1 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 Introduction Background of the Study Statement of the Objectives Significance of the Study Scope and Limitations of the Study Review of Related Literature Methodology Results and Discussion Conclusions Recommendations Bibliography Definition of Terms Appendices Acknowledgment

5 6 6 6 7 7 8 9 14 15 15 16 16 17 18

Lists of Tables

Table 1. Experimental Design Table 2. Mortality of Dog fleas exposed to various concentrations of Atis seed crude extract

9 14

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Lists of Figures

Figure 1. Procedural Design Figure 2. Air drying of seeds Figure 3. Grinding of seeds Figure 4. Soaking of seeds in ethanol solvent Figure 5. Rotary Evaporation Figure 6. Preparation of Materials Figure 7. Applying of Extract RVA 12/3-4/11

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

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5 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 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 hosts 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.

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6 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 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

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7 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

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.

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8 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 fleas saliva that also triggers the hypersensitivity reaction in pets with a flea allergy (PetWellBeing, 2005).

Methodology .Research Design The Completely Randomized Design (CRD) was utilized in the collection of plant samples and dog fleas as test organisms.

Treatment 100% concentration (10mL extract) 50% 12/3-4/11 RVAconcentration(5 mL extract, 5ml water) 25% concentration (2.5 mL extract, 2.5ml water) Negative Control

R1 R1 R1 R1

Replicates R2 R2 R2 R2

R3 R3 R3 R3

9 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.

Table 1 Experimental Design

Figure 1. Procedural Design

Gathering of materials needed Atis seed collection and extraction RVA 12/3-4/11

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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.

Preparation, air drying and soaking of atis seeds

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Figure 2. Air drying of seeds

Figure 3.Grinding of seeds

Figure 4. Soaking 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.

Preparation for Extraction

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Figure 5. Rotary Evaporation 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

Figure 6. Preparation of Materials

Figure 7. Applying of Extract

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 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.

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13 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 tukeysimultaneous comparison by Megastat.

Disposal After the experiments the laboratory apparatus were cleaned and the materials were disposed properly in the laboratory trash cans.

Results and Discussion

Table 2 Mortality of dog fleas exposed to various concentrations of atis crude extract

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14 Mortality R1 10 8 5 R2 8 7 4 R3 9 6 6 Total 27 21 15 Mean* 9a 7b 5c

Treatments 100% concentration 50% concentration 25% concentration

Negative Control 0 0 0 0 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. Conclusion and Recommendation

Conclusion 1. Atis seed methanolic extract is an effective parasiticide against dog fleas in favor of the most concentrated solution. RVA 12/3-4/11

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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.

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 RVA 12/3-4/11

16 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 climates Miami, 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 20052010). 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.

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 0.05

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Acknowledgment

One factor ANOVA Mean 5.25 5.25 5.25 5.25 ANOVA table Source Treatment Error Total SS 134.2 5 6.00 140.2 5 df 3 8 11 MS 44.750 0.750 F 59.67 pvalue 8.10E06 9.0 7.0 5.0 0.0 5.3 n 3 3 3 3 12 Std. Dev 1.00 1.00 1.00 0.00 3.57 T1 T2 T3 negative Total

Post hoc analysis Tukey simultaneous comparison t-values (d.f. = 8) negative T3 T2 0.0 5.0 7.0 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 3.20 0.01 4.38 p-values for pairwise t-tests negative T3 T2 0.0 5.0 7.0 negative 0.0 T3 5.0 .0001 . T2 7.0 9.15E-06 0222 . RVA 12/3-4/11 9.0 T1 1.37E-06 0005 .0222

T1 9.0

T1 9.0

18 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 researchers 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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