Pyrrolizidine Alkaloids and Their N-Oxides

Whilst the N-oxidation of pyrrolizidine alkaloids is a recognized detoxifying mechanism in mammals [26], it has also been shown that ingested N-oxides are indeed toxic, presumably via in vivo reduction to the parent pyrrolizidine alkaloids 

Gas chromatography coupled to mass spectrometry is a widely used analytical method [31] but does require prior reduction of the involatile N-oxides. Additionally, there are problems associated with some alkaloids that require prederivatization to enhance the GC characteristics. These requirements for prederivatization can adversely affect the GG-MS interpretation, especially at trace levels of alkaloids [32]. Gombined SPE-LC-MS approaches have provided methods for qualitative profiling and quantitative analysis of pyrrolizidine alkaloids and their N-oxides. 

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Extraction of the pyrrolizidine alkaloids and their N-oxides from plant sources is a critical first step in LG-MS analysis. Some methods of extraction already described (Section 13.2) consist simply of treatment with aqueous methanol and, following microfiltration (0.45 p.m), immediate LC-MS analysis with no further treatment. However, the aqueous methanol treatment will extract more than just the alkaloids. 

The presence of toxic pyrrolizidine alkaloids in honey has been known for several decades [42]. The potential health concerns associated with pyrrolizidine alkaloids in food [27] and honey [28] in particular demand a rapid, sensitive method of detection in various matrices. The application of LC-MS methods to honey samples extracted using sex SPE cartridges has facilitated the analysis of honey for the presence of pyrrolizidine alkaloids and their N-oxides [34,41]. The SPE and LC-MS analysis has shown that honey attributed to known pyrrolizidine alkaloid-producing sources can have levels in excess of 5000 jrg/kg honey. Further to this, honey attributed to non-pyrrolizidine alkaloid-producing floral sources and unspecified blended honeys can also have significant amounts of alkaloids present. 

Tab. 13.1 Quantities of pyrrolizidine alkaloids and their N-oxides extracted from E. vulgare-derived honey.

AHPA. July 2011. Code of Ethics Business Conduct. Silver Spring, MD American Herbal Products Association. Altamirano, J., S.R. Gratz, and K.A. Wolnik. 2005. Investigation of pyrrolizidine alkaloids and their N-oxides in commercial comfrey-containing products and botanical materials by liquid chromatography electrospray ionization mass spectrometry. /. AOAC Int. 88(2) 406-412. 

A variety of pyrrolizidine alkaloids and their N-oxides have antitumor activity (Wink, 1993b). Herbal material of Senecio vulgaris (Asteraceae) has been used for hundreds of years as a treatment for cancer. The active compounds have been shown to be senecionine (5) and senecionine N-oxide. Indicine N-oxide (16) from Heliotropium indicum (Boraginaceae) has pronounced antitumor activity. Mono-crotaline (8), from several Crotalaria species (Fabaceae), has similar activity (Blasko and Cordell, 1988 Suffness and Cordell, 1985). Alkylation seems to be involved in the antitumor activity. As the A -oxides cannot serve as enamines and hence cannot be directly involved in alkylation, these compounds should only be active to the extent they are converted to the free bases. At least in the case of indicine iV-oxide (16), this does not appear to be the case (Suffness and Cordell, 1985). 

Betteridge K, Cao Y, Colegate SM (2005) An improved method for extraction and LCMS analysis of pyrrolizidine alkaloids and their N-oxides in honey application to Echium vulgare honeys. J Agric Food Chem 53 1894—1902... 

Symphytum officinale (black wort, boneset, bruise wort, comfrey, knitback, knitbone, slippery root) contains pyrrolizidine alkaloids, such as lasiocarpine and symphytine, and their N-oxides, and has repeatedly been associated with hepatotoxicity. 

Two further Heliotropium species have been investigated. The dried leaves of H. arguzioides contain 1.2—2.05% alkaloids, the majority of which is heliotrine (44) 16 same alkaloid occurs with incanine in H. olgae The aerial parts of Paracynoglossum imeretinum (Kusnez.) Popov (fam, Boraginaceae) also contain pyrrolizidine alkaloids so far heliosupine, echinatine (45), and their N-oxides have been identified. ... 

Bull LB, Dick AT (1959) The chronic pathological effects on the liver of the rat of the pyrrolizidine alkaloids heliotrine, lasiocarpine, and their N-oxides. J Pathol Bacteriol 78 483-502... 

In connection with the earlier suggestion that the pyrrolizidine alkaloids are converted into the toxic pyrrole metabolites via their N-oxides, Mattocks has determined the respective toxicities of retrorsine (49) and retrorsine Af-oxide. Although the LD50 of retrorsine and its fV-oxide are comparable when administered orally, retrorsine iV-oxide appears to be much less toxic when injected intraperitoneally. The concentration of pyrrolic metabolites of retrorsine given orally or intraperitoneally reached a maximum in the liver of rats only one hour... 

Additional studies have shown that the oxidation of alkaloid to its N-oxide occurs and competes with pyrrole formation. N-oxides when administered intravenously to rats or when treated with liver microsomal enzymes are not converted to pyrroles. Hence they are non-toxic in the liver, and, by their highly hydrophilic nature, provide a pathway for detoxification. When administered orally to rats and sheep however, N-oxides are converted in the rumen (sheep) or gut (rats) to the parent alkaloids which are then metabolized in the liver causing the normal toxic sequelae. The toxicity (or lack thereof) of the pyrrolizidine N-oxides depends on whether or not they are reduced vivo before reaching the liver (61). After intravenous administration of indicine N-oxide to human cancer patients, free alkaloid has been detected in the blood and subsequent liver damage has been observed (55). 

A considerable number of improvements to existing procedures for the detection, isolation, and separation of pyrrolizidine alkaloid mixtures has been reported. The alkaloids often occur naturally in the form of their N-oxides, and these are usually reduced with zinc in acidic solution to liberate the free bases. An alternative method has been described by Huizing and Malingr 144), which employs a redox polymer on an anion-exchenge resin to give better yields of the free bases. Ion-exchange resins have been used for the extraction (99) and separation 145) of pyrrolozidine alkaloids. 

A very large range of pyrrolizidine alkaloids can theoretically be obtained by combining the known necines and the esterifying acids (= necic acids). So far, more than 500 alkaloids have been found and their structures determined. With the exception of the approximately 35 otonecine alkaloids that cannot form N-oxides, if the N-oxides of these alkaloids are taken into consideration, more than 900 structures are known. 

The mechanism for handling alkaloids in the genus Platyphora is different. Only open-chain pyrrolizidines are accumulated, along with the saponins these insects make themselves (Figure 7.13). The alkaloids are stored as the tertiary amines, not the N-oxides, and only in the haemolymph of larvae and defensive glands of adults. Some alteration of their structures is possible by the insects as shown in Figure 10.6. These species... 

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