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Evaluation of the antimicrobial activity of curry leaves (Murraya koenigii)

Article · January 2018

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 Evaluation of the antimicrobial activity of curry leaves (Murraya koenigii)

Ifeoma Lois Mbachu, Mary-Pearl T. Ojukwu, Rita Omuero and John O. Chikwem

Department of Biology, Lincoln University, PA

Abstract
Methanolic, ethanolic and aqueous extracts of curry leaves (Murraya koenigii) were
tested for their antimicrobial effects on four Gram-positive and four Gram-negative
bacteria and the fungus, Candida albicans using the agar well diffusion technique. Our
results show that aqueous extract of curry leaves exhibited no antimicrobial activity
against all test microorganisms. The methanolic and ethanolic extracts did not inhibit
any of the Gram-negative bacteria; however, they were effective against all four Gram-
positive bacteria. Candida albicans was only inhibited by the ethanolic extract. Further
studies need to be carried out to identify the active antimicrobial component of curry
leaves.

Introduction
The World Health Organization (WHO) reports that drug-resistant microorganisms now
account for an estimated 700,000 deaths a year, and this number could increase to 10
million deaths each year if no action is taken. (1) In 2016 alone, 490,000 people
developed multi-drug resistant tuberculosis globally. (2) Antimicrobial resistance
increases the cost of healthcare with lengthier stays in hospitals, increased mortality
and more intensive care requirement. (2) For example, people with methicillin-
resistant Staphylococcus aureus are 64% more likely to die than people with a non-
resistant form of the infection. (2)
The evolution of drug-resistant microorganisms is partly due to overuse and misuse in
people and animals, and the fact that antimicrobial agents are often given without
professional oversight. (2) One of the strategies for combating antimicrobial resistance
is the development of new drugs; however, the WHO states that declining private
investment and lack of innovation in the development of new antibiotics are undermining
efforts to combat drug-resistant infections. (3) The public is therefore becoming
increasingly aware of problems with overuse, misuse and side-effects of antibiotics.
One of the potential sources of new antimicrobial agents is plants. This is because
plants have been the main source for drugs and other medicines since ancient times.
(4) About 50% of all pharmaceutical products distributed in the United States have plant
origin, even though very few are used as antimicrobials. (4)
Murraya koenigii also known as curry leaves belongs to the family, Rutaceae, and is
highly valued for its characteristic aroma and medicinal value. It is a tropical plant that is
widely used for seasoning and flavoring. (5) The plant has its origin from India,
Pakistan, Sri Lanka, China and Hainan; however, it is widely cultivated in parts of the
United States and Australia. (6) Reports from several studies indicate that Murraya
koenigii is very rich in various organic compounds such as alkaloids, phenolic
compounds, flavonoids, saponins, proteins, sterols and triterpenes. (7)

IHE: Lincoln University Journal of Science; Volume 7: 2018 17

Several studies indicate that Murraya koenigii has both antioxidant and antibacterial
activity against a variety of microorganisms. Methanolic extract of Murraya koenigii has
been reported as demonstrating significant immunomodulatory effect against ovalbumin
and protection against cyclophosphamide induced myelosuppression. (8) Another study
on breast cancer cell lines reports that aqueous extract of Murraya koenigii reduced
tumor size and lung metastasis of 4T1 cells in vitro. (5)
Results from one study indicate that organic extracts of Murraya koenigii showed
antibacterial activity against Gram-positive and Gram-negative bacteria including
Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. (9) Another
report also indicates that the extracts of Murraya koenigii inhibited the growth of
Staphylococcus aureus, Escherichia coli, Streptococcus and Proteus. (10) There are
also reports that ethanolic extracts of Murraya koenigii was effective against
Mycobacterium smegmatis. (11)
The studies cited above indicate that Murraya koenigii shows great potential for use as
an antimicrobial agent. The present study was initiated to evaluate the potential of using
extracts of Murraya koenigii as antimicrobial agents against Gram-positive and Gram-
negative bacteria and the fungus, Candida albicans.
Methods and materials
Curry (Murraya koenigii) leaves were purchased from the Farmer’s Market in Newark,
Delaware. The leaves were dried in a 45oC incubator; then ground in a Warring blender
before storage at 4oC.
Aqueous, methanolic and ethanolic extracts of the curry leaves were obtained by mixing
two grams of ground curry leaves in eight milliliters of each of the solvents (distilled
water; 95% methanol; 95% ethanol) in 50ml tubes. The tubes were placed in a shaking
incubator at 150 rpm and 25oC for 48 hours. After centrifugation at 3,000 rpm at 25oC
for 20 minutes, the supernatant was aseptically pipetted off; and extracts were sterilized
by filtration through 0.22µm Millipore filter. Sterilized extracts were kept in the
refrigerator at 4oC until used.
All microorganisms used in the study were purchased from Ward’s Natural Science,
Rochester, New York. They include four Gram-negative bacteria: Escherichia coli (85W
1860), Salmonella enteritidis (85W 1910), Enterobacter aerogenes (85W 1805) and
Pseudomonas aeruginosa (85W 1903); four Gram-positive bacteria: Staphylococcus
aureus (85W 1941), Staphylococcus epidermidis (85W 1940), Bacillus cereus (85W
1815) and Enterococcus faecalis (85W 1943); and one fungus, Candida albicans (85W
4150).
Overnight cultures of the test microorganisms were each suspended in test tubes
containing sterile saline to a concentration of 0.5 McFarland. For each test
microorganism, three plates of Mueller-Hinton agar were used, one for each solvent.
Sterile cotton swabs were dipped into each suspension, and the soaked swabs were
then used to completely smear the surfaces of Mueller-Hinton agar plates. The plates
were allowed to dry before they were punctured in three spots with 6mm cork-borer.
The agar within the punctured holes were aseptically removed; the center hole was

IHE: Lincoln University Journal of Science; Volume 7: 2018 18

filled with 50µl of appropriate extraction solvent (water; 95% methanol; 95% ethanol)
while the other two holes were filled with 50µl of corresponding extract. The plates were
left at room temperature for one hour before incubation at 37oC for 24 hours. Zones of
inhibition were measured with a caliper and metric ruler.
Extracts showing antimicrobial activity against any of the test microorganism was further
tested to determine the minimum bactericidal concentration. For this test, the extracts
were double diluted (1/4; 1/8; 1/16; 1/32; 1/64; 1/128; 1/256) in the extraction solvent,
before the puncture holes were filled as described above.
Results
The results of this study show that the aqueous extract of Murraya koenigii did not
inhibit the growth of any of the test microorganism.
The methanolic extract of Murraya koenigii did not inhibit the growth of any of the four
Gram-negative bacteria; Escherichia coli, Pseudomonas aeruginosa, Enterobacter
aerogenes and Salmonella enteritidis. The methanolic extract also did not inhibit the
growth of Candida albicans. It however, inhibited the growth of all four Gram-positive
bacteria used in the study.
The ethanolic extract of Murraya koenigii also did not inhibit the growth of any of the
four Gram-negative bacteria; Escherichia coli, Pseudomonas aeruginosa, Enterobacter
aerogenes and Salmonella enteritidis; however, the ethanolic extract inhibited the
growth of all four Gram-positive bacteria. It also inhibited the growth of Candida albicans
at the highest concentration of 12.5mg only.
Results of the minimum bactericidal concentration of the methanolic and ethanolic
extracts of curry leaves on Staphylococcus aureus are shown in Table 1 and Figure 1.
Table 1: Antimicrobial effect of methanol and ethanol extracts of curry leaves on Staphylococcus aureus

1/4 (12.5mg) 1/8 (6.25mg) 1/16 (3.13mg) 1/32 (1.1.56mg) 1/64 (0.78mg) 1/128 (0.39mg) 1/256 (0.195mg)
Methanol (mm) 21 18 17 14 12 10 0
Ethanol (mm) 15 13 12 11 11 0 0

The results show that the methanolic extract was more effective than the ethanolic
extract on Staphylococcus aureus. The zones of inhibition at all dilutions of the
methanolic extract were larger than those of the corresponding ethanolic extract. Also,
the minimum bactericidal concentration of the methanolic extract was smaller (0.39mg)
than that of the ethanolic extract (0.78mg).

IHE: Lincoln University Journal of Science; Volume 7: 2018 19

Figure 1: Minimum bactericidal concentration of methanol and ethanol extracts of curry
leaves on Staphylococcus aureus

25
20
15
10
Methanol (mm)
5
0 Ethanol (mm)

Results of the minimum bactericidal concentration of the methanolic and ethanolic

extracts of curry leaves on Staphylococcus epidermidis are shown in Table 2 and Figure
2. The results also show that the methanolic extract was more effective than the
ethanolic extract in inhibiting the growth of Staphylococcus epidermidis. The zones of
inhibition at all dilutions were wider than those of the ethanolic extracts. The minimum
inhibitory concentration for the methanolic extract was also smaller (0.39mg) than that
of the ethanolic extract (0.78mg).
Table 2: Antimicrobial effect of methanol and ethanol extracts of curry leaves on Staphylococcus epidermidis

1/4 (12.5mg) 1/8 (6.25mg) 1/16 (3.13mg) 1/32 (1.1.56mg) 1/64 (0.78mg) 1/128 (0.39mg) 1/256 (0.195mg)
Methanol (mm) 25 19 18 16 11 8 0
Ethanol (mm) 13 11 11 10 9 0 0

Figure 2: Minimum bactericidal concentration of methanol and ethanol extracts of curry

leaves on Staphylococcus epidermidis

25
20
15
10
Methanol (mm)
5
Ethanol (mm)
0

IHE: Lincoln University Journal of Science; Volume 7: 2018 20

Figure 3: Minimum bactericidal concentration of methanol and ethanol extracts of curry
leaves on Bacillus cereus

20
18
16
14
12
10
8
6 Methanol (mm)
4
2 Ethanol (mm)
0

Table 3: Antimicrobial effect of methanol and ethanol extracts of curry leaves on Bacillus cereus

1/4 (12.5mg) 1/8 (6.25mg) 1/16 (3.13mg) 1/32 (1.1.56mg) 1/64 (0.78mg) 1/128 (0.39mg) 1/256 (0.195mg)
Methanol (mm) 20 16 15 12 10 8 0
Ethanol (mm) 18 17 16 11 11 8 0

Table 3 and Figure 3 show the results of the minimum bactericidal concentration of the
methanolic and ethanolic extracts of curry leaves on Bacillus cereus. The results show
that there was no significant difference between the methanolic and ethanolic extracts in
the inhibition of Bacillus cereus. The zones of inhibition for both extracts were
comparable; also, the minimum bactericidal concentration was identical at 0.39mg.
Table 4: Antimicrobial effect of methanol and ethanol extracts of curry leaves on Enterococcus faecalis

1/4 (12.5mg) 1/8 (6.25mg) 1/16 (3.13mg) 1/32 (1.1.56mg) 1/64 (0.78mg) 1/128 (0.39mg) 1/256 (0.195mg)
Methanol (mm) 22 20 15 14 10 10 8
Ethanol (mm) 15 13 10 8 0 0 0

Table 4 and Figure 4 show the results of the minimum bactericidal concentration of the
methanolic and ethanolic extracts of curry leaves on Enterococcus faecalis. The results
show that the methanolic extract was more effective at all concentrations than the
ethanolic extract. The zones of inhibition of the methanolic extract were wider at all
concentrations; also, the minimum bactericidal concentration of the methanolic extract
was much smaller (0.195mg) than that of the ethanolic extract (1.156mg).

IHE: Lincoln University Journal of Science; Volume 7: 2018 21

Figure 4: Minimum bactericidal concentration of methanol and ethanol extracts of curry
leaves on Enterococcus faecalis.

25

20

15

10 Methanol (mm)
Ethanol (mm)
5

Discussion
The results of this study show that methanolic and ethanolic extracts of curry leaves
have antimicrobial effect on a variety of microorganisms. Our results however do not
agree with those of other researchers regarding the antimicrobial effect on Gram-
negative bacteria.

One study, (9) reported that Murraya koenigii showed antibacterial activity against
Gram-positive and Gram-negative bacteria including Staphylococcus aureus,
Escherichia coli and Pseudomonas aeruginosa. Another study, (10) stated that extracts
of Murraya koenigii inhibited the growth of Staphylococcus aureus, Escherichia coli,
Streptococcus and Proteus. In our study, none of the extracts (aqueous, methanolic and
ethanolic) inhibited the growth of the four Gram-negative bacteria used in this study.

Further studies will be conducted on the antimicrobial ingredient in curry leaves and
how the extracts can be exploited for clinical and other purposes.

Acknowledgements
This study is supported by Lincoln’s Excellent Academic Program in Science – Transformation
(LEAPS-T) funded by the National Science Foundation Grant # 92-1583.

References
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antimicrobial-resistance-globally

IHE: Lincoln University Journal of Science; Volume 7: 2018 22

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