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D

Parasiticidal Effect of Atis (Annona squamosa L.) Seed Crude Extracts against

Dog Fleas (Ctenocephalides canis)

A Community-Based Science Investigation

Researchers:

Adviser:

Iloilo National High School

May 12, 2012

Table of Contents

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Abstract 5

Introduction 6

Background of the Study 6

Statement of the Objectives 6

Significance of the Study 7

Scope and Limitations of the Study 7

Review of Related Literature 8

Methodology 9

Results and Discussion 14

Conclusions 15

Recommendations 15

Bibliography 16

Definition of Terms 16

Appendices 17

Acknowledgment 18

Lists of Tables

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Table 1. Experimental Design 9

Table 2. Mortality of Dog fleas exposed to various concentrations of Atis seed

crude extract

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Lists of Figures

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Figure 1. Procedural Design 10

Figure 2. Air drying of seeds 11

Figure 3. Grinding of seeds

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Figure 4. Soaking of seeds in ethanol solvent 11

Figure 5. Rotary Evaporation 12

Figure 6. Preparation of Materials 12

Figure 7. Applying of Extract 12

Researcher:School:Title: Parasiticidal Effect of Atis (Annona squamosa L.) Seed Crude Extracts against

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Dog Fleas (Ctenocephalides canis)

Abstract

Dog fleas are carrier of several diseases not only to its host animal but also to humans. This has encouraged the screening of Atis scientifically known as Annona squamosa, seed extracts as parasiticide.

Dried seeds of Atis were soaked in methanol and concentrated in vacuo. The crude extract were diluted in distilled water to avail the following concentrations: 100% concentration – 10 mL seed extract, 50% concentration – 5mL seed extract and 5mL distilled water, 25% concentration – 2.5mL seed extract, 7.5mL distilled water and Negative Control - water. The treatments were sprayed on 10 dog fleas per designated glass container. After ten minutes of exposure, the numbers of killed dog fleas were counted.

Results showed that highest mean mortalities of dog fleas were observed: 9 for 100% concentration, 7 for 50% concentration, 5 for 25% concentration and least 0 for the negative control. The parasiticidal activity of the Atis seed extract is concentration-dependent.

The Annona squamosa seed methanol extract is a potential source of bioactive compounds with parasiticidal activity on dog fleas.

The study is directed in three applications: First, a cost-effective parasiticide on dog fleas, second, environment-friendly parasiticide is produced and last, plant propagation of the species is encouraged.

Introduction

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Background of the Study

According to PetWellBeing (2005), Dog fleas (Ctenocephalides canis) are itching and

irritating parasites because they infect including humans. They spread diseases Dipylidium

caninum by feeding on the host’s blood. As noted by the American College of Toxicology

(1983) commercial parasiticide can control the spread of this parasite but it causes hair loss or

accumulation of detergent on the skin surface and the hair follicles due to the presence of

Sodium lauryl sulfate. This has triggered the search for an alternative natural parasiticide.

Atis, scientifically known as Annona squamosa L. is a native to the tropical country

(Huxley, 1992). Its seed contains alkaloids and non-drying oil which is an irritant that kill heads

lice (de Guzman, 1985).

The gathered literature above encouraged the screening of Atis seeds as parasiticide

against Dog fleas.

Statement of the Objectives

General Objective

This study aimed to investigate the parasiticidal effect of Atis seed against Dog fleas.

Specific Objective

1. To determine the insecticidal potency of the different concentrations of Chili fruit

extracts on the mortality of Rice Bugs.

Hypothesis

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1. The different concentrations of Atis seed crude extract potently exhibit parasiticidal effect

based on the mortality of dog fleas.

Significance of the Study

The results of the study are directed in the following: Firstly, the isolation and

purification of the bioactive compounds that exhibit parasiticidal activity. Secondly, the

formulation of potent natural parasiticide that is safe to the environment and practical. Lastly,

open possibility for screening to other pests and insects.

Scope and Limitations of the Study

This study was limited on the evaluation of the different concentrations of Atis seed

crude methanol extract as parasiticide based on the mortality of dog fleas. The isolation and

purification of the bioactive compounds was not conducted. Furthermore, topical application on

the dog and formulation of the parasiticide is not within the scope of the study.

Review of Related Literatures

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According to the Philippine Pharmacopeia (2004), the Atis seed contains alkaloids

responsibloe for its vermicidal and insecticidal properties. Intaranongpai (2006) identified the

active compounds against head lice from the hexane extract of Annona squamosa seeds as oleic

acid and triglyceride.

Fleas were exposed to atis seed extract because they are wingless parasites attacking

warm-blooded vertebrate that could trigger anemia to the host. They can also bring Diplydium

caninum, a tapeworm that lives on cats and dogs. Besides flea bites and the itching associated

with bites, it can spread a number of diseases such as tapeworm, typhus and tularemia. Flea bites

are irritating and this is cause by its saliva which contains enzymes and proteins. It is the flea’s

saliva that also triggers the hypersensitivity reaction in pets with a flea allergy (PetWellBeing,

2005).

Methodology

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Table 1

Experimental Design

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.Research Design

The Completely Randomized Design (CRD) was utilized in the collection of plant

samples and dog fleas as test organisms.

Materials and Tools

The materials used in the study are as follow: 500g of Atis seeds, 100mL of distilled

water, 120 pieces of dog fleas, and 100mL of methanol. The following tools were: 1 piece

100mL graduated cylinder, 1 piece of weighing scale, 1 piece of mortar and pestle, 12 pairs of

gloves, 12 pairs of laboratory gowns, and 12 pieces of masks.

Figure 1. Procedural Design

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Treatment Replicates

100% concentration (10mL extract) R1 R2 R3

50% concentration(5 mL extract, 5ml water) R1 R2 R3

25% concentration (2.5 mL extract, 2.5ml water) R1 R2 R3

Negative Control R1 R2 R3

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Gathering of materials needed

Atis seed collection and extraction

Preparation of treatments’ concentrations

Collection of Dog fleas

Randomly distribution of dog fleas

Exposure of Dog fleas with the designated treatment

Observation for the number of mortality of Dog fleas

Presentation and Analysis of data

Procedure

Identification of the plant

The researcher submitted the plant samples for identification to the Provincial

Environment and Natural Resources Office under the expertise of Mr. Edwin Borja, Forester.

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Figure 4. Soaking of seeds

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Preparation, air drying and soaking of atis seeds

Figure 2. Air drying of seeds Figure 3.Grinding of seeds

Approximately 500g of Atis seeds were collected last May, 2009 and was air dried for

three days. The dried Atis seeds were grinded and soaked in 100mL methanol for another three

(3) days.

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Preparation for Extraction

The samples were filtered using a filter paper and the filtrate was evaporated in vacuo to

avail the concentrated crude extract.

Preparation of materials and Testing of the concentrations

The concentrated samples were stored in amber bottles to prevent degradation of the

bioactive compounds. The Atis crude extract and the distilled water were measured using a

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Figure 5. Rotary Evaporation

Figure 6. Preparation of Materials Figure 7. Applying of Extract

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graduated cylinder in the following dilutions: Treatment A (100% concentration) with 10 mL of

Atis seed extract, Treatment B (50% concentration) with 5 mL of water and 5 mL of Atis seed

extract, and Treatment C (25% concentration) with 2.5 mL of Atis seed extract and 7.5 mL water

and the Negative control, 10mL water only.

Twelve clear glass jars were labelled according to the treatments label and replication.

Tissue papers were completely covered the bottom of the jar to absorb the excess liquid during

application. A total of 120 dog fleas were randomly distributed to all the twelve glass containers.

Each jars having ten dog fleas. The jars were sealed with a screen on the top to avoid the fleas to

fly out. The previously prepared treatments were sprayed spontaneously into the designated

jars. After ten minutes of exposure, the number of dead Dog fleas showing no sign of movement

were noted as dead and utilized as the basis of the data collected.

Statistical Analysis

The data gathered was analyzed using One Way analysis of Variance and tukey-

simultaneous comparison by Megastat.

Disposal

After the experiments the laboratory apparatus were cleaned and the materials were

disposed properly in the laboratory trash cans.

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Results and Discussion

Table 2

Mortality of dog fleas exposed to various concentrations of atis crude extract

TreatmentsMortality

R1 R2 R3 Total Mean*

100% concentration 10 8 9 27 9a

50% concentration 8 7 6 21 7b

25% concentration

Negative Control

5

0

4

0

6

0

15

0

5c

0d

* Column means with common letter superscripts are not significantly different while different letter means signify significant difference at 0.05 level of significance by One-Way ANOVA.

As shown by the data, it was recorded that the 100% concentration of Atis seed extract is

the most potent parasiticide against dog fleas with the mean mortality of 9 followed by the 50%

concentration with the mean of 7 then the 25% concentration with the mean of 5 and lastly the

negative control with the mean of 0. One-way analysis of variance at 0.05 level of significance

confirmed that 100% concentration effect on the mortality of Dog fleas is significantly different.

Having an F-value of 59.67 > critical value of 3.20 shows that the null hypothesis is rejected.

Post hoc, Tukey simultaneous comparison revealed that 100% concentration is the most

significantly different compared to other mortality means of fleas exposed to other treatments.

The presence of alkaloids, oleic acid and triglyceride (Intaranongpai, 2006) contributed to

the positive results. The activity of the extract is concentration – dependent which means the

greater the extract concentration the potent is the parasiticidal activity. The results showed that

Atis seed crude extract can be used as a natural parasiticide against dog fleas.

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Conclusion and Recommendation

Conclusion

1. Atis seed methanolic extract is an effective parasiticide against dog fleas in favor of the

most concentrated solution.

Recommendations

1. Use Atis seed extract as natural parasiticidal against dog fleas.

2. Research about other seeds that may contain bioactive compounds that can effectively

kill dog fleas.

3. Conduct skin test on the dog for possible allergenic reactions.

4. Incorporate the extract in the formation of shampoo for dogs.

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Bibliography

American College of Toxicology (1983). Journal of the American College of Toxicology Volume 2, Number 7, pp. 127-181. Retrieved November 18, 2010 from http://www.hairlossbuddy.com/20/sodium-lauryl-sulfate/

de Guzman, M.S. (1985). Healing wonders of Herbs. Retrieved July 13, 2010 from http://www.stuartxchange.org/Atis.html

Huxley, A. (1992). New RHS Dictionary of Gardening. Retrieved July 13, 2010 from http://en.wikipedia.org/wiki/Sugar-apple

Intaranongpai,J. (2006).SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH Vol 37 No. 3. Retrieved July 13, 2010 from http://www.stuartxchange.org/Atis.html

Morton, J. (1987) Sugar Apple, Fruits of warm climatesMiami, Florida. Retrieved July 13, 2010 from http://www.hort.purdue.edu/newcrop/morton/sugar_apple.html#Toxicity

PetWellbeing, (2005)Dog fleas Dog Fleas (Canine Flea Problem) Atlanta. Retrieved August 25, 2010 from http://pethealth.petwellbeing.com/wiki/Dog_Fleas

Definition of Terms

Atis seed –-- contains alkaloids and a yellow non-drying oil (Philippine Herbal Medicine 2005-

2010). In this research study, atis seed was used as a parasiticide agent against dog fleas.

Dog fleas –-- they cause itching and irritation (PetWellbeing, 2010. In this study, was the test

organism used in the study.

Mortality –-- a measure of the number of deaths in a given sample ( Douglas et al,. 2001).In this

research study, mortality was used to identify if the plant sample affected the dog fleas.

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Appendices

Ho: Atis seed crude extract is not a potent parasiticide against dog fleas.Ha: Atis seed crude extract is a potent parasiticide against dog fleas.Decision: Reject Ho since p-value is less than 0.05One factor ANOVA

Mean n Std. Dev  

5.25 9.0 3 1.00 T15.25 7.0 3 1.00 T25.25 5.0 3 1.00 T35.25 0.0 3 0.00 negative

5.3 12 3.57 Total

ANOVA table

Source SS df MS F p-

value

Treatment 134.25 3 44.750 59.678.10E-

06Error 6.00 8 0.750 Total 140.25 11      

Post hoc analysisTukey simultaneous comparison t-values (d.f. = 8)

negative T3 T2 T10.0 5.0 7.0 9.0

negative 0.0        T3 5.0 7.07      T2 7.0 9.90 2.83    T1 9.0 12.73 5.66 2.83  

critical values for experiment wise error rate:0.05 3.200.01 4.38

p-values for pairwise t-testsnegative T3 T2 T1

0.0 5.0 7.0 9.0 negative 0.0        

T3 5.0 .0001      T2 7.0 9.15E-06 .0222    T1 9.0 1.37E-06 .0005 .0222  

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Acknowledgment

First and foremost, the researches would like thank the Almighty God for His guidance,

blessing and divine assistance to make this study accomplished.

Also, gratitude and appreciation is extended to the following persons:

To Dr. Nordy D. Siason Jr., Principal IV for the opportunity given for the promotion

and support in research.

To Mr. Edwin F. Mosura, Head Research Department for his support and suggestions

on the study

To Mr. Ronilo V. Aponte, the researcher’s adviser for the guidance, assistance, advises

and consideration.

To the researchers’ Family, for their never-ending love and support morally, spiritually

and financially.

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