The Potential Effect of the Football Fruit (Pangium edule) Seed Extract as an Insecticide for Mosquito Culex sp.

Lim Zi En ***, Dr. dr. Sri Andarini**, Dr. dr. Loeki Enggar Fitri * ABSTRACT Mosquito Culex sp. has been a vector of many diseases and it is almost found worldwide. Due to insecticide resistance and the harmful effects of chemical insecticide to human and the environments, natural insecticides are now gaining its popularities. The seed of football fruit (Pangium edule) contains substance such as, cyanide, phenolics, and alkaloids that have been accounts for its toxicity, which could have made it potent as an insecticide. This research is aimed to prove that the football fruit (Pangium edule) has the insecticide potential. The research was a true experimental-post test only control group design which used Culex sp. as sample and was divided into 5 groups with four times repetition. The treatment groups used the football fruit (Pangium edule) seed extract 20%, 40% and 80%. The negative control group was aquadest and the positive control group was malathion. The experiment was done at the duration of time 10 minutes, 20 minutes, 40 minutes, 60 minutes, 100 minutes and 24 hours. From the experiment, the result revealed that higher percentage of Pangium edule seed extract has greater potential as an insecticide. From the Regression analysis, the lowest concentration that is able to kill 50% Culex sp at 100 minutes is the seed extract concentration of 16%, and to kill 100% Culex sp at 100 minutes, the lowest concentration that is needed is 40%. By performing Pearson Correlation test, significant relation between Pangium edule seed extract and the death of Culex sp. (p=0.000) was found. The conclusion of this research is Pangium edule seed extract has insecticide potential against Culex sp. Keywords: Pangium edule seed extract, Culex sp., Insecticide

ABSTRAK Uji Potensi Ekstrak Biji Buah Keluak (Pangium edule) Sebagai Insektisida Terhadap Nyamuk Culex sp. Nyamuk Culex sp. merupakan vektor untuk bermacam-macam penyakit dan bisa ditemukan hampir di seluruh dunia. Penggunaan insektisida alami sudah banyak di kalangan masyarakat. Resistensi nyamuk terhadap insektisida kimiawi dan efek sampingnya banyak terjadi terhadap manusia dan lingkungan. Biji buah keluak (Pangium edule) mengandungi zat-zat seperti sianida, fenolat, dan alkaloid yang toksisitasnya cukup tinggi sehingga berpotensi sebagai insektisida. Penelitian ini bertujuan untuk membuktikan bahwa biji buah keluak (Pangium edule) memiliki potensi sebagai insektisida. Penelitian ini merupakan true experimental-post test only control group yang menggunakan Culex sp. sebagai sampel dibagi menjadi 5 kelompok dengan empat kali pengulangan. Kelompok perlakuan yang digunakan adalah ekstrak biji buah keluak (Pangium edule) 20%, 40% dan 80%. Kontrol negatif yang digunakan adalah aquadest dan kontrol positif merupakan malathion. Percobaan ini dilakukan pada waktu 10 menit, 20 menit, 40 menit, 60 menit, 100 menit dan 24 jam. Dari hasil percobaan, konsentrasi ekstrak biji buah keluak yang lebih tinggi memiliki potensi sebagai insektisida yang lebih besar daripada ekstrak biji buah keluak yang berkonsentrasi rendah. Dari analisis Regresi, konsentrasi terendah yang mampu membunuh 50% Culex sp pada 100 menit adalah ekstrak biji buah keluak dengan konsentrasi 16%, sedangkan untuk membunuh 100% Culex sp pada 100 menit, konsentrasi terendah yang diperlukan adalah 40%. Setelah melakukan uji Korelasi Pearson, terdapat hubungan yang signifikan antara ekstrak biji buah keluak dan kematian Culex sp. (P = 0,000). Kesimpulannya, biji buah keluak (Pangium edule) memiliki potensi sebagai insektisida terhadap nyamuk Culex sp. Kata kunci: ekstrak biji Pangium edule, Culex sp., insektisida

*** Medical Programme of Faculty of medicine of UB ** Public Health Laboratory of Faculty of medicine of UB * Parasitology Laboratory of Faculty of medicine of UB

INTRODUCTION Mosquitoes are some of the most versatile organisms on earth. It is estimated that there are more than 2500 species of mosquitoes worldwide
1.

However, the shortcoming of using any plants to repel mosquitoes is that the oil is not naturally released into the air, the leaves must be crushed before the oil is released, and it does not spread far. There are other natural mosquito repellents that are being researched such as fennel, thyme, clove oil, celery extract, neem oil, soybean, vitamin B1 and garlic
8.

They can

reproduce in virtually any natural or man-made deposit of water, and have been found in mines deep below the surface, in mountain peaks, in highly polluted water bodies, in snow pools, and even in crab holes 2. Conventional insecticides are among the most popular chemical control agents because they are readily available, rapid acting, and highly reliable. Because of their convenience and effectiveness, insecticides quickly became standard practice for pest control during the 1960's and 1970's. Overuse, misuse, and abuse of these chemicals have led to the development of resistance and to a need for more powerful poisons, in a few cases, resulted in long-term environmental consequences
3.

There is an interesting

"nut" that is actually a seed is called "buah keluak" or "kluwak nut" (also spelled kloowak). Kluwak nuts come from the kepayang tree (Pangium edule) of Indonesia & Malaysia, a member of the flacourtia family (Flacourtiaceae). The oily, hard-shelled seeds superficially resemble Brazil nuts. Meaty seeds are edible after the poisonous hydrocyanic acid is removed by soaking and boiling them in water. Fermented kluwak nuts become chocolate-brown, greasy and very slippery. Cooked seeds are used in a number of dishes
9.

Study showed a

Insecticide resistance is expected to directly and profoundly affect the reemergence of vector-borne diseases control
4.

significant correlation between the total phenolic content and its antioxidative and antibacterial activity suggesting that phenolics of the seed extract could be potential natural antioxidant and antibacterial sources
10.The

and where resistance has not contributed to

disease emergence, it is expected to threaten disease

5.

seeds are

The mosquito Culex pipiens is one of the few Vector

widely used for fish preservation, wild dog elimination, rodenticide, molluscicide, and bacteria growth inhibitor 11. Research Design This study is a laboratory experiment with the design of true experimental post-test only. The purpose is to observe the potential insecticidal effect of Pangium edule seed extract on the mosquito Culex sp. Method of Research The experiments are carried out by using 5 treatment groups which is consisted of Malathion 0.28% as the positive control, Aquadest as the negative control and seed extracts of Pangium edule with the concentration of 20%, 40% and 80%. The study was conducted at the Parasitology Laboratory of Medical Faculty of UB between July-August 2011.

insect species in which insecticide resistance, in particular to organophosphate (OP) insecticides products
6.

control is an essential and effective means for controlling transmission of vector-borne diseases, especially in areas where resistance in parasite to drugs is growing. Unlike insecticides, bio-control agents are host specific, safer to the environment, find easy application in the field, are costeffective in production, lack infectivity and pathogenicity in mammals including man and has little evidence of resistance development in target mosquito species 7. It's possible to help reduce mosquito populations around a home through the use of natural or biological means, for example, predatory fish, plant repellents, bacteria and even other mosquitoes can kill the mosquitoes that breed and feed in a given area 8.

Working Method The experiments are carried out by using 5 cages measured 25cm x 25 cm x 25 cm that are placed in a room at room temperature. 25 mosquitoes are put into each cage. Cage 1 is sprayed with Malathion 0.28% as the positive control. Cage 2 is sprayed with aquadest as the negative control. Cage 3, 4 and 5 are sprayed with the seed extracts of Pangium edule with the concentration of 20%, 40% and 80%.

The number of dead mosquitoes in each treatment was calculated after certain duration of time such as 10 minutes, 20 minutes, 40 minutes, 60 minutes, 100 minutes and 24 hours. The data collected will be analysed. Research Results and Data Analysis From the research of potential effect of the football fruit (Pangium edule) seed extract as an insecticide for Mosquito Culex sp., the following results are as follow:

120

Mean Insecticide Potential (%)

100 80 60 40 20 0 10 20 40 60 100 24 hours minutes minutes minutes minutes minutes Duration Of Time

Pangium edule Seed Extract 20% Pangium edule Seed Extract 40% Pangium edule Seed Extract 80% Positive control Negative control

Graph of the mean insecticide potential versus time From the graph above, it can be observed that there are differences in the number of dead Mosquitoes Culex sp. with different concentration of football fruit (Pangium edule) seed extract and duration of time. It can be seen that with an increase in the concentration there is an increase of the insecticide potential. Another important information can be derived from the graph is the LD50 and LD100, and in this research, coincidentally it happened to be the concentration of seed extract below 20% at 100 minutes and 24 hours respectively. Two-Way ANOVA analysis is chosen in this research because there are two different independent variables, namely the concentration of the football fruit (Pangium edule) seed extract (20%, 40% and 80%) and the duration of time (10 minutes, 20 minutes, 40 minutes, 60 minutes, 100 minutes and 24 hours). Before analyzing the data using Two-way ANOVA statistic, there are some criterias that should be fulfilled such as the data distributions which must be normal, the data points must be independent from each other and the variances of the samples are not different. In order to determine how the distribution of data is, Normality tests such as Kolmogorov-Smirnov test and Shapiro-Wilke test are performed. The distribution and variant data of the different potential insecticide effect of pangium edule seed extract based on the number of mosquitoes Culex sp. that died at different duration of time should be tested by using Normality Test and Variant Test. Based on the Kolmogorov-Smirnov Test, it can be seen that the data distribution from the research show a significant value of p=0.075 (p>0.05). Hence, the data has a normal distribution. The significant value from the Test of

Homogeneity of Variance was p=0.067 (p>0.05), so it can be concluded that the variance of the population was homogenous. Since both the Normality test and Variant test have been fulfilled, thus, it is eligible for further TwoWay ANOVA test. Based on the tests of Between-Subject Effects, the significant value (p-value) for time is 0.000 < (0.05) and the significant value (p-value) for the concentration is 0.000 < (0.05). Therefore, it can be concluded that there are significant differences between the varied concentrations and time duration to the total number of dead Mosquito Culex sp. in each experiment. Null hypothesis, H0 is rejected and H1 is accepted. LSD test can be conducted in this research because the null hypothesis is rejected after the two-way ANOVA test. It is performed in order to determine which groups differ from each other. Table: Summary of the analysis of the LSD test
Concentration Negative Control Concentration P. edule seed extract 20% P. edule seed extract 40% P. edule seed extract 80% Positive control Negative Control P. edule seed extract 80% Positive control Negative Control Positive control Negative Control P. edule seed extract 20% Positive control Negative Control P. edule seed extract 20% P. edule seed extract 40% P. edule seed extract 80% Significance 0.000 0.000 0.000 0.000 0.000 0.013 0.000 0.000 0.000 0.000 0.013 0.000 0.000 0.000 0.000 0.000 Explanation Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different Significantly different

Correlation tests are used to assess whether there is a relationship between two or more variables. The Pearson correlation coefficient, r, can take a range of values from +1 to -1. A value greater than 0 indicates a positive association, that is, as the value of one variable increases so does the value of the other variable. A value less than 0 indicates a negative association, that is, as the value of one variable increases the value of the other variable decreases. The variable of time also has a strong correlation with the variable of potential, as r=0.608. In other words, the total number of dead Mosquito Culex sp. increases with the increase in time duration. The variable of concentration has a moderately mild correlation with the variable of potential, r=0.434. In other words, the total number of dead Mosquito Culex sp. will increase with the increase in concentration. Discussion The seed of football fruit contains substance such as, cyanide, lipids, phenolics, oleic acid, linoleic acid, alkaloids and chlorophyll. The active substance of the seed which accounts for its toxicity is cyanide. Cyanide is a rapid in acting poison which can causes death within minutes to hours of exposure. Hydrogen cyanide (HCN) or cyanide ion will binds irreversibly to the iron atom in hemoglobin and thus inhibiting cytochrome oxidase, the terminal enzyme of the mitochondrial electron transport chain
12.

Pangium edule seed extract 20%

As a result,

hemoglobin is unavailable to transport the vital O2 to the body's cells and tissues. It also interferes with ATP (adenosine triphosphate), the main energy storage molecule in the body. These two events combined and rapidly bring the body's metabolism to a halt, resulting in death. It also has a rapid paralyzing effect on most species
13.

Pangium edule seed extract 40% Pangium edule seed extract 80%

Alkaloids are plant metabolites that have a nitrogen-containing chemical ring structure, alkali-like chemical reactivity, and pharmacologic activity. The alkaloids represent a very diverse group of medically significant compounds that include well-known drugs like the opiates
14.

Positive control

It has the ability to mimic the

neurotransmitter acetylcholine where the nerve synapse will be over stimulated and resulted in death 15. Based on the analysis of Two-Way ANOVA test, it can be concluded that there are significant differences between the varied concentrations and time duration to the total number of dead Mosquito Culex sp. in each experiment. From the test, negative control, postive control and the treatment groups of 20%, 40%, and 80% seed extract concentrations produced a significant difference in the total number of dead Mosquito Culex sp. (p=0.000, less than 0.05). The LSD test showed that there were significant difference between the concentration of 20% and 80%. Therefore, it is proven that different concentration can give different insecticidal effect. This may be due to the dissolved active substance is higher at higher concentration. Although the seed extract of the football fruit has the potential as an insecticide and could kill the mosquito up to 100 % in 24 hours but the seed extract of the football fruit still could not compete with malathion as insecticide that could be used by the community. This could be seen from the comparison of the total death of the mosquito by the use of malathion and the football fruit seed in 10 minutes. Although the concentration of the seed extract is higher (20%, 40%, 80%) than the concentration of malathion (0.28%), the seed extract killed mosquito but in a relatively small amount compared to the malathion which can caused higher mortality of mosquito in shorter time. This might be explained as the malathion is more toxic than cyanide which is contained inside the football fruit seed extract. Cyanide contained in the football fruit is not chemically stable hence it is easy to vaporize, and this will decrease the potential of the extract. Further research may be conducted to combat the autohydrolysis properties of the cyanide at room temperature. Other shortcoming or constraint that encountered in this research is that the football fruit which is bought from market might have undergone some degree of fermentation and thus decreased the potential of the extract.

Conclusion The Football fruit (Pangium edule) seed extract has the insecticide potential for mosquito Culex sp. The insecticide potential of the Football fruit will increase with the increase in the seed extract concentration and the longer time duration of exposure of mosquito to the extract will also increase the insecticide potential. LD 50 (Lethal dose 50) or the minimum concentration of the Pangium edule seed extract that will cause 50 % of the adult mosquitoes Culex sp. to die is seed extract concentration of 20% (from this research) which is not too far from the predicted value by the equation derived from regression test that is approximately seed extract concentration of 16%. The Football fruit (Pangium edule) seed extract is less potent than Malathion. Suggestion A research on the toxicity effect of the Pangium edule seed extract to the human and the environment should be done. A research should be conducted to find the substance that could stabilize the active substances in Pangium edule seed extract so that the evaporation process can be slowed down and thus the insecticidal effect on adult mosquitoes Culex sp. can be prolonged. The seed of Football fruit (Pangium edule) should be sought from the plantation site rather than bought from the market to ensure its freshness and hence its toxicity. References 1. Jorge. 2006. The Mosquito, University of Florida IFAS Extension. Publication #ENY-727. p.1-8. 2. OMeara. 2000. Crabhole Mosquito,

Deinocerites Cancer Theobald (Insecta: Diptera: Culicidae).University Of Florida IFAS Extension. Publication #EENY 134. p.1-8. 11. 3. John RM. 2003. Pest Control Tactics, online, retrieved on 30 November 2011, from http://cals.ncsu.edu/course/ent425/text19/co nvchem.html 4. Krogstad. 1996. Malaria as a reemerging disease. Epidemiology Rev 1996;18:77-89. 5. WHO. 1992. Vector Resistance to Insecticides. World Health Organ Tech Rep Ser. 13. 6. Raymond M. & Pasteur N. 1996. Evolution of insecticide resistance in the mosquito Culex pipiens: the migration hypothesis of amplified esterase genes. In: Brown, T. M. (ed.). Molecular Genetics and Evolution of Pesticide Resistance. pp. 9096. American Chemical Society, Washington, DC. 7. Subiah & Brij. 2006. The Challenge of Mosquito Control Strategies: from Primordial to Molecular Approaches, Biotechnology and Molecular Biology Review Vol. 1(2). p. 51-65. 8. Wong. 2006. Natural Mosquito Repellents, online, retrieved on 20 November 2010, from 15. 14. 12.

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Pharmacology. Yuningsih. 2009. Kandungan dan Stabilitas Sianida dalam Tanaman Picung (Pangium edule Reiw.) serta Pemanfaatannya. Balai Besar Penelitian Veteriner. hal.102-107. Isom & Way. 1974. Effect of Oxygen on Cyanide Intoxication VI. Reactivation of Cyanide Inhibited Glucose Catabolism. J Pharmacol Exp Ther 1974; 189:235-43. Lindgren. 1938. The stupefaction of red scale Aonidiella aurantii by hydrocyanic acid. Hiluardia, 11: 213 - 225. Richard AW. 2009. Plant Poisoning, AlkaloidsTropane, online, retrieved on 27 overview John LC. 2008. Encyclopedia of Entomology, 2nd Ed. Springer. p.122-126. Supervisor 1: November 2010, from http://emedicine.medscape.com/article/816657-

____________________ Dr.dr.Loeki Enggar Fitri, M.Kes, Sp, ParK NIP: 19641013 199103 2001

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Antibacterial Activities of Pangium edule