Broad-spectrum Antimicrobial Allelochemicals from Cashew

(Anacardium occidentale) Leaves and Pericarp

Methanolic Crude Extracts

Research Plan
A. Question or Problem being addressed
Chronic infectious diseases experienced by immunosuppressed patients and aged
individuals (Inquirer Washington Bureau, 2012), leading to higher death rate all over the world
with 16.2% (USA TODAY, 2011). Regardless of the efficacy of the known antibiotics, bacteria
are becoming resistant to these antibiotics (Heyes, 2013).
B. Goals/ Expected Outcomes/ Hypotheses
This study aims to determine and evaluate the effectiveness of antimicrobial
allelochemicals in Cashew (Anacardium occidentale) leaves and pericarp against human
pathogens: Escherchia coli, Staphyloccocus aureus,, Pseudomonas aeruginosa, Bacillus cereus,
and Bacillus subtilis.
C.1 Procedures
Collection

Anacardium occidentale leaves and pericarp will be collected from Cashew plantation area in
Guimaras Island, Philippines.

The samples will be washed thoroughly with running water and will be air dried.

Extraction of Anacardium occidentale

Anacardium occidentale leaves and pericarp will be separately soaked in methanol for two days.

Samples will be brought to the University of the Philippines Visayas, Miagao Campus,
Analytical Service Laboratory for in vacuo extraction at 60 0C, 90 rpm for 3 hours.

The solid paste samples will be weighed and utilized to prepare the concentrations.

Preparation of Treatments

The solid paste samples will be diluted in dimethyl sulfoxide (DMSO) to avail the following
treatment: Treatment 1- 400 mg/mL Anacardium occidentale leaves
Treatment 2 - 400 mg/mL Anacardium occidentale pericarp
compared to a positive control (tetracycline) and negative control (DMSO).

Gimoto, Carrol Margareth S. , Gumban, Jan Philip M.

Preparation of Culture Medium

The microbiological laboratory will be prepared by the in charge and all needed materials will be
provided by the institution.

Approximately 38 grams of Mueller-Hinton Agar will be diluted in 300mL of distilled water in

Erlenmeyer flask and will be autoclaved for 15 minutes at 1210C to avail the ideal amount of
medium dispensed in 15 Petri dishes.

The melted agar will be allowed to cool at 600C before dispensing unto the plates.

Preparation of the Bacterial Inocula

Culture slant of microbes ( Escherichia coli, Staphylococcus aureus, Pseudomonas

aeruginosa, Bacillus subtilis and Bacillus cereus ) will be prepared by University of the
Philippines in the Visayas Miag-ao, Iloilo Culture Collection.

Follow a concentration of 13 g/L of nutrient broth

Prepare and dispense the nutrient broth concentration in 5 test tubes.

A loopful of bacteria from the culture slant will be diluted in the designated nutrient broth .

Slowly swirl it to mix.

To test the antibacterial activity, Anacardium occidentale leaves and pericarp crude
extract will be used.

Achieve the McFarland standard per culture by comparing the turbidity of the inocula with
Barium sulfate against black line background.

The antibacterial activity will be studied by agar disc diffusion method inside a sterilized
laminar hood.

Establish the 0.05 McFarland Standard for the bacterial density.

Gimoto, Carrol Margareth S. , Gumban, Jan Philip M.

Microbiological Assay

The Microbiological assay will be conducted in sterile laminar hood of University of the
Philippines Visayas Microbiology Laboratory.

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

The bacterial inoculum will uniformly spread using sterile cotton swab on a sterile Petri dish
Mueller-Hinton agar in a zigzag manner three times beside a lighted alcohol lamp inside the
laminar flow.

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

After all the plates will be swabbed with the bacteria, the discs will be put on the agar on the
designated points.

To test the antibacterial activity, Anacardium occidentale leaves and nut shell crude extracts
using various solvent will be dispensed in disc at 10L in a clockwise manner.

After all the treatments will be dispensed the controls, the Petri plates will be placed inside the
incubator for 18 hours at 350C 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
autoclavable plastics ( Scoville,H, 2012).
All glasswares will be autoclaved and the workplace will be cleaned with Lysol solution
and will be sterilized by UV light for 30 menutes.

Data and Data Gathering Procedures

Results will be based on the diameter of the zone of inhibitions of test treatments and the controls.
Antimicrobial Index will be determined to interpret the extracts 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 bacterial growth will be measured in millimeters.

Gimoto, Carrol Margareth S. , Gumban, Jan Philip M.

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 antimicrobial
Allelochemicals present in Anacardium occidentale leaves and pericarp against five bacteria (E.
coli, Staphyloccocus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus cereus).

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.
Duncans Multiple Range Test (DMRT)- To test the significance of the F-ratio obtained in the
study.

Gimoto, Carrol Margareth S. , Gumban, Jan Philip M.

D. Bibliography

Heyes , J. D., (2013) Natural News. com T.he end of Modern Medicine has arrived, according to the WHO
Retrieved last April 11, 2013
http://www.naturalnews.com/035890_antibiotics_resistance_WHO.html
HubPages, (2013) Microbial | Microbiological assay: Its Definition Uses and methods. Retrieved last
April 11, 2013
http://bheem.hubpages.com/hub/Microbial-Microbiological-assay-Its-Definition-Uses- andmethods
Quinto , E. A., Santos, M. G. (2005)A Guidebook to Plant Screening:Phytochemical and Biological
Retrieved last April 11, 2013
Scoville,H. (2012) How to Dispose of Agar Plates. Retrieved last April 11, 2013
http://www.ehow.com/how_7442542_dispose-agar-plates.html
USA TODAY, (2011 ) WHO's top killers: Heart problems, infectious diseases, cancer. Retrieved last April 11, 2013
http://usatoday30.usatoday.com/news/health/2008-10-27-who-death-report_N.htm

Gimoto, Carrol Margareth S. , Gumban, Jan Philip M.