The antimicrobial activity of methanolic extracts was screened against 3 gram positive and 3 gram negative bacteria using agar well diffusion method.  80 µl of test extract was suspended in the wells. A control well is loaded with equal amount of the solvent i.e. methanol. The plates were then incubated at 37ºC for 24-48 hours. After proper incubation the plates around the extract impregnated wells and this clear zone of growth inhibition was measured in terms of diameter (mm). The inhibition zone is the mean of the three replicates, excluding the well diameter. The (-) sign indicates no activity. Only the aqueous extracts of A. nilotica (leaf and stem) and L. inermis (leaves) were found inhibitory to some of the organisms tested i.e. Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiela pneumoniae and Salmonella typhimurium. Salmonella typhimurium was only inhibited by aqueous extracts of L. inermis (leaves) whereas the rest of the aqueous extracts were found to be inactive against all the test organisms. The maximum activity was shown by aqueous extracts of L. inermis (leaf) against Staphylococcus  aureus that is 1.9 mm diameter of inhibition zone and the minimum was observed in the case of aqueous extracts of Pergularia daemia (stem) i.e. 0.6 mm diameter of inhibition zone against Bacillus subtilis . Salmonella epidermidis was found to most resistance to aqueous extracts of the plant followed by Salmonella typhimurium.Keywords- antimicrobial activity; methanolic extract

Anand, Anand

Madhvi, Madhvi

Tandan, Neeraj

English

International Journal of Phytomedicine

International Journal of Phytomedicine 3 (2011) 549-556 
http://www.arjournals.org/index.php/ijpm/index 
 
Original Research Article 
           
 
Studies on Antibacterial Activity of Methanolic Plant Extracts 
Neeraj1, Madhvi1, Anand1 
 
*Corresponding author: 
 
Neeraj Tandan 
1Department of Biotechnology 
Singhania University Rajasthan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
A B S T R A C T  
The antimicrobial activity of methanolic extracts was screened against 3 gram 
positive and 3 gram negative bacteria using agar well diffusion method.  80 µl 
of test extract was suspended in the wells. A control well is loaded with equal 
amount of the solvent i.e. methanol. The plates were then incubated at 37ºC for 
24-48 hours. After proper incubation the plates around the extract impregnated 
wells and this clear zone of growth inhibition was measured in terms of 
diameter (mm). The inhibition zone is the mean of the three replicates, 
excluding the well diameter. The (-) sign indicates no activity. Only the aqueous 
extracts of A. nilotica (leaf and stem) and L. inermis (leaves) were found 
inhibitory to some of the organisms tested i.e. Bacillus subtilis, Staphylococcus 
aureus, Escherichia coli, Klebsiela pneumoniae and Salmonella typhimurium. 
Salmonella typhimurium was only inhibited by aqueous extracts of L. inermis 
(leaves) whereas the rest of the aqueous extracts were found to be inactive 
against all the test organisms. The maximum activity was shown by aqueous 
extracts of L. inermis (leaf) against Staphylococcus aureus that is 1.9 mm 
diameter of inhibition zone and the minimum was observed in the case of 
aqueous extracts of Pergularia daemia (stem) i.e. 0.6 mm diameter of inhibition 
zone against Bacillus subtilis . Salmonella epidermidis was found to most 
resistance to aqueous extracts of the plant followed by Salmonella 
typhimurium. 
Keyword- antimicrobial activity, methanolic extracts. 
 
Introduction 
Nature has been a source of medicinal agent for 
thousands of years and impressive number of modern 
drugs has been isolated from them. The wide spread 
use of herbal remedies has been documented in 
ancient texts, The Vedas and The Bible. The medicinal 
properties of the plants owes to the presence of 
secondary metabolites like terpenoid, alkaloid, 
flavanoid, phenol etc. present in them. According to 
estimate of WHO (World Health Organization) 80% of 
the world population rely on medicine obtained from 
the plants for their primary health care needs (Santos 
et al. 1995). India is endowed with a rich wealth of 
medicinal plants that make the good contribution to the 
development ancient India materia medica; one of the 
earliest treaties on Indian medicine the charka samhita 
(1000 B.C.) records the use of over 340 drugs of 
vegetable origin. 
 
This revival of interest in plant derived drug is mainly 
due to the current widespread belief that green 
medicine is safe and more dependable compared the 
ISSN: 0975-0185 
 
  
This work is licensed under a Creative Commons Attribution 3.0 License.  
 Neeraj et al. International Journal of Phytomedicine 3 (2011) 549-556 
 
550 
 
costly synthetic drugs many of which have adverse 
side effects. 
Scientific experiments on the antimicrobial properties 
of plant components were first documented in late 19th 
century. Since then a tremendous amount of work had 
been carried out worldwide that include screening of 
various medicinal plants used by traditional healers, for 
their antimicrobial activity and purification as well as 
characterization of the active substance. Till date a lot 
of work had been done in this area and a lot of it is yet 
to be revealed. 
 
Materials and methods 
To study the antibacterial effect of some medicinal 
plants against few bacterial and fungal pathogens.  
 
All the bacterial cultures used in present study were 
obtained in a freeze-dried form from Microbial Type 
Culture Collection, IMTEC Chandigarh. The cultures 
are listed below: 
1. Bacillus subtilis (BS) MTCC No. 121  
2. Staphylococcus aureus (SA) MTCC No. 435 
3. Staphylococcus epidermidis (SE) MTCC No. 
1430  
4. Escherichia coli (EC) MTCC No. 1610  
5. Klebsiela pneumoniae (KP) MTCC No. 661 
6. Salmonella typhimurium (ST) MTCC No. 98
  
Plant material collection: 
Fresh plant material, collected from different locations, 
was washed thoroughly under running tap water and 
then oven dried at 50oC overnight. The dried material 
was then grinded into fine powder and stored in dark 
bottles. 
 
Ten grams of air dried powder was placed in 100 ml of 
organic solvent (methanol) in a conical flask, plugged 
with cotton and then kept on a rotatory shaker at 190-
220 rpm for 24 hours. After 24 hours it was filtered 
through 8 layers of muslin cloth and centrifuged at 
5000g for 15 minutes. The supernatant was collected 
and the solvent was evaporated to make the final 
volume one-fourth of the original. 
 
The bacterial cultures were grown in Nutrient broth at 
37o C until the optical density reaches the absorbance 
of Mc. Farland No. 5 standard i.e. approximately 0.132 
at 600 nm. At this absorbance a concentration of 
108cells/ml was obtained. This suspension was used 
as inoculum for antimicrobial susceptibility testing.  
 
Five antimicrobial agents namely Tetracycline, 
Chloramphenicol, Livofloxacin, Amoxicillin and 
Ofloxacin were tested for their effect on the test 
bacteria at various concentrations (0.2, 0.4, 0.6, 0.8 
and 1.0 mg/l). The tests were performed using agar 
well diffusion method with water as control. The wells 
were loaded with 60 µl of the antimicrobial agents. 
 
The present study consists of evaluating different 
plants for their antibacterial properties. Sixteen plants 
were collected from different locations in Meerut and 
various parts of these plants like leaf, fruit, stem and 
flower were evaluated for their antibacterial potential. 
 
Total 19 extracts were prepared in methanol. All the 
extracts were numbered as shown in the Table 1: 
 
The antimicrobial activity of methanolic extracts was 
screened against 3 gram positive and 3 gram negative 
bacteria using agar well diffusion method. Agar plates were 
prepared and inoculated with the microbial suspension and 
then agar was punched to form wells of 5mm diameter. 80 
µl of test extract was suspended in the wells. A control well 
is loaded with equal amount of the solvent i.e. methanol. 
The plates were then incubated at 37ºC for 24-48 hours. 
After proper incubation the plates around the extract 
impregnated wells and this clear zone of growth inhibition 
was measured in terms of diameter (mm). The antimicrobial 
activity of the extracts was calculated in terms of zone of 
inhibition around the wells loaded with the test tube 
extracts. 
 
 
 
 
 
Table 1: Numbers assigned to different extracts obtained from various plant parts extracted in three different solvents. 
 Neeraj et al. International Journal of Phytomedicine 3 (2011) 549-556 
 
551 
 
S. No. Plant Parts used Methanolic extract no. 
1. Pomegranate Epicarp 1 
2. Sambal 
Sepals 4 
Petals 5 
3. Babool Leaf 8 
4. Aakha Leaf 12 
5. Arandi Leaf 14 
6. Ashoka Leaf 16 
7. Arjun Leaf 17 
8. Neem Leaf 18 
9. Pudina Leaf 19 
10. Sadabahar Leaf 20 
11. Tulsi Leaf 21 
12. Tobacco Leaf 23 
13. Orange Epicarp 2 
14. Mehndi Leaf 9 
15. Jangli Mirchi 
leaf 11 
Stem 10 
16. Aksand 
leaf 6 
Stem 7 
 
 
Result and Discussion  
The results of antimicrobial activity of methanolic 
extracts have been depicted in Table 2. The inhibition 
zone is the mean of the three replicates, excluding the 
well diameter. The (-) sign indicates no activity. Only 
the aqueous extracts of A. nilotica (leaf and stem) and 
L. inermis (leaves) were found inhibitory to some of the 
organisms tested i.e. Bacillus subtilis, Staphylococcus 
aureus, Escherichia coli, Klebsiela pneumoniae and 
Salmonella typhimurium. Salmonella typhimurium was 
only inhibited by aqueous extracts of L. inermis 
(leaves) the rest of the aqueous extracts were found to 
be inactive against all the test organisms. The 
maximum activity was shown by aqueous extracts of L. 
inermis (leaf) against Staphylococcus  aureus (Fig. 
1.1A) that is 1.9 mm diameter of inhibition zone and 
the minimum was observed in the case of aqueous 
extracts of Pergularia daemia (stem) i.e. 0.6 mm 
diameter of inhibition zone against Bacillus subtilis (Fig 
1.1C). Salmonella epidermidis was found to most 
resistance to aqueous extracts of the plant followed by 
Salmonella typhimurium. 
 
 Neeraj et al. International Journal of Phytomedicine 3 (2011) 549-556 
 
552 
 
 
                                     1A 
 
                                     1B 
                                                    
                                                                       1C 
Figure 1- Plates showing zone of inhibition by different aqueous plant extracts on: 1A- Staphylococcus aureus, 1B- 
Escherchia coli, and 1C- Bacillus subtilisMethanolic extracts of Punica granatum ( leaf), Pergularia daemia (stem), 
Acacia nilotica (leaf, stem), Calotropis procera (Leaf, sepals), Ricinus communis (stem, leaf), Citrus aurantium (peels), 
Terminalia arjuna (leaf), Azadirachta indica (leaf), Mentha piperita (leaf), Ocimum (leaf), Catharanthus rescue(leaf), 
Lawsonia inermis (leaf) and Nicotiana rustica (leaf) do not show any inhibitory activity against the test organism. The 
maximum inhibitory activity to be in the case of Toddalia aculeata (leaf) i.e. 2.5 mm diameter against Staphylococcus 
aureus (Fig.2A) and minimum inhibition was observed in the case of Pergularia daemia (leaf) (0.7mm) diameter 
against Klebsiela pneumoniae (Fig.2B) was found to be most resistant followed by E. coli. The rest of the test 
organisms were more or less equally susceptible. 
 
 
    2A        2B  
 Neeraj et al. International Journal of Phytomedicine 3 (2011) 549-556 
 
553 
 
                                                             2C  
Figure 2- Plates showing zone of inhibition by different methanolic plant extracts on: 2A- Staphylococcus aureus, 2B- 
Klebsiela pneumoniae, and 2C- Escherichia coli. 
 
3A    3B  
 
                                        3C  
Figure 3- Plates showing zone of inhibition by different methanolic plant extracts on: 3A- Bacillus subtilis, 3B- 
Salmonella typhimurium, and 3C- Staphylococcus epidermidis. 
 
Analyzing the data it was observed that plant extract 
inhibited the gram positive microorganisms better than 
gram negative microorganisms. The result is in 
agreement with previous report that plant extract are 
active against gram positive bacteria than gram 
negative bacteria [1].The result can be explained that  
the outer membrane of gram positive bacteria known 
to present a barrier to the penetration of numerous 
antibiotic molecule, and the periplasmic space contains 
enzymes that are able to break down foreign 
molecules introduced from outside [2-4] and efflux that 
reduce the cellular level of antibiotics [5].The broad 
spectrum of antibacterial activity found in the study 
may be attributed to the presence of secondary 
metabolite of various chemical types present in the 
plant. Different plants possess different constituents 
and in different concentration which account for 
different antimicrobial effect as also suggested earlier 
[6]. 
 
 Neeraj et al. International Journal of Phytomedicine 3 (2011) 549-556 
 
554 
 
Table 2: Antimicrobial activity of Methanolic and Aqueous extracts of the screened medicinal plants against different 
microbial strains. 
Name of plant Plant part 
Zone of inhibition (mm) 
BS SA SE EC KP ST 
Met Met Met Met Met Met 
Anar 
Leaf - - - - - 0.9 
Epicarp - - - - - - 
Sambal 
Sepal 1.7 - 1.6 1.9 - - 
Petals - - 2.0 - - - 
Babool 
Leaf - - - - - - 
Stem - - - - - - 
Aakha 
Leaf 2.0 - 1.2 - - - 
Stem - - - - - - 
Arahandi 
Stem - - - - - - 
Leaf - - - - - - 
Ashoka Leaf - 1.9 - 1.2 - - 
Arjun Leaf - - - - - - 
Pudina Leaf - - - - - - 
Sadabahar Leaf - - - - - - 
Tulsi Leaf - - - - - - 
Tobacco Leaf - - - - - - 
Orange Epicarp - - - - - - 
Mehandi Leaf - - - - - - 
Jangli Mirchi 
Leaf 1.2 2.2 - - - 1.0 
Stem - 1.0 - - - - 
Aksand 
Leaf 1.8 - - - 0.7 1.5 
Stem - - - - - - 
Neem Leaf - - - - - - 
Bacillus subtilis (BS); Staphylococcus aureus (SA); Staphylococcus epidermidis (SE); Escherichia coli (EC); Klebsiela 
pneumoniae (KP);  Salmonella typhimurium (ST). Zone of inhibition (mm) excluding well diameter. (–) sign 
indicates no activity. 
 
As observed by comparing the Table 2, it was clearly 
indicated that methanolic extracts are more potent anti 
bacterial agents. Successful prediction of botanical 
compounds from plant material is largely dependent on the 
type of solvents used in the extraction procedure. 
Traditional healers use primarily water as solvent but in this 
study, plant extracts in organic solvent (methanol) provided 
more consistent antimicrobial activity. The observation can 
be rationalized in terms of the polarity of compounds being 
extracted by each solvent and, in addition to their intrinsic 
bioactivity, by their ability to dissolve or diffuse in different 
media used in the assay. The result obtained in accordance 
to the preexisting literature [6-8]. 
 
Effect of Antibiotics On The Growth Of Bacteria 
Five antibiotics namely Amoxicillin, Tetracycline, 
Chloramphenicol, Levofloxin and Ofloxacin were used to 
access the susceptibility of test bacteria/organism for them. 
All the test organisms were observed to be susceptible to 
four of the antibiotics used (Amoxicillin, Tetracycline, 
Chloramphenicol, Levofloxin) except Staphylococcus 
epidermidis (resistant to Tetracycline and Amoxicillin) and 
Kleibsella pneumoniae (susceptible to only high 
concentration of Tetracycline and Amoxicillin; (Fig 4.). All 
the strains were resistant to Ofloxacin. 
 
 Neeraj et al. International Journal of Phytomedicine 3 (2011) 549-556 
 
555 
 
                    
        (a)                    (b)                         (c)               (d) 
                                        Fig. 4A            
 
                                       
                                  (a)                   (b)                                        (c)                        (d) 
                                                                         Fig. 4B           
 
                                      
                    (a)                      (b)                                   (d)    
                                                     Fig. 4C                             
 
                                               
(a)    (b)         
                                              Fig. 4D         
 
 
                                       
                                (a)                   (b)                                        (c)                      (d) 
                                                                  Fig. 4E               
 
 Neeraj et al. International Journal of Phytomedicine 3 (2011) 549-556 
 
556 
 
                                                            
(a) (b) 
                         Fig.4F          
Figure 4- Plates showing zone of inhibition by different antibiotics on:  
4A Bacillus subtilis,                     4B Staphylococcus aureus,           4C- Staphylococcus epidermidis, 
4D Escherichia coli,                    4E- Salmonella typhimurium and    4F- Klebsiela pneumonia  
(a): Tetracyclin;                         (b): Chloramphenicol;                      (c): Amoxicillin;        (d): Levofloxacin. 
 
All the active extracts were found to be stable at room temperature up to three months and did not show any reduction 
of activity against the sensitive bacteria as compared to the activities of first day. 
 
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