Lupinine

Last updated
Lupinine
Lupinin.svg
Names
Preferred IUPAC name
[(1R,9aR)-Octahydro-2H-quinolizin-1-yl]methane
Identifiers
3D model (JSmol)
3DMet
ChEBI
ChEMBL
ChemSpider
ECHA InfoCard 100.006.944 OOjs UI icon edit-ltr-progressive.svg
EC Number
  • 207-638-0
KEGG
PubChem CID
UNII
  • InChI=1S/C10H19NO/c12-8-9-4-3-7-11-6-2-1-5-10(9)11/h9-10,12H,1-8H2/t9-,10+/m0/s1 Yes check.svgY
    Key: HDVAWXXJVMJBAR-VHSXEESVSA-N Yes check.svgY
  • InChI=1/C10H19NO/c12-8-9-4-3-7-11-6-2-1-5-10(9)11/h9-10,12H,1-8H2/t9-,10+/m0/s1
    Key: HDVAWXXJVMJBAR-VHSXEESVBK
  • OC[C@H]1[C@@H]2N(CCC1)CCCC2
Properties
C10H19NO
Molar mass 169.268 g·mol−1
Melting point 68 to 69 °C (154 to 156 °F; 341 to 342 K)
Boiling point 269 to 270 °C (516 to 518 °F; 542 to 543 K)
Hazards
GHS labelling:
GHS-pictogram-exclam.svg
Warning
H302, H312, H332
P261, P264, P270, P271, P280, P301+P312, P302+P352, P304+P312, P304+P340, P312, P322, P330, P363, P501
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
X mark.svgN  verify  (what is  Yes check.svgYX mark.svgN ?)
Infobox references

Lupinine is a quinolizidine alkaloid present in the genus Lupinus (colloquially referred to as lupins) of the flowering plant family Fabaceae. [1] The scientific literature contains many reports on the isolation and synthesis of this compound as well as a vast number of studies on its biosynthesis from its natural precursor, lysine. [2] [3] Studies have shown that lupinine hydrochloride is a mildly toxic acetylcholinesterase inhibitor and that lupinine has an inhibitory effect on acetylcholine receptors. [4] [5] The characteristically bitter taste of lupin beans, which come from the seeds of Lupinus plants, is attributable to the quinolizidine alkaloids which they contain, rendering them unsuitable for human and animal consumption unless handled properly. [6] [7] However, because lupin beans have potential nutritional value due to their high protein content, [8] efforts have been made to reduce their alkaloid content through the development of "sweet" varieties of Lupinus. [9] [10]

Contents

Toxicity

Lupinine is a hepatotoxin prevalent in the seeds of leguminous herbs of the genus Lupinus. [9] Lupinine and other quinolizidine alkaloids give a bitter taste to naturally growing lupin flowers. [5] Due to the toxicity of quinolizidine alkaloids, lupin beans are soaked overnight and rinsed to remove some of their alkaloid content. [7] However, when the cooking and rinsing procedure is insufficient, 10 grams of seeds are able to liberate as much as 100 milligrams of lupinine. [11]

The neurotoxicity of lupinine has been known within veterinary medical circles for some time due to the use of lupins as a forage feed for grazing livestock since it has high protein content. [9] It is found to produce lupinosis, which is a morbid, and often fatal condition that results in acute atrophy of liver function and which affects domestic animals such as cattle and sheep. [9] When ingested by humans, quinolizidine alkaloid poisoning causes trembling, shaking, excitation, as well as convulsions. [12] Lupinine, in addition to being orally toxic to mammals, is also an insect antifeedant as well as a growth inhibitor for the grasshopper. [13]

Relative toxicity

Quinolizidine alkaloids in lupins Lupinine Graphic 1.png
Quinolizidine alkaloids in lupins

Lupinine, in comparison to other quinolizidine alkaloids commonly found in lupins, such as lupanine and sparteine, shows a lower toxicity. Lupinine, with a minimal lethal dose of 28–30 mg/kg and a toxic dose of 25–28 mg/kg, is about 85 percent as toxic as d-lupanine and about 90% as toxic as sparteine. The relative toxicity of lupinine with other quinolizidine alkaloids commonly found in lupins is shown in the table below. [14]

Relative Toxicity of Quinolizidine Alkaloids
SubstanceMinimal Lethal Dose (mg/Kg) [14] Toxic Dose (mg/Kg) [14]
Lupinine28-3025-28
Lupanine22-2521-24
Sparteine23-3021-31

Mechanism of action

Structural comparison of lupinine and acetylcholine Lupinine Graphic 5.png
Structural comparison of lupinine and acetylcholine

Studies on the hydrochloride of lupinine have shown it to be a reversible inhibitor of acetylcholinesterases. [4] Lupinine, a nitrogen-containing heterocycle, has a structure similar to the ammonium "head" of the acetylcholinesterase endogenous agonist, acetylcholine. [15] At physiological pH, the amine of lupinine is protonated which leads to ion-ion interaction with the acetylcholinesterase anionic site in the same manner as the ammonium on acetylcholine interacts. [15] Previous studies of reversible onium inhibitors similar to lupinine have shown that the ammonium groups (corresponding to the protonated amine of lupinine) enter the gorge of the active center of the acetylcholinesterase in the region of the Trp84 residue. [4] This leads to the formation of an enzyme-sorption complex with the anionic portion of the acetylcholinesterase located on the active site of lupinine, namely the amine. [15] This complex blocks the access of acetylcholine to the active center which decreases the catalytic hydrolysis and subsequent breakdown of acetylcholine by acetylcholinesterase. [15] Enzyme inactivation leads to an accumulation of acetylcholine in the body, hyperstimulation of both the muscarinic and nicotinic acetylcholine receptors, as well as subsequent disruption of neurotransmission. [16] However, it was found that the time of incubation did not affect the inhibition, leading to the conclusion that lupinine is a reversible inhibitor. [4]

Studies have also shown that lupinine has a binding affinity for both muscarinic and nicotinic acetylcholine receptors. Lupinine was found to have an IC50 value of >500 μM for nicotinic receptors and an IC50 value of 190 μM for muscarinic receptors. However, it has yet to be determined whether this affinity is agonistic or antagonistic in nature. [17]

Synthesis

Biosynthesis of lupinine Lupinine Graphic 2.png
Biosynthesis of lupinine

Biological

Lupinine is naturally biosynthesized from l-lysine in the Lupinus genes of plants along with various other quinolizidine alkaloids. In the biosynthetic process, lysine is first decarboxylated into cadaverine, which is then oxidatively deaminated to the corresponding aldehyde. The aldehyde is then spontaneously cyclized into two tautmers which couple through an aldol type mechanism in which the allylic amine attacks the iminium, forming a dissymmetric dimeric intermediate which is then hydrated. The primary amine is then oxidized and an intramolecular condensation occurs, giving the quinolizidinealdehyde. The aldehyde is then reduced to an alcohol, giving, enantioselectively, (-)- lupinine. [3] [18]

Synthetic

Lupinine has a chiral carbon atom; therefore, total syntheses of lupinine need to be enantioselective for (-)-lupinine in order to provide the biologically active product. The first racemic total synthesis of lupinine occurred in 1937 by Clemo, Morgan, and Raper. [19] Six more total syntheses of lupinine followed between 1940-1956, with the first enantioselective synthesis of lupinine occurring in 1966 by Goldberg and Ragade. [2] Since that initial enantioselective synthesis, there have been numerous total syntheses of both enantio-pure and racemic lupinine. One synthesis, notable because it describes the preparation of all four stereoisomers of lupinine, and containing many references to earlier work in this field, was published by Ma and Ni. [20] Another total synthesis of specific note due to the enantioselectivity and limited number of steps is by Santos et al. In 2010, Santos et al. synthesized enantioselective (-)- lupinine in 36% yield over eight steps using a double Mitsunobu Reaction. [21] First, they employed asymmetric addition of the starting materials using a Lewis acid, followed by treatment with a reducing agent and a base. This gave the (R,R)-alcohol. This configuration was inverted using a Mitsunobu reaction followed by hydrolysis, affording the (R,S) configuration of the alcohol. The alcohol was then reduced with alane, underwent another Mitsunobu reaction, was hydrolyzed to the acid and finally reduced to (-)-lupinine via alane reduction. [21]

Total retrosynthesis of lupinine by Santos et al. Lupinine Graphic 4.png
Total retrosynthesis of lupinine by Santos et al.

Isolation

One of the earliest isolations of lupinine, from Lupinus palmeri collected in Utah, USA, is that reported by Couch, who was able to obtain crystalline lupinine without the use of chromatographic techniques. [22]

Applications

Drawing of a culicine mosquito larva The larva of a Culicine Mosquito hanging down from the surfa Wellcome V0022597.jpg
Drawing of a culicine mosquito larva

Pest control

Lupinine is an insect antifeedant. [13] Studies of its insecticide activity have shown it to be effective against culicine mosquito larvae which are vectors for viruses, filarial worms, and avian malaria. [23] [24]

Botany

Lupins are often found growing with Castilleja (Indian paintbrush) which uses lupins as a host and confers lupinine and other alkaloids to itself. This works in tandem with the increase in nitrogen fixation to increase parasitic reproduction rates and potentially reduce herbivory activity; however, studies have shown mixed results in the efficacy of alkaloid transfer in prevention of herbivory activity. [25]

Regulations

The European Chemicals Agency (ECA) labels lupinine under the hazard statement codes H302, H312, and H332, which indicate that lupinine is harmful if swallowed, harmful in contact with skin, and harmful if inhaled, respectively. It is given a GHS07 labeling which indicates its acute oral toxicity is category 4. [26]

See also

Related Research Articles

Acetylcholine Organic chemical and neurotransmitter

Acetylcholine (ACh) is an organic chemical that functions in the brain and body of many types of animals as a neurotransmitter. Its name is derived from its chemical structure: it is an ester of acetic acid and choline. Parts in the body that use or are affected by acetylcholine are referred to as cholinergic. Substances that increase or decrease the overall activity of the cholinergic system are called cholinergics and anticholinergics, respectively.

Muscarine Chemical compound

Muscarine, L-(+)-muscarine, or muscarin is a natural product found in certain mushrooms, particularly in Inocybe and Clitocybe species, such as the deadly C. dealbata. Mushrooms in the genera Entoloma and Mycena have also been found to contain levels of muscarine which can be dangerous if ingested. Muscarine has been found in harmless trace amounts in Boletus, Hygrocybe, Lactarius and Russula. Trace concentrations of muscarine are also found in Amanita muscaria, though the pharmacologically more relevant compound from this mushroom is the Z-drug-like alkaloid muscimol. A. muscaria fruitbodies contain a variable dose of muscarine, usually around 0.0003% fresh weight. This is very low and toxicity symptoms occur very rarely. Inocybe and Clitocybe contain muscarine concentrations up to 1.6%.

A parasympathomimetic drug, sometimes called a cholinomimetic drug or cholinergic receptor stimulating agent, is a substance that stimulates the parasympathetic nervous system (PSNS). These chemicals are also called cholinergic drugs because acetylcholine (ACh) is the neurotransmitter used by the PSNS. Chemicals in this family can act either directly by stimulating the nicotinic or muscarinic receptors, or indirectly by inhibiting cholinesterase, promoting acetylcholine release, or other mechanisms. Common uses of parasympathomimetics include glaucoma, sjögren syndrome and underactive bladder.

Physostigmine

Physostigmine is a highly toxic parasympathomimetic alkaloid, specifically, a reversible cholinesterase inhibitor. It occurs naturally in the Calabar bean and the Manchineel tree.

Galantamine Neurological medication

Galantamine is used for the treatment of cognitive decline in mild to moderate Alzheimer's disease and various other memory impairments. It is an alkaloid that has been isolated from the bulbs and flowers of Galanthus nivalis, Galanthus caucasicus, Galanthus woronowii, and some other members of the family Amaryllidaceae, such as Narcissus (daffodil), Leucojum aestivum (snowflake), and Lycoris including Lycoris radiata. It can also be produced synthetically.

Epibatidine

Epibatidine is a chlorinated alkaloid that is secreted by the Ecuadoran frog Epipedobates anthonyi and poison dart frogs from the Ameerega genus. It was discovered by John W. Daly in 1974, but its structure was not fully elucidated until 1992. Whether epibatidine is the first observed example of a chlorinated alkaloid remains controversial, due to challenges in conclusively identifying the compound from the limited samples collected by Daly. By the time that high-resolution spectrometry was used in 1991, there remained less than one milligram of extract from Daly's samples, raising concerns about possible contamination. Samples from other batches of the same species of frog failed to yield epibatidine.

Methacholine

Methacholine is a synthetic choline ester that acts as a non-selective muscarinic receptor agonist in the parasympathetic nervous system.

Neuromuscular-blocking drugs block neuromuscular transmission at the neuromuscular junction, causing paralysis of the affected skeletal muscles. This is accomplished via their action on the post-synaptic acetylcholine (Nm) receptors.

Sparteine

Sparteine is a class 1a antiarrhythmic agent; a sodium channel blocker. It is an alkaloid and can be extracted from scotch broom. It is the predominant alkaloid in Lupinus mutabilis, and is thought to chelate the bivalent cations calcium and magnesium. It is not FDA approved for human use as an antiarrhythmic agent, and it is not included in the Vaughan Williams classification of antiarrhythmic drugs.

Methyllycaconitine

Methyllycaconitine (MLA) is a diterpenoid alkaloid found in many species of Delphinium (larkspurs). In common with many other diterpenoid alkaloids, it is toxic to animals, although the acute toxicity varies with species. Early research was focused on identifying, and characterizing the properties of methyllycaconitine as one of the principal toxins in larkspurs responsible for livestock poisoning in the mountain rangelands of North America. Methyllycaconitine has been explored as a possible therapeutic agent for the treatment of spastic paralyses in man, and it has been shown to have insecticidal properties. Most recently, it has become an important molecular probe for studying the pharmacology of the nicotinic acetylcholine receptor.

Histrionicotoxins

Histrionicotoxins are a group of related toxins found in the skin of poison frogs from the family Dendrobatidae, notably Oophaga histrionica, which are native to Colombia. It is likely that, as with other poison frog alkaloids, histrionicotoxins are not manufactured by the amphibians, but absorbed from insects in their diet and stored in glands in their skin. They are notably less toxic than other alkaloids found in poison frogs, yet their distinct structure acts as a neurotoxin by non-competitive inhibition of nicotinic acetylcholine receptors.

Acetylcholinesterase inhibitor Drugs that inhibit acetylcholinesterase

Acetylcholinesterase inhibitors (AChEIs) also often called cholinesterase inhibitors, inhibit the enzyme acetylcholinesterase from breaking down the neurotransmitter acetylcholine into choline and acetate, thereby increasing both the level and duration of action of acetylcholine in the central nervous system, autonomic ganglia and neuromuscular junctions, which are rich in acetylcholine receptors. Acetylcholinesterase inhibitors are one of two types of cholinesterase inhibitors; the other being butyryl-cholinesterase inhibitors. Acetylcholinesterase is the primary member of the cholinesterase enzyme family.

Nifene

Nifene is a high affinity, selective nicotinic α4β2* receptor partial agonist used in medical research for nicotinic acetylcholine receptors, usually in the form of nifene (18F) as a positron emission tomography (PET) radiotracer.

Methoctramine

Methoctramine is a polymethylene tetraamine that acts as a muscarinic antagonist. It preferently binds to the pre-synaptic receptor M2, a muscarinic acetylcholine ganglionic protein complex present basically in heart cells. In normal conditions -absence of methoctramine-, the activation of M2 receptors diminishes the speed of conduction of the sinoatrial and atrioventricular nodes thus reducing the heart rate. Thanks to its apparently high cardioselectivity, it has been studied as a potential parasymphatolitic drug, particularly against bradycardia. However, currently it’s only addressed for research purposes, since the administration to humans is still unavailable.

Anagyrine Chemical compound

Anagyrine is a teratogenic alkaloid commonly found in many species of Lupinus plants. The toxin can cause crooked calf disease if a cow ingests the plant during certain periods of pregnancy.

Autonomic drugs can either inhibit or enhance the functions of the parasympathetic and sympathetic nervous systems. This type of drug can be used to treat a wide range of diseases, such as glaucoma, asthma, urinary, gastrointestinal and cardiopulmonary disorders.

Guanitoxin Chemical compound

Guanitoxin (GNT), formerly known as anatoxin-a(S) "Salivary", is a naturally occurring cyanotoxin commonly isolated from cyanobacteria and causes excess salivation in mammals via inhibition of acetylcholinesterase. Guanitoxin was first structurally characterized in 1989, and consists of a cyclic N-hydroxyguanine organophosphate with a phosphate ester moiety.

Quinolizidine alkaloids

Quinolizidine alkaloids are natural products that have a quinolizidine structure; this includes the lupine alkaloids.

Huprine X

Huprine X is a synthetic cholinergic compound developed as a hybrid between the natural product Huperzine A and the synthetic drug tacrine. It is one of the most potent reversible inhibitors of acetylcholinesterase known, with a binding affinity of 0.026nM, as well as showing direct agonist activity at both nicotinic and muscarinic acetylcholine receptors. In animal studies it has nootropic and neuroprotective effects, and is used in research into Alzheimer's disease, and although huprine X itself has not been researched for medical use in humans, a large family of related derivatives have been developed.

Cholinergic blocking drugs

Cholinergic blocking drugs are a group of drugs that block the action of acetylcholine (ACh), a neurotransmitter, in synapses of the cholinergic nervous system. They block acetylcholine from binding to cholinergic receptors, namely the nicotinic and muscarinic receptors.

References

  1. ↑ Wink, Michael; Meißner, Carsten; Witte, Ludger (January 1995). "Patterns of quinolizidine alkaloids in 56 species of the genus Lupinus". Phytochemistry. 38 (1): 139–153. doi:10.1016/0031-9422(95)91890-d. ISSN   0031-9422.
  2. 1 2 Goldberg, Stanley I.; Ragade, Indukanth (April 1967). "A total synthesis of optically active lupinine without benefit of resolution". The Journal of Organic Chemistry. 32 (4): 1046–1050. doi:10.1021/jo01279a039. ISSN   0022-3263. PMID   6042140.
  3. 1 2 Golebiewski, W. Marek; Spenser, Ian D. (1985-10-01). "Biosynthesis of the lupine alkaloids. I. Lupinine". Canadian Journal of Chemistry. 63 (10): 2707–2718. doi: 10.1139/v85-450 . ISSN   0008-4042.
  4. 1 2 3 4 Rozengart, E. V.; Basova, N. E. (2001-11-01). "Ammonium Compounds with Localized and Delocalized Charge as Reversible Inhibitors of Cholinesterases of Different Origin". Journal of Evolutionary Biochemistry and Physiology. 37 (6): 604–610. doi:10.1023/A:1014414126143. ISSN   0022-0930. S2CID   25689578.
  5. 1 2 Risikobewertung, Bundesinstitut Für (2017). "Risk assessment of the occurrence of alkaloids in lupin seeds" (PDF). OpenAgrar. doi:10.17590/20170530-142504.{{cite journal}}: Cite journal requires |journal= (help)
  6. ↑ Bleitgen, R.; Gross, R.; Gross, U. (1979-06-01). "Die Lupine — ein Beitrag zur Nahrungsversorgung in den Anden". Zeitschrift für Ernährungswissenschaft (in German). 18 (2): 104–111. doi:10.1007/BF02023724. ISSN   0044-264X. PMID   556411. S2CID   82321535.
  7. 1 2 Róbertsdóttir, Anna Rósa (2016). Icelandic herbs and their medicinal uses. Berkeley, California. ISBN   9781623170226. OCLC   910979700.
  8. ↑ Yáñez, Enrique; Ivanović, D.; Owen, D.F.; Ballester, D. (1983). "Chemical and Nutritional Evaluation of Sweet Lupines". Annals of Nutrition and Metabolism. 27 (6): 513–520. doi:10.1159/000176728. ISSN   1421-9697. PMID   6651228.
  9. 1 2 3 4 Introduction to neurobehavioral toxicology : food and environment . Niesink, Raymundus Johannes Maria, 1953-. Boca Raton: CRC Press. 1999. ISBN   978-0849378027. OCLC   39764034.{{cite book}}: CS1 maint: others (link)
  10. ↑ Torres Tello, F.; Nagata, A.; Dreifuss Spiegel, W. (June 1980). "[Methods of eliminating alkaloids from the seeds of Lupinus mutabilis Sweet]". Archivos Latinoamericanos de Nutricion. 30 (2): 200–209. ISSN   0004-0622. PMID   7212919.
  11. ↑ Kirkensgaard, Kristine (2017-12-11). "Lupinine - the chemical stopping you from eating Lupin". natoxaq.ku.dk. Retrieved 2018-04-27.
  12. ↑ Resta, Donatella; Boschin, Giovanna; D'Agostina, Alessandra; Arnoldi, Anna (2008). "Evaluation of total quinolizidine alkaloids content in lupin flours, lupin-based ingredients, and foods". Molecular Nutrition & Food Research. 52 (4): 490–495. doi:10.1002/mnfr.200700206. ISSN   1613-4125. PMID   18324702.
  13. 1 2 Dictionary of plant toxins. Harborne, J. B. (Jeffrey B.), Baxter, Herbert, 1928-, Moss, Gerard P. Chichester: Wiley. 1996. ISBN   978-0471951070. OCLC   34281328.{{cite book}}: CS1 maint: others (link)
  14. 1 2 3 Couch, James (1926). "Relative Toxicity of the Lupine Alkaloids". Journal of Agricultural Research. XXXII: 51–67.
  15. 1 2 3 4 Tilyabaev, Z.; Abduvakhabov, A. A. (1998-05-01). "Alkaloids of Anabasis aphylla and their cholinergic activities". Chemistry of Natural Compounds. 34 (3): 295–297. doi:10.1007/BF02282405. ISSN   0009-3130. S2CID   30640524.
  16. ↑ Colovic, Mirjana B.; Krstic, Danijela Z.; Lazarevic-Pasti, Tamara D.; Bondzic, Aleksandra M.; Vasic, Vesna M. (2013-04-01). "Acetylcholinesterase Inhibitors: Pharmacology and Toxicology". Current Neuropharmacology. 11 (3): 315–335. doi:10.2174/1570159x11311030006. PMC   3648782 . PMID   24179466.
  17. ↑ Schmeller, Thorsten; Sauerwein, Martina; Sporer, Frank; Wink, Michael; Müller, Walter E. (September 1994). "Binding of Quinolizidine Alkaloids to Nicotinic and Muscarinic Acetylcholine Receptors". Journal of Natural Products. 57 (9): 1316–1319. doi:10.1021/np50111a026. ISSN   0163-3864. PMID   7798968.
  18. ↑ Spenser, I. D. (1985-01-01). "Stereochemical aspects of the biosynthetic routes leading to the pyrrolizidine and the quinolizidine alkaloids". Pure and Applied Chemistry. 57 (3): 453–470. doi:10.1351/pac198557030453. ISSN   1365-3075. S2CID   46220343.
  19. ↑ Clemo, G. R.; Morgan, W. McG.; Raper, R. (1937). "199. The lupin alkaloids. Part XII. The synthesis of dl-lupinine and dl-isolupinine". Journal of the Chemical Society (Resumed): 965. doi:10.1039/jr9370000965. ISSN   0368-1769.
  20. ↑ Ma, Shengming; Ni, Bukuo (2004-07-05). "Double Ring-Closing Metathesis Reaction of Nitrogen-Containing Tetraenes: Efficient Construction of Bicyclic Alkaloid Skeletons and Synthetic Application to Four Stereoisomers of Lupinine and Their Derivatives". Chemistry - A European Journal. 10 (13): 3286–3300. doi:10.1002/chem.200305581. ISSN   0947-6539. PMID   15224338.
  21. 1 2 Santos, Leonardo; Mirabal-Gallardo, Yaneris; Shankaraiah, Nagula; Simirgiotis, Mario (2010-12-08). "Short Total Synthesis of (-)-Lupinine and (-)-Epiquinamide by Double Mitsunobu Reaction". Synthesis. 2011 (1): 51–56. doi:10.1055/s-0030-1258356. hdl: 10533/132744 . ISSN   0039-7881.
  22. ↑ J. F. Couch (1934). "Lupine Studies. VIII. The Alkaloids of Lupinus Palmeri, S. Wats". J. Am. Chem. Soc. 56 (11): 2434–2436. doi:10.1021/ja01326a067.
  23. ↑ Campbell, F. L.; Sullivan, W. N.; Smith, C. R. (1933-04-01). "The Relative Toxicity of Nicotine, Anabasine, Methyl Anabasine, and Lupinine for Culicine Mosquito Larvae". Journal of Economic Entomology. 26 (2): 500–509. doi:10.1093/jee/26.2.500. ISSN   1938-291X.
  24. ↑ Molina-Cruz, Alvaro; Lehmann, Tovi; Knöckel, Julia (2013). "Could culicine mosquitoes transmit human malaria?". Trends in Parasitology. 29 (11): 530–537. doi:10.1016/j.pt.2013.09.003. ISSN   1471-4922. PMID   24140295.
  25. ↑ Adler, Lynn S. (2003). "Host Species Affects Herbivory, Pollination, and Reproduction in Experiments with Parasitic Castilleja". Ecology. 84 (8): 2083–2091. doi:10.1890/02-0542. hdl: 10919/46839 . ISSN   0012-9658.
  26. ↑ "Classifications - CL Inventory". www.echa.europa.eu. Retrieved 2018-04-25.
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