Natural Alkamides: Pharmacology, Chemistry and Distribution

Alkamides are a broad and expanding group of bioactive natural compounds found in at least 33 plant families. Despite the relatively simple molecular architecture of alkamides (fig. 1), these natural products show broad structural variability and an important range of biological activities, such as immunomodulatory, antimicrobial, antiviral, larvicidal, insecticidal, diuretic, pungent, analgesic, cannabimimetic and antioxidant activities. Additionally, alkamides are involved in the potentiation of antibiotics and the inhibition of prostaglandin biosynthesis, RNA synthesis and the arachidonic acid metabolism, among others.

anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid cerebral neurotransmitter, alkamides are highly active in the central nervous system (CNS, fig. 2). In general, when alkamide-producing plants are chewed, a pungent taste is released causing itching and salivation. Chloroform is the best solvent for the extraction of alkamides, though both methanol and ethanol have also been used. Pure alkamides are sensitive to oxidation and polymerization of double and triple bonds occur during the drying, handling and storage of these compounds. Notably, alkamides are promising chemical and pharmacological entities that are useful therapeutics for the treatment of several important illnesses. This chapter describes the distribution of alkamides, the chemical aspects used to distinguish these important natural products and the pharmacological properties of the plants from which these compounds are isolated.

Alkamides from the Asteraceae family
The Asteraceae family is characterized by the accumulation of aliphatic alkamides. Aaronsohnia, Achilea, Acmella, Anacyclus, Artemisia, Echinaceae, Heliopsis, Spilanthes, Salmea, Sanvitalia and Wedelia are genera that belong to this alkamide-producing family. These genera share the biogenetic capacity to combine C8 to C18 (with exception of C17) olefinic and acetylenic acid residues with the more widespread Nphenethyl and cyclic amines [piperidinyl (piperidide), 2,3-dehydro-piperidinyl (piperideide), pyrrolidinyl and pyrrolidyl]. However, other minor amides including N-4methylbutyl, N-tyramidyl and O-methyl-tyramidyl residues have also been found ( fig. 3). Currently, the most commonly found alkamides in the Asteraceae family include a C10, C11 and C12 long chain residue acids, which represent approximately 72% of aliphatic alkamides isolated from this family. The second most important group of these natural products includes C14 and C18 long chain residue acids, constituting approximately 13% of Asteraceae alkamides. Most phytochemical and pharmacological studies have been conducted with Achillea, Acmella, Sphilantes, Echinaceae and Heliopsis genera, which will be discussed in subsequent sections.

Achillea genus
The occurrence of alkamides with cyclic amide moieties is confined to the Anthemideae tribe, being Achillea species especially rich in both pyrrolidides and piperidides and their corresponding dehydroderivatives. Apart from the more widespread isobutylamides, this genus is characterized by the frequent occurrence of saturated and unsaturated 5-and 6-ring amides (Greger et al., 1987a(Greger et al., , 1987b. The accumulation of amides with characteristic olefinic and acetylenic patterns is characteristic of this genus. These amides are mainly accumulated in the subterranean parts of these plants (table 1).

Acmella genus
A name frequently used in folk medicine for species containing alkamides is "the tooth herb". These plants exhibit analgesic properties and are frequently used as odontologic agents. For example, Acmella decumbens roots have a pungent taste and when chewed a numbing sensation is felt on the tongue. Acmella radicans is another species also used for the treatment of toothache (Rios-Chavez et al., 2003).
Alkamides from the Acmella genus consist of an N-isobutyl, N-2-methylbutyl or N-phenethyl amine and C8 to C12 acid residues. Of the seven Acmella species that have been chemically analyzed, four species have been observed to produce affinin (spilanthol, N-isobutyl-2E,6Z,8E-decatrienamide, 70), an alkamide with established analgesic properties (Rios et al., 2007). Several affinin analogues are present in extracts from these Acmella species (see table  1), which probably contribute to the analgesic sensation induced by these plants.

Spilanthes genus
For years Spilanthes acmella has been used as traditional folk medicine to treat toothaches, stammering, and stomatitis. Previous studies have demonstrated the diuretic, antibacterial, and anti-inflammatory activities of Spilanthes acmella. Spilanthol (70), the main alkamide isolated from this plant, exhibits antiseptic activity. Additionally, spilanthol (70) is involved in immune stimulation and the attenuation of the inflammatory responses in murine RAW 264.7 macrophages (Wu et al., 2008).

Echinaceae genus
Echinacea is a native herb from North America and Europe that is used as an immunostimulant. Extracts from the Echinacea species are widely used due to the strong belief that the components of the extract stimulate the immune system and help to prevent infections, colds, respiratory infections and influenza. However, the clinical efficacy of this agent has not been proven. E. angustifolia, E. pallida and E. purpurea are three species of Echinacea that are used in commercial preparations with reported alkamide profiles. These species contain complex mixtures of alkamides that are good chemotaxonomic characters (table 1). The major alkamides in E. purpurea roots are the C12-2,4-diene and C12-2,4-dienediyne type, while the C11 diene-diynes were highest in vegetative stems (Binns et al., 2002). E. angustifolia roots are characterized by the presence of di-, tri-and tetraenes in coexistence with mono-and diynes, all of them with variable insaturation degree at the C2, C4, C9 or C10 position. In E. pallida, the major compounds are polienes (also di-, tri-and tetraenes) and diynes (C2 or C2 and C4 unsaturated) Lipophilic alkamides from Echinacea show immunostimulatory activity and have been used for the treatment of cold, flu, respiratory infections and inflammations, making a considerable contribution to the activities attributed to Echinaceae plants (Bauer, 1989a(Bauer, , 1989b(Bauer, , 1990(Bauer, , 1991. Studies on the mechanisms of action of the immunomodulatory activity of Echinacea have indicated that alkylamides can act as cannabinomimetics. Endogenous ligands for cannabinoid receptors such as anandamide ( fig. 2), an animal alkamide that shares structural similarity with the Echinacea alkylamides, can bind to CB2 cannabinoid receptors (LaLone et al., 2010). The cannabinoid receptors CB1 and CB2 have been implicated in the modulation of the CNS and the inflammatory response. CB1 receptors are present in neurons from the central and peripheral nervous system and are concentrated in the brain. CB2 receptors are mainly present in immune cells, such as macrophages.

Heliopsis genus
Heliopsis longipes is a Mexican plant that was broadly used by the Náhuatl civilization as flavoring in food preparation. The stems of this climber are used in traditional medicine as a condiment, buccal anesthetic, analgesic in pain toothache, antiparasitic, anti-inflammatory and antiulcerative agent and to prepare homemade insecticides that, similar to pyrethrins, are toxic and exhibit paralyzing effects. Chewing of a little piece of the Heliopsis longipes stem results in intense salivation and a local analgesic effect (Molina et al., 1996). An ethanolic extract of this plant exhibited antinociceptive effects on acute thermal and chemical inflammation induced nociception in mice with a mechanism partly linked to the lipoxygenase and/or cyclooxygenase systems (Cariño-Cortés et al., 2010). This extract exhibited synergistic interactions with diclofenac in the Hargreaves model of thermal hyperalgesia (Acosta-Madrid et al., 2009). Various unsaturated aliphatic alkamides have also been identified and characterized from the roots of this plant (table 1), such as affinin (70), its most abundant and bioactive alkamide. The analgesic activity of affinin was determined by measuring the release of GABA in mice brain slices (Rios et al., 2007). Furthermore, dosedependent antinociceptive effects have been observed to be a result of the activation of opiodergic, serotoninergic and GABAergic systems (Déciga-Campos et al., 2010).

Aliphatic alkamides from other plant families
Convolvulaceae, Euphorbiaceae, Menispermaceae and Rutaceae are other plant families that produce aliphatic alkamides. N-isobutyl, 2'-hydroxy-N-isobutyl, NH 2 and pyrrolidinyl amine residues have been identified in the structures of alkamides isolated from these plants (table 2).

Convolvulaceae alkamides
Convolvulaceae alkamides are also known as alkaloids MQ. These alkamides are characterized by linear or branched saturated acid residues. All Convolvulaceae alkamides have a pyrrolidinyl residue as the amine group and have been isolated from the Ipomoea and Merremia genera (compounds 130-136).

Euphorbiaceae alkamides
Phyllanthus fraternus is used by traditional healers and tribes in the northern region of India as a folklore remedy for the treatment of malaria and various liver diseases. An aqueous extract of this plant exhibited antioxidant activity, preventing the oxidation of proteins and lipids. Additionally, aqueous extracts of Phyllanthus fraternus protect against allyl alcoholinduced oxidative stress in liver mitochondria (Sailaja & Setty, 2006). Two aliphatic alkamides C 4 isomers , E,E-2,4-octadienamide (137) and E,Z-2,4-decadienamide (138), have been isolated from this plant. Both isomers lack an alkyl residue at the amine group, which is typically joined to an acid residue (Sittie et al., 1998). Instead, these compounds possess an α,β,γ,δ-unsaturated conjugated amide, a feature believed to enhance antiplasmodial activity.
Notably, in vitro assays of these two isomers demonstrated that these compounds possess moderate antiplasmodial activity.

Menispermaceae alkamides
The roots of some species of the Cissampelos genus exhibit significant activity against mechanical, chemical and arthritic pain, increasing the pain threshold and dictating the medicinal value of the plants of this genus. For example, C. glaberrimma is a plant whose bioactivity is a reflection of its alkamide content (alkamides 139-142, Rosario et al., 1996).

Rutaceae alkamides
The fruits of Zanthoxylum integrifoliolum possess a pungent taste. Chemical analysis enabled the isolation and identification of nine isobutylamides (143-151). These amides have a 2E,4Edienamide moiety, including an oxo, diene, tetraene or pentaene acidic fragment (table 2). However, no activity has been reported for these molecules.
Amides have also been isolated from the Glycosmis genus (Rutaceae); however, those isolated from this genus are sulfur-containing amides, a rare group of secondary metabolites that have an aromatic amine residue. Glycosmis alkamides will be discussed in section 3.3 (vide infra).

Aromatic alkamides
Alkamides isolated from Solanaceae, Piperaceae, Brassicaceae and Rutaceae plant families either have one aromatic ring at the amine residue, at the acid residue or both. Capsaicinoids, amides from Lepidium meyenii, and sulfur derivatives from the Glycosmis genus are alkamides with one aromatic ring at the amine residue. Piperine and its analogs are amides with one aromatic residue at the acid fragment. Alkamides that have an aromatic ring at the amine and acid residues are distributed among a large group of plants.

The alkamides from Solanaceae family: Capsaicinoids
Capsicum (also known as "chile" or "chilli") are species used as vegetables, condiments, and for an important number of medicinal preparations. The fruits of Capsicum have been utilized in food preparation, for medicinal applications to tone body muscles after workouts, hot infusions for toothache and muscle pain and aerosols such as Capsicum extracts that are used as personal protection. This species are the source of highly pungent capsacinoids that induce a hot or burning sensation. Capsaicinoids are the major chemical constituents from the following five domesticated species of Capsicum (peppers) genus: C. annuum L., C. baccatum L., C. chinense Jacq., C. frutescens L. and C. pubescens. All of these species have Nvanillylamides (all contain a 4-hydroxy-3-methoxybenzyl amine group) of C8 to C18 fatty acids (table 3).  Some capsaicinoids exhibit strong pungent sensory properties when consumed as part of the diet. Additionally, capsaicinoids possess a variety of biological properties that may affect human health (Kozuke et al., 2010), such as antiviral, antibacterial, antifungal, insecticidal, antioxidative, anti-inflammatory and anticancer activities. Furthermore, capsaicinoids influence neuronal structures that contain substances that are associated with pain transmission and neurogenic inflammation. As a result, these compounds are used as topical analgesics for treating pain. The aforementioned properties are the basis for the use of capsaicinoids to prevent or reduce chronic and age-related pain (Kozuke et al., 2005). Capsaicin (158) and dihydrocapsaicin (157) are notable among natural capsaicinoids because they constitute approximately 90% of the total capsaicinoids in many varieties of peppers. The burning sensation caused by capsaicin is induced by the direct activation of a nonselective cation channel-transient receptor potential, vanilloid 1 (TRPV1), located at the end of sensory nerves. Several physiological activities caused by capsaicin are related to the activation of the TRPV1 receptor. Meghvansi and coworkers have written a review of capsaicinoids in which their ethnopharmacological applications are discussed (Meghvansi et al., 2010). Long acyl chain capsaicinoids exhibiting similar activities to capsaicin, such as anti-inflammatory, antinociceptive and enhanced adrenaline secretion, have been recently reported. The advantages of these compounds are the lack of irritancy or pungency due to the lower accessibility of TRPV1 in the tongue due to higher lipophilicity compared to capsaicin (Kobata et al., 2010).

The alkamides from Lepidium meyenii (Brassicaceae)
The roots from of L. meyenii are used to enhance fertility and sexual behavior in men and women. Additionally, L. meyenii roots serve as a traditional remedy for menopausal symptoms, the regulation of hormone secretion, immunostimulation, memory improvement, as an antidepressant or anticancer agent, and to prevent anemia. Phytochemical analysis of the roots of this plant led to the identification of mmethoxybenzyl and N-benzyl amine residues and macamides, linear C16, C18 or C24 alkamides with one or two double bonds and possible oxidation of C 5 , C 9 or C 13 (table 4).

The alkamides from Glycosmis (Rutaceae)
Sulfur-containing amides (phenethyl/styrylamine-derived amides) form a rare group of secondary metabolites in the Rutaceae family. These amides are only present in the leaves of plants that belong to the Glycosmis genus. Sulfur-containing amides represent a typical chemical profile of this genus. The acid moieties of these alkamides are probably derived from cysteine, which can be oxidized to sulfones and sulfoxides or shortened by β-oxidation (as in ritigalin). With the exception of simple methylamides, the amine residues are characterized by the presence of phenethyl or styryl groups (derived from phenylalanine) that can be linked to different prenyloxy (dambullins) or geranyloxy groups in para position (gerambullins). More recently, a group of similar (methylsulfonyl)propenoic acid amides has been detected in which dopamine is linked to various oxidized geranyl chains (sakerines). Some of these alkamides exhibit pronounced antifungal and/or insecticidal activity (Greger & Zechner, 1996) (table 5).
The compounds produced by the Piperaceae family are pharmacologically very important, as several species of these plants are being used in folkloric medicine in different parts of the world. For example, the roots of plants from the Ottonia genus have a piquant taste and cause intense salivation when are in contact with the mouth. These roots exhibit local anesthetic and hallucinogenic effects and are used in the treatment of toothaches and sore throats. The toothache-relieving reputation of plants that belong to this genus led to the isolation of piperovatine (222), a buccal local anesthesic isobutyl amide isolated from O. corcovadensis. Alkamides isolated from the Ottonia genus contain 1-oxo-5-(3',4'methylenedioxyphenyl)-2E,4E-pentadien-1-yl and 1-oxo-6-(p-methoxyphenyl)-2E,4Ehexadien-1-yl residues as acidic fragments with N-isobutyl or N-3-acetoxy-isobutyl fragments as the amide residues (Antunes et al., 2001;Costa & Mors, 1981, table 6). The Piper species have been used in traditional medicine for thousands of years in China, India and Mexico, among other countries, for the treatment of several diseases and ailments. For example, P. longum is used for treatment of gonorrhea, menstrual and chronic intestinal pain, tuberculosis, sleeping problems, respiratory infections such as coughs, bronchitis and asthma, malarial fever, diarrhea, jaundice and arthritis. The beneficial effects of this species include analgesic and diuretic activities, relaxation of muscle tension, and the alleviation of anxiety.

132
In contrast, P. hispidum and P. tuberculatum exhibit antifungal activity and produce amides with the cis geometry in their side chains, a structural feature quite rare in nature (table 6, Navickiene et al., 2000).
Piperine (E,E isomer of 1-piperolypiperidine, 224) is the major component in the fruits of several species of Piper, particularly P. longum and P. nigrum. This compound showed diverse biological activities such as antioxidant, anti-inflammatory, analgesic, antiplatelet aggregation, antihyperlipidemic, antihypertensive, cytoprotective, antitumor, antimicrobial, hepatoprotective and antidepressant activities. The structure of piperine resembles that of Capsaicin (158, table 3), the pungent component in the majority of the chilli peppers species. Similar to capsaicin, piperine also serves as a natural agonist of the vanilloid receptor (TRPV1 channel), which is involved in the neurotransmission of thermal and nociceptive stimuli.

Other family plants -Alkamides with both fragments including aromatic residues
The cinnamoylbenzylamide tribulusimide (263, fig. 7) and several cinnamoylphenethylamides (table 7) and benzylphenethylamides (table 8) are the condensation products of cinnamic acid and benzylamine derivatives, cinnamic acid and phenethylamine and benzylic acid and phenethylamine, respectively. These alkamides have been isolated from a broad variety of plants that belong to at least 28 families. A selection of these alkamides are shown in   Despite the broad distribution of alkamides with both fragments, including aromatic residues among a wide variety of plant families, the presence of feruloyltyramine (266) is exceptionally important because it is a common compound found in the majority of alkamide-producing plants. The Z-and E-stereoisomers of feruloyltyramine have been isolated and are two of the most frequently characterized alkamides. The second most important alkamide is p-coumaroyltyramine (264), which is isolated also in both stereoisomeric forms, the E-stereoisomer being the most common (table 9).

Conclusion
Alkamides are natural products distributed among several medicinal plants that are a part of at least 33 families. These plants are used for a variety of medicinal purposes in many places throughout the world. Chemical and pharmacological research of these plants have established that alkamides contribute to the notable bioactivity of these plants. Asteraceae, Solanaceae, Rutaceae and Piperaceae are plant families that specialize in the biosynthesis of these natural products. Importantly, alkamides are chemical markers for plants in each family and genus.
Alkamides with both acid and amine aliphatic residues are characteristic compounds produced by the Asteraceae family, especially from the Achillea, Acmella, Spilanthes, Echinaceae and Heliopsis genera. Alkamides with one aromatic residue can be classified in the following two groups: (1) alkamides with an aromatic residue at the amine core and (2) alkamides with an aromatic residue at the acid. The first group has been isolated from the Solanaceae family, specifically from the Capsicum genus for which those alkamides are named "capsaicinoids". Other alkamides that belong to this group have been isolated from the Lepidium (Brassicaceae) and Glycosmis (Rutaceae) genera. Glycosmis alkamides are rare and have characteristic sulfur-containing structures. The second group corresponds to piperine and its analogs. These compounds are characteristic of the Piper genus (Piperaceae). Furthermore, the alkamides with both acid and amine aromatic residues are widely distributed among at least 28 plant families. Feruloyltyramine and p-coumaroyltyramine are the most commonly isolated alkamides that belong to this group of compounds. and arthritic pain. Some alkamides are also consumed to enhance immune response and to relieve colds, respiratory infections and influenza. Anti-inflammatory activity is associated with all of these natural products. Despite the relatively simple structures of alkamides, these compounds have attracted several research groups to study their diversity, distribution and chemical and pharmacological behaviours. Additionally, alkamides have been observed to exhibit many other bioactivities, making these compounds a relatively new and promising family of natural products.

Acknowledgments
To CONACyT (Grant number 79584-Q). I am grateful to Enrique Salazar Leyva for technical assistance. I apologize to all colleagues whose studies were not cited due to space limitations.