Spilanthol: occurrence, extraction, chemistry and biological activities

Alan F. Barbosa Mário G. de Carvalho Robert E. Smith Armando U.O. Sabaa-Srur About the authors

Abstract

Spilanthol (C14H23NO, 221.339 g/mol) is a bioactive compound that is found in many different plants that are used as traditional remedies throughout the world. It is present in Heliopsis longipes and several species in the genus Acmella, including A. oleracea L., also known as paracress and jambu. Its leaves and flowers have sensory properties (pungency, tingling, numbing, mouth-watering) that make it a popular spice and ingredient in several Brazilian dishes. Spilanthol can exert a variety of biological and pharmacological effects including analgesic, neuroprotective, antioxidant, antimutagenic, anti-cancer, anti-inflammatory, antimicrobial, antilarvicidal and insecticidal activities. So, the aim of this review is to present a literature review on the spilanthol that describes its occurrence, chemistry, extraction and biological activities.

Keywords
Acmella oleracea; Alkamides; Bioactivity; Spilanthes oleracea L.; Heliopsis longipes

Introduction

Spilanthol (C14H23NO, 221.339 g/mol) (1) is a bioactive compound that is found in many different plants that are used as traditional remedies throughout the world (Molinatorres et al., 1996; Prachayasittukal et al., 2013; Paulraj et al., 2013; Rios and Olivo, 2014). Its IUPAC name is (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (Molinatorres et al., 1996Molinatorres, J., Salgado-Garciglia, R., Ramirez-Chanez, E., del Rio, R.E., 1996. Purely olefinic alkamides in Heliopsis longipes and Acmella (Spilanthes) oppositifolia . Biochem. Syst. Ecol. 24, 27-43). It is also known as affinin (Prachayasittukal et al., 2013Prachayasittukal, V., Prachayasittukal, S., Ruchiwarat, S., Prachayasittukal, V., 2013. High therapeutic potential of Spilanthes acmella: a review. EXCLI J. 12, 291-312).

The plants in which it is found are often called toothache plants, due to the analgesic effect of spilanthol (Molinatorres et al., 1996Molinatorres, J., Salgado-Garciglia, R., Ramirez-Chanez, E., del Rio, R.E., 1996. Purely olefinic alkamides in Heliopsis longipes and Acmella (Spilanthes) oppositifolia . Biochem. Syst. Ecol. 24, 27-43; Hind and Biggs, 2003Hind, N., Biggs, N., 2003. Plate 460. Acmella oleracea compositae. Curtis's Bot. Mag. 20, 31-39; Wu et al., 2008Wu, L.-C., Fan, N.C., Lin, M-H., Chu, I.-R., Huang, S.J., Han, S.Y., 2008. Anti-inflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators. J. Agric. Food Chem. 56, 2341-2349; Tiwari et al., 2011Tiwari, K.L., Jadhav, S.K., Joshi, V., 2011. An updated review on medicinal herb Genus Spilanthes . J. Chin. Integr. Med. 9, 1170-1178; Dias et al., 2012Dias, A.M.A., Santos, P., Seabraa, I.J., Junior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J. Supercrit. Fluids. 61, 62-70; Sharma et al., 2012Sharma, A., Kumar, V., Rattan, R.S., Kumar, N., Singh, B., 2012. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. Against Plutella xylostella L. Am. J. Plant Sci. 3, 1568-1572; Abeysiri et al., 2013Abeysiri, G.R.P.I., Dharmadasa, R.M., Abeysinghe, D.C., Samarasinghe, K., 2013. Screening of phytochemical, physico-chemical and bioactivity of different parts of Spilantes acmella Murr. (Asteraceae), a natural remedy for toothache. Ind. Crop. Prod. 50, 852-856; Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.; Prachayasittukal et al., 2013Prachayasittukal, V., Prachayasittukal, S., Ruchiwarat, S., Prachayasittukal, V., 2013. High therapeutic potential of Spilanthes acmella: a review. EXCLI J. 12, 291-312; Paulraj et al., 2013Paulraj, J., Govindarajan, R., Palpu, P., 2013. The genus Spilanthes ethnopharmacology, phytochemistry, and pharmacological properties: a review. Adv. Pharmacol. Sci., http://dx.doi.org/10.1155/2013/510298
http://dx.doi.org/10.1155/2013/510298...
; Rios and Olivo, 2014Rios, M.R., Olivo, H.F., 2014. Natural and synthetic alkylamides: applications in pain therapy. In: Atta-Ur-Rahman (Ed.), Studies in Natural Products Chemistry. Else-vier, New York, pp. 79–118.; Dandin et al., 2014Dandin, V.S., Naik, P.M., Murthy, H.M., Park, S.Y., Paek, K.Y., 2014. Rapid regeneration and analysis of genetic fidelity and scopoletin contents of micropropagated plants of Spilanthes oleracea L. J. Hort. Sci. Biotechnol. 89, 79-85; Hajdu, 2014Hajdu, A., 2014. An Ethnopharmacological Survey Conducted in the Bolivian Amazon, and Identification of N-alkylamides and Lignans from Lepidium meyenii and Heliopsis helianthoides var. scabra with Effects on the Central Nervous System. University of Szeged, Szeged, Hungary.). Like other alkamides, it is an amphiphilic compound with a relatively polar amide and a less polar fatty acyl. So, it can be extracted from plants using either methanol, ethanol, supercritical CO2 or hexane (Nakatani and Nagashima, 1992Nakatani, N., Nagashima, M., 1992. Pungent alkamides from Spilanthes acmella L. var. oleracea Clarke. Biosci. Biotechnol. Biochem. 56, 759-762; Sharma et al., 2011Sharma, V., Boonen, J., Chauhan, N.S., Thakur, M., de Spiegeleer, B., Dixit, V.K., 2011. Spilanthes acmella ethanolic flower extract: LC–MS alkylamide profiling and its effects on sexual behavior in male rats. Phytomedicine. 18, 1161-1168; Dias et al., 2012Dias, A.M.A., Santos, P., Seabraa, I.J., Junior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J. Supercrit. Fluids. 61, 62-70; Singh and Chaturvedi, 2012aSingh, M., Chaturvedi, R., 2012. Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilantes acmella Murr. Ind. Crop Prod. 36, 321-328,bSingh, M., Chaturvedi, R., 2012. Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthese acmella Murr. Bioprocess Biosyst. Eng. 35, 943-951; Hajdu, 2014Hajdu, A., 2014. An Ethnopharmacological Survey Conducted in the Bolivian Amazon, and Identification of N-alkylamides and Lignans from Lepidium meyenii and Heliopsis helianthoides var. scabra with Effects on the Central Nervous System. University of Szeged, Szeged, Hungary.; Abeysinghe et al., 2014Abeysinghe, D.C., Wijerathne, S.M.N.K., Dharmadasa, R.M., 2014. Secondary metabolites contents and antioxidant capacities of Acmella oleraceae grown under different growing systems. World J. Agric. Res. 2, 163-167). After being extracted, it can be purified by preparative scale TLC and/or HPLC (Johns et al., 1982Johns, T., Graham, K., Towers, G.H.N., 1982. Molluscicidal activity of affinin and other isobutylamides from the Asteraceae. Phytochemistry. 21, 2737-2738; Ogura et al., 1982Ogura, M., Cordell, G.A., Quinn, M.L., Leon, C., Benoit, P.S., Soejarto, D.D., Farnsworth, N.R., 1982. Ethnopharmacology studies. I. Rapid solution to a problem – oral use of Heliopsis longipes – by means of a multidisciplinary approach. J. Ethnopharmacol. 5, 215-219; Mbeunkui et al., 2011Mbeunkui, F., Grace, M.H., Lategan, C., Smith, P.J., Raskin, I., Lila, M.A., 2011. Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS. J. Chromatogr. B. 879, 1886-1892; Pandey et al., 2011Pandey, V., Chopra, M., Agrawal, V., 2011. In vitro isolation and characterization of biolarvicidal compounds from micropropogated plants of Spilanthes acmela . Parasitol. Res. 108, 297-304; Moreno et al., 2012Moreno, S.C., Carvalho, G.A., Picanço, M.C., Morais, E.G.F., Pereira, R.M., 2012. Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species. Pest Manag. Sci. 68, 386-393; Nakatani and Nagashima, 1992Nakatani, N., Nagashima, M., 1992. Pungent alkamides from Spilanthes acmella L. var. oleracea Clarke. Biosci. Biotechnol. Biochem. 56, 759-762; Hajdu, 2014Hajdu, A., 2014. An Ethnopharmacological Survey Conducted in the Bolivian Amazon, and Identification of N-alkylamides and Lignans from Lepidium meyenii and Heliopsis helianthoides var. scabra with Effects on the Central Nervous System. University of Szeged, Szeged, Hungary.). In addition to its oral analgesic effect, it also has antibacterial effects (Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.). So, either spilanthol or extracts of plants that contain it may be added to toothpaste and used as an oral analgesic in gels (such as Buccaldol® and Indolphar®) and as an anti-wrinkle cream that can substitute for Botox in cosmetic applications (Demarne and Passaro, 2009Demarne, F., Passaro, G., 2009. Use of an Acmella oleracea extract for the botulinum toxin-like effect thereof in an anti-wrinkle cosmetic composition. US Patent No. 7,531,193 B2.; Veryser et al., 2014Veryser, L., Wynendaele, E., Taevernier, L., Verbeke, F., Joshi, T., Tatke, P., De Spiegeleer, B., 2014. N-alkylamides: from plant to brain. Func. Foods Health Dis. 4, 264-275). There are also some anti-aging products (Gatuline®, SYN®-COLL, ChroNOline™) that contain spilanthol. There are about 30 patents that describe products that are made from a variety of Splianthes species (Haw and Keng, 2003Haw, A.B., Keng, C.L., 2003. Micropropagation of Spilanthes acmella L., a bioinsecticide plant, through proliferation of multiple shoots. J. Appl. Hort. 5, 65-68). It is also eaten in foods. The leaves of some of the plants (like S. acmella) that contain spilanthol are used as a spice (Haw and Keng, 2003Haw, A.B., Keng, C.L., 2003. Micropropagation of Spilanthes acmella L., a bioinsecticide plant, through proliferation of multiple shoots. J. Appl. Hort. 5, 65-68; Paulraj et al., 2013Paulraj, J., Govindarajan, R., Palpu, P., 2013. The genus Spilanthes ethnopharmacology, phytochemistry, and pharmacological properties: a review. Adv. Pharmacol. Sci., http://dx.doi.org/10.1155/2013/510298
http://dx.doi.org/10.1155/2013/510298...
). The European Union estimated that the average daily intake of spilanthol was 24 µg/person/day (Veryser et al., 2014Veryser, L., Wynendaele, E., Taevernier, L., Verbeke, F., Joshi, T., Tatke, P., De Spiegeleer, B., 2014. N-alkylamides: from plant to brain. Func. Foods Health Dis. 4, 264-275). It is also possible that spilanthol, like other alkamides, can have important effects on the central nervous system (CNS) and immune system (Gertsch, 2008Gertsch, J., 2008. Immunomodulatory lipids in plants: plant fatty acid amides and the human endocannabinoid system. Planta Med. 74, 638-650; Hajdu, 2014Hajdu, A., 2014. An Ethnopharmacological Survey Conducted in the Bolivian Amazon, and Identification of N-alkylamides and Lignans from Lepidium meyenii and Heliopsis helianthoides var. scabra with Effects on the Central Nervous System. University of Szeged, Szeged, Hungary.; Veryser et al., 2014Veryser, L., Wynendaele, E., Taevernier, L., Verbeke, F., Joshi, T., Tatke, P., De Spiegeleer, B., 2014. N-alkylamides: from plant to brain. Func. Foods Health Dis. 4, 264-275). However, its greatest potential for saving lives and improving human health may be its ability to kill mosquitoes that can spread tropical diseases like malaria and dengue fever (Pandey et al., 2011Pandey, V., Chopra, M., Agrawal, V., 2011. In vitro isolation and characterization of biolarvicidal compounds from micropropogated plants of Spilanthes acmela . Parasitol. Res. 108, 297-304; Spelman et al., 2011Spelman, K., Depoix, D., McCray, M., Mouray, E., Grellier, P., 2011. The traditional medicine Spilanthes acmella, and the alkylamides spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, demonstrate in vitro and in vivo antimalarial activity. Phytother. Res. 25, 1098-1110; Hernández-Morales et al., 2015Hernández-Morales, A., Arvizu-Gómez, J.L., Carranza-Álvarez, C., Gómez-Luna, B.E., Alvardo-Sánchez, B., Ramirez-Chávez, E., Molina-Torres, J., 2015. Larvicidal activity of affinin and its derived amides from Heliopsis longipes A. Gray Blake against Anopheles albimanus and Aedes aegypti. J. Asia-Pacific Entomol. 18, 227–231.). Moreover, it has anti-cancer activity (Soares et al., 2014Soares, C.P., Lemos, V.R., da Silva, A.G., Campoy, R.M., da Silva, C.A.P., Menegon, R.F., Rojahn, I., Joaquim, W.M., 2014. Effect of Spilanthes acmella hydroethanolic extract activity on tumour cell actin cytoskeleton. Cell Biol. Int. 38, 131-135; Mishra et al., 2015Mishra, A., Roy, S., Maity, S., Yadav, R.K., Keshari, A.K., Saha, S., 2015. Antiproliferative effect of flower extracts of Spilanthes paniculata on hepatic carcinoma cells. Int. J. Pharm. Sci. 7, 130-134). So, the purposes of this review are to tell where spilanthol (1) can be found in nature, tell how it can be extracted, describe its chemistry and review its diverse health effects.

Occurrence

Spilanthol (or affinin) (1) can be found in not just Acmella oleracea, but also A. ciliate, A. oppositifolia, A. radicans, A. brachyglossa, A. ciliate, A. oleracea, A. paniculata, A. uliginosa, Welelia parviceps and Heliopsis longipes (Chung et al., 2008Chung, K.-F., Kono, Y., Wang, C.-M., Peng, C.I., 2008. Notes on Acmella (Asteraceae: Heliantheae) in Taiwan. Bot. Stud. 49, 73-82; Prachayasittukal et al., 2013Prachayasittukal, V., Prachayasittukal, S., Ruchiwarat, S., Prachayasittukal, V., 2013. High therapeutic potential of Spilanthes acmella: a review. EXCLI J. 12, 291-312). Many of the articles that describe its presence in H. longipes call it affinin instead of spilanthol (Johns et al., 1982Johns, T., Graham, K., Towers, G.H.N., 1982. Molluscicidal activity of affinin and other isobutylamides from the Asteraceae. Phytochemistry. 21, 2737-2738; Rios et al., 2007Rios, M.Y., Aguilar-Guadarrama, A.B., Gutierrez, M.D., 2007. Analgesic activity of affinin, an alkamide from Heliopsis longipes (Compositae). J. Ethnopharmacol. 110, 364-367; Spelman et al., 2011Spelman, K., Depoix, D., McCray, M., Mouray, E., Grellier, P., 2011. The traditional medicine Spilanthes acmella, and the alkylamides spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, demonstrate in vitro and in vivo antimalarial activity. Phytother. Res. 25, 1098-1110; Déciga-Campos et al., 2012Déciga-Campos, M., Arriaga-Alba, M., Ventura-Martínez, R., Aguilar-Guadarrama, B., Rios, M.Y., 2012. Pharmacological and toxicological profile of extract from Heliopsis longipes and affinin. Drug Dev. Res. 73, 130-137). On the other hand, there is some disagreement in the literature over the name of the genus and species of one of the most important plants that is said to contain spilanthol. Some call it A. oleracea (Moreno et al., 2012Moreno, S.C., Carvalho, G.A., Picanço, M.C., Morais, E.G.F., Pereira, R.M., 2012. Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species. Pest Manag. Sci. 68, 386-393; Simas et al., 2013Simas, N.K., Dellamora, E.C.L., Schripsema, J., Lage, C.L.S., Filho, A.M.O., Wessjohann, L., Porzel, A., Kuster, R.M., 2013. Acetylenic 2-phenylethylamides and new isobutylamides from Acmella oleracea (L.) R.K. Jansen, a Brazilian spice with larvacidal activity on Aedes aegypti . Phytochem. Lett. 6, 67-72; Abeysinghe et al., 2014Abeysinghe, D.C., Wijerathne, S.M.N.K., Dharmadasa, R.M., 2014. Secondary metabolites contents and antioxidant capacities of Acmella oleraceae grown under different growing systems. World J. Agric. Res. 2, 163-167; Castro et al., 2014)Castro, K.N.C., Lima, D.F., Vasconcelos, L.C., Leite, J.R.S.A., Santos, R.C., Neto, A.A.P., Costa-Júnior, L.M., 2014. Acaricide activity in vitro of Acmella oleracea against Rhipicephalus microplus . Parisitol. Res. 113, 3697-3701, but others call it A. oleracea (L.) R. K. Jansen (Simas et al., 2013Simas, N.K., Dellamora, E.C.L., Schripsema, J., Lage, C.L.S., Filho, A.M.O., Wessjohann, L., Porzel, A., Kuster, R.M., 2013. Acetylenic 2-phenylethylamides and new isobutylamides from Acmella oleracea (L.) R.K. Jansen, a Brazilian spice with larvacidal activity on Aedes aegypti . Phytochem. Lett. 6, 67-72; Soares et al., 2014Soares, C.P., Lemos, V.R., da Silva, A.G., Campoy, R.M., da Silva, C.A.P., Menegon, R.F., Rojahn, I., Joaquim, W.M., 2014. Effect of Spilanthes acmella hydroethanolic extract activity on tumour cell actin cytoskeleton. Cell Biol. Int. 38, 131-135; de Alcantara et al., 2014de Alcantara, B.N., Kobayashi, Y.T., Barroso, K.F., da Silva, I.D.R., de Almeida, M.B., Barbosa, W.L.M., 2014. Pharmacognistic analyses and evaluation of the in vitro antimicrobial activity of Acmella oleracea (L.) RK Jansen (Jambu) floral extracts and fractions. J. Med. Plant Res. 9, 91-96), A. oleracea Compositae (Hind and Biggs, 2003Hind, N., Biggs, N., 2003. Plate 460. Acmella oleracea compositae. Curtis's Bot. Mag. 20, 31-39), S. oleracea L. (Martins et al., 2012Martins, C.P.S., Melo, M.T.P., Honório, I.C.G., D’Ávila, V.A., Carvalho Júnior, W.G.O., 2012. Morphological and agronomic characterization of Jambu (Spilanthes oleracea L.) accessions under the conditions of North Minas Gerais State, Brazil. Rev. Bras. Plant. Med. 14, 410-413), S. acmella, (Chung et al., 2008Chung, K.-F., Kono, Y., Wang, C.-M., Peng, C.I., 2008. Notes on Acmella (Asteraceae: Heliantheae) in Taiwan. Bot. Stud. 49, 73-82; Demarne and Passaro, 2009Demarne, F., Passaro, G., 2009. Use of an Acmella oleracea extract for the botulinum toxin-like effect thereof in an anti-wrinkle cosmetic composition. US Patent No. 7,531,193 B2.; Mbeunkui et al., 2011Mbeunkui, F., Grace, M.H., Lategan, C., Smith, P.J., Raskin, I., Lila, M.A., 2011. Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS. J. Chromatogr. B. 879, 1886-1892; Pandey et al., 2011Pandey, V., Chopra, M., Agrawal, V., 2011. In vitro isolation and characterization of biolarvicidal compounds from micropropogated plants of Spilanthes acmela . Parasitol. Res. 108, 297-304; Prachayasittukal et al., 2013Prachayasittukal, V., Prachayasittukal, S., Ruchiwarat, S., Prachayasittukal, V., 2013. High therapeutic potential of Spilanthes acmella: a review. EXCLI J. 12, 291-312; Sana et al., 2014Sana, H., Rani, A.S., Sulakshana, G., 2014. Determination of antioxidant potential in Spilanthes acmella using DPPH assay. Int. J. Curr. Microbiol. Appl. Sci. 3, 219-223; Soares et al., 2014Soares, C.P., Lemos, V.R., da Silva, A.G., Campoy, R.M., da Silva, C.A.P., Menegon, R.F., Rojahn, I., Joaquim, W.M., 2014. Effect of Spilanthes acmella hydroethanolic extract activity on tumour cell actin cytoskeleton. Cell Biol. Int. 38, 131-135; Mishra et al., 2015Mishra, A., Roy, S., Maity, S., Yadav, R.K., Keshari, A.K., Saha, S., 2015. Antiproliferative effect of flower extracts of Spilanthes paniculata on hepatic carcinoma cells. Int. J. Pharm. Sci. 7, 130-134), S. acmella L. var. oleracea Clarke (Nakatani and Nagashima, 1992Nakatani, N., Nagashima, M., 1992. Pungent alkamides from Spilanthes acmella L. var. oleracea Clarke. Biosci. Biotechnol. Biochem. 56, 759-762) and S. acmella Murr. (Asteraceae) (Singh and Chaturvedi, 2012a,b; Abeysiri et al., 2013). At least one article stated that the flower head of S. acmella L. var. oleracea Clarke are yellow, but those of S. acmella are purple (Nakatani and Nagashima, 1992Nakatani, N., Nagashima, M., 1992. Pungent alkamides from Spilanthes acmella L. var. oleracea Clarke. Biosci. Biotechnol. Biochem. 56, 759-762). To add to the confusion, one review article on the genus Spilanthes Jacq stated that “The genus is often confused with the genus Acmella Rich. Ex Pers.”, “Spilanthes species have discoid heads and Acmella species have rayed heads”, and “Spilanthes has a chromosome number of 16, whereas Acmella has 12 or 13” (Paulraj et al., 2013Paulraj, J., Govindarajan, R., Palpu, P., 2013. The genus Spilanthes ethnopharmacology, phytochemistry, and pharmacological properties: a review. Adv. Pharmacol. Sci., http://dx.doi.org/10.1155/2013/510298
http://dx.doi.org/10.1155/2013/510298...
). In complete contrast, another author reported that the inflorescences of Acmella oleracea (L.) R.K. Jansen have discoid heads and a chromosome number of 2n = 68 or 70 (Grubben and Denton, 2004Grubben, G.J.H., Denton, O.A. (Eds.), 2004. Acmella oleracea (L.) R.K. Jansen in Plant Resources of Tropical Africa 2. Vegetables. Grubbe, Backhuys Publishers, Wageningen, NL, p. 35.). Monographs have been written about each genus (Acmella and Spilanthes) (Jansen, 1981Jansen, R.K., 1981. The systematics of Spilanthes (Compositae: Heliantheae) system. Botany. 6, 231-257, 1985Jansen, R.K., 1985. The systematics of Acmella (Asteraceae: Heliantheae) system. Botany. 8, 1-115), but the “toothache plant” was placed in the Acmella genus (Jansen, 1985Jansen, R.K., 1985. The systematics of Acmella (Asteraceae: Heliantheae) system. Botany. 8, 1-115). Some of its common names include jambu, agrião do Pará and paracress (Jansen, 1985Jansen, R.K., 1985. The systematics of Acmella (Asteraceae: Heliantheae) system. Botany. 8, 1-115). The monograph on Acmella warned of false synonyms for A. oleracea that appear on various websites. Some of them state that the “accepted scientific name” is Spilanthes acmella (L.) Murr., but the photos on them clearly show A. oleracea (Jansen, 1985Jansen, R.K., 1985. The systematics of Acmella (Asteraceae: Heliantheae) system. Botany. 8, 1-115). This monograph also stated that the “currently accepted name” for Spilantes acmella (L.) Murr. is Blainvillea acmella (L.) Philipson (Jansen, 1985Jansen, R.K., 1985. The systematics of Acmella (Asteraceae: Heliantheae) system. Botany. 8, 1-115). There is another article that talks about a Mexican plant that they called Acmella (Spilanthes) oppositifolia, while the Nahuatl name was chilcuage (Molinatorres et al., 1996Molinatorres, J., Salgado-Garciglia, R., Ramirez-Chanez, E., del Rio, R.E., 1996. Purely olefinic alkamides in Heliopsis longipes and Acmella (Spilanthes) oppositifolia . Biochem. Syst. Ecol. 24, 27-43). There are also five different species of Acmella in Taiwan that contain spilanthol (Chung et al., 2008Chung, K.-F., Kono, Y., Wang, C.-M., Peng, C.I., 2008. Notes on Acmella (Asteraceae: Heliantheae) in Taiwan. Bot. Stud. 49, 73-82). Finally, there is an article that lists S. acmella and S. oleraceae as being two separate plants (Tiwari et al., 2011Tiwari, K.L., Jadhav, S.K., Joshi, V., 2011. An updated review on medicinal herb Genus Spilanthes . J. Chin. Integr. Med. 9, 1170-1178). Other synonyms include A. ciliata Kunth, Cotula pyretharia L., S. fusca Mart, Bidens fervida Lan and A. uliginosa (Sw.) Cass (Borges, 2009Borges, L.D.S., 2009. Biomassa, teores de nutrients, espinantol e atividade antioxidante em plantas de jambu (Acmella ciliate Knuth) sob abubações mineral e orgânica. UNESP, Botucatu, Brazil.; Costa et al., 2013Costa, S.S., Arumugam, D., Garipey, Y., Rocha, S.C.S., Raghaven, V., 2013. Spilanthol extraction using microwave: calibration curve for gas chromatography. Chem. Eng. Trans. 32, 1783-1788).

Extraction, purification and quantitation

Since spilanthol (1) is amphiphilic, it can be extracted from plants using solvents that range in polarity from hexane (Ramsewak et al., 1999Ramsewak, R.S., Erickson, A.J., Nair, M.G., 1999. Bioactive N-isobutylamides from the flower buds of Spilanthes Acmella . Phytochemistry. 51, 729-732) to methanol:H2O (4:1, v/v) (Abeysinghe et al., 2014Abeysinghe, D.C., Wijerathne, S.M.N.K., Dharmadasa, R.M., 2014. Secondary metabolites contents and antioxidant capacities of Acmella oleraceae grown under different growing systems. World J. Agric. Res. 2, 163-167). There is also an ethanolic extract that is sold in pharmacies (Boonen et al., 2010aBoonen, J., Baert, B., Roche, N., Burvenich, C., de Spiegeleer, B., 2010. Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilantes acmella (Compositae) extracts. J. Ethnopharmacol. 127, 77-84,bBoonen, J., Baert, B., Burvenich, C., Blondeel, P., de Saeget, S., de Spiegeleer, B., 2010. LC–MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behavior of is main bioactive spilanthol. J. Pharm. Biomed. Anal. 53, 243-249). However, to the best of our knowledge no attempt has been made to compare the amount of spilanthol that can be extracted using different methods. Moreover, nobody has ever tried using pressurized liquid extraction with dry methanol, which has been shown to be able to solubilize more material from many fruits and vegetables than other methods, including Soxhlet extraction or ultrasonication (Richter et al., 1996Richter, B.E., Jones, B.A., Ezzell, J.L., Porter, N.L., 1996. Accelerated solvent extraction: a technique for sample preparation. Anal. Chem. 68, 1033-1039; Richards et al., 2014Richards, K.M., Tran, K., Levine, R.A., Luo, R., Maia, G.M., Sabaa-Srur, A.U.O., Maciel, M.I.S., Melo, E.A., de Moraes, M.R., Godoy, H.T., Chaves, M.A., do Sacramento, C.K., Thomas, A.L., Smith, R.E., 2014. Improved extraction of soluble solids from fruits. Nat. Prod. J. 4, 201-210; Levine et al., 2015Levine, R.A., Richards, K.M., Tran, K., Luo, R., Thomas, A.L., Smith, R.E., 2015. Determination of neurotoxic acetogenins in pawpaw (Asimina triloba) fruit by LC–HRMS. J. Agric. Food Chem. 63, 1053-1056). However, some of the previous publications do tell how much material was solubilized. For example, hexane at an unspecified temperature was able to solubilize 10 g of material from 1130 g of lyophilized flowers (Ramsewak et al., 1999Ramsewak, R.S., Erickson, A.J., Nair, M.G., 1999. Bioactive N-isobutylamides from the flower buds of Spilanthes Acmella . Phytochemistry. 51, 729-732). Others used ultrasonication with 60 ml of ethanol:hexane (3:7, v/v) at 50 °C and 30 min to solubilize an unspecified amount of material from 2 g of dried flowers Costa et al., 2013Costa, S.S., Arumugam, D., Garipey, Y., Rocha, S.C.S., Raghaven, V., 2013. Spilanthol extraction using microwave: calibration curve for gas chromatography. Chem. Eng. Trans. 32, 1783-1788). Another group used an unknown amount of ethanol at room temperature to solubilize 106 g (13%) of material from 803 g of dried leaves (Simas et al., 2013Simas, N.K., Dellamora, E.C.L., Schripsema, J., Lage, C.L.S., Filho, A.M.O., Wessjohann, L., Porzel, A., Kuster, R.M., 2013. Acetylenic 2-phenylethylamides and new isobutylamides from Acmella oleracea (L.) R.K. Jansen, a Brazilian spice with larvacidal activity on Aedes aegypti . Phytochem. Lett. 6, 67-72). Others solubilized 15 g from 300 g of flowers using methanol at room temperature (Mbeunkui et al., 2011Mbeunkui, F., Grace, M.H., Lategan, C., Smith, P.J., Raskin, I., Lila, M.A., 2011. Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS. J. Chromatogr. B. 879, 1886-1892). Another group used methanol to solubilize 18.0, 16.6 and 10.2% of the material from dry leaves, stems and flowers, respectively (Abeysiri et al., 2013Abeysiri, G.R.P.I., Dharmadasa, R.M., Abeysinghe, D.C., Samarasinghe, K., 2013. Screening of phytochemical, physico-chemical and bioactivity of different parts of Spilantes acmella Murr. (Asteraceae), a natural remedy for toothache. Ind. Crop. Prod. 50, 852-856). Still others used 2.5 l of ethanol:water (7:3, v/v) to solubilize an unknown amount of material from 426 g of dried flowers (Martins et al., 2012Martins, C.P.S., Melo, M.T.P., Honório, I.C.G., D’Ávila, V.A., Carvalho Júnior, W.G.O., 2012. Morphological and agronomic characterization of Jambu (Spilanthes oleracea L.) accessions under the conditions of North Minas Gerais State, Brazil. Rev. Bras. Plant. Med. 14, 410-413).

Supercritical CO2 with added ethanol and water was also used to try to extract spilanthol from S. acmella flowers, leaves and stems (Dias et al., 2012Dias, A.M.A., Santos, P., Seabraa, I.J., Junior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J. Supercrit. Fluids. 61, 62-70). It was purified from an ethanolic extract using TLC using silica gel plates and hexane:ethyl acetate (2:1, v/v) as the mobile phase (Dias et al., 2012Dias, A.M.A., Santos, P., Seabraa, I.J., Junior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J. Supercrit. Fluids. 61, 62-70). TLC was also used to purify spilanthol from dry A. oleracea flowers that was first extracted with ultrasonication and ethanol:hexane (3:7, v/v) at 50 °C and 30 min (Costa et al., 2013Costa, S.S., Arumugam, D., Garipey, Y., Rocha, S.C.S., Raghaven, V., 2013. Spilanthol extraction using microwave: calibration curve for gas chromatography. Chem. Eng. Trans. 32, 1783-1788). Others used TLC followed by preparative scale HPLC to purify spilanthol from hexane extracts of flowers (Nakatani and Nagashima, 1992Nakatani, N., Nagashima, M., 1992. Pungent alkamides from Spilanthes acmella L. var. oleracea Clarke. Biosci. Biotechnol. Biochem. 56, 759-762). Another group used two preparative scale columns (XAD-16 and Sephadex LH-20) followed by preparative scale TLC to purify spilanthol from leaves (Simas et al., 2013Simas, N.K., Dellamora, E.C.L., Schripsema, J., Lage, C.L.S., Filho, A.M.O., Wessjohann, L., Porzel, A., Kuster, R.M., 2013. Acetylenic 2-phenylethylamides and new isobutylamides from Acmella oleracea (L.) R.K. Jansen, a Brazilian spice with larvacidal activity on Aedes aegypti . Phytochem. Lett. 6, 67-72). Another approach that proved successful was column chromatography on silica gel, followed by TLC (Ramsewak et al., 1999Ramsewak, R.S., Erickson, A.J., Nair, M.G., 1999. Bioactive N-isobutylamides from the flower buds of Spilanthes Acmella . Phytochemistry. 51, 729-732). Finally, centrifugal partition chromatography using a mixture of heptane, ethyl acetate, methanol and water (3:2:3:2, v/v) was used to purify spilanthol (Mbeunkui et al., 2011Mbeunkui, F., Grace, M.H., Lategan, C., Smith, P.J., Raskin, I., Lila, M.A., 2011. Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS. J. Chromatogr. B. 879, 1886-1892).

For quantitation, both HPLC with UV detection and LC–MS have been used (Bae et al., 2010Bae, S.S., Ehrmann, B.M., Ettefagh, K.A., Cech, N.B., 2010. A validated liquid chromatography–electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem. Anal. 5, 438-443; Sharma et al., 2011Sharma, V., Boonen, J., Chauhan, N.S., Thakur, M., de Spiegeleer, B., Dixit, V.K., 2011. Spilanthes acmella ethanolic flower extract: LC–MS alkylamide profiling and its effects on sexual behavior in male rats. Phytomedicine. 18, 1161-1168; Singh and Chaturvedi, 2012aSingh, M., Chaturvedi, R., 2012. Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilantes acmella Murr. Ind. Crop Prod. 36, 321-328,bSingh, M., Chaturvedi, R., 2012. Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthese acmella Murr. Bioprocess Biosyst. Eng. 35, 943-951). Both methods used a C18 column for the separation. One HPLC method used an isocratic mobile phase consisting of 93:7 CH3CN:H2O (v/v), flowing at 0.5 ml/min (Singh and Chaturvedi, 2012aSingh, M., Chaturvedi, R., 2012. Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilantes acmella Murr. Ind. Crop Prod. 36, 321-328,bSingh, M., Chaturvedi, R., 2012. Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthese acmella Murr. Bioprocess Biosyst. Eng. 35, 943-951). The retention time for spilanthol was 7.34 min (Prachayasittukal et al., 2013Prachayasittukal, V., Prachayasittukal, S., Ruchiwarat, S., Prachayasittukal, V., 2013. High therapeutic potential of Spilanthes acmella: a review. EXCLI J. 12, 291-312). Another HPLC method used isocratic elution with CH3CN:H2O (1:1, v/v) flowing at 0.2 min (Bae et al., 2010Bae, S.S., Ehrmann, B.M., Ettefagh, K.A., Cech, N.B., 2010. A validated liquid chromatography–electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem. Anal. 5, 438-443). The retention time was 4.97 min (Bae et al., 2010Bae, S.S., Ehrmann, B.M., Ettefagh, K.A., Cech, N.B., 2010. A validated liquid chromatography–electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem. Anal. 5, 438-443). One LC–MS method used a gradient elution that started with 1:4 CH3CN:H2O (v/v), containing 1% acetic acid and increased to 9:1 CH3CN:H2O (v/v) over 150 min (Sharma et al., 2011Sharma, V., Boonen, J., Chauhan, N.S., Thakur, M., de Spiegeleer, B., Dixit, V.K., 2011. Spilanthes acmella ethanolic flower extract: LC–MS alkylamide profiling and its effects on sexual behavior in male rats. Phytomedicine. 18, 1161-1168). The retention time of spilanthol was 62.37 min (Sharma et al., 2011Sharma, V., Boonen, J., Chauhan, N.S., Thakur, M., de Spiegeleer, B., Dixit, V.K., 2011. Spilanthes acmella ethanolic flower extract: LC–MS alkylamide profiling and its effects on sexual behavior in male rats. Phytomedicine. 18, 1161-1168). The other LC–MS method was validated for quantifying spilanthol in a mixture of unspecified amounts of leaves, flower buds and roots, which were extracted with ethanol:water (19:1, v/v) at room temperature (Bae et al., 2010Bae, S.S., Ehrmann, B.M., Ettefagh, K.A., Cech, N.B., 2010. A validated liquid chromatography–electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem. Anal. 5, 438-443). The combined peak areas due to the [M+H]+ and [2M+H]+ ions with m/z of 222 and 443 were used for quantitation (Bae et al., 2010Bae, S.S., Ehrmann, B.M., Ettefagh, K.A., Cech, N.B., 2010. A validated liquid chromatography–electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem. Anal. 5, 438-443). In addition, fragment ions with m/z of 123, 81, 121, 67 and 149 were also seen. However, the method was validated by simply analyzing spilanthol standards dissolved in an unspecified solvent, showing that a linear calibration curve could be obtained and by testing the repeatability of the analysis of standards. Recoveries of spilanthol that were added to the samples (spiked samples) were not measured. It is also quite likely that the method was not used to actually quantify spilanthol in any samples. There is a table that showed the spilanthol concentrations that were found in extracts of the plant that they called S. acmella but the results were expressed as mg/ml, as if they were concentrations of standards dissolved in solvents. There was no mention of concentrations of spilanthol in units of µg spilanthol per mg of sample (Bae et al., 2010Bae, S.S., Ehrmann, B.M., Ettefagh, K.A., Cech, N.B., 2010. A validated liquid chromatography–electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr. Phytochem. Anal. 5, 438-443). However, a method based on HPLC with UV detection at 237 nm was used to find 3294 µg/g spilanthol per dry weight in the leaves of in vitro plants and 2704 µg/g dry leaves in the leaves of in vivo plants (Singh and Chaturvedi, 2012aSingh, M., Chaturvedi, R., 2012. Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilantes acmella Murr. Ind. Crop Prod. 36, 321-328,bSingh, M., Chaturvedi, R., 2012. Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthese acmella Murr. Bioprocess Biosyst. Eng. 35, 943-951). However, no attempt was made to compare the amount of spilanthol that could be extracted using pressurized liquid extraction, sonication or Soxhlet extraction. It is also quite likely that the concentration of spilanthol is different in different parts of the plant. So, there is clearly a need for an analysis of different parts of genuine A. oleracea.

Chemistry

Spilanthol (1) is an N-alkylamide, many of which have various bioactivities, from helping to protect plants to being an antibacterial, antifungal, analgesic and endocannabinoid agonists (Veryser et al., 2014Veryser, L., Wynendaele, E., Taevernier, L., Verbeke, F., Joshi, T., Tatke, P., De Spiegeleer, B., 2014. N-alkylamides: from plant to brain. Func. Foods Health Dis. 4, 264-275). One article reported that there over 200 alkamides have been found in ten families: Aristolochiaceae, Asteraceae, Brassicaceae, Convolvulaceae, Euphorbiacea, Menispermaceae, Piperaceae, Poaceae, Rutaceae and Solanaceae (Molina-Torres et al., 2004Molina-Torres, J., Salazar-Cabrera, C.J., Armenta-Salinas, C., Ramírez-Sánchez, E., 2004. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduces amides. J. Agric. Food Chem. 52, 4700-4704). Another group reported that over 400 N-alkylamides have been identified in 26 different plant families (Gertsch, 2008Gertsch, J., 2008. Immunomodulatory lipids in plants: plant fatty acid amides and the human endocannabinoid system. Planta Med. 74, 638-650). There is also an alkamide database that has more details in it (Boonen et al., 2012Boonen, J., Bronselaer, A., Nielandt, J., Veryser, L., De Tré, G., De Spiegeleer, B., 2012. Alkamid database: chemistry, occurrence and functionality of plant N-alkylamides. J. Ethnopharmacol. 142, 563-590).

The stereoselective synthesis of spilanthol with a 61% yield has been reported (Ikeda et al., 1984Ikeda, Y., Ukai, J., Ikeda, N., Yamamoto, H., 1984. Facile routes to natural acyclic polyenes syntheses the spilanthol and trail pheromone for termite. Tetrahedron Lett. 25, 5177-5180). It is light yellow with a melting point of 23 °C, a boiling point of 165 °C, a refractive index at 298 °C of 1.5135 and a maximum UV absorption at 228.5 nm (Jacobson, 1957Jacobson, M., 1957. The structure of spilanthol. Chem. Ind. 2, 50-55). Its IR spectrum was reported as having the following major peaks: νmax (film) cm−1: 3340, 3150, 3080, 3020, 1678, 1636, 1550, 1240, 1160, 987, 953 (Nakatani and Nagashima, 1992Nakatani, N., Nagashima, M., 1992. Pungent alkamides from Spilanthes acmella L. var. oleracea Clarke. Biosci. Biotechnol. Biochem. 56, 759-762). It has a monoisotopic molecular weight of 221.177963 Da. So, the positive ion mass spectrum contains a molecular ion [M+H]+m/z = 222 and a fragment [MH−C4H11N]+ with m/z = 149 (loss of isobutyl amine group) as well as a fragment with m/z = 99, that showed the presence of an isobutylamide (Jacobson, 1957Jacobson, M., 1957. The structure of spilanthol. Chem. Ind. 2, 50-55). Its 1H and 13C NMR spectra have been reported (Nakatani and Nagashima, 1992Nakatani, N., Nagashima, M., 1992. Pungent alkamides from Spilanthes acmella L. var. oleracea Clarke. Biosci. Biotechnol. Biochem. 56, 759-762). Chemical shifts are listed in Table 1.

Table 1
1H and 13C NMR chemical shifts (ppm) of spilanthol ( 1 ) in CDCl3 ( Nakatani and Nagashima, 1992Nakatani, N., Nagashima, M., 1992. Pungent alkamides from Spilanthes acmella L. var. oleracea Clarke. Biosci. Biotechnol. Biochem. 56, 759-762 ).

The parts of spilanthol that are important for its analgesic activity, tingling and mouth-watering effects (pharmacophores) are the amide and unsaturated (alkenyl) fatty acyl (Ley et al., 2006Ley, J.P., Krammer, G., Looft, J., Reinders, G., Bertram, H., 2006. Structure–activity relationships of trigeminal effects for artificial and naturally occurring alkamides related to spilanthol. Dev. Food Sci. 43, 21-24; Rios and Olivo, 2014Rios, M.Y., Aguilar-Guadarrama, A.B., Gutierrez, M.D., 2007. Analgesic activity of affinin, an alkamide from Heliopsis longipes (Compositae). J. Ethnopharmacol. 110, 364-367).

Biological activities

Spilanthol has many biological activities (Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.), including analgesic (Molinatorres et al., 1996Molinatorres, J., Salgado-Garciglia, R., Ramirez-Chanez, E., del Rio, R.E., 1996. Purely olefinic alkamides in Heliopsis longipes and Acmella (Spilanthes) oppositifolia . Biochem. Syst. Ecol. 24, 27-43; Hind and Biggs, 2003Hind, N., Biggs, N., 2003. Plate 460. Acmella oleracea compositae. Curtis's Bot. Mag. 20, 31-39; Wu et al., 2008Wu, L.-C., Fan, N.C., Lin, M-H., Chu, I.-R., Huang, S.J., Han, S.Y., 2008. Anti-inflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators. J. Agric. Food Chem. 56, 2341-2349; Cilia-López et al., 2010Cilia-López, V.G., Juárez-Flores, B.I., Aguirre-Rivera, J.R., Reyes-Aguero, J.A., 2010. Analgesic activity of Heliopsis longipes and its effect on the nervous system. Pharm. Biol. 48, 195-200; Tiwari et al., 2011Tiwari, K.L., Jadhav, S.K., Joshi, V., 2011. An updated review on medicinal herb Genus Spilanthes . J. Chin. Integr. Med. 9, 1170-1178; Dias et al., 2012Dias, A.M.A., Santos, P., Seabraa, I.J., Junior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J. Supercrit. Fluids. 61, 62-70; Sharma et al., 2012Sharma, A., Kumar, V., Rattan, R.S., Kumar, N., Singh, B., 2012. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. Against Plutella xylostella L. Am. J. Plant Sci. 3, 1568-1572; Abeysiri et al., 2013Abeysiri, G.R.P.I., Dharmadasa, R.M., Abeysinghe, D.C., Samarasinghe, K., 2013. Screening of phytochemical, physico-chemical and bioactivity of different parts of Spilantes acmella Murr. (Asteraceae), a natural remedy for toothache. Ind. Crop. Prod. 50, 852-856; Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.; Prachayasittukal et al., 2013Prachayasittukal, V., Prachayasittukal, S., Ruchiwarat, S., Prachayasittukal, V., 2013. High therapeutic potential of Spilanthes acmella: a review. EXCLI J. 12, 291-312; Paulraj et al., 2013Paulraj, J., Govindarajan, R., Palpu, P., 2013. The genus Spilanthes ethnopharmacology, phytochemistry, and pharmacological properties: a review. Adv. Pharmacol. Sci., http://dx.doi.org/10.1155/2013/510298
http://dx.doi.org/10.1155/2013/510298...
; Rios and Olivo, 2014Rios, M.R., Olivo, H.F., 2014. Natural and synthetic alkylamides: applications in pain therapy. In: Atta-Ur-Rahman (Ed.), Studies in Natural Products Chemistry. Else-vier, New York, pp. 79–118.; Dandin et al., 2014Dandin, V.S., Naik, P.M., Murthy, H.M., Park, S.Y., Paek, K.Y., 2014. Rapid regeneration and analysis of genetic fidelity and scopoletin contents of micropropagated plants of Spilanthes oleracea L. J. Hort. Sci. Biotechnol. 89, 79-85; Hajdu, 2014Hajdu, A., 2014. An Ethnopharmacological Survey Conducted in the Bolivian Amazon, and Identification of N-alkylamides and Lignans from Lepidium meyenii and Heliopsis helianthoides var. scabra with Effects on the Central Nervous System. University of Szeged, Szeged, Hungary.), antinociceptive (Rios et al., 2007Rios, M.Y., Aguilar-Guadarrama, A.B., Gutierrez, M.D., 2007. Analgesic activity of affinin, an alkamide from Heliopsis longipes (Compositae). J. Ethnopharmacol. 110, 364-367; Déciga-Campos et al., 2012Déciga-Campos, M., Arriaga-Alba, M., Ventura-Martínez, R., Aguilar-Guadarrama, B., Rios, M.Y., 2012. Pharmacological and toxicological profile of extract from Heliopsis longipes and affinin. Drug Dev. Res. 73, 130-137), antioxidant (Abeysiri et al., 2013Abeysiri, G.R.P.I., Dharmadasa, R.M., Abeysinghe, D.C., Samarasinghe, K., 2013. Screening of phytochemical, physico-chemical and bioactivity of different parts of Spilantes acmella Murr. (Asteraceae), a natural remedy for toothache. Ind. Crop. Prod. 50, 852-856), anti-inflammatory (Wu et al., 2008Wu, L.-C., Fan, N.C., Lin, M-H., Chu, I.-R., Huang, S.J., Han, S.Y., 2008. Anti-inflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators. J. Agric. Food Chem. 56, 2341-2349; Hernández et al., 2009Hernández, I., Márquez, L., Martínez, I., Dieguez, R., Delporte, C., Prietoa, S., Molina-Torres, J., Garrido, G., 2009. Anti-inflammatory effects of ethanolic extract and alkamides-derived from Heliopsis longipes roots. J. Ethnopharmacol. 124, 649-652; Dias et al., 2012Dias, A.M.A., Santos, P., Seabraa, I.J., Junior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J. Supercrit. Fluids. 61, 62-70), antimutagenic (Arriaga-Alba et al., 2013Arriaga-Alba, M., Rios, M.Y., Déciga-Campos, M., 2013. Antimutagenic properties of affinin isolated from Heliopsis longipes extract. Pharm. Biol. 51, 1035-1039), anti-wrinkle (Demarne and Passaro, 2009Demarne, F., Passaro, G., 2009. Use of an Acmella oleracea extract for the botulinum toxin-like effect thereof in an anti-wrinkle cosmetic composition. US Patent No. 7,531,193 B2.), antifungal (Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.), bacteriostatic (Molina-Torres et al., 2004Molina-Torres, J., Salazar-Cabrera, C.J., Armenta-Salinas, C., Ramírez-Sánchez, E., 2004. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduces amides. J. Agric. Food Chem. 52, 4700-4704), insecticidal (Kadir et al., 1989Kadir, H.A., Zakaria, M.B., Kechil, A.A., Azirun, M.S., 1989. Toxicity and electrophysiological effects of Spilanthes acmella Murr. extracts on Periplaneta americana L. Pest. Sci. 25, 329-335; Sharma et al., 2012Sharma, A., Kumar, V., Rattan, R.S., Kumar, N., Singh, B., 2012. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. Against Plutella xylostella L. Am. J. Plant Sci. 3, 1568-1572; Moreno et al., 2012Moreno, S.C., Carvalho, G.A., Picanço, M.C., Morais, E.G.F., Pereira, R.M., 2012. Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species. Pest Manag. Sci. 68, 386-393), anti-malarial (Sharma et al., 2012Sharma, A., Kumar, V., Rattan, R.S., Kumar, N., Singh, B., 2012. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. Against Plutella xylostella L. Am. J. Plant Sci. 3, 1568-1572), anti-larvicidal activities against Aedes aegypti and Helicoverpa zea neonates (Ramsewak et al., 1999Ramsewak, R.S., Erickson, A.J., Nair, M.G., 1999. Bioactive N-isobutylamides from the flower buds of Spilanthes Acmella . Phytochemistry. 51, 729-732), and anti-molluscicidal activities (Johns et al., 1982Johns, T., Graham, K., Towers, G.H.N., 1982. Molluscicidal activity of affinin and other isobutylamides from the Asteraceae. Phytochemistry. 21, 2737-2738). There have also been reports on its activities as an anticonvulsant, antioxidant, aphrodisiac, pancreatic lipase inhibitor, antimicrobial agent, antinociceptive agent, diuretic, vasorelaxant, anti-human immunodeficiency virus, toothache relief and as an anti-inflammatory agent (Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.). It can be absorbed through the skin, endothelial gut, oral mucosa and blood–brain barrier (Boonen et al., 2010aBoonen, J., Baert, B., Roche, N., Burvenich, C., de Spiegeleer, B., 2010. Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilantes acmella (Compositae) extracts. J. Ethnopharmacol. 127, 77-84,bBoonen, J., Baert, B., Burvenich, C., Blondeel, P., de Saeget, S., de Spiegeleer, B., 2010. LC–MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behavior of is main bioactive spilanthol. J. Pharm. Biomed. Anal. 53, 243-249; Veryser et al., 2014Veryser, L., Wynendaele, E., Taevernier, L., Verbeke, F., Joshi, T., Tatke, P., De Spiegeleer, B., 2014. N-alkylamides: from plant to brain. Func. Foods Health Dis. 4, 264-275). It can enhance the ability of caffeine, fortestosterone and five mycotoxins to penetrate the skin (De Spiegeleer et al., 2013De Spiegeleer, B., Boonen, J., Malysheva, S.V., Di Mavungu, J.D., De Saeger, S., Roche, N., Blondeel, P., Taevernier, L., Veryser, L., 2013. Skin penetration enhancing properties of the plant N-alkylamide spilanthol. J. Ethnopharmacol. 148, 117-125). So, it is important to make sure that formulations containing spilanthol are not contaminated with mycotoxins (De Spiegeleer et al., 2013De Spiegeleer, B., Boonen, J., Malysheva, S.V., Di Mavungu, J.D., De Saeger, S., Roche, N., Blondeel, P., Taevernier, L., Veryser, L., 2013. Skin penetration enhancing properties of the plant N-alkylamide spilanthol. J. Ethnopharmacol. 148, 117-125). It also improved male sexual performance in rats as indicated by penile erection, mounting frequency, intromission frequency, ejaculation frequency that lasted even 14 days after discontinuing its administration (Sharma et al., 2011Sharma, V., Boonen, J., Chauhan, N.S., Thakur, M., de Spiegeleer, B., Dixit, V.K., 2011. Spilanthes acmella ethanolic flower extract: LC–MS alkylamide profiling and its effects on sexual behavior in male rats. Phytomedicine. 18, 1161-1168).

The antinociceptive activity of spilanthol was studied in detail (Déciga-Campos et al., 2010Déciga-Campos, M., Rios, M.Y., Aguilar-Guadarrama, A.B., 2010. Antinociceptive effect of Heliopsis longipes extract and affinin in mice. Planta Med. 76, 665-670). Intraperitoneal administration of 30 mg/kg spilanthol produced an antinociceptive dependent-dose effect when assessed in mice submitted to acetic acid and capsaicin tests. Spilanthol-induced antinociception was blocked by naltrexone, p-chlorophenylalanine and flumazenil. So, its antinociceptive effect may be due to the activation of opiodergic, serotoninergic and GABAergic systems. Moreover, the antinociceptive effect decreased when mice were pretreated with 1H-[1,2,4]oxadiazolo[1,2-a]quinoxalin-1-one and glibenclamide. This supports the idea that the nitric oxide-K+ channels pathway could be involved in the mechanism of action (Déciga-Campos et al., 2010Déciga-Campos, M., Rios, M.Y., Aguilar-Guadarrama, A.B., 2010. Antinociceptive effect of Heliopsis longipes extract and affinin in mice. Planta Med. 76, 665-670). Subsequently, the same group found that spilanthol not only had a antinociceptive effect, but it also modified anxiety behavior and prolonged the time of sodium pentobarbital-induced hypnosis. They also found that spilanthol decreased the time of clonic and tonic seizures that were induced by pentylenetetrazole (PTZ) (Déciga-Campos et al., 2012Déciga-Campos, M., Arriaga-Alba, M., Ventura-Martínez, R., Aguilar-Guadarrama, B., Rios, M.Y., 2012. Pharmacological and toxicological profile of extract from Heliopsis longipes and affinin. Drug Dev. Res. 73, 130-137).

Analgesic activity was studied by evaluating the inhibition of acetic acid induced writhing in mice (Ogura et al., 1982Ogura, M., Cordell, G.A., Quinn, M.L., Leon, C., Benoit, P.S., Soejarto, D.D., Farnsworth, N.R., 1982. Ethnopharmacology studies. I. Rapid solution to a problem – oral use of Heliopsis longipes – by means of a multidisciplinary approach. J. Ethnopharmacol. 5, 215-219). Spilanthol was administered orally in aqueous solutions at doses ranging from 2.5 to 10.0 mg/kg. It exhibited an ED50 of 6.98 mg/kg. The analgesic activity of spilanthol was attributed to increased GABA release in the temporal cerebral cortex (Ogura et al., 1982Ogura, M., Cordell, G.A., Quinn, M.L., Leon, C., Benoit, P.S., Soejarto, D.D., Farnsworth, N.R., 1982. Ethnopharmacology studies. I. Rapid solution to a problem – oral use of Heliopsis longipes – by means of a multidisciplinary approach. J. Ethnopharmacol. 5, 215-219). In another study, spilanthol caused GABA to be released 0.5 min after being administered at a concentration of 1 × 10−4 M. One other study found that spilanthol displayed analgesic action similar to ketorolac (Cilia-López et al., 2010Cilia-López, V.G., Juárez-Flores, B.I., Aguirre-Rivera, J.R., Reyes-Aguero, J.A., 2010. Analgesic activity of Heliopsis longipes and its effect on the nervous system. Pharm. Biol. 48, 195-200). Also, its stimulating effect on the nervous system of adult mice was comparable to caffeine (Cilia-López et al., 2010Cilia-López, V.G., Juárez-Flores, B.I., Aguirre-Rivera, J.R., Reyes-Aguero, J.A., 2010. Analgesic activity of Heliopsis longipes and its effect on the nervous system. Pharm. Biol. 48, 195-200).

The antimutagenic activity of spilanthol was demonstrated by its ability to reduce 2AA- and NOR-induced mutations inTA98 and TA102 strains of Salmonella Typhimurium (Arriaga-Alba et al., 2013Arriaga-Alba, M., Rios, M.Y., Déciga-Campos, M., 2013. Antimutagenic properties of affinin isolated from Heliopsis longipes extract. Pharm. Biol. 51, 1035-1039). Spilanthol (25 and 50 µg/plate) significantly reduced the frameshift mutations that were generated by 2-aminoanthracene (2AA) (40%) and reduced the oxidative DNA damage generated by norfloxacin (NOR) (37–50%) (Arriaga-Alba et al., 2013Arriaga-Alba, M., Rios, M.Y., Déciga-Campos, M., 2013. Antimutagenic properties of affinin isolated from Heliopsis longipes extract. Pharm. Biol. 51, 1035-1039).

The antioxidant power of spilanthol and extracts of A. oleracea have also been studied (Abeysiri et al., 2013Abeysiri, G.R.P.I., Dharmadasa, R.M., Abeysinghe, D.C., Samarasinghe, K., 2013. Screening of phytochemical, physico-chemical and bioactivity of different parts of Spilantes acmella Murr. (Asteraceae), a natural remedy for toothache. Ind. Crop. Prod. 50, 852-856). One study found 5.29, 1.42 and 3.42 mg of trolox equivalents per g of dry leaves, stems and flowers (Abeysiri et al., 2013Abeysiri, G.R.P.I., Dharmadasa, R.M., Abeysinghe, D.C., Samarasinghe, K., 2013. Screening of phytochemical, physico-chemical and bioactivity of different parts of Spilantes acmella Murr. (Asteraceae), a natural remedy for toothache. Ind. Crop. Prod. 50, 852-856). It also found 7.59, 1.65 and 5.34 mg of gallic acid equivalents per gram dry weight (mg GAE/g DW) of total phenolic compounds (Abeysiri et al., 2013Abeysiri, G.R.P.I., Dharmadasa, R.M., Abeysinghe, D.C., Samarasinghe, K., 2013. Screening of phytochemical, physico-chemical and bioactivity of different parts of Spilantes acmella Murr. (Asteraceae), a natural remedy for toothache. Ind. Crop. Prod. 50, 852-856). A different study found 9.2, 10.3 and 7.7 mg of trolox equivalents per g of dry arial parts of A. oleracea grown three different ways: in the field, with hydroponics and as a callus, respectively (Abeysinghe et al., 2014Abeysinghe, D.C., Wijerathne, S.M.N.K., Dharmadasa, R.M., 2014. Secondary metabolites contents and antioxidant capacities of Acmella oleraceae grown under different growing systems. World J. Agric. Res. 2, 163-167). The same study found 11.0, 11.5 and 9.9 mg GAE/g DW total phenolics in A. oleracea grown in the field, with hydroponics and as a callus, respectively (Abeysinghe et al., 2014Abeysinghe, D.C., Wijerathne, S.M.N.K., Dharmadasa, R.M., 2014. Secondary metabolites contents and antioxidant capacities of Acmella oleraceae grown under different growing systems. World J. Agric. Res. 2, 163-167). The total flavonoid content was 11.3, 12.3 and 7.4 mg rutin equivalents per gram of dry weight in A. oleracea grown in the field, with hydroponics and as a callus, respectively.

The anti-inflammatory activity of dried flowers was demonstrated on the commonly used lipopolysaccharide-activated murine macrophage model, RAW 264.7 (Wu et al., 2008Wu, L.-C., Fan, N.C., Lin, M-H., Chu, I.-R., Huang, S.J., Han, S.Y., 2008. Anti-inflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators. J. Agric. Food Chem. 56, 2341-2349). These macrophages produce nitric oxide (NO) to mediate inflammation, through an inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Spilanthol inhibited the production of iNOS and COX-2 and the mRNA that code for them. It was also suggested that spilanthol attenuates the inflammatory responses in murine RAW 264.7 macrophages partly due to the inactivation of NF-κB. This down regulates the production of proinflammatory mediators. Spilanthol also had an anti-inflammatory effect on the arachidonic acid model with ED50 = 1.2 mg/ear (Wu et al., 2008Wu, L.-C., Fan, N.C., Lin, M-H., Chu, I.-R., Huang, S.J., Han, S.Y., 2008. Anti-inflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators. J. Agric. Food Chem. 56, 2341-2349). In a different study using the phorbol myristate acetate model, spilanthol showed an anti-inflammatory dose-dependent effect with ED50 = 1.3 mg/ear (Hernández et al., 2009Hernández, I., Márquez, L., Martínez, I., Dieguez, R., Delporte, C., Prietoa, S., Molina-Torres, J., Garrido, G., 2009. Anti-inflammatory effects of ethanolic extract and alkamides-derived from Heliopsis longipes roots. J. Ethnopharmacol. 124, 649-652).

Extracts containing spilanthol have been used to treat toothaches, stomatitis and skin diseases such as swimmer's eczema (Boonen et al., 2010aBoonen, J., Baert, B., Roche, N., Burvenich, C., de Spiegeleer, B., 2010. Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilantes acmella (Compositae) extracts. J. Ethnopharmacol. 127, 77-84,bBoonen, J., Baert, B., Burvenich, C., Blondeel, P., de Saeget, S., de Spiegeleer, B., 2010. LC–MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behavior of is main bioactive spilanthol. J. Pharm. Biomed. Anal. 53, 243-249). Extracts and spilanthol are in buccal mucosa preparations that are indicated for a painful mouth and minor mouth ulcers. Several spilanthol containing preparations for buccal use are commercially available (Boonen et al., 2010aAbeysinghe, D.C., Wijerathne, S.M.N.K., Dharmadasa, R.M., 2014. Secondary metabolites contents and antioxidant capacities of Acmella oleraceae grown under different growing systems. World J. Agric. Res. 2, 163-167,bBoonen, J., Baert, B., Burvenich, C., Blondeel, P., de Saeget, S., de Spiegeleer, B., 2010. LC–MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behavior of is main bioactive spilanthol. J. Pharm. Biomed. Anal. 53, 243-249). Also, spilanthol has been incorporated in tooth pastes and mouth rinses. The objective is to provide a lasting fresh minty flavor; it also increases salivation, which improves appetite. The spilanthol present also has a mild anesthetic effect thus enabling people with toothache to brush comfortably (Hatasa and Iioka, 1973Hatasa, S., Iioka, I., 1973. Spilanthol-containing compositions for oral use. U.S. Patent No. 3,720,762.). There is also a patent for manufacturing toothpastes or other oral compositions with spilanthol-rich essential oils (Shimada and Gomi, 1995Shimada, T., Gomi, T., 1995. Spilanthol-rich essential oils for manufacturing tooth-pastes or other oral compositions. JP patent 07090294.). A mouthwash contained ethanol 10.0, 85% glycerin 8.0, 65% sorbitol 2.0, chlorohexidine gluconate 0.05, triclosan 0.003, menthol 0.01, peppermint oil 0.01, sodium saccharin 0.001, spilanthol-rich essential oil 0.01 wt.% and balance purified water (Shimada and Gomi, 1995Shimada, T., Gomi, T., 1995. Spilanthol-rich essential oils for manufacturing tooth-pastes or other oral compositions. JP patent 07090294.).

Also, spilanthol in A. oleracea L. extracts inhibited contractions in subcutaneous muscles, notably those of the face, and can be used as an anti-wrinkle product (Demarne and Passaro, 2009Demarne, F., Passaro, G., 2009. Use of an Acmella oleracea extract for the botulinum toxin-like effect thereof in an anti-wrinkle cosmetic composition. US Patent No. 7,531,193 B2.). As a result, many anti-aging products containing spilanthol such as Gatuline®, SYN®-COLL and ChroNOline™ are available.

The antifungal and bacteriostatic activities of spilanthol and other alkamides from the roots of H. longipes were also studied (Molina-Torres et al., 2004Molina-Torres, J., Salazar-Cabrera, C.J., Armenta-Salinas, C., Ramírez-Sánchez, E., 2004. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduces amides. J. Agric. Food Chem. 52, 4700-4704). Four of the assayed fungi showed growth inhibition of 100% due to the presence of spilanthol: Sclerotium rolfsii, S. cepivorum, Phytophthora infestans, and Rhizoctonia solani AG-3 and AG-5. Spilanthol also inhibited the growth of Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae at concentrations as low as 25 µg/ml (Molina-Torres et al., 2004Molina-Torres, J., Salazar-Cabrera, C.J., Armenta-Salinas, C., Ramírez-Sánchez, E., 2004. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduces amides. J. Agric. Food Chem. 52, 4700-4704). In another study, spilanthol in S. calva was found to have antifungal activity against the fungi Fusarium oxysporum and Trichophyton mentagrophytes (Rai et al., 2004Rai, M.K., Varma, A., Pandey, A.K., 2004. Antifungal potential of Spilanthes calva after inoculation of Piriformospora indica . Mycoses. 47, 479-481). This antifungal activity was enhanced when S. calva was inoculated with the root endophyte Piriformospora indica, which also increased the concentration of spilanthol in the roots of S. calva (Rai et al., 2004Rai, M.K., Varma, A., Pandey, A.K., 2004. Antifungal potential of Spilanthes calva after inoculation of Piriformospora indica . Mycoses. 47, 479-481).

Spilanthol was also shown to be useful as an insecticide (Kadir et al., 1989; Spelman et al., 2011; Sharma et al., 2012). It killed the diamondback moth, Plutella xylostella L, which is one of the most destructive pests that attack cruciferous vegetables, such as broccoli (Sharma et al., 2012Sharma, A., Kumar, V., Rattan, R.S., Kumar, N., Singh, B., 2012. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. Against Plutella xylostella L. Am. J. Plant Sci. 3, 1568-1572). Spilanthol was also able to kill the tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), which attacks solanaceous plants and has become a serious threat to tomatoes in the Mediterranean region (Moreno et al., 2012Moreno, S.C., Carvalho, G.A., Picanço, M.C., Morais, E.G.F., Pereira, R.M., 2012. Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species. Pest Manag. Sci. 68, 386-393). Electrophysiological studies indicated immediate hyperexcitation followed by complete inhibition of the cockroach cercal nerve activity. Spilanthol exhibited the highest toxicity to Tuta absoluta, with the lowest LD50 (0.13 µg mg−1). Furthermore, spilanthol was approximately five times more toxic than permethrin and approximately 321 times more potent than Azadirachta indica extract. On the other hand, spilanthol was not toxic to two beneficial insects, the predator Solenopsis saevissima (Smith) (Hymenoptera: Formicidae) and the pollinator, tetragonisca angustula (Latr.) (Hymenoptera: Apidae: Melipninae) (Moreno et al., 2012Moreno, S.C., Carvalho, G.A., Picanço, M.C., Morais, E.G.F., Pereira, R.M., 2012. Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species. Pest Manag. Sci. 68, 386-393). Even more important, spilanthol has been shown to be toxic to the mosquitoes (Plasmodium falciparum) that carry malaria (Spelman et al., 2011Spelman, K., Depoix, D., McCray, M., Mouray, E., Grellier, P., 2011. The traditional medicine Spilanthes acmella, and the alkylamides spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, demonstrate in vitro and in vivo antimalarial activity. Phytother. Res. 25, 1098-1110). It had an IC50 of 16.5 µg/ml and 41.4 µg/ml on P. falciparum strain PFB and IC50 of 5.8 µg/ml and 16.3 µg/ml for the chloroquine resistant P. falciparum K1 strain, respectively. Further investigations revealed that at relatively low concentrations, spilanthol and the water extract of S. acmella reduced the parasitemia 59 and 53% in mice infected with P. yoelii yoelii 17XNL at 5 and 50 mg/kg, respectively. This parasite is used to infect mice in an animal model of malaria. These results provide evidence supporting the antimalarial activities of S. acmella and spilanthol (Spelman et al., 2011Spelman, K., Depoix, D., McCray, M., Mouray, E., Grellier, P., 2011. The traditional medicine Spilanthes acmella, and the alkylamides spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, demonstrate in vitro and in vivo antimalarial activity. Phytother. Res. 25, 1098-1110). Finally, another group reported the ability of extracts of S. acmella Murr. to kill the American cockroach, Periplaneta americana L. (Kadir et al., 1989Kadir, H.A., Zakaria, M.B., Kechil, A.A., Azirun, M.S., 1989. Toxicity and electrophysiological effects of Spilanthes acmella Murr. extracts on Periplaneta americana L. Pest. Sci. 25, 329-335). The potency was found to be 1.3, 2.6 and 3.8 times more toxic than carbaryl, bioresmethrin and lindane, respectively (Kadir et al., 1989Kadir, H.A., Zakaria, M.B., Kechil, A.A., Azirun, M.S., 1989. Toxicity and electrophysiological effects of Spilanthes acmella Murr. extracts on Periplaneta americana L. Pest. Sci. 25, 329-335).

Spilanthol is also active against Aedes aegyptii larvae, which can spread the viruses that cause dengue fever, chikungunya, and yellow fever as well as Helicoverpa zea neonates (corn earworm) at concentrations of 12.5 and 250 mg/ml, respectively (Ramsewak et al., 1999Ramsewak, R.S., Erickson, A.J., Nair, M.G., 1999. Bioactive N-isobutylamides from the flower buds of Spilanthes Acmella . Phytochemistry. 51, 729-732). Spilanthol, at 7.5 ppm concentration, caused 100% motility of eggs, larvae, and pupae of Anopheles, Culex, and Aedes mosquitoes at lower doses; it is also effective against eggs and pupae (Saraf and Dixit, 2002Saraf, D.K., Dixit, V.K., 2002. Spilanthes acmella Murr.: study on its extract spilanthol as larvicidal compound. Asian J. Exp. Sci. 16, 9-19). The insecticidal activity of Heliopsis longipes roots against Anopheles albimanus and Aedes aegypti was determined (Hernández-Morales et al., 2015Hernández-Morales, A., Arvizu-Gómez, J.L., Carranza-Álvarez, C., Gómez-Luna, B.E., Alvardo-Sánchez, B., Ramirez-Chávez, E., Molina-Torres, J., 2015. Larvicidal activity of affinin and its derived amides from Heliopsis longipes A. Gray Blake against Anopheles albimanus and Aedes aegypti. J. Asia-Pacific Entomol. 18, 227–231.). A concentration of 7 mg/l of ethanolic extract caused 100% of larval mortality for A. albimanus, and had the same effect on A. aegypti larvae. This effect could be attributed to spilanthol. The conjugated double bonds present in its structure were found to be necessary to maintain larvicidal activity. This study demonstrated the potential of H. longipes for controlling the larval stage of A. albimanus and A. aegypti, transmitter vectors of malaria and dengue fever, respectively (Hernández-Morales et al., 2015Hernández-Morales, A., Arvizu-Gómez, J.L., Carranza-Álvarez, C., Gómez-Luna, B.E., Alvardo-Sánchez, B., Ramirez-Chávez, E., Molina-Torres, J., 2015. Larvicidal activity of affinin and its derived amides from Heliopsis longipes A. Gray Blake against Anopheles albimanus and Aedes aegypti. J. Asia-Pacific Entomol. 18, 227–231.).

Others explored Spilanthes acmella Murr. for insecticidal activity (Sharma et al., 2012Sharma, A., Kumar, V., Rattan, R.S., Kumar, N., Singh, B., 2012. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. Against Plutella xylostella L. Am. J. Plant Sci. 3, 1568-1572). The seed extract and spilanthol were toxic to Plutella xylostella. An activity of 95–100% was observed at a dose of 2 g/l of spilanthol, while 60–70 and 80–90% mortality was seen in crude seed extracts prepared in methanol and hexane at a dose of 5 g/l after 48 h exposure. LC50 values of 1.49, 5.14, 5.04, 11.75 g/l were observed for spilanthol, crude methanolic seed extract, hexane extracts and deltamethrin, respectively. These findings indicated the potential of S. acmella and spilanthol for controlling P. xylostella and other insects of agricultural importance (Sharma et al., 2012Sharma, A., Kumar, V., Rattan, R.S., Kumar, N., Singh, B., 2012. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. Against Plutella xylostella L. Am. J. Plant Sci. 3, 1568-1572). Spilanthol also has strong molluscicidal activity against Physa occidentalis (LD50 of 100 µM) and the cercariae of the fluke (Johns et al., 1982Johns, T., Graham, K., Towers, G.H.N., 1982. Molluscicidal activity of affinin and other isobutylamides from the Asteraceae. Phytochemistry. 21, 2737-2738). At a concentration of 50 mg/l in water at 21° snails were inactive after 60 min and dead within 18 h. At 150 mg/l (the solubility limit for spilanthol) cercarial emergence ceased and the snails showed immobility after 30 min. Cercariae ceased to move after five set and convulsed after 1 min (Johns et al., 1982Johns, T., Graham, K., Towers, G.H.N., 1982. Molluscicidal activity of affinin and other isobutylamides from the Asteraceae. Phytochemistry. 21, 2737-2738).

Spilanthol also can also stimulate the growth of roots in Arabidopsis thaliana seedlings (Campos-Cuevas et al., 2008Campos-Cuevas, J.C., Pelagio-Flores, R., Raya-González, J., Méndez-Bravo, A., Ottiz-Castro, R., López-Bucio, J., 2008. Tissue culture of Arabidopsis thaliana explants reveals a stimulatory effect of alkamides on adventitious root formation and nitric oxide accumulation. Plant Sci. 174, 165-173). Although the effects of spilanthol was similar to those produced by auxins on adventitious root development, the ability of shoot explants to respond to spilanthol was found to be independent of auxin signaling. These results suggest a role for spilanthol in regulating adventitious root development, probably operating through the NO signal transduction pathway (Campos-Cuevas et al., 2008Campos-Cuevas, J.C., Pelagio-Flores, R., Raya-González, J., Méndez-Bravo, A., Ottiz-Castro, R., López-Bucio, J., 2008. Tissue culture of Arabidopsis thaliana explants reveals a stimulatory effect of alkamides on adventitious root formation and nitric oxide accumulation. Plant Sci. 174, 165-173).

Spilanthol was also shown to inhibit CYP P450 enzymes, with IC50 values of 25, 16.1 and 13.5 µg/ml for CYP1A1/2, CYP2D6 and CYP3A4, respectively (Rodeiro et al., 2009Rodeiro, I., Donato, M.T., Jimenez, N., Garrido, G., Molina-Torres, G., Menendez, R., Castell, J.V., Gómez-Lechón, M., 2009. Inhibition of human P450 enzymes by natural extracts used in traditional medicine. Phytother. Res. 23, 279-282). These results suggest that spilanthol inhibits the major human P450 enzymes involved in drug metabolism and could induce potential herbal–drug interactions (Smith, 2014Smith, R.E., 2014. Medicinal Chemistry – Fusion of Traditional and Western Medicine, 2nd ed. Bentham Science, Sharjah, U.A.E. 192-196). On the other hand, CYP1A1/2 inhibition could be associated with decreased carcinogenic risk. Although, in vitro inhibition of P450s does not necessarily lead to relevant in vivo effects, these results recommend a cautious evaluation of the potential clinical consequences derived from the consumption of these products, particularly for long-term treatments (Rodeiro et al., 2009Rodeiro, I., Donato, M.T., Jimenez, N., Garrido, G., Molina-Torres, G., Menendez, R., Castell, J.V., Gómez-Lechón, M., 2009. Inhibition of human P450 enzymes by natural extracts used in traditional medicine. Phytother. Res. 23, 279-282).

In conclusion, spilanthol is a secondary metabolite with high industrial potential as well as several biological properties and health effects. It can be found, extracted and purified from A. oleracea and H. longipes. A. oleracea is used as a spice and a food in the northern part of Brazil. It is also used as a treatment for treating toothaches, so it is called the toothache plant. Spilanthol may also have analgesic (Molinatorres et al., 1996Molinatorres, J., Salgado-Garciglia, R., Ramirez-Chanez, E., del Rio, R.E., 1996. Purely olefinic alkamides in Heliopsis longipes and Acmella (Spilanthes) oppositifolia . Biochem. Syst. Ecol. 24, 27-43; Hind and Biggs, 2003Hind, N., Biggs, N., 2003. Plate 460. Acmella oleracea compositae. Curtis's Bot. Mag. 20, 31-39; Cilia-López et al., 2010Cilia-López, V.G., Juárez-Flores, B.I., Aguirre-Rivera, J.R., Reyes-Aguero, J.A., 2010. Analgesic activity of Heliopsis longipes and its effect on the nervous system. Pharm. Biol. 48, 195-200; Tiwari et al., 2011Tiwari, K.L., Jadhav, S.K., Joshi, V., 2011. An updated review on medicinal herb Genus Spilanthes . J. Chin. Integr. Med. 9, 1170-1178; Dias et al., 2012Dias, A.M.A., Santos, P., Seabraa, I.J., Junior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J. Supercrit. Fluids. 61, 62-70; Sharma et al., 2012Sharma, A., Kumar, V., Rattan, R.S., Kumar, N., Singh, B., 2012. Insecticidal toxicity of spilanthol from Spilanthes acmella Murr. Against Plutella xylostella L. Am. J. Plant Sci. 3, 1568-1572; Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.; Prachayasittukal et al., 2013Prachayasittukal, V., Prachayasittukal, S., Ruchiwarat, S., Prachayasittukal, V., 2013. High therapeutic potential of Spilanthes acmella: a review. EXCLI J. 12, 291-312; Paulraj et al., 2013Paulraj, J., Govindarajan, R., Palpu, P., 2013. The genus Spilanthes ethnopharmacology, phytochemistry, and pharmacological properties: a review. Adv. Pharmacol. Sci., http://dx.doi.org/10.1155/2013/510298
http://dx.doi.org/10.1155/2013/510298...
; Wu et al., 2008Wu, L.-C., Fan, N.C., Lin, M-H., Chu, I.-R., Huang, S.J., Han, S.Y., 2008. Anti-inflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators. J. Agric. Food Chem. 56, 2341-2349; Rios and Olivo, 2014Rios, M.R., Olivo, H.F., 2014. Natural and synthetic alkylamides: applications in pain therapy. In: Atta-Ur-Rahman (Ed.), Studies in Natural Products Chemistry. Else-vier, New York, pp. 79–118.; Dandin et al., 2014Dandin, V.S., Naik, P.M., Murthy, H.M., Park, S.Y., Paek, K.Y., 2014. Rapid regeneration and analysis of genetic fidelity and scopoletin contents of micropropagated plants of Spilanthes oleracea L. J. Hort. Sci. Biotechnol. 89, 79-85; Hajdu, 2014Hajdu, A., 2014. An Ethnopharmacological Survey Conducted in the Bolivian Amazon, and Identification of N-alkylamides and Lignans from Lepidium meyenii and Heliopsis helianthoides var. scabra with Effects on the Central Nervous System. University of Szeged, Szeged, Hungary.), antinociceptive (Rios et al., 2007Rios, M.Y., Aguilar-Guadarrama, A.B., Gutierrez, M.D., 2007. Analgesic activity of affinin, an alkamide from Heliopsis longipes (Compositae). J. Ethnopharmacol. 110, 364-367; Déciga-Campos et al., 2012Déciga-Campos, M., Arriaga-Alba, M., Ventura-Martínez, R., Aguilar-Guadarrama, B., Rios, M.Y., 2012. Pharmacological and toxicological profile of extract from Heliopsis longipes and affinin. Drug Dev. Res. 73, 130-137), antioxidant (Abeysiri et al., 2013Abeysiri, G.R.P.I., Dharmadasa, R.M., Abeysinghe, D.C., Samarasinghe, K., 2013. Screening of phytochemical, physico-chemical and bioactivity of different parts of Spilantes acmella Murr. (Asteraceae), a natural remedy for toothache. Ind. Crop. Prod. 50, 852-856), anti-inflammatory (Wu et al., 2008Wu, L.-C., Fan, N.C., Lin, M-H., Chu, I.-R., Huang, S.J., Han, S.Y., 2008. Anti-inflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators. J. Agric. Food Chem. 56, 2341-2349; Hernández et al., 2009Hernández, I., Márquez, L., Martínez, I., Dieguez, R., Delporte, C., Prietoa, S., Molina-Torres, J., Garrido, G., 2009. Anti-inflammatory effects of ethanolic extract and alkamides-derived from Heliopsis longipes roots. J. Ethnopharmacol. 124, 649-652; Dias et al., 2012Dias, A.M.A., Santos, P., Seabraa, I.J., Junior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. J. Supercrit. Fluids. 61, 62-70), antimutagenic (Arriaga-Alba et al., 2013Arriaga-Alba, M., Rios, M.Y., Déciga-Campos, M., 2013. Antimutagenic properties of affinin isolated from Heliopsis longipes extract. Pharm. Biol. 51, 1035-1039), anti-wrinkle (Demarne and Passaro, 2009Demarne, F., Passaro, G., 2009. Use of an Acmella oleracea extract for the botulinum toxin-like effect thereof in an anti-wrinkle cosmetic composition. US Patent No. 7,531,193 B2.), antifungal (Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.), bacteriostatic (Molina-Torres et al., 2004Molina-Torres, J., Salazar-Cabrera, C.J., Armenta-Salinas, C., Ramírez-Sánchez, E., 2004. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduces amides. J. Agric. Food Chem. 52, 4700-4704), insecticidal (Kadir et al., 1989; Sharma et al., 2012; Moreno et al., 2012), anti-malarial (Soares et al., 2014Soares, C.P., Lemos, V.R., da Silva, A.G., Campoy, R.M., da Silva, C.A.P., Menegon, R.F., Rojahn, I., Joaquim, W.M., 2014. Effect of Spilanthes acmella hydroethanolic extract activity on tumour cell actin cytoskeleton. Cell Biol. Int. 38, 131-135), anti-larvicidal against Aedes aegypti and Helicoverpa zea neonates (Ramsewak et al., 1999Ramsewak, R.S., Erickson, A.J., Nair, M.G., 1999. Bioactive N-isobutylamides from the flower buds of Spilanthes Acmella . Phytochemistry. 51, 729-732), and anti-molluscicidal (Johns et al., 1982Johns, T., Graham, K., Towers, G.H.N., 1982. Molluscicidal activity of affinin and other isobutylamides from the Asteraceae. Phytochemistry. 21, 2737-2738). There have also been reports on its activities as an anticonvulsant, antioxidant, aphrodisiac, pancreatic lipase inhibitor, antimicrobial agent, antinociceptive agent, diuretic, vasorelaxant, anti-human immunodeficiency virus, toothache relief and anti-inflammatory (Dubey et al., 2013Dubey, S., Maity, S., Singh, M., Saraf, S.A., Saha, S., 2013. Phytochemistry, pharmacology and toxicology of Spilanthes acmella: a review. Adv. Pharmacol. Sci., Article ID 423750.). The biological activities are listed in Box 1.

Box 1
Biological activities of spilanthol.

However, the human toxicity of spilanthol has not been thoroughly tested, even though A. oleracea and H. longipes have been consumed for a long time. Also, the concentrations of spilanthol in different parts of these plants have not been determined.

Acknowledgements

The authors want to thank the Fundação Carlos Chagas de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ), CNPq, and to CAPES for scholarships and financial support.

References

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Publication Dates

  • Publication in this collection
    Jan-Feb 2016

History

  • Received
    2 July 2015
  • Accepted
    31 July 2015
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