Steroidal glycoalkaloids in Solanum species : consequences for potato breeding and food safety

Tuberiferous and nontuberiferous wild <u>Solanum</u> species are increasingly being used in potato breeding as a source of genes for disease and pest resistances and for other valuable characteristics. A disadvantage of <u>Solanum</u> species, from a consumers point of view, is that they contain steroidal glycoalkaloids (SGAs), which are natural toxins occurring in all parts of these plants. The SGAs consist of a C ₂₇ -steroidal alkaloid (SA) and a sugar moiety, often a tri- or tetrasaccharide. The tubers of the cultivated potato usually contain small quantities of one type of SGAs, the solanidine glycosides.Evidence has been presented that utilization of wild <u>Solanum</u> species can result in the introduction of hazardous levels of solanidine glycosides into the cultivated potato. However, little is known of the qualitative and quantitative SGA composition of wild <u>Solanum</u> species used in potato breeding. Such information on hybrid offspring or on cultivars containing germplasm from wild species is entirely lacking.The aim of the studies described in this thesis was to evaluate the possible health hazards of the SGAs from the genus <u>Solanum</u> and to develop and apply methods for analysis of the SGA compositions of <u>Solanum</u> species. The collected information was placed into the perspectives of potato breeding and of food safety, in order to point out possible consequences of introducing undesired levels or types of SGAs into the cultivated potato.In Chapter I, the literature on the distribution and accumulation of solanidine glycosides in the cultivated potato and on the biosynthesis of the SGAs is reviewed. It was shown that many factors during the growth and post-harvest period of potatoes can lead to levels of solanidine glycosides exceeding 200 mg/kg fresh weight. This information is important also with respect to introduction of SGAs from wild <u>Solanum</u> species into the household potato, as alien SGAs will almost certainly accumulate similarly, because the biosynthetic pathways of the various SGAs are closely related.The objective of Chapter II was to evaluate the toxicity of the SGAs, based on the data available in the literature, in order to assess possible consumer hazards and to derive safe levels for household potatoes. The data showed that acute poisoning in man may occur due to consumption of potatoes with a solanidine glycoside content above 200 mg/kg fresh weight. This level is only two to three times higher than levels that are considered normal for tubers of current cultivars. Because of the virtual absence of chronic toxicity data, an adequate 'no-adverse effect level' for potato SGAs could not be assessed. Consequently an acceptable daily intake (ADI) figure for man or an acceptable level for potatoes could not be derived. It was concluded that the SGA content of new household potato cultivars should not be allowed to rise above the average level of the current cultivars; preferably it should be lower. SGAs alien to <u>S. tuberosum</u> must not be introduced into the household potato as acceptable levels have not been established.The methods for SGA analysis described in the literature are not adequate for determining -qualitatively and quantitatively- the SGA composition of breeding material. Therefore, a comprehensive, quantitative and efficient method applicable to diverse plant material, was developed.In Chap ter III, a new hydrolysis technique employing a two-phase system is described. The technique was developed in order to prevent the losses of aglycones which do usually occur during conventional hydrolysis of different SGAs. The two-phase system consists of an aqueous acid phase, in which the glycosides are hydrolysed, and an immiscible nonpolar organic phase, which serves as a protective phase for the unstable nonpolar aglycones. Using the technique quantitative recoveries of various SAs after simultaneous hydrolysis of different SGAs were obtained.In Chapter IV a capillary gas chromatography (GC) method is described, which enabled -for the first time- the separation of the Δ5-and 5α-aglycone pair solanidine and demissidine as well as separation of the other SAs studied. Derivatization of the SAs was not required. The capillary GC method in combination with the bisolvent extraction and two- phase hydrolysis described in Chapter III offered prospects for quantitative analysis of complex SGA compositions of <u>Solanum</u> species in a single run.Chapter V deals with the development of a method that enabled an unambiguous differentiation during capillary GC between peaks of unknown SAs and peaks of closely related nonnitrogen-containing (non-N) compounds such as sterols and steroidal sapogenins. The method involves a splitting device that connects a capillary column to a dual detector system, for flame ionization detection (FID) and N-specific detection (NPD), respectively. This system is connected to a two-channel system for interactive processing of the two sets of data. The application of hydrogen as carrier gas was introduced to achieve a short analysis duration. As tubers of wild <u>Solanum</u> species appeared to vary strongly in SGA contents, identification by retention times was not possible. Therefore a retention index system for the SAs was introduced. The retention indices were sufficiently reproducible, even for identification of the closely eluting Δ5-and 5α-aglycone pair solanidine and demissidine. Application of NPD/FID response ratios together with retention indices proved to be useful in the identification and characterization of SAs. Costly spectrometric techniques will have to be employed for characterization only when new SAs are detected.Chapter VI deals with an evaluation of the developed methods using diverse plant tissues of wild <u>Solanum</u> and <u>Lycopersicon</u> species, cultivars and hybrid progeny. The results showed that the methods were useful for qualitative and quantitative analysis of the SAs in the diverse samples. A potato extract spiked with 20 SAs, including three Δ5-and 5α-aglycone pairs, could be analysed in a single GC run. A comparison with procedures described in the literature showed that the procedure developed for sample preparation followed by capillary GC should be applied, because literature procedures for sample preparation can lead to losses of various SGAs or SAs, and other chromatographic techniques do not offer the efficiency required for separation of the various SAs.In Chapter VII, the SGA compositions of a large number of wild <u>Solanum</u> species used in potato breeding, are presented. The tubers of most of the species contained high concentrations of solanidine glycosides. Some species contained glycosidic-bound demissidine, solasodine and/or tomatidine. Unidentified compounds were also detected, which were most probably SAs as was shown by their NPD/FID response ratios. The total SGA contents of the tubers of the wild species varied from 123 to 7348 mg/kg. In order to place these contents in a realistic perspective, tubers of cultivars, corresponding in small size with and grown under the same conditions as the tubers of the wild species, were analysed. The contents of solanidine glycosides of these tubers were two to three times higher than those of field-grown normal tubers. Thus, even when this factor was taken into consideration, the SGA contents of the tubers of several wild species were extremely high. This study also revealed that samples of some wild species contained high or low levels of one or many other glycosidic-bound SAs, and that the SGA composition can vary within species and between different organs of one plant. The results further suggested that the SGA synthesis can be organ specific or regulated by light and that translocation of SGAs from leaves to tubers and <u>vice</u><u>versa</u> did not occur. It was concluded that wild crossing parents should be analysed as to their SA composition, before they are used in a breeding programme.A procedure developed for the characterization of unidentified SAs present in plant extracts with complex SA compositions, is described in Chapter VIII. The combined results of GC-mass spectrometry (MS) applying electron impact and chemical ionization, and high-resolution MS, of the application of retention indices and NPD/FID response ratios, and of a comparison of the degree of dehydration of SAs in different twophase hydrolysis systems, were used. This study revealed that the SAs all possessed a solanidane skeleton; they were characterized as substituted, e.g. hydroxylated or methylated, forms; dehydrogenated forms; or substituted saturated forms of solanidine. Most of these SAs had not been reported before. The complete SA composition, including the minor new SAs, of <u>Solanum</u> (sub)species used in potato breeding, is presented in this chapter and the importance of analysis of the minor SAs is indicated.<u>S. vernei</u> is widely being used in potato breeding. Although the SGA composition of its tubers had not been reported, cultivars containing <u>S. vernei</u> germplasm have been released and an increasing number will be released in the future.Chapter IX describes the identification and characterization of the SAs of <u>S. vernei</u> tubers produced under various growth conditions, and deals with a tentative study on the transmission of these SAs to hybrid offspring. High levels of solanidine and solasodine glycosides were found together with tomatidenol and novel glycosidic-bound SAs, amongst which the 22R,25R epimer of solanidine, a structural configuration not reported before for naturally occurring solanidanes. It was revealed that the SA composition can vary significantly in tubers grown under different cultivation conditions.In the tubers of <u>S. vernei</u> offspring, high levels of solanidine glycosides were found but the newly identified and characterized SAs of <u>S. vernei</u> were not detected. Solasodine was also found in the offspring, even in tubers of cultivars obtained after several times backcrossing, but fortunately, the levels were low. However, it can not be totally excluded that <u>S. vernei</u> offspring may synthesize hazardous levels of solasodine glycosides under particular growth or post-harvest conditions, and in this respect the possible teratogenic potency of solasodine should be kept in mind.In conclusion it can be stated that the ingestion by the public of the amount of solanidine glycosides should not be allowed to rise but should preferably be reduced. SGAs alien to <u>S. tuberosum</u> should not be introduced into the household potato. The basis for the production of a potato crop safe for consumption is, to grow current cultivars and to breed new ones which accumulate low levels of only solanidine glycosides under various growth and post-harvest conditions. Utilization of germplasm from wild <u>Solanum</u> species must be approached with caution. Therefore it is recommended to analyse the SGAs of potential wild crossing parents before they are used in a breeding programme, in order to select the genotypes that combine a desired trait with the least unfavourable SCA composition. Depending on the SGA composition of the parents, the offspring should be monitored too.For analysis of SGA compositions, the newly developed procedures for separation, quantification and identification/characterization, described in this thesis, must be applied, as the conventional methods described in the literature do not meet the required standards.The accumulation of SGAs in new cultivars containing wild-species germplasm should be studied under various environmental conditions before such cultivars are registered. The guideline of 60-70 mg solanidine glycosides per kg fresh tuber recommended in the literature from the viewpoint of consumer safety (see Chapter II), should be used in potato breeding until an adequate acceptable level for these compounds has been established.