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Biological investigation of ethanolic extract of Spilanthes calva DC
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Shimana Nasrin, Md. Golam Hossain*
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Pharmacy Discipline, Life science school, Khulna University, Khulna-9208, Bangladesh
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Runing title: Pharmacological profiles of Spilanthes calva DC.
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*Corresponding author:
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Dr. Md. Golam Hossain
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Professor, Department of Pharmacy
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School of Life Science, Khulna University, Khulna-9208, Bangladesh
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Cell: +88-01732642220.
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E-mail: ghossain99@yahoo.com
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ABSTRACT
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Phytochemical evaluation of the ethanolic extracts of Spilanthes calva DC indicated the
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presence of alkaloids, steroids, tannins, gums, and absence of carbohydrates, flavonoids,
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saponins. The pharmacological interests of these compounds, coupled with the use of this
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plant in traditional medicine tended to investigate Spilanthes calva DC for possible
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analgesic, antidiarrheal, and anti-oxidant activities. Ethanolic extracts of Spilanthes calva
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DC (250 and 500 mg/kg) exhibited significant inhibition of writhing reflex 12.42% (P<
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0.01) and 59.32% (P< 0.001), respectively while the standard drug diclofenac-Na showed
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75.52% (P< 0.001) at a dose of 25 mg/kg body weight.
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diarrhoeal mice, the ethanolic extracts of Spilanthes calva DC increased the latent period
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and reduced the number of stools at the doses of 250 mg/kg & 500 mg/kg comparing with
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standard drug Loperamide. By spraying 0.02% DPPH solution of ethanol on TLC plate
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which developed in non-polar, medium polar and polar medium, the extract showed
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yellow spot that indicates the presence of anti-oxidant components in the Spilanthes calva
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DC. The plant extract showed potent antioxidant capacity. The IC50 for ascorbic acid was
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approximately 1 µg/ml whereas the plant extract showed 500 µg/ml. Our results show that
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Spilanthes calva DC has potent antidiarrheal, analgesic and anti-oxidant activities.
In the castor oil-induced
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Keywords: Antioxidant, antibacterial, analgesic, Spilanthes calva DC.
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INTRODUCTION
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Spilanthes calva DC is an annual herb, stem usually glabrous, sometimes sparsely
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pubescent, up to 55 cm tall. Leaves ovate, petiolate, petioles up to 1.5 cm long, laminae
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1.0-5.5×0.5-3.5 cm, obtuse or acute at the apex, abruptly or gradually attenuate at the base,
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margins entire or undulate, serrate or sub-crenate, usually glabrous on both surfaces, rarely
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sparsely pubescent on both surfaces. Generally it grows in open sunny places in jhums,
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sandstones, dry soil, riversides, waste places and open clearings. It is widely found in
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Bangladesh, Brazil, West Indies, China, India, South Asia, Thailand, Malaysia, New
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Caledonia and tropical Africa.
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Aerial parts of the plants contain palmitic and stearic acids, tetra-triacontanoic acid,
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sitosterol, stigmasterol and sitosterol glucoside. Flower heads contain a local anaesthetic
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amide, spilanthol, and three other amides, a sterol, a non-reducing polysaccharide. The
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plant is used in the treatment of Allergy, Foot mud sore, Hysteria, Insanity, Stomachache,
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Toothache, Traumatic paralysis, Rheumatism, Scabies. The plant possesses α- and β-
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amyrin esters and sitosterol glucoside. The roots, flower heads and whole aerial part yield
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spilanthol. (Ghani 2003).The essential oil of fresh plant of S. calva from southern India
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was investigated by GC and GC/MS and more than 45 components have been identified
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including (E)-2-hexenol, 2-tridecanone, germacrene D. hexanol, β-caryophyllene and (Z)-
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3-hexenol as significantly dominating compounds. A system for biocontrol of malaria and
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filarial mosquito vectors has been developed using crude hexane extract obtained from
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flowering heads of Spilanthes spp. Cent percent mortalities was achieved against the late
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third/early fourth instar larvae of Anopheles stephensi Liston, A. culicifacies species C and
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C. quinquefasciatus using minimal does of plant extract (Pandey et al 2007).
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The influence of Piriformospora indica was investigated on the antifungal principle of
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Spilanthes calva. An antifungal efficacy was shown by aqueous and petroleum ether
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extracts of S. calva against Fusarium oxysporum and Trichophyton mentagrophytes. The
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petroleum ether extract of S. calva was more effective than the aqueous extract in
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inoculated as well as uninoculated plants. The antifungal activity of the plant was
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enhanced due to the increase in spilanthol content after inoculation of P. indica. Antifungal
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potential of Spilanthes calva after inoculation of Piriformospora indica (Rai et al 2004).
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Since Bangladesh is a country of low economic growth. So, scientific exploration and
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standardization of potential crude drugs is an urgent need to revolutionize our drug sector.
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The presence of diverse bioactive metabolites like alkaloids, glycosides, flavonoids,
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tannins etc. in plants has formed the therapeutic basis of herbal medications.
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emphasis is given on the biological and phytochemical screening of medicinal plants for
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further exploration of their active constituents. Besides, Bangladesh imports a large
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quantity of pharmaceutical raw materials including medicinal plants and semi processed
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plant products to produce drugs and medicines. This huge foreign exchange can be saved
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if the manufactures to satisfy their needs utilize the indigenous medicinal plants or their
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semi-processed products. From the literature survey we found no scientifically elegant
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studies supporting traditional uses of the plant has yet been reported. Therefore, to judge
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the traditional use of this medicinal plant, this work was investigated to evaluate the anti-
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oxidant, analgesic, anti-diarrhoel effects of Spilanthes calva DC.
Thus
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MATERIALS AND METHODS
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Collection and Identification: The plant Spilanthes calva DC was collected from Khulna
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University, Bangladesh, during the month of 14th December, 2009 on the day time. The
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plant was identified by the experts of Bangladesh National Herbarium, Mirpur, Dhaka
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(Accession no: DACB - 34992) and a voucher specimen was also deposited there.
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Preparation of plant extract: The collected plant parts were separated from undesirable
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materials or plants or plant parts. They were air dried for three weeks.
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were ground into a fine powder with the help of a suitable grinder (Capacitor start motor,
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Wuhu motor factory, China). The powder was stored in an airtight container and kept in a
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cool, dark and dry place until analysis commenced. A glass jar with plastic cover was
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taken and washed thoroughly. The jar was rinsed with ethanol and dried. Then 150gm of
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the dried powder was taken in the jar. After that 95% ethanol (750ml) was poured into the
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jar up to 1 inch height above the sample surface as it can sufficiently cover the sample
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surface. The plastic cover with aluminum foil was closed properly to resist the entrance of
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air into the jar. This process was performed for 8 days. The jar was shaken and stirring
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several times during the process to get better extraction. After the extraction process the
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extract was filtered by a piece of clean, white cotton material. Then it was filtered through
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Whatman filter paper. The filtrate was collected in a beaker. The filtrate (ethanolic
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extracts) obtained was evaporated by Rotary Evaporator and after that it was kept under
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ceiling fan to evaporate the ethanol completely. It rendered a gummy concentrate (5.2
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gm) of greenish black color. The gummy concentrate was designated as crude extract of
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ethanol. The yield value was 3.46%.
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Antidiarrheal activity test: To prepare suspension of the test samples at the doses of 250
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& 500 mg/kg per body weight, 250 & 500 mg of ethanolic extract were measured
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respectively. The extract was triturated in unidirectional manner by the addition of small
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amount of tween-80. After proper mixing of extract and tween-80, the distilled water was
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slowly added. The final volume of the suspensions was made 10.0 ml. For the preparation
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standard drug 3 mg of Loperamide was taken & was triturated in unidirectional manner by
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the addition of small amount of tween-80. After proper mixing, distilled water was slowly
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added up to the final volume 10ml. The amount to be administered to get desired
The plant parts
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concentration= (Body weight of mice × 0.01) ml. For the test fresh mice were randomly
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chosen from the animal laboratory of Khulna University, Khulna, Bangladesh, and divided
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into four groups having five mice in each. Of the experimental groups, group-I or the
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control received only distilled water containing distilled water. Group-II or the positive
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control received standard anti-motility drug, Loperamide at a dose of 3 mg/kg-body
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weight as oral suspension. The test groups were treated with suspension of ethanolic
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extracts of Spilanthes calva DC. at the oral dose of 250 mg/kg-body weight and 500
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mg/kg-body weight. The mice were fed with the samples 40 minutes (for proper
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absorption) prior to the oral administration of castor oil. After that the mice were fed
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castor oil at a dose of 0.5 ml. Individual animals of each group were placed in separate
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cages having adsorbent paper beneath and examined for the latent period and after that the
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presence of diarrhea every hour for four hours was observed. Number of stools or any
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fluid material that stained the adsorbent paper were counted at each successive hour during
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the 4-hour period and were noted for each mouse. At the beginning of each hour new
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papers were placed for the old ones.
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In vitro Anti-oxidant Activity test (Qualitative and Quantitative analysis) Qualitative
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test: A little amount of ethanolic extract of plant was dissolved in ethanol and diluted
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suitably and was applied on the two different TLC plate by spotter. A small amount of
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ascorbic acid was also dissolved in ethanol and diluted suitably and was applied on the
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former TLC plate by spotter at the same way. The plate was then kept in a TLC jar
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containing a solvent system. A filter paper was kept in the TLC jar and was closed the jar
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properly. After about 5 minutes spotted TLC plate was kept into the jar dipping into the
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solvent system. When the solvent system was reached at the desired level then it was
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removed from TLC jar and was subjected for air drying.
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were developed into the solvent system of n-hexane and Acetone (2:1). This is a non-polar
At first two chromatograms
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solvent system. After drying chromatograms were observed under UV detector at longer
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wavelength (306 nm) and shorter wavelength (256 nm) and colored spots was marked by
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pencil. Spots which was observed into longer wavelength was manifested by the symbol (
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) and spots which was observed into shorter wavelength was evident by the symbol < >.
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Later than one of the chromatograms were taken and 0.02 % DPPH solution of ethanol
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was sprayed on it by a spray gun. Yellow color was formed on the chromatogram. Other
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chromatogram was treated with 10% H2SO4 at the same way. Then the plate was heated
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on hot plate and was developed. Then the sample was compared with standard. Another
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two chromatograms were developed into the solvent system of Chloroform, Methanol and
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Water (40:10:1) and also two chromatogram were developed using the solvent system
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Chloroform and Methanol (5:1). This is polar solvent system. Then by the similar
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procedure described above was applied to compare the sample with standard.
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Quantitative analysis: DPPH radical scavenging assay: At first 6 test tubes were taken
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to make aliquots of 6 conc. (1, 5, 10, 50, 100, 500 and μg/ml).Plant extract and ascorbic
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acid were weighed 2 times and dissolved in ethanol to make the required concentrations
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by dilution technique. Here ascorbic acid was taken as ‘+’ve control. DPPH was weighed
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and dissolved in ethanol to make 0.004% (w/v) solution. To dissolve homogeneously
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magnetic stirrer was used. After making the desired concentrations 1ml plant extracts or
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standard of different concentration solution were taken into different test tubes and 3 ml of
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reagent solution was added into each of the test tubes.The room temperature was recorded
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and kept the test tubes for 30 minutes in light to complete the reactions. DPPH was also
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taken as blank in a test tube. After 30 minutes, absorbances of each test tube were
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determined by UV spectrophotometer at 517 nm. IC50 was determined from % inhibition
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vs concentration graph. The formula used for % inhibition ratio was % inhibition = (Blank
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OD-Sample OD/ Blank OD) × 100.
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Posphomolybdenum method: 0.6 ml plant extracts or standard of different concentration
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solution were taken into different test tubes and 6ml of reagent solution was added into
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each of the test tubes. The test tubes were incubated at 95⁰C for 90 minutes to complete
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the reaction. The absorbance of the solutions was measured at 695 nm using a
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spectrophotometer against blank after cooling to room temperature. A typical blank
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solution contained 6 ml of reagent solution and the appropriate volume (0.6 ml) of the
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same solvent for the sample and incubated under the same conditions as the rest if the
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samples solution. The antioxidant activity is expressed as the number of equivalents of
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ascorbic acid.
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Analgesic activity test: Young Swiss-albino mice aged 4-5 weeks; average weight ranges
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from 20-28 gm were used for the experiment. The mice were purchased from the animal
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Research Branch of the International Centre for Diarrhoeal Disease and Research,
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Bangladesh (ICDDRB). They were kept in standard environmental condition for one
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week in the animal house of Pharmacy Discipline, Khulna University, Bangladesh for
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adaptation after their purchase & the tests were performed on their. Experimental animals
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were randomly selected and divided into four groups denoted as group-I, group-II, group-
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III, group- IV consisting of 5 mice in each group. Each group received a particular
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treatment i.e. control, positive control and the two doses of the extract. Each mouse was
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weighed properly and the doses of the test samples and control materials were adjusted
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accordingly. Test samples, control and Diclofenac-Na were given orally by means of a
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feeding needle. A thirty minutes interval was given to ensure proper absorption of the
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administered substances. Then the writhing inducing chemical, acetic acid solution (0.7%,
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10 ml/kg) was administered intraperitonealy to each of the animals of a group. After an
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interval of five minutes, which was given for absorption of acetic acid, number of squirms
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(writhing) was counted for 15 minutes.
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RESULTS
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Phytochemical test: Ethanolic extracts of Spilanthes calva DC. showed the presence of
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the functional group tannin, alkaloid, steroid, gum and shows the absence of reducing
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sugar, flavonoid, saponin group.
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Antidiarrheal activity test: In the castor oil-induced diarrhoeal mice, the ethanolic
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extracts of Spilanthes calva DC. At the doses of 250 mg/kg & 500 mg/kg, increase the
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latent period and reduce the number of stools comparing with standard drug Loperamide
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(3 mg/kg) (Fig 1).
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Analgesic activity test: Ethanolic extracts of Spilanthes calva DC. (250 and 500 mg/kg)
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exhibited significant inhibition of writhing reflex 12.42% (P< 0.01) and 59.32% (P<
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0.001) respectively while the standard drug diclofenac inhibition was found to be 75.52%
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(P< 0.001) at a dose of 25 mg/kg body weight (Fig 2).
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Anti-oxidant Activity: Yellow color was formed which indicates the antioxidant
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property of the ethanolic extracts of Spilanthes calva DC. The total antioxidant capacity
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by the extract demonstrated was concentration dependent. The extract was likely to have
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the capacity reduction of Mo (VI) to Mo (V) by the antioxidant compound (Fig 3 and
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Table 1).
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DISCUSSION
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The phytochemical evaluation shows the presence of alkaloids, steroids, tannin, gums
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compounds in extract of Spilanthes calva DC. Our results also show that the ethanolic
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extracts of Spilanthes calva DC have potent analgesic activity. Traditionally Spilanthes
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calva DC. has been used in the treatment of toothache (Ghani, 2003). The plant contains
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α- and β-amyrin esters and sitosterol glucoside. The mixture of the two pentacyclic
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triterpenes α-amyrin and β-amyrin given by intraperitoneal (i.p.) or oral (p.o.) routes,
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causes dose-related and significant antinociception against the visceral pain in mice
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produced by i.p. injection of acetic acid. Moreover, i.p., p.o., intracerebroventricular
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(i.c.v.), or intrathecal (i.t.) administration of α,β-amyrin inhibited both neurogenic and
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inflammatory phases of the overt nociception caused by intraplantar (i.pl.) injection of
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formalin. The antinociception caused by the mixture of compounds seems to involve
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mechanisms independent of opioid, α-adrenergic, serotoninergic, and nitrergic system
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mediation, since it is not affected by naloxone, prazosin, yohimbine, dl-p-
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chlorophenylalanine methyl ester, or l-arginine. Interestingly, the i.p. administration of
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α,β-amyrin reduces the mechanical hyperalgesia produced by i.pl. injection of
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carrageenan, capsaicin, bradykinin, substance P, prostaglandin E2, 8-Br-cAMP, and TPA
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in rats. However, the mixture of compounds failed to alter the binding sites of
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[3H]bradykinin, [3H]resiniferatoxin, or [3H]glutamate in vitro. It is concluded that the
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mixture of triterpene α-amyrin and β-amyrin produced consistent peripheral, spinal, and
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supraspinal antinociception in rodents, especially when assessed in inflammatory models
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of pain. The mechanisms involved in their action are not completely understood but seem
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to involve the inhibition of protein kinase A- and protein kinase C-sensitive pathways. So,
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further investigations are necessary to find out the active constituent responsible for this
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effect.
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Our result demonstrated that the ethanolic extracts of Spilanthes calva DC possess potent
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anti-diarrheal activity. Further study is needed to isolate the active constituents responsible
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for this effect.
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Our result also showed that the ethanolic extracts of Spilanthes calva DC possess
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significant anti-oxidant activity. Both from the qualitative and quantitative analysis we
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find the data supporting this. By the % inhibion of free radicals in DPPH Scavanging
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method and no of equivalent of ascorbic acid neutralizing effect in Posphomolybdenum
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method we see that it has significant antioxidant effect. The IC50 (inhibitory conc. 50%)
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for ascorbic acid is about 1 µg/ml and for the sample it is about 750 µg/ml. It is now well
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established that free radicals (e.g. super oxide, hydroxyl radical, nitric oxide) and other
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reactive species (e g. hydrogen per oxide, single oxygen, hypochlorous acid) contribute to
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the pathology of many disorders including atherogenesis, neurodegeneration, chronic
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inflammation, and function of the immune system. Recent studies suggest that the
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inflammatory tissue damage is due to the liberation of reactive oxygen species from
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phagocytes invading the inflammation site. Therefore, the substance which relieves pain or
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inflammation may act by neutralizing (anti-oxidant) the oxidative group present in the
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inflammatory site. In my previous investigation the ethanolic extract of Spilanthes calva
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DC reduces marked degree of pain in acetic acid induced pain in mice. From the previous
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data it can be assumed that the analgesic activity of the extract was may be due to
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reduction in production of arachidonic acid from phospholipids or may inhibit the enzyme
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system which may responsible for the production of prostaglandins. So, from the previous
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data on analgesic activity of the extract favor the presence of the antioxidant group present
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in Spilanthes calva DC. Further investigations are necessary to find out the active
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constituent responsible for this effect. Finally, from the different investigations we found
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that the plant extract has strong analgesic, anti-diarrheal and antioxidant effects. Further
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investigation is required to find out the chemical constituents responsible for these effects.
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CONCLUSION
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Our observation indicates that the ethanolic extracts of Spilanthes calva DC possess potent
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analgesic, anti-diarrheal and anti-oxidant activities. Therefore, Spilanthes calva DC may
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be a potential source of compounds with analgesic, anti-oxidant and antidiarrhoeal
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activities and may contribute to national/ global economy if active compounds are
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isolated.
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CONFLICT OF INTEREST STATEMENT
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We declare that we have no conflict of interest.
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ACKNOWLEDGEMENTS
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Authors are grateful to the authority of the Khulna University for giving the opportunity to
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conduct such experiment and providing necessary chemical, instrument and utility
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support. Authors also like to express their cordial thanks to the experts of Bangladesh
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National Herbarium who helped for the identification of the plant.
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REFERENCES
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1. Ghani A (2003) Medicinal Plants of Bangladesh. The Asiatic Society of Bangladesh.
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2nd ed. pp. 1-17.
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2. Jeripa B and Chowdhury JU (2008) Essential oil from inflorescense of Spilanthes calva
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DC. Bangladesh J Bot. 37(2): 217-218.
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3. Rai MK, Varma A, Pandey AK (2004) Antifungal potential of Spilanthes calva after
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inoculation of Piriformospora indica. Department of Biotechnology, Amravati University,
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Amravati, Maharashtra, India. 47(11-12):479-81.
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4. Firoz A, Ishrat ZS, Ahmed AR and Samir KS (2007) Antinociceptive, antidiarrhoeal
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5. Longanga OA, Vercruysse A and Foriers A (2000) Contribution to the ethnobotanical,
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treatment of dysentery and diarrhoea in Lomela area, Democratic Republic of Congo
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(DRC). J Ethnopharmacol 71: 411-423.
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6. Uddin SJ, Shilpi JA, Alam SMS, Alamgir M, Rahman MT and Sarker SD (2005)
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Antidiarrhoeal activity of the methanol extract of the barks of Xylocarpus molucensis in
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castor oil and magnesium sulphateinduced diarrhoea models in mice. J Ethnopharmacol
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101: 139-143.
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Holarrhena floribunda leaves. African Journal of Biotechnology 9(3):340-346.
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8. Pandey V, Agrawal V, Raghavendra K and Dash AP (2007) Strong larvicidal activity of
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three species of Spilanthes (Akarkara) against malaria (Anopheles stephensi Liston,
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Anopheles culicifacies, species C) and filaria vector (Culex quinquefasciatus Say).
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Department of Botany, University of Delhi, Delhi, 110007, India. 102(1):171-4.
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9. Yasuda I, Takeya K and Itokawa H (1980) The geometric structure of spilanthol. J
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Chem Pharm Bull. 28:2251–2253.
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10. Alam MA., Nyeem MAB., Awal MA., Mostofa M., Alam MS., and Mostafizeur
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Rhaman. Antioxidant and hepatoprotective action of the crude ethanolic extract of the
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flowering top of Rosa damascene. Oriental Pharmacy and Experimental Medicine 2008
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8(2):164-170.
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Fig 1 Effects of ethanolic extracts of Spilanthes calva DC on the latent period and number of stool in
castor oil-induced diarrheal mice model. N=5; *p< 0.01, ***p<0.001, **** P<0.0005.
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N u m b e r o f w rith in g
30
% o f w r ith in g in h ib itio n
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Fig 2 Effects of ethanolic extracts of Spilanthes Calva DC on acetic acid induced writhing in mice. N=5;
***p<0.001, **** P<0.0005.
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After applying DPPH on the TLC plate
A
CH3OH: CHCl3 :H2O =40:10:1(DPPH)
nHexane: Acetone =2:1(DPPH)
CHCl3: CH3OH =5:1(DPPH)
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343
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346
347
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Ascorbic acid
Plant Extract
Ascorbic acid
Plant Extract
Ascorbic acid
Plant Extract
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After applying H2SO4 on the TLC plate
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B
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CH3OH: CHCl3: H2O =40:10:1(H2SO4)
n Hexane: Acetone =2:1(H2SO4)
CHCl3: CH3OH =5:1(H2SO4)
Ascorbic acid
Ascorbic acid
Ascorbic acid
352
353
354
355
356
357
358
359
Plant Extract
Plant Extract
Plant Extract
360
361
Fig 3 Antioxidant effects of ethanolic extracts of Spilanthes Calva DC.
362
363
364
365
366
367
368
16
369
370
Table 1 Total antioxidant capacity of Spilanthes calva DC Vs Ascorbic acid
Concentration
(µg/ml)
Mean absorbance
Extract
of
Mean absorbance
Ascorbic acid
of
5
0.0595
0.11
0.540
25
0.06
0.143
0.419
50
0.0685
0.236
0.290
100
0.085
0.444
0.191
200
0.1145
0.73
0.156
500
0.221
1.5895
0.139
Number of Equivalent
Number of Equivalent = Absorbance of Extract/Absorbance of Ascorbic acid
371
372
17