ANTIFUNGAL ACTIVITY OF PURE LEMON EXTRACT AGAINST CANDIDA ALBICANS

Lemon is a citrus fruit known to have several health benefits on the human body. This study aims to know the effectivity of pure Lemon extract against Candida albicans. The pure Lemon extract sample which contains citric acid with a pH of 2 would be used as the antifungal agent. In the experiment, three agar plates containing Candida albicans samples were incubated for 24 hours. Ten wells were made on the first and second agar plates, while a single well was made on the third agar plate. Pure Lemon extract were incorporated in each well of the first and second agar plate.

INTRODUCTION

Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans.
An increase in pH promotes growth at pH 2-10 in an alkaline environment, as well as acidic environment. Lower pH forms less pathogenic yeast.
Candida growth is inhibited at pH 4 or below which inhibits its hyphae production.

Candida albicans is one of the main organisms of denture-induced stomatitis due to its ability to adhere and form biofilms on oral tissues and denture surfaces. Biofilm grows
extensively on acrylic resin denture material and removal is a significant challenge by both chemical and mechanical methods. Candida albicans produce toxins that are hazardous to patient’s health, causing the patient to have candidiasis.

Pure Lemon extract is an acid with a pH of roughly 2.2 that makes it highly
antimicrobial. It contains 5% to 6% citric acid. Its citric acid property can easily dissolve tarnish making it a very effective material in cleaning, eliminating bacteria and suppress fungus.

Canesten thrush cream is an antifungal medicine used to treat infections. The active ingredient is clotrimazole which kills the Candida fungi by interfering with its cell membrane.
Clotrimazole works by stopping the fungi from producing a substance called ergosterol, which is
an essential component of fungal cell membranes. The disruption in production of ergosterol
causes holes to appear in the fungal cell membrane.

The present study will evaluate the antifungal activity of pure Lemon extract on Candida albicans.
In our previous experiment, we tried to use lemon extract as a denture cleanser or mouthwash aid if we confirmed that Candida albicans can adhere to soft liner.
Unfortunately it doesn’t have the capacity, which leads us to do the antimicrobial assay of pure Lemon extract.

STATEMENT OF THE PROBLEM

• This study aims to answer the question “Is it effective to use pure Lemon extract as an antifungal agent against Candida albicans?”

OBJECTIVES

• General Objective: To determine the effectivity of pure Lemon extract against Candida albicans Specific Objective: 1. To verify the effectivity of pure Lemon extract at pH 2 against Candida albicans. 2. To measure the surface area of Candida albicans after exposure to pure Lemon extract.

HYPOTHESIS

• There is a significant effect of pure Lemon extract against Candida albicans

SCOPE AND LIMITATIONS

The researchers studied the antifungal properties of pure Lemon extract against Candida albicans. The results were gathered by measuring the clearing zones on the agar plate.
The study is limited to pure Lemon extract with pH 2 concentration and from the protocol of University of the Philippines Diliman laboratory to check the result within 24 hours only. It is also conducted via in vitro type of experimental research.

SIGNIFICANCE OF THE STUDY

The purpose of the study is to determine the effectivity of pure Lemon extract against Candida albicans. Pure Lemon extract can be used as alternative over-the-counter mouthwash and denture cleanser since Candida albicans is one of the most common fungi present in the oral cavity and in denture.
It can also promote alternative treatment for patients with Candida albicans related disease. This study provides additional knowledge on other uses of pure lemon extract.

DEFINITION OF TERMS

• Agar plate – petri dish that contains a growth medium (typically agar plus nutrients) used to culture microorganisms.
• Clotrimazole – active ingredient in antifungal medication
• Cork borer – used to punch holes on agar plates to perform well diffusion assays.
• Inoculate – to introduce a microorganism into a suitable situation for growth.

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• pH – measure of the acidity or basicity of an aqueous solution. Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline.
• Pipette – slender tube attached to or incorporating a bulb, for transferring or measuring out small quantities of liquid.
• Streaking technique – use to isolate a pure strain from a single species of microorganisms, other than bacteria.

CONCEPTUAL FRAMEWORK

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MATERIALS AND METHODS

PREPARATION OF SAMPLES

• Fresh lemons were washed in a running water, cut in half, and then squeezed in a sterile bottle.
• Seeds should be separated from the extract. Acidity of the lemon extract was determined through pH paper.
• Microbial suspension was prepared from 24-hour old culture of the test organism. The suspending medium used was 0.1% peptone water.

MATERIALS

• Pure Lemon extract Candida albicans Canesten solution (100 µl) Glucose Yeast peptone agar (GYP) Cotton swab Cork borer (10mm diameter) Sterilized bottle Petri dish Applicator stick

METHODS

Pre-poured Glucose yeast peptone agar (GYP) plates about 3 mm thick, in 140x20mm
Petri dishes were inoculated with the microbial suspension by swabbing the agar surface.
The cotton swab on an applicator stick was dipped into the microbial suspension, rotated several times and pressed firmly on the inside wall of the tube above the fluid level to remove excess inoculums from the swab. The swab was streaked over the entire agar surface.

This procedure was repeated two more times, rotating the plate 60° each time to ensure even distribution of the inoculums. Ten (10) wells were made on the agar plate using a cork borer (10mm diameter). Two hundred (200) µg of the sample was placed in each well using pipettes.
Two plates were used for a total of 20 wells. For the positive control, a single well was made on the center of another agar plate and 100 µl of Clotrimazole “Canesten” solution was placed in it.

Diameter of Clearing zone – Diameter of well
AI = ---------------------------------------------------------------
Diameter of well

RESULTS AND ANALYSIS

a
No clearing zone, no inhibition
b
Contains 1% clotrimazole

The results show that the Lemon extract, tested in 20 replicates, did not inhibit the growth of Candida albicans.
The controlled group (Canesten), on the other hand, showed inhibition of
Candida albicans. It had 35 mm clearing zone and an average of 2.5 antimicrobial index.

DISCUSSION, CONCLUSION, AND RECOMMENDATIONS

Lemon is known to have citric acid with a pH of 2.2 that makes it anti-microbial. It can
disrupt biofilm through a sequestering mechanism in which calcium ions breaks that leads to
anti-biofilm activity. In the experiment, even though lemon is at pH 2 which is highly acidic, it
doesn’t have the antifungal capacity against Candida albicans.

At the end of the experiment, even though lemon at a pH of 2 is not effective as an
antifungal agent for Candida albicans because the said microorganism undergo fermentation
process does citric acid cannot destruct the soft lipoprotein membrane and unable to expose the nucleus means not causing cell death.

CONCLUSION

From the collected data, the results indicated that lemon extract was not effective as an antifungal agent against Candida albicans. It did not inhibit the growth and no clearing zone seen. Further studies could be conducted to check the effectiveness of lemon extract.

RECOMMEDATION

After conducting a research about the antifungal activity of Lemon extract against
Candida albicans, the researchers found out that Lemon extract cannot inhibit the growth of
Candida albicans. Therefore, the researchers suggest further in vitro study on the effectivity of
Lemon against Candida albicans. The use of other substances that will inhibit the growth of
Candida albicans is also advisable.

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