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POTENTIAL ANTIBACTERIAL ACTIVITIES OF PLANTS

Introduction
Bacterial disease can be defined as any form of illness brought about by bacteria. Usually, there are
millions of bacteria living on our body. Bacteria that causes harmful infections are called pathogenic
bacteria (Haines 2013). A few examples of pathogenic bacteria would be Escherichia coli,
Helicobacter pylori, Staphylococcus aureus and Pseudomonas aeruginosa which can cause serious
and life-threatening complications such as bacteremia and toxic shock syndrome (Haines 2013).
Researches and studies are done over many years and this bring about the emergence of
antibiotics. Many fatal and infectious diseases were able to be brought under control, saving
millions of lives. Researchers even thought that synthetic antibiotics might be the solution to cure
infectious disease and they were discouraged in making serious efforts to develop drugs from
simple natural compounds (Thormar 2011).
However, rising in number of resistance to antibiotics is alarming and that new antibacterial
sources are being done now, such as investigation on plants. Infectious diseases cause premature
death, leading the chart with almost 50,000 fatality every day (Mahida and Mohan 2007).
Necessary steps were taken to reduce resistance problem, such as control the consumption of
antibiotics, researches for better understanding of genetic mechanism of resistance and
development of new, synthetic or natural drugs (Nascimento et al. 2000).
Recently, there have been a renewed interest in antibacterial effects of natural compounds
(Thormar 2011). Plant extracts and essential oils were screened for antibacterial activity and bioassay guided fractionation of active extracts were done to isolate the active constituent from the
selected plants (Mohamed et al. 2010).

Plants selected for use in antibacterial


Table 1 shows the plants containing antibacterial activities selected for this dissertation (Cetin et al
2009; DerMarderosian and Beutler 2010):
Name

Scientific

Family

name
Garlic

Allium

Plants part
used

Liliaceae

Bulb

Sativum
Lemon

Citrus limon

Active constituent and chemicals

Volatile oil (0.5%), alliin, (allicin when


converted by allinase).

Rutaceae

Fruit, juice,

Volatile oil (2.5%), limonene, alpha-

peels and

terpinene, alpha-pinene, citral,

seeds

coumarins, mucilage, pectins,


bioflavonoids.

Peppermint

Mentha x

Lamiaceae

piperita

Leaves and
stem

Menthol (29-48%), menthone (20-31%),


menthyl acetate (3-10%), caffeic acid,
flavonoids, tannins.

Thyme

Thymus

Lamiaceae

vulgaris

Leaves and
flowering
tops

Wormwood

Artemisia

Asteraceae

Whole herb

absinthium

Carvacrol, Thymol, p-Cymene


Linalool, -Terpinene-Caryophyllene,
-Terpineol ,rosmarinic acid
Volatile oil (contains thujone),
anabsinthin, absinthin, resins, organic
acids

Table 1

Garlic

Figure 1 (Govan 2005)

Figure 2 (Albert 2011)

Lemon

Figure 3(Parentinghealthybabies.com 2014)

Figure 4 (Cheung 2013)

Peppermint

Figure 5 (The Apothecary in Inglewood 2014)

Thyme

Figure 6 (Stakich 2014)

Figure 7(GourmetGarden 2014)

Figure 8 (SweetAddictions.net 2010)

Wormwood

Figure 9 (PureBalance, 2014)

Figure 10 (Grieve 2014)

Screening methods
There are a few common screening methods used for antibacterial properties of plant extracts.
1. One of the methods used is the disc-diffusion method. This method uses paper discs and are
impregnated with plant extracts and put onto an inoculated agar medium. After incubation, the
diameter around the disc where bacteria growth is stopped or inhibited is measured. The
parameter is known as zone of growth inhibition. There are several disadvantages using this
method such as essential oil unable to diffuse through the agar due to the hydrophobic nature of
the essential oil (Thormar 2011). Another drawback is that this test is qualitative test, as zone of
growth inhibition is only measured in mm, and Minimum Inhibitory concentration cannot be
determined (MichiganStateUniversity 2011).
2. Another method used is the agar- or broth-dilution method. This method offers a better
parameter such as allowing the calculation of Minimum Inhibitory Concentration (MIC). MIC is
simply defined as lowest concentration of the plant drugs that inhibit growth of test bacteria and
more useful than zone of growth inhibition since MIC allows establishment of safe and effective
final concentration in formulated drugs (Thormar 2011).
3. The 96-well plate assays are also used to screen for antibacterial activity and also to determine
the MIC of the drugs. Final concentrations of 100% (undiluted), 50%, 25%, 12.5%, and 6.25% of
serial dilutions of the extracts were prepared using sterile water. Then, bacteria of 108 CFU/mL
were added to each experimental well. The plates were incubated at 37C for 24 hours.

P-iodonitrotetrazolium violet (INT) marker solution was added and presence of antibacterial
activity is indicated by changes of colour from clear to red. MIC is determined by means of digital
images in a computer (Frey and Meyers 2010).

Screening of selected plant examples


The garlics were first dried and pulverized into powder form. Then, the active constituent were
extracted by using 80% ethanol and deionized water. Paper disc diffusion method was used where
Whatmann No.1 Filter paper was cut into 5mm diameter. The disc absorbed maximum volume of
0.052 mL. The ethanol extracts were reconstituted in Di Methyl Suphoxide (DMSO) and
500mgml1 , 250mgml1 , 75mgml1and 35.25 mgml1 were produced by double dilution method.
Dilution of bacterial culture was done by serial dilution from 101to 105. A sterile swab stick was
used on 103and 104 dilution, to seed the nutrient agar plates. The discs were impregnated with
the garlic extract of 1000mgml1, 500mgml1 , 250mgml1, 75mgml1 and 35.25 mgml1 in
triplicates and put onto the seeded nutrient plates. The zone of inhibition were calculated after
incubation of 24 hours (Ekwenye and Elegalam 2005).
Antibacterial activity in Lemon was done using fresh lemon juice. Agar well diffusion method was
used in this experiment. 50 l of inoculums was spread uniformly on nutrient agar plates and after
5 minutes, a 6mm diameter well was made with a cork borer. 50 l of lemon juice and the standard
ampicillin (2.5mg/ml) were transferred to the well with sterile syringe. Then, the plates are
incubated for 25 hours at 37C. The zone of inhibition was measured and the MIC was determined
by using UV spectrophotometer (Bansode and Chavan 2012) .
Antibacterial activity of peppermint was conducted by standard disc diffusion method. Essential oil,
decoction and aqueous infusion were prepared for this studies. 6mm diameter sterilised discs were
soaked in 1ml of each of the infusion, oil, decoction and juice for 1-2 minutes. Mueller-Hinton agar
and broth were used for preparation of inoculum. Isolated colonies of bacteria were inoculated in
the broth and incubated. Next, cotton swab is used to dip in bacterial broth suspension and
inoculate the surface of the agar plates. Then, prepared discs of extracts were placed onto the agar
plates and incubated at 35-37C for 24 hours (Saeed et al. 2006).
Antibacterial activity of Thyme was performed by using powdered thyme in hexane, ethyl-acetate,
methyl alcohol 80% and distilled water by maceration. Bacterial strains, collected from clinical
specimen, sputum and urine, were activated by transferring of Brain Heart Infusion (BHI) into

nutrients broth followed by incubation at 37C for 24 hours. Agar dilution method is used to
determine antibacterial activity and MIC. Serial concentrations of thyme extracts were obtained
from 0.02 mgml1 up to 13 mgml1 . Petri plates of BHI containing various concentration of
extracts were inoculated with bacterial strains and spread on solid agar plates. Then, the agar
containing extracts and bacterial strain were incubated for 24 hours. The lowest concentration
extracts that completely inhibit growth of tested bacteria was determined to be the MIC (El-Safey
and Salah 2011).
Antibacterial activity of Wormwood were done to determine the MIC using broth dilution method.
Brain heart infusion (BHI) broth with Tween 80 detergent was used. Bacterial strains were
suspended in the BHI broth to get a final density of 107 cfu ml1 . 40 l Wormwood essential oils
with various dilutions were added to tubes containing the BHI and bacterial strains. The tubes were
then incubated with incubator shaker and the lowest concentration of essential oil used, without
visible growth, was considered the MIC (Taherkhani et al. 2013).

Side effects of the plants


Plants

Side effects

Garlic

Burning sensation in mouth and stomach

(WebMD 2014)

Heartburn

Bad breath

Body odor

Lemon

Loss of gloss in teeth

(DerMarderosian and Beutler

Change in enamel colour

2010)

Irregular dental tissue of tooth enamel

Peppermint

Heartburn

(Medicinenet 2014)

Nausea and vomiting

Uncommon:

Thyme
(Chamberlins.com 2011)

Flushing

Severe abdominal pain

Mouth sores

Headache

Wormwood

Dizziness

Low blood pressure

Reduced heart rate

Heartburn

Muscle weakness

Can worsen inflammation in urinary tract infections

Thujone found in essential oil of wormwood can cause:

(DerMarderosian and Beutler

Absinthism

2010)

Digestive disorders

Restlessness

Numbness of the extremities

Loss of intellect

Conclusion
The experimental results of garlic extracts are shown in Table 2:
Plant extracts

Bacterial strains tested

Zone of Inhibition(mm)

Garlic ethanol extracts (Crude

Escherichia coli

solution 100%) (Ekwenye and

Salmonella typhi

Garlic Aqueous extracts

Escherichia coli

14.3 0.54

(Gull et al. 2012)

Pseudomonas aeruginosa

18.3 0.72

Staphylococcus aureus

19.3 1.08

Elegalam 2005).

Shigella

13 0.47

Salmonella typhi

15.6 0.56

Table 2
The experimental results of lemon extracts are shown in Table 3:
Plant extracts

Bacterial strains tested

Zone of Inhibition(mm)

Lemon juice (Crude solution

Escherichia coli

100%) (Bansode et al. 2012)

Salmonella parathypi b

Shigella sonnei

Lemon peels ethanol extracts

Escherichia coli

20

(Pandey et al. 2011)

Pseudomonas aeruginosa

19

Staphylococcus aureus

21

Table 3
The experimental results of peppermint extracts are shown in Table 4:
Plant extracts

Bacterial strains tested

Zone of Inhibition(mm)

Peppermint oil

Escherichia coli

13

(Saeed et al. 2006)

Pseudomonas aeruginosa

12

Salmonella typhi

10.33

Salmonella parathypi b

11

Peppermint juice

Escherichia coli

12.26

(Saeed et al. 2006)

Pseudomonas aeruginosa

11.56

Salmonella typhi

9.5

Salmonella parathypi b

Table 4
The experimental results of thyme extracts are shown in Table 5:
Plant extracts

Bacterial strains tested

Zone of Inhibition(mm)

Thyme essential oil

Escherichia coli

22.55.5

(Kon and Rai 2012)

Staphylococcus aureus

22.71.6

Thyme aqueous extract

Escherichia coli

(Bassam et al. 2004)

MRSA

12

Pseudomonas aeruginosa

Table 5
The experimental results of Wormwood extracts are shown in Table 6:
Plant extracts

Bacterial strains tested

Minimal inhibitory
concentration(mg/ml)

Wormwood essential oil

Escherichia coli

(Taherkhani et al. 2013).

Staphylococcus aureus

Pseudomonas aeruginosa
Plant extracts

Bacterial strains tested

2.5
Zone of inhibition (mm)

Wormwood essential oil

Escherichia coli

15

(Taherkhani et al. 2013).

Staphylococcus aureus

32

Pseudomonas aeruginosa

21

Table 6

Based on the results above, all selected plant extracts have shown a significant antibacterial
properties to certain Gram-positive and Gram-negative bacteria. Garlic shows a better antibacterial
properties in the aqueous extracts compared to ethanol extracts, with the highest zone of
inhibition against Gram-positive Staphylococcus aureus. The antibacterial properties of allicin found
in garlic has been shown to have a wide spectrum of antibacterial activity against Gram-negative
and Gram-positive bacteria (Ankri and Mirelman 1999).
In lemon, most of the antibacterial potential lies within the peel, where the zone of inhibition
against the test bacteria showed significant difference compared to lemon juice extracts. The most
susceptible bacterial strains is shown to be Staphylococcus aureus, with the highest zone of
inhibition of 21mm. Lemon peels contain flavonoids, terpenes, ascorbic acid, corydaline alkaloids,
hypericin and other components that contributes to overall antibacterial properties of lemon
extracts (Dhanavade et al. 2011).
The antibacterial properties of peppermint oil and peppermint juice show good inhibition with high
zone of inhibition. From the results, peppermint has shown excellent antibacterial properties in the
extracts of the stem and leaves, E.coli is the most susceptible with slightly higher zone of inhibition
(Saeed et al. 2006).
Thyme essential oil shows a significant inhibitory activity against E.coli and S.aureus with high
degree of zone of inhibition. Thyme aqueous extract showed less activity with lower zone of
inhibition compared to thyme essential oil. The results showed that thyme oil antibacterial
attribute could be due to the presence of Carvacrol and Thymol in Thyme extracts (Cetin et al.
2009).
Wormwood essential oil has shown to have a potent antibacterial effects, with the most
susceptible bacteria being Staphylococcus aureus (MIC: 1 mg/ml and inhibitory diameter 32mm). It
also shows good activity against E.coli and P.aeruginosa with significant inhibitory effects
(Taherkhani et al. 2013).
In conclusion, plant drugs have been proven to show a wide spectrum of activities against bacteria.
With the alarming increase in antibiotic resistant bacteria, plants and herbs potential as
antibacterial should not be overlooked as they can be a good source for potential drug candidate.

(2012 words)

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