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*Corresponding author: E-mail: mervemeliha@comu.edu.tr; 
 
 
 

Asian Journal of Immunology 
 
4(1): 175-179, 2021; Article no.AJI.85117 
 

 
 

 

 

Antimicrobial Efficacy of Four Different Extracts of 
Plantago major: An In vitro Study 

 
G. Akkuş a and M. M. HIZ-ÇİÇEKLİYURT b* 

 
a 
Department of Biology, Faculty of Arts & Science, Çanakkale Onsekiz Mart University, Turkey. 

b
 Department of Medical Biology, Faculty of Medicine, Çanakkale Onsekiz Mart University, Turkey. 

 
Authors’ contributions  

 
This work was carried out in collaboration between both authors. Author GA is a student responsible 

for all antimicrobial assays and performed the statistical analysis and wrote the first draft of the 
manuscript. Author MMÇ wrote the protocol, managed the analyses of the study and the literature 

searches. Both authors read and approved the final manuscript. 
 

Article Information 
 

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Received 27 October 2021  
Accepted 29 December 2021 
Published 30 December 2021 

 
 

ABSTRACT 
 

Aims: Plantago major is frequently used in traditional treatment for upper respiratory tract diseases 
such as pneumonia, cough, pharyngitis, and skin, eye, and urinary tract infections. This study aims 
to evaluate the antimicrobial activity and minimum effective dose of hexane, methanol, ethanol, and 
water extract of Plantago major leaves.  
Study design:  In vitro experimental study. 
Methodology: P. major leaves was crushed into a fine powder and dissolved in different solvents 
(hexane, methanol, ethanol, or water) using a Soxhlet device, then the extracts were purified by 
evaporation of the solvent. All extracts were analyzed for antibacterial and antifungal properties 
using broth dilution method depending on MIC value determined according to the solvent-
microorganism-time trio in DDM. 
Results: The in vivo test showed that all methods to extract of Plantago major leaves have activity 
against all test microorganisms. Both hexane and water extract showed the same activity on 
Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa, C. albicans and Candida 
tropicalis at 4 mg/ml. The lowest activity of Plantago major’s (PM's) hexane and water extract was 
on Escherichia coli and Pseudomonas vulgaris bacteria as 8 mg/ml. Methanol and ethanol extracts 
of PM showed higher activity than hexane and water extract. PM ethanol extract showed 
antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas 
aerouginosa at 2ml/mL, and Bacillus subtilis Escherichia coli and Proteus vulgaris at 4 ml/mL. 

Original Research Article 



 
 
 
 

Akkuş and HIZ-ÇİÇEKLİYURT; AJI, 4(1): 175-179, 2021; Article no.AJI.85117 
 

 

 
176 

 

Conclusion: The results nominate the Plantago major extract has potential antimicrobials and 
antifungals. However, the development of antimicrobial agents requires purifying the active bio 
components by high throughput techniques to achieve effective activity as positive controls. 

 

 
Keywords: Plantago major; antimicrobial activity; MIC. 
 

ABBREVIATIONS 
 
Here is the Definitions section. This is an optional 
section.  
 
TB :  Boiling point 
PM  :  Plantago Major 
 

1. INTRODUCTION  
 
Plantago major (P. major) is a widely used 
medicinal plant in folk medicine [1]. The plant 
contains bioactive components such as 
flavonoids, polysaccharides, terpenoids, lipids, 
iridoid glycosides, and caffeic acid derivatives 
[2,3]. Due to its rich components, the plant was 
used to treat various medical conditions such as 
coughs, infection, fever, bleeding, and 
inflammation [3,4]. Currently, animal studies 
focus on using the Plantago major (PM) on 
different medicinal conditions. Parhizgar and 
colleagues have demonstrated the protective 
effect of PM extract in the presence of kidney 
damage. Parhizgar has shown glomerular 
filtration rate (GFR), urine osmolarity, and urinary 
excretion rate of potassium were increased via 
the treatment of PM extract of kidney tissue-
damaged rats [5]. In another study, 
hydroalcoholic extract of Plantago major also had 
a protective role in doxorubicin-induced 
nephropathy. These two animal studies have 
been demonstrated the protective role of PM 
extract on the renal system. Boskabadi et al. [6] 
have been shown potent relaxant effects of PM 
extract on Tracheal Smooth Muscles of rats. The 
randomized, double-blind placebo-controlled 
clinical trial done by Jazayeri and colleagues has 
achieved elevated liver enzymes and a better 
prognosis for Nonalcoholic Fatty Liver Disease 
by using 2 gr of PM supplementation twice daily 
for 12 weeks [7]. Depending on the therapeutic 
properties shown in both animals and clinical 
studies, PM extract protects the kidneys and liver 
against toxicity.  
 
In another way of view, PM extract is not harmful 
to animals and humans by its protective role, 
thus potent bioactive compound for drug 
development. PM extracts were widely used for 

antibiotics, antioxidants, analgesics, and wound 
healer purposes from ancient times. The 
antibacterial activity of PM extract was evaluated 
by Sharma et al. They found no activity for 
periodontal pathogens by the Kirby-Bauer disc 
diffusion technique [8]. However, none of the 
studies were performed to evaluate PM extract's 
antimicrobial activity by MIC method against 
standard test microorganisms. Thus, this study 
investigated the bioactivity of a Plantago major 
against pathogens: Staphylococcus aureus, 
Enterococcus faecalis, Bacillus subtilis, 
Pseudomonas aeruginosa, Proteus vulgaris as 
antimicrobial, and Candida albicans and Candida 
tropicalis as antifungal. 
 

2. MATERIALS AND METHODS 
 

2.1 Chemicals  
 
The medium used for this study was Nutrient 
Broth for bacterial, and RPMI-1640 medium with 
L-glutamine for fungi were purchased from 
Sigma-Aldrich (Hamburg, Germany). The 
microbial lines were purchased from American 
Type Culture Collection (ATCC®, Manassas, VA, 
USA). The antibiotics and antifungal 
pharmaceuticals were used as a positive control 
as follows: Ampicillin (Mustafa Nevzat 
Pharmaceuticals, Turkey); Gentamicin (Bilim 
Pharmaceuticals, Turkey); Fluconazole (Pfizer 
Pharmaceuticals, Roerig Division (New York, 
N.Y.). All other analytical grade chemicals used 
without further purification were purchased from 
Sigma-Aldrich (Hamburg, Germany). 
 

2.2 Preparation of Plant Extract 
 
The plants leaves were washed with distilled 
water, dried in the shade with continuous airflow, 
and then grounded into powder as 1-3 mm 
pieces. The powder of PM was randomized 
aliquoted into 20 mg using filter paper 
(Whatmann No.1) and then was macerated with 
200 ml solution for 8 hours in Soxhlet (Wisd, 
Wise Therm) for each solution. The extraction 
step was done with four different solvents as 

follows: hexane (C₆H₁₄, TB:69ºC), methanol 
(CH3OH, TB:64,7 ºC), ethanol (C2H5OH, TB: 



 
 
 
 

Akkuş and HIZ-ÇİÇEKLİYURT; AJI, 4(1): 175-179, 2021; Article no.AJI.85117 
 

 

 
177 

 

78.4ºC and water (H2O, TB:100ºC). After this 
step, all extracts were evaporated until dried at 
50 ºC to obtain a solvent-free extract, which will 
be stored in the refrigerator (0-4 ºC) until 
experiments. Each extract was redissolved in 
Dimethyl sulfoxide (DMSO) to yield a final 
concentration of 32 mg/mL. All of them were 
sterilized by a membrane filter (0.2 µm) before 
use [9].  
 

2.3 Antimicrobial and Antifungal Activity 
Assay 

 
Antimicrobial activity of extracts was applied to 
Gram-positive bacteria cultures: Staphylococcus 
aureus (ATCC 6538), Enterococcus faecalis 
(ATCC 292112), Bacillus subtilis (ATCC 6633), 
whereas gram-negative bacterial cultures 
Escherichia coli (ATCC 25922), Pseudomonas 
aeruginosa (27853), Proteus vulgaris (ATCC 
13315). Antifungal activity has been studied 
against Candida albicans (ATCC 60193) and 
Candida tropicalis (ATCC 13803). 

Antimicrobial activity analyses of four different 
extracts of Plantago major were performed using 
Broth microdilution methods as recommended by 
Clinical & Laboratory Standards Institu      -     
                                         
                                        
                                                  
                             1.5 × 10

8 
CFU/mL. 

The inoculums were pipetted in each well. The 
plant extracts were dissolved in dimethyl 
sulfoxide (DMSO), and after two times 
concentrated extracts at 32-0,156 mg/ml 

concentration were pipetted in each well of 
microtiter plates. In brief, the wells were filled 
with 100 µL culture suspension as nutrient broth 
(Sigma-Aldrich, Germany) for antimicrobial 
activity and RPMI-1640 medium with L-Glutamin 
(Sigma-Aldrich, Germany) for antifungal activity 
as a growth medium. As per protocol, all extracts 
have been applied to wells 32 mg/mL and diluted 
1/2 respectively. From 1-12 wells, it was from 32 
to 0.156 mg/mL. The standard antibiotics and 
fungicide were used as Ampicillin, Gentamicin, 
and Fluconazole 2 mg/ml, respectively as 
control. The aerobic incubation condition was 
applied at 37 °C 18-24 h for bacteria and 48 h for 
fungi. The minimum inhibitory concentration 
(MIC) was calculated by visible inhibition of the 
microbial growth at the lowest concentration. 
 

3. RESULTS AND DISCUSSION 
 
Table 1 and Table 2 are summarized the 
antibacterial and antifungal effects of different 
extracts of Plantago Major. The antimicrobial 
activity of hexane and aqueous extracts of PM 
against all test microorganisms except 
Enterococcus faecalis was the same. 
 
The results revealed that the efficiency of both 
ethanol and methanol extracts of PM was similar 
to all test microorganisms. Methanol and ethanol 
showed the highest activity (2 mg/mL) in S. 
aureus, E. faecialis and P. aeruginosa bacteria 
and C. albicans yeast however had low activity 
on B.subtilis, E. coli P. vulgaris bacteria, and C. 
tropicalis yeast. 

 
Table 1. Antimicrobial activity of different extracts of Plantago major 

 

MIC (mg/ml) 

Test Materials Gram (+) Gram (-) 

S
ta

p
h

y
lo

c
o

c
c
u

s
 

a
u

re
u

s
 

A
T

C
C

 6
5

3
8

 

E
n

te
ro

c
o

c
c
u

s
 

fa
e
c
a
li

s
 

A
T

C
C

 2
9

2
1
1

2
 

B
a
c
il
lu

s
 s

u
b

ti
li
s

 

A
T

C
C

 6
6

3
3

 

E
s
c
h

e
ri

c
h

ia
 c

o
li

 

A
T

C
C

 2
5

9
2
2

 

P
s
e
u

d
o

m
o

n
a

s
 

a
e
ro

u
g

in
o

s
a

 

A
T

C
C

 2
7

8
5
3

 

P
ro

te
u

s
 v

u
lg

a
ri

s
 

A
T

C
C

 1
3

3
1
5

 

PM-nHexOH 4 2 4 8 4 8 
PM-MeOH 2 2 4 4 2 4 
PM-EtOH 2 2 4 4 2 4 
PM-H2O 4 4 4 8 4 8 
Gentamicin 10

-3
 10

-3
 8.10

-3
 16.10

-3
 8.10

-3
 16.10

-3
 

Ampicilin 1610
-3

 16.10
-3

 16.10
-3

 32.10
-3

 32.10
-3

 16.10
-3

 
DMSO - - - - - - 

 



 
 
 
 

Akkuş and HIZ-ÇİÇEKLİYURT; AJI, 4(1): 175-179, 2021; Article no.AJI.85117 
 

 

 
178 

 

Table 2. Antifungal activity of different extracts of Plantago major 
 

MIC (mg/ml) 

Test Materials 

C
a
n
d

id
a

 a
lb

ic
a

n
s
 

A
T

C
C

 6
0
1
9

3
 

C
a
n
d

id
a

 t
ro

p
ic

a
lis

 

A
T

C
C

 1
3
8
0

3
 

PM-nHexOH 4 4 
PM-MeOH 2 4 
PM-EtOH 2 4 
PM-H2O 4 4 
Fluconasole 625. 10

-3
 25. 10

-1
 

DMSO - - 

 
In addition, the results (Tables 1-2) show that 
methanol and ethanol extracts showed higher 
activity than hexane and water extract. It is 
known that hexane has polar solubility, methanol 
and ethanol have semi-polar (methanol is more 
polar than ethanol), and water has apolar 
solubility. These differences explain that ethanol 
and methanol would be better solvents for PM, 
and bioactive compounds were more easily 
resolved in ethanol and methanol. The four types 
of PM extracts exhibited inhibitory effects against 
test microorganisms; however, these 
antimicrobial activities were below the MIC value 
of positive controls (ampicillin gentamicin and 
fluconazole). thus, we can conclude that PM 
extracts show antimicrobial activity but are not as 
effective as standard therapeutics. 

 
This study provides further documentation of the 

applicability of the Plantago major extract against 

microorganisms. Previous studies have shown 

that PM has no antimicrobial activity against 

primary plaque colonizers or periodontal 

pathogens [8]. However, we clearly showed that 

PM ethanol extract showed antimicrobial activity 

against Staphylococcus aureus, Enterococcus 

faecalis, Pseudomonas aerouginosa at 2ml/mL, 

and Bacillus subtilis Escherichia coli and Proteus 

vulgaris at 4 ml/mL. In addition to ethanol extract, 

hexane, methanol, and water extract showed 

antimicrobial activity. Together with this, all 

extracts showed antifungal activity against 

Candida albicans and Candida tropicalis. In a 

previous study, the Kirby-Bauer disc diffusion 

technique was used to investigate the 

antimicrobial activity, which is not recommended 

as a reference method. 

In contrast, we clearly showed antimicrobial 
activity by the MIC method, which is a gold 
standard used in clinics to calculate the 
antimicrobial activity at routine practice. In 
addition, Sharma and colleagues prepared their 
text extract with maceration; thus, the 
preparation procedure should affect the 
concentration of bioactive compounds in total. 
Similar to our study, Ferrazzano and colleagues 
[12] demonstrated a significant antimicrobial 
effect of Plantago lanceolata and evaluated the 
plant extract as a natural anti-cariogenic agent. 
Although Ferrazzano et al. studied different 
species, both plants have the same genus and 
have similar phytochemicals. The broth dilution 
MICs for Plantago major extracts tested 
against C. albicans and C. tropicalis showed 
significant inhibition, which means PM extracted 
by different solvents might be used with its 
antifungal activity. In summary, we have provided 
documentation of PM extracts with antimicrobial 
and antifungal activity; therefore, PM extracts 
should be a good candidate for drug 
development.  
 

4. CONCLUSION 
 

This study demonstrated the antimicrobial activity 
of Plantago major against all test 
microorganisms. In addition, n-EtOH and n-
MeOH extract of PM showed better antimicrobial 
and antifungal activity when compared to hexane 
and aqueous extracts. The difference in 
antimicrobial efficiency at different extracts 
clearly showed that the extraction method is 
important to obtain bioactive molecules. Despite 
significant improvements in synthetic molecules 
in the pharmaceutical industry, the growing 
antibiotic resistance problem still requires finding 



 
 
 
 

Akkuş and HIZ-ÇİÇEKLİYURT; AJI, 4(1): 175-179, 2021; Article no.AJI.85117 
 

 

 
179 

 

alternative antimicrobial agents. In this frame, the 
discovery of plant-based antibiotics is also 
essential to reduce the side effects of synthetic 
antimicrobials. Thus, we recommended testing 
different extract types and using gold standards 
as antimicrobial activity determination. The 
results of our study show that PM extracts are 
effective as an antimicrobial agent. However, the 
active component of the plant extracts should be 
enhanced, or active components should be 
purified by high-throughput techniques to 
develop bio-based pharmaceuticals.  
 

COMPETING INTERESTS 
 

Authors have declared that no competing 
interests exist. 
 

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in any medium, provided the original work is properly cited. 

 

 

 

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