IAETSD JOURNAL FOR ADVANCED RESEARCH IN APPLIED SCIENCES ISSN NO: 2394-8442

Evaluation of Antioxidant and antibacterial activity of Callistemon

spp. plant extract
Pratiksha Joshi1, Komal Adhane1, Sushma Chature1, and Neha Chavan2
Department of Biotechnology,Shivchhatrapati college,Aurangabad-431001,MS,India.
1pratiksha.v.joshi96@gmail.com,1adhanekomal@gmail.com,1sushmachature358@gmail.com and
2chavanneha08@gmail.com

Abstract: Callistemon species are commonly known as ‘Bottlebrush’ plant belongs to family Myrtaceae that has great
medicinal importance. Callistemon spp. is common ornamental small tree or shrub native to Australia. Callistemon shows various
types of activities such as free radical scavenging activity, antifungal, antidiabetic, antithrombin, antibacterial, insecticidal and
herbicidal activity. The present work describes in-vitro antioxidant activity and antibacterial activity of methanolic leaves
extract of Callistemon spp. Antioxidant activity of methanolic leaves extract of Callistemon was determined by DPPH free
radical scavenging activity. Callistemon extract showed good percent inhibition at 500µg/ml concentration. The flowers
extract of Callistemon also showed significant antibacterial activity against bacterial cultures which is comparable with standard
antibiotics.

Key words – Callistemon spp., Antioxidant activity, DPPH free radical scavenging activity, Antibacterial activity.

I. INTRODUCTION
Callistemon is a genus of 34 species of shrubs belongs to family Myrtaceae. They are woody aromatic shrubs widely distributed
in the wet tropics, notably Australia, South America and tropical Asia.Callistemon spp. are commonly referred to as bottlebrush
because of their cylindrical, brush like flowers resembling traditional bottlebrush. Callistemon spp. have attractive narrow
foliage and white papery bark (4). Leaves lanceolate sometimes broadly so, up to 7.5 cm long, with prominent vein, midrib
and oil gland; flowers, crimson with dark red anthers, in 10 cm long spikes; capsules depressed-globose(1).In India Callistemon
spp introduced from Australia and cultivated in gardens for its beautiful flower, uncommonly pretty foliage, gorgeous shade
and large amount of Nectar.(5). Callistemon flowers were used as food source by Australian aborigines. The flowers were
sucked for their Nectars and used to make sweet drinks. Callistemon sp. also had role as traditional bush medicines for
Australian aborigines. The leaves were used to cure respiratory tract infections (2). Moreover, Antistaphylococcal,
nematicidal, larvicidal, pupicidal,antithrombotic activities of genus callistemon, and antioxidant activities have been
documented (5). In China, Callistemon spp., specially Callistemon viminalis are used in traditional medicine pills for treating
hemorrhoids. Callistemon are also used as weed control and as Bioindicators for environmental management. Ecologically,
Callistemon sp. as farm tree is planted for forestry plantations or ornamental purposes. Callistemon known as traditional and
folk medicine have been reported to have antihelmenthic, antibacterial, antidiabetic, antiinflamentary, anticough, and
antibronchitis activity.The plant has been used by tribal communities of India for the treatment of gastrointestinal
disorders,pain,and infectious diseases (2).

Reactive oxygen species (ROS) such as superoxide anion radicals, hydroxyl radicals and hydrogen peroxide are formed as a
byproduct during normal oxygen metabolism. But their excessive production leads to damage of proteins, DNA and lipids
which is associated with the chronic degenerative diseases including cancer, coronary artery disease, hypertension and
diabetes etc. (9). The role of antioxidants is to neutralize the excess of free radicals, to protect the cells against their harmful
effects and to contribute to disease prevention. A direct relationship between oxidant activity and phenolic content of plant
extracts has also been reported (11).

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Different pathogenic microorganisms are responsible for a number of diseases of human being. Although modern science
has developed many types of antibiotic against these microbes but generation of resistance is a global alarming issue.
Therefore scientific research focuses on plant species to explore new and potential antimicrobial agents, nowadays (9).
Therefore present study focuses on evaluation of antioxidant as well as antioxidant potential of Callistemon spp.

II. METHODOLOGY

 Collection of Plant materials: Callistemon spp. leaves and flowers were procured from Garden near Shivchhatrapati College,
Aurangabad and immediately transported to Laboratory.
 Preparation of Callistemon spp.leaves and flower extract : Collected plant material was dried under shade and dried
material ground to a coarse powder.Crude methanolic leaves and flowers extract of Callistemon spp. was prepared by Soxhlet
extraction method (3).10g of leaves and flower powder packed into a thimble and run in a soxhlet extractor separately.It was
extracted with 400 ml of methanol for a period of about 48 hrs and 22 cycles or till the solvent in the siphon tube of an
extractor become colorless. After this, the extract was filtered and the solvent was allowed to evaporate over night. Then
the extract was stored at 40 C for further analysis.
 DPPH free radical scavenging activity of CLE : Antioxidant activity of CLE was evaluated using DPPH free radical
scavenging activity.In this,0.1 ml of crude methanolic Callistemon leaves extract solution(concentration ranging from 25-500
µg/ml) was used and to this solution, 2 ml of methanol and freshly prepared 0.25 ml of methanolic soluition of 1mM DPPH
( 2,2-diphenyl-1 picrylhydrazyl radical) were added. Reaction mixtures were allowed to stand for 30 mins at room temperature
under dark condition.The absorbance of reaction mixtures were taken at 517 nm.Ascorbic acid was used as positive
control.(7,10)The scavenging activities were expressed as percentage inhibition and calculated using the formula :

% Inhibition = ABlank – ASample / ABlank × 100

A Percent inhibition versus concentration curve was plotted and the concentration of sample required for 50% inhibition
(IC50 ) was determined.(8)

 Antibacterial activity of CFE :

Five bacterial cultures were acquisized from MTCC, Chandigarh and NCIM, Pune viz. Escherichia coli, Salmonella typhi,
Staphylococcus aureus,Proteus vulgaris,Pseudomonas aeruginosa.. These microbes were used in the test evaluation of antimicrobial
assay of Callistemon spp.flower extract. The antibacterial activity was done using agar well diffusion method All bacterial
cultures were inoculated in nutrient broth tubes and incubated at 370 C for 24h. Cell densities of all culture suspensions were
adjusted as per 0.5 McFarland standards and then used for assay. 0.1 ml of each grown culture was added to soft agar and
mixed well. This seeded soft agar was then plated on basal agar plates. All plates were allowed to solidify. After solidification
plates were punched using cork borer to obtained 5 wells of 6mm diameter. In one well 15 µl of methanol was added as a
control and in other four wells of same plates,15 µl of different concentrations of Callistemon flowers extract
(0.25%,0.50%,0.75% and 1%) were added. All plates were kept in refrigerator for diffusion and then incubated at 370 C for
24hrs (15).

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III. RESULT AND DISCUSSION

Collection of Plant materials: In this study Callistemon leaves and flowers were collected from botanical garden, as shown
in Fig.1.

Fig.1.Collected Callistemon plant

Preparation of Callistemon spp.leaves and flower extract:

The extracts of leaves and flowers were prepared in methanol by Soxhlet extractor.

DPPH free radical scavenging activity of CLE:

The Callistemon leaves extract was evaluated for their capacity to scavenge DPPH free radical along with ascorbic acid as
positive control(Fig.2,3.4.&5). The DPPH free radical scavenging capacity may serves as indicator of its antioxidant
capacity(Yildirim et al.,2000).The methanolic extract of leaves of Callistemon exhibited pronounced antioxidant activity i.e.
66.30% (IC50 Value : 197.76) at concentration of 500 ug/ml,which is a comparable to scavenging activity of 100 ug/ml
concentration of ascorbic acid (Table.1).The potent antioxidant activity of methanolic Callistemon leaves extract can be a
result of high amount of flavonoids & phenolic compounds (Alothman et al. 2009; Isabelle et al. 2010; Hossain et al.
2011).Nearly similar result of antioxidant activity of Callistemon leaves extract was reported by Arvin Kumar et al. (2015) at
200 ug/ml concentration which is 64.60% and S.Chowdhary et al. (2012) with highest percent inhibition of 61.64%.

Fig.2. DPPH free radical scavenging activity of CLE

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DPPH Scavenging Activity

100
% Inhibition y = 0.0863x +
50 47.353
0 % Inhibition
0 200 400 600
Concentration in µg/ml

Fig.3.DPPH Scavenging activity of Ascorbic acid

DPPH Scavenging Activity

100
% Inhibition

y = 0.0671x +
50 36.752
0 % Inhibition
0 500 1000
Concentration in µg/ml

Fig.4: DPPH Scavenging activity of Callistemon leaves extract

Name of the Concentration Absorbance (517 % Inhibition IC50 (µg/ml)
sample (µg/ml) nm)
Control 0.092
25 0.061 33.69
50 0.059 35.86
100 0.048 47.82
Callistemon leaves 200 0.040 56.52 197.76
Extract
300 0.037 59.78
400 0.034 63.04
500 0.031 66.30
25 0.059 35.86
50 0.042 54.34
100 0.033 64.13
Ascorbic acid 200 0.027 70.65
(Standard) 30.81
300 0.023 75.00
400 0.018 80.43
500 0.012 86.95
Table.1.DPPH free radical scavenging activity

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Antioxidant Activity
100

Percent Inhibition
Callistemon
50 leaves
extract
0 Ascorbic
1 2 3 4 5 6 7 acid
Concentration in µg/ml
Fig .5. Comparison of antioxidant activity of CLE and Ascorbic acid

Antibacterial activity of Callistemon flowers extract(CFE)

The Callistemon flowers extract exhibited fairly significant antibacterial activity against all tested bacterial cultures(Fig.6).
Zones were more prominent with higher concentrations of extract than that of lower.(Table.2). No inhibition was observed
around well having methanol as control. The highest zone of inhibition by CFE was found against S.aureus which is 20 mm.
The CFE showed antibacterial activity against all bacteria used in study. This observation is particularly noteworthy because
plants extracts are known to be more active against all Gram positive than Gram negative bacteria and the flowers extract
notably exhibited activity against all bacterial species tested here. However Gram positive bacteria were more susceptible
than Gram negative bacteria.This is agreement with previously reported result by Cock Ian Edwin(2012). The values of
Inhibition zone diameter obtained in this study against S.aureus were more than that found by Udita Tiwari (2014).

Sr.No. Bacterial cultures Zone of inhibition in mm

0.25% 0.5% 0.75% 1.0%
1. Staphylococcus.aureus 16 17 19 20
2. Escherichia coli 15 15 16 16
3. Proteus vulgaris 15 16 17 17
4. Salmonella typhi 13 14 14 16
5. Pseudomonas aeruginosa - 14 15 16
Table .2. Zones of inhibition shown by CFE

Fig.6.Antibacterial activity of CFE

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