Plants affecting the CNS

1 I. True

Amino acid derivates which contain nitrogen in the heterocycle

2 I.1. alkaloids

Tropane rings are biosynthesised from ornithine amino acid Me = (-CH 3) Two major tropane groups: • Solanaceous alkaloids • alkaloids

Natural occurence in species of , Erythroxylaceae, Convolvulaceae Major alkaloids are: S and R (S) (R) (one of its hyoscyamine enantiomers is atropine ), (syn: hyoscin), 3 scopolamine Determination of tropane alkaloids

Vitali – Morin colour reaction

1 g sample from the + 20 ml fuming H2SO4 solution Evaporated to dryness on a water-bath To the residue add 0,5 ml 3% (w/v) KOH in methanol  bright purple colouration  changes to red  fades to colourless

4 Mechanism of action Inhibition of muscarinic cholinergic receptors (competitive antagonists of acetylcholine) Site of action : smooth and cardiac muscle, gland cells, in peripheral ganglia and CNS Alkaloids bounds reversible to receptors Solanaceous alkaloids are , reducing the activity of the parasympathetic nervous system and promoting sympathetic predominance Symptoms are developed in 30-60 minutes, lasts for 2 hours (intensive phase), later decrease for 10-12 hours (except in the eyes: 72 hours) Alkaloids are excreted with urine 5 Major symptoms 1. Xerostomia (dry mouth) 2. (dilated pupils) 3. Tachycardia (high heart rate) 4. Rapid breathing 5. Spasm, seizures 6. Death (respiratory failure)

6 stramonium – jimsonweed Solanaceae – nightshade f. Distribution: Mexico, but naturalised in many countries Annual weed Disturbed, nutrient (nitrogen) rich fields, from early summer Used to „treat” roaring (respiratory obstruction) horses before selling them Psychoactive effects  jimson weed (Jamestown, Virginia) Treatment for asthma

7 8 9 Seed is dominated by numerous pock-like depressions The surface of the seed is covered evenly with minute bumps Unmature seeds have a sweet taste! 10 Poisoning The whole plant is poisonous for all species ! Level of toxins vary among plant organs: ~ 0,2-0,6% Toxins accumulate in tissues! Generally seeds admixed into the meal It’s rarely grazed due to the bitter taste and unpleasant smell Most susceptible: pigs Less susceptible: rabbits , poultry

11 Case studies Schulman & Bolton (1998): high proportion of seed in the forage

Binev et al. (2006): ensiled maize with Datura

Soler-Rodríguez et al. (2006): lucerne hay with Datura

12 Naudé et al. (2004): hay samples contamined with Datura

13 Other Datura species

Datura innoxia – downy thorn-apple

14 – henbane Solanaceae – nightshade f. Distribution: continental Eurasia, India, N- Annual/biennial weed On waste ground and also as a cereal weed Formerly used to steal chickens, applied at witch rituals, against toothache or in beers as a flavouring As a medicine: antispasmodic, bronchodilator effect

15 16 17 Seeds are compressed and scrobiculate (pitted): having numerous shallow grooves or depressions

18 Poisoning The whole plant is poisonous for all species ! Symptoms are similar to Datura poisoning Mainly humans are affected

19 belladonna – deadly nightshade Solanaceae – nightshade f. Distribution: , N-Africa, W- Perennial, (often growing as a ) In shady forests from early summer Atropos – one of the three Greek fates of destinies

Bella donna (in Italian) – beautiful lady Formerly it was used as a love potion Atropine sulphate is a mydriatic for eye examinations, antispasmodic effect 20 21 22 23 Seeds are ornamented by numerous pock-like depressions

24 Poisoning

The whole plant is poisonous for all species Symptoms are similar to Datura , but poisonings are rare Average toxin content (in the above-ground parts): 0,2-2% Tolerant species: rabbit, birds, guinea pigs Susceptible species: pigs LD: for kids 2-5 berries, for adults 10-20

25 Other European species with Datura-like symptoms Scopolia carniolica – European scopolia officinarum -

26 Coca alkaloids Erythroxylum cocoa – coca shrub

with a effect

27 I.2. Benzylisoquinoline alkaloids

Natural occurences mainly in species of Papaveraceae family , but also species in

Aristolochiaceae and thyrosine isoquinoline Liliaceae Synthesis: isoquinoline backbone from thyrosine amino acid Most important alkaloids: , codeine, papaverine, rhoedine Commercial tests available (Mandelin reagent)

28 Mechanism of action – (morphine)

Opioids bound to an receptor of a neurone Blocking Ca 2+ ions entering the cells K+ ion efflux Hyperpolarization  inhibition of action potential  neurons less likely to fire  paralysing effect, suppression of transmission of pain signals 29 Symptoms – opioids (morphine)

Pinpoint pupils Spasm of the stomach or intestinal tract, vomiting Paralysis of limbs  stumbling walk Slow and labored breathing Urinary retention, constipation Coma, hallucinations, death

30 Papaver rhoeas – field poppy Papaveraceae – poppy f. Distribution: cosmopolitan (worldwide) Agricultural weed (cereals); from spring Annual Pappa (in Latin) = food, milk Rhoeas (in Greek) = red Toxic agents are rhoeadine papaverine alkaloids (about 25 different molecules, the major is rhoeadine – nearly 50% of the total) and papaverine 31 32 33 Small, kidney-shaped seeds with a dark, greyish brown colour

34 Poisoning

The whole plant is poisonous (especially its milk) for all species Can be admixed in hay and meals Poisonings are rare Toxins can be excreted in the milk  a potential risk for offsprings Mainly cattle, horses and sheeps are sensitive Slow recovery after poisoning

35 Other Papaver species Papaver somniferum – poppy

Somniferum (in Latin) – „sleep bringing” Dried latex = opium The mixture of about 50 different alkaloids (morphine, codeine, papaverine, etc.) Painkilling effect János Kabay – extraction of morphine from poppy straw

36 37 I.3. Piperidine alkaloids Natural occurence in species of Apiaceae family Synthesis: piperidine nucleus biosynthesised from lysine amino acid (true alkaloid) or from acetate (pseudoalkaloid) Most important molecules: , coniceine, lobeline Coniine + NaOH (KOH)  unpleasant smell, similar to mice urine

38 Mechanism of action – coniine In the peripheral nervous system coniine blocks the receptors on the post-synaptic membrane of neuromuscular junctions  blocking movements Depresses the cranial nerve III.  depressing motor functions The central nervous system is not immediately affected

Sypmtoms Symptoms develop within 0,5-2 hours Uncontrolled urination Heart rate first slow, becomes rapid Trembling, problems in movement (paralysis of limbs) Respiration becomes slow, labored, and irregular Person remains conscious and aware until respiratory paralysis 39 maculatum – hemlock Apiaceae – carrot f. Distribution: Europe Disturbed habitats, on nutrient (nitrogen) rich soils from summer Biennial Maculatum (in Latin) = spotted 8 different alkaloids, mainly coniine , coniceine Death of Socrates – infusion of hemlock plant

40 41 42 43 Fruit is dry but does not split open when ripe Ridged, compressed laterally and smooth

44 Poisoning The whole plant is poisonous , but mainly the green fruits (alkaloid content up to 2-3%) All species are susceptible, mainly cattle Can be admixed into hay, silage or meal Drying can decrease toxicity About 100-500 g green can be lethal

45 Case studies

Gelay et al. (1992)

Binev et al. (2007) 46 II. Terpenoids

47 II.1. Diterpene pseudoalkaloids Natural occurence mainly in species of Ranunculaceae, Asteraceae, Euphorbiaceae, Lamiaceae families Biosynthesised from geranyl- geranyl pyrophosphate (GGPP )

 kaurene (C20 ) with a nitrogenic atom  pseudoalkaloid

(with C19 skeleton) Major types are and Taxus alkaloids No quick tests but determination of aconitine is possible from blood and urine  see Beike et al.

(2004) 48 Mechanism of action - aconitine In the cardiac and skeletal muscle cell membranes, aconitine makes the voltage-gated Na + ion channels permanently open  high Na + levels in muscle cells inhibits Na +/Ca 2+ exchangers  higher Ca 2+ and Na + ion availability within the muscle cell  cell membrane cannot be repolarized  paralysis In neurons , aconitine depolarizes both the membranes of presynaptic and postsynaptic cells by opening their voltage- gated Na + ion channels Due to the strong depolarization, the permeability of the membrane for K+ and Ca 2+ ions is increased  Ca 2+ ions stimulate the release of Ach into the synaptic gap 49 Symtomps of poisoning

Paresthesia (sensation of burning) Tingling and numbness in the mouth, burning feeling in the rumen Vomiting Motoric weakness , slowing movements Heart and respiration steadily fail (asphyxia ) Ventricular arrhythmias, asystole, cardiac arrest , death

50 Aconitum lycoctonum – wolfsbane Ranunculaceae – buttercup f.

Distribution: Middle and North Europe Over 250 Aconitum species mainly in the northern hemisphere Perennial In mountanious forests or gardens from mid-summer Formerly used as an arrow poison Dermal absorption of the alkaloids (aconitine , mesaconitine, hypaconitine) is possible

LD 50 : 1 mg/bwkg (mice) 1-2 mg/bwkg (human) 51 52 53 Poisoning The whole plant is poisonous for all species , but mainly the tubers and the seeds (even it’s dried) Animal poisonings are rare Mainly on alpine grasslands or in gardens

54 Puschner et al. (2002) Species with similar poisonous effects  see Seminar 8 Delphinium spp. – Aconitum napellus – cultivated larkspur species monkshood

55 II.2. Steroidal triterpenoid pseudoalkaloids For the biosynthesis of steroid skeleton  see seminar 4. Steroidal skeleton bounds with heterocyclic rings  pseudoalkaloids Natural occurences in members of Liliaceae and Solanaceae plant families About 153 compounds have already been isolated from ~30 species Detection of Solanum alkaloids : adding formaldehyde (CH2O) and sulfuric acid (H2SO4)  the red-violet color indicates alkaloids No quick tests for Veratrum alkaloids 56 Mechanism of action and symptoms - Solanum alkaloids

Inhibition of acetylcholinesterase  ACh accumulates  Na + channels stay open  no possibility of a new action potencial to be triggered  no more muscle contractions  paralysis Saponin effect : disrupts cell membranes Symptoms are similar to potato poisoning  see seminar 3.

57 Solanum nigrum – black nightshade Solanaceae – nightshade f.

Distribution: Eurasia, but naturalised worldwide Annual Disturbed habitats, on soils rich in nutrient (nitrogen), crop fields, or in gardens from summer Agricultural weed Nigrum (in Latin) = black (berries) Solanine glycoalkaloid (aglycone: solanidine)

58 59 Poisoning The whole plant is poisonous for all species (especially for kids) Toxins are poorly absorbed from the gastrointestinal tract and eliminated rapidly by urinal and fecal excretion Most cases reporting mild toxicity following ingestion

LD50 : 30-40 mg/bwkg (mouse), 20 mg/bwkg (rabbit)

60 Solanum dulcamara – bittersweet nightshade Solanaceae – nightshade f.

Distribution: Eurasia, but naturalised worldwide In fresh forests, hedges and marshes from late autumn Dulcis (in Latin) = sweet, amara (in Latin) = bitter Solanine glycoalkaloid (aglycone: solanidine)

61 62 Misidentification – confused taxa Solanum dulcamara

Lycium spp. – goji , wolfberry

63 Other speices

Solanum pseudocapsicum – Jerusalem cherry

64 Case studies

Lima et al. (2014)

65 Mechanism of action and symptoms – Veratrum alkaloids

Jervine – inhibits smoothened (SMO) protein  inhibition of hedgehog signaling pathway (controlls embryo developement) Protoveratrine - opens the Na + channels of neurones permanently For embryos: teratogenic and mutagenic effect  birth defects For adults: symptoms are similar to Aconitum poisoning with hemorrhages

66 Veratrum album – white hellebore Melanthiaceae – death camas f. Distribution: Europe, W-Asia

On lush grasslands, from early cholesterol summer Perennial Album (in Latin) = white (flowers) Steroidal alkaloids (the basic steroidal skelet changes in many steps): protoveratrine, jervine, veratridine Supposed death of Alexander the Great Ethnoveterinary – against skin parasites 67 68 69 70 Poisoning

The whole plant is poisonous (mainly the rhizome) for all species Sheeps are susceptible Plants can be nibbled on pastures , or admixed in the hay Symptoms develop in 1-2 hours

LD50 : 0,1mg/bwkg (rabbit), 1-2 g rhizome/forage kg (ruminants)

71 72 Other Veratrum species Veratrum nigrum – black hellebore

73 Case study

Mainly bioassay experiments

74 II.3. Non-steroidal triterpenoids

Aglycons biosynthesised from squalene, but pentacyclic molecules squalene Many of them are glycosides forming an amphipathic molecule (=saponin) Distribution: non taxa specific compounds (origin: Saponaria species) For detailed mechanism and symptoms see seminar 3. (forage legumes)

75 Agrostemma githago – corncockle Caryophyllaceae – pink f. Distribution: in the continental region Weed of wheat; from summer Annual Toxic agents are triterpenoid saponins (aglycone: githagenin) and agrostin (RIP protein) In Europe now it’s only locally distributed  increased use of agrostemma saponin herbicides 76 77 Angular-oval shape, often flattened on one side Covered in rows of raised tubercles (wart- like projections) that increase in height away from hilum

78 Poisoning

The whole plant is poisonous, but mainly the seeds (toxin content can reach 5-7%) Poisonous for all species Seeds can admixed into meals Symptoms are developed in 1-2 hours Acute poisoning: respiratory depression , vomiting, diarrhea, salivation and paralysis No recent studies about the LD value, but doses higher that 2-3 g seeds are considered toxic for humans 79 Case study

Less published data about poisonings

80 III. Thiaminase enzyme

81 Mechanism of action Enzymes cleave the thiamine (vitamin B1) molecule and render it biologically inactive When thiamine molecule is cleaved the body is incapable of restoring it  thimanine deficiency (humans: beri-beri)

Vitamine B1 has a pivotal role in carbohydrate and fatty acid metabolism acting as a coenzyme in the pyruvate dehydrogenase enzyme complex that produces acetyl-CoA from pyruvate Low levels of acetyl-CoA can lead to metabolic coma and death Source of thiaminase: in fish, shellfish, ferns, bacteria Fish thiaminases are heat sensitive, but the fern-type may not be completely destroyed by cooking 82 Symptoms Weakness, decreased production, milk with bluish color Enlarged heart, low heart beat In ruminants: cerebrocortical necrosis (CCN) (polyencephomalacia PEM) – behavioral disorders Brain becomes inflamed and edematous

83 Equisetum arvense – field horsetail Equisetaceae – horsetail f. A fern plant Distribution: throughout the arctic and temperate regions An agriculture weed, on disturbed grasslands Perennial Brown (fertile) shoots appears in spring, green (steril) shoots in summer Rich in silicon dioxide (silica ) Used to treat urinary infections or to polish pewter

84 85 86 Poisoning Silica crystals can irritate the digestive tract Ruminants are fairly resistant to thiamine deficiency, but young animals can be sensitive LD: over 20% in the hay (horses) – symptoms develop in 2-5 weeks Diets rich in whole grains can trigger the growth of certain thiaminase-producing bacteria (Clostridium sporogenes, Bacillus spp.) in the rumen

87 Other Equisetum species

Equisetum palustre – marsh horsetail

88 Other species with thiaminase content

Pteridium aquilinum – bracken fern Marsilea drummondii - nardoo

89 Cyprinus carpio - carp Clupea harengus – atlantic herring

Mytilus galloprovincialis - mediterranean mussel Penaeus spp. - prawns

90