INO), a mono-ester pyrrolizidine alkaloid, apparently seems to be a compound of interest for its low hepatotoxic properties and its high anti-tumor activity. Therefore the production of these pyrrolizidine alkaloids by biotechnical techniques might be of interest. In vitro-grown cells of comfrey were tested for their feasibility as a model system for the production of these alkaloids. For this purpose biogenetic properties of cell suspension cultures and regenerating callus of a Symphytum officinale cytotype have been surveyed, with emphasis on the analysis of precursors in the pyrrolizidine alkaloid biogenesis.
Symphytum: Summary and Conclusions
Because some of the pyrrolizidine alkaloids from Boraginaceae have useful medical applications, including cytostatic effects, an attempt has been made to investigate the usefulness of in vitro-grown cells from Symphytum officinale for the production of related alkaloids.
The induction of primary calli from various organs of Symphytum officinale (2n = 24) plants on solid MS and B5 medium supplemented with appropriate growth regulators is described. The callus cultures were subcultured on B5 medium. Cell suspension cultures were obtained from B5-grown calli by transfer to liquid B5 medium. Cells from calli as well as from cell suspension cultures were induced to regenerate whole plants on solid MS medium, supplemented with indole-acetic acid and benzyladenine. Plants regenerated from short-term cultures were identical in morphology and chromosome number with plants from which cultures were initiated. Production of pyrrolizidine alkaloids ceased on prolonged subculturing of suspension cultures. However, regenerated plants produced again the alkaloids usually found.
Microdensitometry of Feulgen-stained fixed protoplasts from callus cells of Symphytum officinale (2n = 24 cytotype) did not show great deviation in nuclear DNA content from protoplasts derived from leaf material from the same cytotype. Therefore it was concluded that the loss of expression of alkaloid biogenesis was not a result of genetic instability, but rather an effect of an alteration of cell metabolism.
It was demonstrated that the polyamine biogenesis in Symphytum officinale did not deviate substantially from that described for tobacco cell cultures. The abundance of the supposed alkaloid precursors was not likely to be the limiting factor in the alkaloid biogenesis. In addition, the supply of polyamines and inhibitors of the polyamine catabolism did not give rise to the alkaloid synthesis. Isoputreanine-lactam, a metabolite of spermidine and spermine, did not appear to be an alkaloid precursor either. Cell suspension cultures of Symphytum officinale were able to acetylate lycopsamine. There was evidence that an oxygenation of the necine moiety of the alkaloid might also occur.
N-(4-amino-butan-l-one)-2-pyrrolidone, a compound found in shoot regenerating calli, was conceived as a precursor in pyrrolizidine alkaloid biogenesis (unpubl.). It may be formed via a condensation of two y-aminobutyric acid (GABA) molecules; GABA has been detected in cell suspension of Symphytum officinale. A hypothetical pathway for the pyrrolizidine alkaloid biogenesis in Symphytum officinale is given. So far, it was not possible to initiate alkaloid synthesis in cell suspension cultures of Symphytum officinale. There are indications, however, that photomixotrophic cultures may be essential for the induction of the alkaloid biogenesis.
Selections from the book: “Medicinal and Aromatic Plants III”, 1991.