Pyrrolizidine alkaloids (PAs), sometimes referred to as necine bases, are a group of naturally occurring alkaloids based on the structure of pyrrolizidine. Their use dates back centuries and is intertwined with the discovery, understanding, and eventual recognition of their toxicity on humans and animals. [1]
PAs were first discovered in plants in the 19th century, but their toxic effects were not immediately recognized. [2] Instead, many PA-containing plants were traditionally used for medicinal purposes in various cultures around the world. For example, herbs containing PAs were used in traditional Chinese medicine and by Native American tribes for their purported therapeutic properties. [3] It has been estimated that 3% of the world's flowering plants contain pyrrolizidine alkaloids. [4] Honey can contain pyrrolizidine alkaloids, [5] [6] as can grains, milk, offal and eggs. [7] To date (2011), there is no international regulation of PAs in food, unlike those for herbs and medicines. [8]
In the early to mid-20th century, researchers began to observe and document cases of livestock poisoning linked to the consumption of PA-containing plants. [9] These observations led to the recognition of PAs as potent hepatotoxic and genotoxic compounds. [10]
In response to growing concerns about PA exposure, regulatory agencies around the world began to establish guidelines and regulations to limit PA levels in food, herbal products, and animal feed. [11] These regulations aim to protect human and animal health by minimizing PA exposure and mitigating the risk of toxicity.
Despite regulatory efforts, the issue of PA exposure remains relevant today. Ongoing research continues to explore various aspects of PA toxicity, including the identification of new PA-containing plants, the development of sensitive analytical methods, and the assessment of human health risks associated with PA exposure. [12] Additionally, efforts to raise awareness among healthcare professionals, herbal product manufacturers, and the general public about the risks of PA exposure are ongoing.
PAs are a group of naturally occurring compounds found in a wide range of plant species. These alkaloids are secondary metabolites synthesized by plants primarily as a defense mechanism against herbivores, insects, and pathogens. [13]
The biosynthesis of PAs was discovered to occur through the first pathway-specific enzyme Homospermidine synthase. [14]
The polyamines putrescine and spermidine are derived from the basic amino acid arginine. Subsequently, homospermidine synthase exchanges the 1,3-diamonopropane by putrescine and forms symmetric homospermidine. Oxidation of homospermidine by copper-dependent diamine oxidases initiates cyclization to pyrrolizidine-1-carbaldehyde, which is reduced, to 1-hydroxymethylpyrrolizidine. Desaturation and hydroxylation ultimately form retronecine, which is acylated with an activated necic acid, for instance with senecyl-CoA2 as in the example shown below. [15]
Pyrrolizidine alkaloids (PAs) are preferably found in the plant families Asteraceae (tribes Eupatorieae and Senecioneae), Boraginaceae (many genera), Fabaceae (mainly the genus Crotalaria), and Orchidaceae (nine genera). More than 95% of the PA-containing species investigated thus far belong to these four families. [14]
PAs are compounds made up of a necine base, a double five-membered ring with a nitrogen atom in the middle, and one or two carboxylic esters called necic acids. [17] Four major necine bases are described, with Retronecine and its enantiomer Heliotridine being the largest group, and highly toxic. Another group is the platynecine, the difference between these groups is its saturated base, which makes it less toxic. [18] Most bases have a 1,2-unsaturated base. Another difference in the groups is with Otonecine, which cannot form N-oxides, due to the methylation of the nitrogen atom. [16]
The alcohol groups on the necine bases can make esters in a wide variety of forms. Among the possibilities are mono-esters, like Floridine and Heliotrine, and di-esters either with an open or closed ring structure, like Usaramine and Lasiocarpine. In total more than 660 PAs and PA N-oxides have been identified in over 6000 plants. [11]
There are multiple ways to synthesize PAs and their derivatives. A flexible strategy would be to start with a Boc (tert-Butoxycarbonyl) protected pyrrole molecule and use specific reaction for synthesis into the desired compound. [19]
PAs are commonly introduced into the body via oral ingestion through contaminated food or traditional medicine, notably borage leaf, comfrey and coltsfoot. [20] It can readily form salts with nitrates, chlorides and sulphates, which facilitate the uptake in the gastrointestinal tract. After which they travel to the liver via the portal vein. [21] [22]
Metabolites form mostly in the liver. Here esterases can hydrolyze the PAs to reduce the compound into its necine acids and bases, both forms are non-toxic for humans and do not damage the body. However, cytochrome P450 (CYP450) also metabolizes PAs, this enzyme can form pyrrolic esters (EPy), these are hepatotoxic due to their high reactivity. The EPy can also be hydrolyzed into alcoholic pyrroles, which are mutagenic and carcinogenic. [24] [23]
Since this mostly happens in the liver, this is the most affected organ. Other affected organs are the lungs and kidneys. The EPy can escape the liver, and travel through the Disse space into the bloodstream.
The electrophilic nature of pyrroles makes it an easy target for nucleophilic attack from nucleic acids and protein. If bound by glutathione it can become a non-toxic conjugate and be excreted via the kidneys. [24] [25]
A second detoxifying pathway is the formation of the N-Oxide. [26] [27] In the liver and lungs of certain mammal species enzymes called monooxygenase can prevent aromatization of the double 5-ring and in turn prevent the formation of the pyrrole-protein adduct. [20]
The toxicity consequences resulting from the metabolism of PAs in humans primarily revolve around hepatotoxicity and genotoxicity. [1]
PAs are metabolized in the liver through CYP450-mediated pathways. This metabolic process leads to the formation of reactive intermediates, such as pyrrolic metabolites, which can covalently bind to proteins in the liver, forming pyrrole-protein adducts. These adducts impair the function of essential liver proteins, leading to hepatotoxicity. The severity of liver damage correlates with the level of pyrrole-protein adduct formation. Hepatotoxicity induced by PAs can manifest as liver injury, inflammation, necrosis, HSOS (Hepatic Sinusoidal Obstruction Syndrome) and even liver failure in severe cases. [16] [28] The pathogenesis of PAs-induces HSOS is shown by Xu.
Genotoxicity is another consequence of PA metabolism. The reactive metabolites formed during PA metabolism can also bind to DNA, leading to the formation of DNA adducts. These adducts can induce mutations and DNA damage, increasing the risk of cancer development and other adverse health effects. Genotoxicity is particularly concerning as it can lead to long-term health consequences, including carcinogenesis. [29]
The toxicity of PA metabolites can vary depending on the specific PA compound and its chemical structure. Different PAs may undergo metabolic activation to varying degrees, resulting in differences in toxicity. For example, retronecine-type PAs like monocrotaline are known to be highly hepatotoxic, while other types may exhibit lower toxicity or different toxicological profiles. [30]
Next to its toxicological effects, PAs have long been researched for their potential beneficial effects. [18] Traditional medicinal plants have long been known to contain PAs, the exact effect of the PAs regarding beneficial effect of the plants is debated. [31] Among these traditional medicines is the root of the Ligularia Achyrotricha of Tibet. [32] Several pharmacological effects have been found among these effects are Anti-Microbial Activity, [33] [34] Antiviral Activity [35] and Antineoplastic Activity, [36] [37] Acetylcholinesterase inhibition [38] [39] and gastric ulcers treatment. [40]
Anti-Microbial activity of several PAs have been identified as having mild to strong effect against bacteria: E. coli and P. Chrysogenum. [33] In particular Lasiocarpine and 7-angeloyl heliotrine were found to have significant activity against these microbes. Derivatives of PAs have been found to induce cell death in these bacteria by attacking bacterial cell membranes. Retronecine derivatives have been found slow the growth rate of several strains of the fungus Fusarium oxysporum. [34]
Antiviral Activity has been found in Haliotridine derivates. [35] However, effects are not consistent across PA compounds, derivates significantly differ in activity between different viral pathogens. As a result, it is difficult to determine an exact PA with an effect on a specific virus. Several PAs have been found with significant inhibition of growth in the following viruses Coxsackie, Poliomyelitis, Measles and Vesicular stomatitis.
Antineoplastic activity, specifically against leukaemia, has been found in retronecine derivatives like Indicine. [36] A 1984 study by L. Letendre treated 22 leukaemia patients with Indicine, this resulted in a significant observed antineoplastic response with four complete remissions and five partial remissions. An observed adverse side effect of the treatment was observed in 5 patients who died of hepatic toxicity likely caused by the medication. Two different dose levels were tested on children: 2 g/m2/ day for 5 consecutive days (14 patients) and 2,5 g/m2/ day for 5 consecutive days (17 patients). [37] Therapeutic effect was determined based on these doses and deemed to have a limited antileukemic effect below a dose of 3 g/m2/ day. However, this study also found severe hepatotoxic responses to be common at these doses.
Four known PAs: 7-O-Angeloyllycopsamine N-oxide, echimidine N-oxide, echimidine and 7-O-Angeloylretronecine have been clinically shown to inhibit Acetylcholinesterase (AChE) . [38] AChE inhibitors have been used as one of the treatments for Alzheimer's disease. [39] The effect of these compounds was significant in the reduction of AChE production and thus a potential alternative in the fight against Alzheimer's.
PAs like senecionine, integerrimine, retrorsine, usaramine and seneciphylline have been shown to cause an increase in both the levels of gastrin and the expression of Epidermal Growth Factor (EGF). [40] These two compounds aid in the repair of the stomach after gastric ulcers. A high concentration of said compounds can reduce lesions in the stomach. This may aid in treatment after operation to the stomach.
The toxicological effects of PAs have been studied on animals. Retronecine derivatives are known to cause a toxic response in the livers of livestock like cows. [41] Symptoms tend to start with a change in rough hair coat and depression. When Pregnant livestock is exposed to PAs an effect can be seen on the foetus, mainly stillbirth and accumulation in the foetus. The main lethal responses in adult livestock exhibit necrosis, HSOS and megalacytosis. Additional to the short-term effect PAs have been found to lead to carcinogenic growths on the long term. The carcinogenic effect is caused by formation of DNA adducts, [20] because of metabolic reactions. No minimum dosage for the carcinogenic effect is currently known. However, there have been studies to determine the Lowest dose for an adverse effect, also known as LOAEL. [42] LOAEL and LD50 (oral) for 40 PAs have been experimentally found out. These values can be seen in the Table below. The found low LD50 values clearly show the relatively high toxicity of PAs, however no significant relation was found between the LD50 and LOAEL.
| Type | Compound | LD50 (g/kg) | LOAEL (g/kg) |
| Retronecine types | Retrorsine∗ | 0.320 | 0.001 |
| Clivorine | 0.386 | 0.002 | |
| Riddelliine | 0.616 | 0.015 | |
| Senecionine | 0.127 | 0.001 | |
| Usaramine | 0.264 | 0.002 | |
| Jacobine | 0.461 | 0.003 | |
| Monocrotaline∗ | 0.731 | 0.002 | |
| Seneciphylline | 0.264 | 0.002 | |
| Integerrimine | 0.254 | 0.002 | |
| Senecivernine | 0.592 | 0.004 | |
| Jacoline | 0.230 | 0.001 | |
| Trichodesmine | 0.324 | 0.004 | |
| Fulvine | 0.369 | 0.002 | |
| Angularine | 0.559 | 0.009 | |
| Crotananine | 0.592 | 0.004 | |
| 7-Acetylintermedine | 0.559 | 0.009 | |
| 7-Acetyllycopsamine | 0.356 | 0.003 | |
| Echimidine | 0.616 | 0.015 | |
| Echiumine | 0.122 | 0.001 | |
| Lycopsamine | 0.239 | 0.001 | |
| Intermedine | 0.264 | 0.002 | |
| Indicine | 0.264 | 0.002 | |
| Retronecine∗ | 0.242 | 0.001 | |
| Lasiocarpine | 0.555 | 0.001 | |
| Heliosupine | 0.708 | 0.002 | |
| Heleurine | 0.616 | 0.015 | |
| Supinine | 0.215 | 0.001 | |
| Callimorphine | 0.559 | 0.009 | |
| Heliotrine | 0.056 | 0.001 | |
| Echinatine | 0.250 | 0.003 | |
| Rinderine | 0.486 | 0.001 | |
| Platynecine types | Platyphylline∗ | 0.443 | 0.002 |
| Trachelanthamine | 0.391 | 0.001 | |
| Heliocoromandaline | 0.246 | 0.004 | |
| Heliocurassavicine | 0.404 | 0.001 | |
| Otonicine types | Acetylanonamine | 0.230 | 0.001 |
| Senkirkine | 0.275 | 0.001 | |
| Otosenine | 0.106 | 0.001 | |
| Petasitenine | 0.264 | 0.002 | |
| Otonecine | 0.467 | 0.001 |
PAs are also used as a defense mechanism by some organisms such as Utetheisa ornatrix . Utetheisa ornatrix caterpillars obtain these toxins from their food plants and use them as a deterrent for predators. PAs protect them from most of their natural enemies. The toxins stay in these organisms even when they metamorphose into adult moths, continuing to protect them throughout their adult stage. [43]
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Symphytum is a genus of flowering plants in the borage family, Boraginaceae, known by the common name comfrey. There are 59 recognized species. Some species and hybrids, particularly S. officinale, Symphytum grandiflorum, and S. × uplandicum, are used in gardening and herbal medicine. They are not to be confused with Andersonglossum virginianum, known as wild comfrey, another member of the borage family.
Hepatotoxicity implies chemical-driven liver damage. Drug-induced liver injury is a cause of acute and chronic liver disease caused specifically by medications and the most common reason for a drug to be withdrawn from the market after approval.
Genotoxicity is the property of chemical agents that damage the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, but some genotoxic substances are not mutagenic. The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. Cells prevent expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.
Toxication, toxification or toxicity exaltation is the conversion of a chemical compound into a more toxic form in living organisms or in substrates such as soil or water. The conversion can be caused by enzymatic metabolism in the organisms, as well as by abiotic chemical reactions. While the parent drug are usually less active, both the parent drug and its metabolite can be chemically active and cause toxicity, leading to mutagenesis, teratogenesis, and carcinogenesis. Different classes of enzymes, such as P450-monooxygenases, epoxide hydrolase, or acetyltransferases can catalyze the process in the cell, mostly in the liver.
Sudan I is an organic compound, typically classified as an azo dye. It is an intensely orange-red solid that is added to colourise waxes, oils, petrol, solvents, and polishes. Sudan I has also been adopted for colouring various foodstuffs, especially curry powder and chili powder, although the use of Sudan I in foods is now banned in many countries, because Sudan I, Sudan III, and Sudan IV have been classified as category 3 carcinogens by the International Agency for Research on Cancer. Sudan I is still used in some orange-coloured smoke formulations and as a colouring for cotton refuse used in chemistry experiments.
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A hepatotoxin is a toxic chemical substance that damages the liver.
Petasites japonicus, also known as butterbur, giant butterbur, great butterbur and sweet-coltsfoot, is an herbaceous perennial plant in the family Asteraceae. It is native to China, Japan, Korea and Sakhalin and introduced in Europe and North America. It was introduced to southern British Columbia in Canada by Japanese migrants.
Symphytum officinale is a perennial flowering plant in the family Boraginaceae. Along with thirty four other species of Symphytum, it is known as comfrey. To differentiate it from other members of the genus Symphytum, this species is known as common comfrey or true comfrey. Other English names include boneset, knitbone, consound, and slippery-root. It is native to Europe, growing in damp, grassy places. It is locally frequent throughout Ireland and Britain on river banks and ditches. It occurs elsewhere, including North America, as an introduced species and sometimes a weed. The flowers are mostly visited by bumblebees. Internal or long-term topical use of comfrey is discouraged due to its strong potential to cause liver toxicity.
A loline alkaloid is a member of the 1-aminopyrrolizidines, which are bioactive natural products with several distinct biological and chemical features. The lolines are insecticidal and insect-deterrent compounds that are produced in grasses infected by endophytic fungal symbionts of the genus Epichloë. Lolines increase resistance of endophyte-infected grasses to insect herbivores, and may also protect the infected plants from environmental stresses such as drought and spatial competition. They are alkaloids, organic compounds containing basic nitrogen atoms. The basic chemical structure of the lolines comprises a saturated pyrrolizidine ring, a primary amine at the C-1 carbon, and an internal ether bridge—a hallmark feature of the lolines, which is uncommon in organic compounds—joining two distant ring carbons. Different substituents at the C-1 amine, such as methyl, formyl, and acetyl groups, yield loline species that have variable bioactivity against insects. Besides endophyte–grass symbionts, loline alkaloids have also been identified in some other plant species; namely, Adenocarpus species and Argyreia mollis.
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Pyrrolizidine alkaloidosis is a disease caused by chronic poisoning found in humans and other animals caused by ingesting poisonous plants which contain the natural chemical compounds known as pyrrolizidine alkaloids. Pyrrolizidine alkaloidosis can result in damage to the liver, kidneys, heart, brain, smooth muscles, lungs, DNA, lesions all over the body, and could be a potential cause of cancer. Pyrrolizidine alkaloidosis is known by many other names such as "Pictou Disease" in Canada and "Winton Disease" in New Zealand. Cereal crops and forage crops can sometimes become polluted with pyrrolizidine-containing seeds, resulting in the alkaloids contaminating flour and other foods, including milk from cows feeding on these plants.
Pyrrolizidine alkaloid sequestration by insects is a strategy to facilitate defense and mating. Various species of insects have been known to use molecular compounds from plants for their own defense and even as their pheromones or precursors to their pheromones. A few Lepidoptera have been found to sequester chemicals from plants which they retain throughout their life and some members of Erebidae are examples of this phenomenon. Starting in the mid-twentieth century researchers investigated various members of Arctiidae, and how these insects sequester pyrrolizidine alkaloids (PAs) during their life stages, and use these chemicals as adults for pheromones or pheromone precursors. PAs are also used by members of the Arctiidae for defense against predators throughout the life of the insect.
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Riddelliine is a chemical compound classified as a pyrrolizidine alkaloid. It was first isolated from Senecio riddellii and is also found in a variety of plants including Jacobaea vulgaris, Senecio vulgaris, and others plants in the genus Senecio.
Gynura bicolor, hongfeng cai 紅鳳菜, Okinawan spinach or edible gynura, is a member of the chrysanthemum family (Asteraceae). It is native to China, Thailand, and Myanmar but grown in many other places as a vegetable and as a medicinal herb.
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Echites umbellatus is a flowering climber, belonging to subfamily Apocynoideae of the family Apocynaceae and has the English common name devil's potato. It was first described in 1760 by Dutch botanist, Nikolaus Joseph von Jacquin. The species grows in parts of Florida, Tabasco, Yucatán Peninsula, Belize, Honduras, Cayman Islands, Cuba, Hispaniola, Jamaica, Leeward Islands, Bahamas, Turks and Caicos Islands, and the Colombian islands in the Western Caribbean.
Monocrotaline (MCT) is a pyrrolizidine alkaloid that is present in plants of the Crotalaria genus. These species can synthesise MCT out of amino acids and can cause liver, lung and kidney damage in various organisms. Initial stress factors are released intracellular upon binding of MCT to BMPR2 receptors and elevated MAPK phosphorylation levels are induced, which can cause cancer in Homo sapiens. MCT can be detoxified in rats via oxidation, followed by glutathione-conjugation and hydrolysis.