molecules Review Pyrrolizidine Alkaloids: Biosynthesis, Biological Activities and Occurrence in Crop Plants Sebastian Schramm, Nikolai Köhler and Wilfried Rozhon * Biotechnology of Horticultural Crops, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Liesel-Beckmann-Straße 1, 85354 Freising, Germany; [email protected] (S.S.); [email protected] (N.K.) * Correspondence: [email protected]; Tel.: +49-8161-71-2023 Academic Editor: John C. D’Auria Received: 20 December 2018; Accepted: 29 January 2019; Published: 30 January 2019 Abstract: Pyrrolizidine alkaloids (PAs) are heterocyclic secondary metabolites with a typical pyrrolizidine motif predominantly produced by plants as defense chemicals against herbivores. They display a wide structural diversity and occur in a vast number of species with novel structures and occurrences continuously being discovered. These alkaloids exhibit strong hepatotoxic, genotoxic, cytotoxic, tumorigenic, and neurotoxic activities, and thereby pose a serious threat to the health of humans since they are known contaminants of foods including grain, milk, honey, and eggs, as well as plant derived pharmaceuticals and food supplements. Livestock and fodder can be affected due to PA-containing plants on pastures and fields. Despite their importance as toxic contaminants of agricultural products, there is limited knowledge about their biosynthesis. While the intermediates were well defined by feeding experiments, only one enzyme involved in PA biosynthesis has been characterized so far, the homospermidine synthase catalyzing the first committed step in PA biosynthesis. This review gives an overview about structural diversity of PAs, biosynthetic pathways of necine base, and necic acid formation and how PA accumulation is regulated. Furthermore, we discuss their role in plant ecology and their modes of toxicity towards humans and animals. Finally, several examples of PA-producing crop plants are discussed. Keywords: Borago officinalis; Crassocephalum; Copper-dependent diamine oxidase; Gynura bicolor; Homospermidine synthase; Lolium perenne; Necic acids; Necine bases; Pyrrolizidine alkaloid biosynthesis; Senecionine 1. Introduction Pyrrolizidine alkaloids (PAs) are heterocyclic organic compounds synthesized by plants that are thought to act as defense compounds against herbivores [1]. Estimates indicate that approximately 6.000 plant species worldwide, representing 3% of all flowering plants, produce these secondary metabolites. In particular, members of the Asteraceae, Boraginaceae, Heliotropiaceae, Apocynaceae, and some genera of the Orchidaceae and the Fabaceae are PA producers [2]. Reported concentrations vary greatly, from trace amounts to up to 19% dry weight, and are considered to be dependent on a number of factors including the developmental stage, tissue type, environmental conditions, and extraction procedures [3]. PAs consist of a necine base esterified with a necic acid. The necine base typically includes pyrrolizidine, a bicyclic aliphatic hydrocarbon consisting of two fused five-membered rings with a nitrogen at the bridgehead [4] (Figure1). Loline alkaloids may be formally considered as PAs since they also possess a pyrrolizidine system, although it contains an ether bridge linking carbon 2 (C-2) and carbon 7 (C-7). While stricto sensu PAs are exclusively formed in plants, lolines are synthesized by Molecules 2019, 24, 498; doi:10.3390/molecules24030498 www.mdpi.com/journal/molecules Molecules 2019, 24, 498 2 of 44 Molecules 2018, 23, x FOR PEER REVIEW 2 of 45 endophytic fungal symbionts of the genus Epichloë [5]. In addition, their biosynthesis is distinct from by endophytic fungal symbionts of the genus Epichloë [5]. In addition, their biosynthesis is distinct PAs [5–7]. Thus, lolines will be discussed only peripherally in this review. from PAs [5–7]. Thus, lolines will be discussed only peripherally in this review. FigureFigure 1. 1.Core Core structures structures and and examples examples for for pyrrolizidine, pyrrolizidine, loline, loline, indolizidine, indolizidine, quinolizidine, quinolizidine, tropane tropane andand granatane granatane alkaloids. alkaloids. In In contrast contrast to to the the other other alkaloids alkaloids pyrrolizidine pyrrolizidine alkaloids alkaloids appear appear mainly mainly as as NN-oxides,-oxides, as as shown shown for for the the example example of of senecionine- senecionine-NN-oxide.-oxide. TheThe fused fused bicyclic bicyclic system system of of PAs PAs resembles resembles indolizidine indolizidine and and quinolizidine quinolizidine alkaloids, alkaloids, which which containcontain aa fivefive andand aa six-membered six-membered ringring oror twotwo six-memberedsix-membered rings,rings, respectivelyrespectively [[8]8] (Figure(Figure1 ).1). TropaneTropane and and granatane granatane alkaloids alkaloids also showalso ashow similar a structuresimilar consistingstructure ofconsisting a five and of a six-membereda five and a ringsix-membered or two six-membered ring or two rings, six-membered respectively rings, [9]. respectively However, in [9]. contrast However, to necine in contrast bases, theto necine rings ofbases, tropane the andrings granatane of tropane alkaloids and granatane are bridged alkaloids rather are thanbridged fused. rather While than severalfused. While tropane several and quinolizidinetropane and alkaloidsquinolizidine including alkaloids atropine including (the racemic atropine mixture (the racemic of (±)-hyoscyamine) mixture of (±)-hyoscyamine) and sparteine areand used sparteine in medicine are used [9], in PAs medicine are mainly [9], PAs known are mainly for their known hepatotoxic for their and hepatotoxic potentially and carcinogenic potentially propertiescarcinogenic [10]. properties Nevertheless, [10]. some Nevertheless, PAs show some interesting PAs show pharmacological interesting propertiespharmacological that are properties currently underthat are investigation currently under (see Section investigation 5.3)[10 ,11(see]. WhileChapter tropane 5.3) [10,11]. and quinolizidine While tropane alkaloids and quinolizidine are usually presentalkaloids in plantsare usually in their present free forms, in plants PAs are in mainlytheir free present forms, as NPAs-oxides are (Figuremainly 1present), which as are N highly-oxides water-soluble(Figure 1), which and consideredare highly water-soluble less toxic than and the considered free PAs. less toxic than the free PAs. 2.2. Structural Structural Diversity Diversity of of Pyrrolizidine Pyrrolizidine Alkaloids Alkaloids WithinWithin the the combination combination of of a a set set of of necine necine bases bases (Figures (Figure2 2; and Figure3) and 3) aand considerable a considerable number number of necicof necic acids acids (Figure (Figure4), an enormous4), an enormous structural structural diversity diversity of PAs can of bePAs obtained. can be Thisobtained. is further This amplified is further byamplified modifications, by modifications, including N -oxidationincluding ofN the-oxidation tertiary of nitrogen the tertiary of the necinenitrogen base, of hydroxylationthe necine base, of thehydroxylation necine base and/orof the necine the necic base acid, and/or and the acetylation necic acid, of hydroxyand acetylation groups of of hydroxy the acid moiety.groups Thus,of the itacid is notmoiety. surprising Thus, thatit is severalnot surprising hundreds that of several different hund PAsreds have of already different been PAs identified have already and eachbeen yearidentified new variantsand each are year described. new variants are described. 2.1.2.1. Diversity Diversity of of Necine Necine Bases Bases InIn addition addition to to the the pyrrolizidine pyrrolizidine ring ring system system most most necine necine bases bases possess possess a a hydroxymethyl hydroxymethyl group group atat positionposition 1 (Figure 22),), which is is a aconsequence consequence of ofthe the biosynthetic biosynthetic pathway pathway (see (seeChapter Section 3.1). 3.1 Since). Since1-hydroxymethylpyrrolizidine 1-hydroxymethylpyrrolizidine contains contains two twochiral chiral centers, centers, carbons carbons C-1 C-1 and and C-8, C-8, in totaltotal fourfour compoundscompounds exist: exist: The The enantiomers enantiomers (-)/(+)-trachelanthamidine (-)/(+)-trachelanthamidine and and (-)/(+)-isoretronecanole (-)/(+)-isoretronecanole (Figure (Figure2B). Among2B). Among them, (-)-trachelanthamidinethem, (-)-trachelanthamidine and (-)-isoretronecanole and (-)-isoretronecanole are most are frequently most frequently found, for found, instance, for asinstance, the necine as basethe necine of trachelanthamine base of trachelanthamine (Figure5C) and(Figure the nervosines5C) and the [ 12 nervosines] (Figure5 E),[12] respectively. (Figure 5E), Examplesrespectively. forPAs Examples containing for (+)-trachelanthamidinePAs containing (+)-tr andachelanthamidine (+)-isoretronecanole and (+)-isoretronecanole are acetyllaburnine [ 13are] (Figureacetyllaburnine5G) and madhumidine [13] (Figure A 5G) [ 14], and respectively. madhumid Theine most A frequent[14], respectively. modification The of saturated most frequent necine basesmodification is hydroxylation of saturated at C-7. necine However, bases theis hydroxylation positions C-2 andat C-7. C-6 However, are also occasionally the positions hydroxylated. C-2 and C-6 Necineare also bases occasionally like (-)-platynecine, hydroxylated. possessing Necine bases hydroxy like groups(-)-platynecine, on C-7 and possessing C-8, and hydroxy (-)-rosmarinecine