Journal of Phytology 2011, 3(3): 68-76 ISSN: 2075-6240

Phytopharmacology Available Online: www.journal-phytology.com

REGULAR ARTICLE

ANTIMICROBIAL, ANTIOXIDANT ACTIVITY

AND PHYTOCHEMICAL SCREENING OF
TECOMA STANS (L.) JUSS. EX KUNTH
Govindappa M1*, Sadananda TS1, Channabasava R1, Jeevitha MK2,
Pooja KS2 and Vinay B. Raghavendra3
1Department
of Biotechnology, Shridevi Institute of Engineering & Technology,
Sira Road, Tumkur-572 106, Karnataka, India,
2Department of Biotechnology, Shridevi PG Center, Sira Road, Tumkur-572 106, Karnataka, India
3Department of Biotechnology, Teresian PG Center, Siddartha Nagar, Mysore-570 011, India

SUMMARY
The ethanol, methanol and water extracts of Tecoma stans effective against tested bacteria
(Pseudomonas fluorescens, Clavibacter michiganensis sub sp. michiganensis, Xanthomonas
axanopodis pv. malvacearum, Staphylococcus aureus, E. coli, Pseudomonas aeruginosa and
Klebsiella pneumonia) and fungi (all species of Aspergillus and Alternaria). Phytochemical
analysis revealed the presence of alkaloids, flavonoids, saponins, phenols, steroids,
anthraquinones and tannins. The three extract fractions have showed highest total
Phenolic content (177-216 mg gallic acid equivalent/g). These three solvent fractions
possessed strong radical scavenging activity from FRAP and DPPH. It was ranged from
1433.75 to 3841.17 g/ml. The results indicate that this plant is a potential candidate to be
used as an antimicrobial and antioxidant.
Key words: Tecoma stans, Antimicrobial, antioxidant, phytochemicals
Govindappa M et al. Antimicrobial, Antioxidant Activity and Phytochemical Screening of Tecoma stans (L.) Juss. ex Kunth. J Phytol 3/3 (2011) 68-76.
*Corresponding Author, Email: dravidateja07@yahoo.co.in, Tel.: +91-9686114847, +91-816-2212626, Fax: +91-816-2212629

1. Introduction
The increase in prevalence of multiple against various diseases (Kumar et al., 2004;
drug resistance has showed down the Sheeja and Kuttan, 2007; Mukherjee et al.,
development of new synthetic antimicrobial, 2007). The rapid emergence of multiple drug
antioxidative drugs and the new drug is resistance strains of pathogens to current
necessary to search for new antimicrobial, antimicrobial agents has generated an urgent
antioxidant and anti-inflammatory from intensive for new antibiotics from medicinal
alternative sources. Phytochemicals from plants. Many medicinal plants have been
medicinal plants showing antimicrobial and screened extensively for their antimicrobial
antioxidant activities have the potential of potential worldwide (Kaur and Arora, 2009;
filling this need because of structures are Mothana et al., 2009; Adedapo et al., 2009a).
different from those of the more studied and Free radicals which have one or more
their of the more action may too very likely unpraired electrons (superoxide, hydroxyl,
differ (Fabricant and Fanworth, 2001). In this peroxyl) are produced in normal or
growing interest, many of the Phytochemical pathological cell metabolism and the
bioactive compounds from a medicinal compounds that can scavenge free radicals
plants have shown many pharmacological have great potential in ameliorating the
activities (Prachayasittikul et al., 2008; Chen diseases and pathological cells (Halliwell,
et al., 2008; Pesewu et al., 2008; Turker and 1995; Squadriato and Peyor, 1998; Gulcin et
Usta, 2008). Screening of various bioactive al., 2001). Antioxidants thus play an
compounds from plants has lead to the important role to protect the human body
discovery of new medicinal drug which have against damage by reactive oxygen species.
efficient protection and treatment roles in Free radicals or Reactive Oxygen Species
 Govindappa M et al./J Phytol 3/3 (2011) 68-76

(ROS) are produced in vivo from various attachment. The extract was lyophilized
biochemical reactions and also from the under 5 μm Hg pressure and stored at -200C.
respiratory chain as a result occasional The experimental were carried out using an
challenges. These free radicals are the main appropriate amount of lyophilized material.
culprits in lipid peroxidation. Plants
congaing bioactive compounds have been Phytochemical analysis
reported to possess strong antioxidant Phytochemical analysis was carried out
properties. for saponins, flavonoids, steroids, phenol,
Tecoma stans (Bignoniaceae) known as anthroquinone, alkaloids (Obdoni and
yellow elder is an erect shrub or small tree. Ochuko, 2001) and tannins (Kaur and Arora,
The plant has been used for a variety of 2009) were performed as described by the
purposes in herbal medicine, treating authors. Wagner’s and Heger’s reagents was
diabetes and digestive problems. Extracts used for alkaloid foam test for saponins, Mg-
from Tecoma stans leaves have been found to HCl and Zn-HCl for flavonoids, acetic
be inhibit the growth of the yeast infection. anhydride and sulphuric acid for steroids,
Marzouk et al. (2006) have studied the chloride and gelatin for tannins, ferric
anticancer activity of Tecoma stans and chloride for phenol, hexane and diluted
antioxidant constituents. Alanso-Castro et al. ammonia for anthraquinones test. All these
(2010) have reported that the Tecoma stans experiments were carried out for distilled
extracts exhibited antidiabetic activity. water, ethanol and methanol extracts
Senthilkumar et al. (2010) have reported that individually.
the extracts having antibacterial activity on
human pathogenic bacteria. Determination of total Phenolic content
In present study was aimed to examine Total Phenolic Content (TPC) in extracts
the total Phenolic content and Phytochemical was determined by Folin-Ciocalteu’s
analysis of ethanol, methanol and water colorimetric method as described by
extract of Tecoma stans were screened for Adedapo et al. (2009b). Each solvent
antimicrobial and antioxidant properties extracted solution (0.3ml in triplicate) was
using standard methods. The findings from mixed with 1.5 ml of 10% Folin-Ciocalteu’s
this work may add to the overall value of the reagent and 1.2 ml of 7.5% (W/V) sodium
medicinal potential of the plant. carbonate. The mixture was kept in the dark
for 30 min and absorbance was measured at
2. Materials and Methods 765 nm. Quantification was done on the basis
The plant was collected in November of a standard curve of gallic acid. The results
2009 from our college campus (Shridevi were expressed as gallic acid equivalent
Institute of Engineering & Technology, Sira (GAE) i.e. mg gallic acid/100ml. All tests
Road, Tumkur, Karnataka, India). The plant were performed in triplicate.
was identified by their vernacular names and
later it was compared with the herbarium of Determination of antimicrobial activity
Antimicrobial assay
Department of Studies in Botany, Manasa
Pseudomonas fluorescens, Clavibacter
Gangothri, University of Mysore, Mysore
michiganensis sub sp. michiganensis,
and Government Ayurvedic College, Mysore,
Xanthomonas oryzae pv. oryzae, Xanthomonas
India.
axanopodis pv. malvacearum and strains of
Staphylococcus aureus, E. coli, Pseudomonas
Extract Preparation
Plant parts were air dried at room aeruginosa and Klebsiella pneumonia bacteria
temperature for 4 weeks to get consistent were obtained from stock cultures presented
weight. The dried parts were later ground to at -80°C at Department of Studies in Applied
powder. 100 g of wet and dried samples Botany, Seed pathology and Biotechnology,
were extracted with distilled water, ethanol University of Mysore, Manasa Gangothri,
and methanol (600-800C, 200ml) separately Mysore, Karnataka, India and Department of
for 2 days in water both with a shaking Studies in Biotechnology and Microbiology,
Bangalore University, Gnana Bharathi,
 Govindappa M et al./J Phytol 3/3 (2011) 68-76

Bangalore, India respectively. Two Gram FRAP assay

positive bacteria tested were Clavibacter FRAP reagents was freshly prepared by
michiganensis sub sp. michiganensis, mixing 25 mL acetate buffer (300 mM, pH
Staphylococcus aureus and six Gram negative 3.6), 2.5 mL 2,4,6-tris (2-pyridyl)-S-triazine
bacterias tested were Pseudomonas fluorescens, (TPTZ) solution (10 mM TPTZ in 40 mM/L
Xanthomonas oryzae pv. oryzae, Xanthomonas HCl) and 2.5mL FeCl3 (20 mM) water
axanopodis pv. malvacearum, E. coli, solution. Each sample (150 µL) (0.5 mg/mL)
Pseudomonas aeruginosa and Klebsiella dissolved in methanol was added in 4.5 mL
pneumonia. All bacteria were grown on of freshly prepared FRAP reagent and stirred
nutrient agar media. and after 5 min, absorbance was measured at
Fungi (Aspergillus flavus, Aspergillus niger, 593nm, using FRAP working solution as
Alternaria carthami, Alternaria helianthi, blank (Szollosi and Szollosi Varga, 2002;
Cercospora carthami, Fusarium solani, Fusarium Tomic et al., 2009). A calibration curve of
oxysporum, Fusarium verticilloides and ferrous sulfate (100-1000 µmol/L) was used
Nigrospora oryzae were obtained from and results were expressed in µmol Fe2+/mg
Department of Studies in Applied Botany, dry weight extract. The relative activity of
Seed Pathology and Biotechnology, the samples was compared to L-ascorbic acid
University of Mysore, Manasa Gangothri, and BHT. The assay was repeated thrice for
Mysore, Karnataka, India and Department of each solvent extracts.
Studies in Microbiology, Bangalore
University, Gnana Bharathi, Bangalore, India DDPH radical assay
respectively. All fungi were grown on potato The effect of different solvent extracts on
dextrose agar medium. DPPH radical was estimated using the
method of Liyana-Pathirana and Shahidi
Paper disc method (2005). DPPH solution was freshly prepared
Diameter of zone of inhibition was by dissolve 24mg DPPH in 100ml of each
determined using the paper disc diffusion solvent, stored at -200C before use. 150µl of
method as described by Lai et al. (2009) and sample (10µl sample + 140µl distilled water)
Adedapo et al. (2008). A swab of the bacteria is allowed to react with 2850µl of DPPH
or fungi suspension containing 1x108 reagent (190µl reagent + 2660µl distilled
CFU/ml was spread on to Petri plates water) for 24h in the dark condition.
containing nutrient agar media separately. Absorbance was measured at 515nm.
Each solvent extracts were dissolved in each Standard curve is linear between 25 to
solvent to final concentration of 10mg/ml. 800µM ascorbic acid. Results expressed in
Sterile filter paper discs (6mm in diameter) µm AA/g fresh mass. Additional dilution
impregnated with 1mg of plant extracts were needed if the DPPH value measured will
placed on culture plates separately for over the linear range of the standard curve.
bacteria and fungi. The plates were Mix 10ml of stock solution in a solution of
incubated at 37°C for 24h. The standard 45ml of solvents, to obtain an absorbance of
chloromphenicol (10μg) for bacteria and 1.1±0.02 units at 517nm using
carbendazim for fungi discs were used as spectrophotometer (Katalinic et al., 2006). All
positive controls. Antimicrobial activity was determinations were performed in triplicate.
indicated by the presence of clear inhibition The percentage inhibition of DPPH radical
zone around the discs. The assay was by the samples was calculated according to
repeated thrice and mean of three formula of Yen and Duh (1994),
experiments was recorded. % inhibition= [{Abs control- Abs
sample}/Abs control] x 100,
Determination of antioxidant activity Where Abs control is the absorbance of the
In order to investigate the antioxidant DPPH radical+ ethanol, Abs sample is the
properties of the examined extracts, ferric ion absorbance of DPPH radical+ sample
reducing antioxidant power (FRAP) and 2, 2- extract/standard.
diphenyl-1-picrylhydrazyl (DPPH) assay.
 Govindappa M et al./J Phytol 3/3 (2011) 68-76

Statistical analysis extracts inhibited the growth of all most all

Analysis of variance (ANOVA) was used the isolates of bacteria and fungi. The
to determine the significance of difference methanol and ethanol extract showed more
between treatment groups (p<0.05). Means potent against E. coli, Xanthomonas axanopodis
between treatment groups were compared pv. malvacearum, Clavibacter michiganensis sub
for significance using Duncan’s new Multiple sp. michiganensis, Staphylococcus aureus,
Range post test. Klebsiella pneumoniae, Pseudomonas aeruginosa,
Pseudomonas fluorescens and moderate activity
3. Results observed in Xanthomonas oryzae pv. oryzae. All
Antimicrobial assay the solvent extracts exhibited high activity on
The antimicrobial activities of methanol all species of Aspergillus and Alternaria. All
and ethanol extracts of T. stans gave different the extracts did not showed any effect on
zones of inhibition on the organisms tested species of Fusarium and Nigrospora oryzae.
(Table 1). The methanolic and ethanolic

Table 1. Zone of inhibition (in mm) of antimicrobial activity y disc diffusion method using different solvent extract
of Tecoma stans
Microorganisms Samples
Methanol Ethanol water chloromphenicol
Bacterial pathogens
Klebsiella pneumonia 10±1 9±1 7±1 18±

Escherichia coli 15±2 14±2 2±1 20±

Staphylococcus aureus 11±1 11±1 2±1 18±2
Pseudomonas aeruginosa 10±1 9±1 6±1 18±
Pseudomonas fluorescens 9±1 9±1 4±1 21±
Clavibacter michiganensis sub sp. michiganensis 13±2 13±2 6±1 16±
Xanthomonas oryzae pv. oryzae 6±1 5±1 2±1 16±
Xanthomonas axanopodis pv. malvacearum 15±2 16±2 6±1 22±
Fungal pathogens Carbendazim
Aspergillus niger 16±2 14±2 5±1 21±
Aspergillus flavus 16±2 11±2 2±1 21±
Alternaria carthami 9±1 8±1 4±1 19±
Alternaria helianthi 6±1 6±1 2±1 21±
Cercospora carthami 8±1 6±1 4±1 21±
Fusarium solani 2±1 2±1 2±1 19±
Fusarium verticilloides 2±1 2±1 2±1 20±
Nigrospora oryzae 4±1 3±1 2±1 20±
+: Presence; -: absence, repeated the each experiments three times for each replicates

Phytochemical analysis all solvent extracts. The phytochemicals

The phytochemical screening showed that strongly present in the ethanol and methanol
the different solvent extracts of T. stans, the extracts. But the water extract yielded less
tannin, flavonoids, phenol, alkaloids, steroids, quantity of phytochemicals (Table-2).
anthraquinones and saponins were present in
 Govindappa M et al./J Phytol 3/3 (2011) 68-76

Table 2: Phytochemical analysis for the different solvent extracts of T. stans

Extracts Alkaloid Flavonoi Saponin Phenol Steroid anthroquinone Tannin
s ds s s s s s
Methan +++ +++ +++ +++ +++ +++ +++
ol
Ethanol +++ +++ +++ +++ +++ +++ +++
Water + + + + + + +
+++ Strong; ++ medium; +poor Presence; -: absence, repeated the experiments three times for
each replicates, Classification was based on observation of colour intensity and amount of precipitate

Table 3: Total Phenolic Content and total antioxidant activity from different solvent extract of T. stans
Extracts FRAP ( mol/L) TPC
(mg gallic acid/g plant extract)
Methanol 3841.17±66.34a 216+16a
Ethanol 3416.24±62.71b 206+09a
Water 1433.75+14.22d 177+12b
Ascorbic acid 1648.52±17.46c -
BHT 64.84±2.97e -
All the data is three replicates of each samples
Phenolic antioxidant coefficient calculated as the ratio FRAP (µM/L)

Total phenol contents and antioxidant of T. trans had higher activity than that of
activity water extract. At a concentration of
Total Phenolic Content (TPC) was 0.1mg/ml, the scavenging activity of ethanol
determined using the Folin-Ciocalteau and methanol extracts reached 56.88% and
reagent and expressed in terms of mg gallic 58.92% respectively while at the
acid equivalent (GAE)/ g ml extract. The concentration, that of water was 39.23%.
more TPC was observed in methanol (216) Though the DPPH radical scavenging
followed by ethanol (206) and water (177) abilities of the extract were less than those of
extracts (Table 3). ascorbic acid (98%) and BHT (97.8%) at 0.1
The antioxidant activity of the ethanol mg/ml, the study showed that the extracts
and methanol crude extract and its various have the proton donating ability and could
fractions, as measured by the ability to serve as free radical inhibitors or scavenging,
scavenge DPPH free radicals, was compared acting possibly as primary antioxidants
with the standards/ ascorbic acid and (Figure-1).
Butylated Hydroxy Toluene (BHT). It was
observed that ethanol and methanol extracts

Figure 1. DPPH scavenging activities of the different solvent extracts of T. stans

The reducing ability of the extract was in ethanol and methanol extracts of T. stans
the range of 1433.75- 3841.17 μm Fe (II)/mg were estimated from their ability to reduce
(Table 3). The antioxidant potentials of the TPRZ-Fe (III) complex to TPTZ-Fe (II). The
 Govindappa M et al./J Phytol 3/3 (2011) 68-76

FRAP values for the ethanol, methanol and The anthraquinones have also shown as
water extract of T. stans were significantly antimicrobial (Comini et al., 2011) and
lower that of ascorbic acid but higher that of antioxidant (Gow-Chin et al., 2000)
BHT. properties. Strong presence of tannins in all
extracts may explain its potent bioactivities
4. Discussion are known to possess potent antimicrobial
In recent years, the search for activities (Kaur and Arora, 2009) and
phytochemicals possessing antimicrobial and antioxidants (Zhang and Lin, 2008). The
antioxidant properties have been on the rise Saponins from plant extracts have already
due to their potential use in the therapy of reported as potent antimicrobial (Mandal et
various chronic and infectious diseases. al., 2005) and antioxidant activity (Gulcin et
Epidemiology and experimental studies have al., 2004).
implicated oxidative cellular damage arising The present investigation has shown that
from an imbalance between free radical the ethanol and methanol extract of T. stans
generating and scavenging systems as the have active phytochemicals which are able to
primary cause of cardiovascular, diseases, inhibit plant and animal pathogenic bacteria
cancer, aging etc (Halliwell, 1996). Due to and fungi. The ethanol and methanol extract
risk of adverse effects encountered with the fractions showed significantly antimicrobial
use of synthetic antibiotics, medicinal plants activity against all Gram-positive and Gram-
may offer an alternative source for negative bacteria and different fungi tested.
antimicrobial agent with significant activity Strong antioxidant properties were
against pathogenic and infective confirmed in the ethanol and methanol
microorganisms. In addition, a number of extract fractions. These activities may be due
antibiotics have lost their effectiveness due to to strong occurrence of polyphenolic
the development of resistant strains, mostly compounds such as flavonoids, tannins,
through the expression of resistance genes alkaloids, steroids, phenols and Saponins.
(Berahou et al., 2007). The antioxidant activity was comparable
Results of our findings confirmed the use with standard ascorbic acid and BHT. These
of T. stans as traditional medicine. We found findings provide scientific evidence to
strong antimicrobial and antioxidant support traditional medicinal uses and
activities specifically in the ethanolic and indicate a promising potential for the
methanolic extracts of T. stans. High TPC development of an antimicrobial and
values found in ethanolic and methanolic antioxidant agent from T. stans plant. This
extracts (11.32 and 11.64 mg GAE/g extract) medicinal plant by in vitro results appear as
imply the role of phenolic compounds in interesting and promising and may be
contributing these activities. Plant phenolic effective as potential sources of novel
compounds have been found to possess antimicrobial and antioxidant drugs.
potent antioxidants (Adedapo et al., 2009b;
Adesegun et al., 2009; Lai et al., 2010) and Acknowledgements
antimicrobial (Kaur and Arora, 2009; Alcaraz We thank Dr MR Hulinaykar, Managing
et al., 2000; Lai et al., 2010). Trustee, Sri Shridevi Charitable Trust (R.)
The flavonoids from plant extracts have and Dr MA Venkatesh, Principal, SIET,
been found to possess antimicrobial and Tumkur, India for encouragement, Dr. P
antioxidants properties in various studies Sharanappa, DOS in Biosciences, University
(Lin et al., 2008; Lopez-Lazaro, 2009; Yoshida of Mysore, Hassan, India for assistance in
et al., 2009; Amaral et al., 2009). The presence identifying plant. Dr. S Lokesh, DOS in
of alkaloids have shown as antimicrobial Applied Botany, Seed Pathology and
(Erdemoglu et al., 2007) and antioxidant Biotechnology, University of Mysore,
(Maiza-Benabdesselam et al., 2007) activity. Manasa Gangothri, Mysore and Dr. DC
Plant based steroids also possess as Mohan, DOS in Microbiology, Bangalore
antimicrobial (Shihabudeen et al., 2010) and University, Jnana Bharathi, Bangalore for
antioxidant (Koduru et al., 2007) extracts.
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