Antibacterial activities of selected plants

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 bio-

assay 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

name

Family Plants part

used

Active constituent and chemicals

Garlic Allium

Sativum

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

converted by allinase).

Lemon Citrus limon Rutaceae Fruit, juice,

peels and

seeds

Volatile oil (2.5%), limonene, alpha-

terpinene, alpha-pinene, citral,

coumarins, mucilage, pectins,

bioflavonoids.

Peppermint Mentha x

piperita

Lamiaceae Leaves and

stem

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

menthyl acetate (3-10%), caffeic acid,

flavonoids, tannins.

Thyme Thymus

vulgaris

Lamiaceae Leaves and

flowering

tops

Carvacrol, Thymol, p-Cymene

Linalool, γ-Terpineneβ-Caryophyllene,

α-Terpineol ,rosmarinic acid

Wormwood Artemisia

absinthium

Asteraceae Whole herb 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) Figure 6 (Stakich 2014)

Thyme

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 37°C 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

500mgml−1, 250mgml−1, 75mgml−1and 35.25 mgml−1were produced by double dilution method.

Dilution of bacterial culture was done by serial dilution from 10−1to 10−5. A sterile swab stick was

used on 10−3and 10−4 dilution, to seed the nutrient agar plates. The discs were impregnated with

the garlic extract of 1000mgml−1, 500mgml−1, 250mgml−1, 75mgml−1 and 35.25 mgml−1 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 µl of inoculums was spread uniformly on nutrient agar plates and after

5 minutes, a 6mm diameter well was made with a cork borer. 50 µl 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 37°C. 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 35-37°C 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 37°C for 24 hours. Agar dilution method is used to

determine antibacterial activity and MIC. Serial concentrations of thyme extracts were obtained

from 0.02 mgml−1 up to 13 mgml−1. 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 107cfu ml−1. 40 µl 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

(WebMD 2014)

Burning sensation in mouth and stomach

Heartburn

Bad breath

Body odor

Lemon

(DerMarderosian and Beutler

2010)

Loss of gloss in teeth

Change in enamel colour

Irregular dental tissue of tooth enamel

Peppermint

(Medicinenet 2014)

Heartburn

Nausea and vomiting

Uncommon:

Flushing

Severe abdominal pain

Mouth sores

Thyme

(Chamberlins.com 2011)

Headache

Dizziness

Low blood pressure

Reduced heart rate

Heartburn

Muscle weakness

Can worsen inflammation in urinary tract infections

Wormwood

(DerMarderosian and Beutler

2010)

Thujone found in essential oil of wormwood can cause:

Absinthism

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

solution 100%) (Ekwenye and

Elegalam 2005).

Escherichia coli 8

Salmonella typhi 8

Garlic Aqueous extracts

(Gull et al. 2012)

Escherichia coli 14.3 ± 0.54

Pseudomonas aeruginosa 18.3 ± 0.72

Staphylococcus aureus 19.3 ± 1.08

Shigella 13 ± 0.47

Salmonella typhi 15.6 ± 0.56

Table 2

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


Lemon juice (Crude solution

100%) (Bansode et al. 2012)

Escherichia coli 5

Salmonella parathypi b 6

Shigella sonnei 6

Lemon peels ethanol extracts

(Pandey et al. 2011)

Escherichia coli 20

Pseudomonas aeruginosa 19

Staphylococcus aureus 21

Table 3

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


Peppermint oil

(Saeed et al. 2006)

Escherichia coli 13


Salmonella typhi 10.33


Peppermint juice

(Saeed et al. 2006)

Escherichia coli 12.26

Pseudomonas aeruginosa 11.56

Salmonella typhi 9.5


Table 4

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


Thyme essential oil

(Kon and Rai 2012)

Escherichia coli 22.5±5.5

Staphylococcus aureus 22.7±1.6

Thyme aqueous extract

(Bassam et al. 2004)

Escherichia coli 6

MRSA 12


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

(Taherkhani et al. 2013).

Escherichia coli 5


Pseudomonas aeruginosa 2.5

Plant extracts Bacterial strains tested Zone of inhibition (mm)

Wormwood essential oil


Escherichia coli 15



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


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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Antibacterial activities of selected plants

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