Received June 26, 2011; accepted September 16, 2011; published online November 15, 2011.
Full-text LinkOut at PubMed. Journal title in PubMed: Zhong Xi Yi Jie He Xue Bao.

Correspondence: Shailesh Kumar Jadhav, PhD, Professor; Tel: +91-77-12263022; E-mail: shailesh_07@sify.com

     The World Health Organization has estimated that 80% of the population of developing countries relies on plant-based traditional medicines to maintain their primary health care needs. High treatment costs and side effects along with drug resistance are major problems associated with synthetic drugs. Since traditional medicine is not only easily accessible but also affordable, there is an increased emphasis on the use of plants to treat human diseases. Therefore, the global markets are turning to plants as a potential and realistic source of ingredients for healthcare products.
     Secondary metabolites are found to be the source of various phytochemicals that could be used directly or as intermediates for the production of pharmaceuticals, as additives in cosmetic, food or drink supplements. In recent years, the aim of searching for novel treatments against a variety of illnesses has lead to the discovery of many new compounds from plants. Nowadays, researchers and academicians focus on the active principle responsible for the pharmacological activity of traditionally used medicinal plants. One such medicinal plant genus is Spilanthes, which has great market potential due to its numerous medicinal uses^[1]. The present review highlights the up-to-date phytochemistry and medicinal uses and in vitro conservation method of this medicinally useful plant.

1  General introduction
     Genus Spilanthes belongs to the Asteraceae family and is widely distributed in tropical and sub-tropical regions of the world^[1]. In India, plants of this genus are reported from some of the regions of South India, Chhattisgarh and Jharkhand^[2]. There are recent reports of their presence in Jhalarapatan in Jhalawar district of state Rajasthan^{[3, 4]}. The plant genus grows naturally in damp areas, near lakes or ponds and near sewage discharge areas. It is commonly known as the toothache plant, paracress, eyeball plant and spot plant.
     Around 60 species of genus Spilanthes have been reported from different regions of the world^[5]. Out of these, five species occur in India namely S. acmella Murr., S. acmella L. var. oleracea Clarke, S. calva L., S. paniculata L., and S. mauritiana L.^[2]. S. calva, S. paniculata, and S. mauritiana grow wildly. S. oleracea is rare in occurrence whereas S. acmella is an acutely threatened species^[6]. The genus Spilanthes is also mentioned as Acmella in some of the literature. S. americana, S. ocymifolia, S. alba, S. uliginosa, S. nervosa, S. urens, S. paraguyensis, S. oppositifolia, S. macraei, S. ciliate, S. costata, S. repens and S. beccabunga are examples of the known important species of genus Spilanthes which grow in different parts of the world.
     Plants of genus Spilanthes are generally around 40 to 60 cm tall. The stems are prostrate in some of the species and erect in others, with opposite, triangular or lanceolate leaves and dentate or almost entire margins. The flower heads are yellow, cone-like or ovoid, long-peduncled and solitary. The roots are long and tapering and also emerge from the nodal regions in prostrate plants. In some of the species, the heads have a dark red spot in the center. The seeds are small and grey, black in color. This plant genus grows throughout the year in tropical regions with rich soil and organic compost. Regular watering and shady conditions are essential for the proper growth of the plant genus. Genus Spilanthes has no serious disease problems although it may occasionally suffer from spider mite damage. It has poor vegetative propagation and a low rate of germination^[7]. However, stem cuttings are often used for its propagation.

2  Phytochemistry of Spilanthes
     Phytochemicals are the chemical compounds that occur naturally in plants and have protective or disease-preventive properties, but are not essential nutrients. However, an analysis of phytochemicals is vital to make proper use of any medicinal plant.
     Different phytochemicals like alkaloids, glycosides, flavanoids, tannins, anthraquinones, saponins and cardiac glycosides have been reported in Acmella calva^[8]. Alkamides are the most abundant phytochemicals present in genus Spilanthes which account for most of its biological activity. Alkamides have been identified in several species of Spilanthes including S.acmella, S. americana, S. oppositifolia, S. ocymifolia, S. ciliata, S. calva and S. mauritiana. Alkamides are bitter in taste and could stimulate salivation.
     Several secondary volatile metabolites like sesquiterpenes, alkamides and oxygenated compounds have been isolated and identified in S. americana^[9]. However, the most abundant alkamide found was spilanthol. The molecular formula of spilanthol was determined as (2E,6Z,8E)-N-isobutylamide-2,6,8-decatrienamide^[10]. It is present in the roots, flower heads and whole aerial parts of genus Spilanthes^[11], and the flower heads and root part of the plant genus have been reported to be especially rich in this active principle content^[12]. Spilanthol has a strong pungent taste; it may produce local astringency and anaesthetic effects^[13].
     The structure of spilanthol and three other alkamides isolated from the flower head of S. acmella var. oleraceae has been determined by spectroscopic methods^[14]. In another study, structures of the pungent alkamides of S. acmella L. were determined by using nuclear magnetic resonance spectroscopy and high-performance liquid chromatography-mass spectrometry (HPLC-MS) with atmospheric-pressure chemical ionization and electron-impact ionization^[15]. The structure of some alkamides are given in Figure 1.
Recently, the LC-electrospray ionisation-MS method has been developed for the identification and quantification of spilanthol in S. acmella (L.) Murr.^[16].
     Different compounds reported in genus Spilanthes along with their nature are shown in Table 1.

Figure 1  Structure of some alkamides present in Spilanthes

Table 1  Secondary metabolites in Spilanthes

Spilanthes species	Reported secondary metabolites and their chemical nature
S. acmella	N-isobutylamide: spilanthol[17], undeca-2E,7Z,9E-trienoic acid isobutylamide[17], undeca-2E-en-8,10-diyonic acid isobutylamide[17], 2E-N-(2-methylbutyl)-2-undecene-8,10-diynamide[14], 2E,7Z-N-isobutyl-2,7-tridecadiene-10,12-diynamide[14], 7Z-N-isobutyl-7-tridecene-10,12-diynamide[14] Phytosterol: β-sitosterol[18], stigmasterol[18, 19], α- and β-amyrins[18] Essential oil: limonene[20], β-caryophyllene[20], (Z)-β-ocimene[20], germacrene D[20], myrcene[20] Hydrocarbon: mixture of C22 to C35 normal hydrocarbons[21] Alcohol: myricyl alcohol[18, 19]
S. alba	N-isobutylamide: spilanthol[22], undeca-2E,7Z,9E-trienoic acid isobutylamide[22], acetylenic amides, acetylenes[22]
S. oleracea	N-isobutylamide: spilanthol[23], 2-methyl-butylamide[23], (Z)-non-2-en-6,8-diynoic acid isobutylamide[23], (Z)-dec-2-en-6,8-diynoic acid isobutylamide[23]
S. ocymifolia	Phenylethylamide: N-2-phenylethylcinnamamide[24]
S. calva	Flavonoid glucoside: tetrahydroxdihydrochalcone 3′-0-glucoside[25]
S. leiocarpa	Eudesmanolide[26]
S. americana	Sesquiterpene: α- and β-bisabolenes[9], caryophyllene[9], cadinenes[9] N-isobutylamide: N-(isobutyl)-2E,6Z,8E-decatrienamide[9], N-(isobutyl)-6Z,83-decadienamide[9] Phenylethylamide: N-(2-phenelethyl)2E,6Z,8E-decatrienamide[9]

3  Ethnomedical uses
     Various medical beliefs and practices of indigenous cultures are associated with the genus Spilanthes. Traditionally, the whole Spilanthes plant is used in the treatment of dysentery. In Cameroon, the plant is used as a snakebite remedy and in the treatment of articular rheumatism^[27]. It is supposed to be useful in cases of tuberculosis^[28]. In Germany, the plant extract is used for the treatment of soreness or bruising^[29]. In India, S. acmella flower heads are used to treat stammering in children. Other traditional uses of Spilanthes according to the parts of the plant used are presented in Table 2.

Table 2  Traditional uses of different parts of Spilanthes

Part used	Traditional uses
Flowers	Relieve toothache[30], in stomatitis and throat complaints[30], cure paralysis of tongue[31], remedy for stammering[31], as spice[32]
Leaves	Stimulant[33], sialagogue[7], local anaesthetic[33], in bacterial and fungal skin diseases[34], as diuretic and lithotriptic[4], as salad[35]
Roots	Purgative and laxative[27], in cold and flu[27], in headache, asthma and rheumatism[19]

4  Pharmacological activities
     The genus Spilanthes includes many plant species that are being used for the treatment of various disorders including life-threatening diseases. Researchers have done different in vivo and in vitro pharmacological screenings to authenticate the traditional uses. These studies have revealed the potential of the plant to be developed as a curative agent from natural resources. Table 3 shows a detailed description of reported pharmacological activities on different Spilanthes species.

Table 3  Different pharmacological activities reported on various Spilanthes species

Pharmacological activity	Species	Part used	Type of extract	Models used	Reference number
Diuretic	S. acmella	Flowers	CWE	Hydrated rats	36
	S. acmella	Leaves	Petroleum ether, chloroform and ethanol	Hydrated Wistar albino rats	37
Antimalarial, larvicidal	S. acmella Murr.	Flowers	Ethanol	Anopheles, Aedes, Culex larvae	38
	S. acmella, S. calva,S. paniculata	Flowers	Hexane	A. stephensi, A. culicifacies, C. quinquefasciatus larvae	39
	S. mauritiana	Aerial parts	Methanol extract	Aedes aegypti larvae	40
	S. mauritiana	Leaves	Crude powder	A. gambiae, Culex larvae	41
Insecticidal	S. calva	Leaves and flowers	Petroleum ether, ethyl acetate and methanol	Helopeltis theivora	42
	S. acmella Murr.	Leaves and flowers	Aqueous	Chilo partellus	43
	S. acmella	NA	NA	Periplaneta Americana	44
Vasorelaxant	S. acmella Murr.	Aerial parts	Chloroform, hexane, ethyl acetate, methanol	Phnenylephrine-induced rat	45
Antioxidant	S. acmella Murr.	Aerial parts	Chloroform, hexane, ethyl actate, methanol	2,2-Diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase (SOD) assay	45
	S. acmella	Leaves, stems	Methanol	DPPH, SOD assay	46
Antinociception, antihyperalgesic	S. acmella	Flowers	CWE	Formalin test of nociception and carrageenan-induced thermal hyperalgesia in rats	47
	Acmella uliginosa (Sw.) Cass	Flowers	Methanol	Chemicals (acetic acid-induced abdominal constriction and formalin-, capsaicin-, glutamate-induced paw-licking test) and thermal models (hot-plate test) of nociception in mice	48
Anti-inflammatory	S. acmella	Aerial parts	Aqueous	Carragenan-induced paw edema in rats	49
	S. acmella	Aerial parts	Ethanol	Lipopolysaccharide-activated murine macrophage model	50
Antimicrobial	S. paniculata	Leaves	NA	Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa,Candida albicans and Microsporum gypseum	51
	S. americana	Whole plant	Aqueous, ethanol and hexane	Staphylococcus aureus, Streptococcus hemolytic, Bacillus cereus, Pseudomonas aeruginosa and Escherichia coli	52
	S. mauritiana	Roots and flowers	NA	Staphylococcus, Enterococcus, Pseudomonas, Escherichia and Klebsiella, Salmonella	53
	S. acmella Linn.	Flower heads	Petroleum ether	Fusarium oxysporium, F. moniliformis, Aspergillus niger and A. paraciticus	54
	S. mauritiana	Roots and flowers	NA	Candida species and Aspergillus species	55
	S. calva	Roots	Methanol	Oral microflora: Streptococcus mutans, Lactobacillus acidophilus and Candida albicans	56
Immunomodulatory	S. acmella Murr.	Leaves	Ethanol	Macrophage function in mice	57
	S. acmella	Leaves	Ethanol	Neutrophil adhesion test in rat	58
HIV-1 protease inhibitor	S. acmella L.	Whole plant	Chloroform, methanol and water	In vitro HIV-1 protease solution assay method	59
Convulsant	S. acmella	Whole plant	Hexane	Electroencephalo-graph behaviour of rats	60
Antiviral	S. americana	Flowers	NA	NA	32
Antimutagenic	S. calva	NA	Chloroform	Ames Salmonella/microsome assay	61
Pancreatic lipase-inhibitory	S. acmella	Flowers	Ethanol	In vitro test	62
Antihepatoxic	S. ciliata	Whole plant	Ethanol	Paracetamol-induced hepatic damage in rats	63
Antipyretic	S. acmella Murr.	NA	Aqueous	Aspirin-treated rats	64
Local anaesthetic	S. acmella Murr.	NA	Aqueous	Xylocaine-induced guinea pig and frog	64
Aphrodisiac	S. acmella L. Murr.	Flowers	Ethanol	Nitric oxide release in human corpus cavernosum cell line and penile erection in rats	65
Analgesic	S. acmella	Aerial parts	Aqueous	Acetic acid-induced writhing response in albino mice	49

CWE: cold water extract; NA: data not available.

5  Marketed preparations
     Genus Spilanthes is well known for its "folklore remedy". It is a potential medicinal plant which is also used for culinary purposes by tribes in various parts of the world. However, apart from its use by tribes, it is commercially cultivated and marketed in different parts of the world that indicates the increasing economic potential of this herb. Application of genus Spilanthes contributing to its economic potential includes its use in pharmaceuticals, as a nutritional supplement^[66] and beauty care cosmetics^[66-68], etc. A recent cosmetic product "Gatuline" claims that topical application of the extract of genus Spilanthes (Acmella oleracea) could reduce muscle tension and decrease facial wrinkles. Gatuline is being used as a non-injectable, cheap and easy-to-apply herbal Botox replacement (a popular cosmetic injectable preparation used to prevent the development of wrinkles)^[69].

6  Tissue culture studies
     With the increasing worldwide demand for plant-derived medicines, there has been a simultaneous increase in the demand for raw materials. However, the increasing human and livestock populations have affected the status of wild plants, particularly those used for making herbal medicines. Due to the multifold uses, genus Spilanthes is being overexploited by local populations as well as pharmaceutical companies. In addition, until now, little information is available on the biosynthetic pathway of alkamides. It has therefore become imperative to develop methods for large-scale propagation, conservation, and optimizing production of secondary metabolites. In this context, plant tissue culture is a useful tool for the conservation and rapid propagation of medicinally important and endangered plants.
     An efficient micropropagation protocol has been developed for S. acmella L. using seedling leaf explants through callus organogenesis. The best green and compact callus was obtained on 1 μmol/L naphthyl acetic acid and 10 μmol/L benzyladenine (BA) on the 15th day. The callus differentiated an average of 12.90±0.32 shoot buds in 50% cultures^[70].
     S. acmella has been micropropagated using leaf explants which gave 20.00±0.47 shoots per explant with 3.0 mg/L BA and 1.0 mg/L indoleacetic acid^[71].
     S. mauritiana DC. was successfully grown in vitro through axillary bud culture; 1.0 μmol/L BA and 0.1 μmol/L naphthaleneacetic acid resulted in maximum shooting response with minimal callusing^[72].
     The aseptic axillary buds of S. acmella L. formed multiple shoots within five weeks when cultured on Murashige and Skoog medium supplemented with 2.0 mg/L N⁶-BA^[73].
The nodal explants of S. acmella when placed vertically in a Erlenmeyer flask (250, 500, or 1 000 mL) produced more multiple shoots than those cultured in 350 mL jam bottles and 500 mL tex-Z flask. It has been also observed that temperatures above 28 ℃ caused abnormalities of in vitro plantlets^[34].
     An efficient plant regeneration protocol was developed for S. calva using nodal segments grown on Murashige and Skoog medium containing 4.54 μmol/L thidiazuron which showed a better growth response and produced 22.3±0.3 shoots per explant after 35 d.
     S. acmella (L.) Murr. can be conserved at low temperature (4 ℃) up to 60 d by encapsulating shoot tips using 3% sodium alginate and 100 mmol/L calcium chloride and maximum percent response for the conversion of encapsulated shoot tips into plantlets was obtained on growth regulator-free full-strength liquid MS medium^[74].
     In a gas chromatography-MS analysis, spilanthol was detected in the mother plant, flower heads and in vitro plantlets with similar retention time. Thus in vitro culture technique can become an alternative way to extract this valuable compound rather than from plants in the field^[75].

7  Conclusion
     Genus Spilanthes is a plant of choice for many health-related disorders. Its traditional uses have been proved pharmacologically. The active ingredient spilanthol is proposed to be responsible for most of its biological activities. There are many other alkamides and secondary compounds reported from the plant, which can be investigated for the similar effects. Despite the fact that similar alkamides are reported from many other species of Spilanthes, S. acmella has been more extensively explored and found more potent pharmacologically. However, there is a paradox as some of the workers reported the convulsant activity of hexane extract of S. acmella. Thus there is a need for optimisation and demarcation of dose, responsible for its convulsant activity and other pharmacological activities which will be a step towards a safe and effective use of the plant.

8  Acknowledgements
     The authors are thankful to Council of Scientific and Industrial Research (CISR), New Delhi, India for providing financial assistance.

9  Competing interests
     The authors declare that they have no competing interests.