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INTRODUCTION
According to the Inquirer Washington Bureau (2012), they have predicted that major
health problems will come from the widespread occurrence of chronic infectious diseases that
will greatly affect the elderly and those with low immunity. These infectious diseases lead to
increased death rate all over the world based from the latest report (USA Today News, 2011).
Diseases like whooping cough, bubonic plague and tuberculosis caused by bacteria are some
specific reasons of this death rate (Mann,2009). Broad spectrum antibiotic are used as an
effective drug against a wide range of infectious microorganisms which includes both Gram
positive and Gram negative bacteria (SRS Pharmaceuticals PVT. LTD, 2010). But despite of this
augmentation, the World Health Organization reported that bacteria are becoming resistant to
these antibiotics (Heyes, 2013). This is because antibiotics has been over-prescribed and are used
for a long period of time (Natural News, 2013).
On the other hand, terrestrial plant community is best described by its composition and
dynamics. Plants belonging to terrestrial community indicates their potential role in supplying
different nutritional values and medicinal uses (Shurin et al, 2005) due to the presence of
secondary metabolites with broad biological activities (Elleuch et al., 2009).
Anacardium occidentale, commonly known as Cashew contain anacardic acid found not
only in the cashew pericarp oil but also in the nut and the fruit juice possessing antioxidant
activities (Kubo et al., 2005). The liquid gained usually distinguish as Cashew Nuts Shell Liquid
(CNSL) has a broad application useful in both medical purposes and development of drugs,
antioxidants, fungicide, and other chemicals (Andrade et al, 2011). The extracts were evaluated
to exhibit strong antimicrobial properties against gram-positive bacteria (Himejima et al, 1991).

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In this study, the broad-spectrum antibacterial properties of Anacardium occidentale
leaves and pericarp methanolic crude extracts were evaluated against human pathogens
(Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and
Bacillus cereus). This leads to the isolation, identification and elucidation of potent
allelochemicals for drug development.

MATERIALS AND METHODS

Collection of Cashew Leaves and Pericarp
Anacardium occidentale leaves and pericarp were collected from Cashew plantation area
in Guimaras last May 2013. Collected samples were washed thoroughly and air dried at room
temperature for two days.
Extraction of Anacardium occidentale
Anacardium occidentale leaves and pericarp were then separately soaked in methanol for
two days. Samples were 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 were weighed and utilized to prepare the concentrations.
Preparation of Treatments
The ---g of solid paste sample from the leaves and the pericarp were diluted in dimethyl
sulfoxide (DMSO) to avail the following treatments: Treatment 1- 400 mg/mL Anacardium
occidentale leaves and Treatment 2 400 mg/mL Anacardium occidentale leaves compared to a
positive control (tetracycline) and negative control (DMSO).

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Preparation of Culture Medium
The microbiological laboratory was prepared by the in charge and all needed materials
were provided by the institution. Approximately 38 grams of Mueller-Hinton Agar was diluted in
300mL of distilled water in Erlenmeyer flask and autoclaved for 15 minutes at 1210C to avail the
ideal amount of medium dispensed in 15 petri dishes. The melted agar was 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) were provided by the University of the
Philippines in the Visayas, Miag-ao, Iloilo Culture Collection. Following a concentration of 13
g/L of nutrient broth was prepared and dispensed in 5 test tubes. Aseptically, a loopful of bacteria
from the culture slant was diluted in the designated nutrient broth and slowly swirled to mix. The
McFarland standard per culture was achieved by comparing the turbidity of the inocula with
Barium sulfate against black line background. If the turbidity is cloudy than the standard,
nutrient broth were added. The bacterial inocula were separately and 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 hood to maintain sterility.

Microbiological Assay
DMSO (-)

Tetracycline
(+)
Figure 1. Designation of treatments in the plates.

Cashew leaves extract

Cashew pericarp
extract

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After all the plates were swabbed with the bacteria, the discs were put on the agar on the
designated points. The agar plugs were removed and sterilized in Lysol solution.
In vitro antibacterial activity test was carried out by disk diffusion method. To test the
antibacterial activity, Anacardium occidentale leaves and nut shell crude extracts using various
solvent were dispensed in disc at 10L in a clockwise manner. After all the treatments were
dispensed the controls, the Petri plates were placed inside the incubator for 18 hours at 350C
temperature. Results were based on the diameter of the zone of inhibitions of test treatments and
the controls. Antimicrobial Index was determined to interpret the extracts activities. By
comparing the areas of zone of inhibition of test with standard the concentration and potency of
test samples are determined (HubPages, 2013). Inhibition of the bacterial growth was measured
in millimeters.
Data and Data Gathering Procedures
After an overnight incubation, the Petri plates were inspected for structure of zones of
inhibition around the filter paper discs.
Diameter of Zone of inhibition
The diameter of the zones were measured by means of a ruler and the average data was
taken from the data in the x and y axis of the zone. The data was plotted in tabulated form and
treated statistically to determine the antimicrobial Allelochemicals present in Anacardium
occidentale leaves and nut shell against five bacteria namely, E. coli, Staphyloccocus aureus,
Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus cereus.
Statistical Data Analysis Procedure
The data obtained from the study were subjected to the following descriptive and
inferential statistical treatments using the Statistical Package for Social Sciences (SPSS)

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Software. The statistical tools used in this study were: Mean- The mean was used to express the
average diameter of the zone of inhibition developed in each treatment in this study. Standard
deviation- To determine the dispersion between the mean. ANOVA- used to determine the
significant difference of three or more groups means set at 0.05 level of significance. Duncans
Multiple Range Test (DMRT) compare significant differences of means of various groups or
treatments in the study.
Plant identification
Collected plant samples of cashew leaves and pericarp were brought to the University of
the Philippines, Miag-ao, Iloilo for plant identification.

RESULTS
The diameter of the clear zone of inhibition around the impregnated disk dispensed on the
surface of the agar indicates antibacterial activities of the extracts being tested. These results are
compared to the two standards; one is the positive control tetracycline a known antibiotic and a
negative control, dimethyl sulfoxide: the diluent used to prepare the various concentrations. The
quantitative data is expressed in millimeter and further analyze of its the potency by determining
the Antimicrobial Index (AI). The closer the value of AI to 1 the more potent is the sample. This

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is the basis for further bio-directed antibacterial assay for the detection of secondary metabolites
as antibiotic.
The antibacterial activities of the samples against E. coli showed that the cashew leaves
and the pericarp methanol crude extracts exhibited resistant activities comparable with the
DMSO. Tetracycline being a known antibacterial agent exhibited 16.67 mm and 1.78 AI..
One way analysis of variance and Duncans test revealed that there is a significant
difference in the mean zone of inhibition of Tetracycline compared to the extracts and negative
control against E. coli having a p- value of .000<0.05 level of significance.
The antibacterial activities of the samples against Pseudomonas aeruginosa showed that the
cashew pericarp methanol crude extract exhibited the highest antibacterial activities having a
mean of 33.00 mm and AI of 4.50 followed by Tetracycline having a mean of 20.67 mm and
negative activity exhibited by the cashew leaves and the negative control-DMSO.
One way analysis of variance and Duncans test revealed that there is a significant
difference in the mean zone of inhibition exhibited by Cashew pericarp extract against
Pseudomonas aeruginosa having a p- value of .000<0.05 level of significance.
The antibacterial activities of the samples against Staphylococcus aureus showed that the
Cashew pericarp methanol crude extract revealed the highest antibacterial activity having a mean
of 30.00 mm followed by Tetracycline having a mean of 19.67 mm while negative activity
exhibited by the cashew leaves and the negative control DMSO.
One-way analysis of variance and Duncans test revealed significant difference in the
mean zone of inhibition of Cashew pericarp extract against Staphylococcus aureus having a pvalue of .000<0.05 level of significance.

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The antibacterial activities of the samples against Bacillus cereus showed that the
Cashew pericarp methanol crude extract revealed the highest antibacterial activity having a mean
of 43.33 mm followed by Tetracycline having a mean of 25.00 mm while negative activity
exhibited by the Cashew leaves and DMSO.
One-way analysis of variance and Duncans test revealed significant difference in the
mean zone of inhibitions against Bacillus cereus having a p- value of .000<0.05 level of
significance.
The antibacterial activities of the samples against Bacillus subtilis showed that the
Cashew pericarp methanol crude extract revealed the highest antibacterial activities having a
mean of 46.00 mm followed by Tetracycline having a mean of 39.00 mm while negative activity
exhibited by the Cashew leaves and DMSO.
One-way analysis of variance and Duncans test revealed significant difference in the
mean zone of inhibitions exhibited by the cashew pericarp against Bacillus subtilis having a pvalue of .000<0.05 level of significance.

Samples

Samples Clear Zone Diameter

(mm)
1

Cashew Leaves
Escherichia coli

Cashew pericarp

DMSO (-)
Tetracyline (+)

0
0
0
17

0
0
0
17

0
0
0
16

Mean*
0b
0b
0b
16.67a

0
35
0
20

0
34
0
17

0
30
0
25

0c
33.00a
0c
20.67b

SD

AI**

0
0
0

0
0
0
1.78

0
4.50
0
2.45

p value = .000

Cashew Leaves
Pseudomonas
aeruginosa

Cashew pericarp

DMSO (-)
Tetracyline (+)

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p value = .000

Cashew Leaves
Staphylococcus
aureus

Cashew pericarp

DMSO (-)
Tetracyline (+)

0
30
0
31

0
25
0
15

0
35
0
13

0c
30.00a
0c
19.67b

0
50
0
20

0
50
0
35

0
30
0
20

0c
43.33a
0c
25.00b

0
45
0
35

0
35
0
37

0
58
0
45

0c
46.00a
0c
39.00b

0
4.00
0
2.28

p value = .000

Cashew Leaves
Cashew pericarp

Bacillus cereus

DMSO (-)
Tetracyline (+)

0
6.22
0
3.17

p value = .000

Cashew Leaves
Bacillus subtilis
Table 1

Cashew pericarp

DMSO (-)
Tetracyline (+)

0
6.67
0
5.50

p value = .000

Antibacterial activities of Cashew leaves and shell extracts on selected human pathogens
compared to the control and its antimicrobial index (AI)
* Column means with common letter superscripts per bacterium are not significantly different at 0.05 level
of significance by One-way ANOVA and Duncans test.
**Antimicrobial Index = 1 or > than

is potent antimicrobial agent

Figure 2. Revealed clear zones of inhibition around the impregnated disk of the samples used:
(A) E. coli, (B) P. aeruginosa, (C) S. aureus, (D) B. cereus and (E) B. subtilis.

DISCUSSION
The zones of inhibitions were observed around the discs, this indicated antibacterial
activities of the plant leaves and pericarp extracts. The sensitivity of the different test organisms
to methanolic extracts of the leaves and pericarp of Anacardium occidentale was shown by zone
of inhibitions after 18 hours of incubation(As illustrated in Figure 1).
The absence of zone of inhibitions around each discs signified resistance. It was observed
that the crude extracts of Anacardium occidentale leaves did not inhibit the growth of any of the
test bacteria. While the Anacardium occidentale pericarp extract exhibited potent or strong
antibacterial properties against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus,
Bacillus cereus, and Bacillus subtilis higher than the positive control tetracycline. This
indicates that the Cashew pericarpl is rich in allelochemicals or secondary metabolites that
exhibited the antibacterial properties.

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According to Nandi (2013), the cashew shell contains reddish brown oil and high zinc
concentration. This concurs with the work of Kannan et al. (2009) showing that it has various
range of biological properties and distinct range of phytochemicals present. Contrastingly to the
study of Agedah et al. (Bioline, 2010), the leaves extract exhibited antibacterial activities against
E. coli and S. aureus.
The antibacterial assay of Cashew pericarp and leaves provide strong indication that the
pericarp contains allelochemicals with broad-spectrum antibacterial activities. This result also
encourages the isolation, characterization and elucidation of allelochemicals with potent
antibacterial activities. Considering that today a lot of diseases are attributed to the prevalence of
infections caused by these range of human pathogens. The formulation and production of end
products for wide array of medicinal, medical and industrial uses from the Cashew pericarp is
also expected from this providing baseline information for in-depth research.

CONCLUSIONS
The cashew pericarp methanolic crude extract contain allelochemicals with potent
antibacterial properties against Gram positive and Gram negative human pathogens; S. aureus, P.
aeruginosa, B. cereus and B. subtilis except E. coli. On the other hand, the cashew leaves
methanolic crude extract exhibited negative antibacterial properties against all tested human
pathogens. Results lead to formulation of several products with wide array of medicinal uses and
identification of the potent allelochemicals.

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References Cited
Andrade, T. , Arajo, B., Cit, A. M. , Silva, J., Richter M. F. , Ferraz, A. (2011)
Antioxidant properties and chemical composition of technical Cashew Nut Shell
Liquid (tCNSL). Retrieved last September 7, 2013 from
http://academic.research.microsoft.com/Publication/49339824/antioxidantproperties-and-chemical-composition-of-technical-cashew-nut-shell-liquid-tcnsl
Elleuch, L. , Shaaban, M. , Smaoui, S. , Mellouli, L. , Rebai, I. K., Ben Fguira, L. F. , Shaaban,
K. A. , and Laatsch, H. ( 2009) Bioactive Secondary Metabolites from a New Terrestrial
Streptomyces sp. TN262. Retrieved last September 7, 2013 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128704/
Heyes , J. D., (2013) The end of Modern Medicine has arrived, according to
the WHO
Retrieved April 11, 2013 from
http://www.naturalnews.com/035890_antibiotics_resistance_WHO.html

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Himejima M. and Kubo I. (1991) Antibacterial Agents from the Cashew Anacardium
occidentale (Anacardiaceae) Nut Shell Oil. Retrieved last September 7, 2013 from
http://www.aseanbiotechnology.info/abstract/21000029.pdf
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-Usesand-methods
Kubo, Masuoka, Ha, Tsujimoto , (2005) Antioxidant activity of Anacardic acids. Retrieved last
April 11, 2013
http://www.agronavigator.cz/userfiles/File/Agronavigator/Kvasnickova/anacardic
%20acids.pdf

Mann, D., (2009) Study: Showerheads may deliver blast of bacteria. Retrieved last
September 7, 2013 from

http://edition.cnn.com/2009/HEALTH/09/14/showerhead.bacteria/index.
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-deathreport_N.htm
Shurin, J. B., Gruner G. S., and Hillebrand H. (2005) All wet or dried up? Real
differences between aquatic and terrestrial food webs. Retrieved last
September 7, 2013 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1560001/

ACKNOWLEDGEMENT
Utmost gratitude is extended to the following persons:

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Mrs. Gloria Carmencita S. Gimoto, Mr. Dione H. Gumban and Mrs. Rosene M. Gumban,
parents of the researchers.
Mr. Leandro Gamarcha and Mrs. Ma. Theresa Romey of the College of Arts and
Sciences, UPV- Miag-ao Campus.
Mrs.Rose Ebangcol of Sibunag, Guimaras for providing the samples.
Mr. Ronilo V. Aponte and Mrs. Branche A. Sumido, Research Advisers
Ms. Grace Mamon and Mr. Neil John Joquio, the researchers grammarian and
statistician. Mrs. Toni Dinah Cheer Fernandez, Department Head of School of the Future (SOF),
Iloilo National High School. Mr. Edwin F. Mosura, Research Head- Research Department, Iloilo
National High School. Dr. Nordy D. Siason Jr. Principal IV- Iloilo National High School
Most of all, to the Almighty God for the strength and wisdom provided to the researchers.