Occurrence in the Solanaceae Tables 31 and

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Our knowledge of the occurrence and distribution of nicotinoids is based on (i) traditional isolation and structure elucidation procedures until the 1960s and (ii) in more recent times on different chromatographic methods, predominantly GC/MS analysis. Nicotianoideae

The accumulation of large amounts of nicotine and/or its congeners is confined to four solanaceous genera belonging to two clades of the subfamily Nicotianoideae (Nicotianeae clade: Nicotiana; Cyphanthera clade: Crenidium, Cyphanthera, Duboisia).

Nicotianeae clade. Nicotiana is the fifth largest solanaceous genus and one of the most comprehensively studied flowering plant genera at all. It comprises 77 naturally occurring species [geographic distribution: America 49 spp., Australia 25 spp., Pacific islands (S-Melanesia) 1 sp., Namibia (SW-Africa) 1 sp., cultivated almost worldwide 1 sp.] (Chase et al. 2003 and references therein). Inferred from extensive phylogenetic analyses with multiple plastid DNA regions including 75 naturally occurring species, the genus is assumed to have evolved in southern South America east of the Andes and later dispersed to Africa, Australia, and southwestern North America (Clarkson et al. 2004). According to Goodspeed (1954) who recognized 60 species (several new species have been described since) the genus is divided into three subgenera (Rustica, Tabacum, Petunioides) and subdivided into 14 sections (see Table 3.4). None of these subgenera turned out to be monophyletic in a comprehensive phylogenetic study of the ITS nrDNA involving 66 species (Chase et al. 2003). However, most of these sections were coherent, others clearly polyphyletic. The genus as a whole seems to be monophyletic though this is supported in the analysis only in a limited manner (bootstrap percentage: 71). However, the least diverged taxa from the Nicotiana, the genera of Anthocercidae are more divergent from any species of Nicotiana than any of the latter is from other congeneric species. This fact is interpreted by the authors as a further support that Nicotiana is monophyletic. One year later the same authors published a new sectional classification of the genus based on the data from the former study (Knapp et al. 2004). Most of Goodspeed's sections are upheld by molecular analysis. Several species have been transferred to other sections. Only naturally occurring species have been included. The new classification is integrated in Table 3.4.

As already mentioned, most of the commercial tobaccos produced in the world belong to Nicotiana tabacum L., which is assumed to be an allotetraploid, natural hybrid of two wild species, N. sylvestris (maternal genome) and N. tomentosiformis (paternal genome), also supported by phylogenetic molecular analysis based on ITS regions of nuclear ribosomal DNA (Chase et al. 2003). Alternatively, though less likely the latter is assumed to be N. otophora. However, a molecular analysis based on five genes encoding putrescine N-methyltransferase supported the hypothesis of Kenton et al. (1993) that the progenitors are N. sylvestris and an introgressed hybrid between N. tomentosiformis and N. otophora (Riechers and Timko 1999 and references therein). There are innumerable cultivars, e.g., over 1500 entries in a United States Department of Agriculture inventory. The only other species used on a limited scale is N. rustica (Tso 1999), which is assumed to be a hybrid of N. undulata and N. paniculata (Wolters 1994; Hansel 2004) though N. knightiana instead of N. paniculata might be an alternative candidate; both belong to the section Paniculatae (Chase et al. 2003). Tobacco is the most widely grown commercial non-food crop in the world, produced in at least 117 countries. In 2004, total world production of tobacco leaves was estimated at 6.5 million metric tons (FAO, statistical data bases). The tremendous commercial importance of tobacco plants has led to an unusual knowledge about their secondary metabolites: About 3000 constituents were identified and characterized in tobacco leaf and some 4000 in smoke. The decisive constituent, nicotine, ranges in concentration from 0.5 to 8% (dry weight) in the major cultivated tobacco species, N. tabacum and N. rustica (Enzell et al. 1977; Leffingwell 1999). However, there are also reports on cultivars with even higher proportions, e.g., N. tabacum up to 10% and N. rustica up to 18% (Wolters 1994).

It seemed reasonable to suppose that other, wild species of the genus are also able to synthesize nicotinoids. Furthermore, such wild species might be useful for improving tobacco germplasm. Thus, early studies led to reports on the occurrence of nicotine and its congeners already since the forties of the past century (e.g., Jackson 1941; Shmuk and Borozdina 1941; Smith and Smith 1942). Two extensive studies on 60 and 64 species, respectively, based on capillary GC, clearly demonstrated that the occurrence of nicotinoids is a consistent trait in the genus Nicotiana (Saitoh et al. 1985; Sisson and Severson 1990). Both reports included the same 60 species with additional four in the second one (N. thyrsiflora, N. palmeri, N. sanderae, N. linearis). They represented all 14 sections of the genus recognized at that time. The first report was focused on separated samples from leaves and roots; the second one compared samples from greenhouse and field-grown plants. These two reports show remarkably corresponding results concerning the alkaloid profile. Nicotine and nornicotine have been detected in all 60 of the common species of both reports, anabasine and anatabine in almost all of them; these four metabolites are documented with their relative percentage composition in every species. This refutes the observations of Smith and Abashian (1963), who found nicotine and nornicotine to be completely absent in some Nicotiana species. The diverging results were achieved apparently due to the different sensitivity of the methods used (GC vs TLC). Anabasine could not be detected in samples of N. sanderae and N. linearis, possibly due to the very low total alkaloid content of both species (falling below the analytic detection limit). This is also true for anatabine and N. linearis. Additionally the second report included data on the presence of two minor pyridylpyrrolidines: myosmine, detected in only six species (<1%), and N'-acetylnornicotine, detected in 45 species (70%).

Table 3.4 shows the combination of the data of both reports mentioned above. Nicotine turned out to be a major alkaloid in 54 Nicotiana species (84%) and the principal component in 28 species (44%). Nornicotine was a major component of 32 species (50%) but predominating, i.e., without nicotine or another compound as a second main alkaloid, in only eight species (12%). With the exception of N. alata, N. maritima, and N. africana, the concentrations of nicotine surmounted those of nornicotine in the roots of all species (95%). However, this was the case in only 36 out of 60 species (60%) for the leaves. (See also Sect. 3.3.4).

There were only three species (5%) with anabasine as the principal alkaloid (N. debneyi, N. glauca, N. petunioides) though it was at least one of two or three major alkaloids (nicotine and/or nornicotine) in another 13 species (20%). However, considering only the roots anabasine was found predominating in 7 species (N. glauca, N. solanifolia, N. benavidesii, N. cordifolia, N. debneyi, N. maritima, N. hesperis). Ontogenetic variation of the alkaloid profile was observed for N. glauca (Lovkova et al. 1976). Nicotine was the main nicotinoid in 15-day-old seedlings whereas anabasine prevailed in 48-day-old plants. Anatabine turned out to be always a minor component with the exception of N. otophora where it represented the principal alkaloid in the leaves (roots: nicotine).

The total-alkaloid content in leaves (dry weight) varied in a wide range from 0.003% (N. alata) to 2.96% (N. sylvestris); the corresponding values in roots varied from 0.027% (N. langsdorffii) to 2.48% (N. velutina) (Saitoh et al. 1985). The total-alkaloid content of the roots was higher than in the leaves for 43 out of 60 species (72%) but there were very interesting exceptions, e.g., N. sylvestris (leaves: 2.96% vs roots: 0.786%), N. attenuata (2.227% vs 0.248%), N. tabacum (1.146% vs 0.218%), N. langsdorffii (0.258% vs 0.027%). In the roots of most species the concentrations of the two pyridylpiperidine-type nicotinoids were surmounting those of the leaves. However, there were some exceptions again, e.g., N. glauca or N. tabacum with higher concentrations of anabasine in the leaves than in the roots as well as of anatabine in N. hesperis.

Field-grown plants were found to contain significantly higher total-alkaloid levels than greenhouse plants, e.g. N. rustica 2.56% vs 0.51%. The range for the former turned out to be between 0.07% (N. forgetiana) and 2.87% (N. arentsii), for the latter between 0.10% (N. forgetiana) and 1.91% (N. excelsior). Remarkably, nicotine accounted for nearly the entire alkaloid fractions in samples with the highest total-alkaloid levels. There was only one exception, N. noctiflora with anabasine as the predominant alkaloid (total alkaloids: 1.70%). In both large reports the nicotine content of N. sylvestris was found to be much higher than the one of N. tomentosiformis. This is interesting because they are supposed to be the parents of N. tabacum as already mentioned. In the case of the presumable parents of the s econd cultivated tobacco species, N. rustica, there were diverging results with regard to the content of nicotine: N. undulata was in the lead according to the results of Sisson and Severson; however, Saitoh et al. had opposite results, i.e., in favour of N. paniculata. As has been shown for N. tabacum nicotine is present in all parts of the plants including the generative organs like flowers (all parts, e.g., stigma, stamen, ovary, petal), seeds, immature and mature capsules (Saitoh et al. 1985).

Another study on leaves from 40 Nicotiana species should be mentioned which provides details on the occurrence of further congeners in addition to the main alkaloids (Sarychev and Sherstyanykh 1985). Accordingly, 2,3'-dipyridyl could be detected in 29, nicotyrine in 19, and N'-methylanabasine in 10 species. This study also comprised information on the occurrence of simple bases like pyridine, 3-acetylpyridine, 3-cyanopyridine, a-picoline, 6-picoline, quinoline, and isoquino-line. However, since these simple bases are typical pyrolytic degradation products of nornicotine and myosmine, respectively (Balasubrahmanyam and Quin 1962), it can be assumed that they may also have been artefacts in the study mentioned above.

Cyphanthera clade. This clade comprises three genera (Crenidium, Cyphanthera, Duboisia) endemic to Australia. The members of this clade are unique in that they accumulate nicotinoids as well as tropane alkaloids and - at least some of them -also simple pyrrolidine alkaloids (for details see Sects. 3.1.2 and 3.4.2, respectively, as well as Table 3.1). It is remarkable that neither tropanes nor simple pyrrolidines were ever found in the well-studied genus Nicotiana. The genus Duboisia comprises four species. They accumulate the alkaloids differently in the plant: In a first study on their location in D. hopwoodii it was reported that the tropanes are more or less restricted to the roots whereas the nicotinoids turned out to be concentrated in its leaves (Kennedy 1971). This contrasts with D. leichhardtii which accumulated the nicotinoids predominantly in its roots and tropanes in its leaves. The situation concerning D. myoporoides is more complicated due to the existence of three natural chemical varieties (chemovars), one of them with nicotinoids as dominant alkaloids (Mortimer and Wilkinson 1957). Finally, there is only one report on the tropane alkaloid content of the fourth species, D. arenitensis (Griffin and Lin 2000); whether this species is also able to synthesize nicotinoids is unknown. All chemovars of D. myoporoides are characterized by the occurrence of (i) tropane alkaloids (scopolamine, hyoscyamine), (ii) simple pyrrolidine alkaloids (hygrine), and (iii) nicotinoids (Griffin and Lin 2000). However, they differ in their dominant alkaloid, first discovered for varieties with different tropane alkaloids (Loftus Hills et al. 1954a). Seven decades after the discovery of tropane alkaloids in D. myoporoides (Gerrard 1880; Ladenburg 1880), nicotine (0.7-0.9% dry weight) and nornicotine (0.2-0.3%) were identified in the leaves of this species (cultivated from seeds from New Caledonia) besides scopolamine [syn.: Hyoscine; (0.25-0.55%)] (Loftus Hills et al. 1953). The isolation of nicotine and anabasine from a sample collected at a certain locality (Acacia Plateau) in the wild of South Queensland was documented (Mortimer 1957; Mortimer and Wilkinson 1957); these nicotinoids represent the principal alkaloids of the sample, thus establishing a novel chemovar. In a more recent study four leaf collections from natural stands of differing l ocations in Queensland representing high and low altitudes in semitropical and tropical regions were analyzed (Gritsanapan and Griffin 1991). The findings of

1957 could be confirmed. In contrast to the remaining three samples (major alkaloid: Scopolamine) the one from the Acacia Plateau could be characterized as a distinct nicotinoid ('pyridine') variety again. Finally, anatabine was also isolated from D. myoporoides cultivated in Japan (Kitamura et al. 1980). In a study on ontoge-netic variations during the first year of growth only traces of nornicotine and anabasine had been detected in cotyledons; after two months the leaves contained nicotine, nor-nicotine, and anabasine; maximum of nicotine content was measured after four months. Afterwards it decreased until a constant value has been reached (Kitamura et al. 1985).

Nicotine content in the leaves of D. leichhardtii was reported to be very low; however, the roots contained relatively large concentrations (Kennedy 1971; Endo and Yamada 1985). The production of the nicotinoid has also been observed in different studies with root and cell cultures of this species (Kagei et al. 1980; Yamada and Endo 1984; Endo and Yamada 1985; Leete et al. 1990).

According to early monographs (Husemann et al. 1884 and references therein; Remington and Wood 1918), Gerrard and Petit, independently from each other (1879), had succeeded in isolating minute quantities of an alkaloid from the leaves of D. hopwoodii, the 'pituri' plant (see Sect. 3.4.6, "Ethnobotany"), which was named 'piturine'. Gerrard as well as Petit thought 'piturine' to be identical with nicotine. Though Ladenburg pointed out that the boiling point of 'piturine' and nicotine, respectively, was identical and even similar results in comparative pharmacological studies were obtained by Ringer and Murrell (Husemann et al. 1884 and references therein), other authors claimed to be sure that their results distinguish 'piturine' from nicotine, e.g., Senft (1911). However, the assumption of Gerrard/Petit could be confirmed by Rothera (1911) and Petrie (1917b) as well as by Hicks and Späth in their studies on nicotine and its nor congener in the thirties of the past century as already mentioned above (Sect. 3.3.1). Both alkaloids were detected as constituents in the leaves of a large number of specimens of D. hopwoodii by Bottomley et al. (1945). A comprehensive study of leaf and root collections of this species led, e.g., for samples from central Australia being used for the isolation of nicotine, nornico-tine, myosmine, and N-formylnornicotine as well as to the identification of cotinine, N-acetylnornicotine, anabasine, anatabine, anatalline and "bipyridyl" (2,3' -dipyridyl) by GC/MS analysis from the leaves (Luanratana and Griffin 1982). The roots contained nicotine, nornicotine, and N-formylnornicotine besides tropane alkaloids. The leaves may show large concentrations of nicotine (up to 5.3%) (Bottomley et al. 1945) and nornicotine (up to 4.1%), respectively. Both are potential principal alkaloids. This indicates that the leaves of D. hopwoodii are qualitatively and quantitatively equivalent to those of Nicotiana tabacum with regard to the nicotinoid profile. Empirical experience had led Australian aborigines during the nineteenth century to use the cured leaves of this Duboisia species for the preparation of a narcotic stimulant ('pituri'), applied preferentially as "chewing tobacco" (Watson et al. 1983; see also Sect. 3.3.6 "Ethnobotany and Ethnomedicine").

Nicotinoids were also identified as constituents of three Cyphanthera species (out of eight). Nicotine turned out to be the principal alkaloid of C. tasmanica in leaves and roots though tropane alkaloids were also present. Furthermore, it was a major alkaloid in the leaves of C. anthocercidea accompanied by nornicotine and anabasine as well as tropanes. Nornicotine and anabasine were the alkaloids which could be characterized in the aerial parts of C. racemosa (no tropanes). However, the alkaloid content was rather low in this species (aerial parts: 0.01% compared with 0.21% and 0.17%, respectively, for C. anthocercidea and C. tasmanica). A further species, C. frondosa, also capable of synthesizing nicotine and anabasine, is supposed to be a hybrid of C. albicans and Duboisia myoporoides. Since C. albicans itself had turned out to be a nicotinoid-negative species the capability of C. frondosa to synthesize these alkaloids might be a heritage of its second (nico-tinoid-positive) parent. Anabasine was the sole nicotinoid (besides tropanes) of the monotypic genus Crenidium, represented by C. spinescens (Evans and Ramsey 1983; El Imam and Evans 1984). Other Subfamilies

There are some reports on the erratic occurrence of nicotine or certain congeners in the remaining solanaceous subfamilies. This is the case for Petunia violacea, Petunioideae (minute concentrations).

Cestroideae. Nornicotine (major alkaloid), nicotine, and anabasine were detected in Salpiglossis sinuata (Salpiglossis clade) (Schröter 1958, 1963). The same alkaloids were found as constituents of Streptosolen jamesonii (Browallieae clade) (Schröter 1963). Nicotine and its nor congener were isolated also from the leaves of Cestrum diurnum and C. nocturnum (Cestreae clade), in addition cotinine and myosmine from the latter species only. This was the first report on the occurrence of cotinine and myosmine outside of the genus Nicotiana. However, all these alkaloids were present in such small concentrations that the toxicity attributed to both Cestrum species should be based on different constituents (steroidal saponins, see Sect. 7.7) (Halim et al. 1971).

Solanoideae. Furthermore, there are some reports on the occurrence of nicotine in this subfamily in extremely low concentrations, e.g., 0.0005-0.002% in dried leaves of tomato, Solanum lycopersicum sub nom. Lycopersicon esculentum (Solaneae clade) (Wahl 1952), of deadly nightshade, Atropa belladonna (Hyoscyameae clade), of three Datura spp. (Datureae clade) (Wahl 1953). Alternatively, it has been discussed that these previous data might reflect a contamination of the samples (e.g., due to smokers in the laboratories) instead of genuine production in the plant or a wrong identification (Gemeinholzer and Wink 2001). Recently, the nicotine content of edible nightshades, fruits of peppers and pepperonis (Capsicum annuum, Capsiceae clade), tomatoes (Solanum lycopersicum), aubergines (S. melongena) and tubers of potatoes (S. tuberosum), Solaneae clade, has been determined (Siegmund et al. 1999). The authors had paid special attention to the avoidance of any contamination with environmental nicotine, e.g., tobacco smoking. Thus, their results may be interpreted as genuine occurrence of nicotine in all these species. The GC/MS-detected the presence of nicotine in flowers of

Brugmansia candida was supposed to be at least in part - beside tropanes - responsible for the addiction induced by consumption of flowers by humans (Gambaro and Roses 1989). Finally, Withania somnifera (Physaleae clade, Withaninae subc-lade) is reported to contain nicotine (Majumdar 1952, 1955).

The unequivocally nicotine-positive GC/MS results with numerous convolvula-ceous species (Sect. 3.3.3) may be an indication for a genuine distribution of this plesiomorphic character also throughout the Solanaceae though only in minute concentrations. Another indication might be that all solanaceous taxa mentioned above to contain small amounts of nicotine have turned out to synthesize also calystegines (see Sect. 3.5). Nicotine and calystegines share the N-methyl-A1-pyrrolinium cation as a precursor in their biosynthetic pathway. The fact that Nicotiana tabacum is lacking calystegines is not inconsistent with this indication; apparently tobacco has lost this plesiomorphic characters as it has lost the ability to synthesize simple pyrrolidines.

Chemotaxonomic Relevance. The monotypic Nicotianeae subclade and the Australian endemic traditionally recognized tribe Anthocercideae G.Don form a sister pair (Garcia and Olmstead 2003 and references therein; Clarkson et al. 2004). This tribe is identical with the traditionally recognized subfamily Anthocercidoideae (G.Don) Tetenyi used by Hunziker (2001). The Cyphanthera subclade is the most advanced of this tribe/subfamily, thus showing a maximum distance to the Nicotianeae subclade within the Nicotianoideae clade (see Fig. 2.2). Nevertheless, the occurrence of nicotinoids in combination with their accumulation seems to be a consistent trait of these both subclades involving Nicotiana on one hand as well as Crenidium, Cyphanthera, and Duboisia on the other. In contrast the remaining monotypic subclades between the Nicotianeae subclade and the Cyphanthera subclade, i.e., those involving the phytochemically well-checked genera Symonanthus, Anthocercis, Grammosolen, and Anthotroche, apparently lack nico-tinoids. This may be interpreted as a loss of the ability to synthesize them, since they may be considered unequivocally as plesiomorphic characters present also, e.g., in the sister family Convolvulaceae.

Based on the results of their study already reported above, Saitoh et al. (1985) concluded that no clear-cut correlation between alkaloid pattern and classification of the genus Nicotiana exist. In contrast to this conclusion Sisson and Severson (1990) interpreted their results, also already mentioned above, as evidence for the association between alkaloid characteristics and the phylogenetic classification of the species in the genus. Thus, in the section Paniculatae (data for six species) total alkaloid levels were in the medium range relative to the other sections. Five species in this section produced mainly nicotine. The species in the section Tomentosae (data for five species) turned out to be characterized by low and medium total alkaloid levels and nornicotine was the predominant alkaloid in all five species. The section Alatae (data for seven species) showed mainly very low total alkaloid levels and nicotine as the principal alkaloid. Section Noctiflorae (data for three species) was characterized by the predominance of anabasine. The largest section Suaveolentes (data for 22 spp.), almost exclusively of Australian origin, could be divided in three groups of species according to their different distribution in (i) the arid central and northern regions with high total-alkaloid levels and nicotine as the principal alkaloid, (ii) the south-central and eastern regions with total-alkaloid levels in the medium range and nornicotine as the predominant alkaloid, and (iii) the western regions with low total-alkaloid levels and nicotine as well as nornicotine making up the largest proportion.

The occurrence and distribution of nicotinoids in the remaining subfamilies of the Solanaceae did not offer further chemotaxonomic results of significance. This is not very surprising due to the fact that their biosynthesis is very close to the primary metabolism. Consequently, the simple occurrence of nicotinoids is of no chemotaxonomic or phylogenetic relevance as has been documented by plotting their occurrence on a molecular phylogenetic framework of the solanaceous genera (Gemeinholzer and Wink 2001) in contrast to their accumulation in certain taxa.

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