Abstract

      In common plants have been identified to
relieve various diseases. Spilanthes
acmella is a vital medicinal plant which is found widely mainly in India
and South American regions. A range of extracts and active metabolites from
different parts of this plant is found to contain useful pharmacological
activities. Traditionally  known as tooth
ache plant, it  reduces the pain allied
with tooth aches and is found to induce saliva secretion. Literature survey
proposed that it has numerous pharmacological actions, which comprises of
antifungal, antipyretic, local anaesthetic, bioinsecticide against insects of
agricultural importance, antioxidant, analgesic, antimicrobial, vasorelaxant,
anti-human immune deficiency virus, tooth ache relief and anti-inflammatory
effects. The researchers these days are emphasizing on estimation and
characterization of plants constituents against a number of diseases based on
their traditional claims. Extraction of the bioactive  constituents from plants  has always been a demanding task for the
researchers. This review would support researchers to search scientific information
on Spilanthes acmella in the future.

      Key words:
Spilanthes acmella, tooth ache plant,
antifungal, antipyretic, local anaesthetic, bioinsecticide, antioxidant,
analgesic, antimicrobial, vasorelaxant, anti-human immune deficiency virus,
anti-inflammatory effects.

 

 

 

 

 

 

 

 

 

 

      INTRODUCTION

      The haphazard use of chemical pesticides
has risen up serious environmental pollutions, genetic resistance by pests and
toxic residues in stored products etc. Therefore, there is a necessity to
develop botanical pesticides which are effective, biodegradable, provide
broad-spectrum of activity which do not leave any detrimental effects on environment
1.There is a rising demand for herbal medicines and their increased
recognition in international market because of potent pharmacological potential
and high therapeutic value had been proving to be a boon to the people.
Conversely, struggles are needed to explore, standardise, and validate
ayurvedic medicines for their potency, safety, and efficacy so as to bring them
to market as main line therapeutics. For the past few decades, many plant
species have been screened and plant-based products from a variety of sources
have been identified and developed. The Asteraceae is one of the largest
vascular plant families, with 30,000 species and over 1100 genera. Almost whole
of these plant types display antimicrobial activity due to the production of sesquiterpene
as principal secondary metabolites1. The genus Spilanthes has found to
contain 35 tropical species, of which three of them are reported from India. Spilanthes acmella, a vital medicinal
plant is prominently distributed in the tropical and subtropical regions around
the world . It is known for decades to contain rich source of therapeutic and
medicinal constituents. The main constituents of this plant such as
“Spilanthol” and “Acmellonate”, are sometimes used to  reduce the pain related ailments such as
toothache and can potentially  induce
saliva secretion 1, 2. Someother traditional applications of this herb are
the following: Spilanthes acmella has
been well accepted for its uses as spices, as antiseptic, antimalarial,
antibacterial, antifungal treatment, and as remedy for toothache, flu, cough,
rabies diseases, and tuberculosis2,4.

      TRADITIONAL USES

      This plant is widespread among the
ancient tribal community; special food item is prepared from this plant during
religious festival. To be particular, this plant is a famous folklore remedy
for tooth ache and for throat and gum infections6. It is believed to be
useful in cases of tuberculosis4. The flowers are crushed and applied at the
site of toothache, particularly in “Irula tribe of Hasanur hills in Erode
district of Tamilnadu” where it is known by the local name “Mandal Poo Chedi”
7. Apart from TamilNadu, root paste of the plant is used in throat problems
in Chindwara and Betul district of MadhyaPradesh 8. The plant is also known
to be used as panacea(Sumatra), asstimulant, for toothache (Sudan), for
stomatitis(Java), and for wound healing(India) 9. In India, Spilanthes acmella flower heads are
usually applied to treat stammering in children. Leaves and flowers of the
plant are moreover used to treat leucorrhoea in females among people of tribes
in Bangladesh9. In Cameroon, this plant is employed as a snake bite remedy
and in the treatment of articular rheumatism10. The whole plant paste of Spilanthes acmella is also found to be
used as “poisonous sting” in Chittagong hill tracts of Bangladesh where it is
also known as Jhummosak 11.

      PHYTOCHEMISTRY

      It is essential to explore the
phytochemical constituents of any medicinal plant in order to establish a
relationship between chemistry and pharmacology of the plant. Numerous studies
have been carried out for structural determination and chemical analysis of
pungent alkamides from Spilanthes acmella.
The most important pungent constituent reported in this plant S. acemella is “spilanthol,” which is an
isobutylamide and is popular for its insecticidal properties12,13. Spilanthol
had found to show 95 – 100 percent mortality against the 2nd instar larvae of Plutella xylostella  when it was compared to the crude extracts of
methanol (60% – 70%) and  hexane (70 –
80) after 48 hours of exposure. The extracts from Spilanthes acmella plant have
been predominantly acknowledged for toxicity against different mosquito species
such as Anopheles, Culex and Aedes.
The flower head and root part of the plant have been reported to be the
prosperous source of active principles. Triterpenoids is also one among the
important compounds 14. Spilanthol is chemically N-isobutylamide which is
found to be bitter in taste and has capabilities to stimulate salivation. The
molecular formula of spilanthol is determined as (2E, 6Z,
8E)-N-isobutylamide-2,6,8-decatrienamid15.Spilanthol possess strong pungent
taste; it may also produce local astringency and anaesthetic effects in
general. Spilanthes acmella is also
found to contain certain  important
secondary metabolites. Spilanthol can be concentrated using ethanol extract,
which has once been found to include 9.04% of total N-alkylamides yet 88.84%
spilanthol16.

      PHARMACOLOGICAL ACTIONS

      Spilanthes acmella as
already mentioned is known to have multiple pharmacological actions which are
summarized in Table1.

      Local Anaesthetic Activity

      Spilanthes
acmella has been evaluated for its local anaesthetic activity using two
different animal models: (i) intracutaneous wheal produced in guinea pigs using
nupercaine as a standard (appropriate for determining degree of anaesthesia)
and (ii) plexus anaesthesia in frog using cocaine as a standard (used for
determining inception of anaesthesia).The mean onset of local anaesthetic
action was very effective which may perhaps be attributed to the presence of
alkylamides 17.

 

 

      Antipyretic Effects

      One such antipyretic effects of Spilanthes acmella was evaluated by
yeast induced method which is generally used for the induction of pyrexia 18
and also indicated the presence of 
flavonoids which are prominent inhibitors of lipo-oxygenase or
cyclo-oxygenase 19. The dose had been found to differ accordingly in varied
studies. Multiple workers used different concentrations and different doses of
yeast.

      Anti-Inflammatory/Analgesic
Activity

      The anti-inflammatory action of Spilanthes acmella has been carried out
by the researchers using carrageena. It was found to induce hind paw disorder.
It is a standard phlogistic agent to study anti-inflammatory activity. The
extract was found to produce considerable dose-dependent inhibition of paw
oedema which was less than the standard drug. The analgesic activity of Spilanthes acmella using acetic acid had
been found to induce abdominal constriction demonstrated by tail flick
method.The aqueous extract produced better results as compared to tail flick
method which meant that the plant can be explored as peripherally acting
analgesic. The activity was attributed to the presence of flavonoids which are
potent inhibitors of prostaglandins at later stages of acute inflammation 20.

      Antifungal Activity

      Spilanthes
acmella has a significant activity against four different fungal species
such as Aspergillus niger, Aspergillus
parasiticus, Fusarium oxysporum, and Fusarium moniliformi, which had been
demonstrated using  different
concentrations of flower head extracts
 by Raniand Murty(2006). All the
concentrations of the test solution were found to inhibit the fungal species
with varying degrees of sensitivity. The maximum zone of inhibition was found
to be for highest concentration and increased proportionally with the dose.
Among the test organisms, high inhibition zones were observed in F. oxysporium
and F. moniliformis followed by A.niger and A.paraiticus 21.

      Vasorelaxant and Antioxidant Activity

      The plant extracts were found to elicit
vasorelaxation activity through partially endothelium induced nitric oxide and
prostaglandin-I2 in a dose-dependent manner. Considerably, the ethyl acetate
extract is the most potent antioxidant in the diphenylpicrylhydrazine
assayDPPH Assay and exhibited immediate vasorelaxation in nanogram levels.
The chloroform extract displays the highest vasorelaxation with the highest
antioxidant concentration. Antioxidant potentiality of leaf extracts of Spilanthes acmella was also calculated
recently by the researchers and they found that it was due to the presence of tannins,
flavonoids and phenolic compounds 27, 28.

      Bioinsecticide
and Convulsant Activity.

      The genus Spilanthes is specified to
consist of 42 known species and several insecticidal compounds have been
reported in Spilanthesmauritiana, S. alba, S. ocymifolia, S. oleracea, and Spilanthes acmella 2,12,32. Hexanic
extract of Spilanthes acmella plant
in rats was reported to induce full tonic-clonic convulsions accompanied by
typical electrographic seizures in the electroencephalogram25.

Table1. Pharmacological actions of Spilanthes acmella.

Serial
no

Pharmacological
activity

Parts
of plant used

Experimental
models

Animals
used

References

1.

Local anaesthetic

Whole plant

Intracutaneous wheal in guinea
pigs and plexus anaesthesia in frog

Guinea pig, frog

19

2.

Antipyretic activity

Whole plant

Yeast induced pyrexia

Albino rats

19

3.

Anti-inflammatory activity

Whole plant, leaves

Carrageenan induced paw oedema

Albino rats

22,31

4.

Analgesic activity

Whole plant

Tail flick method, acetic acid
induced abdominal constriction

Albino rats

22

5.

Antifungal activity

Flower

–

–

23

6.

Vasorelaxant activity

Flower

Partially endothelium induced
nitric oxide and PGI2

Albino rats

30,31

7.

Bioinsecticidal

Whole plant, leaves

–

–

2,12,32

 

 

      TOXICOLOGY

      Insecticidal Toxicity of Spilanthol.

      Spilanthol extract from the flower heads
of Spilanthes acmella had been
an  active  compound against Plutella xylostella 36. The extracts from Spilanthes acmella were most toxic against different mosquito
species (i.e.,Anopheles,Culex, and Aedes).The insecticidal property was
attributed to spilanthol and alkamides. Ovicidal, pupacidal and insecticidal
activities of the ethanolic extract from Spilanthes acmella were estimated at a
dose of 7.5 ppm concentration against Culex,
Anopheles, and Aedes mosquito37. The hexane extract of dried flower buds
of Spilanthes acmella  was found active against Aedes aegypti larvae. Spilanthol was shown to be toxic against
adults of P. americana. It is one of
the most potent compound when compared with conventional insecticides such as
carbaryl, lindane, and bioresmethrin with a potency found to be 1.3, 3.8, and
2.6times, respectively36.

      PLANT EXTRACTION PROCEDURE

      The plant extraction common techniques
are basically maceration, infusion, percolation, digestion, decoction,
microwave-assisted extraction, ultrasound extraction (sonication), hot
continuous extraction (Soxhlet), supercritical fluid extraction,
aqueous-alcoholic extraction by fermentation, counter current extraction and
distillation techniques (water distillation, steam distillation, phytonic
extraction (with hydro fluorocarbon solvents). Few latest extraction methods employed
for the aromatic plants are solid phase micro extraction, protoplast
extraction, headspace trapping  and micro
distillation.

      The basic parameters which can possibly
influence the quality of an extract are: 

·          
Plant part used as starting material

·          
Extraction procedure

·          
Solvent used for extraction

     Plant phytochemical effects tends to
depend on:

·          
The environment of the plant material

·          
Its origin

·          
Moisture content

·          
Degree of processing

·          
Particle size 

      The quality and the secondary metabolite
concentration of an extract can be significantly affected due to variations in
extraction methods that usually depends upon: 

·          
Type of extraction

·          
Time of extraction

·          
Temperature

·          
Nature of solvent

·          
Solvent concentration

·          
Polarity

      Plant material

      Plant based natural constituents can be
derived from any  part of the plant like
stem ,bark, flowers, leaves, roots, fruits, seeds, etc.

Table 2.  Solvents used for active component extraction

Water

Ethanol

Methanol

Chloroform

Ether

Acetone

Anthocyanins Starches Tannins Saponins
Terpenoids Polypeptides Lectins 

Tanins
Polyphenols
Polyacetylenes
Flavonols
Terpenoids
Sterols
Alkaloids

Anthocyanins Tannins Saponins
Terpenoids
Lactones
Flavones
Phenols
Polyphenols

Terpenoids
Flavonoids

Alkaloids
Terpenoids
Coumarins
Fatty acids

Flavonols
 Phenols

 

          EXTRACTION PROCEDURES

          Plant tissue homogenization:

      Plant tissue homogenization in solvent is
a widely used technique. Dried or wet, fresh plant parts are grinded in a
blender to fine particles, put in a certain quantity of solvent and  either shaken vigorously for 5 – 10 min or
left for 24 h. It is then filtered. The filtrate may then be  dried under reduced pressure and which is
then redissolved in the solvent to estimate the concentration. Some researchers
however centrifuged the filtrate for clarification of the extract 39.

1.        
Soxhlet
extraction:

When the desired
compound has a limited solubility in a solvent , Soxhlet extraction is
performed and also employed when the impurity is being insoluble in that
solvent. If the desired compound has a high solubility in a solvent then a
simple filtration can be used to separate the compound from the insoluble
substance. The advantage of soxhlet extraction is that instead of many portions
of warm solvent being passed through the sample, just one batch of solvent is
recycled. This method was not intented to be used for thermolabile compounds
since prolonged heating may lead to degradation of compounds 40.

2.        
Maceration:

Maceration is the
process where in the whole or coarsely powdered plant-drug is subjected to be
in contact with the solvent in a stoppered container for a defined period of
time with frequent agitation until a soluble matter is dissolved. This method
is best suitable for use in thermolabile drugs 41. 

3.        
Decoction:

      Water soluble and heat stable
constituents from crude drug can be extracted by boiling it in water for 15
minutes, followed by cooling, straining and passing sufficient cold water
through the drug so as to produce the required volume 42.

4.        
Percolation:

      A percolator (a narrow, cone-shaped
vessel open at both ends) is generally used for extracting active ingredients
in the preparation of tinctures and fluid extracts. The solid ingredients are
moistened with an appropriate amount of menstruum and allowed to stand for
approximately 4 h in a well closed container, after which the mass is packed
and the top of the percolator is closed. Additional quantity of menstruum is
added to form a shallow layer above the mass, and the mixture is allowed to
macerate in the closed percolator for 24 h. The outlet of the percolator is
opened and the liquid contained within is then allowed to drip gradually. The
marc is then pressed and the expressed liquid is added to the percolate. Ample
amount of menstruum is further added so as to produce desired volume, finally
the mixed liquid is either clarified by filtration or by standing which is
followed by decanting 43.

5.        
Sonication:

      Ultrasound with frequencies ranging from
20 kHz to 2000 kHz is used for this method , which increases the permeability
of cell walls and produces cavitation.Though the process is useful in certain
extraction processess, its large-scale application is limited due to the higher
costs. Disadvantage is occasional but known deleterious effect of ultrasound
energy (more than 20 kHz) may be present on the desired constituents of
medicinal plants via formation of free radicals and subsequently undesirable
changes may result  in the drug molecules
43. 

 

            PHYTOCHEMICAL ASSAYS

1.       
Detection
of proteins and amino acids

Millon’stest :

To
perform Millon’s test, 2ml millon’s reagent was added to the extract which
forms a white precipitate would gradually turn red upon gentle heating that
indicated the presence of protein.

Xanthoproteic Test:

The
extracts were treated with few drops of concentrated nitric acid. Formation of
yellow colour confirmed the presence of proteins on performing this test.

Ninhydrin Test:

To
the extract, 0.25% w/v Ninhydrin reagent was added and then boiled for few
minutes. Development of blue colour confirmed the presence of amino acid.  

2.       
Detection
of carbohydrates

Extracts
were dissolved individually in 5 ml distilled water and filtered. The filtered
extract was used to indicate the presence of carbohydrates.  

Molisch’s Test:

Extract
obtained was treated with 2 drops of alcoholic ?-naphthol solution in a test
tube. The Development of violet ring at the junction indicated the presence of
Carbohydrates. 

Benedict’s test:
Extract was treated with Benedict’s reagent and gently heated. Orange red
precipitate indicated the presence of reducing sugars.  

Fehling’s test:

Fehling
A and Fehling B reagent were added in equal quantities from which 2ml was
withdrawn and added with the crude extract and gently boiled until a brick red
precipitate appeared at the bottom of the test tube which confirmed the
presence of reducing sugars.

Iodine test:

Extract
was mixed with 2ml of iodine solution. A dark blue or purple coloration
indicated the presence of the carbohydrate.

3.       
Detection
of alkaloids

Crude
extract was dissolved individually in dilute hydrochloric acid and filtered to
obtain the clarified extract.  

Mayer’s Test:

The
extract was treated with Mayer’s reagent (Potassium Mercuric Iodide). Finally
formation of a yellow coloured precipitate indicated the presence of
alkaloids. 

Wagner’s Test:

The
extract was treated with Wagner’s reagent (Iodine in Potassium Iodide).
Formation of brown/reddish precipitate indicated the presence of
alkaloids. 

Dragendroff’s Test:

The
extract was treated with Dragendroff’s reagent (solution of Potassium Bismuth
Iodide) and formation of red precipitate indicated the presence of
alkaloids. 

Hager’s Test:

The
extract was treated with Hager’s reagent (saturated picric acid solution). The
existence of alkaloids had been confirmed by the formation of yellow coloured
precipitate.

 

 

4.       
Detection
of glycosides

Liebermann’s
test :

The
extract was mixed with 2ml of chloroform and 2ml of acetic acid and the mixture
was cooled in ice. Carefully concentrated H2SO4 was added to the above mixture.
A colour change from violet to blue and to green colour confirmed the presence of
steroidal nucleus, i.e., glycone portion of glycoside.

Salkowski’s
test:

 
The extract was mixed with 2ml of chloroform and 2ml of concentrated
H2SO4 was added carefully and then shaken gently. A reddish brown colour
solution indicated the presence of steroidal ring, i.e., glycone portion of the
glycoside.

Keller-kilani test:

The
extract was mixed with 2ml of glacial acetic acid containing 1-2 drops of 2%
solution of FeCl3 and the mixture was then poured into another test tube which
contained 2ml of concentrated H2SO4. A brown ring formation at the interphase
indicated the presence of cardiac glycosides.

5.       
Detection
of flavonoids

      Shinoda
test:

The
extract had been mixed with few fragments of magnesium ribbon and concentrated
HCl was added drop wise accordingly. Pink scarlet colour which appeared after
few minutes indicated the presence of flavonoids.

6.       
Detection
of saponins

The
extract obtained was mixed with 5ml of distilled water in a test tube and it was
shaken vigorously. The formation of stable foam was measured and taken as an
indicator for the presence of saponins.

7.       
Detection
of phenols 

             Ferric Chloride Test:

             Extracts were treated with 3-4
drops of ferric chloride solution. Formation of bluish black colour indicated
the presence of phenols.  

8.       
Detection
of tannins 

Gelatin Test:

To
the extract obtained, 1% gelatin solution containing sodium chloride was added.
Formation of white precipitate at the bottom indicated the presence of tannins.

9.       
Detection
of diterpenes

      Copper
acetate Test:

      Extracts were dissolved in water and
treated with 3-4 drops of copper acetate solution. Formation of emerald green
colour indicated the presence of diterpenes 19, 21, 22.

10.   
Detection
of steroid 

             Extract had been mixed with 2ml of
chloroform and concentrated H2SO4 was added side by side. The formation of red
colour at the bottom most chloroform layer indicated the presence of steroids.
Another test was performed by mixing crude extract with 2ml of chloroform. Then
2ml of each of concentrated H2SO4 and acetic acid were
added into the mixture. The development of a greenish coloration indicated the
occurrence of steroids. 

 

      CONCLUSION

      Spilanthes acmella is
a familiar plant in Indian traditional system of medicine which consists of
multiple pharmacological actions and minor side effects. In this review, we
emphasized on ethnobotany, phytochemistry, pharmacology, toxicology, plant
material extraction procedure and phytochemical assays in a eloquent manner.
Extracts and phytoconstituents isolated from this plant have shown to be
effective in   pharmacological response,
which includes anticonvulsant, analgesic, anti-inflammatory, vasodilation,
diuretic, and antimalarial effects.The utmost traditional use of this plant is
to reduce tooth ache in the most prevalent regions. Many researchers proposed
that whole plant has local anaesthetic, anti-inflammatory, antioxidant,
aphrodisiac, antinociception, immunomodulator, and insecticidal effect. On the
other hand, flower part has been made known to produce diuretic,
vasorelaxation, antifungal and pancreatic lipase inhibition properties. Its
multiple traditional use and pharmacological responses has allowed us to write a
review of Spilanthes acmella. This
review will give all the scientific information in a concise manner to the
scientific society.