## 4 Biosynthesis and Health Implications of Pyrrolizidine Alkaloids
### Biosynthesis of Pyrrolizidine Alkaloids  
The PA-content in plant material varies considerably, from trace amounts up to 19% based on dry weight, and this variability depends on numerous factors: genetic variation and heredity, tissue type and developmental stage of the plant, environmental conditions and extraction procedures [48]. In plant tissues, PAs are mainly detected as the corresponding N-oxides (PANOs) except for the seeds of _Crotalaria_ spp. or in Orchidaceae [48]. Despite numerous warnings regarding PA contamination, currently, knowledge regarding their biosynthesis is still limited, while the intermediates were well defined via feeding experiments [51]. The first studies regarding the biosynthesis of PA date back to the 1960s with [PERSON] and [PERSON] [52] who conducted the first feeding experiments with radiolabeled precursor. Subsequently, these studies have been continued and deepened, and in the 1990s, the homospermidine synthase (HSS, EC 2.5.1.44) was identified as the first pathway-specific enzyme of PA biosynthesis [51] as seen in Figure 4. In several PA-producing species, HSS presents a highly different spatiotemporal gene expression, it is specific for distinctive tissues or cells in relation to the phenological stage of the plant [53]. The various chemical structures of PAs are characteristics of specific taxa, although there are some overlaps. The sencecionin-type PAs (jacobine, jacoline, jaconine, terrosine, sencecionine, and senciphylline) are typical of the genera _Sentecio_ and _Crotalaria_. Lycospamine-type PAs (echimidine, lycospamine, and vulgarine) are present in the Boraginaceae family and Eupatorieae tribe. Heliotrine-type PAs (europine, heliotrine, and lasiocarpine) are characteristic of the genus _Heliprotropin_. Monocrolatine-type PAs (6 ulvene, monocrolatine, and tricodesmine) were detected mainly in the genus _Crotalaria_, while triangularine-type PAs were detected in the Senecioneae tribe and Boraginaceae family. Phalaenopsin-type PAs are found in the Orchidaceae family [54]. The PA distribution is highly dynamic and the diversification in PA accumulation in different organs may indicate a defensive strategy against herbivores. Furthermore, PA expression is influenced by nutrient and water supply, as well as herbivore infestation [49]. Asteraceae species synthesize the PAs exclusively in the roots; for example, in _Senceio_, the sencecionine N-oxide derives from the backbone structure of macrocyclic PAs of the sencecionine type, which is biosynthesized in the roots and then translocated to shoots through the phloem, where it is further functionalized to produce species-specific PA patterns [55], while biosynthetic localization of PAs in in the Boraginaceae are more variable. Figure 3: Pyrrolizidine alkaloid (PA)-producing plants. (**a**) _Anchusa officinalis_, (**b**) _Borago officinalis_, (**c**) _Echium italicum_, (**d**) _Symphytum officinal_, (**e**) _Heliotropium europneum_, and (**f**) _Lithospermum officinal_ representative species of Boraginaceae family; (**g**) _Eupatorium cannabinoid_, (**h**) _Pétasites hybridus_, (**i**) _Senecio vulgaris_, (**j**) _Tussilago farfura_ from Asteraceae family. So far, the site of biosynthesis is species dependent; it can be is located in the entire shoot (e.g., _H. indictum_), in roots (e.g., _S. officiala_), or both roots and shoot (e.g., _C. official_) [55]. The chemical structure of PAs consists of a bicyclic necine base to which aliphatic mono- or di-carboxylic acids, the necic acids, are generally linked via esterification. PAs can occur in plants as tertiary amines or N-oxides (PANOs), which can easily be converted back to the tertiary amines via a reduction reaction [56]. Depending on the presence or not of a double bond between C1 and C2 of the necine base, PAs can be grouped into 1,2-unsaturated PAs and saturated PAs.