The effect of different concentrations of Bay leaves (Syzygium polyanthum)

extract on the inhibition of Salmonella typhii (In vitro)

Yunita Lidya Istiqomah

ABSTRACT

In Indnesia Bay leaves (Syzygium polyanthum) are used as traditional medicine in treatment of diabetes mellitus and stress. The active component (Syzygium polyanthum) has been proven to exhibit antibacterial properties. The objective of this study was to determine the influence of different concentrations of Bay leaves extract on the inhibition of Salmonella typhii. Bay leaves extract at concentrations of 20%, 40%, 60%, 80%, and 100% was prepared. Fifty milliliters of each concentration were dropped into holes of 6 millimeters in diameter on a MHA agar that has been inoculated with Salmonella typhii. Distilled water was used as a control. After 24 hours of incubation, the inhibition diameters were measured and analyzed. The statistical results of the one-way analysis of variance (ANOVA) illustrated that the different concentrations of Bay leaves extract had a significant influence on the inhibition of Salmonella typhii. This was followed with the Least Significant Difference (LSD) which implied that there were significant differences between all the concentrations of Bay leaves extract used in this experiment. The conclusion of this study was that Bay leaves extract exhibited antibacterial effect towards Salmonella typhii and different concentration of Bay leaves extract

influenced the inhibition of Salmonella typhii. Key words: Syzygium polyanthum, Salmonella typhii, antibacterial

INTRODUCTION

Worldwide, typhoid fever affects roughly 17 million people annually, causing nearly 600,000 deaths. The causative agent, Salmonella enterica typhi (referred to as Salmonella typhi from now on), is an obligate parasite that has no known natural reservoir outside of humans.1 S. typhi has a combination of characteristics that make it an effective pathogen. This species contains an endotoxin typical of Gram negative organisms, as well as the Vi antigen which is thought to increase virulence.2 It also produces and excretes a protein known as invasin that allows non-phagocytic cells to take up the bacterium, where it is able to live intracellularly. It is also able to inhibit the oxidative burst of leukocytes, making innate immune response ineffective.3 As the public awareness for oral hygiene increases, many people are now turning to traditional medicine for a solution. The Bay leaves originates from Indonesia.5 In Indonesia, this plant is referred to as daun salam.7 Over centuries, this plant has provided leaves as a range of healing properties.8

In a study to determine the most effective method for

inhibiting other gram negatives bacteria, such as Eschericia colli, it shows that Bay leaves could reduce the ability of Eschericia colli colonize in 20% extract. 11 From the above, it is deduced that although the anti-bacterial effect of Bay leaves has
been proven, there is still a grey area concerning its effective

concentration against the Salmonella typhii specifically. The objective of this study is to determine the
effectiveness of the Bay leaves extract as an anti-bacterial against the growth of Salmonella typhii.

MATERIALS

AND METHOD

This was a laboratory experimental study. The

extractions of Bay leaves extract were carried out at the Chemical Laboratory in University of 17 August. The materials used in this experiment are Bay leaves

aqueous extract ( 20%, 40%, 80%, and 100%),

distilled water, Brain Heart Infusion (BHI) as a culture medium, Muller Hinton Agar (MHA) and Cultured

Salmonella typhii. Among the instruments used are

Petri dishes (10 cm in diameter), test tubes, test tube rack, and water bath, sliding calipers, sterile loop, incubator, autoclave and micropipettes. One kilogram of matured and complete Bay leaves is selected. One litre of distilled water was added to the leaves and then it was blended in

After 24 hours of incubation, the Petri dishes were observed for inhibition zones around each hole. This translucent area around the hole where there was no Salmonella typhii growth is also referred to as the radical zone. The required area was from the edge of the hole to the outer border of bacterial inhibition. The diameter is measured using a sliding caliper with a precision of 0.01 mm.
Each measurement was taken three times to ensure

the blender. The mixture was put through a Buchner filter

to separate the filtrate and the residue. The filtrate was then evaporated over a water-bath at 60 C to obtain concentrated

extract. The extract was then ready for dilution.13 Based on previous experiments carried out by Hulbert,15 in this study the concentrations of Bay leaves extract used are 20%, 40%, 80%, and 100%. Distilled water was used to dilute the Bay leaves extract
and the various concentration of Bay leaves were prepared

higher accuracy. The horizontal diameter of the inhibition zone was measured, followed by the vertical diameter and finally the diameter between the first two lines at an angle of 45. The average of the three measurements for each
zone was recorded. The procedure was repeated for all three

Petri dishes. The results obtained were then systematically documented in a form of a table.
Statistical analysis of the data was carried out using the

by the mass of extract per volume. The dilution details are presented below: Group A: for the control only 100 ml of distilled water; Group B: for 20%, 20 mg of leaves extract and distilled water added little by little to make the volume 100 ml; Group C: for 40%, 40 mg of Bay leaves extract and distilled water added little by little to make the volume 100 ml; Group D: for 80%, 80 mg of Bay leaves extract and distilled water added little by little to make the volume 100 ml; Group E: for 100%, 100 mg of Bay leaves extract and distilled water added little by little to make the

One-way analysis of variance (ANOVA). In this test, the calculated F ratio was compared to the value from the F distribution table at a confidence level of 95%.17 ANOVA is to see if the different concentrations and control have a significant influence on the inhibition of Salmonella typhii. The ANOVA was then followed by a post-hoc comparison, in this case, the Least Significant Difference (LSD). LSD was used to further speculate if there was a significant difference between the various concentrations.17

volume 100 ml.

The bacterial sensitivity test was carried out to determine
RESULTS

the antibacterial effect of the Bay leaves extract. In this experiment the bacterial sensitivity test was carried out using the diffusion techniques.16 This technique was used by most laboratories to test routinely for bacterial sensitivity. The sample of Salmonella typhii strain for this experiment was obtained from the Microbiology department of the Sultan Agung University. The Salmonella

A line diagram is plotted to see the relationship between

the inhibition diameters and extract concentrations. The chart shows that the increase of Salmonella typhii inhibition diameter corresponds with the increase of Bay leaves extract concentration. The diagram representing these results is presented in figure 1.
The data is first analyzed with the normality tests. The

typhii was cultured on

Mueller Hinton Agar. After 24 hours of incubation at 37 C, 5 colonies were transferred into 2 ml of Brain Heart Infusion (BHI). Its turbidity was compared to the standard Brown III solution. Using a sterile loop of about 4 mm in diameter, the suspension was inoculated three times on the centre of a MHA plate. A sterile cotton wool pad is then

results of the test show that the probability is 0.85. This value is greater then 0.05, thus implying that the acquired data is normally distributed (table 1).

D iam et er o f In h ibit on Z on e (mm)

used to spread the inoculum evenly over the plate.

Six holes were then punctured on the MHA each six milimetres in diameter. Fifty microlitres extract of each concentration was dripped into holes. For the control, fifty microlitres of distilled water was used. Similarly, two other Petri dishes were prepared as well. After incubation for 24 hours at 37 C, the inhibition zones are measured with a

14 12 10 8 6 4 2 0 20 40 80 100
Consentration of Bay leaves Extract (%)

sliding caliper.
Figure 1.

Relationship between the inhibition diameters of Salmonella typhii and Bay leaves extract.

Table 1. Statistical results of the normality test

Kolmogorov-Smirnov Radical Zone Diameters 0.190 Df 54 Sig 0.850

The normality test is then followed by the ANOVA. In

this test, the calculated F ratio is compared to the value from the F distribution table at a confidence level of 95%. The ANOVA speculates if the different concentrations of Bay leaves extract have a significant influence on the inhibition of Salmonella typhii. The results of this

analysis are presented in table 2.

is larger as the concentration of Bay leaves extract is increased. The statistical analysis carried out using the ANOVA proves that the different concentrations have significant influence on the growth of Salmonella typhii. These results also coincide with the results in the research of Hulbert .15 Hulbert 15 documented a study using Bay leaves to inhibit the growth of E. coli. Although the bacteria used in the both the experiment are different, all the results correspond with the theory that flavonoids the active component of Bay is of the phenol group and that phenols substances can destroy bacterial cell walls which will inevitably inhibit the growth of bacteria.18 The breakdown of cell wall disturbs osmotic pressure and leads to cell death.19
The statistical analysis summarized in table 2 proves

Table 2. Statistical results of the One-way analysis of varience

(ANOVA) Source Ss Df Ms F Between Groups Within Groups 3328.267 48

Total 68983.000 53

P 251.114 6495.874 0.038 0.001

that the different concentration of Bay leaves extract have

1255.568 5

a significant influence on the growth of Salmonella typhii. The post hoc test shows that the probability between the concentration of 80% and 100% is 0.011. Although this value is statistically significant, it is higher that the probability of all the other concentration. The different value confirms that there is less statistical difference between the concentration of 80% and 100%. This gives evidence that the effectiveness of Bay leaves extract on the Salmonella typhii at concentration 80% and 100% is clinically almost the same.
Nevertheless, it is still not viable to determine the effective concentration of the Bay leaves extract to inhibit

The results of the ANOVA show that the probability is

0.001; this value is less than the 0.05 confidence level. The interpretations of the ANOVA illustrates that the different concentration of Bay leaves extract has a significant influence on the inhibition of Salmonella typhii. To further speculate if there is a significant difference between the various concentrations, the ANOVA test is then followed by a post-hoc comparison, the Least Significant Difference (LSD). These results imply that there are significant differences between all the concentrations of

Bay leaves extract used in this experiment (table 3).

Table 3. Result (mm)

the growth of Salmonella typhii. The effective concentration is the concentration where beyond that value the inhibition diameter is constant or decreases. In this experiment it can only be approximated that the effective concentration is above 80%. The value is indistinct hence, future experiments with a narrower range are necessary to determine the effective concentration of Bay leaves extract to inhibit the growth of Salmonella typhii.
In this experiment, the Bay leaves are extracted using

Repeating Con (-) 20% 40% 60% 80% 100% 1 2 3 7 9 12 13 19 20 12 19 22 11 18 22

Mean

- 9,333 12 18,67 21,

distilled water as the dissolvent, therefore the active component that is extracted is less. The use of organic dissolvent such as ethanol is more effective because theoretically the active component belongs to the phenol group and phenols would dissolve better in organic dissolvent.11 Ethanol extractions may also be able to show the effective concentration of Bay leaves extract in the inhibition of Salmonella typhii which could not be determined in this experiment. However, ethanol extractions are more complicated and expensive, therefore in this study, distilled water is used as it is more economical.
From this study it can be concluded that the Bay leaves

DISCUSSION

The results of this study exemplifies that the Bay

leaves extract has antibacterial activity towards Salmonella typhii. The inhibition area that is formed

extract has antibacterial properties towards the Salmonella typhii. This could be further developed as an alternative method to prevent typhoid fever. In this experiment, the extracting method has been kept simple without the use of ethanol and sophisticated methods. The experiment

is performed keeping in mind that the ultimate aim is to

find a cheaper and safe method to curb typhoid fever in developing

countries.

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