Khan et al. BMC Complementary and Alternative Medicine (2015) 15:127
DOI 10.1186/s12906-015-0646-z
RESEARCH ARTICLE
Open Access
Alkanna tinctoria leaves extracts: a prospective
remedy against multidrug resistant human
pathogenic bacteria
Usman Ali Khan1†, Hazir Rahman1*†, Muhammad Qasim1, Anwar Hussain2, Azizullah Azizllah3, Waheed Murad3,
Zakir Khan4, Muhammad Anees1 and Muhammad Adnan3
Abstract
Background: Plants are rich source of chemical compounds that are used to accomplish biological activity.
Indigenously crude extracts of plants are widely used as herbal medicine for the treatment of infections by people
of different ethnic groups. The present investigation was carried out to evaluate the biological potential of Alkanna
tinctoria leaves extract from district Charsadda, Pakistan against multidrug resistant human pathogenic bacteria
including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.
Methods: Anti-multi-drug resistant bacterial activity of aqueous, chloroform, ethanol and hexane extracts of
Alkanna tinctoria leaves were evaluated by well diffusion method. Minimum inhibitory concentrations (MICs) and
minimum bactericidal concentrations (MBCs) of different extracts were determined. Moreover qualitative
phytochemicals screening of the studied extracts was performed.
Results: All four selected bacteria including A. baumannii, E. coli, P. aeruginosa and S. aureus were categorized as
multi-drug resistant (MDR) as they were found to be resistant to 13, 10, 19 and 22 antibiotics belonging to different
groups respectively. All the four extract showed potential activity against S. aureus as compare to positive control
antibiotic (Imipenem). Similarly among the four extracts of Alkanna tinctoria leaves, aqueous extract showed best
activity against A. baumannii (10 ± 03 mm), P. aeruginosa (12 ± 0.5 mm), and S. aureus (14 ± 0.5 mm) as compare to
Imipenem. The MICs and MBCs results also showed quantitative concentration of plant extracts to inhibit or kill
MDR bacteria. When phytochemicals analysis was performed it was observed that aqueous and ethanol extracts
showed phytochemicals with large number as well as volume, especially Alkaloides, Flavonoides and Charbohydrates.
Conclusion: The undertaken study demonstrated that all the four extracts of Alkanna tinctoria leaves exhibited
considerable antibacterial activity against MDR isolates. Finding from the current study will be helpful for further
elucidation of lead molecules from Alkanna tinctoria leaves for future therapeutic use against MDR pathogens.
Keywords: Alkanna tinctoria leaves extracts, Anti-MDR bacterial activity, MICs and MBCs, Phytochemicals analysis
Background
Plants act as reservoirs for wide variety of secondary metabolites including alkaloids, flavonoids, tannins and terpenoids which possess therapeutic properties [1]. The
antimicrobial potential of plants have been studied by a
large number of researchers across the globe [2,3].
* Correspondence: hazirrahman@hotmail.com
†
Equal contributors
1
Department of Microbiology, Kohat University of Science and Technology,
Kohat, Pakistan
Full list of author information is available at the end of the article
According to World Health Organization (WHO) in 91
countries there are nearly 2000 medicinal plants [4]. Medicinal plants are still being used by rural communities with
increasing popularity for treating or preventing various infections. In Pakistan limited data is available on the therapeutic uses of medicinal plants. Among these medicinal
plants Alkanna tinctoria is under investigation as having
several therapeutic uses [5]; however has not been documented for its anti-multi-drug resistant bacterial activity
and phytochemicals content.
© 2015 Khan et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Khan et al. BMC Complementary and Alternative Medicine (2015) 15:127
Microorganisms resistant to more than two groups of
antibiotics are regarded as MDR [6]. Recent emergence
of antibiotic resistance trigger research to explore medicinal plants for their antimicrobial protection against
MDR pathogens because there is need to discover new
antibiotic therapies to combat resistance to older drugs
[7,8]. Hospital acquired infections caused by MDR bacteria is major challenge for clinician [4].
The present explored anti-MDR bacterial activity of
Alkanna tinctoria leaves from district Charsadda,
Pakistan. Moreover, phytochemicals analysis of the
Alkanna tinctoria leaves was also performed. Finding
from the present study will be helpful for elucidation of
bioactive molecules from Alkanna tinctoria leaves which
can be used to treat infections caused by MDR bacterial
pathogens.
Methods
The undertaken study was accomplished to check the
antibacterial activity and phytochemicals content of
Alkanna tinctoria leaves against MDR clinical bacterial
isolates. The already characterized clinical isolates on
the bases of 16rRNA gene sequencing including A. baumannii and E. coli were taken from patients having urinary tract infections while P. aeruginosa and S. aureus were
isolated from patients having wound infections admitted
in the tertiary care hospital of Peshawar. The isolates were
reconfirmed on the basis of culture, microscopy and biochemical characteristics. An informed written consent was
taken from patient to use the samples for the current
study. Ethical approval was granted by the Departmental
Research Ethics Committee of Kohat University of Science
and Technology (DREC KUST).
Page 2 of 6
(aqueous, chloroform, ethanol and hexane) and kept it for
21 day. T she plant extracts were then filtered with a sterilized filter paper. The filtrate plant’s extracts were then
processed in rotary evaporator at 125 rpm on 44°. After
evaporation solidified extracts were kept in sterile small
air tight glass bottles. Then 250 mg of each extract was
added in 10 ml DMSO achieving the concentration of
25 mg/ml. The tubes were kept on rotary shaker at
125 rpm for 1 hour to obtain homogeneous mixture [12].
Anti-MDR bacterial activity of Alkanna tinctoria leaves
Activity of Alkanna tinctoria leaves were evaluated
against selected MDR bacteria by using well diffusion
method [9]. In each plate four extracts (aqueous, chloroform, ethanol and hexane) of Alkanna tintoria leaves were
poured in each well separately. DMSO was incorporated
as negative control. For positive control a standard antibiotic disc (Imipenem) was applied on the medium in the
centre of the plate. All the plates were sealed and incubated at 37°C for 24 hours. Results were measured by
noted zone of inhibition in millimeters (mm) around the
wells and disc [13].
Determination of minimum inhibitory concentrations
(MICs) and minimum bactericidal concentrations (MBCs)
The susceptibility of the clinical isolates to commonly
used antibiotics was determined on Mueller-Hinton agar
(MHA) using disc diffusion method [9]. The results were
interpreted as resistant (R), intermediate sensitive (I) and
sensitive (S) as described by Clinical and Laboratory
Standard Institute (CLSI formerly NCCLS) guidelines
2007 [10].
Briefly, the stock extracts (100 mg/ml) were prepared in
nutrient broth and serially diluted as 50 mg/ml, 25 mg/ml,
12.5 mg/ml and 6.25 mg/ml. Then 100 μl of overnight
bacterial broth cultures were matched to 0.5 McFarland
standard and inoculated in all the tubes. One tube was inoculated with nutrient broth and bacterial culture but
without extract as positive control while tubes having nutrient broth and extract but without bacterial culture was
incorporated as negative control. The tubes were incubated at 37°C for 24 hours. The lowest concentration at
which no growth appeared was considered as MIC.
MBC was processed with subsequent steps of MICs.
Each selected MIC’s tubes content were inoculated on
prepared nutrient agar plates. The plates were incubated
at 37°C for 18 hours. The lowest concentration at which
no growth appeared was considered as MBC of specific
plant against selected bacteria [14,15].
Collection and processing of Alkanna tinctoria
Phytochemicals screening
The Alkanan tinctoria leaves were collected from
Charsadda region of Pakistan and identified at the
Herbarium of Botany Department, KUST, where the
voucher specimen was deposited under reference number 10050/ATL. The grinding process was performed as
described [11]. Alkanna tinctoria leaves were first
washed, dried and then chopped into small pieces. After
slicing the plant was dried and mashed into powder.
After the plant grinding process, 1000 g of dried plant
powder was soaked in 4 liter of each selected solvent
Phytochemicals analysis of secondary metabolites such as
Alkaloids, Bufadienoloides, Charbohydrate, Flavonoids,
Proteins, Resins, Saponins, Tannins, Phenolics, Triterpenoides, Steroids, Gallotannins and Pseudotannins was carried out according to the common phytochemical
methods as described by Trease and Evans [16].
Antibiotic susceptibility pattern of clinical isolates
Data analysis
All the experiments were repeated 3 times. Experimental
data was reported as mean ± standard error.
Khan et al. BMC Complementary and Alternative Medicine (2015) 15:127
Results
Antibiotic susceptibility of pathogenic bacterial isolates
was determined. A. baumannii showed resistance to
about thirteen antibiotics widely used against this pathogen while E. coli was resistant to about ten antibiotics.
When P. aeruginosa susceptibility pattern was checked
it showed resistant to almost nineteen antibiotics while
S. aureus was resistant to twenty two antibiotics including Vancomycin (Table 1).
Page 3 of 6
After confirmation of MDR pathogens, aqueous,
chloroform, ethanol and hexane extracts of Alkanna
tinctoria leaves were evaluated in vitro against A. baumannii, E. coli, P. aeruginosa and S. aureus. The concentration of all plant extracts were set to 25 mg/ml.
Average zone of inhibition were recorded for all experiments performed three times. It was observed that
Alkanna tinctoria showed maximum medicinal potential
against S. aureus as compared to other three bacteria.
Table 1 Evaluation of multi-drug resistant bacterial isolates
Antibiotics
Bacteria (zone diameter nearest whole mm)
A. baumannii
E. coli
P. aeruginosa
S. aureus
R
I
S
R
I
S
R
I
S
R
I
S
Amoxicillin
0
-
-
0
-
-
0
-
-
0
-
-
Amoxycillin/Clavulanic acid
-
15 ± 1
-
0
-
-
7±0
-
-
6.5 ± 0
-
-
Ampicillin
7 ± 0.28
-
-
0
-
-
0
-
-
0
-
-
Aztreonam
7 ± 0.11
-
-
0
-
-
0
-
-
0
-
-
Cefepime
-
-
24 ± 0.5
15 ± 0.5
-
-
-
15 ± 0.3
-
-
15 ± 0.5
-
Cefixime
-
-
20 ± 0.5
-
-
19 ± 0.7
-
18 ± 0.3
-
10 ± 0
-
-
Cefotaxime
7±0
-
-
14 ± 0.5
-
-
0
-
-
7±0
-
-
Cell wall synthesis inhibitors
Ceftazidime
-
19 ± 0.7
-
14 ± 0.5
-
-
7 ± 03
-
-
-
15 ± 0.3
-
Ceftriaxone
12 ± 0.5
-
-
-
16 ± 0.5
-
7±0
-
-
10 ± 0.3
-
-
Cephradine (only for S. aureus)
NA
NA
NA
NA
NA
NA
NA
NA
NA
6.5 ± 0
-
-
Fosfomycin
-
-
22 ± 0.5
-
-
24 ± 0.5
-
-
30 ± 1
-
-
-
Imipenem
10 ± 0.5
-
-
-
-
23 ± 0.7
10 ± 0
-
-
-
15 ± 0.7
-
Meropenem
10 ± 0.5
-
-
8±0
-
-
7±0
-
-
7±0
-
-
Oxacillin (only for S. aureus)
NA
NA
NA
NA
NA
NA
NA
NA
NA
0
-
-
Pipracillin/Tazobactam
-
-
27 ± 1
-
-
18 ± 1
14 ± 0.5
-
-
14 ± 0.3
-
-
Vancomycin (only for S. aureus)
NA
NA
NA
NA
NA
NA
NA
NA
NA
9 ± 0.3
-
-
0
-
-
-
-
12 ± 0.3
-
-
11 ± 0.3
10 ± 0
-
-
Amikacin
-
15 ± 0.3
-
-
15 ± 0.5
-
9
-
-
7±0
-
-
Chloramphenicol
NA
NA
NA
NA
NA
NA
12 ± 0.5
-
-
9±0
-
-
Cell membrane inhibitors
Colistin sulphate
Protein synthesis inhibitors
Doxycycline
-
13 ± 0.5
-
-
-
14 ± 0.5
7 ± 0.3
-
-
10 ± 0.3
-
-
Erythromycin
0
-
-
7±0
-
-
7±0
-
-
8±0
-
-
Gentamycin
9 ± 0.28
-
-
-
-
15 ± 0.5
9±0
-
-
10 ± 0.3
-
-
Tetracycline
7±0
-
-
-
-
18 ± 0.3
0
-
-
7±0
-
-
Tigecyclin
-
-
19 ± 0.7
-
18 ± 0.5
-
-
15 ± 0.5
-
-
15 ± 0.5
-
DNA inhibitors
Ciprofloxacin
15 ± 0.5
-
-
-
-
22 ± 0.5
12 ± 0.5
-
-
11 ± 0.3
-
-
Nitrofurantoin
-
15 ± 0.5
-
8 ± 0.3
-
-
0
-
-
8±0
-
-
0
-
-
-
11 ± 0
-
0
-
-
0
-
-
Folate pathway inhibitors
Trimethoprim/Sulphamethoxazole
± = standard error of given value, – = not observed, NA = not applicable R = resistant, I = intermediate sensitive and S = sensitive.
Khan et al. BMC Complementary and Alternative Medicine (2015) 15:127
Page 4 of 6
Table 2 Antibacterial activity of Alkanna tinctoria (leaves) against MDR pathogens
MDR bacteria
Aqueous extract (mm)
Chloroform
extract (mm)
Ethanol
extract (mm)
Hexane
extract (mm)
+Ve IPM (mm)
-Ve DMSO (mm)
A. baumannii
10 ± 0.3
9 ± 0.3
11 ± 0.5
10 ± 0.3
10 ± 0.5
0
E. coli
11 ± 0.5
14 ± 0.5
14 ± 0.5
13 ± 0.3
23 ± 0.7
0
P. aeruginosa
12 ± 0.5
9 ± 0.3
11 ± 0.3
10 ± 0.3
10 ± 0
0
S. aureus
14 ± 0.5
19 ± 0.7
19 ± 0.5
15 ± 0.5
15 ± 0.7
0
± = standard error of given value, +Ve IPM = Imipenem as positive control and -Ve DMSO = Dimethyl sulfoxide as negative control.
Chloroform and ethanolic extracts showed highest zone
of inhibition (19 mm) and aqueous extract showed
14 mm inhibitory zone against S. aureus. Zone of inhibition of the aqueous, chloroform, ethanol and hexane extracts of Alkanna tinctoria leaves against A. baumannii,
E. coli, P. aeruginosa appeared in the range of 9 mm to
14 mm as shown in Table 2.
The MICs were calculated to check the bacteriostatic
effect of plant extracts. The range of MICs of selected
four plant extracts against selected MDR isolates were in
between 6.25 mg/ml to 25 mg/ml (Table 3).
Aqueous extract inhibited A. baumannii and S. aureus
at minimum concentration of 12.5 mg/ml, while E. coli
and P. aeruginosa were inhibited at lower concentration
of 25 mg/ml. MIC of hexane extract against all four
MDR bacteria were recorded as 25 mg/ml.
The MBCs were calculated to check the bactericidal
effect of plant extracts against MDR pathogens. MBCs
of most of the extracts the plant found to be higher than
100 mg/ml which was the maximum concentration used
for the experiment (Table 3).
After checking the qualitative and quantitative measurement of antibacterial activity of Alkanna tinctoria leaves
the phytochemicals contents were analyzed. Alkaloids
were detected in all four extracts of Alkanna tinctoria
leaves but aqueous and ethanol extracts contained in
higher amount than chloroform and hexane extracts
which showed less amount. Bufadienoloides and Resins
were present in moderate quantity. Flavonoides and
Table 3 Minimum inhibitory concentration (MICs) and
minimum bactericidal concentration (MBCs) of Alkanna
tinctoria (leaves) against MDR isolates
MDR
bacteria
Aqueous
extract
(mg/ml)
Chloroform
extract
(mg/ml)
Ethanol
extract
(mg/ml)
Hexane
extract
(mg/ml)
MICs MBCs MICs MBCs MICs MBCs MICs MBCs
A.baumannii 12.5
>100
25
>100
25
>100
25
>100
25
>100
25
>100
25
>100
25
>100
P.aeruginosa 25
>100
25
>100
12.5
>100
25
>100
>100
12.5
>100
12.5
>100
25
>100
E. coli
S. aureus
12.5
>100 shows concentration greater than 100, + = no bacterial growth observed
and - = bacterial growth observed.
Gallotannins were only present in hexane and aqueous extracts respectively. Carbohydrates, Phenolics and Triterpenoides were not detected in any of the four extracts.
Proteins and Tannins were found to be in lower concentration. Steroides and Pseudotannins were appeared in higher
and moderate volumes except ethanol and aqueous extracts
which lower to undetectable amount respectively. Saponins
of all extracts of this plant were in lower volume (Table 4).
Discussion
Medicinal plants are the rich source of traditional medicines and have been used to treat infectious diseases.
The universality and efficacy of medicinal plants is evident from their continued use [17]. Alkanna tinctoria
leaves is a commonly used as plant herbal therapy for
treating different infections including its frequent traditional use in Charsadda, Pakistan.
In the study Alkanna tinctoria leaves were selected for
the investigation of its qualitative and quantitative potential of antibacterial activity, and further phytochemicals
analysis. The aqueous, chloroform, ethanol and hexane
Table 4 Phytochemicals composition of Alkanna tinctoria
(leaves)
Phytochemicals
Aqueous
extract
Chloroform
extract
Ethanol
extract
Hexane
extract
Alkaloides
+++
+
+++
+
Bufadienoloides
+
++
++
++
Charbohydrates
_
_
_
_
Flavonoides
_
_
_
+++
Gallotannins
+++
_
_
_
Phenolics
_
_
_
_
Proteins
+
+
+
+
Pseudotannins
_
++
++
++
Resins
+
++
++
++
Saponins
+
+
+
+
Steroides
+++
+++
+
+++
Tannins
_
+
+
+
Triterpenoides
_
_
_
_
- sign shows not detected, + shows compound present in small amount;
++ shows compound present in moderate amount; +++ shows compound
present in higher amount.
Khan et al. BMC Complementary and Alternative Medicine (2015) 15:127
extracts were screened against MDR bacteria including A.
baumannii, E. coli, P. aeruginosa and S. aureus. Tchana
et al., (2014) performed similar type of study in Cameroon,
in which it was confirmed that medicinal plants had the
ability to encountered MDR bacteria [18].
It was observed that Alkanna tinctoria leaves extracts
showed convincing zone of inhibition against selected
MDR pathogens. S. aureus appeared to be more sensitive
than other three bacteria, this can be the key of previous
work of Ghosh and Gaba on Alkanna tinctoria wound
healing ability [19]. Quick wound healing can be due to
the inhibition of S. aureus an important pathogen in
wound infections. Aqueous, chloroform and ethanolic extracts showed highest zone of inhibition against S. aureus,
and moderate zone of inhibition against A. baumannii, E.
coli and P. aeruginosa. Unlikely hexane extract did not exhibit as much antibacterial activity against all MDR pathogens. Alkanna tinctoria was previously reported as red dye
as well as antibacterial [20,21]. As for as literature mining
is concerned, the antibacterial activity of Alkanna tinctoria
leaves extracts of aqueous, chloroform, ethanol and hexane against MDR bacterial isolates were not observed till
now in the research literature, as well as phytochemicals
analysis of Alkanna tinctoria leaves is described here for
the first time. Moreover bacteriostatic and bacteriocidal
activity Alkanna tinctoria leaves was calculated for the
first time against MDR pathogens.
By comparing results of inhibitory zone, bacteriostatic
concentration and bactericidal concentration of all four
extracts of Alkanna tinctoria leaves it was obvious that
aqueous and ethanol extracts has potential activity against
selected MDR pathogens. When phytochemicals analysis
was performed it was observed that aqueous and ethanol
extracts showed phytochemicals with large number as well
as concentration, especially alkaloides, flavonoides and
charbohydrates. This shows good solibility of phytochemicals in tested solvents. In phytochemicals results it was observed that the extracts which showed large number of
phytochemicals exhibited potential antibacterial activity.
One interesting observation we found that the antibacterial activity of aqueous extract against P. aeruginosa, and
chloroform and ethanolic extracts against S. aureus
showed better activity as compared to broad spectrum
antibiotic Imipenem.
Conclusion
The current study demonstrated that Alkanna tinctoria
leaves extracts were efficient against MDR isolates. Phytochemicals analysis showed that Alkanna tinctoria leaves
have versatile biochemical molecules which may inhibit or
kill drug resistant human pathogenic bacteria. Further indepth analysis will be helpful for future elucidation of bioactive molecules to treat infectious diseases caused by
resistant bacteria.
Page 5 of 6
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
UK carried out the experiments, analyzed the data and drafted the
manuscript. HR designed the study, helped in the experiments, interpreted
the results and drafted the manuscript. MQ, ZK and MA analyzed the data
and drafted the manuscript. AA, WM, AH and MA contributed in plant
selection, results interpretation and discussion. All authors read and
approved the final manuscript.
Acknowledgements
We are thankful to the Department of Microbiology, Kohat University of
Science and Technology, Kohat for providing clinical isolates.
Author details
1
Department of Microbiology, Kohat University of Science and Technology,
Kohat, Pakistan. 2Department of Botany, Abdul Wali Khan University, Mardan,
Pakistan. 3Department of Botany, Kohat University of Science and
Technology, Kohat, Pakistan. 4Department of Pharmacy, University of Lahore,
Punjab, Pakistan.
Received: 6 November 2014 Accepted: 13 April 2015
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