EXCLI J EXCLI Journal 1611-2156 Leibniz Research Centre for Working Environment and Human Factors 2012-407 Doc291 Review article High therapeutic potential of Spilanthes acmella: A review Prachayasittikul Veda 1 Prachayasittikul Supaluk * 2 Ruchirawat Somsak 3 Prachayasittikul Virapong 1 Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology,Mahidol University, Bangkok 10700, Thailand Laboratory of Medicinal Chemistry, Chulabhorn Research Institute and ChulabhornGraduate Institute, Bangkok 10210, Thailand *To whom correspondence should be addressed: Supaluk Prachayasittikul, Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology,Mahidol University, Bangkok 10700, Thailand; Telephone: 662-441-4376, Fax: 662-441-4380, E-mail: supaluk@swu.ac.th 04 04 2013 2013 12 291 312 29 11 2012 24 03 2013 Copyright © 2013 Prachayasittikul et al. 2013

This is an Open Access article distributed under the following Assignment of Rights http://www.excli.de/documents/assignment_of_rights.pdf. You are free to copy, distribute and transmit the work, provided the original author and source are credited.

 This article is available from http://www.excli.de/vol12/prachayasittikul_04042013_2_proof.pdf

Spilanthes acmella, a well known antitoothache plant with high medicinal usages, has been recognized as an important medicinal plant and has an increasingly high demand worldwide. From its traditional uses in health care and food, extensive phytochemical studies have been reported. This review provides an overview and general description of the plant species, bioactive metabolites and important pharmacological activities including the preparation, purification and in vitro large-scale production. Structure-activity relationships of the bioactive compounds have been discussed. Considering data from the literature, it could be demonstrated that S. acmella possesses diverse bioactive properties and immense utilization in medicine, health care, cosmetics and as health supplements. As a health food, it is enriched with high therapeutic value with high potential for further development.

Spilanthes acmella bioactive metabolites bioactivities structure-activity relationships in vitro production Introduction

Plants contain a diverse group of highly valuable and readily available resource of bioactive metabolites, e.g. alkaloids, tannins, essential oils and flavonoids (Jagan Rao et al., 2012[35]; Prachayasittikul et al., 2008[65], 2009[68], 2010[67]), which have been used in medicinal practices for a long time (Tiwari et al., 2011[108]). Thus far, medicinal plant is an alternative medicine (Consult, 2003[21]) that is still in use and is a popular choice for primary health care(Vanamala et al., 2012[111]). However, if improperly used plants can also be toxic (Perry et al., 2000[63]). The World Health Organization has estimated that about 80 % of the population in developing countries are unable to afford drugs and rely on traditional medicines especially those that are plant-based (Elumalai et al., 2012[27]) such as India (Jain et al., 2006[36]; Little, 2004[47]), Sri Lanka (Ediriweera 2007[26]), Bangladesh (Rahmatullah et al., 2010[75]), China and Japan (Little, 2004[47]) including Thailand (Phongpaichit et al., 2005[64]; Sawangjaroen et al., 2006[89]).

The practice of botanical healing slowly disappeared from western countries with the introduction and advent of science and technology (Tyagi and Delanty, 2003[110]). However, the uses of traditional medicine dramatically increased in Europe and North America in the last 50 years (Tyagi and Delanty, 2003[110]).

Herbal medicines have been utilized for many purposes, particularly in medical care as antiasthmatics (86.79 %), anti-rheumatics (62 %) (Jain et al., 2006[36]), diuretics (60.22 %) (Kumar et al., 2010[42]; Vanamala et al., 2012[111]), antiinflammation (29.62 %), anticancer (9.75 %), antidiabe-tics (8.33 %), antimicrobials, antifungals, antioxidants, antiallergy, analgesics, antiobesity and antihypertention. In dental care it has been employed as anticariogenic (Ferrazzano et al., 2011[29]), analgesic (Abascal and Yarnell, 2010[2]; Kroll, 1995[40]), local anesthetic (Abascal and Yarnell, 2010[2]), wound healing agents (Abascal and Yarnell, 2001[1], 2010[2]; Jagan Rao et al., 2012[35]), antiinflammation (Abascal and Yarnell 2001[1], 2010[2]) and recurrent aphthous stomatitis treatment (Abascal and Yarnell 2010[2]). It has also been used for beauty care (Artaria et al., 2011[6]; Demarne and Passaro, 2009[24]) and as health food e.g. curcumin (Curcuma longa Linn.) (Kohli et al., 2005[38]), ginger (Zingiber officinale) (Kubra and Rao, 2011[41]), lemon grass (Cymbopogon citrates Stapf) (Nanasombat and Teckchuen, 2009[56]), green shallot (Allium cepa var. aggregatum) (Rabinowitch and Kamenetsky, 2002[74]), garlic (Allium sativum L.) (Borek, 2010[14]), holy basil (Ocimum sanctum Linn.) (Singh et al., 1996[96]), sweet basil (Ocimum basilicum L.) (Lee et al., 2005[43]), hairy basil (Ocimum basilicum L.f. var. citratum Back.) (Chanwitheesuk et al., 2005[20]) and kitchen mint (Mentha cordifolia Opiz.) (Özbek and Dadali, 2007[60]).

Recently, health foods, herbs as well as dietary supplements enriched with medicinal ingredients such as antioxidants and bioactive metabolites have drawn considerable attention worldwide, especially herbs that are used as food and traditional medicine (Tyagi and Delanty, 2003[110]). Our concern centers around medicinal plants bearing bioactive compounds, which are employed as therapeutics and health care (Abascal and Yarnell, 2010[2]). Therefore, Spilanthes acmella Murr. is a plant of great interest owing to its known reputation as an antitoothache plant and hold tremendous medicinal usages. This review focuses on the general background, therapeutic uses, bioactive compounds and large-scale production.

General

Spilanthes (Compositae or Asteraceae) is a genus comprising of over 60 species that are widely distributed in tropical and subtropical regions of the world, such as Africa, America, Borneo, India, Sri Lanka and Asia (Sahu et al., 2011[86]; Tiwari et al., 2011[108]). S. acmella is native to Brazil and is cultivated throughout the year as ornamental or medicinal plant. It is an annual or short-lived herb that is 40-60 centimeters tall. It is grown in damp area (Tiwari et al., 2011[108]; Wongsawatkul et al., 2008[115]) and has low rate of germination or poor vegetative propagation (Tiwari et al., 2011[108]). Its flowers and leaves have pungent taste and when touched it is accompanied by tingling sensation and numbness (Wongsawatkul et al., 2008[115]). The plant species has been used commonly as a folk remedy, e.g. for toothache, rheumatic and fever (Wongsawatkul et al., 2008[115]), as fresh vegetable (Tiwari et al., 2011[108]) as well as spice for Japanese appetizer (Leng et al., 2011[46]).

 Traditional Uses

The whole plants (e.g. flowers, leaves, roots, stems and aerial parts) of Spilanthes have been used in health care (Leng et al., 2004[45]; Ospina De Nigrinis et al., 1986[59]; Purabi and Kalita, 2005[73]; Research, 1976[80]; Rios-Chavez et al., 2003[83]; Senthilkumar et al., 2007[92]; Tiwari and Kakkar, 1990[107]) and food (Barman et al., 2009[9]; Boonen et al., 2010[12]; Wu et al., 2008[117]). Particularly, S. acmella or S. oleracea (paracress or eyeball plant), is a well-known antitoothache plant (Sahu et al., 2011[86]) and has been used as traditional medicine for many purposes (Table 1(Tab. 1)) (References in Table 1: Medical: Bunyapraphatsara and Chokechareunporn, 1999[17]; Farnsworth and Bunyapraphatsara, 1992[28]; Yadav and Singh, 2010[118]; Haw and Keng, 2003[32]; Tiwari et al., 2011[108]; Sahu et al., 2011[86]; Leng et al., 2011[46]; Yuliana et al., 2011[120]; Dental: Haw and Keng, 2003[32]; Tiwari et al., 2011[108]; Savadi et al., 2010[88]; Abascal and Yarnell, 2001[1]; Sahu et al., 2011[86]; Shimada and Gomi, 1995[95]; Beauty care cosmetics: Belfer, 2007[10]; Demarne and Passaro, 2009[24]; Schubnel, 2007[91]). So far, various Thai medicinal plants have been used for the remedy of toothache as well as used in dental applications (Table 2(Tab. 2)) (References in Table 2: Thiengburanathum, 1999[106]; Boonkird et al., 1982[13]; Matchacheep, 1991[48]; Thanaphum and Muengwongyard, 2006[105]).

 Bioactive Metabolites

Extensive phytochemical investigations of S. acmella had previously been reported. It constitutes a diverse group of compounds. Major isolates were lipophilic alkylamides or alkamides bearing different number of unsaturated hydrocarbons (alkenes and alkynes), such as spilanthol (1) or affinin (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (Gokhale and Bhide, 1945[31]; Ramsewak et al., 1999[76]) and amide derivatives 2-8 (Figure 1(Fig. 1)). In general, when alkamides are chewed, a pungent taste is released and causes itch and salivation (Rios, 2012[81]). Alkamides are structurally related to animal endocannabinoids and is highly active in the central nervous system. Particularly, anandamide (N-arachidonoyl-ethanolamine, 9) is an endogenous cannabinoid cerebral neurotransmitter (Figure 1(Fig. 1)).

Spilanthol was first isolated in 1945 from the flower head ethanol (EtOH) extract of S. acmella. In early 1903, it was first obtained from the different plant species, S. acmella L.var. oleracea Clarke (Gokhale and Bhide, 1945[31]). Aside from being found in S. acmella, spilanthol was also found in other plant species as shown in Table 3(Tab. 3) (Rios, 2012[81]).

The synthesis of spilanthol was reported in multistep and afforded low overall yields. However, an efficient synthetic method had been developed (Wang et al., 1998[113]). Thus far, the spilanthol is commercially available in form of alcoholic (65 % EtOH) extract or A. Vogel Spilanthes.

In addition, phytosterols (e.g. β-sitosterol, stigmasterol, α- and β-amyrins), essential oils (e.g. limonene and β-caryophyllene), sesquiterpenes, α- and β-bisabolenes and cadinenes, flavonoid glucoside and a mixture of long chain hydrocarbons (C22-C35) were reported (Sahu et al., 2011[86]; Tiwari et al., 2011[108]).

In recent years, other bioactive metabolites 10 - 15 (Figure 2(Fig. 2)) have been isolated from the aerial part of S. acmella, namely vanillic acid (10), trans-ferulic acid (11), trans-isoferulic acid (12), scopolelin (13), 3-acetylaleuritolic acid (14) and β-sitostenone (15) (Prachayasittikul et al., 2009[68]).

 Bioactivity

The Spilanthes genera have been used for the treatment of various disorders including life-threatening diseases. Diverse pharmacological activities of this plant species were previously reported (Sahu et al., 2011[86]; Tiwari et al., 2011[108]). Selected bioactivities of S. acmella are summarized below.

Antipyretic activity

Many medicinal plants have long been used as antipyretics, e.g. S. acmella (flower and aerial aqueous) extracts (Chakraborty et al., 2010[19]). In general, pyrexia or fever is caused by a secondary impact of infection, tissue damage, inflammation, graft rejection, malignancy and other diseases (Elumalai et al., 2012[27]). These impacts initiate the formation of pro-inflammatory mediators or in particular cytokines (i.e. interleukin 1β, α, β, and TNF-α). This results in an increase of prostaglandin E2 (PGE2) synthesis and ultimately increases the body temperature (Elumalai et al., 2012[27]). The studies showed that S. acmella (aerial aqueous extract) displayed antipyretic activity against Brewer's yeast-induced pyrexia. The antipyretic activity of the plant species can be attributed to flavonoids (Narayana et al., 2001[57]; Trease and Evans, 1972[109]), which were predominant inhibitors of either cyclooxygenase (COX) or lipoxygenase (LOX) (Sadavongvivad and Supavilai, 1977[85]). Flavonoids are known to target prostaglandins in the late phase of acute inflammation and pain (Chakraborty et al., 2004[18]).

Antiinflammatory activity

Spilanthol is the main constituent isolated from many parts of S. acmella such as flower 85 % EtOH extract (Wu et al., 2008[117]), root hexane extract (Wagner, 1989[112]) and also from other plants such as Heliopsis longipes root EtOH extract (Hernández et al., 2009[33]). Traditional usages of S. acmella flowers have been reported as antiinflammatory agent (Sharma, 2003[94]). Previous investigations demonstrated that spilanthol exerted antiinflammatory action via inhibition of NF-κB pathway; afforded reduction in mRNA level and protein expression of COX-2 and iNOS; and also induced free radical scavenging activity (Wu et al., 2008[117]). Most antiinflammatory medicinal plants possessed LOX and COX inhibitors such as Asteraceae, Apiaceae, Lamiaceae and Fabaceae (Schneider and Bucar, 2005[90]). Plant species affording such properties is the H. longipes root extract and its isolated spilanthol (Hernández et al., 2009[33]). The antiinflammatory activity of spilanthol can be attributed to its dual inhibition of COX and LOX owing to the similar structures of spilanthol and arachidonic acid, in which the arachidonic acid is a precursor of prostaglandin and leukotriene syntheses (Hernández et al., 2009[33]). Interestingly, the H. longipes extract displayed stronger antiinflammatory activity than that of the spilanthol. This was possibly due to synergistic effects of the containing compounds in the plant extract (Hernández et al., 2009[33]). Moreover, EtOH extract from the leaves of S. acmella exhibited significant antiinflammatory activity against acute (carragenan induced rat paw edema method), sub-acute (granuloma pouch method) and chronic (adjuvant arthritis method) inflammation (Barman et al., 2009[9]) but has been shown to be less than that of aspirin. The observed antiinflammatory activity originates from the inherent flavonoids that are found in the plant extracts (Chakraborty et al., 2004[18]). Mechanisms of acute inflammation consists of two phases involve with histamine, serotonin and kinin, released in the first hour (Ganesh et al., 2008[30]) and with prostaglandin-like substances that are released in the second and third hours. Therefore the antiinflammatory action of S. acmella may take part in the later phase via inhibition of COX enzyme (Brooks and Day, 1991[16]).

A recent study in 2012 has shown that triterpenoids, namely β-sitosterol and β-sitostenone isolated from Leucosidea sericea (Rosaceae), exhibited antiinflammatory activity via inhibitions of COX-1 and COX-2. The study employed indomethacin as a standard drug and the results showed that β-sitosterol displayed stronger antiinflammatory activity than the standard drug (Nair et al., 2012[54]).

Analgesic activity

A number of antitoothache plants has been recognized, S. acmella is one of these plants that has been used in pain relief. The studies showed that S. acmella EtOH leaves extracts exerted significant centrally (e.g. tail flick method) and peripherally (e.g. Writhing test) analgesic activities (D' Armour and Smith, 1941[22]; Witkin et al., 1961[114]). The mechanism of action was possibly due to the presence of flavonoids in the plant extract (Chakraborty et al., 2004[18]) which decreases prostaglandins, PGE2 and PGF2 that are known to be involved in pain perception (Jyothi et al., 2008[37]). In addition, cold aqueous extract of S. acmella flowers also displayed antinociceptive activity against persistent pain and antihyperalgesic activity. The mechanism of action was possibly through inhibition of prostaglandins by spilanthol-containing extract (Ratnasooriya and Pieris, 2005[78]).

Another study of antitoothache plant H. longipes revealed that it was used as local anesthetic and analgesic in Mexican indigenous medicine and the results showed that its stem acetone extract and spilanthol from root displayed dose-dependent antinociceptive effect in mice as assessed by Writhing and capsaicin tests (Déciga-Campos et al., 2010[23]). Possible mechanisms of action of the spilanthol and H. longipes extract may be attributed to the activation of opioidergic, serotoninergic and GABAergic systems as well as the K+channel opening facilitated by nitric oxide (NO) induced cGMP production (Acosta-Madrid et al., 2009[3]; Rios et al., 2007[82]). Partial participation of cGMP and K+channel in the antinociceptive activity of spilanthol was proposed for several drugs (Bermúdez-Ocaña et al., 2006[11]; Hernandez-Pacheco et al., 2008[34]; Ortiz et al., 2006[58]).

Local anesthetic activity

S. acmella is known to be constituted of pungent alkamide-like spilanthol that causes numbness and tingle. Local anesthetic activity was studied in animal models through intracutaneous wheal in guinea pigs and plexus anesthesia in frogs (Chakraborty et al., 2010[19]). S. acmella aerial aqueous extract exhibited significant activity that could be due to the presence of alkamides (Chakraborty et al., 2010[19]). However, its onset of action was slower than that of xylocaine, the standard drug.

The well-recognized local anesthetics are comprised of mostly amide compounds such as xylocaine (lidocaine). Its mechanism of action involves the blockage of voltage-gated Na+ channels. By the same analogy, the alkamides of S. acmella extracts produced local anesthetic action presumably through the blockage of Na+ channels. Isobutylamide and piperovatine of other antitoothache plant (Piper piscatorum) were reported to display local anesthetic activity through the same mechanism of action (McFerren et al., 2002[50]).

Antimicrobial activity

Ethyl acetate (EtOAc) and methanol (MeOH) extracts from the leaves of S. acmella exhibited the strongest antimicrobial activity among the tested extracts using the well diffusion method against Klebsiella pneumoniae (Arora et al., 2011[5]). The EtOAc extract had two-fold higher activity than that of doxycycline, the standard drug, whereas the MeOH extract showed comparable activity with doxycycline. This could be due to the fact that the plants contain flavonoids, tannins, and other phytochemicals, which are well-known antimicrobials. On the other hand, aerial parts of EtOAc and MeOH extracts from S. acmella tested by the agar dilution method were shown to be inactive antimicrobials whereas its chloroform (CHCl3) extract displayed antimicrobial activity against Streptococcus pyogenes with MIC of 256 μg/mL (Prachayasittikul et al., 2009[68]). In addition, hexane and CHCl3 extracts exhibited antifungal activity (MIC 256 μg/mL) against Saccharomyces cerevisiae (Prachayasittikul et al., 2009[68]).Moreover, isolated fractions of CHCl3 and EtOAc extracts selectively inhibited the growth of Corynebacterium diphtheriae NCTC 10356 with MIC range 64-256 μg/mL (Prachayasittikul et al., 2009[68]). Another study showed that leaves/flowers MeOH extract of the plant species displayed no antimicrobial action (disk diffusion method) (Nanasombat and Teckchuen, 2009[56]).

Medicinal plants for treatments of oral cavity infections, dental caries and periodontal diseases were reported (Rosas-Piñón et al., 2012[84]). The most frequently used were species from many families: Myrthaceae (17.8 %), Punica (15.1 %), Compositae (11.3 %), Asteraceae (9.7 %), Piperaceae (8.6 %), Anacardiaceae (7.3 %), Fagaceae (6.9 %), Labiateae (5.4 %), Leguminosae (5.1 %), Butalaceae (0.5 %) and others (6.4 %). Dental caries and periodontal diseases are two major dental pathologies affecting humankind that arises from colonization and accumulation of oral microorganisms especially Streptococcus mutans and Porphyromonas gingivalis (Rosas-Piñón et al., 2012[84]). Antibacterial activity of Compositae plant extracts against oral microorganisms is shown in Table 4(Tab. 4) (Rosas-Piñón et al., 2012[84]). The data showed that S. mutans was the most sensitive to plant extracts while P. gingivalis was the most resistant (Rosas-Piñón et al., 2012[84]).

Antifungal activity

Several parts of S. acmella were tested for antifungal activity (Table 5(Tab. 5)) (References in Table 5: EtOAc: Arora et al., 2011[5]; Petroleum ether: Rani and Murty, 2006[77]; CHCl3: Phongpaichit et al., 2005[64]; Hexane,CHCl3 EtOAc,MeOH: Prachayasittikul et al., 2009[68]) and the studies showed that S. acmella leaves (EtOAc and aqueous) extracts exhibited better antifungal activity than the standard drug (fluconazole) against Rhizopus arrhigus and Rhizopus stolonifer (Arora et al., 2011[5]).The leaves extract also displayed weak activity against Aspergillus niger and Penicillium chrysogenum (Arora et al., 2011[5]). The whole plant CHCl3 extract was shown to be active antifungal against opportunistic fungal infection (e.g. Microsporum gypseum and Cryptococcus neoformans) in AIDS patients (Phongpaichit et al., 2005[64]). S. acmella flower head petroleum ether extract exerted antifungal activity against A. niger, A. parasiticus, Fusarium moniliformis and F. oxysporium (Rani and Murty, 2006[77]). The antifungal activity of S. acmella extracts may be due to the presence of spilanthol and alkamides (Nakatani and Nagashiwa, 1992[55]), non-volatile sesquiterpenoids and saponins (Krishnaswami et al., 1975[39]; Mukharya and Ansari, 1986[51]). In addition, aerial parts of S. acmella extracts (hexane and CHCl3) exhibited activity against Saccharomycese cerevisiae (Prachayasittikul et al., 2009[68]).

Antimalarial activity

S. acmella is a traditional medicine used in Africa and India for the treatment of malaria (Spelman et al. 2011[100]). Pharmacological study showed that spilanthol (1) and acetylenic alkamide (undeca-2E-ene-8,10-diynoic acid isobutylamide or UDA) (3), isolated from the root EtOH extract of S. acmella, displayed antimalarial activity against two strains of Plasmodium falciparum (PFB strain originated from Brazil and chloroquine resistant, K1 strain originated from Thailand). Both compounds had a reported antimalarial activity with IC50 in the range of 5.8-41.4 μg/mL in which the spilanthol was the most potent compound. It was reported that semi-purified compounds of S. acmella, isolated by centrifugal partition chromatography (CPC) and electrospray ionization-ion trap-time of flight-mass spectrometry (ESI-IT-TOF-MS), showed significantly higher antiplasmodial activity as indicated by the lower IC50 value (Mbeunkui et al. 2011[49]). This could be a result from synergistic effects of N-alkylamides in the tested compounds. Moreover, regenerated S .acmella (in vitro) root hexane extract exhibited 100 % larvicidal activity affording the lowest values of LC50 and LC90 against malaria and filarial vectors (Pandey and Agrawal, 2009[61]). It was suggested that the regenerated plant species contained higher active principle content than those that are field grown. In addition, the studies demonstrated the potential of S. acmella for the treatment and prevention of malaria (Bae et al., 2010[7]).

Antioxidant activity

Antioxidant activity of S. acmella extracts obtained from polar and nonpolar solvents were investigated. It was found that S. acmella flower EtOAc extract displayed the highest free radical scavenging activity (DPPH and ABTS assays) when compared to the other tested extracts (Wu et al., 2008[117]). On the other hand, leaves and flowers of S. acmella MeOH extracts showed weak antioxidant activity (Nanasombat and Teckchuen, 2009[56]). The aerial parts of S. acmella were also investigated (Prachayasittikul et al., 2009[68]; Wongsawatkul et al., 2008[115]). The tested extracts (hexane, CHCl3, EtOAc and MeOH) exhibited antioxidant activity as indicated by DPPH and SOD assays. The EtOAc and MeOH extracts were shown to be the most potent antioxidants (DPPH). This could be due to the presence of phenolic and coumarin compounds that are present in the extracts (Prachayasittikul et al., 2009[68]). In addition, fractions isolated from CHCl3 extract exerted potent SOD activity, which may be attributed to the presence of triterpenoids, stigmasterol and its glucosides (Prachayasittikul et al., 2009[68]). Interestingly, the fractions from the MeOH extract which displayed strong and potent antioxidant activity as well as being shown to exhibit antimicrobial activity (Prachayasittikul et al., 2009[68]). Other medicinal plants with antioxidant activity also showed antimicrobial actions, e.g. Saraca thaipingensis (Leguminosae) (Prachayasittikul et al., 2012[72]), Polyalthia cerasoides (Annonaceae) (Prachayasittikul et al., 2010[67]) and Hydnophytum formicarum Jack. (Rubiaceae) (Prachayasittikul et al., 2008[65]).

Vasorelaxant activity

S. acmella extracts were studied for their vascular effects using rat thoracic aorta (Wongsawatkul et al., 2008[115]). The results showed that the tested extracts exhibited vasorelaxant activity via partial endothelium-induced NO and PGI2 in dose dependent manner. EtOAc extract displayed immediate vasorelaxant and the most potent antioxidant (DPPH) activities. Similar vasorelaxant and antioxidant (SOD) activities were also observed in the CHCl3 extract of the plant species (Prachayasittikul et al., 2009[68]; Wongsawatkul et al., 2008[115]). These bioactivities can be attributed to the presence of phenolic and triterpenoids (Prachayasittikul et al., 2009[68]). The other plant species of Compositae, the Eclipta prostrata Linn. were also shown to possess vasorelaxant and antioxidant activities (Prachayasittikul et al., 2010[69]). In addition, analogs of nicotinic acid (vitamin B3) and orotic acid (vitamin B13) were reported to afford vasorelaxants and antioxidants (Prachayasittikul et al., 2010[70][71]).

The plant species have been used as a powerful aphrodisiac in traditional medicinal practice for cases of sexual deficiency or depressed desire as it has been shown to improve sexual function in man (Sharma et al., 2011[93]). The study showed that S. acmella EtOH flower extract improved sexual behavior. It was suggested that alkamides may mimic the action of testosterone or stimulate the secretion of testosterone. In addition, the contribution of NO in vasorelaxation (Wongsawatkul et al., 2008[115]) may be involved in enhancing sexual performance as penile erection is directly controlled by NO (Sharma et al., 2011[94]). The study suggested possible development of S. acmella EtOH extract as therapeutics for stimulating male sexual activity (Sharma et al., 2011[94]).

Moreover, S. acmella extract is an active component in body and beauty care cosmetics as a fast-acting muscle relaxant that may be essential in accelerating the repair of functional wrinkles as well as stimulate, reorganize and strengthen the collagen network and has thus been utilized for anti aging purposes in the form of anti wrinkle cream formulations (Prachayasittikul et al., 2009[68]). Other plant species such as the Zanthoxylum bungeanum fruit husks extract are known to be rich in spilanthol that has been found to exert anti wrinkle effect owing to its capacity to relax subcutaneous muscles and act as a topical-lifting agent for wrinkles (Artaria et al., 2011[6]).

Diuretic activity

Naturally occurring diuretics such as caffeine are known to be present in coffee, tea and cola. So far in Ayurvedic practice, many indigenous drugs have been claimed to have diuretic effect. The study of S. acmella EtOH leaves extract revealed diuretic effect possibly arising from tannin, steroid and carotenoid (Vanamala et al., 2012[111]). In addition, flower cold aqueous extract of the plant species exhibited strong diuretic activity (Kumar et al., 2010[42]). The effect may be attributed to its alkaloids. It was suggested that the extract acted as a loop diuretic, which is the most powerful of all diuretics (Ratnasooriya et al., 2004[79]). However, several other diuretic plants from different families have been reported to contain triterpenoids, steroids, saponins, alkaloids, flavonoids, phenolics, glycosides and bis-benzylisoquinolines (Vanamala et al., 2012[111]).

Immunostimulant activity

S. acmella leaves have been used traditionally as tonic, treatment of rheumatism, gout and sialogogue as well as being claimed to possess immunostimulant activity (Savadi et al., 2010[88]). The investigation was performed using various experimental models. The EtOH leaves extract showed significant immunomodulatory activity by increasing macrophage count with the maximum number of cells on the 15th day (Savadi et al., 2010[88]). The leaves of S. acmella contained various compounds such as alkamides, pungent amides, carbohydrates, tannins, steroids, carotenoids, essential oils, sesquiterpenes and amino acids (Amal and Sudhendu 1998[4]; Lemos et al., 1991[44]; Nagashima and Nakatani, 1992[52]; Nagashima and Nobuji, 1991[53]; Tiwari and Kakkar, 1990[107]). It was reported that spilanthol was involved in immune stimulation and attenuation of inflammatory response in murine Raw 264.7 macrophages (Wu et al., 2008[117]). In addition, some alkamides are being consumed as to enhance immune response, for example, to relieve colds, respiratory infections and influenza (Rios, 2012[81]).

Structure-activity relationship

As stated previously, S. acmella extract and its isolates (e.g. spilanthol, flavonoids and triterpenoids) are known to be involved in many bioactivities. Particularly, antipyretic, antiinflammatory and analgesic activities arise from the capacity of compounds to inhibit COX enzymes that lead to the inhibition of prostaglandin syntheses. The well-known drug such as aspirin has been used as a nonsteroidal antiinflammatory drug, analgesics and antipyretics. Its mechanism of action had been proposed (Brenner and Stevens, 2010[15]) to irreversibly inhibit COX enzymes that are homodimeric proteins containing two identical active sites with serine residues at positions 530 (COX-1) and 516 (COX-2), forming covalent bond. Ultimately, acetyl groups of the aspirin were added to COX enzymes via nucleophilic attack of serine (OH group) to electrophilic center (carbonyl group) of the drug (Figure 3(Fig. 3)).

Considering the structures of spilanthol, contains amide carbonyl group, flavonoid, coumarin and triterpenoid (β-sitostenone), which all have electrophilic centers that could interact with serine residues of COX enzymes. Thus, the possible mode of action of spilanthol is presumably a result from the addition of serine (OH) to the carbonyl group of spilanthol with subsequent loss of amine moiety as shown in Figure 4(Fig. 4).

Based on the functional moiety of the compounds, therefore, similar enzymatic nucleophilic addition of serine (OH) to electrophilic carbonyl groups of coumarin and β-sitostenone could possibly be proposed. Besides the carbonyl group, an alcohol function of the compound such as β-sitosterol has been shown to be a stronger antiinflammatory agent than the β-sitostenone, and even stronger than the standard drug, indomethacin (Nair et al., 2012[54]). This could be due to the loss of the OH group from β-sitosterol as H2O molecules, thus forming carbocation (electrophilic center) that further reacted with the nucleophilic serine (OH) as described previously.

Another important correlation that has been observed for the compounds is aside from the antioxidant activity they also possess vasorelaxant activity (Prachayasittikul et al., 2010[69]; Wongsawatkul et al., 2008[115]). This could be attributed to the fact that antioxidant compounds inhibited the formation of peroxynitrite as a result from the reaction of NO (as superoxide scavenger) with

In Vitro Micropropagation

To date, S. acmella, with high medicinal values, is increasingly demanded worldwide as a plant-derived medicine (Tiwari et al., 2011[108]). It has been recognized as one of the most important medicinal plants of the world (Singh and Chaturvedi, 2012[96]). However, S. acmella has been itemized as an endangered plant species due to the low rate of germination and poor vegetative propagation (Rios-Chavez et al., 2003[83]), including limited availability of information of the biosynthetic pathway of alkamides (Tiwari et al., 2011[108]).

To increase the supply of S. acmella, in vitro micropropagation has been recently proposed to be a reliable and routine approach for large-scale production (Sahu et al., 2011[86]; Tiwari et al., 2011[108]). The method is a useful tool for rapid cultivation of S. acmella which provides high yield and consistent production or quality of bioactive metabolites irrespective of seasons and regions (Singh and Chaturvedi, 2012[96]) as well as conservation of genetic fidelity, long term storage and cost effectiveness (Sahu et al., 2011[86]). So far, a number of studies has been reported for successful in vitro micropropagation of S. acmella through leaf, axillary bud, and shoot tip (Sahu et al., 2011[86]). The content of spilanthol was found to be higher than the mother plant or those that are field grown (Singh and Chaturvedi, 2012[97]). Importantly, the produced spilanthol (in vitro) showed strong (100 %) antilarvicidal activity against malaria and filarial vectors (Pandey and Agrawal, 2009[61]). The methods employed different culture media, mostly using Murashige and Skoog media (MS) in combination with other growth regulators or auxins as shown in Table 6(Tab. 6) (References in Table 6: S. acmella: Singh and Chaturvedi, 2012[96]; S. acmella: Singh and Chaturvedi, 2012[97]; S. acmella: Leng et al., 2011[46]; S. acmella: Pandey et al., 2011[62]; S. acmella: Yadav and Singh, 2011[119]; S. acmella: Pandey and Agrawal, 2009[61]; S. acmella: Singh et al., 2009[98]; S. acmella: Saritha and Naidu, 2008[87]; S. acmella: Leng et al., 2004[45]; S. acmella: Haw and Keng, 2003[32]; S. calva: Tiwari et al., 2011[108]; S. mauritiana DC: Bais et al., 2002[8]).

 Conclusion

It is very fascinating that S. acmella, starting from the simple antitoothache plant to highly valuable annual herb, possesses multifunctional roles as indigenous medicine for therapeutics in health care, beauty care and cosmetics as well as health food or supplements enriched with numerous antioxidants. The most abundant isolates of the plant species were lipid alkamides, especially, the spilanthol along with other bioactive metabolites e.g. phenolic, flavonoid, coumarin and triterpenoid compounds.

Pharmacological studies revealed that such compounds exhibited an array of diverse bioactivities. Considering the data, some conclusions could be drawn that the S. acmella extracts and its constituting compounds such as spilanthol and flavonoids have been shown to possess inhibitory activity toward PG synthesis. It could be presumably proposed that these compounds share a common functional group with electrophilic center, interacting with COX enzymes through nucleophilic addition of serine residues. As a result, the syntheses of PG were inhibited subsequently contributing to the observed antiinflammatory, antipyretic and analgesic activities. In addition, spilanthol has been shown to reduce NO release and thereby inhibit inflammatory mediators and attenuating the expression of COX-2 and iNOS. This could be attributed to the immunostimulant activity of S. acmella in its traditional usages.

S. acmella exerted vasorelaxant and antioxidant activities, which is beneficial for its lifting effect as fast acting muscle relaxant in anti wrinkle and anti aging applications. The participation of NO in vasorelaxation makes S. acmella a powerful aphrodisiac in traditional medicine for improving sexual performance in men.

Structure-activity relationship of vasorelaxant and antioxidant activities of nicotinic acid derivatives were elucidated by molecular modeling. The studies provided insights on the essential molecular descriptors governing the observed biological activities. Such findings provide useful insights for the design and synthesis of robust bioactive compounds.

To supply the market demand of S. acmella as a plant-derived medicine, its preparation, purification and in vitro propagation have been discussed herein.

In brief, it could be demonstrated that S. acmella is a medicinal plant enriched with compounds having high therapeutic value that can be further developed for applications in medicines, health care, cosmetics, supplements and health food.

 Notes

Supaluk Prachayasittikul and Virapong Prachayasittikul (Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand; Telephone: 662-441-4376, Fax: 662-441-4380; virapong.pra@mahidol.ac.th) contributed equally as corresponding authors.

 Acknowledgements

This project is supported by the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative. V.P. thanks Asst. Prof. Dr. Chanin Nantasenamat for the proofreading of this manuscript.

 Abascal K Yarnell E Herbs for treating periodontal disease Alternat Comple-mentary Ther 2001 7 216 220 Abascal K Yarnell E Treatment for recurrent aphthous stomatitis Alternat Complement Ther 2010 16 100 106 Acosta-Madrid II Castañeda-Hernández G Cilia-López VG Cariño-Cortés R Pérez-Hernández N Fernández-Martínez E Interaction between Heliopsis longipes extract and diclofenac on the thermal hyperalgesia test Phytomedicine 2009 16 336 341 Amal MK Sudhendu M Analysis of free amino acid content in pollen of nine Asteraceae species of known allergenic activity Ann Agric Environ Med 1998 5 17 20 Arora S Vijay S Kumar D Phytochemical and antimicrobial studies on the leaves of Spilanthes acmella J Chem Pharm Res 2011 3 145 150 Artaria C Maramaldi G Bonfigli A Rigano L Appendino G Lifting properties of the alkamide fraction from the fruit husks of Zanthoxylum bungeanum Int J Cosmetic Sci 2011 33 328 333 Bae SS Ehrmann BM Ettefagh KA Cech NB A validated liquid chromatography-electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr Phytochem Analysis 2010 21 438 443 Bais HP Green JB Walker TS Okemo PO Vivanco JM In vitro propagation of Spilanthes mauritiana DC., an endangered medicinal herb, through axillary bud cultures In Vitro Cell Dev Biol Plant 2002 38 598 601 Barman S Sahu N Deka S Dutta S Das S Antiinflammatory and analgesic activity of leaves of Spilanthes acmella (ELSA) in experimental animal models Pharmacologyonline 2009 1 1027 1034 Belfer WA Cosmetic compositions comprising peptides and Acmella oleracea extract to accelerate repair of functional wrinkles US Pat 2007 2007048245 Bermúdez-Ocaña DY Ambriz-Tututi M Pérez-Severiano F Granados-Soto V Pharmacological evidence for the participation of NO–cyclic GMP–PKG–K+ channel pathway in the antiallodynic action of resveratrol Pharmacol Biochem Behav 2006 84 535 542 Boonen J Baert B Roche N Burvenich C De Spiegeleer B Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilanthes acmella (Compositae) extracts J Ethnopharmacol 2010 127 77 84 Boonkird S Sadakorn J Sadakorn T Thai plants 1982 Bangkok Animate Print and Design 25, 30, 32, 37, 261 Borek C Watson RR Preedy RR Garlic and aging: current knowledge and future considerations Bioactive foods in promoting health 2010 San Diego, CA Academic Press 221 234 Brenner GM Stevens CW Brenner GM Stevens CW Drugs for pain, inflammation, and arthritic disorders Pharmacology 2010 2nd Philadelphia, PA Saunders 336 337 Brooks PM Day RO Nonsteroidal antiinflammatory drugs — differences and similarities New Engl J Med 1991 324 1716 1725 Bunyapraphatsara N Chokechareunporn O Tradition medicinal plants 1999 Bangkok Prachachon Chakraborty A Devi BRK Rita S Sharatchandra K Singh TI Preliminary studies on antiinflammatory and analgesic activities of Spilanthes acmella Murr. in experimental animal models Indian J Pharmacol 2004 36 148 150 Chakraborty A Devi BRK Thokchom I Sanjebam R Khumbong S Preliminary studies on local anesthetic and antipyretic activities of Spilanthes acmella Murr. in experimental animal models Indian J Pharmacol 2010 42 277 279 Chanwitheesuk A Teerawutgulrag A Rakariyatham N Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand Food Chem 2005 92 491 497 Consult M Complementary and alternative medicine. Clinical topic tours 2003 St. Louis Elsevier D'Armour FE Smith DL A method for determining loss of pain sensation J Pharmacol Exp Ther 1941 72 74 79 Déciga-Campos M Rios MY Aguilar-Guadarrama AB Antinociceptive effect of Heliopsis longipes extract and affinin in mice Planta Med 2010 76 665 670 Demarne F Passaro G Use of an Acmella oleracea extract for the botulinum toxin-like effect thereof in an anti wrinkle cosmetic composition US patent 2009 US 7,531193 B2 Dias AMA Santos P Seabra IJ Júnior RNC Braga MEM De Sousa HC Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction J Supercrit Fluids 2012 61 62 70 Ediriweera ERHSS A review on medicinal uses of weeds in Sri Lanka Trop Agric Res Ext 2007 10 11 16 Elumalai A Pendem N Eswaraiah MC Naresh V An updated annual review on antipyretic medicinal plants (Jan-Dec. 2011) Int J Univers Pharm Life Sci 2012 2 207 215 Farnsworth N Bunyapraphatsara N Thai medicinal plants recommended for primary healthcare system 1992 Bangkok Prachachon Ferrazzano G Amato I Ingenito A Zarrelli A Pinto G Pollio A Plant polyphenols and their anticariogenic properties: A review Molecules 2011 16 1486 1507 Ganesh M Vasudevan M Kamalakannan K Kumar AS Vinoba M Ganguly S Antiinflammatory and analgesic effects of Pongamia glabra leaf gall extract Pharmacologyonline 2008 1 497 512 Gokhale V Bhide B Chemical investigation of Spilanthes acmella (Murr.) J Indian Chem Soc 1945 22 250 252 Haw AB Keng CL Micropropagation of Spilanthes acmella L., a bio-insecticide plant, through proliferation of multiple shoots J Appl Hort 2003 5 65 68 Hernández I Márquez L Martínez I Dieguez R Delporte C Prieto S Antiinflammatory effects of ethanolic extract and alkamides-derived from Heliopsis longipes roots J Ethnopharmacol 2009 124 649 652 Hernandez-Pacheco A Araiza-Saldana CI Granados-Soto V Mixcoatl-Zecuatl T Possible participation of the nitric oxide-cyclic GMP-protein kinase G-K+ channels pathway in the peripheral antinociception of melatonin Eur J Pharmacol 2008 596 70 76 Jagan Rao N Subash KR Sandeep Kumar K Role of phytotherapy in gingivitis: A review Int J Pharmacol 2012 8 1 5 Jain JB Kumane SC Bhattacharya S Medicinal flora of Madhya Pradesh and Chattisgarh-A review Indian J Tradit Knowl 2006 5 237 242 Jyothi G William MC Ravi KB Krishna MG Antinociceptive and antiinflam-matory activity of methanolic extract of leaves of Shorea Robusta Pharmacologyonline 2008 1 9 19 Kohli K Ali J Ansari MJ Raheman Z Curcumin: A natural antiinflammatory agent Indian J Pharmacol 2005 37 141 147 Krishnaswami NR Prasanna S Seahadri TR Vedantham TNC α- and β-Amyrin esters and sitosterol glucoside from Spilanthes acmella Phytochemistry 1975 14 1666 1667 Kroll D Using alternative therapies in your dental practice: Holistic dentistry making gains Alternat Complement Ther 1995 233 237 Kubra IR Rao LJM An impression on current developments in the technology, chemistry, and biological activities of ginger (Zingiber officinale Roscoe) Crit Rev Food Sci Nutr 2011 52 651 688 Kumar BNS Swamy BMV Swamy A Murali A A review on natural diuretics Res J Pharm Biol Chem Sci 2010 1 615 634 Lee S-J Umano K Shibamoto T Lee K-G Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties Food Chem 2005 91 131 137 Lemos TLG Pessoa ODL Matos FJA Alencar JW Craveiro AA The essential oil of Spilanthes acmella Murr J Essent Oil Res 1991 3 369 370 Leng T Haw A Keng CL Effect of reduced N6-benzyladenine, explant type, explant orientation, culture temperature and culture vessel type on regeneration of adventitious shoot and in vitro plantlets of Spilanthes acmella J Plant Biol 2004 47 15 20 Leng TC Ping NS Lim BP Keng CL Detection of bioactive compounds from Spilanthes acmella (L.) plants and its various in vitro culture products J Med Plant Res 2011 5 371 378 Little JW Complementary and alternative medicine: Impact on dentistry Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004 98 137 145 Matchacheep S Herbs 1991 Bangkok Praepittaya 61, 213, 236 Mbeunkui F Grace MH Lategan C Smith PJ Raskin I Lila MA Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS J Chromatogr B 2011 879 1886 1892 McFerren MA Cordova D Rodriguez E Rauh JJ In vitro neuropharmacological evaluation of piperovatine, an isobutyl-amide from Piper piscatorum (Piperaceae) J Ethnopharmacol 2002 83 201 207 Mukharya DKK Ansari AH Olean-12-en-3-O-ß-D-Galactopyranosyl (1-4)-O-a-l- rhamnopyranoside: a new triterpenoidal saponin from the roots of Spilanthes acmella (Murr) Indian J Chem 1986 26 81 87 Nagashima M Nakatani N LC-MS analysis and structure determination of pungent alkamides from Spilanthes acmella L. flowers Lebens Wiss Technol 1992 25 417 421 Nagashima M Nobuji N Two sesquiterpenes from Spilanthes acmella L Chem Express 1991 6 993 996 Nair JJ Aremu AO Van Staden J Anti-inflammatory effects of Leucosidea sericea (Rosaceae) and identification of the active constituents S Afr J Bot 2012 80 75 76 Nakatani N Nagashiwa M Pungent alkamides from Spilanthes acmella L. Var. Clark Biosci Biotech Biochem 1992 56 759 762 Nanasombat S Teckchuen N Antimicrobial, antioxidant and anticancer activities of Thai local vegetables J Med Plants Res 2009 3 443 449 Narayana KR Reddy MS Chaluvadi MR Krishna DR Bioflavonoids classification, pharmacological, biochemical effects and therapeutic potential Indian J Pharmacol 2001 33 2 16 Ortiz MI Medina-Tato DA Sarmiento-Heredia D Palma-Martinez J Granados-Soto V Possible activation of the NO-cyclic GMP-protein kinase G-K+ channels pathway by gabapentin on the formalin test Pharmacol Biochem Behav 2006 83 420 427 Ospina De Nigrinis L Olarte J Nunez O Phytopharmacologic study of flower lipid soluble fractions from Spilanthes americana (Mutis). Part 1. Phytochemical study Rev Columb Cien Quim Farm 1986 15 37 47 Özbek B Dadali G Thin-layer drying characteristics and modelling of mint leaves undergoing microwave treatment J Food Eng 2007 83 541 549 Pandey V Agrawal V Efficient micropropagation protocol of Spilanthes acmella L. possessing strong antimalarial activity In Vitro Cell Dev Biol Plant 2009 45 491 499 Pandey V Chopra M Agrawal V In vitro isolation and characterization of biolarvicidal compounds from micropropagated plants of Spilanthes acmella Parasitol Res 2011 108 297 304 Perry E Milner L Houghton P From ancient texts to modern phytotherapy: plants in mind. Herbal medicine: a concise overview for professionals 2000 Oxford Butterworth & Heinemann Phongpaichit S Subhadhirasakul S Wattanapiromsakul C Antifungal activities of extracts from Thai medicinal plants against opportunistic fungal pathogens associated with AIDS patients Mycoses 2005 48 333 338 Prachayasittikul S Buraparuangsang P Worachartcheewan A Isarankura-Na-Ayudhya C Ruchirawat S Prachayasittikul V Antimicrobial and antioxidative activities of bioactive constituents from Hydnophytum formicarum Jack Molecules 2008 13 904 921 Prachayasittikul S Manam P Chinwor-rungsee M Isarankura-Na-Ayudhya C Ruchirawat S Prachayasittikul V Bio-active azafluorenone alkaloids from Polyalthia debilis (Pierre) Finet & Gagnep Molecules 2009 14 4414 4424 Prachayasittikul S Saraban P Cherdtra-kulkiat R Ruchirawat S Prachayasittikul V New bioactive triterpenoids and antimalarial activity of Diospyros rubra Lec EXCLI J 2010 9 1 10 Prachayasittikul S Suphapong S Wora-chartcheewan A Lawung R Ruchirawat S Prachayasittikul V Bioactive metabolites from Spilanthes acmella Murr Molecules 2009 14 850 867 Prachayasittikul S Wongsawatkul O Suksrichavalit T Ruchirawat S Prachayasittikul V Bioactivity evaluation of Eclipta prostrata linn: A potential vasorelaxant Eur J Sci Res 2010 44 167 176 Prachayasittikul S Wongsawatkul O Worachartcheewan A Nantasenamat C Ruchirawat S Prachayasittikul V Eluci-dating the structure-activity relationships of the vasorelaxation and antioxidation properties of thionicotinic acid derivatives Molecules 2010 15 198 214 Prachayasittikul S Wongsawatkul O Worachartcheewan A Ruchirawat S Prachayasittikul V Vasorelaxation and superoxide scavenging activities of orotic acid Int J Pharmacol 2010 6 375 380 Prachayasittikul S Worachartcheewan A Yainoy S Lomchoey N Kittiphatcharin P Ruchirawat S Antioxidant and antimicrobial activities of Saraca thaipingensis Cantley ex Prain Asian Pac J Trop Biomed 2012 2 S796 S799 Purabi D Kalita MC In vitro clonal propagation and organogenesis in Spilanthes acmella (L) Murray: a herbal pesticidal plant of North-East India J Plant Biochem Biotech 2005 14 69 71 Rabinowitch HD Kamenetsky R Rabinowitch HD Currah L Shallot (Allium cepa, Aggregratum group) Allium crop science: recent advances 2002 New York CABI Publishing 409 410 Rahmatullah M Rahman MA Hossan MS Taufiq-Ur-Rahman M Jahan R Mollik MAH A pharmacological and phyto-chemical evaluation of medicinal plants used by the Harbang clan of the Tripura tribal community of Mirsharai area, Chittagong district, Bangladesh J Alternat Complement Med 2010 16 769 785 Ramsewak RS Erickson AJ Nair MG Bioactive N-isobutylamides from the flower buds of Spilanthes acmella Phytochemistry 1999 51 729 732 Rani SA Murty SU Antifungal potential of flower head extract of Spilanthes acmella Linn Afr J Biomed Res 2006 9 67 69 Ratnasooriya WD Pieris KPP Attenuation of persistent pain and hyperalgesia by Spilanthus acmella flowers in rats Pharm Biol 2005 43 614 619 Ratnasooriya WD Pieris KPP Samaratunga U Jayakody JRAC Diuretic activity of Spilanthes acmella flowers in rats J Ethnopharmacol 2004 91 317 320 Research CoSI The wealth of India, raw materials 1976 2 New Delhi Publication and Information Directorate, Council of Scientific Industrial Research 11 12 Rios MY Natural alkamides: Pharmacology, chemistry and distribution. Drug discovery research in pharmacognosy. Vallisuta O, Olimat SM InTech 2012 107 144 Rios MY Aguilar-Guadarrama AB Gutiérrez MdC Analgesic activity of affinin, an alkamide from Heliopsis longipes (Compositae) J Ethnopharmacol 2007 110 364 367 Rios-Chavez P Ramirez-Chavez E Armenta-Salinas C Molina-Torres J Acmella radicans var. radicans: In vitro culture establishment and alkamide content In Vitro Cell Dev Biol Plant 2003 39 37 41 Rosas-Piñón Y Mejía A Díaz-Ruiz G Aguilar MI Sánchez-Nieto S Rivero-Cruz JF Ethnobotanical survey and antibacterial activity of plants used in the Altiplane region of Mexico for the treatment of oral cavity infections J Ethnopharmacol 2012 141 860 865 Sadavongvivad C Supavilai P Three monohydroxycoumarins from Alyxia lucida Phytochemistry 1977 16 1451 Sahu J Jain K Jain B Sahu RK A review on phytopharmacology and micropro-pagation of Spilanthes acmella Pharmacologyonline newslett 2011 2 1105 1110 Saritha KV Naidu CV Direct shoot regeneration from leaf explants of Spilanthes acmella Biol Plant 2008 52 334 338 Savadi R Yadav R Yadav N Study on immunomodulatory activity of ethanolic extract Spilanthes acmella Murr. Leaves Indian J Nat Prod Resour 2010 1 204 207 Sawangjaroen N Phongpaichit S Subhadhirasakul S Visutthi M Srisuwan N Thammapalerd N The antiamoebic activity of some medicinal plants used by AIDS patients in southern Thailand Parasitol Res 2006 98 588 592 Schneider I Bucar F Lipoxygenase inhibitors from natural plant sources. Part 1: Medicinal plants with inhibitory activity on arachidonate 5-lipoxygenase and 5-lipoxygenase/cyclooxygenase Phytother Res 2005 19 81 102 Schubnel L A different approach to lifting efficacy based on a natural active ingredient SOFW J 2007 133 Senthilkumar P Paulsamy S Vijayakumar KK In vitro regeneration of the medicinal herb of Nilgiri shola, Acmella calva L. from leaf derived callus Plant Tissue Cult Biotechnol 2007 17 109 114 Sharma R Medicinal plants of India - an encyclopedia 2003 Delhi Daya Publishing House Sharma V Boonen J Chauhan NS Thakur M De Spiegeleer B Dixit VK Spilanthes acmella ethanolic flower extract: LC-MS alkylamide profiling and its effects on sexual behavior in male rats Phytomedicine 2011 18 1161 1169 Shimada T Gomi T Spilanthol-rich essential oils for manufacturing toothpastes or other oral compositions JP Pat 1995 JP Pat. 07090294 Singh M Chaturvedi R Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthes acmella Murr Bioprocess Biosyst Eng 2012 35 943 951 Singh M Chaturvedi R Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr Industrial Crops and Products 2012 36 321 328 Singh S Majumdar DK Rehan HMS Evaluation of antiinflammatory potential of fixed oil of Ocimum sanctum (Holybasil) and its possible mechanism of action J Ethnopharmacol 1996 54 19 26 Singh S Rai M Asthana P Pandey S Jaiswal VS Jaiswal U Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species Acta Physiol Plant 2009 31 649 653 Spelman K Depoix D McCray M Mouray E Grellier P 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 2011 25 1098 1101 Stashenko EE Puertas MA Combariza MY Volatile secondary metabolites from Spilanthes americana obtained by simultaneous steam distillation-solvent extraction and supercritical fluid extraction J Chromatogr A 1996 752 223 232 Suksrichavalit T Prachayasittikul S Nantasenamat C Isarankura-Na-Ayudhya C Prachayasittikul V Copper complexes of pyridine derivatives with superoxide scavenging and antimicrobial activities Eur J Med Chem 2009 44 3259 3265 Suksrichavalit T Prachayasittikul S Piacham T Isarankura-Na-Ayudhya C Nantasenamat C Prachayasittikul V Copper complexes of nicotinic-aromatic carboxylic acids as superoxide dismutase mimetics Molecules 2008 13 3040 3056 Sun L Rezaei KA Temelli F Ooraikul B Supercritical fluid extraction of alkylamides from Echinacea angustifolia J Agric Food Chem 2002 50 3947 3953 Thanaphum V Muengwongyard P Herbal trees 2006 Bangkok Samcharoenphanit 406, 409 Thiengburanathum W The encyclopedia of Thai herbs 1999 Bangkok Auksorn Pittaya 97, 320, 325, 375, 415, 475, 511, 559, 586, 598, 609, 619, 623, 645, 646, 788, 842 Tiwari H Kakkar A Phytochemical examination of Spilanthes acmella Murr J Indian Chem Soc 1990 67 784 785 Tiwari KL Jadhav SK Joshi V An updated review on medicinal herb genus Spilanthes Chin J Integr Med 2011 9 1170 1178 Trease G Evans W Phenolic compounds and tannins 1972 London Ballilliere Trindall Tyagi A Delanty N Herbal remedies, dietary supplements, and seizures Epilepsia 2003 44 228 235 Vanamala U Elumalai A Eswaraiah MC Shaik A An updated review on diuretic plants-2012 Int J Pharm Biol Arch 2012 3 29 31 Wagner H Search for new plant constituents with potential antiphlogistic and antiallergic activity Planta Med 1989 55 235 241 Wang Z Lu X Lei A Zhang Z Efficient preparation of functionalized (E,Z) dienes using acetylene as the building block J Org Chem 1998 63 3806 3807 Witkin LB Heubner CF Galdi F O'Keefe E Spitaletta P Plummer AJ Pharmacology of 2-amino-indane hydrochloride (SU-8629): a potent non-narcotic analgesic J Pharmacol Exp Ther 1961 133 400 408 Wongsawatkul O Prachayasittikul S Isarankura-Na-Ayudhya C Satayavivad J Ruchirawat S Prachayasittikul V Vasorelaxant and antioxidant activities of Spilanthes acmella Murr Int J Mol Sci 2008 9 2724 2744 Worachartcheewan A Prachayasittikul S Pingaew R Nantasenamat C Tantimongcolwat T Ruchirawat S Antioxidant, cytotoxicity, and QSAR study of 1-adamantylthio derivatives of 3-picoline and phenylpyridines Med Chem Res 2012 21 3514 3522 Wu LC Fan NC Lin MH Chu IR Huang SJ Hu CY Antiinflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators J Agric Food Chem 2008 56 2341 2349 Yadav K Singh B Micropropagation of Spilanthes acmella Murr. – An important medicinal plant Nature and Science 2010 8 5 11 Yadav K Singh N In vitro flowering of shoots regenerated from cultured nodal explants of Spilanthes acmella Murr.- an ornamental medicinal herb An U O Fasc Biol 2011 18 Yuliana ND Jahangir M Korthout H Choi YH Kim HK Verpoorte R Comprehensive review on herbal medicine for energy intake suppression Obes Rev 2011 12 499 514 Traditional uses and applications of <italic>S. acmella</italic> Thai medicinal plants for dental application Spilanthol from plant species Antibacterial activity<sup>a </sup>of Compositae plants Antifungal activity of <italic>S. acmella</italic> <italic>In vitro</italic> production of<italic> Spilanthes</italic> Structure of spilanthol and derivatives Bioactive metabolites isolated from <italic>S. acmella</italic> Proposed mechanism of action of aspirin Possible mode of action of spilanthol