Antioxidant and Antibacterial Activities of Ethanolic Extract of
Therapeutically Important Plant, Anacyclus pyrethrum
Deepak K. Singh
Department of Botany, University of Delhi, Delhi-110007.
E mail: firstname.lastname@example.org
This study was performed to evaluate the antioxidant and antibacterial activity of ethanolic extract of Anacyclus
pyrethrum. Antioxidant activity of seed, seedling, market sample(root), in vivo and in vitro leaves and root
ethanolic extract of different dilution (0.5-10mg/l) were assessed and compared by using DPPH(2, 2-diphenyl-
1-picrylhydrazyl) free radical scavenging method. Highest antioxidant activities were 97.88% and 96.01% in
market sample of root and in vitro root respectively. Antibacterial activity of in vitro and in vivo root was
performed by “Disk diffusion method”. The extracts made of different plant sample of A. pyrethrum showed
immense action against selected bacteria Corynebacterium pollutisoli, Pontibacter mucosus and Algoriphagus
2roseus. Maximum area of inhibition was recorded 97.86 mm against Algoriphagus roseus.
Anacyclus pyrethrum, a perennial, procumbent
herb of the family Asteraceae, resembling chamomile
plant, is indigenous to North Africa (Annalakshmi et al.
2012). It is cultivated on an experimental basis in
Jammu and Kashmir from seeds imported from
Algeria, Spain, Morocco and other Mediteranean
countries (Puri 2003; Kurian and Sankar 2007). In local
dialect (Hindi) it is known as ‘Akarkara’, a name also
shared by Acmella oleracea (Syn. Spilanthes acmella,
S. oleracea). Anacyclus pyrethrum has been
extensively used in the traditional systems of medicine
for its rejuvenating properties and as a sex stimulant
(Puri 2003). Organic extract of its root has been shown
to have insecticidal, antimycotic, anti-inflammatory
and anti-bacterial properties (Crombie 1955). It is
often recommended for treatment of liver disorders
(Lamnauer 2005). When mixed with olive oil it is
reported to cure rheumatism, cold, epilepsy and
Therapeutically significant plant is any plant
which contain bioactive compound synthesized during
secondary metabolism, used due to its remedial
properties or can be used as a precursor to synthesize
useful medicine (Hossain et al. 2012). Impressive
number of modern drugs have been successfully
isolated from medicinal plants (Owolabi et al. 2007).
Worlds 80% inhabitants rely on traditional system of
medicine for their primary health care (Farnswort et al.
1985). Diseases curing potential of any plant against
chronic alilments may be due to presence of secondary
metabolites such as alkaloids, flavonoids, glycosides,
phenolics, terpenoids etc. (Cowan 1999).
paralysis (Venkatakrishnabhatt et al. 1988;
Annalakshmi et al. 2012). Powdered herbs or root
forms a good sniff to treat chronic catarrh of the heads
or nostrils by promoting flow of viscid humor, saliva,
nasal mucous and tears (Khare 2004; Kurian and
Sankar 2007; Annalakshmi et al. 2012). Dried pellitory
roots having a pungent taste are used as masticators to
allay toothache and headache (Crombie 1952). The
diluted essential oil from the roots is used for gargling
and in the preparation of tooth powders (Kurian and
Sankar 2007; Annalakshmi et al. 2012). Patients are
prescribed to chew the roots on a daily basis for several
months to get relief from paralyses of the tongue,
mouth diseases, rheumatic or neurologic infections of
the head and face (Khare 2004; Lamnauer 2005;
Kurian and Sankar 2007; Annalakshmi et al. 2012). In
India, tobacco is often chewed along with roots of
Anacyclus pyrethrum and Acmella calva to reduce the
mutagenic action of tobacco by inhibiting nitrosation
(Boonen et al. 2012). The powder of whole plant or its
roots is mixed with hog's grease to make an ointment,
which is an excellent approved remedy in the lethargy
(fibromyalgia) and also helps in gout, sciatica and
epilepsy (Puri 2003; Khare 2004; Lamnauer 2005).
The decoction of the root is useful as a gargle in
relaxation of the uvula, odontalgia, tonsilitis and
pharyngitis (Annalakshmi et al. 2012). Present reports
deals with the screening of therapeutically significant
plant, Anacyclus pyrethrum for the evaluation of
antioxidant and antibacterial activities in ethanolic
extract of different plant samples.
The Botanica 67: 67-71. 2017
THE BOTANICA 67
DPPH free Radical Scavenging Assay:
Plant material : In vivo leaves and roots of Anacyclus
pyrethrum were collected from Deoban, Chakrata
Uttarakhand (Latitude: 77°52.541ˈE, Longitude:
30°45.149ˈN, Altitude: 2622m) in the month of
September 2012, while in vitro leaves and roots were
obtained from MS basal medium following the protocol
of Singh et al. (2015). The herbarium specimen of one of
the collected plant was deposited in the University of
Delhi herbarium (Voucher specimen no. DUH 13751).
Leaves and roots of in vitro developed plant
maintained on MS medium and those from the native
habitat were dried for 48 hr in an oven maintained at
°50 C. Dried plant sample were then pulverized in liquid
nitrogen. 100 and 200 mg powder of each sample were
added individually to 5ml of ethanol contained in 50 ml
Falcon tubes and incubated on a rotary shaker for 12 hr at
200 rpm and maintained at 25 C. The tubes were then
centrifuged at 10,000g for 20 min and supernatant was
filtered through Millipore filters (0.22µm). The filtrate
containing plant extracts to be analyzed were stored at
°4 C till further use.
MATERIALS AND METHODS
Preparation of ethanolic crude plant extracts
Luria Bertani (LB) agar media was prepared and pH was
adjusted to 7.5 with 0.11NaOH or 0.1N HCl.
Approximately 25ml of medium was poured into 90mm
sterile petridish. Mother plates of each culture were too
maintained during subculture.
Bacteria used were Corynebacterium pollutisoli,
Pontibacter mucosus and Algoriphagus roseus. These
bacterial cultures were obtained from Department of
Zoology, University of Delhi, India.
The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical
scavenging antioxidant potential of seeds, seedling,
market sample (roots), in vitro and in vivo roots and
leaves ethanolic extract was determined by the method
of Brand-Williams et al. (1995) with slight modification.
The stock solution of the reagent, DPPH was made by
dissolving 24 mg of it into 100mL of methanol and stored
at -23 C until use for the experiments. 1ml from the stock
of DPPH was added to 1ml of extract of different
concentrations (0.5, 1, 5 and 10mg/ml). Mixer of 1ml
methanol and 1ml DPPH solution was used as control.
Resulting mixture was shaken with vortex and incubated
in dark for 30min at room temperature. Change in
Preparation of growth media
Antibacterial activities of varying concentration
(0, 20 and 40mg/l) of ethanolic extract of in vitro and in
vivo roots were tested using "disc diffusion
technique"(Doughari et al. 2008). 150 μl of the test
cultures of bacterial suspension were spreaded on the top
of the solid LB media with the help of L shaped spreader
(Hi Media, India) and allowed to dry for 10 min.
Different concentration (0, 20, 40mg/ml) of extract
loaded sterile discs (6mm sterile disc, Hi media, India)
were placed on the surface of the LB medium cultured
with individual bacterial species. Antibacterial activities
of the plant extract were assessed by measuring clear
zone of inhibition around sterile discs.
Screening of plant sample for their antibacterial
absorbance of the reacting mixture was recorded with the
spectrophotometer at 593nm. Antioxidant activities of
different plant sample were expressed as the percentage
of inhibition and evaluated using the following formula:
Experiment design, culture condition and data
analysis: Three sterile discs soaked in 0, 10, 20mg/l of
ethanolic plant extract were inoculated per petriplate.
Bacterial culture plates were incubated at 28 ± 2 °C for
48 hr. Area of inhibition around each disc was measured
in milli meters after 48hr of incubation. Each experiment
was repeated twice and the results were analyzed using
one way ANOVA test and significant difference between
each treatment evaluated by the Duncan's multiple
range test at P= 0.05 using SPSS (version 22) software
package. The values followed by different superscript(s)
in each of the tables are significantly different.
Antioxidant activity of extracts from seeds,
seedling, market sample (roots), in vitro and in vivo
roots, and leaves was compared by DPPH radical
scavenging method at four concentrations of each
sample. In all the cases, antioxidant activity increased
with the increase in concentration of the crude extract.
Among all, the roots of the market sample had the
highest antioxidant activity, closely followed by roots of
the in vitro raised plants. The extracts of the whole seeds
had minimum radical scavenging capability (Table 1).
Scavenging activity of plant samples towards DPPH
Absorbance of the control-Absorbance of the sample% radical scavenging activity = 100
Absorbance of the control