Solanum aviculare Forst., Solanum laciniatum Ait. (Poroporo): In Vitro Culture and the Production of Solasodine
The interest in solasodine-bearing plants is primarily due to the potential conversion of solasodine to pharmaceutically important compounds. The steroidal glycoalkaloids of solasodine type have become increasingly important as starting material for the synthesis of corticosteroids and contraceptives until the mid 1970’s, these compounds were prepared almost exclusively from diosgenin, but the shortage of this material promoted the search for alternative sources.
Recent fluctuations in supply of diosgenin have had social and political rather than agricultural causes, but nevertheless its production is ultimately limited. As chemical synthesis is at present fraught with problems, an undesirable pressure is placed on natural plant populations. Field production is also faced with many difficulties. One way to alleviate these complications would be the in vitro production of the required compounds.
Solasodine has been found to be a suitable starting material for the degradation sequence leading to key intermediates for the synthesis of steroids. This compound has been established to occur in relatively high concentrations in a number of Solarium species. Studies indicate that from species recognized as particularly interesting, S. aviculare is one of the most promising. Some species have been studied from the point of view of in vitro production, S. aviculare and S. laciniatum again being among them.
Solarium aviculare Forst., Solarium laciniatum Ait.
S. aviculare Forst., first mentioned by Forster (1786), belongs to the tribe Solanaeae from the section Archesolanum. The genus Solanum is the widest of the family Solanaceae (order Solanales) in subclass Asterideae of the Dicotyledons. Some of its properties were first described again by Forster (1786). In earlier literature S. aviculare Forst. appears also under the following synonyms: S. laciniatum Ait., S. reclinatum L’Herit, S. vescum F. Muell., S. pinnatifidum Lam., S. dispar Loisel. (Vilmorin 1896).
In favourable climates S. aviculare is a hardy, short-lived perennial, glabrous or subglabrous shrub 1 to 3 m in height. It has a spindle-shaped main root, usually with many dividing branches, mostly 20-30 cm deep in the soil. Leaves are entire or laciniate, often on the same plant; entire leaves 10-20×1.5-4.5 cm, narrow- to linear-lanceolate, longacuminate, narrowly cuneate at base, decurrent on the short petiole; laciniate leaves with one to three pairs of narrowly triangular to linear lanceolate, longacuminate lobes.
The natural range of S. aviculare extends from the Papua New Guinea Highlands of 1300-2700 m in elevation, through Australia and the islands of the southwest Pacific to coastal New Zealand locations near Kaikoura (42½ °S) and some of its off-shore islands. A few plants have been found in sheltered pockets even on Banks Peninsula at 45 °S. The natural range of S. laciniatum extends from the southern tip of New Zealand (46 °S) to as far north as Adelaide (35 °S).
S. aviculare Forst., together with S. laciniatum Ait., are both called poroporo by the New Zealand Maoris, because the two species are difficult to distinguish by means of vegetative characteristics. Tuzson (1954) recognized that poroporo may be an economic source of solasodine and initiated intensive research.
There has been considerable confusion in the identification of poroporo species. The basis for distinguishing between them are the caryological studies of Baylis (1954). As S. aviculare has chromosome number n = 23 and S. laciniatum has n = 46, these species cannot be hybridized with each other unless some ploidy manipulation is utilized. Based on this difference, Stary and Storchova-Burianova (1962) estimated that many plant samples presented in Europe as S. aviculare are actually S. laciniatum. At the same time, but independently, Foldesi et al. showed the same mistake by Hungarian workers and it seems likely that similar confusion also misled others.
A number of taxonomic keys are now available to identify poroporo species. It is assumed that S. laciniatum developed from S. aviculare by polyploidization. This is supported by the close familiarity of the morphological and vegetative characteristics. Baylis (1963), in his cytogenetical studies, writes about a species complex in relation to S. aviculare, which is a row of polyploidic species with the basic chromosome number of n = 23. This species complex is spreading from New Guinea through Australia to New Zealand.
Production of Solasodine in Some Members of Solanaceae
Extensive studies were made to find the best source for commercial production of Solasodine Schreiber (1968) lists 167 Solarium species, 52 of which contain solasodine aglycone. Some of them accumulate it in the whole plant, while some tropical species concentrate the alkaloids in the fruits only. According to Rip-perger and Schreiber (1981) solamargine and solasonine are the predominant alkaloids, appearing in about 60% of the species tested. Bradley et al. (1978) made a survey of Australian Solarium plants potentially useful as sources of Solasodine.
Weiler et al. (1980) followed the taxonomic distribution of Solasodine in the genus Solarium. Its occurrence has been investigated in small leaf samples of herbarium specimens of over 250 species. The values obtained ranged from 0% to 4.3% of Solasodine in dry mass, the latter for one S. laciniatum sample. Only nine species contained more than 2% DM in the leaf. These are as follows: S. laciniatum, S. troyanum, S. suaveolens, S. rugosum, S. remyanum, S. pinnatum, S. jasminifolium, S. aviculare and S. agrimonifolium.
From the review of Mann (1978), it appears that over 2% DM of Solasodine in fruits can be found in S. aculeatissimum, S. eleagifolium, S. incanum, S. jubatum, S. khasianum, S. laciniatum, S. marginatum, S. platanifolium, S. sodomeum, S. trachycypium and S. trilobatum.
Seasonal changes could also play an important role in dictating alkaloid content. When comparing the content in berries, the losses during ripening must be taken into account, as Solasodine is either degraded or exported from the fruits. A few species, however, retain significant amounts of Solasodine in ripe fruits.
S. khasianum Clarke is the best known from this group. According to Mahato et al. (1980), it contains solasonine, solamargine and khasianine. This bushy, thorn-leaved plant grown either as annual or as a short-lived perennial in India contains Solasodine in fruits ranging from 2.5% to 5.4% DM. Yaniv et al. (1980) showed that there is a positive correlation between seed number, fruit weight and Solasodine content. Yaniv et al. (1984) studied the effect of water stress on growth and development of S. khasianum and on the Solasodine content of its fruits. Perez-Medina et al. (1964) reported 20% of glycoalkaloids in the greenish liquid inside the pericarp of S. mammosum fruits. Khan and Ikram (1983) obtained 5.2% of Solasodine in fruit of selected S. aviculare comparing to the lower yield given by S. mammosum, S. nigrum, S. pseudocapsicum and S. khasianum.
Ntahomvukiye et al. (1983) described the preparation of Solasodine at 1.7% yield from berries of S. adoense. Telek (1979) obtained a good yield from S. mammosum and S. khasianum berries (1.1% and 2% DM respectively). Leonart and Moreira (1984) analyzed Solasodine in fruits of S. brusquense. Solasonin and solasurin were found equal to more than 1.1% DM. S. erianthum studied by Moreira et al. (1980) has notable productivity in fruit (5.9% DM). The fruits of S. asperum and S. paludosum contain 0.67% and 0.24% DM respectively.
Focussing on the taxa native to the Northern temperate climatic zone, Mathe and Mathe (1979) found that S. dulcamara can be suitable for production in Hungary. This and other related species were analyzed for alkaloid and protein content, because of the possibility of profitable double exploitation.
Until now it seems that only poroporo is worth considering for foliar production of Solasodine, but many other authors not cited here look for further possible sources. Some of the species suggested by Weiler et al. (1980) might in future replace poroporo by showing better characteristics.
Conclusions and Prospects
Selection and optimalization techniques allow fairly good yield improvement, but as other sources of steroids are still in a good competitive position, solasodine production by in vitro cultures is far from being of an economic standard.
The methods for controlling the derepression of metabolic genes, to direct the precursor flow from primary to secondary metabolism, are likely to provide the main thrust of future work. In addition to tumour transformation and immobilization techniques, some positive results can also be expected from the exploitation of artificially elicited stress metabolism. Plants of S. aviculare in conditions of stress enhance their production of antifungal metabolites, including Solasodine. This has not yet been tested with tissue cultures of S. aviculare. Co-cultivation of S. laciniatum cell suspension with a cyanobacterium enhances the productivity of Solasodine, but it is not clear yet if this is due to the triggering of a defence mechanism or an improvement in the nitrogen nutrition.
Newly developed culture techniques [S. aviculare cells were grown with a good yield of biomass in plastic bags using a device described by Kybal and Sikyta (1985)], or more effective fermenters, new growth regulators and other factors may improve the cultures and this, together with a higher yield of products, will bring about a decrease in the price per product unit obtained by in vitro methods.
As long as the prices of products obtained in vivo exhibit a steady increase, these values have to meet somewhere, for high unit price substances sooner, for others later. In the long term even solasodine production in vitro may become economically feasible.
Aseptic plantlets from surface sterilized seeds grow well on hormone-free medium of Murashige and Skoog, in modification of Linsmaier and Skoog (1965). Primary explants from leaves, stems or roots readily form callus on LS medium containing 30 mg/l sucrose, 0.4mg/l thiamine and 100 mg/l inositol, and supplemented with 1.75 mg/l IAA and 215 mg/l kin. The best total yield of solasodine was obtained on the same medium supplemented by 0.225 mg/l 2,4-D and 0.215 mg/l kin. BAP (2-5 mg/l) stimulates the shoot organogenesis in both poroporo species.
T.E. Macek, Medicinal and Aromatic Plants II (1989)