3.2.1. Tiglic Acid and Related C5 Necic Acids

C5-acids of the tiglic acid type are frequently observed as building blocks of secondary plant metabolites. For instance, tiglic acid is the moiety of meteloidine and other tropane alkaloids formed by *Datura* species [95]. Feeding experiments in *Datura meteloides* showed that tiglic acid is derived from isoleucine [96]. Similar results were also obtained in *Cynoglossum officinale* for angelic acid, which is present as ester in the PA heliosupine [31]. Feeding of *Datura meteloides* with radiolabeled 2-methylbutanoic acid showed that the radioactivity was efficiently incorporated into the tiglic acid moiety of meteloidine, identifying 2-methylbutanoic acid as a precursor [97]. The same pathway was also established for carabid beetle [98]. Data from mammals [99] suggest that 3-hydroxy-2-methylbutyric acid acts as intermediate between 2-methylbutanoic acid and tiglic acid, and that the intermediates appear as coenzyme A thioesters. These steps are in fact part of the common L-isoleucine degradation pathway and thus it is not surprising that these metabolites including tiglyl-CoA are present in most plant tissues. McGaw and Woolley showed that angelic acid is derived by *cis*-*trans* isomerization from tiglic acid in *Cynoglossum officinale* [100]. These data suggest a pathway for biosynthesis of tiglic and angelic acid starting from L-isoleucine (Figure 11).

**Figure 11.** Biosynthesis of tiglic and angelic acid. L-Isoleucine is desaminated and the obtained ketocarboxylic acid decarboxylated to 2-methylbutyric acid, which is likely accompanied by linking with coenzyme A to yield 2-methylbutyryl-CoA. This intermediate is hydroxylated and subsequently dehydrated to form tiglyl-CoA, which can be isomerized to angelyl-CoA. The activated tiglic and angelic acid residues are finally transferred to necine bases and coenzyme A is released.

## 3.2.2. C7 Necic Acids

The more complex acids are generated by specific biosynthetic pathways. The observation that many of these acids include one or two C5 units resembles isoprenoids, although with unusual linkage and oxygenation patterns, encouraging the idea that they might be derived from mevalonate [101]. However, feeding experiments with 14C-labeled acetate, acetoacetate and mevalonate did not result in specific incorporation into necic acids, excluding these compounds as direct precursors for necic acids. In contrast, feeding experiments with 14C-labeled amino acids provided compelling evidence that necic acids are derived from L-isoleucine and L-valine. Additionally, [14C]-L-threonine and a few further amino acids were found to be efficiently incorporated, but only since they acted as precursors for L-isoleucine or L-valine.

The PA heliosupine appearing in *Cynoglossum officinale* consists of the necine base (-)-heliotridine esterified on the 7-hydroxy group with angelic acid (which is synthesized as shown in Figure 12) and on the 9-hydroxy group with echimidinic acid, a C7-acid (Figure 12A). Feeding experiments revealed that echimidinic acid is derived from L-valine and an additional C2 unit of unknown origin [102].

Trachelanthic acid, which is structurally very similar to echimidinic acid, is also derived from L-valine, as revealed by feeding of 14C-labeled amino acids. These results were further supported by an isotopologue study by feeding a root culture of *Eupatorium clematideum* with a mixture of 13C-labeled and unlabeled glucose [103]. Subsequently, trachelanthamine and the amino acids L-arginine, L-proline, and L-valine were isolated and the 13C labelling pattern analyzed by NMR spectroscopy. The 13C pattern of the necine base was in agreement with that of L-arginine, while the major part of trachelanthic acid (shown in red in Figure 12B) fitted to that observed for L-valine. The remaining two carbons showed a pattern as reconstituted for hydroxyethyl-TPP. This confirmed the conclusions for echimidinic acid. However, it must be emphasized that the result that the 13C pattern of the C2 unit corresponds to that of hydroxyethyl-TPP does not mean that this compound is indeed the direct precursor. In contrast, there might be a number of metabolites in-between. Thus, the direct precursor of the C2 unit remains to be determined.

**Figure 12.** Biosynthesis of C7 necic acids. (**A**) Echimidinic acid present in heliosupine is derived from L-valine (red) and a C2 unit of unknown origin (green). Angelic acid esterifying the 7-hydroxy group of (-)-heliotridine is derived from L-isoleucine (blue) as shown in Figure 11. (**B**) Trachelanthic acid is also derived from L-valine (red) and a C2 unit (green) that is likely derived from hydroxyethyl thiamine pyrophosphate. Adapted from References [70,103].

#### 3.2.3. Monocrotalic Acid and Related Compounds

Biosynthesis of monocrotalic acid was analyzed in the Fabaceae *Crotalaria retusa* and *Crotalaria spectabilis* by feeding of 14C-labeled acetate, DL-alanine, L-threonine, and L-isoleucine [104]. While minute incorporation was observed for acetate and DL-alanine, L-threonine and L-isoleucine were incorporated into monocrotaline to significant levels. Degradation studies showed that the necine base had incorporated little radioactivity while the necic acid monocrotalic acid contained most of the activity. More specifically, most activity was observed in fragments consisting of C-1, C-2 and C-6 and of C-3 and C-7, while the fragment consisting of C-4, C-5, and C-8 had incorporated comparatively little activity (Figure 13A). Thus, the right hand part of monocrotalic acid is derived from L-isoleucine while the origin of the left hand part remains elusive. Another study found, in addition to efficient incorporation of 14C-labeled L-isoleucine and L-threonine, a significant incorporation of L-valine [105]. Since no degradation studies were performed, it remains unknown whether L-valine serves as a precursor for the left hand part.

The structure of trichodesmic acid is very similar to monocrotalic acid, except that a methyl group is replaced by an isopropyl group (Figure 13B). Thus, it was also expected that the right hand part of trichodesmic acid is derived from L-leucine (and L-threonine), which could be confirmed by feeding studies. In addition, 14C-labeled L-valine and L-leucine were also efficiently incorporated, particularly into the right hand part [106]. The fact that both amino acids were incorporated to a similar level suggests that they are converted to a common precursor prior to incorporation into trichodesmic acid. However, the pathway and mechanism remain to be elucidated.

**Figure 13.** Biosynthesis of necic acids forming 11-membered rings. (**A**) The right hand part of monocrotalic acid is formed from L-leucine (and its precursor L-threonine). The C3 left hand unit is of unknown origin. (**B**) The right hand side of trachelanthic acid is derived from L-leucine (and its precursor L-threonine) while the left hand side seems to be formed from L-valine or L-leucine. Dotted lines indicate the both sides. Adapted from Reference [70].

#### 3.2.4. Senecic Acid and Senecic Acid-Derived Compounds

Among the necic acids forming 12-membered macrocyclic PAs biosynthesis of senecic acid is best characterized. Since seneciphyllic acid and isatinecic acid are derived from senecic acid, results obtained for these compounds will be discussed together with that for senecic acid. Feeding experiments of *Senecio douglasii* with 14C-labeled acetate, acetoacetate and mevalonate showed that these compounds were inefficiently incorporated into seneciphylline, and that the radioactivity was randomly distributed between the necine base retronecine and the necic acid seneciphyllic acid. This indicated that none of the tested compounds are direct precursors of the alkaloid. In contrast, L-threonine was efficiently incorporated and the radioactivity was found selectively in the necic acid part. Subsequent experiments showed that L-threonine was first converted to L-isoleucine prior to incorporation into seneciphyllic acid, identifying isoleucine as the direct precursor. In addition, evidence was provided that only C-2 to C-5 of L-leucine were incorporated into seneciphyllic acid, while C-1 (the carboxy group) was lost [107]. Feeding experiments using *Senecio magnificus* provided similar results for senecic acid and provided compelling evidence that the acid is derived from two molecules of L-isoleucine, both losing their C-1 during biosynthesis [108,109]. From the four possible isoleucine stereomers, L/D-isoleucine and L/D-alloisoleucine, only L-isoleucine was efficiently incorporated [110]. The loss of the carboxy groups raised the question for the five-carbon intermediate. Possible candidates were 2-methylbutanoic acid, which is an intermediate of L-isoleucine degradation and thus are present in most plant tissues, and 2-methyl-3-oxobutanoic acid and angelic acid. The latter was selected since it has the same configuration as the double bond in senecioic acid. Feeding resulted in incorporation of 0.06% (*S*)-[14C]-2-methylbutanoate, which was a slightly lower level than for L-[14C]-leucine, where incorporation rates of 0.1 to 0.4% were observed. However, analysis of the necic acid and the necine base showed random distribution of radioactivity. The two other compounds were incorporated in only trace amounts. Based on these results the idea that one of those compounds is a precursor of senecic

acid was discarded. Next, 14C-labeled 2-amino-3-methylenepentanoic acid (β-methylenenorvaline) was tested and efficient incorporation (0.07–0.11%) was observed with essentially only senecioic acid possessing the radio label. This observation suggested this compound as a possible intermediate (Figure 14A). Unfortunately, it could not be investigated whether the 14C-label was incorporated in either or both halves of senecic acid [111].

**Figure 14.** Biosynthesis of senecic acid. (**A**) Feeding experiments with 14C-labeled compounds showed that senecic acid is formed from two L-isoleucine molecules accompanied by loss of both carboxy carbons. 2-Amino-3-methylenepentanoic acid might be an intermediate, although it is not clear whether for one or both halves of senecic acid. Carbons are numbered according to Reference [112]. (**B**) Feeding with L-isoleucine stereospecifically labeled with 3H (tritium, T) at C-4 showed that only the *4R* label was retained upon incorporation. (**C**) 2-Aminobutanoic acid is converted *in planta* to L-isoleucine. Due to 2-ketobutanoic acid as intermediate the initial stereochemistry of 2-aminobutyric acid is irrelevant. A label (13C or 3H; red) at C-3 and C-4 is retained and found on position C-5 and C-6 in L-isoleucine. (**D**) The 13C-labeled C-4 and C-5 (blue) of L-leucine (obtained in planta from labeled 2-aminobutanoic acid) and most of the 2H (deuterium, D; red) is retained upon incorporation into senecic acid. (**E**) Possible mechanism for formation of senecic acid. CoA, coenzyme A; L-Glu, L-glutamic acid; NAD+/NAHD, oxidized/reduced nicotinamide adenine dinucleotide; OG, 2-oxoglutaric acid; and Py, pyridoxalphosphate.

The most enigmatic step in biosynthesis of senecic acid and related compounds is the mechanism for uniting C-13 and C-14 (Figure 14A). C-13 corresponds to C-4 carbons of the isoleucine precursor forming the right hand part of senecic acid. Studies with L-leucine stereospecifically labeled at C-4 with 3H showed that the pro*-R* hydrogen was retained, while the pro*-S* hydrogen was lost on both L-isoleucine molecules required for synthesis of senecic acid. This demonstrates that both carbons cannot be oxidized further than the alkene or carbinol level [113]. The observation that 2-aminobutanoic acid is efficiently converted to isoleucine in feeding experiments [113] (Figure 14C) enabled feeding studies with stable isotope labelling and analysis of the produced alkaloids by NMR [112]. Feeding of *Senecio pleistocephalus* and root cultures of *Senecio vulgaris* with [3,4-13C2]-2-aminobutanoic acid confirmed that C-3 and C-4 of 2-aminobutyric acid carbons are the precursors of C-20 and C-21 in the left hand side of senecic acid and of C-13 and C-19 in the right hand side (Figure 14D). Analysis of alkaloids obtained by feeding of [3,4-2H5]-2-aminobutanoic acid revealed that three 2H were retained on the methyl groups of both C-21 and C-19, and one 2H was retained on each C-13 and C-20. These results show that the C-3 and C-4 positions of 2-aminobutanoic acid corresponding to C-5 and C-6 of L-isoleucine are exclusively and equally incorporated intact into the two halves of senecic acid. These results are in agreement with a mechanism proposed by Bale, where the left hand part of senecic acid is derived from L-isoleucine by conversion to β-methylenenorvaline, which reacts with pyridoxal phosphate to a Schiff's base (Figure 14E). Deprotonation would generate a mesomeric anion with a nucleophilic character at C-6 (numbering corresponding to that of L-isoleucine). This nucleophile might react with a suitable, yet unknown electrophile [111]. From the experiments with 3H-labeled L-isoleucine and 2H-labeled 2-aminobutanoic acid, it is clear that the electrophile cannot possess a carbonyl group at C-4 while an alkene would be in agreement with those results. An attractive candidate is tiglyl-coenzyme A since this compound is a metabolite of L-isoleucine degradation [114] and thus present in most tissues. In addition, the C-C double bond is activated due to the neighboring keto group. Attack of the nucleophile on tiglyl-CoA would create the C-13/C-14 bond. Subsequent release of the pyridoxal phosphate coenzyme would create the C-16/C-20 double bond, which is either directly or further metabolized (e.g., as epoxide, diol, etc.) almost invariably present in necic acids of the senecic acid type. It must be emphasized that feeding experiments using (*S*)-[14C]-2-methylbutanoate, the precursor of tiglyl-CoA in the L-isoleucine degradation pathway, did not give conclusive results since high incorporation was observed, but the incorporation pattern was unspecific [111]. However, it might be worth reinvestigating a possible role of tiglyl-CoA in senecic acid biosynthesis by state of the art isotopologous NMR-based techniques.

First evidence for conversion of senecionine to other senecionine-type PAs came from feeding of [ 14C]-putrescine to *Senecio vernalis* root cultures, which resulted in rapid incorporation in senecionine *N*-oxide. However, the labeled senecionine *N*-oxide was progressively converted within 10 days to senkirkine, an otonecine-type PA [28]. Pulse-chase feeding experiments of *Senecio erucifolius* root cultures with [14C]-putrescine showed rapid incorporation of radioactivity in senecionine *N*-oxide and revealed absence of any significant alkaloid turnover with the exception of a slow but progressive conversion of labeled senecionine *N*-oxide to its dehydrogenation product, seneciphylline *N*-oxide [115]. Using whole *Senecio erucifolius* plants, in addition to formation of seneciphylline *N*-oxide, conversion of senecionine *N*-oxide to *O*-acetyl seneciphylline, and of erucifoline to *O*-acetyl erucifoline and to eruciflorine was also observed, indicating that the stem might be crucial for these modifications [116]. These data clearly demonstrate that the first product of PA biosynthesis in *Senecio* is senecionine *N*-oxide, which is further converted to a bouquet of senecionine-like alkaloids or their *N*-oxides (Figure 15). Such reactions mainly include simple one or two-step reactions like hydroxylations, acetylations, desaturations and epoxidations. In addition, it is tempting to speculate that the epoxide ring of jacobine and similar compounds might undergo further metabolization by hydrolysis or addition of hydrochloric acid to yield jacoline *N*-oxide or jaconine *N*-oxide, respectively. Thus, senecionine-like alkaloids are derived by modification of senecionine *N*-oxide rather than combination of necine bases with different necic acids.

**Figure 15.** Diversification of senecionine-like PAs. Modifications of the senecionine *N*-oxide structure are shown in red. Adapted from Reference [116].

#### **4. Regulation of Pyrrolizidine Alkaloid Levels and Biosynthesis**

PA biosynthesis is known to be regulated differently during plant development. For example, in *Eupatorium cannabium* HSS expression is restricted to young roots with close correlation to plant growth [82]. HSS is only detectable in newly grown, white roots until the produced biomass peaks and flowers open. When these plant parts die off at the end of the vegetation period, de novo alkaloid biosynthesis is required at the beginning of the next growth period. In contrast, plants of the species *Symphytum officinale* can activate a second site of HSS expression once inflorescence development begins in leaves subtending emerging flowers [117]. The same study could also demonstrate that not only HSS but the whole PA biosynthetic pathway are active in these young leaves. This second site of PA biosynthesis allows for drastically increased PA levels within the inflorescences ensuring optimal protection of the reproductive structures against herbivores.

Furthermore, PA expression is influenced by nutrient and water supply as well as herbivore infestation [118–120]. On the effects of nutrient availability, a general proposal is that a higher NPK supply reduces the PA content in *Senecio jacobaea*, *Senecio vulgaris,* and *Senecio aquaticus*, presumably due to dilution effects in shoots and roots, whereas PA levels in flowers are not affected [118]. These findings are further supported by results of PA analysis in *Senecio jacobaea* and *Senecio aquaticus* on marginal, sandy soils showing that low nutrient supply increases relative PA amounts [119]. Nevertheless, the same study found a significant change in PA composition on fertilized soils, as they reported a high rise in jacobine contents, while the total PA amount remained constant under nutrient rich conditions. One reason for increased jacobine levels could be the role of jacobine in insect herbivore resistance. Research on root herbivore damage in *Senecio jacobaea* has revealed no change in total PA content in the whole plant, but a translocation of PAs from shoot to the root, mostly of *N*-oxides, also causing the

ratio of *N*-oxide to free PAs to drop from 2:1 to 1:1 in the shoot [120]. Moreover, plants stabilize free PA levels in aerial plant parts by converting PA *N*-oxide into free bases [120].

There is substantial evidence for the genetic control and heritability of PA biosynthesis. A study by Vrieling et al. [121] using a diallel cross of *Senecio jacobaea* revealed that 48% of the variation in total PA content was caused by genotypic variation. Half-sib analysis from natural progenies combined with drought and nutrient deficiency treatments indicated that 85–90% of the differences in total PA concentration were caused by additive genetic variation. Heritability for individual PAs was significant as well. van Dam et al. [122] estimated that 33–43% of the variation in PA levels between selfed families of *Cynoglossum officinale* is due to broad-sense heritability and found that the inducibility of PA production by wounding of leaves differs significantly between families. Macel et al. [123] evaluated the variations in PA profiles within clonal families of *Senecio jacobaea* and found significant differences in the relative percentages and absolute concentrations of individual PAs between clonal families. The variation in PA composition within clonal families was smaller than the variation among families, indicating a strong genetic influence. Furthermore, Joosten et al. [51] found that the presence of pyrrolizidine alkaloids in the tertiary amine form in *Senecio jacobaea* is genotype-dependent as well. Pelser et al. [124] reconstructed the evolutionary history of qualitative PA variation in the *Jacobaea* section of *Senecio*. They concluded that the variations in PA profiles are not caused by simple gain and loss of PA-specific genes, but rather by changes in transient expression of PA biosynthesis genes, since the large intra and interspecific variation in PA distribution seems to be largely incidental and nearly all of the PAs identified in the *Jacobaea* section are also present in species of other sections of the genus.

Hybridization can result in the occurrence of novel PA structures through inter-specific epistatic interactions between enzymes and substrates, as demonstrated by Kirk et al. [119]. They observed that F1 hybrids between *Senecio jacobaea* and *Senecio aquaticus* produce florosenine, which is not present in parent populations. Since florosenine is formed by *O*-acetylation of otosenine, the capacity of *Senecio aquaticus* to synthesise otosenine and the ability of *Senecio jacobaea* for PA acetylation could be combined in the hybrids. A study by Cheng et al. [125] using F1 and F2 hybrids of *Senecio jacobaea* and *Senecio aquaticus* showed that hybrid roots contained acetylated otosenine-like PAs, while roots of the parental lines did not. Additionally, shoots of F2 hybrids exhibited an over-expression of otosenine-like PAs with contents reaching >20% of total PAs. In contrast, otosenine-like PAs have not been previously reported as a major fraction of the PA bouquet in *Senecio jacobaea* or *Senecio aquaticus*. Furthermore, some F2 hybrids contained higher relative proportions of erucifoline-like PAs in shoots compared to the parental genotypes where jacobine or senecionine-type PAs dominated. These findings indicate that hybridization contributes to the increase of the structural diversity of PAs.

There is little knowledge about the regulation of PA biosynthesis by plant hormones. Methyl-jasmonate is thought to play a part because of its role as an elicitor of induced responses and anti-herbivory resistance. Wei et al. [126] conducted a study with *Senecio jacobaea* and *Senecio aquaticus* grown aseptically on medium containing methyl jasmonate. In treated *Senecio jacobaea* plants, the total concentration of PAs increased in shoots but decreased in roots. A similar non-significant trend was observed for *Senecio aquaticus*. The application of methyl jasmonate leads to a strong shift from senecionine to erucifoline-like PAs, while the jacobine and otosenine-like PAs were not affected. This indicates that methyl jasmonate does not necessarily induce *de novo* synthesis, but rather leads to reallocation of certain PAs from roots to shoots and a conversion of PA structures. Sievert et al. [127] tested the influence of methyl jasmonate on PA levels and on the transcript levels of homospermidine synthase in *Heliotropium indicum*, *Symphytum officinale*, and *Cynoglossum officinale,* but could not detect any significant effects. The only case where a clear influence of methyl jasmonate on PAs was observed was in hairy root cultures of *Echium rauwolfii* [128]. Root culture medium, supplemented with 100 μM of methyl jasmonate, lead to a 19-fold increase of total PAs, while the flavonoid quercetin boosted the PA accumulation 6-fold at 50 μM. When the root cultures were pre-incubated with salicylic acid, the inducing effect of both compounds could be suppressed.

Regarding PA transport within the plant it has been shown for *Senecio vernalis* that translocation from roots to shoots occurs through phloem cells, located opposite to specialized HSS-expressing cells of the endodermis and the adjoining cortex parenchyma [81,129]. These cells are likely to execute the entire biosynthesis of PAs which can then be transported as *N*-oxides through the pericycle into the phloem, from there into the shoot and finally to the sites of storage, thereby uncoupling sites of PA synthesis from sites of activity. PA *N*-oxides are more soluble than free PA bases and are thus very phloem mobile, enabling transport between different storage tissues when necessary [130]. Also, the shoot is suggested to harbor the ability to diversify the chemical composition of PAs by simple hydroxylation, epoxidation, dehydrogenation, or *O*-acetylation reactions [89,116].

In a study by van Dam et al. [131], the inducibility of PA production after leaf damage was tested in two different plants species, namely *Senecio jacobaea* and *Cynoglossum officinale*, by cutting off 50% of their leaf area. PA concentrations were measured at different time points after damage and the cut-off leaf tips were used as controls for diurnal fluctuations. In *Senecio jacobaea* leaf damage significantly decreased PA concentrations. PA levels reached a minimum 12 h after the treatment and went back to initial values 24 h after. PA levels in *Cynoglossum officinale* steadily increased with time after damage. The authors hypothesize that the different responses might be due to adaptations related to the type and severity of herbivory occurring under natural conditions as *Senecio jacobaea* is adapted to initiate regrowth after severe defoliation by specialist herbivores. Therefore, a decrease of PAs in the leaves after damage could be caused by a reallocation of defense resources for future regrowth. *Cynoglossum officinale* on the other hand is not adapted to severe herbivory and does not recover as quickly following damage. Thus, it might be advantageous for this species to boost its chemical defense in order to reduce herbivore-inflicted damage.

#### **5. Biological Activity**

#### *5.1. Role in Plant Ecology*

The parallel evolvement of PA synthesis in phylogenetically unrelated plant families suggests an evolutionary advantage from PA presence. One important aspect in this context is, as for the evolution of many other secondary metabolites, a defense against insect herbivores [132,133]. Support for this hypothesis is found by studying insects specialized on PA-containing plants, which are found in a number of families, such as Lepidoptera, Coleoptera, Orthoptera, and Homoptera [134]. Additionally, grazing animals are known to only consume PA plants in times of low feed supply and generalist insect herbivores are suggested to be deterred by different PAs whereas adapted insects are thought to be attracted [131,134–136]. The general PA metabolism in insects is similar to human PA metabolism, including the negative physiological effects. Various studies regarding the effects of single PAs and general PA content on both specialist and generalist herbivore feeding have been conducted. Wei et al. [137] and Macel et al. [138] showed that the influence on different generalist species depends on PA-type and concentration. While Macel et al. [138] only observed effects under certain dietary conditions, Wei et al. [137] performed bioassays with *Senecio jacobaea* x *Senecio aquaticus* F2 hybrids with varying PA contents to draw conclusions about the correlation between the presence and abundance of different PAs and feeding damage. The results indicate that, for instance, thrips prefer leaves with lower jacobine-like PA contents, while for slugs, low senecionine-like PA levels are important. Furthermore, single PAs of both types were correlated to feeding behavior. These results are particularly interesting because presence of a C-13/C-19 double bond seems to play an important role in generalist herbivore resistance [137]. However, also jacobine *N*-oxide, although not carrying the C-13/C-19 double bond, has been shown to deter western flower thrips (*Frankliniella occidentalis*) and higher amounts were shown to be a characteristic trait of thrips resistant PA plants [90,139].

There are several reports stating that PA *N*-oxides are less bioactive against insect herbivores than the corresponding free bases [131,140–142]. Contrarily, PAs are mostly present in plants as *N*-oxides [129] with some jacobine-like PAs occurring up to 50% as free base in *Senecio jacobaea* [51] and *Crassocephalum crepidioides* [50]. An advantage of *N*-oxides is their higher solubility resulting in more efficient storage and transport [130,143,144]. Another possible explanation suggested by Liu et al. [145] are synergistic effects of PA *N*-oxides with other plant metabolites. They showed that PA free bases and chlorogenic acid act antagonistically on western flower thrips (*Frankliniella occidentalis*) mortality, while in contrast, PA *N*-oxides showed synergistic interactions with chlorogenic acid on thrips mortality. In the absence of chlorogenic acid, PA free bases decreased thrips survival more severely than PA *N*-oxides, but when chlorogenic acid was added, this effect was reversed. Thus, the bioactivity of individual PAs seems to be influenced by the natural chemical background in which they occur. This aspect is further supported by a study of Liu et al. [146], which demonstrated that fractions of a methanol extract from *Senecio jacobaea* all showed a higher survival rate of western flower thrips than the whole extract. Additionally, the expected combined effect of the single fractions on survival, assuming no interaction, was lower than that of the methanol extract. Furthermore, retrorsine and retrorsine *N*-oxide were added alone and in combination to the five fractions; the effects on thrips survival depended on the fraction to which the PAs were added. These studies highlight the relevance of synergistic effects of PAs and other plant metabolites on herbivores. In contrast, different PAs (free amines and *N*-oxides) do not show synergistic effects on one another [141].

While the correlation of PA content and feeding is generally negative for generalists, the exact opposite is reported for specialists, indicating an attraction by PA in plants. In fact, several cases of adaption and utilization of PAs by insects have been reported [1,147,148]. The oviposition of *Tyria jacobaea*, a well-studied member of the Lepidoptera, also called the cinnabar moth, was shown to be positively influenced by the concentration of jacobine-like PAs [136]. This finding suggests an advantage for *Tyria jacobaea* larvae from PA ingestion. Frequently, PAs are sequestered and stored as *N*-oxides in specialist beetles, moths, butterflies and grasshoppers [1,147–149]. With the storage of PA *N*-oxides insects have developed a strategy of using plant defense chemicals for their own defense against predators. An astonishing example for this adaption are neotropic Ithomiinae butterflies which, unlike moths, neither feed on PA plants nor sequester them from larvae stage on, but rather take up PAs solely through nectar or withered twigs [135,138]. This habit protects them from the spider *Nephila clavipes*, which cuts out butterflies that had previously ingested PAs, of her own net [150,151]. In *Utetheisa ornatrix* the male butterflies are able to transfer their PA storage during mating onto females, who then utilize them to protect their eggs [152]. Sequestered PAs play an important role in the mating process of some tribes of the Lepidoptera family, because they serve as a precursor for male pheromones [135,153]. Another impressive adaption was found in *Tyria jacobaea* and *Creatonotos transiens*. These two arctiids are able to synthesize their own PAs by esterifying a necine base of plant origin with a necic acid derived from isoleucine by their own metabolism [42,154,155]. Kubitza et al. [156] provided a high-resolution crystal structure of the flavin-dependent monooxygenase from the African locust (*Zonocerus variegatus*). This locust expresses three flavin-dependent monooxygenase isoforms contributing to a counterstrategy against PAs. By *N*-oxidation of PAs and accumulation of PA *N*-oxides within its hemolymph the locust circumvents the chemical plant defense and uses PA *N*-oxides to protect itself against predators. By such mechanisms specialized insects may gain advantages from PA-containing plants. Macel and Klinkhamer [157] reported positive correlations between the jacobine *N*-oxide and free base content of *Senecio jacobaea* with damage from specialist insect herbivores. Moreover, a study by Joshi and Vrieling [158] reported higher jacobine concentrations in *Senecio jacobaea* growing in invasive areas, indicating fitness advantage of lower PA amounts in areas with specialist herbivores compared to areas with exclusively generalist insects, wherein higher contents are advantageous.

Livshultz et al. [77] studied the evolution of PAs in species of the Apocynaceae family, which are larval host plants for PA-adapted butterflies of the Danainae family. The phylogenetic analysis showed a monophyletic origin of the HSS sequences early in the evolution of one Apocynaceae lineage. They found HSS orthologues, pseudogenes and multiple losses of HSS amino acid motifs in several non-PA producing species consistent with multiple independent losses of PAs. This indicates a selection for the loss of PA biosynthesis by PA-adapted specialist herbivores in the Apocynaceae family.

Despite their contribution in defense against insects, PAs have also been reported to influence interactions with symbiotic and pathogenic fungi. An inhibitory effect of PAs was measurable on *Fusarium* and *Trichoderma*, with PA mixtures exhibiting the highest inhibition [159]. Interestingly, in the same study an enhancement of growth by PAs was quantifiable for strains isolated from *Senecio jacobaea* indicating specialization. PA chemotypes of *Senecio jacobaea* also influence the diversity of fungal communities in soil, as shown by Kowalchuk et al. [119]. It was possible to distinguish fungal communities associated with high-PA jacobine chemotypes from low PA samples as well as senecionine/seneciphylline chemotypes. Additionally, a trend towards lower diversity in the rhizosphere of high-PA plants was observed compared to low-PA plants. This shows that PA chemotypes of *Senecio jacobaea* influence fungal communities in the rhizosphere, with jacobine types selecting more intensely than senecionine/seneciphylline types. In another study, artificial root colonization of *Senecio jacobaea* by *Rhizophagus irregularis* increased concentrations of senecionine, jacoline *N*-oxide, jaconine *N*-oxide, and usaramine *N*-oxide in roots but not in shoots. Only the amount of senecionine was significantly correlated with root length colonized [160]. On the other hand, Reidinger et al. [161] observed a negative correlation between natural colonization levels of roots of *Senecio jacobaea* by vesicles and the concentrations of both jacoline and total PAs. The authors suggest that the natural variations in PA concentrations between individual plants might have affected arbuscular mycorrhizal fungi colonization.

In the Fabaceae member *Crotalaria* PA production can also be influenced by root nodulation as demonstrated by Irmer et al. [162]. Only nodulating *Crotalaria spectabilis* plants infected with their rhizobial partner produce the PA monocrotaline, which is not regarded as being functionally involved in the symbiosis. The absolute amounts of PA per plant were highest in leaves, followed by nodules, roots, and stems, while the concentration was highest in the nodules (1.97 mg/g dry weight), exhibiting a 10-fold higher concentration than in leaves (0.21 mg/g dry weight). A plant derived HSS sequence was identified suggesting that the plant and not the microbiont is the PA producer. HSS transcripts were only detectable in nodules, indicating that they are the only location of alkaloid biosynthesis and the source from which the PAs are transported to above ground parts of the plant.

Plants which are not producing PAs can still accumulate them if they grow on soil containing decomposing PA-containing plants according to a study by Nowak et al. [163]. Various plant species commonly used as herbal teas or spices, i.e., melissa (*Melissa officinalis*), peppermint (*Mentha x piperita*), chamomile (*Matricaria chamomilla*), and parsley (*Petroselinum crispum*), were grown on soil mulched with 1 g of dried *Senecio jacobaea* plant material to investigate the uptake of PAs. Seven days after application, all mulched plants exhibited marked concentrations of PA in their leaves while the untreated controls were PA-free. The maximum PA levels in peppermint, melissa and chamomile were 0.1–0.15 mg/kg dry weight, whereas PA levels in parsley were up to five times higher (>0.5 mg/kg). Fourteen days after mulching, PA concentrations severely decreased, especially the *N*-oxide forms. The composition of the imported PAs in parsley, chamomile and melissa, showed a similar pattern to that of *Senecio jacobaea* with erucifoline being the most abundant alkaloid, followed by seneciphylline and jacobine, whereas in peppermint erucifoline occurred only in traces. This observation is probably due to species dependent differences in PA degradation or modification in the acceptor plants. While PA free bases can pass the membranes of the roots by simple diffusion, the uptake of PA *N*-oxides might be catalyzed by transporters normally responsible for the uptake of other compounds. Since the flowers of treated chamomile plants did not contain any PAs it was assumed that the transport to the leaves of acceptor plants happens via the xylem. These findings are significant for the production of herbal teas, spices, and plant derived pharmaceuticals as PA-contaminations can occur due to decomposing PA-containing plants in the fields.

#### *5.2. Toxicity of Pyrrolizidine Alkaloids and Mechanisms for their Detoxification*

Toxicity and pharmacology of PAs has recently been discussed in a detailed review by Moreira et al. [10]. Thus, we summarize toxicity here only very briefly and focus more on detoxification mechanisms.

PAs are one of the most important classes of naturally occurring toxins due to their wide distribution and high risk of unwitting consumption of contaminated natural products like grains, honey, milk, herbal teas, and medicines [3,164–171]. Numerous studies have demonstrated the hepatotoxic [172], genotoxic [173], cytotoxic [173,174], tumorigenic [175], and neurotoxic [176] potential of naturally occurring PAs. Possible symptoms of PA toxicity are hepatic veno-occlusive disease, liver cirrhosis, megalocystosis and cancer. Furthermore, they can cause chronic pulmonary arterial hypertension and congenital anomalies [3,177]. Several cases of poisonings and poisoning outbreaks caused by PA contaminated food have been documented [3,178]. The two main sources of intoxication for humans are the consumption of cereal grain contaminated with seeds from PA-containing weeds and the use of alkaloid-forming herbs or herbal remedies for medicinal and dietary purposes [17,177].

The degree of harm caused by a specific PA relies on its necine base structure, since toxic PAs, like the retronecine, otonecine and heliotridine-types, generally carry a 1,2-unsaturated necine base. The first step of metabolic activation of PAs after ingestion and absorption is dehydrogenation catalyzed by cytochrome P450 monooxygenases (CYP3A4 and CYP2C19) of the liver (Figure 16) [179]. In retronecine and heliotridine-type PAs hydroxylation of the necine base occurs at the C-3 and C-8 position to form 3- or 8-hydroxynecine derivatives followed by spontaneous dehydration [180–182]. For otonecine-type PAs, oxidative *N*-demethylation of the necine base occurs, followed by ring closure and dehydration [183,184]. Those reactions lead to the formation of reactive dehydropyrrolizidine intermediates, also known as pyrroles or pyrrolic esters, followed by the formation of pyrrolizinium ions. The electrophilic metabolites are capable of binding nucleophilic -OH, -SH, or -NH functional groups on physiologically important macromolecules like glutathione (GSH), DNA and proteins [173,185–189]. Nucleophilic substitution of one or two glutathione molecules produces the GSH conjugates 7-, 7,9-di- and 9-glutathionyl-6,7-dihydro-1-hydroxymethyl-5*H*-pyrrolizine for excretion [173,183,190,191]. These GSH conjugates are considered to be detoxification metabolites but it has been reported that they also act as reactive metabolites, causing DNA adduct formation [172]. Additionally, glutathione *S*-transferases (GSTs) can mediate these reactions leading to species-dependent toxicity effects, as it has been observed that guinea pig hepatic GSTs catalyze GSH conjugation of the pyrrolic metabolites of jacobine whereas rat hepatic GST did not affect the reaction [192]. GSH conjugation is one of the most important detoxification reactions of PAs. If the levels of reactive intermediates are high enough, the cellular GSH pool can be depleted causing severe oxidative damage of liver tissues [193–195]. Dehydro-PAs, including 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5*H*-pyrrolizine (DHP) [196,197], their C-7 and C-9 hydrolysis product, and the dehydration product (3*H*-pyrrolizin-7-yl)methanol [198] are all reactive alkylating species. They can bind to liver cellular proteins and DNA resulting in the formation of DNA and/or protein adducts as well as DNA-protein cross links, which are all associated with genotoxicity and carcinogenicity ultimately leading to veno-occlusive disease and/or tumors [175,199,200].

**Figure 16.** Metabolic activation of pyrrolizidine alkaloids. PAs are oxidized by cytochrome P450 enzymes (CYP), which converts them to the reactive and toxic pyrrole derivatives. Adapted from Reference [11].

A study on the in vitro metabolism of lasiocarpine in liver microsomes of humans, pigs, rats, mice, rabbits, and sheep was compared and a tendency towards species-dependent toxicity was observed [201]. The highest levels of reactive metabolites like (3*H*-pyrrolizin-7-yl)methanol and GSH conjugates were detected in human microsomes, followed by pig, rat and mouse, all known to be susceptible to PA intoxication. The microsomes of rabbit and sheep, known to be more PA-resistant, exhibited lower metabolite levels. Interestingly, the scale of PA toxicity also depends on age and gender, since male rats are more prone to clavinorine and isoline intoxication than female rats [194,202,203] and young mice are more susceptible to retrorsine in comparison to adult mice [204].

One mode of detoxification catalyzed by flavin-containing monooxygenase (FMOs) is the *N*-oxidation of the necine bases of retronecine and heliotridine-type PAs to form PA *N*-oxides [205,206]. Otonecine-type PAs on the other hand cannot form PA *N*-oxides due to methylation of the necine base nitrogen. PA *N*-oxides can be conjugated for excretion and are therefore generally regarded as detoxification products, although they may also be converted back to their toxic parent PAs [181,207–209]. As it was demonstrated for senecionine, the susceptibility of an animal species to PA exposure depends on the rate of FMO-catalyzed conversion of the PA into its *N*-oxide leading to detoxification as well as the P450-catalyzed formation of DHP leading to toxicity [210]. In susceptible species such as rats, cattle, horses, and chickens, a high glutathione–DHP conjugation is observed. However, in sheep, a resistant species, low rates of glutathione conjugation combined with high rates of FMO-catalyzed conversion of senecionine into its *N*-oxide occur.

Another mode of detoxification in mammal metabolism is the cleavage of the necine base-necine acid bond, mediated by unspecific esterases followed by phase II-conjugation and excretion. This process was shown to contribute to guinea pig resistance to PA toxicity and rat susceptibility, as guinea pigs have higher liver esterase activity in contrast to rats [211–214].

Recently, *N*-glucuronidation has been identified as a potential new metabolic pathway in humans and several animal species for the activation and detoxication of PAs [215].

PAs with a saturated necine base like platynecine-type PAs are known to be nontoxic. It has been demonstrated that in hepatocytes treated with platyphylline the metabolites being formed are mainly dehydroplatyphylline carboxylic acid and in lower amounts, platyphylline epoxide as well as platyphylline *N*-oxide [216]. Disparate in comparison to unsaturated PAs, which are activated to reactive pyrrolic esters causing the formation of hepatotoxic protein and/or DNA adducts, the saturated structure of the platynecine-type PAs causes the P450-mediated oxidative metabolism to generate nonreactive polar compounds that are water-soluble and easily excreted.

#### *5.3. Beneficial Properties of Certain PAs*

Despite the toxic properties of 1,2-unsaturated PAs and their negative implications on human and animal health, some PAs have the potential to be used for the treatment of diseases and infections. One of the most consequential and positive characteristics of these PAs is their glycosidase inhibitory activity [217,218], potentially leading to antidiabetic, anticancer, fungicidal, and antiviral effects [219]. Glycosidases are a class of enzymes involved in selective hydrolysis of sugar bonds in glycoconjugates as well as the covalent attachment of carbohydrates to lipids, proteins, and peptides on the cell surfaces [220–223]. Disfunctional glycosidases can reduce the proliferation of malignant cells and tumor growth [219,224]. Furthermore, inhibited glycosidases can cause the formation of disorganized oligosaccharides, thereby interrupting cell–cell and cell–virus recognition [219,225]. Thus, certain PAs may be used for the treatment of cancer, diabetes, and infections [226,227]. Some of the most potent glycosidase inhibitors are polyhydroxylated PAs since they mimic the pyranosyl and furanosyl core of the natural glycosidase substrates [226,228]. It has been demonstrated that the polyhydroxylated PAs alexine from *Alexa leiopetala* and australine from *Castanospermum australe* exhibit a strong glycosidase inhibition [229–231] and show promising potential to be used as chemotherapeutic [219], antiviral [228], and immunomodulatory drugs.

A pair of PAs with antitumor as well as antibiotic properties are (-)-clazamycin A and (+)-clazamycin B, which were isolated from *Streptomyces puniceus* [232,233]. These compounds show activity against *Herpes simplex* virus in vitro [233], as well as a strong inhibition of *Pseudomonas* species [232]. Moreover, in some fungus species, PAs with potent activity against Gram-positive and Gram-negative bacteria, as well as tumor cells have been isolated [234,235].

#### **6. Occurrence of Pyrrolizidine Alkaloids in Crop Plants**

While many plant species are PA producers, few of them are used by humans, which is likely related to PA toxicity. Among them, most are used in traditional medicine for treatment of diverse diseases, which is discussed in detail in a number of review articles [16,17,177,236–239]. However, there are also several PA-producing plants used as food or as forage crops. While intake of medicinal plants is usually minute, vegetables and herbs may be consumed at substantial amounts, making presence of PAs very critical. Thus, we will focus here on plant species that are consumed as food or used as livestock feed in different regions of the world.

## *6.1. Borago officinalis*

Borage (*Borago officinalis*), which originated from the Mediterranean area, is a common annual garden herb used in salads and teas and is widely cultivated for seed oil production. The oil contains high amounts of the essential fatty acid γ-linolenic acid (18–25%), and is therefore used for nutritional, medical, and cosmetic purposes. Larson et al. [240] first identified the pyrrolizidine alkaloids lycopsamine and amabiline (Figure 17) in fresh and dried leaves and stems of *B. officinalis* with a total alkaloid amount of less than 0.001% dry weight. They also showed that in roots the alkaloids are mainly present as the free base, while fresh leaves contain mainly *N*-oxides. Lüthy et al. [241] did not only detect lycopsamine and amabiline but also intermedine, supinine and the isomer pair acetyllycopsamine/acetylintermedine in the range of 2-10 mg/kg in vegetative tissues. Dodson et al. [92] determined thesinine as the only alkaloid in the flowers and the major alkaloid in seeds. Mature seeds were found to contain thesinine and minute amounts of lycopsamine. Immature seeds contained only thesinine. The first glycosylated pyrrolizidine alkaloid reported in plants, namely thesinine-4 -*O*-β-D-glucoside, was discovered in dried and defatted borage seeds [53]. Seeds contain approximately 0.1% to 0.5% thesinine-4 -*O*-β-D-glucoside.

Wretensjö and Karlberg [242] analyzed crude borage oil as well as borage oil from different processing stages and found no pyrrolizidine alkaloids present above a detection limit of 20 μg/kg. Additionally, they could demonstrate by spiking experiments with crotaline that the pyrrolizidine content in crude borage oil was reduced overall by a factor of approximately 30,000 in the refining process. Additionally, Vacillotto et al. [243] could not detect any pyrrolizidine alkaloids above a detection limit of 0.2 μg/kg in seed oil.

**Figure 17.** Pyrrolizidine alkaloids found in *Borago officinalis* (borage): (**A**) Flower of *B. officinalis*. Young flowers show a bright blue color while older flowers exhibit, because of a lower pH, a slightly reddish coloration. (**B**) Leaves of *B. officinalis* contain the supinidine-type PAs amabiline and supinine and the retronecine-type lycopsamine and intermedine as well as their 7-acetylated derivatives. Seeds contain mainly the (-)-isoretronecanol-type PA thesinine and its glucoside thesinine-4 -*O*-*β*-D-glucoside.

#### *6.2. Crassocephalum crepidioides*

The genus *Crassocephalum* belongs to the Asteraceae family (subfamily Asteroideae, tribe Senecioneae) [244] and includes approximately 100 plant species growing in Africa, tropical and subtropical regions of Asia and Australia and locally in America [245,246]. Both *Crassocephalum crepidioides* (Figure 18A) and *Crassocephalum rubens* are erect annual herbs used as leafy vegetables [245,247–249].

*C. crepidioides* and *C. rubens* are used fresh or dried for preparation of sauces, soups and stews [250]. *C. crepidioides* is also eaten raw, for example in Australia in salad dishes [245]. A factor making particularly *C. crepidioides* interesting for food production is its high yield of up to 25-27 t/ha per year even on soils with low nutrient supply [245]. Moreover, it has desirable nutritional properties since dried leaves contain about 27% protein, 8% fiber, 42% carbohydrates, and 3% lipids [248,251]. These values suggest that *C. crepidioides* is a good source of fiber while the relatively high amount of carbohydrates makes it a good source of energy. *C. crepidioides* and *C. rubens* contain also high levels of minerals including potassium, magnesium, calcium, phosphorous, iron, copper and manganese [251]. Both plant species are also used in traditional medicine due to supposed medicinal properties, such as antibiotic, antioxidant, anti-fungal, anti-inflammatory, anti-diabetic and blood regulative effects. They are included in traditional treatment of hepatic insufficiency, intestinal worms, stomach disorders and open wounds [245,252,253]. Furthermore, in Africa *C. crepidioides* is traditionally part of diets of pregnant and breast-feeding women as it is believed to prevent anemia and stimulate

milk production [252]. In addition to human consumption and application in traditional medicine, *C. crepidioides* is also used as livestock feed, for instance for poultry [245].

Based on brine shrimp lethality tests and application of Mayer's reagent Adjatin et al. considered both *C. crepidioides* and *C. rubens* as safe for human consumption [254]. In sharp contrast, Asada et al. reported isolation of significant amounts of jacobine and jacoline (Figure 18C) from *C. crepidioides* [255]. The reason for these divergent results might be the low sensitivity of Mayer's reagent for PAs, which are in fact usually detected with Dragendorff's reagent, or with higher sensitivity and specificity, by chromatographic techniques. Indeed, presence of high amounts of jacobine in *C. crepidioides* was recently confirmed by cation exchange chromatography [50]. Interestingly, the content of jacobine depended highly on the tissue. In young leaves jacobine was present at levels exceeding 200 mg/kg fresh weight while it was below 1 mg/kg fresh weight in old leaves. In addition, the jacobine content was under developmental control since six-week-old plants showed contents of approximately 27 mg/kg fresh weight while 10-week-old plants had contents of 260 mg/kg. Also, the growth conditions had a significant impact since incubator-grown plants had high levels while jacobine was below the detection limit (1 mg/kg) in greenhouse grown plants [50]. Production of alkaloids has also been reported for *C. rubens* [256]. These results clearly show that *C. crepidioides* and *C. rubens* cannot be considered safe for human consumption and that PA-free varieties have to be identified prior to utilization of these species as food or forage crops.

**Figure 18.** *Crassocephalum crepidioides*. *C. crepidioides* plant (**A**) and flowers (**B**). (**C**) The retronecine-type PAs jacobine and jacoline were reported in *C. crepidioides*. Pictures shown in (**A**) and (**B**): Courtesy of N. Adebimpe Adedej.

#### *6.3. Gynura*

*Gynura*, a genus of the Asteraceae family, contains several edible species native to Asia like *G. bicolor*, *G. divaricata* and *G. procumbens* which are used in Asian cuisine and traditional medicine. The species *G. bicolor* for example, has great nutraceutical potential, containing high levels of phenolic acids, flavonoids, carotenoids and anthocyanins and exhibits anti-oxidative as well as anti-inflammatory properties [257]. Furthermore, it can be used for the treatment of diabetes mellitus [258] and cancer [259,260]. Even though the use of *Gynura* may have numerous beneficial effects on health, its consumption is not without risk because of the presence of PAs. Intergerrimine and usaramine were first identified in *G. divaricata* by Roeder et al. [261]. Chen et al. [262] compared the PA profiles of *G. bicolor* and *G. divaricata* collected from five different provinces of China. They found 27 PAs consisting of five different types in the two species. Known PAs that were identified are retrorsine, spartioidine, seneciphylline, integerrimine, senecionine, senkirkine, retrorsine *N*-oxide, senecionine *N*-oxide, and seneciphylline *N*-oxide (Figure 19). Additionally, 18 other unidentified PAs were detected in both species. *G. divaricata* collected in the Jiangsu province showed the highest concentration (39.7 μg/g) of retronecine type PAs while *G. bicolor* from Fujian contained the lowest concentration (1.4 μg/g). *G. divaricata* always contained higher amounts of retronecine type PAs in comparison to *G. bicolor* from the same locality and harvest time. Both species collected from Jiangsu contained the most diverse PA profile, while *G. divaricata* from Fujian showed the lowest variety of

PAs. A weak cytotoxic activity was detected in alkaloid extracts of both species collected from Jiangsu. These results show that the amount and variety of PAs in *Gynura* depends on the species as well as the growing region, indicating that environmental factors likely play a role in PA accumulation in *Gynura*. Another *Gynura* species traditionally used in Chinese medicine is *Gynura segetum*, also known as Jusanqi. It has been shown to have anti-angiogenic [263] as well as antioxidative and anti-inflammatory properties [264]. Its medicinal use is risky though as there have been several reports of hepatic veno-occlusive disease linked to the consumption of *G. segetum* [265–268]. Liang and Roeder were the first to characterize senecionine from *G. segetum* [269]. The presence of senecionine was confirmed by Yuan et al. [270], who could additionally detect seneciphylline as well as seneciphyllinine and *E*-seneciphylline. Qi et al. [271] analyzed whole plants of *G. segetum* collected from Zhejang province of China and could unequivocally identify seneciphylline, senecionine, seneciphyllinine, and seneciphyllinine *N*-oxide using HPLC / ITMS. 16 further PA or PANO structures were tentatively assigned, among them *E*-seneciphylline, *E*-seneciphylline *N*-oxide, senecicannabine, senecicannabine *N*-oxide, a yamataimine isomer, jacoline, and tetrahydrosenecionine, which has been reported for the first time from a natural source. Considering the present research about PAs in *Gynura* indicates that caution is required concerning its use for dietary and medicinal purposes.

**Figure 19.** Pyrrolizidine alkaloids observed in *Gynura bicolor*. (**A**) *G. bicolor* plant. (**B**) *G. bicolor* leaves from the upper (green) and lower (ruby-colored) side. (**B**) Senecionine, seneciphylline and retrorsine were reported in *G. bicolor* in both, their free forms (shown above) and as *N*-oxides, while intergerrimine, spartiodine, and senkirkine were only found as free alkaloids.

#### *6.4. Lolium*

*Lolium* species, commonly known as ryegrass, are important pasture grass species serving as main feed for grazing livestock in numerous countries and regions, for example Australia, New Zealand, North America and Europe. These species can have toxic effects on mammals that graze them and are related to a syndrome known as ryegrass staggers. Therefore, their chemical composition has been analyzed intensively.

Interestingly, the originally identified toxins, lolitrem B [272], ergovaline [273], and loline alkaloids are produced by endophytic fungi [5]. The loline alkaloids represent a special class of PAs, which are characterized by the presence of an ether bridge (Figure 1). In contrast to classical PAs, which are produced by plants, lolines are metabolic products of fungi, particularly of the genus *Epichloë* [5,274,275].

In addition, several *Lolium* species can also produce PAs of the thesinine type themself. Koulman et al. [54] were the first to isolate and elucidate the stereoisomers *E*-thesinine-*O*-4 α-rhamnoside and *Z*-thesinine-*O*-4 -α-rhamnoside from *Lolium perenne* (Figure 20), thereby providing

the first report of PAs in the Poaceae family which are synthesized by the plant itself. Furthermore, they could detect those two compounds together with the aglycone and a hexoside in *Festuca arundinacea*, a species of grasses closely related to *L. perenne*. In *L. perenne* the highest concentrations of *E*/*Z*-thesinine-*O*-4 -α-rhamnoside were found in sheath tissue, followed by blade tissue while immature tissue contained the lowest amounts. Infection with the endophytic fungus *Neotyphodium lolii* increased the concentrations only slightly. The authors analyzed plants grown in a greenhouse and found variations in the ratios of *E*-thesinine-*O*-4 -α-rhamnoside to *Z*-thesinine-*O*-4 -α-rhamnoside between 0.2 and 1. When clonal material was grown in a climate room under controlled conditions the variation was severely reduced, indicating a strong environmental influence on the accumulation of *E*/*Z*-thesinine-rhamnoside. Since the identified alkaloid conjugates do not possess a C-1/C-2 unsaturation and a hydroxy group at C-7, they likely exhibit a limited toxicity to mammals. The common occurrence of these PAs in several widely used commercial *L. perenne* and *F. arundinacea* cultivars further demonstrates their non-toxicity or low toxicity to grazing livestock, but detoxification by rumen bacteria might play a role as well [276]. It is currently unknown if the thesinine-rhamnoside PAs play any role in insect deterrence or if they enhance plant fitness by any other means. Wesseling et al. [91] analyzed various Pooideae species grown under controlled conditions and found that PA biosynthesis is restricted to a small group of species producing only a narrow range of thesinine conjugates. The three closely related outbreeding *Lolium* species *L. perenne*, *L. multiflorum*, and *L. rigidum* were shown to accumulate PAs while the inbreeding *L. temulentum* and *L. remotum* do not. Furthermore, *F. arundinacea* contained PAs while in the closely related *F. pratensis* they were absent. Potential explanations for this pattern might be the independent evolution of PA biosynthesis in *F. arundinacea* or a secondary loss in the inbreeding *Lolium* species. Hybridization of PA producing *Lolium* species with *Festuca* could be another possibility of how PA production was acquired in *Festuca*. Additionally, the inter and intra-specific PA patterns are highly variable. *L. rigidum* contained the highest average amounts of PAs, followed by *L. perenne* and *F. arundinacea*, while *L. multiflorum* showed the lowest concentrations. *F. arundinacea* was the only species which contained the aglycone thesinine in significant amounts. In *L. perenne* and *L. multiflorum* PAs were restricted to only a few of the tested individuals, while all samples of *L. rigidum* and *F. arundinacea* were tested positive. It seems like PA biosynthesis is a constitutive trait in some species, while in others it occurs only infrequently and might possibly be induced by external stimuli or occur in later developmental stages. Gill et al. [76] identified two putative HSS genes in perennial ryegrass, one of them (*LpHSS1*) occurring only sporadically. A significant association of absence of the *LpHSS1* gene with lower levels of thesinine-rhamnoside PAs was found. This provides the possibility of developing genetic markers for future breeding efforts in regard to PA presence and enables investigations about the role of PAs in deterring herbivore pests. Further HSS-like gene sequences were identified in other Poaceae species, including tall fescue, wheat, maize, and sorghum. Therefore, PA biosynthesis might be even more widespread in the grass family than it is known to date with unknown PA structures still to be discovered.

**Figure 20.** Pyrrolizidine alkaloids reported for *Lolium* species. (**A**) *Lolium perenne* (ray grass). (**B**) *Lolium multiflorum* (Italian rye-grass). (**C**) Structures of *E-* and *Z-*thesinine-4 -*O*-α-L-rhamnoside. Pictures shown in (A) and (B): Courtesy of Leo Michels.

**Author Contributions:** Conceptualization, W.R., S.S.; writing and original draft preparation W.R., S.S. and N.K.; figure preparation, W.R. and S.S.

**Funding:** The publication costs were covered by the German Research Foundation (DFG) and the Technical University of Munich (TUM) in the framework of the Open Access Publishing Program.

**Acknowledgments:** Thanks to N. Adebimpe Adedej for providing pictures used in Figure 18 and for help with editing. Thanks to Leo Michels for providing pictures used in Figure 20. Thanks to Brigitte Poppenberger and Veronica Ramirez for critical reading of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
