High Therapeutic Potential of Spilanthes Acmella: a Review

Home , Acmella, Acmella oleracea, Eclipta, Heliopsis, Heliopsis longipes, Spilanthes

EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Review article:

HIGH THERAPEUTIC POTENTIAL OF SPILANTHES ACMELLA: A REVIEW

Veda Prachayasittikul1, Supaluk Prachayasittikul2*,Somsak Ruchirawat3, Virapong Prachayasittikul1*

1 Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand 2 Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand 3 Laboratory of Medicinal Chemistry, Chulabhorn Research Institute and Chulabhorn Graduate Institute, Bangkok 10210, Thailand

* corresponding authors: 1 E-mail: [email protected]; Telephone: 662-441-4376, Fax: 662-441-4380 2 E-mail: [email protected]; Telephone: 662-441-4376, Fax: 662-441-4380

ABSTRACT 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.

Keywords: Spilanthes acmella, bioactive metabolites, bioactivities, structure-activity relationships, in vitro production

INTRODUCTION The World Health Organization has esti- mated that about 80 % of the population in Plants contain a diverse group of highly developing countries are unable to afford valuable and readily available resource of drugs and rely on traditional medicines es- bioactive metabolites, e.g. alkaloids, tan- pecially those that are plant-based (Eluma- nins, essential oils and flavonoids (Jagan lai et al., 2012) such as India (Jain et al., Rao et al., 2012; Prachayasittikul et al., 2006; Little, 2004), Sri Lanka (Ediriweera 2008, 2009b 2010a), which have been used 2007), Bangladesh (Rahmatullah et al., in medicinal practices for a long time 2010), China and Japan (Little, 2004) in- (Tiwari et al., 2011). Thus far, medicinal cluding Thailand (Phongpaichit et al., plant is an alternative medicine (Consult, 2005; Sawangjaroen et al., 2006). 2003) that is still in use and is a popular The practice of botanical healing slowly choice for primary health care(Vanamala disappeared from western countries with et al., 2012). However, if improperly used the introduction and advent of science and plants can also be toxic (Perry et al., 2000). technology (Tyagi and Delanty, 2003). 291 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

However, the uses of traditional medicine employed as therapeutics and health care dramatically increased in Europe and (Abascal and Yarnell, 2010). Therefore, North America in the last 50 years (Tyagi Spilanthes acmella Murr. is a plant of great and Delanty, 2003). interest owing to its known reputation as Herbal medicines have been utilized for an antitoothache plant and hold tremen- many purposes, particularly in medical dous medicinal usages. This review focus- care as antiasthmatics (86.79 %), anti- es on the general background, therapeutic rheumatics (62 %) (Jain et al., 2006), diu- uses, bioactive compounds and large-scale retics (60.22 %) (Kumar et al., 2010; production. Vanamala et al., 2012), antiinflammation (29.62 %), anticancer (9.75 %), antidiabe- General tics (8.33 %), antimicrobials, antifungals, Spilanthes (Compositae or Asteraceae) antioxidants, antiallergy, analgesics, anti- is a genus comprising of over 60 species obesity and antihypertention. In dental care that are widely distributed in tropical and it has been employed as anticariogenic subtropical regions of the world, such as (Ferrazzano et al., 2011), analgesic Africa, America, Borneo, India, Sri Lanka (Abascal and Yarnell, 2010; Kroll, 1995), and Asia (Sahu et al., 2011; Tiwari et al., local anesthetic (Abascal and Yarnell, 2011). S. acmella is native to Brazil and is 2010), wound healing agents (Abascal and cultivated throughout the year as ornamen- Yarnell, 2001, 2010; Jagan Rao et al., tal or medicinal plant. It is an annual or 2012), antiinflammation (Abascal and short-lived herb that is 40-60 centimeters Yarnell 2001, 2010) and recurrent aph- tall. It is grown in damp area (Tiwari et al., thous stomatitis treatment (Abascal and 2011; Wongsawatkul et al., 2008) and has Yarnell 2010). It has also been used for low rate of germination or poor vegetative beauty care (Artaria et al., 2011; Demarne propagation (Tiwari et al., 2011). Its flow- and Passaro, 2009) and as health food e.g. ers and leaves have pungent taste and curcumin (Curcuma longa Linn.) (Kohli et when touched it is accompanied by tin- al., 2005), ginger (Zingiber officinale) gling sensation and numbness (Wong- (Kubra and Rao, 2011), lemon grass sawatkul et al., 2008). The plant species (Cymbopogon citrates Stapf) (Nanasombat has been used commonly as a folk remedy, and Teckchuen, 2009), green shallot (Alli- e.g. for toothache, rheumatic and fever um cepa var. aggregatum) (Rabinowitch (Wongsawatkul et al., 2008), as fresh and Kamenetsky, 2002), garlic (Allium sa- vegetable (Tiwari et al., 2011) as well as tivum L.) (Borek, 2010), holy basil (Oci- spice for Japanese appetizer (Leng et al., mum sanctum Linn.) (Singh et al., 1996), 2011). sweet basil (Ocimum basilicum L.) (Lee et al., 2005), hairy basil (Ocimum basilicum Traditional uses L.f. var. citratum Back.) (Chanwitheesuk The whole plants (e.g. flowers, leaves, et al., 2005) and kitchen mint (Mentha roots, stems and aerial parts) of Spilanthes cordifolia Opiz.) (Özbek and Dadali, have been used in health care (Leng et al., 2007). 2004; Ospina De Nigrinis et al., 1986; Recently, health foods, herbs as well as Purabi and Kalita, 2005; Research, 1976; dietary supplements enriched with medici- Rios-Chavez et al., 2003; Senthilkumar et nal ingredients such as antioxidants and al., 2007; Tiwari and Kakkar, 1990) and bioactive metabolites have drawn consid- food (Barman et al., 2009; Boonen et al., erable attention worldwide, especially 2010; Wu et al., 2008).Particularly, S. ac- herbs that are used as food and traditional mella or S. oleracea (paracress or eyeball medicine (Tyagi and Delanty, 2003). Our plant), is a well-known antitoothache plant concern centers around medicinal plants (Sahu et al., 2011) and has been used as bearing bioactive compounds, which are traditional medicine for many purposes

292 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

(Table 1). So far, various Thai medicinal ticularly, anandamide (N-arachidonoyl- plants have been used for the remedy of ethanolamine, 9) is an endogenous canna- toothache as well as used in dental applica- binoid cerebral neurotransmitter (Fig- tions (Table 2). ure 1). Spilanthol was first isolated in 1945 Bioactive metabolites from the flower head ethanol (EtOH) ex- Extensive phytochemical investigations tract of S. acmella. In early 1903, it was of S. acmella had previously been reported. first obtained from the different plant spe- It constitutes a diverse group of com- cies, S. acmella L.var. oleracea Clarke pounds. Major isolates were lipophilic al- (Gokhale and Bhide, 1945). Aside from kylamides or alkamides bearing different being found in S. acmella, spilanthol was number of unsaturated hydrocarbons (al- also found in other plant species as shown kenes and alkynes), such as spilanthol (1) in Table 3 (Rios, 2012). or affinin (2E,6Z,8E)-N-isobutyl-2,6,8- The synthesis of spilanthol was reported decatrienamide (Gokhale and Bhide, 1945; in multistep and afforded low overall Ramsewak et al., 1999) and amide deriva- yields. However, an efficient synthetic tives 2–8 (Figure 1). In general, when al- method had been developed (Wang et al., kamides are chewed, a pungent taste is re- 1998). Thus far, the spilanthol is commer- leased and causes itch and salivation (Rios, cially available in form of alcoholic (65 % 2012). Alkamides are structurally related EtOH) extract or A. Vogel Spilanthes. to animal endocannabinoids and is highly active in the central nervous system. Par-

Table 1: Traditional uses and applications of S. acmella Health care Treatment Plant References extract

Medical Rheumatism, fever leaves, Bunyapraphatsara and Chokechareunporn, Diuretics flowers 1999; Farnsworth and Bunyapraphatsara, 1992 Flu, cough, rabies diseases, Yadav and Singh, 2010 Tuberculosis, antimalarials, Haw and Keng, 2003 Antibacterials Antifungals, skin diseases leaves Tiwari et al., 2011 Immunomodulatory Sahu et al., 2011 Antiscorbutic Leng et al., 2011; Sahu et al., 2011 Local anesthetics Tiwari et al., 2011 Digestive Leng et al., 2011; Sahu et al., 2011 Obesity control flowers Yuliana et al., 2011 (lipase inhibitor) Snake bite whole plant Tiwari et al., 2011 Dental Toothache leaves, Haw and Keng, 2003; Tiwari et al., 2011 flower Toothpaste leaves Savadi et al., 2010 Periodontal disease flower Abascal and Yarnell, 2001; Sahu et al., 2011; heads, roots Shimada and Gomi, 1995 Recurrent aphthous stomati- leaves Abascal and Yarnell, 2010 tis Beauty care Fast acting muscle relaxant whole plant Belfer, 2007 cosmetics Anti wrinkle Demarne and Passaro, 2009; Schubnel, 2007

293 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Table 2: Thai medicinal plants for dental application Plant species Common Thai name Part used Treatment and usage family name

1Barleria lupulina Linn. Barleria Saled-pang-porn whole plant toothache, oral ulceration, oral Acanthaceae diseases

1Cocos nucifera Linn. Coconut Ma-phrao oil from coconut toothache Arecaceae shell, root 1Helianthus annuus Linn. Sunflower Tan-tawan flower head toothache Asteraceae 1Averrhoa carambola Star fruit Ma-fuang fruit toothache, scurvy, oral ulceration Linn. Averrhoaceae 1Spilanthes acmella Murr. Paracress Phak-krad leaf, flower, root, fever, toothache Compositae whole plant potential local anesthetic 1Citrullus lanatus Mats & Watermelon Tang-mo fruit toothache, oral ulceration Nakai Cucurbitaceae 1Ocimum canum L., Holy basil, Mang-lak whole plant whole plant O. basilicum L. Sweet basil Labiatae 2Ocimum sanctum Linn. Holy basil Kra-prao leaf Toothpaste and mouthwash Labiatae ingredients 3Mentha cordifolia Kitchen mint Sa-ra-nhae leaf toothache Opiz.ex Fresen Labiatae 4Cinnamomum bejolghota Cinnamon Oub-choei Root, bark dissolve sputum, toothpaste, (Buch.-Ham.) mouthwash and chewing gum Lauraceae ingredient 2Cinnamomum camphora Camphor tree Kara-boon leaf, seed toothache, gingivitis (Linn.) Presl Lauraceae 1Tinospora crispa Linn. Tinospora Bora-ped leaf, flower periodontitis, toothache Menispermaceae stem 1Streblus asper Lour. Toothbrush Khoi stem bud toothache, gingivitis, antimicrobial Moraceae tree in oral cavity, toothpaste ingredient 1Psidium guajava Linn. Guava Fah-rhang leaf, fruit, leaf toothache, halitosis, scurvy, gin- Myrtaceae givitis, toothpaste ingredient

2Syzygium aromaticum Clove Kan–plu flower toothache, scurvy, toothpaste Linn.Eugenia caryophyl- and mouthwash ingredients lus (Sprenge) Bullock et Harrison Myrtaceae 2Murraya paniculata Linn. Orange jas- Keaw leaf toothache Jack mine Rutaceae 1Solanum melongena Egg plant Ma-khau-yao stem, root, flower toothache, oral ulceration Linn. Solanaceae

1(Thiengburanathum, 1999), 2(Boonkird et al., 1982),3 (Matchacheep, 1991), 4 (Thanaphum and Muengwongyard, 2006)

In addition, phytosterols (e.g. β- In recent years, other bioactive metabo- sitosterol, stigmasterol, α- and β-amyrins), lites 10 – 15 (Figure 2) have been isolated essential oils (e.g. limonene and β- from the aerial part of S. acmella, namely caryophyllene), sesquiterpenes, α-and β vanillic acid (10), trans-ferulic acid (11), bisabolenes and cadinenes, flavonoid glu- trans-isoferulic acid (12), scopolelin (13), coside and a mixture of long chain hydro- 3-acetylaleuritolic acid (14) and β- carbons (C22-C35) were reported (Sahu et sitostenone (15) (Prachayasittikul et al., al., 2011; Tiwari et al., 2011). 2009b).

294 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Figure 2: Bioactive metabolites isolated from S. acmella

Antipyretic activity Many medicinal plants have long been used as antipyretics, e.g. S. acmella (flower Figure 1: Structure of spilanthol and deriva- and aerial aqueous) extracts (Chakraborty tives et al., 2010). In general, pyrexia or fever is caused by a secondary impact of infection, tissue damage, inflammation, graft rejec- Table 3: Spilanthol from plant species tion, malignancy and other diseases Tribe Genus Species (Elumalai et al., 2012). These impacts ini- tiate the formation of pro-inflammatory Ecliptinae Less Welelia parviceps mediators or in particular cytokines (i.e. Galinsoginae B. and Acmella ciliata interleukin 1β, α, β, and TNF-α). This re- H. oleracea oppositifolia sults in an increase of prostaglandin E2 radicans (PGE2) synthesis and ultimately increases Zinniinae B. and H. Heliopsis longipes the body temperature (Elumalai et al., 2012). The studies showed that S. acmella (aerial aqueous extract) displayed antipy- Bioactivity retic activity against Brewer’s yeast- The Spilanthes genera have been used induced pyrexia. The antipyretic activity of for the treatment of various disorders in- the plant species can be attributed to flavo- cluding life-threatening diseases. Diverse noids (Narayana et al., 2001; Trease and pharmacological activities of this plant Evans, 1972), which were predominant species were previously reported (Sahu et inhibitors of either cyclooxygenase (COX) al., 2011; Tiwari et al., 2011). Selected bi- or lipoxygenase (LOX) (Sadavongvivad oactivities of S. acmella are summarized and Supavilai, 1977). Flavonoids are below. known to target prostaglandins in the late

295 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

phase of acute inflammation and pain nisms of acute inflammation consists of (Chakraborty et al., 2004). two phases involve with histamine, seroto- nin and kinin, released in the first hour (Ganesh et al., 2008) and with prostaglan- Antiinflammatory activity din-like substances that are released in the Spilanthol is the main constituent iso- second and third hours. Therefore the anti- lated from many parts of S. acmella such inflammatory action of S. acmella may as flower 85 % EtOH extract(Wu et al., take part in the later phase via inhibition of 2008), root hexane extract (Wagner, 1989) COX enzyme (Brooks and Day, 1991). and also from other plants such as Heliop- A recent study in 2012 has shown that sis longipes root EtOH extract (Hernández triterpenoids, namely β-sitosterol and β- et al., 2009). Traditional usages of S. ac- sitostenone isolated from Leucosidea seri- mella flowers have been reported as anti- cea (Rosaceae), exhibited antiinflam- inflammatory agent (Sharma, 2003). Pre- matory activity via inhibitions of COX-1 vious investigations demonstrated that and COX-2. The study employed indo- spilanthol exerted antiinflammatory action methacin as a standard drug and the results via inhibition of NF-κB pathway; afforded showed that β-sitosterol displayed stronger reduction in mRNA level and protein ex- antiinflammatory activity than the standard pression of COX-2 and iNOS; and also drug (Nair et al., 2012). induced free radical scavenging activity

(Wu et al., 2008). Most antiinflammatory medicinal plants possessed LOX and COX Analgesic activity inhibitors such as Asteraceae, Apiaceae, A number of antitoothache plants has Lamiaceae and Fabaceae (Schneider and been recognized, S. acmella is one of these Bucar, 2005). Plant species affording such plants that has been used in pain relief. The properties is the H. longipes root extract studies showed that S. acmella EtOH and its isolated spilanthol (Hernández et leaves extracts exerted significant centrally al., 2009). The antiinflammatory activity of (e.g. tail flick method) and peripherally spilanthol can be attributed to its dual inhi- (e.g. Writhing test) analgesic activities (D' bition of COX and LOX owing to the simi- Armour and Smith, 1941; Witkin et al., lar structures of spilanthol and arachidonic 1961). The mechanism of action was pos- acid, in which the arachidonic acid is a sibly due to the presence of flavonoids in precursor of prostaglandin and leukotriene the plant extract (Chakraborty et al., 2004) syntheses (Hernández et al., 2009). Inter- which decreases prostaglandins, PGE2 and estingly, the H. longipes extract displayed PGF2 that are known to be involved in stronger antiinflammatory activity than pain perception (Jyothi et al., 2008). In ad- that of the spilanthol. This was possibly dition, cold aqueous extract of S. acmella due to synergistic effects of the containing flowers also displayed antinociceptive ac- compounds in the plant extract (Hernández tivity against persistent pain and antihy- et al., 2009). Moreover, EtOH extract from peralgesic activity. The mechanism of ac- the leaves of S. acmella exhibited signifi- tion was possibly through inhibition of cant antiinflammatory activity against prostaglandins by spilanthol-containing acute (carragenan induced rat paw edema extract (Ratnasooriya and Pieris, 2005). method), sub-acute (granuloma pouch Another study of antitoothache plant H. method) and chronic (adjuvant arthritis longipes revealed that it was used as local method) inflammation (Barman et al., anesthetic and analgesic in Mexican indig- 2009) but has been shown to be less than enous medicine and the results showed that that of aspirin. The observed antiinflamma- its stem acetone extract and spilanthol tory activity originates from the inherent from root displayed dose-dependent an- flavonoids that are found in the plant ex- tinociceptive effect in mice as assessed by tracts (Chakraborty et al., 2004). Mecha- Writhing and capsaicin tests (Déciga-

296 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Campos et al., 2010). Possible mechanisms whereas the MeOH extract showed compa- of action of the spilanthol and H. longipes rable activity with doxycycline. This could extract may be attributed to the activation be due to the fact that the plants contain of opioidergic, serotoninergic and GA- flavonoids, tannins, and other phytochemi- BAergic systems as well as the K+channel cals, which are well-known antimicrobials. opening facilitated by nitric oxide (NO) On the other hand, aerial parts of EtOAc induced cGMP production (Acosta-Madrid and MeOH extracts from S. acmella tested et al., 2009; Rios et al., 2007). Partial par- by the agar dilution method were shown to ticipation of cGMP and K+channel in the be inactive antimicrobials whereas its chlo- antinociceptive activity of spilanthol was roform (CHCl3) extract displayed antimi- proposed for several drugs (Bermúdez- crobial activity against Streptococcus py- Ocaña et al., 2006; Hernandez-Pacheco et ogenes with MIC of 256 μg/mL al., 2008; Ortiz et al., 2006). (Prachayasittikul et al., 2009b). In addition, hexane and CHCl3 extracts exhibited antifungal activity (MIC 256 μg/mL) Local anesthetic activity S. acmella is known to be constituted of against Saccharomyces cerevisiae pungent alkamide-like spilanthol that caus- (Prachayasittikul et al., 2009b).Moreover, and EtOAc ex- es numbness and tingle. Local anesthetic isolated fractions of CHCl3 tracts selectively inhibited the growth of activity was studied in animal models Corynebacterium diphtheriae NCTC through intracutaneous wheal in guinea 10356 with MIC range 64-256 μg/mL pigs and plexus anesthesia in frogs (Prachayasittikul et al., 2009b). Another (Chakraborty et al., 2010). S. acmella aeri- study showed that leaves/flowers MeOH al aqueous extract exhibited significant extract of the plant species displayed no activity that could be due to the presence antimicrobial action (disk diffusion meth- of alkamides (Chakraborty et al., 2010). od) (Nanasombat and Teckchuen, 2009). However, its onset of action was slower Medicinal plants for treatments of oral than that of xylocaine, the standard drug. cavity infections, dental caries and perio- The well-recognized local anesthetics dontal diseases were reported (Rosas- are comprised of mostly amide compounds Piñón et al., 2012). The most frequently such as xylocaine (lidocaine). Its mecha- used were species from many families: nism of action involves the blockage of + Myrthaceae (17.8 %), Punica (15.1 %), voltage-gated Na channels. By the same Compositae (11.3 %), Asteraceae (9.7 %), analogy, the alkamides of S. acmella ex- Piperaceae (8.6 %), Anacardiaceae tracts produced local anesthetic action pre- + (7.3 %), Fagaceae (6.9 %), Labiateae sumably through the blockage of Na (5.4 %), Leguminosae (5.1 %), Butalaceae channels. Isobutylamide and piperovatine (0.5 %) and others (6.4 %). Dental caries of other antitoothache plant (Piper piscato- and periodontal diseases are two major rum) was reported to display local anes- dental pathologies affecting humankind thetic activity through the same mecha- that arises from colonization and accumu- nism of action (McFerren et al., 2002). lation of oral microorganisms especially

Streptococcus mutans and Porphyromonas Antimicrobial activity gingivalis (Rosas-Piñón et al., 2012). Anti- Ethyl acetate (EtOAc) and methanol bacterial activity of Compositae plant ex- (MeOH) extracts from the leaves of S. ac- tracts against oral microorganisms is mella exhibited the strongest antimicrobial shown in Table 4 (Rosas-Piñón et al., activity among the tested extracts using the 2012). The data showed that S. mutans was well diffusion method against Klebsiella the most sensitive to plant extracts while P. pneumoniae (Arora et al., 2011). The gingivalis was the most resistant (Rosas- EtOAc extract had two-fold higher activity Piñón et al., 2012). than that of doxycycline, the standard drug,

297 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Antifungal activity Antimalarial activity Several parts of S. acmella were tested S. acmella is a traditional medicine used for antifungal activity (Table 5) and the in Africa and India for the treatment of ma- studies showed that S. acmella leaves laria (Spelman et al. 2011). Pharmacologi- (EtOAc and aqueous) extracts exhibited cal study showed that spilanthol (1) and better antifungal activity than the standard acetylenic alkamide (undeca-2E-ene-8,10- drug (fluconazole) against Rhizopus diynoic acid isobutylamide or UDA) (3), arrhigus and Rhizopus stolonifer (Arora et isolated from the root EtOH extract of S. al., 2011).The leaves extract also displayed acmella, displayed antimalarial activity weak activity against Aspergillus niger and against two strains of Plasmodium falcipa- Penicillium chrysogenum (Arora et al., rum (PFB strain originated from Brazil and 2011). The whole plant CHCl3 extract was chloroquine resistant, K1 strain originated shown to be active antifungal against op- from Thailand). Both compounds had a portunistic fungal infection (e.g. Micro- reported antimalarial activity with IC50 in sporum gypseum and Cryptococcus the range of 5.8-41.4 μg/mL in which the neoformans) in AIDS patients spilanthol was the most potent compound. (Phongpaichit et al., 2005). S. acmella It was reported that semi-purified com- flower head petroleum ether extract exert- pounds of S. acmella, isolated by centrifu- ed antifungal activity against A. niger, A. gal partition chromatography (CPC) and parasiticus, Fusarium moniliformis and F. electrospray ionization-ion trap-time of oxysporium (Rani and Murty, 2006). The flight-mass spectrometry (ESI-IT-TOF- antifungal activity of S. acmella extracts MS), showed significantly higher an- may be due to the presence of spilanthol tiplasmodial activity as indicated by the and alkamides (Nakatani and Nagashiwa, lower IC50 value (Mbeunkui et al. 2011). 1992), non-volatile sesquiterpenoids and This could be a result from synergistic ef- saponins (Krishnaswami et al., 1975; fects of N-alkylamides in the tested com- Mukharya and Ansari, 1986). In addition, pounds. Moreover, regenerated S .acmella aerial parts of S. acmella extracts (hexane (in vitro) root hexane extract exhibited and CHCl3) exhibited activity against Sac- 100 % larvicidal activity affording the charomycese cerevisiae (Prachayasittikul lowest values of LC50 and LC90 against et al., 2009b). malaria and filarial vectors (Pandey and Agrawal, 2009). It was suggested that the Table 4: Antibacterial activitya of Compositae regenerated plant species contained higher plants active principle content than those that are S. mutans P. gingivalis field grown. In addition, the studies demonstrated the potential of S. acmella Compositae H2O EtOH H2O EtOH extract extract extract extract for the treatment and prevention of malaria (Bae et al., 2010).

Cirsium mex- >1000 >1000 >1000 >1000 icanum DC. Antioxidant activity Iostephane 67.5 125 125 250 Antioxidant activity of S. acmella ex- heterophylla tracts obtained from polar and nonpolar (Cav.) Benth. Heterotheca 125 32.5 500 125 solvents were investigated. It was found inuloides that S. acmella flower EtOAc extract dis- Cass. played the highest free radical scavenging Coreopsis 250 62.5 >1000 >1000 activity (DPPH and ABTS assays) when mutica DC. compared to the other tested extracts (Wu Calendula 125 250 500 500 officinalis et al., 2008). On the other hand, leaves and aAntibacterial activity was determined by MIC flowers of S. acmella MeOH extracts value (μg/mL), using microdilution method. showed weak antioxidant activity H2O denoted as an aqueous

298 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

(Nanasombat and Teckchuen, 2009). The Vasorelaxant activity aerial parts of S. acmella were also investi- S. acmella extracts were studied for gated (Prachayasittikul et al., 2009b; their vascular effects using rat thoracic aor- Wongsawatkul et al., 2008). The tested ta (Wongsawatkul et al., 2008). The results extracts (hexane, CHCl3, EtOAc and showed that the tested extracts exhibited MeOH) exhibited antioxidant activity as vasorelaxant activity via partial endotheli- indicated by DPPH and SOD assays. The um-induced NO and PGI2 in dose depend- EtOAc and MeOH extracts were shown to ent manner. EtOAc extract displayed im- be the most potent antioxidants (DPPH). mediate vasorelaxant and the most potent This could be due to the presence of phe- antioxidant (DPPH) activities. Similar vas- nolic and coumarin compounds that are orelaxant and antioxidant (SOD) activities present in the extracts (Prachayasittikul et were also observed in the CHCl3 extract of al., 2009b). In addition, fractions isolated the plant species (Prachayasittikul et al., from CHCl3 extract exerted potent SOD 2009b; Wongsawatkul et al., 2008). These activity, which may be attributed to the bioactivities can be attributed to the pres- presence of triterpenoids, stigmasterol and ence of phenolic and triterpenoids its glucosides (Prachayasittikul et al., (Prachayasittikul et al., 2009b). The other 2009b). Interestingly, the fractions from plant species of Compositae, the Eclipta the MeOH extract which displayed strong prostrata Linn. were also shown to possess and potent antioxidant activity as well as vasorelaxant and antioxidant activities being shown to exhibit antimicrobial activ- (Prachayasittikul et al., 2010b). In addi- ity (Prachayasittikul et al., 2009b). Other tion, analogs of nicotinic acid (vitamin B3) medicinal plants with antioxidant activity and orotic acid (vitamin B13) were report- also showed antimicrobial actions, e.g. ed to afford vasorelaxants and antioxidants Saraca thaipingensis (Leguminosae) (Pra- (Prachayasittikul et al., 2010c, d). chayasittikul et al., 2012), Polyalthia cera- The plant species have been used as a soides (Annonaceae) (Prachayasittikul et powerful aphrodisiac in traditional medici- al., 2010a) and Hydnophytum formicarum nal practice for cases of sexual deficiency Jack. (Rubiaceae) (Prachayasittikul et al., or depressed desire as it has been shown to 2008). improve sexual function in man (Sharma et al., 2011). The study showed that S. acmella EtOH flower extract improved sexual

Table 5: Antifungal activity of S. acmella Plant extract Tested Method Microorganism Inhibition References part zone or MIC EtOAc leaves well diffusion R. arrhigus 23a Arora et al., 2011 Aqueous leaves well diffusion R. stolonifer 25a EtOAc leaves well diffusion A. niger 16 a EtOAc, MeOH, leaves well diffusion P. chrysogenum 14,12,15a petroleum ether Petroleum ether flower agar cup A. niger 20 a Rani and Murty, heads bioassay A. parasiticus 18 a 2006 F. oxysporium 23 a F.monilifermis 21 a b CHCl3 whole modified M. gypseum 256 Phongpaichit et plants agar dilution C. neoformans 128 b al., 2005 method b Hexane,CHCl3 aerial agar dilution S. cerevisiae 256 Prachayasittikul et EtOAc,MeOH parts method inactive - al., 2009b a inhibition zone (mm.), b MIC(μg/mL)

299 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

behavior. It was suggested that alkamides and bis-benzylisoquinolines (Vanamala et may mimic the action of testosterone or al., 2012). stimulate the secretion of testosterone. In addition, the contribution of NO in vaso- Immunostimulant activity relaxation (Wongsawatkul et al., 2008) S. acmella leaves have been used tradi- may be involved in enhancing sexual per- tionally as tonic, treatment of rheumatism, formance as penile erection is directly con- gout and sialogogue as well as being trolled by NO (Sharma et al., 2011). The claimed to possess immunostimulant activ- study suggested possible development of S. ity (Savadi et al., 2010). The investigation acmella EtOH extract as therapeutics for was performed using various experimental stimulating male sexual activity (Sharma et models. The EtOH leaves extract showed al., 2011). significant immunomodulatory activity by Moreover, S. acmella extract is an ac- increasing macrophage count with the tive component in body and beauty care th maximum number of cells on the 15 day cosmetics as a fast-acting muscle relaxant (Savadi et al., 2010). The leaves of S. ac- that may be essential in accelerating the mella contained various compounds such repair of functional wrinkles as well as as alkamides, pungent amides, carbohy- stimulate, reorganize and strengthen the drates, tannins, steroids, carotenoids, es- collagen network and has thus been uti- sential oils, sesquiterpenes and amino acids lized for anti aging purposes in the form of (Amal and Sudhendu 1998; Lemos et al., anti wrinkle cream formulations (Pracha- 1991; Nagashima and Nakatani, 1992; yasittikul et al., 2009b). Other plant spe- Nagashima and Nobuji, 1991; Tiwari and cies such as the Zanthoxylum bungeanum Kakkar, 1990). It was reported that spilan- fruit husks extract are known to be rich in thol was involved in immune stimulation spilanthol that has been found to exert anti and attenuation of inflammatory response wrinkle effect owing to its capacity to re- in murine Raw 264.7 macrophages (Wu et lax subcutaneous muscles and act as a top- al., 2008). In addition, some alkamides are ical-lifting agent for wrinkles (Artaria et being consumed as to enhance immune al., 2011). response, for example, to relieve colds, respiratory infections and influenza (Rios, 2012). Diuretic activity Naturally occurring diuretics such as caffeine are known to be present in coffee, Structure-activity relationship tea and cola. So far in Ayurvedic practice, As stated previously, S. acmella extract many indigenous drugs have been claimed and its isolates (e.g. spilanthol, flavonoids to have diuretic effect. The study of S. ac- and triterpenoids) are known to be in- mella EtOH leaves extract revealed diuret- volved in many bioactivities. Particularly, ic effect possibly arising from tannin, ster- antipyretic, antiinflammatory and analgesic oid and carotenoid (Vanamala et al., 2012). activities arise from the capacity of com- In addition, flower cold aqueous extract of pounds to inhibit COX enzymes that lead the plant species exhibited strong diuretic to the inhibition of prostaglandin synthe- activity (Kumar et al., 2010). The effect ses. The well-known drug such as aspirin may be attributed to its alkaloids. It was has been used as a nonsteroidal anti- suggested that the extract acted as a loop inflammatory drug, analgesics and antipy- diuretic, which is the most powerful of all retics. Its mechanism of action had been diuretics (Ratnasooriya et al., 2004). How- proposed (Brenner and Stevens, 2010) to ever, several other diuretic plants from dif- irreversibly inhibit COX enzymes that are ferent families have been reported to con- homodimeric proteins containing two iden- tain triterpenoids, steroids, saponins, alka- tical active sites with serine residues at po- loids, flavonoids, phenolics, glycosides sitions 530 (COX-1) and 516 (COX-2),

300 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

forming covalent bond. Ultimately, acetyl Based on the functional moiety of the groups of the aspirin were added to COX compounds, therefore, similar enzymatic enzymes via nucleophilic attack of serine nucleophilic addition of serine (OH) to (OH group) to electrophilic center (car- electrophilic carbonyl groups of coumarin bonyl group) of the drug (Figure 3). and β-sitostenone could possibly be pro- Considering the structures of spilanthol, posed. Besides the carbonyl group, an al- contains amide carbonyl group, flavonoid, cohol function of the compound such as β- coumarin and triterpenoid (β-sitostenone), sitosterol has been shown to be a stronger which all have electrophilic centers that antiinflammatory agent than the β- could interact with serine residues of COX sitostenone, and even stronger than the enzymes. Thus, the possible mode of ac- standard drug, indomethacin (Nair et al., tion of spilanthol is presumably a result 2012). This could be due to the loss of the from the addition of serine (OH) to the OH group from β-sitosterol as H2O mole- carbonyl group of spilanthol with subse- cules, thus forming carbocation (electro- quent loss of amine moiety as shown in philic center) that further reacted with the Figure 4. 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., 2010b; Wongsawatkul et al., 2008). 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 O2 , thus improving NO-induced vasorelaxation.

Figure 3: Proposed mechanism of action of aspirin

Figure 4: Possible mode of action of spilanthol

301 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

In addition, it has been observed that S. its wide range of medicinal applications as acmella extracts provided both antioxidant well as its increasing demand for the mar- activity and antimicrobial action. So far, ket (Tiwari et al., 2011). Spilanthol is con- many other medicinal plants also showed stituent in many parts of the plant species: such correlation (Prachayasittikul et al., flower heads, leaves, aerial parts, stems 2008, 2012). It was reported that com- and roots. In general, chemical constituents pounds with antimicrobial activity also can be isolated by conventional chroma- displayed antioxidant activity as well tography and identified by spectroscopic (Suksrichavalit et al., 2008, 2009). Such methods, NMR, IR, HPLC and LC-MS activity relationship could be possibly ex- (Prachayasittikul et al., 2008, 2009a, plained by the fact that these compounds 2010a; Tiwari et al., 2011). may enhance bacterial killing by synergis- To obtain a large-scale spilanthol with tically converting superoxide radical to higher yields and purities, therefore more hydrogen peroxide (H2O2) in which accu- effective methods are required. Recently, mulation of H2O2 exhibited harmful effect supercritical fluid extraction (SFE) has to bacterial cells as well as participating in been proven to be the most effective ex- the ultimate formation of hydroxyl radical traction process for spilanthol from all through Fenton’s reaction (Suksrichavalit parts of S. acmella (e.g. flowers, leaves et al., 2008). and stems) (Dias et al., 2012). The ad- Molecular modeling of vasorelaxant vantage of SFE provides ready-to-use and antioxidant activities had been previ- product with contamination-free green ex- ously reported (Prachayasittikul et al., tract and solvent independence (Dias et al., 2010c). It was found that dipole moment 2012). Previously, other plant species such (μ) is a useful molecular descriptor for as- as S. americana (Stashenko et al., 1996) sessing the vasorelaxant and antioxidant and Echinacea angustifolia (Sun et al., activities where compounds with high μ 2002) were validated using the SFE meth- correspondingly had high antioxidant od. It was found that the method was effi- (SOD) activity. This suggested that elec- cient for selective extraction of spilanthol tron withdrawing group is crucial for su- from S. americana flowers and leaves. peroxide scavenging (SOD) activity. The High pressure liquid chromatography- explanation is that molecules having high μ electrospray ionization-mass spectrometry induce a positive charge that is highly ca- (HPLC-ESI-MS) was employed as a rapid pable to scavenge the superoxide anion and effective identification and quantifica- (Prachayasittikul et al., 2010c; Wora- tion method for spilanthol from S. acmella chartcheewan et al., 2012). Thus, the com- (e.g. whole plants, leaves, flowers, stems pound with high antioxidant activity facili- and roots) EtOH extracts (Bae et al., 2010). tates the induced NO which plays the im- Furthermore, the obtained spilanthol was portant role in vasorelaxation. The radical shown to be stable in EtOH extracts for scavenging (DPPH) activity can be as- well over six months when even stored at sessed from the ionization potential (IP) room temperature (Bae et al., 2010). where low IP value is an indicator of good CPC is another technique used for antioxidant activity as it has a higher prob- quantitative isolation of N-alkylamides ability of losing an electron in scavenging from S. acmella MeOH flower extract the radical (Prachayasittikul et al., 2010c). (Mbeunkui et al., 2011). Structures of the isolates were identified by ESI-IT-TOF- MS and validated by 1H-and13C-NMR Preparation and purification analysis. The CPC offered high recovery A combination of bioactive compounds of the target compounds and high- has been found in S. acmella. Especially, throughput as compared with other tradi- spilanthol is the most abundant alkamide tional separation methods, for example, accounting for the diverse bioactivities and column chromatography and thin layer

302 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

chromatography. The study demonstrated CONCLUSION the potential of CPC for large-scale isola- It is very fascinating that S. acmella, tion of major N-alkylamides from S. ac- starting from the simple antitoothache mella (Mbeunkui et al., 2011). plant to highly valuable annual herb, possesses multifunctional roles as indigenous In vitro micropropagation medicine for therapeutics in health care, To date, S. acmella, with high medicinal beauty care and cosmetics as well as health values, is increasingly demanded world- food or supplements enriched with numer- wide as a plant-derived medicine (Tiwari ous antioxidants. The most abundant iso- et al., 2011). It has been recognized as one lates of the plant species were lipid alka- of the most important medicinal plants of mides, especially, the spilanthol along with the world (Singh and Chaturvedi, 2012a). other bioactive metabolites e.g. phenolic, However, S. acmella has been itemized as flavonoid, coumarin and triterpenoid com- an endangered plant species due to the low pounds. rate of germination and poor vegetative Pharmacological studies revealed that propagation (Rios-Chavez et al., 2003), such compounds exhibited an array of di- including limited availability of infor- verse bioactivities. Considering the data, mation of the biosynthetic pathway of al- some conclusions could be drawn that the kamides (Tiwari et al., 2011). S. acmella extracts and its constituting To increase the supply of S. acmella, in compounds such as spilanthol and flavo- vitro micropropagation has been recently noids have been shown to possess inhibito- proposed to be a reliable and routine ap- ry activity toward PG synthesis. It could be proach for large-scale production (Sahu et presumably proposed that these com- al., 2011; Tiwari et al., 2011). The method pounds share a common functional group is a useful tool for rapid cultivation of S. with electrophilic center, interacting with acmella which provides high yield and COX enzymes through nucleophilic addi- consistent production or quality of bioac- tion of serine residues. As a result, the syn- tive metabolites irrespective of seasons and theses of PG were inhibited subsequently regions (Singh and Chaturvedi, 2012a) as contributing to the observed antiinflamma- well as conservation of genetic fidelity, tory, antipyretic and analgesic activities. In long term storage and cost effectiveness addition, spilanthol has been shown to re- (Sahu et al., 2011). So far, a number of duce NO release and thereby inhibit in- studies has been reported for successful in flammatory mediators and attenuating the vitro micropropagation of S. acmella expression of COX-2 and iNOS. This through leaf, axillary bud, and shoot tip could be attributed to the immunostimulant (Sahu et al., 2011). The content of spilan- activity of S. acmella in its traditional us- thol was found to be higher than the moth- ages. er plant or those that are field grown S. acmella exerted vasorelaxant and an- (Singh and Chaturvedi, 2012b). Important- tioxidant activities, which is beneficial for ly, the produced spilanthol (in vitro) its lifting effect as fast acting muscle re- showed strong (100 %) antilarvicidal activ- laxant in anti wrinkle and anti aging appli- ity against malaria and filarial vectors cations. The participation of NO in vaso- (Pandey and Agrawal, 2009). The methods relaxation makes S. acmella a powerful employed different culture media, mostly aphrodisiac in traditional medicine for im- using Murashige and Skoog media (MS) in proving sexual performance in men. combination with other growth regulators or auxins as shown in Table 6.

303 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Table 6: In vitro production of Spilanthes Plant Induction Culture media Method References species S. acmella acallus formations BAP;2,4D,NAA/MS leaf disc explant Singh and (cell biomass) (cell suspension Chaturvedi, 2012a culture) S. acmella a,bshoots via direct BAP/MS leaf disc explant Singh and organogenesis BAP,NAA/MS Chaturvedi, 2012b BAP,IAA/MS S. acmella c - 2,4-D/MS callus cell Leng et al., 2011 suspension S. acmella plant flowers BA,NAA/MS leaf explant Pandey et al., 2011 S. acmella shoots BAP/MS cultured nodal re- Yadav and Singh, flowerings BAP,IAA/MS generated shoot 2011 S. acmella dshoot buds BA,NAA/MS seedling leaf ex- Pandey and plant Agrawal, 2009 S. acmella eshoot generations BAP/MS nodal segment Singh et al., 2009 shoot tips Sodium algena- algenate- te/CaCl2 encapsulated S. acmella shoots BA, indole acetic leaf explant Saritha and Naidu, acid/MS 2008 S. acmella multiple shoots MS nodal explant Leng et al., 2004

S. acmella multiple shoots BA/MS aseptic bud Haw and Keng, 2003

S. calva shoots thidiazuron/MS nodal segment Tiwari et al., 2011 S. mauri- shoots BA,NAA axillary bud Bais et al., 2002 tiana DC aIn vitro plant produced higher spilanthol than the mother plant (field grown). bPloidy stability is similar to the field grown plant. cSpilnathol ( in vitro) had similar retention time to the mother plant and flower head. dIn vitro plant possessed strong larvicidal activity. eGenerated shoots can be stored at 4 °C for 60 days. BA = N6- benzyladenine, BAP = N6- benzylaminopurine, IAA = indole 3- acetic acid, MS = Murashige and Skoog medium, NAA = α-naphthalene acetic acid, 2,4-D = 2,4-dichlorophenoxy acetic acid

Structure-activity relationship of vaso- that can be further developed for applica- relaxant and antioxidant activities of nico- tions in medicines, health care, cosmetics, tinic acid derivatives were elucidated by supplements and health food. molecular modeling. The studies provided insights on the essential molecular de- scriptors governing the observed biological ACKNOWLEDGEMENTS activities. Such findings provide useful in- This project is supported by the Office sights for the design and synthesis of ro- of the Higher Education Commission and bust bioactive compounds. Mahidol University under the National Re- To supply the market demand of S. ac- search Universities Initiative. V.P. thanks mella as a plant-derived medicine, its Asst. Prof. Dr. Chanin Nantasenamat for preparation, purification and in vitro prop- the proofreading of this manuscript. agation have been discussed herein. In brief, it could be demonstrated that S. acmella is a medicinal plant enriched with compounds having high therapeutic value

304 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

REFERENCES Barman S, Sahu N, Deka S, Dutta S, Das S. Antiinflammatory and analgesic activity Abascal K, Yarnell E. Herbs for treating of leaves of Spilanthes acmella (ELSA) in periodontal disease. Alternat Comple- experimental animal models. Pharmaco- mentary Ther 2001;7:216-20. logyonline 2009;1:1027-34.

Abascal K, Yarnell E. Treatment for Belfer WA. Cosmetic compositions recurrent aphthous stomatitis. Alternat comprising peptides and Acmella oleracea Complement Ther 2010;16:100-6. extract to accelerate repair of functional

wrinkles US Pat. 2007;2007048245. Acosta-Madrid II, Castañeda-Hernández G, Cilia-López VG, Cariño-Cortés R, Bermúdez-Ocaña DY, Ambriz-Tututi M, Pérez-Hernández N, Fernández-Martínez E Pérez-Severiano F, Granados-Soto V. et al. Interaction between Heliopsis Pharmacological evidence for the longipes extract and diclofenac on the participation of NO–cyclic GMP–PKG–K+ thermal hyperalgesia test. Phytomedicine channel pathway in the antiallodynic 2009;16: 336-41. action of resveratrol. Pharmacol Biochem

Behav 2006;84:535-42. Amal MK, Sudhendu M. Analysis of free amino acid content in pollen of nine Boonen J, Baert B, Roche N, Burvenich C, Asteraceae species of known allergenic De Spiegeleer B. Transdermal behaviour activity. Ann Agric Environ Med 1998;5: of the N-alkylamide spilanthol (affinin) 17-20. from Spilanthes acmella (Compositae)

extracts. J Ethnopharmacol 2010;127:77- Arora S, Vijay S, Kumar D. Phytochemical 84. and antimicrobial studies on the leaves of

Spilanthes acmella. J Chem Pharm Res Boonkird S, Sadakorn J, Sadakorn T. Thai 2011;3:145-50. plants (pp 25, 30, 32, 37, 261). Bangkok:

Animate Print and Design, 1982: Artaria C, Maramaldi G, Bonfigli A,

Rigano L, Appendino G. Lifting properties Borek C. Garlic and aging: current know- of the alkamide fraction from the fruit ledge and future considerations.In: Watson husks of Zanthoxylum bungeanum. Int J RR, Preedy RR (eds.): Bioactive foods in Cosmetic Sci 2011;33:328-33. promoting health (pp 221-34). San Diego,

CA: Academic Press, 2010. Bae SS, Ehrmann BM, Ettefagh KA, Cech

NB.A validated liquid chromatography- Brenner GM, Stevens CW. Drugs for pain, electrospray ionization-mass spectrometry inflammation, and arthritic disorders. In: method for quantification of spilanthol in Brenner GM, Stevens CW (eds.): Pharma- Spilanthes acmella (L.) Murr. Phytochem cology, 2nd Analysis 2010;21:438-43. ed. (pp 336-7). Philadelphia, PA: Saunders, 2010. Bais HP, Green JB, Walker TS, Okemo PO, Vivanco JM. In vitro propagation of Brooks PM, Day RO. Nonsteroidal anti- Spilanthes mauritiana DC., an endangered inflammatory drugs — differences and medicinal herb, through axillary bud similarities. New Engl J Med 1991;324: cultures. In Vitro Cell Dev Biol Plant 1716-25. 2002;38:598-601. Bunyapraphatsara N, Chokechareunporn O. Tradition medicinal plants. Bangkok: Prachachon, 1999.

305 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Chakraborty A, Devi BRK, Rita S, Elumalai A, Pendem N, Eswaraiah MC, Sharatchandra K, Singh TI. Preliminary Naresh V. An updated annual review on studies on antiinflammatory and analgesic antipyretic medicinal plants (Jan-Dec activities of Spilanthes acmella Murr. in 2011). Int J Univers Pharm Life Sci experimental animal models. Indian J 2012;2: 207-15. Pharmacol 2004;36:148-50. Farnsworth N, Bunyapraphatsara N. Thai Chakraborty A, Devi BRK, Thokchom I, medicinal plants recommended for primary Sanjebam R, Khumbong S. Preliminary healthcare system. Bangkok:Prachachon, studies on local anesthetic and antipyretic 1992. activities of Spilanthes acmella Murr. in experimental animal models. Indian J Ferrazzano G, Amato I, Ingenito A, Pharmacol 2010;42:277-9. Zarrelli A, Pinto G, Pollio A. Plant polyphenols and their anticariogenic Chanwitheesuk A, Teerawutgulrag A, properties: A review. Molecules Rakariyatham N. Screening of antioxidant 2011;16:1486-507. activity and antioxidant compounds of some edible plants of Thailand. Food Ganesh M, Vasudevan M, Kamalakannan Chem 2005;92:491-7. K, Kumar AS, Vinoba M, Ganguly S et al. Antiinflammatory and analgesic effects of Consult M. Complementary and alternative Pongamia glabra leaf gall extract. medicine. Clinical topic tours. St. Louis: Pharmacologyonline 2008;1:497-512. Elsevier, 2003. Gokhale V, Bhide B. Chemical investig- D'Armour FE, Smith DL. A method for ation of Spilanthes acmella (Murr.). J determining loss of pain sensation. J Indian Chem Soc 1945;22:250-2. Pharmacol Exp Ther 1941;72:74-9. Haw AB, Keng CL. Micropropagation of Déciga-Campos M, Rios MY, Aguilar- Spilanthes acmella L., a bio-insecticide Guadarrama AB. Antinociceptive effect of plant, through proliferation of multiple Heliopsis longipes extract and affinin in shoots. J Appl Hort 2003;5:65-8. mice. Planta Med 2010;76:665-70. Hernandez-Pacheco A, Araiza-Saldana CI, Demarne F, Passaro G.Use of an Acmella Granados-Soto V, Mixcoatl-Zecuatl T. oleracea extract for the botulinum toxin- Possible participation of the nitric oxide- like effect thereof in an anti wrinkle cyclic GMP-protein kinase G-K+ channels cosmetic composition. US patent 2009;US pathway in the peripheral antinociception 7,531193 B2. of melatonin. Eur J Pharmacol 2008;596:70-6. Dias AMA, Santos P, Seabra IJ, Júnior RNC, Braga MEM, De Sousa HC. Hernández I, Márquez L, Martínez I, Spilanthol from Spilanthes acmella Dieguez R, Delporte C, Prieto S et al. flowers, leaves and stems obtained by Antiinflammatory effects of ethanolic selective supercritical carbon dioxide extract and alkamides-derived from extraction. J Supercrit Fluids 2012;61:62- Heliopsis longipes roots. J 70. Ethnopharmacol 2009;124:649-52.

Ediriweera ERHSS. A review on medicinal Jagan Rao N, Subash KR, Sandeep Kumar uses of weeds in Sri Lanka. Trop Agric K. Role of phytotherapy in gingivitis: A Res Ext 2007;10:11-6. review. Int J Pharmacol 2012;8:1-5.

306 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Jain JB, Kumane SC, Bhattacharya S. Leng T, Haw A, Keng CL. Effect of Medicinal flora of Madhya Pradesh and reduced N6-benzyladenine, explant type, Chattisgarh-A review. Indian J Tradit explant orientation, culture temperature Knowl 2006;5:237-42. and culture vessel type on regeneration of adventitious shoot and in vitro plantlets of Jyothi G, William MC, Ravi KB, Krishna Spilanthes acmella. J Plant Biol 2004;47: MG. Antinociceptive and antiinflam- 15-20. matory activity of methanolic extract of leaves of Shorea Robusta. Pharmacology- Leng TC, Ping NS, Lim BP, Keng CL. online 2008;1:9-19. Detection of bioactive compounds from Spilanthes acmella (L.) plants and its Kohli K, Ali J, Ansari MJ, Raheman Z. various in vitro culture products. J Med Curcumin: A natural antiinflammatory Plant Res 2011;5:371-8. agent. Indian J Pharmacol 2005;37:141-7. Little JW. Complementary and alternative Krishnaswami NR, Prasanna S, Seahadri medicine: Impact on dentistry. Oral Surg TR, Vedantham TNC. α- and β-Amyrin Oral Med Oral Pathol Oral Radiol Endod esters and sitosterol glucoside from 2004;98:137-45. Spilanthes acmella. Phytochemistry 1975;14:1666-7. Matchacheep S. Herbs(pp 61, 213, 236). Bangkok: Praepittaya, 1991. Kroll D.Using alternative therapies in your dental practice: Holistic dentistry making Mbeunkui F, Grace MH, Lategan C, Smith gains. Alternat Complement Ther 1995; PJ, Raskin I, Lila MA. Isolation and 233-7. identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers Kubra IR, Rao LJM. An impression on using centrifugal partition chromatography current developments in the technology, and ESI-IT-TOF-MS. J Chromatogr B chemistry, and biological activities of 2011;879:1886-92. ginger (Zingiber officinale Roscoe). Crit Rev Food Sci Nutr 2011;52:651-88. McFerren MA, Cordova D, Rodriguez E, Rauh JJ. In vitro neuropharmacological Kumar BNS, Swamy BMV, Swamy A, evaluation of piperovatine, an isobutyl- Murali A. A review on natural diuretics. amide from Piper piscatorum (Piperaceae). Res J Pharm Biol Chem Sci 2010;1:615- J Ethnopharmacol 2002;83:201-7. 34. Mukharya DKK, Ansari AH. Olean-12-en- Lee S-J, Umano K, Shibamoto T, Lee K- 3-O-ß-D-Galactopyranosyl (1-4)-O-a-l- G. Identification of volatile components in rhamnopyranoside: a new triterpenoidal basil (Ocimum basilicum L.) and thyme saponin from the roots of Spilanthes leaves (Thymus vulgaris L.) and their acmella (Murr). Indian J Chem 1986;26: antioxidant properties. Food Chem 81-7. 2005;91:131-7. Nagashima M, Nakatani N. LC-MS Lemos TLG, Pessoa ODL, Matos FJA, analysis and structure determination of Alencar JW, Craveiro AA. The essential pungent alkamides from Spilanthes oil of Spilanthes acmella Murr. J Essent acmella L. flowers. Lebens Wiss Technol Oil Res 1991;3:369-70. 1992;25: 417-21.

307 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Nagashima M, Nobuji N. Two sesquiter- Pandey V, Agrawal V. Efficient micro- penes from Spilanthes acmella L. Chem propagation protocol of Spilanthes acmella Express 1991;6:993-6. L. possessing strong antimalarial activity. In Vitro Cell Dev Biol Plant 2009;45:491- Nair JJ, Aremu AO, Van Staden J. Anti- 9. inflammatory effects of Leucosidea sericea (Rosaceae) and identification of the active Pandey V, Chopra M, Agrawal V. In vitro constituents. S Afr J Bot 2012;80:75-6. isolation and characterization of biolarvicidal compounds from micro- Nakatani N, Nagashiwa M. Pungent propagated plants of Spilanthes acmella. alkamides from Spilanthes acmella L. Var. Parasitol Res 2011;108:297-304. Clark. Biosci Biotech Biochem 1992;56:759-62. Perry E, Milner L, Houghton P. From ancient texts to modern phytotherapy: Nanasombat S, Teckchuen N. Antimicro- plants in mind. Herbal medicine: a concise bial, antioxidant and anticancer activities overview for professionals (pp 1-18). of Thai local vegetables. J Med Plants Res Oxford: Butterworth & Heinemann, 2000. 2009;3:443-9. Phongpaichit S, Subhadhirasakul S, Narayana KR, Reddy MS, Chaluvadi MR, Wattanapiromsakul C. Antifungal Krishna DR. Bioflavonoids classification, activities of extracts from Thai medicinal pharmacological, biochemical effects and plants against opportunistic fungal therapeutic potential. Indian J Pharmacol pathogens associated with AIDS patients. 2001;33:2-16. Mycoses 2005;48:333-8.

Özbek B, Dadali G. Thin-layer drying Prachayasittikul S, Buraparuangsang P, characteristics and modelling of mint Worachartcheewan A, Isarankura-Na- leaves undergoing microwave treatment. J Ayudhya C, Ruchirawat S, Food Eng 2007;83:541-9. Prachayasittikul V. Antimicrobial and antioxidative activities of bioactive Ortiz MI, Medina-Tato DA, Sarmiento- constituents from Hydnophytum Heredia D, Palma-Martinez J, Granados- formicarum Jack. Molecules 2008;13:904- Soto V. Possible activation of the NO- 21. cyclic GMP-protein kinase G-K+ channels pathway by gabapentin on the formalin Prachayasittikul S, Manam P, Chinwor- test. Pharmacol Biochem Behav rungsee M, Isarankura-Na-Ayudhya C, 2006;83:420-7. Ruchirawat S, Prachayasittikul V. Bio- active azafluorenone alkaloids from Ospina De Nigrinis L, Olarte J, Nunez O. Polyalthia debilis (Pierre) Finet & Gagnep. Phytopharmacologic study of flower lipid Molecules 2009a;14:4414-24. soluble fractions from Spilanthes americana (Mutis). Part 1. Phytochemical study. Prachayasittikul S, Suphapong S, Wora- Rev Columb Cien Quim Farm 1986;15:37- chartcheewan A, Lawung R, Ruchirawat S, 47. Prachayasittikul V. Bioactive metabolites from Spilanthes acmella Murr. Molecules 2009b;14:850-67.

308 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Prachayasittikul S, Saraban P, Cherdtra- Rahmatullah M, Rahman MA, Hossan MS, kulkiat R, Ruchirawat S, Prachayasittikul Taufiq-Ur-Rahman M, Jahan R, Mollik V. New bioactive triterpenoids and MAH. A pharmacological and phyto- antimalarial activity of Diospyros rubra chemical evaluation of medicinal plants Lec. EXCLI J 2010a;9:1-10. used by the Harbang clan of the Tripura tribal community of Mirsharai area, Prachayasittikul S, Wongsawatkul O, Chittagong district, Bangladesh. J Alternat Suksrichavalit T, Ruchirawat S, Pracha- Complement Med 2010;16:769-85. yasittikul V. Bioactivity evaluation of Eclipta prostrata linn: A potential vaso- Ramsewak RS, Erickson AJ, Nair MG. relaxant. Eur J Sci Res 2010b;44:167-76. Bioactive N-isobutylamides from the flower buds of Spilanthes acmella. Prachayasittikul S, Wongsawatkul O, Phytochemistry 1999;51:729-32. Worachartcheewan A, Nantasenamat C, Ruchirawat S, Prachayasittikul V. Eluci- Rani SA, Murty SU. Antifungal potential dating the structure-activity relationships of flower head extract of Spilanthes of the vasorelaxation and antioxidation acmella Linn. Afr J Biomed Res 2006;9: properties of thionicotinic acid derivatives. 67-9. Molecules 2010c;15:198-214. Ratnasooriya WD, Pieris KPP. Attenuation Prachayasittikul S, Wongsawatkul O, of persistent pain and hyperalgesia by Worachartcheewan A, Ruchirawat S, Spilanthus acmella flowers in rats. Pharm Prachayasittikul V. Vasorelaxation and Biol 2005;43:614-9. superoxide scavenging activities of orotic acid. Int J Pharmacol 2010d;6:375-80. Ratnasooriya WD, Pieris KPP, Samaratunga U, Jayakody JRAC. Diuretic Prachayasittikul S, Worachartcheewan A, activity of Spilanthes acmella flowers in Yainoy S, Lomchoey N, Kittiphatcharin P, rats. J Ethnopharmacol 2004;91:317-20. Ruchirawat S et al. Antioxidant and antimicrobial activities of Saraca thaipin- Research CoSI. The wealth of India, raw gensis Cantley ex Prain. Asian Pac J Trop materials. New Delhi: Publication and Biomed 2012;2:S796-9. Information Directorate, Council of Scientific Industrial Research, 1976:2;11- Purabi D, Kalita MC. In vitro clonal 2. propagation and organogenesis in Spilanthes acmella (L) Murray: a herbal Rios MY. Natural alkamides: Pharmaco- pesticidal plant of North-East India. J Plant logy, chemistry and distribution. Drug Biochem Biotech 2005;14:69-71. discovery research in pharmacognosy. Vallisuta O, Olimat SM. InTech 2012: Rabinowitch HD, Kamenetsky R. Shallot 107-144. (Allium cepa, Aggregratum group). In: Rabinowitch HD, Currah L (eds.). Allium Rios MY, Aguilar-Guadarrama AB, crop science: recent advances (pp 409-10). Gutiérrez MdC. Analgesic activity of New York: CABI Publishing, 2002. affinin, an alkamide from Heliopsis longipes (Compositae). J Ethnopharmacol 2007;110:364-7.

309 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Rios-Chavez P, Ramirez-Chavez E, Schubnel L. A different approach to lifting Armenta-Salinas C, Molina-Torres J. efficacy based on a natural active Acmella radicans var. radicans: In vitro ingredient. SOFW J 2007;133. culture establishment and alkamide content. In Vitro Cell Dev Biol Plant Senthilkumar P, Paulsamy S, Vijayakumar 2003;39:37-41. KK. In vitro regeneration of the medicinal herb of Nilgiri shola, Acmella calva L. Rosas-Piñón Y, Mejía A, Díaz-Ruiz G, from leaf derived callus. Plant Tissue Cult Aguilar MI, Sánchez-Nieto S, Rivero-Cruz Biotechnol 2007;17:109-14. JF. Ethnobotanical survey and antibacterial activity of plants used in the Altiplane Sharma R. Medicinal plants of India - an region of Mexico for the treatment of oral encyclopedia. Delhi: Daya Publishing cavity infections. J Ethnopharmacol 2012; House, 2003. 141:860-5. Sharma V, Boonen J, Chauhan NS, Thakur Sadavongvivad C, Supavilai P. Three M, De Spiegeleer B, Dixit VK. Spilanthes monohydroxycoumarins from Alyxia acmella ethanolic flower extract: LC-MS lucida. Phytochemistry 1977;16:1451. alkylamide profiling and its effects on sexual behavior in male rats. Phytomedi- Sahu J, Jain K, Jain B, Sahu RK. A review cine 2011;18:1161-9. on phytopharmacology and micropropagation of Spilanthes acmella. Pharmaco- Shimada T, Gomi T. Spilanthol-rich logyonline newslett 2011;2:1105-10. essential oils for manufacturing toothpastes or other oral compositions. JP Pat. 1995; Saritha KV, Naidu CV. Direct shoot JP Pat. 07090294. regeneration from leaf explants of Spilanthes acmella. Biol Plant 2008;52: Singh M, Chaturvedi R. Evaluation of 334-8. nutrient uptake and physical parameters on cell biomass growth and production of Savadi R, Yadav R, Yadav N. Study on spilanthol in suspension cultures of immunomodulatory activity of ethanolic Spilanthes acmella Murr. Bioprocess extract Spilanthes acmella Murr. Leaves. Biosyst Eng 2012a;35:943-951. Indian J Nat Prod Resour 2010;1:204-7. Singh M, Chaturvedi R. Screening and Sawangjaroen N, Phongpaichit S, quantification of an antiseptic alkylamide, Subhadhirasakul S, Visutthi M, Srisuwan spilanthol from in vitro cell and tissue N, Thammapalerd N. The antiamoebic cultures of Spilanthes acmella Murr. activity of some medicinal plants used by Industrial Crops and Products 2012b;36: AIDS patients in southern Thailand. 321-8. Parasitol Res 2006;98: 588-92. Singh S, Majumdar DK, Rehan HMS. Schneider I, Bucar F. Lipoxygenase Evaluation of antiinflammatory potential inhibitors from natural plant sources. Part of fixed oil of Ocimum sanctum 1: Medicinal plants with inhibitory activity (Holybasil) and its possible mechanism of on arachidonate 5-lipoxygenase and 5- action. J Ethnopharmacol 1996;54: 19-26. lipoxygenase/cyclooxygenase. Phytother Res 2005;19:81-102.

310 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Singh S, Rai M, Asthana P, Pandey S, Thiengburanathum W. The encyclopedia Jaiswal VS, Jaiswal U. Plant regeneration of Thai herbs (pp 97, 320, 325, 375, 415, from alginate-encapsulated shoot tips of 475, 511, 559, 586, 598, 609, 619, 623, Spilanthes acmella (L.) Murr., a 645, 646, 788, 842). Bangkok: Auksorn medicinally important and herbal pesticidal Pittaya, 1999. plant species. Acta Physiol Plant 2009;31: 649-53. Tiwari H, Kakkar A. Phytochemical examination of Spilanthes acmella Murr. J Spelman K, Depoix D, McCray M, Indian Chem Soc 1990;67:784-5. Mouray E, Grellier P. The traditional medicine Spilanthes acmella, and the Tiwari KL, Jadhav SK, Joshi V. An alkylamides spilanthol and undeca-2E-ene- updated review on medicinal herb genus 8,10-diynoic acid isobutylamide, Spilanthes. Chin J Integr Med 2011;9: demonstrate in vitro and in vivo anti- 1170-8. malarial activity. Phytother Res 2011; 25:1098-101. Trease G, Evans W. Phenolic compounds and tannins. London: Ballilliere Trindall, Stashenko EE, Puertas MA, Combariza 1972. MY. Volatile secondary metabolites from Spilanthes americana obtained by Tyagi A, Delanty N. Herbal remedies, simultaneous steam distillation-solvent dietary supplements, and seizures. Epilep- extraction and supercritical fluid sia 2003;44:228-35. extraction. J Chromatogr A 1996;752:223- 32. Vanamala U, Elumalai A, Eswaraiah MC, Shaik A. An updated review on diuretic Suksrichavalit T, Prachayasittikul S, plants-2012. Int J Pharm Biol Arch 2012;3: Piacham T, Isarankura-Na-Ayudhya C, 29-31. Nantasenamat C, Prachayasittikul V. Copper complexes of nicotinic-aromatic Wagner H. Search for new plant carboxylic acids as superoxide dismutase constituents with potential antiphlogistic mimetics. Molecules 2008;13:3040-56. and antiallergic activity. Planta Med 1989;55:235-41. Suksrichavalit T, Prachayasittikul S, Nantasenamat C, Isarankura-Na-Ayudhya Wang Z, Lu X, Lei A, Zhang Z. Efficient C, Prachayasittikul V. Copper complexes preparation of functionalized (E,Z) dienes of pyridine derivatives with superoxide using acetylene as the building block. J scavenging and antimicrobial activities. Org Chem 1998;63:3806-7. Eur J Med Chem 2009;44:3259-65. Witkin LB, Heubner CF, Galdi F, O'Keefe Sun L, Rezaei KA, Temelli F, Ooraikul B. E, Spitaletta P, Plummer AJ. Supercritical fluid extraction of Pharmacology of 2-amino-indane alkylamides from Echinacea angustifolia. hydrochloride (SU-8629): a potent non- J Agric Food Chem 2002;50:3947-53. narcotic analgesic. J Pharmacol Exp Ther 1961;133:400-8. Thanaphum V, Muengwongyard P. Herbal trees (pp 406, 409). Bangkok: Samcharo- enphanit, 2006.

311 EXCLI Journal 2013;12:291-312 – ISSN 1611-2156 Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013

Wongsawatkul O, Prachayasittikul S, Yadav K, Singh B. Micropropagation of Isarankura-Na-Ayudhya C, Satayavivad J, Spilanthes acmella Murr. – An important Ruchirawat S, Prachayasittikul V. medicinal plant. Nature and Science Vasorelaxant and antioxidant activities of 2010;8:5-11. Spilanthes acmella Murr. Int J Mol Sci 2008;9:2724-44. Yadav K, Singh N. In vitro flowering of shoots regenerated from cultured nodal Worachartcheewan A, Prachayasittikul S, explants of Spilanthes acmella Murr.- an Pingaew R, Nantasenamat C, Tantimong- ornamental medicinal herb. An U O Fasc colwat T, Ruchirawat S et al. Antioxidant, Biol 2011;18. cytotoxicity, and QSAR study of 1- adamantylthio derivatives of 3-picoline Yuliana ND, Jahangir M, Korthout H, and phenylpyridines. Med Chem Res 2012; Choi YH, Kim HK, Verpoorte R. 21: 3514-22. Comprehensive review on herbal medicine for energy intake suppression. Obes Rev Wu LC, Fan NC, Lin MH, Chu IR, Huang 2011;12: 499-514. SJ, Hu CY et al. Antiinflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators. J Agric Food Chem 2008;56:2341-9.

312