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Antibacterial and antifungal activity of isatis tinctoria L. (Brassicaceae) using the micro-plate method

Authors:
  • Korea Research Institute of Bioscience and Biotechnology (KRIBB)
  • Capital University of Science and Technology (CUST)

Abstract

Isatis tinctoria L. has well-documented history as conventional therapeutic herb. In present study its crude extract was examined for broad-spectrum antimicrobial activity using micro-titer plate method. Four different plant parts were extracted with 14 different solvents. All fractions were analyzed against seven bacterial and four fungal strains. Ethyl acetate, chloroform, n-hexane and acetone showed maximum antibacterial activity with minimum IC50 value (≤200 µg/ml). Leaves>branches> roots>flower is the order of different parts based on antibacterial activity. Although, in some cases like against Klebsiella pneumonia and Micrococcus luteus the flower showed better results as compared to other parts. Roots showed better results against Staphylococcus aureus and Pseudomonas aeruginosa. Extracts showed better antimicrobial activity as compared to antibiotics (cefotaxime). The activity of the extracts against gram positive was better than gram negative. For antifungal activity, ethyl acetate > n-hexane-ethyl acetate (1:1) > chloroform> acetone was the order of the fraction with increasing growth inhibition rate. All the parts (except branches) were observed having antifungal activity. The most resistant strains found in this study were Mucor mycosis, none of the fraction have more than 30% inhibition on used concentration. Plant crude extract being having broad spectrum antimicrobial activity is suggested for pre-clinical and clinical trials.
Pak. J. Bot., 49(5): 1949-1957, 2017.
ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF ISATIS TINCTORIA L.
(BRASSICACEAE) USING THE MICRO-PLATE METHOD
IKRAM ULLAH1, ABDUL WAKEEL1, ZABTA KHAN SHINWARI1, 2*, SOHAIL AHMAD JAN1*,
ALI TALHA KHALIL1 AND MUHAMMAD ALI1
1Department of Biotechnology, Quaid-i-Azam University Islamabad, 45320-Pakistan
2Qarshi University Lahore, Pakistan
*Corresponding author’s email: shinwari2008@gmail.com; sjan.parc@gmail.com
Abstract
Isatis tinctoria L. has well-documented history as conventional therapeutic herb. In present study its crude extract was
examined for broad-spectrum antimicrobial activity using micro-titer plate method. Four different plant parts were extracted
with 14 different solvents. All fractions were analyzed against seven bacterial and four fungal strains. Ethyl acetate,
chloroform, n-hexane and acetone showed maximum antibacterial activity with minimum IC50 value (≤200 µg/ml).
Leaves>branches> roots>flower is the order of different parts based on antibacterial activity. Although, in some cases like
against Klebsiella pneumonia and Micrococcus luteus the flower showed better results as compared to other parts. Roots
showed better results against Staphylococcus aureus and Pseudomonas aeruginosa. Extracts showed better antimicrobial
activity as compared to antibiotics (cefotaxime). The activity of the extracts against gram positive was better than gram
negative. For antifungal activity, ethyl acetate > n-hexane-ethyl acetate (1:1) > chloroform> acetone was the order of the
fraction with increasing growth inhibition rate. All the parts (except branches) were observed having antifungal activity. The
most resistant strains found in this study were Mucor mycosis, none of the fraction have more than 30% inhibition on used
concentration. Plant crude extract being having broad spectrum antimicrobial activity is suggested for pre-clinical and
clinical trials.
Key words: Anti-bacterial, Anti-fungal, Isatis tinctoria L., Resistant strains, Therapeutics agents
Introduction
Isatis tinctoria L. is a well-known medicinal plant
commonly known as Woad, belongs to family of
Brassicaceae. It is distributed in Europe, Russia, China,
Japan, Iran, Afghanistan, Pakistan, Germany and England.
Woad is locally known as “Tora panrra” is well known for
its insecticidal effects; it is placed around the natural
products as a barrier to protect them from insects and
reptiles. It was prescribed for the cure of inflammatory
problem, snake bites, tumors, injuries, ulcers and
hemorrhoids; Mohsenzadeh et al., 2009). The compounds
isolated from Isatis tinctoria L. have antifungal and anti-
myco-bacterial activity (Honda et al., 1980).
Several classes of modern antibiotics are available for
killing these bacterial strains to get rid of the diseases
caused by them, but there is also exponential increase in
the resistant of these bacteria strains to drugs. To solve
the resistant problem, the best way is to use herbal
preparation and phytochemicals (Gilani et al., 2007;
Shinwari et al., 2009). Other secondary metabolites are
carboxylic acid both aliphatic and aromatic, different
derivatives of glycosides, peptides, isoprenoids (Hartleb
et al., 1995), flavonoids and anthranoids (Wu et al.,
1997). The procedure used for the separation of
pharmaceutically active compound from a complex
mixture of metabolites by using a suitable solvent is
called extraction. The solvent penetrate mixed and
dissolve the same polarity containing compounds. These
extracts can be used as solution, suspension, semi-solid,
or as a powder (Ncube et al., 2008). The quality of extract
mainly depends up on: type and part of plant used, solvent
(nature, concentration and polarity) used for extraction
and appropriate procedure (Ncube et al., 2008). Different
parts of the plant like branches, flowers, leaves, roots,
fruits, seeds and barks etc. are selected either randomly or
on the basis of conventional and traditional knowledge
(Parekh et al., 2006; Gilani et al., 2010). The compounds
that can slow down or completely stop oxidative damage
by scavenge free radicals are called anti-oxidants. These
antioxidants are reducing agents and donate hydrogen by
receiving oxygen (Anokwuru et al., 2011; Yamagishi et
al., 2011). Fruits, vegetables and cereals are a rich source
of antioxidants (Mertens-Talcott et al., 2006). Synthetic
antioxidants are available but natural source having no or
less side effect are preferred (Meenakshi et al., 2011).
Phenols, flavonoids and tannins are medicinal plants
based antioxidants being focused by the scientists
(Upadhyay et al., 2010). In the present study we used
fourteen different solvent extraction systems to find out
active fraction as anti-microbial from four different parts
of Isatis tinctoria L.
Materials and Methods
Antimicrobial activity of Isatis tinctoria L. was
performed using micro-plate method. A total of 56
fractions, extracted from four different parts (branches,
flowers, leaves and roots) with fourteen different solvents
were analyzed against seven bacterial and four fungal
strains.
Bacterial culture preparation: A bacterial colony was
picked from the starter culture and placed in freshly
prepared nutrient broth. The culture was incubated for 12
hours. After incubation, the culture was stored in the
refrigerator at 4oC. Before each assay the culture was
transferred to incubator for 14 hrs. For assay the culture
was diluted in the ratio of 1 to 10 in nutrient broth
(Sultanbawa et al., 2009).
IKRAMULLAH ET AL.,
1950
Bacterial test strains used
Gram positive: (1) Bacillus subtilis (2) Micrococcus
luteus (3) Staphylococcus aureus.
Gram negative: (4) Escherichia coli (5) Klebsiella
pneumoniae (6) Pseudomonas aeruginosa (7) Salmonella
typhimurium.
Assay procedure: The assay was performed in 96-well
plate. In the first row 7.5µl (4mg/ml) of each sample was
added and then diluted down in the column using three fold
serial dilution to get the final concentration of 100, 33.3,
11.1 and 3.7µg/ml (Fig. 1). The Dimethyl sulfoxide
(DMSO) and cefotaxime were taken as negative and
positive control respectively. After samples dilutions,
195µl inoculums (of each strain mentioned earlier) freshly
diluted were added to each well containing either sample,
DMSO or cefotaxime. Three empty wells were inoculated
only with culture. The plate were incubated for 30 minutes
and reading were taken using (ELX800, BIOTEK) micro-
plate reader at 630nm (Sultanbawa et al., 2009). After
calculating percent inhibition, IC50 was also calculated for
each sample by using method of Anderson et al. (2001).
Antifungal assay: The antifungal activity was carried out
using micro-titer plate method with a little modification in
the micro-spectrophotometric assay (Broekaert et al.,
1990). Fungal strains selected for assay were; Mucor
mycosis, Aspergillus nigar, Aspergillus fumagatus and
Aspergillus flavus. Crude extracts (20mg/ml) at the rate of
20μl were added to each well in micro-plate. Then 20μl of
amphotericin (an antifungal) as positive control, DMSO
and only culture were used as negative control. Then
100μl of the freshly diluted culture was added to each
well, containing either, amphotericin or DMSO (Satish et
al., 2007). Plate were incubated for 30 minutes at 27C0,
and readings were taken using (ELX 800, BIOTEK)
micro-titer plate reader at 630 nm. Percent inhibition was
calculated using formula;
% Inhibition= [(ΔC – ΔT)/ΔC] x 100.
ΔC represent corrected (48 hours reading 30 min
reading) absorbance of control, ΔT is corrected
absorbance of test sample (Broekaert et al., 1990).
Results
An antibacterial activity was evaluated against 14
different extracts each from leaves, flower, branches, and
flowers of Isatis tinctoria L. All the extracts were
analyzed against human pathogens. All the pathogens
were clinical isolates in which two strains, Pseudomonas
and Klebsiella were isolated from burn samples of patient
under treatment in Pakistan institute of medical sciences
(PIMS), Islamabad, Pakistan. Assays were performed
using modified technique of microtiter 96-well plate
method. The readings were taken on 630nm wave length
rays using (ELX 800, BIOTEK) micro-plate reader. All
the experiments were performed in triplicate and IC50 was
calculated using Excel sheet 2013 for each bacterial
strain. Using Origin-Pro 8.5 graphs were prepared from
IC50 calculated for each strain.
Anti-Escherichia coli activity: Four different
concentrations of crude extracts: 100, 33.34, 11.1 and
3.7µg/ml were tested against each strain. IC50 values were
calculated using MS Excel Sheet 2013 shown in (Fig. 1).
The best activity even better than Cefotaxime (used as a
standard) reported on used concentrations was of ethyl
acetate’s extracts from branches, flowers, leaves.
Chloroform’s extracts from roots were observed having
maximum activity with lower IC50 value. Acetone-water
(1:1) combination’s extracts from branches and flowers;
methanol-water (1:1) combination’s extracts from leaves
and ethanol’s extracts from roots were reported having
poor activity on the concentrations used (Fig. 2).
Anti-Bacillus subtilis activity: Cefotaxime used as
standard caused maximum inhibition with only 70µg/ml
IC50 value. The chloroform fraction of branches, leaves,
roots and ethyl acetate fraction of branches and leaves
showed maximum inhibition with 117, 111, 110, 110 and
111 (µg/ml) IC50 values respectively (Fig. 2).
Anti-Salmonella typhi activity: On the basis of IC50 value
many of the extracts resulted better inhibition than
Cefotaxime (used as a standard) on different concentration
used in assay. The IC50 value for present standard was
calculated 100 µg/ml. Some of the extracts from each part
showed lower than 100 µg/ml IC50 value (Fig. 3).
Anti-Staphylococcus aureus activity: Very diverse results
were reported from the activity against S. aureus with an IC50
value ranges from less than 100µg/ml to more than
5000µg/ml. Cefotaxime (used as positive control) was found
having an IC50 value of 200µg/ml, which greater than many
extracts. The significantly (p<0.05) maximum inhibition
with lowest IC50 value from branches was noted with ethyl
acetate fraction. The best fraction is represented with
subscript (g). Other fraction are also nominated with same
letter, that shows similar activity with no significant (p<0.05)
difference. Significantly (p<0.05) maximum inhibition form
flower, leaves and root was reported for ethyl acetate
fraction. The minimum inhibition with maximum IC50 values
was reported: methanol’s fraction from branches with more
than 800 µg/ml; methanol’s fraction from flowers with more
than 1000µg/ml; methanol-ethyl acetate (1:1) combination’s
fraction from leaves and water’s fraction with more than
5000 µg/ml from roots (Fig. 4).
Anti-Klebsiella pneumonia activity: In the present study
the K. pneumonia showed resistant to Cefotaxime (3rd
generation broad spectrum antibiotic) used as positive
control. The IC50 value calculated for Cefotaxime was
more than 700µg/ml, except branches and flowers.
Cefotaxime showed very weak activity whereas highest
IC50 values were reported in leaves and roots. The
significantly (p<0.05) best inhibitory activities were
reported for: ethyl acetate’s fractions from all plant parts
(branches, flowers, leaves and roots). The significantly
(p<0.05) minimum inhibitory activities with maximum
IC50 values were reported for: acetone-water (1:1)
combinations’ fractions and Cefotaxime from branches;
methanol-water (1:1) combinations’ fraction from
flowers; acetone-water (1:1) combinations’ fraction after
Cefotaxime from leaves and methanol-water (1:1)
combinations’ after Cefotaxime from roots (Fig. 5).
ANTIMICROBIAL ACTIVITY OF ISATIS TINCTORIA L.
1951
0
500
1000
1500
2000 Branches
f
f
f
f e e
e
d d
d
d
c
c
b
a
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
0
200
400
600
800
1000 Flowers
f
f f
f
f e
e
e d d
c
b
b
b
a
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
0
200
400
600
800
1000
1200
d
d
d
d d
c
c
c
c
c
b
b
b b
a
IC50 Value(µg/ml)
IC50 Value(µg/ml)
IC50 Value(µg/ml)
IC50 Value(µg/ml)
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Leaves
0
200
400
600
800
1000
1200
Anti E. Coli activity
Extracts with different solvents Extracts with different solvents
Extracts with different solvents
Extracts with different solvents
Roots
ed
d
d d d
d
c
c
c
b
b
b
b
a
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Fig. 1. Anti-Escherichia coli activity of 14 fraction with respective solvents from each branches, flowers, leaves and roots. Graphs
indicate IC50 values µg/ml bars with standard deviation bar having similar subscripts letters indicate no significant (p<0.05) difference,
while different subscripts letters shows significant (p<0.05) difference among different extracts. The highest inhibitory effects are shown
with last alphabet in alphabetical order. The minimum inhibitory fractions with maximum IC50 values are nominated with (a).
0
100
200
300
400
500
600
f
e
edd
d
d
c
c
c
b b
a
a
a
IC50 Value(µg/ml)
Branches
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
100
200
300
400
500
600
700
800
900
f
e
e
d
d
d
d c
c
c
c
b
a a
a
IC50 Value(µg/ml)
IC50 Value(µg/ml)
Flowers
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
200
400
600
800
1000
1200
f
e
e
e
e
e
e
e
d
d
c
c
c
b
a
IC50 Value(µg/ml)
Leaves
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
200
400
600
800
1000
Anti B. subtilis activity
f
f
e
e
e d
d
d
c
c
c
c
c
b
a
Roots
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
Fig. 2. Anti-Bacillus subtilis activity of 14 fraction with respective solvents from each branches, flowers, leaves and roots. Graphs indicate
IC50 values µg/ml bars with standard deviation bar having similar subscripts letters indicate no significant (p<0.05) difference, while
different subscripts letters shows significant (p<0.05) difference among different extracts. The highest inhibitory effects are shown with
last alphabet in alphabetical order. The minimum inhibitory fractions with maximum IC50 values are nominated with (a).
IKRAMULLAH ET AL.,
1952
40
60
80
100
120
140
160
180
200
IC50 Value(µg/ml)
IC50 Value(µg/ml)
g
f f
f
e e e e e e
d
c
b
b
a
Branches
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
40
60
80
100
120
140
160
180
f
ff
ee
e
dd
d
d
d
c
c
b
a
Flowers
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
100
200
300
400
500
600
f
f
f
f
f
f
fe
dd
c
c
c
b
a
IC50 Value(µg/ml)
Leaves
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
40
60
80
100
120
140
160
180
200
220
240
g
gff
e
e
e
cc
b
bb
b
a
a
Anti S. typhi activity
IC50 Value(µg/ml)
Roots
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
Fig. 3. Anti-Salmonella typhi activity of 14 fraction with respective solvents from each branches, flowers, leaves and roots. Graphs
indicate IC50 values µg/ml bars with standard deviation bar having similar subscripts letters indicate no significant (p<0.05) difference,
while different subscripts letters shows significant (p<0.05) difference among different extracts. The highest inhibitory effects are shown
with last alphabet in alphabetical order. The minimum inhibitory fractions with maximum IC50 values are nominated with (a).
0
200
400
600
800
1000
g g g g g
g
f
e e
d d
c
b
b
a
IC50 Value(µg/ml)
Branches
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
200
400
600
800
1000
1200
g
g f
f f f
f
f
e e
d
d
c
b
a
IC50 Value(µg/ml)
Flowers
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
200
400
600
800
1000
g
g
g
g f
f e
e e
e
d
c c
b
a
IC50 Value(µg/ml)
Leaves
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
1000
2000
3000
4000
5000
6000
e e e e e e e
e
e e d
c c
b
a
Anti S. aureus activity
IC50 Value(µg/ml)
Staphylococcus aureus
Roots
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
Fig. 4. Anti-Staphylococcus aureus activity of 14 fraction with respective solvents from each branches, flowers, leaves and roots. Graphs
indicate IC50 values µg/ml bars with standard deviation bar having similar subscripts letters indicate no significant (p<0.05) difference,
while different subscripts letters shows significant (p<0.05) difference among different extracts. The highest inhibitory effects are shown
with last alphabet in alphabetical order. The minimum inhibitory fractions with maximum IC50 values are nominated with (a).
ANTIMICROBIAL ACTIVITY OF ISATIS TINCTORIA L.
1953
Anti-Micrococcus luteus activity: M. luteus was found
susceptible to both cefotaxime (positive control) and
many of the fractions from all parts. More than half
fractions having an IC50 value less than 100 µg/ml. IC50
value for cefotaxime is only 78 µg/ml, while only
42µg/ml in ethyl acetate’s fraction from flowers were
reported. The ethyl acetate’s fraction from flowers
showed significantly (p<0.05) maximum inhibitory effect
(Fig. 6).
Anti-Pseudomonas aeruginosa activity: The activities of
some fractions against P. aeruginosa were not appreciable.
These fractions had IC50 values more than 700, 1000, 800
and 1200 (µg/ml) in branches, flowers, leaves and roots
respectively. Besides having such high IC50 values in some
fraction more than half fraction having IC50 values less 174
µg/ml, which is the IC50 values for Cefotaxime. The
chloroform fraction from leaves showed best inhibition on
given concentrations that’s why having 32 µg/ml IC50
values. Methanol’s and ethyl acetate’s fractions from
branches, (h)ethyl acetate fractions of flowers and
chloroform’s fraction from leaves and roots showed
significantly maximum activities (Fig. 7).
Anti-fungal activity: Four fungal strains were selected to
test the anti-fungal activity of I. tinctoria extracts. The
response of different parts toward fungal strains was
much diverged, over all fractions from braches were very
poor, with only one fraction of methanol-acetone (1:1)
combination showed more than 20% inhibition. The most
resistant strains reported in this study were Mucor
mycosis. None of the fraction has more than 30%
inhibition on used concentration (Table 1).
All the values were nominated with alphabetical order
from top to bottom. Fraction of ethyl acetate from flowers,
acetone’s fraction from leaves against Aspergillus nigar;
ethyl acetate’s fraction from leaves against Aspergillus
fumagatus; n-hexane’s, chloroform’s and ethyl acetate’s
fractions from leaves, ethyl acetate’s fraction from flowers
against Aspergillus flavus are fraction with best activity
(Table 1). The experimental fungal strains used in this study
were observed having significant (p<0.05) diversity in
susceptibility toward used concentrations of (56) fractions.
On the basis increasing susceptibility strains can be arranged
like: (Mucor mycosis < Aspergillus nigar < Aspergillus
fumagatus < Aspergillus flavus). On the basis of increasing
(%) inhibition of fungal growth capability of plants parts can
arranged as: (Branches< Roots< Flowers< Leaves). Just like
antibacterial activity; chloroform’s, ethyl acetate and
acetone’s fractions were very consistent as anti-fungal.
Discussion
Ethyl acetate fraction showed consistency except in
branches against Salmonella typhi. Chloroform, acetone
and n-hexane alone and combination were also consistent
respectively. Leaves were the part with outstanding
consistency, in the above consistent solvents. Branches,
roots and flower were consistent respectively. Although in
some cases like against Klebsiella pneumoniae and
Micrococcus luteus the flower showed better results.
Roots showed better results against S. aureus and P.
aeruginosa. The activity of positive control (cefotaxime)
was very consistent against B. subtilis M. luteus, S. typhi,
Pseudomonas aeruginosa, Escherichia coli, and
Staphylococcus aureus respectively. The worse activity of
positive control was against K. pneumonia. In all cases
the extracts showed the best results as compared to
antibiotics (cefotaxime). The activity of the extracts
against gram positive was better than gram negative
Our results are similar to the findings Sartoratto et al.
(2004) those reported that the aqueous and methanolic
extracts of many Brazilian medicinal plants shows better
antibacterial activity. The antibacterial activity against E.
coli and other gram-negative have also been reported
(Bakht et al., 2017; Yagoub, 2008). The antibacterial
activity may be due to the presence of alkaloids
(inhibiting cell division) in the extracts. Methanolic
extract inhibition of E. coli, P. aeruginosa and S. aureus
was reported but the concentration was very high (Djeussi
et al., 2013). Present study proved that ethyl acetate and
chloroform showed best results than water and methanolic
extracts. Our study was in accord once with Tane et al.
(2005) who recorded that Flavonoids, alkaloids and
diterpenoids of plant compounds had antibacterial
activity. A micro-plate method also prove that methanol’s
extracts have complete bacterial growth inhibition less
than 1mg/ml, which contained grifolic acid (Langfield et
al., 2004). The activity of organic solvents extracts is far
better than aqueous extracts (De Boer et al., 2005). The
best activity against gram-positive was also reported
earlier while contradictory to other published data which
reported good activity of extract against Gram-negative
(Awodele et al., 2013). Previous study also proves the
importance of our study being active against both Gram-
positive and Gram-negative. The broad spectrum activity
was previously reported ranging from 390-6250 μg/ml.
The good activity of the extracts as compare to positive
control of present study is also reported in literature (Aziz
et al., 2017; Pendota et al., 2015). The EC50/IC50 was
calculated for the first time for gentamicin against P.
aeruginosa (Soothill et al., 1992). The IC50 was
calculated for 50% growth inhibition of Bacillus
megaterium of plasma protein (Anderson et al., 2001).
For antifungal activity 4 mg/ml concentrations of all
(56) fraction were used. Four parts (branches, flowers,
leaves and roots) were used and all the parts (except
branches) were observed having antifungal activity.
Ethyl acetate > n-hexane-ethyl acetate (1:1) combination
> chloroform> acetone was the order of the fraction with
increasing rate growth inhibition, supported for
chloroform extract earlier (Ertas et al., 2005). The
fungicidal compounds were studied in ethyl acetate
fraction of Toddalia asiatica (L.) a conventional
medicinal plant (Duraipandiyan et al., 2009). The
maximum inhibitory activity for methanol was reported
for lantana leaves and flowers which supported by our
studies that the maximum A. flavus growth inhibition of
methanol fraction was (35%) of I. tinctoria (leaves)
(Bokhari, 2009). Medicinal plants play important role
for controlling of many important bacterial and fungal
diseases (Habiba et al., 2016; Khan & Shinwari, 2016;
Qasim et al., 2016; Hussain et al., 2015; Jan et al., 2015;
Shinwari et al., 2013).
IKRAMULLAH ET AL.,
1954
Fig. 5. Anti Klebsiella pneumoniae activity of 14 fraction with respective solvents from each branches, flowers, leaves and roots. Graphs
indicate IC50 values µg/ml bars with standard deviation bar having similar subscripts letters indicate no significant (p<0.05) difference,
while different subscripts letters shows significant (p<0.05) difference among different extracts. The highest inhibitory effects are shown
with last alphabet in alphabetical order. The minimum inhibitory fractions with maximum IC50 values are nominated with (a).
0
200
400
600
800
1000
1200
e e
e
e
e d
c c c
b
e d c
b
a
Branches
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
50
100
150
200
250
300
350
400
e
e
d
d
c
c
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
d
f
g
e f
f
e
c
b
a
Flowers
0
100
200
300
400
500
600
700
800
IC50 Value(µg/ml)
Anti M. luteus activity
g g
g
g
g
g f
f e d d
d c b
a
Leaves
IC50 Value(µg/ml)
IC50 Value(µg/ml)
IC50 Value(µg/ml)
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
50
100
150
200
250
300
e e
e f
f f
f
e
d
c c
b
a
a a
Roots
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
Fig. 6. Anti-Micrococcus luteus activity of 14 fractions with respective solvents from each branches, flowers, leaves and roots. Graphs
indicate IC50 values µg/ml bars with standard deviation bar having similar subscripts letters indicate no significant (p<0.05) difference,
while different subscripts letters shows significant (p<0.05) difference among different extracts. The highest inhibitory effects are shown
with last alphabet in alphabetical order. The minimum inhibitory fractions with maximum IC50 values are nominated with (a).
ANTIMICROBIAL ACTIVITY OF ISATIS TINCTORIA L.
1955
IKRAMULLAH ET AL.,
1956
ANTIMICROBIAL ACTIVITY OF ISATIS TINCTORIA L.
1957
0
100
200
300
400
500
600
700
800
h
h g
g
g
f f
e
e
d d d
c
b
a
IC50 Value(µg/ml)
Anti P. aeruginosa activity
Branches
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
200
400
600
800
1000
1200
f f f f f f
e e e e
d d
c
b
a
IC50 Value(µg/ml)
Flowers
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
200
400
600
800
1000
1200
g f
f f f
e d d d d d
c
b
a
IC50 Value(µg/ml)
Leaves
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
0
200
400
600
800
1000
1200
1400
f
f e
e
e e
e e e
d
d c c
b
a
IC50 Value(µg/ml)
Roots
Met,Eac
Met,Chf
n-Hex,Eac
n-Hex,Eth
Wat
Met
Eth
Ace
Eac
Chf
n-Hex
Ceft
Met,Wat
Ace,Wat
Met,Ace
Extracts with different solvents
Fig. 7. Anti-Pseudomonas aeruginosa activity of 14 fraction with respective solvents from each branches, flowers, leaves and roots.
Graphs indicate IC50 values µg/ml bars with standard deviation bar having similar subscripts letters indicate no significant (p<0.05)
difference, while different subscripts letters shows significant (p<0.05) difference among different extracts. The highest inhibitory effects
are shown with last alphabet in alphabetical order. The minimum inhibitory fractions with maximum IC50 values are nominated with (a).
Conclusion
Isatis tinctoria L. is one of the important medicinal
plant shows great potential to control many lethal diseases.
Our results showed that this plant species has the strong
potenitials to control the growth and other activity of many
lethal bacterial and fungal species such as Klebsiella
pneumonia, Micrococcus luteus, Staphylococcus aureus,
Pseudomonas aeruginosa, Mucor mycosis, Aspergillus
nigar, etc. However the susceptibility and tolerance
response vary with type of microbe, type of plant parts used
and with type of different extracts. The M. mycosis strain
showed more resistant to all plant parts as compared to
other tested organisms. The present study reported toxic
activities of this important plant species against many lethal
microbes and serve as model to test other plant species
against these pathogens.
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