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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
2
3
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 Chulabhorn
Graduate Institute, Bangkok 10210, Thailand
* corresponding authors:
1
E-mail: virapong.pra@mahidol.ac.th; Telephone: 662-441-4376, Fax: 662-441-4380
2
E-mail: supaluk@swu.ac.th; 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
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) such as India (Jain et al.,
2006; Little, 2004), Sri Lanka (Ediriweera
2007), Bangladesh (Rahmatullah et al.,
2010), China and Japan (Little, 2004) including Thailand (Phongpaichit et al.,
2005; Sawangjaroen et al., 2006).
The practice of botanical healing slowly
disappeared from western countries with
the introduction and advent of science and
technology (Tyagi and Delanty, 2003).
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; Prachayasittikul et al.,
2008, 2009b 2010a), which have been used
in medicinal practices for a long time
(Tiwari et al., 2011). Thus far, medicinal
plant is an alternative medicine (Consult,
2003) that is still in use and is a popular
choice for primary health care(Vanamala
et al., 2012). However, if improperly used
plants can also be toxic (Perry et al., 2000).
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However, the uses of traditional medicine
dramatically increased in Europe and
North America in the last 50 years (Tyagi
and Delanty, 2003).
Herbal medicines have been utilized for
many purposes, particularly in medical
care as antiasthmatics (86.79 %), antirheumatics (62 %) (Jain et al., 2006), diuretics (60.22 %) (Kumar et al., 2010;
Vanamala et al., 2012), antiinflammation
(29.62 %), anticancer (9.75 %), antidiabetics (8.33 %), antimicrobials, antifungals,
antioxidants, antiallergy, analgesics, antiobesity and antihypertention. In dental care
it has been employed as anticariogenic
(Ferrazzano et al., 2011), analgesic
(Abascal and Yarnell, 2010; Kroll, 1995),
local anesthetic (Abascal and Yarnell,
2010), wound healing agents (Abascal and
Yarnell, 2001, 2010; Jagan Rao et al.,
2012), antiinflammation (Abascal and
Yarnell 2001, 2010) and recurrent aphthous stomatitis treatment (Abascal and
Yarnell 2010). It has also been used for
beauty care (Artaria et al., 2011; Demarne
and Passaro, 2009) and as health food e.g.
curcumin (Curcuma longa Linn.) (Kohli et
al., 2005), ginger (Zingiber officinale)
(Kubra and Rao, 2011), lemon grass
(Cymbopogon citrates Stapf) (Nanasombat
and Teckchuen, 2009), green shallot (Allium cepa var. aggregatum) (Rabinowitch
and Kamenetsky, 2002), garlic (Allium sativum L.) (Borek, 2010), holy basil (Ocimum sanctum Linn.) (Singh et al., 1996),
sweet basil (Ocimum basilicum L.) (Lee et
al., 2005), hairy basil (Ocimum basilicum
L.f. var. citratum Back.) (Chanwitheesuk
et al., 2005) and kitchen mint (Mentha
cordifolia Opiz.) (Özbek and Dadali,
2007).
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). Our
concern centers around medicinal plants
bearing bioactive compounds, which are
employed as therapeutics and health care
(Abascal and Yarnell, 2010). 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; Tiwari et al.,
2011). 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; Wongsawatkul et al., 2008) and has
low rate of germination or poor vegetative
propagation (Tiwari et al., 2011). Its flowers and leaves have pungent taste and
when touched it is accompanied by tingling sensation and numbness (Wongsawatkul et al., 2008). The plant species
has been used commonly as a folk remedy,
e.g. for toothache, rheumatic and fever
(Wongsawatkul et al., 2008), as fresh
vegetable (Tiwari et al., 2011) as well as
spice for Japanese appetizer (Leng et al.,
2011).
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; Ospina De Nigrinis et al., 1986;
Purabi and Kalita, 2005; Research, 1976;
Rios-Chavez et al., 2003; Senthilkumar et
al., 2007; Tiwari and Kakkar, 1990) and
food (Barman et al., 2009; Boonen et al.,
2010; Wu et al., 2008).Particularly, S. acmella or S. oleracea (paracress or eyeball
plant), is a well-known antitoothache plant
(Sahu et al., 2011) and has been used as
traditional medicine for many purposes
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ticularly, anandamide (N-arachidonoylethanolamine, 9) is an endogenous cannabinoid cerebral neurotransmitter (Figure 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). Aside from
being found in S. acmella, spilanthol was
also found in other plant species as shown
in Table 3 (Rios, 2012).
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). Thus far, the spilanthol is commercially available in form of alcoholic (65 %
EtOH) extract or A. Vogel Spilanthes.
(Table 1). So far, various Thai medicinal
plants have been used for the remedy of
toothache as well as used in dental applications (Table 2).
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-2,6,8decatrienamide (Gokhale and Bhide, 1945;
Ramsewak et al., 1999) and amide derivatives 2–8 (Figure 1). In general, when alkamides are chewed, a pungent taste is released and causes itch and salivation (Rios,
2012). Alkamides are structurally related
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
extract
References
Medical
Rheumatism, fever
Diuretics
Flu, cough, rabies diseases,
Tuberculosis, antimalarials,
Antibacterials
Antifungals, skin diseases
Immunomodulatory
Antiscorbutic
Local anesthetics
Digestive
Obesity control
(lipase inhibitor)
Snake bite
Toothache
leaves,
flowers
Bunyapraphatsara and Chokechareunporn,
1999; Farnsworth and Bunyapraphatsara, 1992
Yadav and Singh, 2010
Haw and Keng, 2003
leaves
Tiwari et al., 2011
Sahu et al., 2011
Leng et al., 2011; Sahu et al., 2011
Tiwari et al., 2011
Leng et al., 2011; Sahu et al., 2011
Yuliana et al., 2011
Dental
Toothpaste
Periodontal disease
Beauty care
cosmetics
Recurrent aphthous stomatitis
Fast acting muscle relaxant
Anti wrinkle
flowers
whole plant
leaves,
flower
leaves
flower
heads, roots
leaves
Tiwari et al., 2011
Haw and Keng, 2003; Tiwari et al., 2011
whole plant
Belfer, 2007
Demarne and Passaro, 2009; Schubnel, 2007
293
Savadi et al., 2010
Abascal and Yarnell, 2001; Sahu et al., 2011;
Shimada and Gomi, 1995
Abascal and Yarnell, 2010
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Table 2: Thai medicinal plants for dental application
Plant species
family
Common
name
Thai name
Part used
Treatment and usage
1
Barleria lupulina Linn.
Acanthaceae
Barleria
Saled-pang-porn
whole plant
toothache, oral ulceration, oral
diseases
1
Cocos nucifera Linn.
Arecaceae
1
Helianthus annuus Linn.
Asteraceae
1
Averrhoa carambola
Linn.
Averrhoaceae
1
Spilanthes acmella Murr.
Compositae
1
Citrullus lanatus Mats &
Nakai
Cucurbitaceae
1
Ocimum canum L.,
O. basilicum L.
Labiatae
2
Ocimum sanctum Linn.
Labiatae
3
Mentha cordifolia
Opiz.ex Fresen
Labiatae
4
Cinnamomum bejolghota
(Buch.-Ham.)
Lauraceae
2
Cinnamomum camphora
(Linn.) Presl
Lauraceae
1
Tinospora crispa Linn.
Menispermaceae
1
Streblus asper Lour.
Moraceae
Coconut
Ma-phrao
toothache
Sunflower
Tan-tawan
oil from coconut
shell, root
flower head
Star fruit
Ma-fuang
fruit
toothache, scurvy, oral ulceration
Paracress
Phak-krad
Watermelon
Tang-mo
leaf, flower, root,
whole plant
fruit
fever, toothache
potential local anesthetic
toothache, oral ulceration
Holy basil,
Sweet basil
Mang-lak
whole plant
whole plant
Holy basil
Kra-prao
leaf
Kitchen mint
Sa-ra-nhae
leaf
Toothpaste and mouthwash
ingredients
toothache
Cinnamon
Oub-choei
Root, bark
Camphor tree
Kara-boon
leaf, seed
dissolve sputum, toothpaste,
mouthwash and chewing gum
ingredient
toothache, gingivitis
Tinospora
stem
Toothbrush
tree
Bora-ped
leaf, flower
periodontitis, toothache
Khoi
stem bud
1
Psidium guajava Linn.
Myrtaceae
Guava
Fah-rhang
leaf, fruit, leaf
toothache, gingivitis, antimicrobial
in oral cavity,
toothpaste ingredient
toothache, halitosis, scurvy, gingivitis, toothpaste ingredient
2
Clove
Kan–plu
flower
toothache, scurvy, toothpaste
and mouthwash ingredients
Orange jasmine
Keaw
leaf
toothache
Egg plant
Ma-khau-yao
stem, root, flower
toothache, oral ulceration
Syzygium aromaticum
Linn.Eugenia caryophyllus (Sprenge) Bullock et
Harrison
Myrtaceae
2
Murraya paniculata Linn.
Jack
Rutaceae
1
Solanum melongena
Linn.
Solanaceae
1
2
3
toothache
4
(Thiengburanathum, 1999), (Boonkird et al., 1982), (Matchacheep, 1991), (Thanaphum and Muengwongyard, 2006)
In addition, phytosterols (e.g. βsitosterol, stigmasterol, α- and β-amyrins),
essential oils (e.g. limonene and βcaryophyllene), sesquiterpenes, α- and βbisabolenes and cadinenes, flavonoid glucoside and a mixture of long chain hydrocarbons (C22-C35) were reported (Sahu et
al., 2011; Tiwari et al., 2011).
In recent years, other bioactive metabolites 10 – 15 (Figure 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 βsitostenone (15) (Prachayasittikul et al.,
2009b).
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COOH
COOH
HO
HO
OCH3
OCH3
10
11
COOH
H3CO
H3CO
HO
OH
O
O
13
12
COOH
O
CH3CO
14
O
15
Figure 2: Bioactive metabolites isolated from
S. acmella
Antipyretic activity
Many medicinal plants have long been
used as antipyretics, e.g. S. acmella (flower
and aerial aqueous) extracts (Chakraborty
et al., 2010). 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). 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). The studies showed that S. acmella
(aerial aqueous extract) displayed antipyretic activity against Brewer’s yeastinduced pyrexia. The antipyretic activity of
the plant species can be attributed to flavonoids (Narayana et al., 2001; Trease and
Evans, 1972), which were predominant
inhibitors of either cyclooxygenase (COX)
or lipoxygenase (LOX) (Sadavongvivad
and Supavilai, 1977). Flavonoids are
known to target prostaglandins in the late
Figure 1: Structure of spilanthol and derivatives
Table 3: Spilanthol from plant species
Tribe
Genus
Species
Ecliptinae Less
Galinsoginae B. and
H.
Welelia
Acmella
Zinniinae B. and H.
Heliopsis
parviceps
ciliata
oleracea
oppositifolia
radicans
longipes
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; Tiwari et al., 2011). Selected bioactivities of S. acmella are summarized
below.
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nisms of acute inflammation consists of
two phases involve with histamine, serotonin and kinin, released in the first hour
(Ganesh et al., 2008) 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).
A recent study in 2012 has shown that
triterpenoids, namely β-sitosterol and βsitostenone 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).
phase of acute inflammation and pain
(Chakraborty et al., 2004).
Antiinflammatory activity
Spilanthol is the main constituent isolated from many parts of S. acmella such
as flower 85 % EtOH extract(Wu et al.,
2008), root hexane extract (Wagner, 1989)
and also from other plants such as Heliopsis longipes root EtOH extract (Hernández
et al., 2009). Traditional usages of S. acmella flowers have been reported as antiinflammatory agent (Sharma, 2003). 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). Most antiinflammatory
medicinal plants possessed LOX and COX
inhibitors such as Asteraceae, Apiaceae,
Lamiaceae and Fabaceae (Schneider and
Bucar, 2005). Plant species affording such
properties is the H. longipes root extract
and its isolated spilanthol (Hernández et
al., 2009). 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). 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). 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) 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). Mecha-
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; Witkin et al.,
1961). The mechanism of action was possibly due to the presence of flavonoids in
the plant extract (Chakraborty et al., 2004)
which decreases prostaglandins, PGE2 and
PGF2 that are known to be involved in
pain perception (Jyothi et al., 2008). 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).
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 (Déciga296
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Campos et al., 2010). 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; Rios et al., 2007). Partial participation of cGMP and K+channel in the
antinociceptive activity of spilanthol was
proposed for several drugs (BermúdezOcaña et al., 2006; Hernandez-Pacheco et
al., 2008; Ortiz et al., 2006).
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., 2009b). In addition, hexane and CHCl3 extracts exhibited
antifungal activity (MIC 256 μg/mL)
against
Saccharomyces
cerevisiae
(Prachayasittikul et al., 2009b).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., 2009b). Another
study showed that leaves/flowers MeOH
extract of the plant species displayed no
antimicrobial action (disk diffusion method) (Nanasombat and Teckchuen, 2009).
Medicinal plants for treatments of oral
cavity infections, dental caries and periodontal diseases were reported (RosasPiñón et al., 2012). 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). Antibacterial activity of Compositae plant extracts against oral microorganisms is
shown in Table 4 (Rosas-Piñón et al.,
2012). The data showed that S. mutans was
the most sensitive to plant extracts while P.
gingivalis was the most resistant (RosasPiñón et al., 2012).
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). S. acmella aerial aqueous extract exhibited significant
activity that could be due to the presence
of alkamides (Chakraborty et al., 2010).
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).
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). The
EtOAc extract had two-fold higher activity
than that of doxycycline, the standard drug,
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Antimalarial activity
S. acmella is a traditional medicine used
in Africa and India for the treatment of malaria (Spelman et al. 2011). Pharmacological study showed that spilanthol (1) and
acetylenic alkamide (undeca-2E-ene-8,10diynoic 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-TOFMS), showed significantly higher antiplasmodial activity as indicated by the
lower IC50 value (Mbeunkui et al. 2011).
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). 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).
Antifungal activity
Several parts of S. acmella were tested
for antifungal activity (Table 5) 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).The leaves extract also displayed
weak activity against Aspergillus niger and
Penicillium chrysogenum (Arora et al.,
2011). 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). S. acmella
flower head petroleum ether extract exerted antifungal activity against A. niger, A.
parasiticus, Fusarium moniliformis and F.
oxysporium (Rani and Murty, 2006). The
antifungal activity of S. acmella extracts
may be due to the presence of spilanthol
and alkamides (Nakatani and Nagashiwa,
1992), non-volatile sesquiterpenoids and
saponins (Krishnaswami et al., 1975;
Mukharya and Ansari, 1986). In addition,
aerial parts of S. acmella extracts (hexane
and CHCl3) exhibited activity against Saccharomycese cerevisiae (Prachayasittikul
et al., 2009b).
Table 4: Antibacterial activitya of Compositae
plants
S. mutans
P. gingivalis
Compositae
H2O
extract
EtOH
extract
H2O
extract
EtOH
extract
Cirsium mexicanum DC.
Iostephane
heterophylla
(Cav.) Benth.
Heterotheca
inuloides
Cass.
Coreopsis
mutica DC.
Calendula
officinalis
>1000
>1000
>1000
>1000
67.5
125
125
250
125
32.5
500
125
250
62.5
>1000
>1000
125
250
500
500
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). On the other hand, leaves and
flowers of S. acmella MeOH extracts
showed
weak
antioxidant
activity
a
Antibacterial activity was determined by MIC
value (μg/mL), using microdilution method.
H2O denoted as an aqueous
298
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(Nanasombat and Teckchuen, 2009). The
aerial parts of S. acmella were also investigated (Prachayasittikul et al., 2009b;
Wongsawatkul et al., 2008). 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., 2009b). 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.,
2009b). 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., 2009b). Other
medicinal plants with antioxidant activity
also showed antimicrobial actions, e.g.
Saraca thaipingensis (Leguminosae) (Prachayasittikul et al., 2012), Polyalthia cerasoides (Annonaceae) (Prachayasittikul et
al., 2010a) and Hydnophytum formicarum
Jack. (Rubiaceae) (Prachayasittikul et al.,
2008).
Vasorelaxant activity
S. acmella extracts were studied for
their vascular effects using rat thoracic aorta (Wongsawatkul et al., 2008). 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.,
2009b; Wongsawatkul et al., 2008). These
bioactivities can be attributed to the presence of phenolic and triterpenoids
(Prachayasittikul et al., 2009b). The other
plant species of Compositae, the Eclipta
prostrata Linn. were also shown to possess
vasorelaxant and antioxidant activities
(Prachayasittikul et al., 2010b). In addition, analogs of nicotinic acid (vitamin B3)
and orotic acid (vitamin B13) were reported to afford vasorelaxants and antioxidants
(Prachayasittikul et al., 2010c, d).
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). The study showed that S. acmella EtOH flower extract improved sexual
Table 5: Antifungal activity of S. acmella
Plant extract
Tested
part
leaves
leaves
leaves
leaves
Method
Microorganism
well diffusion
well diffusion
well diffusion
well diffusion
flower
heads
agar cup
bioassay
CHCl3
whole
plants
Hexane,CHCl3
EtOAc,MeOH
aerial
parts
modified
agar dilution
method
agar dilution
method
EtOAc
Aqueous
EtOAc
EtOAc, MeOH,
petroleum ether
Petroleum ether
a
References
R. arrhigus
R. stolonifer
A. niger
P. chrysogenum
Inhibition
zone or MIC
23a
25a
16 a
14,12,15a
A. niger
A. parasiticus
F. oxysporium
F.monilifermis
M. gypseum
C. neoformans
20 a
18 a
23 a
21 a
256 b
128 b
Rani and Murty,
2006
S. cerevisiae
inactive
256 b
-
Prachayasittikul et
al., 2009b
inhibition zone (mm.), b MIC(μg/mL)
299
Arora et al., 2011
Phongpaichit et
al., 2005
EXCLI Journal 2013;12:291-312 – ISSN 1611-2156
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and bis-benzylisoquinolines (Vanamala et
al., 2012).
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)
may be involved in enhancing sexual performance as penile erection is directly controlled by NO (Sharma et al., 2011). The
study suggested possible development of S.
acmella EtOH extract as therapeutics for
stimulating male sexual activity (Sharma et
al., 2011).
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., 2009b). 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).
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). 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). 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; Lemos et al.,
1991; Nagashima and Nakatani, 1992;
Nagashima and Nobuji, 1991; Tiwari and
Kakkar, 1990). 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). In addition, some alkamides are
being consumed as to enhance immune
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,
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).
In addition, flower cold aqueous extract of
the plant species exhibited strong diuretic
activity (Kumar et al., 2010). 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). However, several other diuretic plants from different families have been reported to contain triterpenoids, steroids, saponins, alkaloids, flavonoids, phenolics, glycosides
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) 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),
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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).
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.
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 βsitosterol has been shown to be a stronger
antiinflammatory agent than the βsitostenone, and even stronger than the
standard drug, indomethacin (Nair et al.,
2012). 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., 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
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
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In addition, it has been observed that S.
acmella extracts provided both antioxidant
activity and antimicrobial action. So far,
many other medicinal plants also showed
such correlation (Prachayasittikul et al.,
2008, 2012). It was reported that compounds with antimicrobial activity also
displayed antioxidant activity as well
(Suksrichavalit et al., 2008, 2009). Such
activity relationship could be possibly explained by the fact that these compounds
may enhance bacterial killing by synergistically converting superoxide radical to
hydrogen peroxide (H2O2) in which accumulation of H2O2 exhibited harmful effect
to bacterial cells as well as participating in
the ultimate formation of hydroxyl radical
through Fenton’s reaction (Suksrichavalit
et al., 2008).
Molecular modeling of vasorelaxant
and antioxidant activities had been previously reported (Prachayasittikul et al.,
2010c). It was found that dipole moment
(μ) is a useful molecular descriptor for assessing the vasorelaxant and antioxidant
activities where compounds with high μ
correspondingly had high antioxidant
(SOD) activity. This suggested that electron withdrawing group is crucial for superoxide scavenging (SOD) activity. The
explanation is that molecules having high μ
induce a positive charge that is highly capable to scavenge the superoxide anion
(Prachayasittikul et al., 2010c; Worachartcheewan et al., 2012). Thus, the compound with high antioxidant activity facilitates the induced NO which plays the important role in vasorelaxation. The radical
scavenging (DPPH) activity can be assessed from the ionization potential (IP)
where low IP value is an indicator of good
antioxidant activity as it has a higher probability of losing an electron in scavenging
the radical (Prachayasittikul et al., 2010c).
its wide range of medicinal applications as
well as its increasing demand for the market (Tiwari et al., 2011). Spilanthol is constituent in many parts of the plant species:
flower heads, leaves, aerial parts, stems
and roots. In general, chemical constituents
can be isolated by conventional chromatography and identified by spectroscopic
methods, NMR, IR, HPLC and LC-MS
(Prachayasittikul et al., 2008, 2009a,
2010a; Tiwari et al., 2011).
To obtain a large-scale spilanthol with
higher yields and purities, therefore more
effective methods are required. Recently,
supercritical fluid extraction (SFE) has
been proven to be the most effective extraction process for spilanthol from all
parts of S. acmella (e.g. flowers, leaves
and stems) (Dias et al., 2012). The advantage of SFE provides ready-to-use
product with contamination-free green extract and solvent independence (Dias et al.,
2012). Previously, other plant species such
as S. americana (Stashenko et al., 1996)
and Echinacea angustifolia (Sun et al.,
2002) were validated using the SFE method. It was found that the method was efficient for selective extraction of spilanthol
from S. americana flowers and leaves.
High pressure liquid chromatographyelectrospray ionization-mass spectrometry
(HPLC-ESI-MS) was employed as a rapid
and effective identification and quantification method for spilanthol from S. acmella
(e.g. whole plants, leaves, flowers, stems
and roots) EtOH extracts (Bae et al., 2010).
Furthermore, the obtained spilanthol was
shown to be stable in EtOH extracts for
well over six months when even stored at
room temperature (Bae et al., 2010).
CPC is another technique used for
quantitative isolation of N-alkylamides
from S. acmella MeOH flower extract
(Mbeunkui et al., 2011). Structures of the
isolates were identified by ESI-IT-TOFMS and validated by 1H-and13C-NMR
analysis. The CPC offered high recovery
of the target compounds and highthroughput as compared with other traditional separation methods, for example,
column chromatography and thin layer
Preparation and purification
A combination of bioactive compounds
has been found in S. acmella. Especially,
spilanthol is the most abundant alkamide
accounting for the diverse bioactivities and
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EXCLI Journal 2013;12:291-312 – ISSN 1611-2156
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chromatography. The study demonstrated
the potential of CPC for large-scale isolation of major N-alkylamides from S. acmella (Mbeunkui et al., 2011).
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.
In vitro micropropagation
To date, S. acmella, with high medicinal
values, is increasingly demanded worldwide as a plant-derived medicine (Tiwari
et al., 2011). It has been recognized as one
of the most important medicinal plants of
the world (Singh and Chaturvedi, 2012a).
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),
including limited availability of information of the biosynthetic pathway of alkamides (Tiwari et al., 2011).
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; Tiwari et al., 2011). 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, 2012a) as
well as conservation of genetic fidelity,
long term storage and cost effectiveness
(Sahu et al., 2011). 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). The content of spilanthol was found to be higher than the mother plant or those that are field grown
(Singh and Chaturvedi, 2012b). Importantly, the produced spilanthol (in vitro)
showed strong (100 %) antilarvicidal activity against malaria and filarial vectors
(Pandey and Agrawal, 2009). 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.
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EXCLI Journal 2013;12:291-312 – ISSN 1611-2156
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Table 6: In vitro production of Spilanthes
Plant
species
S. acmella
Induction
Culture media
Method
References
a
callus formations
(cell biomass)
BAP;2,4D,NAA/MS
Singh and
Chaturvedi, 2012a
S. acmella
a,b
S. acmella
c
BAP/MS
BAP,NAA/MS
BAP,IAA/MS
2,4-D/MS
leaf disc explant
(cell suspension
culture)
leaf disc explant
Leng et al., 2011
S. acmella
S. acmella
plant flowers
shoots
flowerings
d
shoot buds
BA,NAA/MS
BAP/MS
BAP,IAA/MS
BA,NAA/MS
S. acmella
e
S. acmella
shoots
S. acmella
multiple shoots
BAP/MS
Sodium algenate/CaCl2
BA, indole acetic
acid/MS
MS
callus cell
suspension
leaf explant
cultured nodal regenerated shoot
seedling leaf explant
nodal segment
algenateencapsulated
leaf explant
S. acmella
multiple shoots
S. calva
S. mauritiana DC
shoots
shoots
S. acmella
shoots via direct
organogenesis
-
shoot generations
shoot tips
Singh and
Chaturvedi, 2012b
Pandey et al., 2011
Yadav and Singh,
2011
Pandey and
Agrawal, 2009
Singh et al., 2009
nodal explant
Saritha and Naidu,
2008
Leng et al., 2004
BA/MS
aseptic bud
Haw and Keng, 2003
thidiazuron/MS
BA,NAA
nodal segment
axillary bud
Tiwari et al., 2011
Bais et al., 2002
a
In 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 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.
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.
304
EXCLI Journal 2013;12:291-312 – ISSN 1611-2156
Received: November 29, 2012, accepted: March 24, 2013, published: April 04, 2013
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;1:1027-34.
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