INTRODUCTION

Background of the Study

Candidiasis is a common healthcare-associated infection. It is a fungal infection caused

by yeasts from the genus Candida. Specifically, Candida albicans is the predominant cause of

the disease. Its yeast is a part of the gut flora, a group of microorganisms that live in your mouth and

intestine. (Hidalgo, 2015)

According to Richards (2013), Candida albicans is the cause of many undesirable symptoms

ranging from fatigue and weight gain, to joint pain and gas. Almost everyone has Candida albicans in

their gut, and a significant proportion of us may have Candidiasis, or an overgrowth of Candida. Candida

albicans starts to cause trouble when there is some change in the body that allows it to overgrow. This

change could be anything from a few courses of antibiotics, a prolonged diet rich in carbohydrates and

sugar, or even something as common as a lengthy period of stress at work. Overgrowth of Candida

albicans produces toxins that your body’s immune system can struggle to cope with, particularly if one is

under stress or otherwise weakened.

On the other hand, according to the site PlantVillage.org, lemongrass, Cymbopogon

citratus, is a perennial grass in the family Poaceae grown for its fragrant leaves and stalks which

are used as a flavoring. The grass grows in dense clumps and has several stiff stems and slender

blade-like leaves which droop towards the tips. The leaves are blue-green in color, turning red in

the fall and emit a strong lemon fragrance when damaged. Lemongrass produces large compound

flowers on spikes when grown in the tropics, but rarely flowers when grown in more Northern

latitudes.

1
 A similar study, conducted by de Bona da Silva, et al (2008), tested the Antifungal

activity of Lemongrass oil and citral against Candida spp. and showed positive results. Thus, this

study was conducted to specifically test the efficacy of Lemongrass leaves extract alone as it is

one of the perennial plants in the locality, making it very accessible to people, against one

particular Candida spp. – Candida albicans.

Statement of the Problem

The study aimed to determine and evaluate the antifungal activity of Lemongrass (Cymbopogon

citratus) leaves extract against Candida albicans.

Specifically, the study aimed to:

 Determine if there is a significant difference between the zone of inhibition of Cymbopogon

citratus leaves extract at100%, 95%, and 90% concentrations respectively and Nystatin against C.

albicans.

Hypothesis

There is no significant difference between the zone of inhibition of Cymbopogon citratus leaves

extract at100%, 95%, and 90% concentrations respectively and Nystatin against C. albicans.

2
Significance of the Study

Testing the antifungal activity of Cymbopogon citratus leaves extract against Candida albicans is

beneficial for:

1. The People. This study can help people in financial aspects. It would be a cheaper, feasible,

and easy to obtain alternative for the commercial drug.

2. Medical Practitioners. This study can help in giving advice for alternative remedies than

expensive ones. It can help the people especially students about the benefits of lemongrass

and the nature of Candida albicans.

3. Future Researchers. This study can give way for future researchers to test the anti-fungal or

antibacterial activity potential of lemongrass to other pathogenic microorganisms. And

likewise explore the benefits of the other by-product of lemongrass leaves extract which is the

hydrosol.

Scope and Limitations

The researcher focused on the antifungal activity of Lemongrass (Cymbopogon citratus) leaves

extract against Candida albicans. Other species of Candida were not included in the study. The

lemongrass leaves were extracted through condensation and separated through distillation with assistance

of personnel in EcoAgri at Bacolod City, Negros Occidental. Portions of the 100% oil extract was diluted

in distilled water to obtain 95% and 90% concentrations respectively and the efficacy of the three

concentrations were tested through a specific dosage (20 µL/plate). The results were compared to the

efficacy of the commercial drug against Candida albicans – Nystatin placed at 20.0 µL/disk.

Furthermore, the researcher conducted the study in Villanueva Clinical Laboratory, Bacolod City,

Negros Occidental.

3
 Methodology

Flowchart

COLLECTION OF MATERIALS

EXTRACTION OF LEMONGRASS
(Cymbopogon citarus) Leaves

PREPARATION OF TREATMENTS

CULTURE OF FUNGUS

EXPERIMENT

DATA COLLECTION AND ANALYSIS

RESULTS

4
Procedure

Collection of Materials

The lemongrass leaves were collected from Eco-Agri, Bacolod City and were cut into 1 -

1.5 inches. On the other hand, the culture of the fungi was obtained from Riverside Medical

Hospital.

Extraction of Lemongrass

The lemongrass leaves were extracted at Eco-Agri, Bacolod City, Negros Occidental

through the hydro-steam distiller. The lemongrass leaves extract was measured through the

graduated cylinder to prepare the concentrations.

Preparation of Treatments

The extract samples were diluted in distilled water and obtained the following treatments:

Treatment 1 - 100% Cymbopogon citratus leaves compared to a positive control Nystatin,

Treatment 2 – 95% Cymbopogon citratus leaves compared to a positive control Nystatin,

Treatment 3 – 90%Cymbopogon citratus leaves compared to a positive control Nystatin.

5
Preparation of Culture Medium

The microbiological laboratory was prepared by the in-charge and all needed materials

were provided by the institution. Seeded agar plates were prepared by pouring 20 mL of SDA

into each sterile plate. The melted agar plate was allowed to cool at 60 degrees Celsius before

dispensing into plates.

Preparation of the Fungal Inocula

The cultures of Candida albicans were cultivated on 4% Sabouraud Dextrose Agar at 25

degrees Celsius for 48 hours. Antifungal activity was tested using Lemongrass leaves extract.

Sensitivity test was achieved. The antifungal activity was studied by agar disc diffusion method.

Seeded agar plates were prepared by pouring 20 mL of SDA into each sterile plate. After

solidification of medium, each plate was overlaid with 5ml of SDA. Lemongrass leaves extract

was applied on filter paper disks (20.0µL/disk) of 6mm in diameter.

Nystatin solution (0.3 mg/mL) was used as antifungal reference substance (20.0µL/disk).

The disks was placed on the surface of seeded agar plates (one disk for plate).All plates were

incubated at 35 degrees Celsius ±2 degrees Celsius for 24 hours.

6
Microbiological Assay

The microbiological assay was conducted in Villanueva Clinical Laboratory. The alcohol

lamp was lighted to keep the sterility of the area. The fungal inoculum was uniformly spread

using sterile cotton swab on the sterile Petri dish SDA in zigzag manner three times beside a

lighted alcohol lamp inside the laminar flow. In vitro antifungal activity test was carried out by

disk diffusion method. After the plates were swabbed with the fungus, the discs were placed on

the agar on the designated plates.

After all the treatments were dispensed, the controls and the Petri plates were placed

inside the incubator for 18 hours at 35 degrees Celsius temperature. After an overnight

incubation, the Petri plates were inspected for structure of zones of inhibition around the filter

paper discs.

Analysis of Data

The data were subjected to One-way Analysis of Variance (ANOVA) at 0.05 level of

significance. One way ANOVA was used to determine if there is a significant difference on three

different concentrations of Lemongrass leaves extract against Candida albicans.

Disposal

The used gel-like agars were scraped from the Petri dishes and disposed in autoclave

plastics (Scoville, 2012). All glasswares were autoclaved and the workplace were cleaned with

Lysol solution and were sterilized by UV light for 30 minutes.

7
 RESULTS AND DISCUSSIONS

Results

Table 1. Sensitivity Test Results

Agent Plate 1 Plate 2 Plate 3 Average

100% Lemongrass Leaves
Extract 16 mm 15 mm 17mm 16 mm
95% Lemongrass Leaves
Extract 16 mm 15 mm 18 mm 16.33 mm
90% Lemongrass Leaves
Extract 15 mm 15 mm 15 mm 15 mm
Nystatin 18 mm 17 mm 18 mm 17.67 mm

Table 1 shows the measurement of the zone of inhibition of the three treatments (100%,

95%, and 90% respectively) in three plates, given Nystatin for the positive control and

lemongrass leaves extract for the experimental.

Table 2A. Statistical analysis of the zone of inhibition (in mm) obtained between and among
lemongrass leaves extract concentrations
95% Confidence
Std. Std. Interval for Mean
N Mean Minimum Maximum
Deviation Error Lower Upper
Bound Bound
100% lemongrass
3 16.0000 1.00000 .57735 13.5159 18.4841 15.00 17.00
extract
95% lemongrass
3 16.3333 1.52753 .88192 12.5388 20.1279 15.00 18.00
extract
90% lemongrass
3 15.3333 .57735 .33333 13.8991 16.7676 15.00 16.00
extract
Nystatin 3 17.6667 .57735 .33333 16.2324 19.1009 17.00 18.00
Total 12 16.3333 1.23091 .35533 15.5512 17.1154 15.00 18.00

8
 Table 2B. Analysis of Variance for zone of inhibition (mm)
between and among lemongrass leaves extract concentrations

Sum of Mean
Squares Df Square F Sig.
Between 1.556 2 .778 .636 .562
Groups
Within Groups 7.333 6 1.222
Total 8.889 8

Table 2A and 2B above show the statistical analysis using One-Way ANOVA that was

conducted to compare the zones of inhibition of lemongrass leaves extract against Candida

albicans in 100%, 95% and 90% concentrations respectively. Using 95% level of confidence, if

p<0.05, then there is a significant difference between variables. However, results showed that

p>0.05 [F(2, 6)=0.636, p=0.562]. Therefore, it can be assumed that there is no significant

difference between the zones of inhibition against Candida albicans of the three levels of

concentration of lemongrass leaves extract.

Table 3A. Statistical analysis of the zone of inhibition (in mm) obtained between lemongrass
leaves extract and Nystatin

95% Confidence
Std. Std. Interval for Mean
N Mean Minimum Maximum
Deviation Error Lower Upper
Bound Bound
100% lemongrass
3 16.0000 1.00000 .57735 13.5159 18.4841 15.00 17.00
extract
95% lemongrass
3 16.3333 1.52753 .88192 12.5388 20.1279 15.00 18.00
extract
90% lemongrass
3 15.3333 .57735 .33333 13.8991 16.7676 15.00 16.00
extract
Nystatin 3 17.6667 .57735 .33333 16.2324 19.1009 17.00 18.00
Total 12 16.3333 1.23091 .35533 15.5512 17.1154 15.00 18.00

9
 Table 3B. Analysis of Variance of zone of inhibition obtained between
lemongrass leaves extract and Nystatin

Sum of Mean
df F Sig.
Squares Square
Between Groups 8.667 3 2.889 2.889 .102
Within Groups 8.000 8 1.000
Total 16.667 11

Table 3A and 3B show the statistical analysis using One-way ANOVA that was

conducted to compare the zones of inhibition of lemongrass leaves against Candida albicans in

100%, 95% and 90% concentrations and Nystatin. Using 95% level of confidence, if p<0.05,

then there is a significant difference between variables. However, results showed that

p>0.05[F(3, 8)=2.889, p=0.102].Therefore it can be assumed that there is no significant

difference between the antifungal activity of three levels of concentration of lemongrass extract

and Nystatin against Candida albicans,

10
Discussions

The antifungal activity of lemongrass leaves extract was tested against the Candida

albicans. Lemongrass leaves contains borneol, estragole, methyleugenol, geranyl acetate,

geraniol, beta-myrcene, limonene, piperitone, citronellal, carene-2, alpha-terpineole, pinene,

farnesol, proximadiol, (+)-cymbodiacetal and its main component is citral which has a chemical

structure of C10 H16 O.

In a similar study conducted by de Bona da Silva and colleagues (2008), where he tested

the antifungal activity of the commercially obtained lemongrass oil and citral supplied by Sigma-

Aldrich from Germany against Candida species. Results showed the efficacy of lemongrass oil

and citral against 8 different species of Candida. On the other hand, in this study, lemongrass

leaves was extracted through the hydro-steam distiller. These grasses were planted and grown

following the standards set by the DOST to ensure quality of the plant.

According to a site called PubChem, Nystatin is a polyene antifungal drug to which

many molds and yeasts are sensitive, including Candida spp. Nystatin has some toxicity

associated with it when given intravenously, but it is not absorbed across intact skin or mucous

membranes. It is considered a relatively safe drug for treating oral or gastrointestinal fungal

infections having a molecular formula of C47H75NO17.

According to a site called ScopeMed, similarly lemongrass leaves extract has citral that

acts as a fungicidal agent because it is able to form a charge transfer complex with an electron

donor of fungal cells, resulting in fungal death.

11
 CONCLUSIONS AND RECOMMENDATIONS

Conclusions

The statistical analysis using One-Way ANOVA shows that there is no significant

difference between the zone of inhibition of Cymbopogon citratus leaves extract at100%, 95%, and 90%

concentrations respectively and Nystatin against C. albicans.

With this, the researcher concludes that lemongrass leaves extract is an effective

alternative for the commercial drug Nystatin against Candida albicans.

Recommendations

Based on the results, the researcher would like to recommend the future researchers to:

1. Find another plant that can be potentially used as antifungal against Candida albicans.

2. Test the antibacterial and antifungal (other fungi species) potentials of Cymbopogon

citratus leaves extract.

3. Test the water content (hydrosol) of the lemongrass after distillation.

4. Future researchers may research more on antifungal and antibacterial potentials of other

lemongrass species.

5. Lemongrass leaves extract may be mixed with carrier oil for safe application on skin in

case skin is sensitive

12
 ACKNOWLEDGEMENT

The study served as a great achievement. Along with this one had gained and

realized a lot of things from this experience. The study tested one’s patience, determination and

faith. It is a product of sleepless nights, struggles, and hard work. The researcher would like to

express her heartfelt gratitude to the people who helped her with the study, to the ones who gave

their time and effort and also to the ones who cheered her up during those difficult times.

First, the researcher would like to thank the Almighty God for the knowledge, strength,

patience and faith. For the guidance and protection in times of travel. Without his help this study

would not be a success.

Second, to her ever supportive parents. Thank you for the support and effort. For

6providing her needs especially financially. In times of fears and struggles they were there to

comfort, give advices, and cheer her up. The researcher deeply thank them for their love and

care.

To Ms. Ma. Theresa L. Lopez, consultant, for sharing her knowledge and wisdom to

improve the study; to Mr. Nelson C. Cabalo for the assistance in the extraction of Lemongrass

leaves. The researcher would also like to thank Ms. Kalvin Joy V. Bauno and her family for

sharing her knowledge and for her help with the errands in research; to her teachers for being

considerate during the times that she was not able to attend some of their classes due to various

research appointments. Also, the researcher would like to extend her thanks to Ms. Sheena

Balinsoy for her help with the results of the study, for making the statistical analysis and

interpretation of results. Also to Ms. Verena and Vernalin Contiga for sharing their knowledge

and extending their help for the Research Plan.

13
 Lastly, the researcher would like to express her gratitude to her Research Adviser, Ms.

Madonna B. Decena. Thank for the guidance and support during the whole process. Thank you

for being considerate and understanding. This study will not be accomplished without her

encouragement and knowledge that she had shared. To the Principal, Dr. Michael G.

Divinagracia, thank you for the understanding, for being supportive in all research studies, and

for the encouragement to do well in research.

14
 BIBLIOGRAPHY

Thesis/Dissertation

Binga-an, P.B (2015). Antibacterial Activity of Carabao Grass (Paspalum conjugatum

P.J.Bergius) on Escherechia coli(Unpublished Science Investigatory Project). Negros

Occidental National Science High School, Victorias City

Journal Articles

Lopes, G., Pinto, E., Andrade, P., and Valentao, P. (2013) Antifungal Activity of Phlorotannins

against Dermatophytes and Yeasts: Approaches to the Mechanism of Action and

Influence on Candida albicans Virulence Factor

http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0072203

Ramage, G., Jose, A., Sherry, L., Lappin, D., Jones, B., and Williams, C. (2013)Liposomal

Amphotericin B Displays Rapid Dose-Dependent Activity against Candida albicans

Biofilms, 57(5): 2369–2371.

Silva Cde B, Guterres SS, Weisheimer V, Schapoval EE. (2008) Antifungal activity of the

lemongrass oil and citral against Candida spp., 12(1):63-6.

Yousef, A., (2013) Antifungal Activity of Volatiles from Lemongrass (Cymbopogon citratus)and

Peppermint (Mentha piperita) Oils Against Some Respiratory Pathogenic Species of

Aspergillus, (2013)2(6): 261-272

15
Web Pages

HealthCentral (n.d.). Candida Albicans - HealthCentral Encyclopedia. Retrieved July 2, 2016 from

http://www.healthcentral.com/encyclopedia/hc/candida-albicans-3168573/

Richards, L. (2013). The Candida Diet. Retrieved July 2, 2016 from

https://www.thecandidadiet.com/what-is-candida-albicans/

Medical Health Guide. (n.d.) Lemongrass Herbal Medicine. Retrieved July 23, 2016 from

http://www.medicalhealthguide.com/herb/lemongrass.htm#a

Plant Village. (n.d.) Lemongrass Cymbopogon citratus. Retrieved July 23, 2016

https://www.plantvillage.org/en/topics/lemongrass/diseases_and_pests_description_uses_

propagationv

Sigma-Aldrich. (n.d.). Lemongrass Cymbopogon. Retrieved July 23, 2016 from

http://www.sigmaaldrich.com/life-science/nutrition-research/learning-center/plant-

profiler/cymbopogon.html

16
 APPENDIX

Swabbing of Fungus Materials to be used

Plates applied with Treatments Incubation of Plates

Hydro steam Distiller

17
 Lemongrass leaves Lemongrass leaves in hydro steam distiller

Filtering of Lemongrass
Lemongrass leaves extract
leaves extract

Zone of Inhibitions

18
 “Antifungal Activity of Lemongrass (Cymbopogon citratus) Leaves Extract against Candida

albicans”

Research Plan

Proponents: Alesca Fair L. Malicay

A. Question or problem being addressed

Candidiasis is a common healthcare-associated infection. It is a fungal infection caused

by yeasts from the genus Candida. Specifically, Candida albicans is the predominant cause of the

disease. Lemongrass, Cymbopogon citratus, is a perennial grass in the family Poaceae grown for

its fragrant leaves and stalks which are used as a flavoring. Lemon grass grows to as high as 1

meter. With the increasing demand for the antifungal remedies, finding alternative and

more practical antifungals using Lemongrass (Cymbopogon citratus) leaves can provide

accessible and cheaper remedies to the ones available in the market

B. Goals/ Expected Outcomes/ Hypotheses

This study aims to determine and evaluate the antifungal activity of Lemongrass

(Cymbopogon citratus) leaves extract against Candida albicans.

C. 1 Procedures

Collection

 Lemongrass leaves will be collected from Eco-Agri, Brgy. Pahanocoy, Bacolod

City.

 Lemongrass leaves will be cut by 1 – 1.5 inches.

 Candida albicans will be obtained from Riverside Hospital, Bacolod City.

19
Extraction of Lemongrass

 Lemongrass leaves will be extracted in Eco-Agri, Brgy. Pahanocoy, Bacolod City.

 Lemongrass leaves will be extracted through the hydro steam distiller

 The lemongrass leaves extract will be measured through the graduated cylinder to

prepare the concentrations.

Preparation of Treatments

 The extract samples will be diluted in distilled water to obtain the following

treatments:

Treatment 1 - 100% Cymbopogon citratus leaves compared to a positive control

Nystatin. Treatment 2 – 95% Cymbopogon citratus leaves compared to a positive

control Nystatin. Treatment 3 – 90%Cymbopogon citratus leaves compared to a

positive control Nystatin.

Preparation of Culture Medium

 The microbiological laboratory will be prepared by the in charge and all needed

materials will be provided by the institution.

 Seeded agar plates will be prepared by pouring 20 mL of SDA into each sterile

plate.

 The melted agar will be allowed to cool at 60 degrees Celsius before dispensing

into plates.

20
Preparation of the Fungal Inocula

 The cultures of Candida albicans will be cultivated on Sabouraud Dextrose of 4%

Agar (SDA) at 25 degrees Celsius for 48 hours.

 Antifungal activity will be tested using Lemongrass leaves extract.

 Sensitivity test will be achieved

 The antifungal activity will be studied by agar disc diffusion method.

 Seeded agar plates will be prepared by pouring 20 mL of SDA into each sterile

plate.

 After solidification of medium, each plate will be overlaid with 5ml of SDA.

 Lemongrass leaves extract will be applied on filter paper disks (20.0 µL/disk) of 6

mm in diameter.

 Nystatin solution (0.3 mg/mL) will be used as antifungal reference substance

(20.0µL/disk).

 The disks will be placed on the surface of seeded agar plates (one disk for plate).

 All plates will be incubated at 35 degrees Celsius ±2 degrees Celsius for 24 hours.

Microbiological Assay

 The microbiological assay will be conducted in Villanueva Clinical Laboratory.

 The alcohol lamp will be lighted to keep the sterility of the area.

 The fungal inoculum will be uniformly spread using sterile cotton swab on the

sterile Petri dish SDA in zigzag manner three times beside a lighted alcohol lamp

inside the laminar flow.

 In vitro antifungal activity test will be carried out by disk diffusion method.

21
  After all the plates will be swabbed with the fungus, the discs will be placed on

the agar on the designated plates.

 After all the treatments will be dispensed the controls and the Petri plates will be

placed inside the incubator for 18 hours at 35 degrees Celsius temperature.

 After an overnight incubation, the Petri plates will be inspected for structure of

zones of inhibition around the filter paper discs.

Disposal

 The used gel-liked agars will be scraped from the Petri dishes and disposed in

autoclave plastics (Scoville,H, 2012).

 All glass wares will be autoclaved and the workplace will be cleaned with Lysol

solution and will be sterilized by UV light for 30 minutes.

Data and Data Gathering Procedures

 Results will be based on the diameter of the zone of inhibitions of test treatments

and the controls. Antifungal Index will be determined to interpret the extract’s

activities.

 By comparing the areas of zone of inhibition of test with standard concentration

and potency of test samples will be determined. (HubPages, 2013)

 Inhibition of the fungal growth will be measured in millimeters.

22
Diameter of Zone of Inhibition

 The diameter of the zones will be measured by means of a ruler and the average

data will be taken from the data in the x and y axis of the zone.

 The data will be plotted in tabulated form and treated statistically to determine the

antifungal activity of Lemongrass leaves extract against Candida albicans.

Statistical Data Analysis Procedure

 The data obtained from the study will be subjected to the following descriptive

and inferential statistical treatments using the Statistical Package for Social

Sciences (SPSS) Software.

The statistical tools will be used in this study are:

Mean – The mean will be used to determine the average scores of the results of

the treatments in this study.

Standard deviation – To determine the dispersion between the mean.

ANOVA will be used to determine the difference between two or more means set

at 0.05 level of significance.

Duncan’s Multiple Range Test (DMRT) – To test the significance of the F-ratio

obtained in the study.

23
D. Bibliography

The Telegraph (n.d.) Retrieved April 20, 2016

htttp://www.telegraph.co.uk/lifestyle/wellbeing/healthadvice/4711185/Lemongras

s-and-tea-treeoil-are-powerful-solutions.html

Silva Cde B, Guterres SS, Weisheimer V, Schapoval EE. (2008) Antifungal activity of

lemongrass oil and citral against Candida spp., 12(1):63-6.

Richards, L. (2013). The Candida Diet. Retrieved April 20, 2016

http://www.thecandidadiet.com/what-is-candida-albicans

HubPages, (2013) Microbial ‫ ׀‬Microbiological assay: Its Definition Uses and methods.

Retrieved April 20, 2016

http://bheem.hubpages.com/hub.Microbial-Microbiological-assay-Its-Definition-

Uses-and-methods

Scoville,H. (2012) How to Dispose of Agar Plates. Retrieved April 20, 2016

http://www.ehow.com/how_7442542_dispose-agar-plates.html

24