IDENTIFICATION OF SOURSOP SEEDS (Annona muricata L.)EXTRACT AS A CANDIDATE AGAINST THE Aedes aegypti L. MUSQUITO DBD VECTOR CONTROL

IDENTIFICATION OF SOURSOP SEEDS (Annona muricata L.)EXTRACT AS A CANDIDATE AGAINST THE Aedes aegypti L. MUSQUITO DBD VECTOR CONTROL Sarah Zaidan, Ratna Djamil, Siti Nuraini Faculty of Pharmacy, Pancasila University Jln. Srengseng Sawah Jagakarsa, Pasar Minggu, Jakarta Selatan 12640

Abstract Aedes aegypti L. mosquitos are the disease vectors of dangue hemorrhagie fever (DHF), caused by dengue virus which is transmitted by Ae. Aegypti mosquito. The effort to control Ae. aegypti vector have been done in so many times, including chemical, physical, and biological control method. Multilevel extraction by kinetic maceration have been done with soursop seeds (Annona muricata L.) with the solvent of n-hexane, ethyl acetate and 70% of ethanol. Subsequently, the obtained extract is tested phytochemical screening along with the powder and larvicidal activity against Ae. aegypti. The results of phytochemical screening of the powder and 70% ethanol extract of soursop seeds have obtained the compound of saponin, triterpenoid and coumarin. In the n-hexane extract have obtained triterpenoids and in ethyl acetate extract which is found triterpenoids and coumarin. Based on the activity test against the larva of Ae. aegypti from n-hexane extract, ethyl acetate and ethanol 70% of soursop seeds sequentially, show LC50 values were about 198,610 ppm, 74,798 ppm and 67,042 ppm. Soursop seeds extract that has the highest activity is 70% ethanol extract. These indicate that the chemical compounds which is found in soursop seeds have a potential as a larvicides. Keywords: Aedes aegyti L., Annona muricata L., soursop seeds, larvasida Abstrak Aedes aegypti L. adalah nyamuk vektor penyakit demam berdarah dengue (DBD), yang menularkan virus dengue. Upaya untuk mengendalikan vektor A. aegypti telah sering dilakukan, termasuk pengendalian kimia, fisik, dan dengan kontrol biologis. Multi level ekstraksi dengan maserasi kinetik telah dilakukan terhadap biji sirsak (Annona muricata L.) dengan pelarut n-heksana, etil asetat dan 70% etanol. Selanjutnya, ekstrak yang diperoleh diuji skrining fitokimia dan aktivitas larvasida terhadap A. aegypti. Hasil skrining fitokimia dari serbuk dan ekstrak etanol 70% biji sirsak diperoleh senyawa saponin, triterpenoid dan kumarin. Dalam ekstrak nheksana diperoleh triterpenoid dan di ekstrak etil asetat ditemukan triterpenoid dan kumarin. Berdasarkan uji aktivitas terhadap larva A. aegypti dari ekstrak n-heksana, etil asetat dan etanol 70% biji sirsak secara berurutan, diperoleh nilai LC50 berturutturut sebesar 198.610 ppm, 74.798 ppm dan 67.042 ppm. Ekstrak biji sirsak yang memiliki aktivitas tertinggi adalah ekstrak etanol 70%. Hal ini menunjukkan bahwa senyawa kimia yang ditemukan dalam biji sirsak memiliki potensi sebagai larvasida.

Volume 8, No.2, Agustus 2015

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Kata kunci: Aedes aegypti L., Annona muricata L., biji sirsak, larvasida. Introduction Mosquitoes are insects that are often found in tropical countries such as Indonesia. Besides disturbing human life, the presence of mosquitoes act as vectors of some diseases. In Indonesia, a disease transmitted by mosquitoes is still a health problem because of the high mortality rate. Some diseases transmitted by mosquito vectors such as filariasis, malaria and dengue fever (DBD)(Kaihena et al., 1979; Susanti et al., 2013). Aedes aegypti L. mosquitoes are diurnal, or active during the morning and afternoon. Ae. aegypti mosquitoes carrying dengue virus causes dengue which is obtained from infected individuals and multiply in the body and the salivary glands of mosquitoes (Susanti et al., 2013). Dengue disease not only in children but in all ages. DBD has been becoming known in Indonesia in 1968 in Surabaya and Jakarta, and then continue to expand as the spread of dengue endemic area. The number of cases of dengue and widely spread is increasing along with the increasing mobility and population density. There are 150,000 cases of dengue in 2007 and continued to increase until 2010. In addition, WHO reported more than 35% of the population living in urban areas are affected by the disease. Until now there is no specific vaccine to treat dengue fever, and the only control vector for controlling the spread of the disease (Susanti et al., 2013; Hadi dan Upik, 2005; WHO, 2005; Palgunadi et al., 2011). Vector control mosquitoes until today, still put emphasis on the use of insecticides, for example synthetic larviciding. Larvicides in general have a higher efficacy and the results can be seen quickly. The use of continuous and repetitive can cause environmental pollution and resistance against the target organism. This encourages biological larviciding as controlling mosquito vectors (Biolarvasida). These biological larvicides are safer for humans, readily available and environmentally friendly (Susanti et al., 2013; Rosmayanti, 2014). Biological larvicides are useful for the improvement of local natural resources. Local plant showing a potential biological is larvicides generally from families Annonaceae, including soursop (Annona muricata L.). Empirically, it has been various research done on the soursop as larvicides. The plant parts potentially as larvicides are seed (semen) (Mulyawati AP, 2010). Soursop seed (with shell beans) have larvicidal activity against A. aegypti with LC50 value of 244.27 ppm for the ethanol extract of the seeds of soursop (Rosmayanti, 2014; Taslimah, 2014). In addition, Ward et al, reported that seeds of soursop and sugar apple seed (without seed coat) affect on mortality Chrysomya bezziana fly larvae (Wardhana AH et al., 2006; Wardhana AH et al, 2004). The main active compounds from the seeds of the soursop is annonacin and squamocin belonging to asetogenin compound. Squamocin annonacin compound of the family Annonaceae to have toxicity properties and are quite effective against insects of the order Diptera (A. aegypti L.) which are cytotoxic, and neurotoxic. Asetogenin compounds can inhibit the action of the enzyme NADH in the mitochondria, causing the death of larvae, as well as toxic contact and stomach poison to insects (Rosmayanti, 2014; Wardhana et al., 2006; Wardhana et al., 2004).


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This research is continuing efforts to obtain biological larvicides. In this study, it is conducted qualitative identification through screening phytochemicals and the activity of seed extracts of soursop (Annona muricata L.) (without seed coat) against Ae. aegypti. Extract obtained from extraction method using a multi-storey solvents with different polarities i.e. n-hexane, ethyl acetate and ethanol 70% and seen the highest larvicidal activity of the three fractions of the extract. Extract larvicidal activity test using Standard Methods of Pesticide Efficacy Testing Households and Vector Control (Departemen Pertanian, 2011). The data seen LC50 values were obtained by probit analysis using Probit Analysis Program Epa Used For Calculating LC / EC Values Version 1.5. Methods Plant material Soursop seeds (Annona muricata L.) were obtained from Balittro (Research Institute for Spices and Medicinal Plants) Cimanggis, Bogor. Determination at Bogoriense Herbarium, Research Center, LIPI Cibinong , Bogor. Extraction Sun dried of soursop seed (Annona muricata L.) were crushed and blended into a fine powder. Powdered crude drug was extracted by maceration kinetic in stages using different solvent polarity in n-hexane, ethyl acetate, and ethanol 70% at room temperature until perfectly extracted, then filtered with cotton and proceed with filter paper, and pulp. Each extract was separated and concentrated by vacuum rotary evaporator at a temperature of 450C to obtain a viscous extract of n-hexane, ethyl acetate and ethanol 70%. Identification with phytochemical screening Phytochemical screening performed on pollen and seed extract of soursop with Farnsworth method in Biological and phytochemical screening of Plant sorsop seed was conducted to identify the qualitative content of secondary metabolites. Flavonoids Two grams of soursop seed powder or 0,67 g of n-hexane extract and ethyl acetate extract; 0,15g of extract ethanol 70% is boiled with 100 ml of hot water for 5 minutes, then filtered with filter paper, 5 mL filtrate of extract solution coupled with a bit of powdered zinc or magnesium and 1 mL of 2 N HCl and 5 mL amyl alcohol . Flavonoids compounds would pose orange to red (Fransworth NR, 1966). Saponins Entering 10 ml sample into a test tube and shake for 30 seconds and observe what happens. If the foam is formed solid (not dissapear for 30 seconds) the identification showed the presence of saponins (Fransworth NR, 1966). Coumarin 2.12 grams of powder simplisia or 0,67 g of n-hexane extract and ethyl acetate extract; 0,15g of extract ethanol 70% were included in the test tube and added 10 ml


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of chloroform, heated 20 minutes on waterbath and then cooled. After it is filtered with filter paper, the filtrate sunk in waterbath until dry. The residue was added 10 mL of hot water, then cooled and put into a test tube, added 0.5 mL of 10 % ammonia solution and then observed under UV light at a wavelength of 365 nm (blue or green fluorescence showed the presence of cumarin (Fransworth NR, 1966). Volatile oil Two grams of sour sop seed powder and 0,67 g extract put into a test tube, then added 10 mL of petroleum ether, at the mouth of the tube fitted with a mouthpiece that was given cotton and moistened with water, then heated above waterbath for10 minutes. Furthermore, after the water cold then filtered with a filter paper. The obtained filtrate was evaporated in the vaporizer cup, the residue was dissolved in 5 mL ethanol and then filtered with filter paper. If residues smell aromatic, indicate the content of volatile oils (Fransworth NR, 1966). Kuinon Five ml of solution experiments inserted into a test tube, addd a few drops of 1 N sodium hydroxide solution. Red color arising was indicate a compounds of quinine (Fransworth NR, 1966). Steroids/Triterpenes The 1.10 g of powder or sour sop seed extract: 0,33 g extract of n-hexane; 0.34 g of ethyl acetate extract; 0,67 g of ethanol extract 70% extract, macerated with 20 mL ether for 2 hours, then filtrated the solution, and total of 5 mL of the extract solution was evaporated to dryness, then added with a reagent Lieberman- Burchard. Arising green-red color indicates compounds terpenoids or steroids (Fransworth NR, 1966). Tannin Two g of sour sop seed powder or 0,67 g of n-hexane and ethyl acetate; and 0,15g of extract ethanol 70% added 100 mL of water, boiled for 15 Minutes, cooled and filtered, further divided to 5 mL filtrate each (reaction tubes): added a few drops of solution of iron (III) chloride 1 %, changes blue or blackish green and added a few drops of 1 % solution of gelatin to form white results indicates the compounds of tannins . To the second filtrate was added 15 mL reagent Stiasny (formaldehyde 30% - hydrochloric acid = 2 : 1), the result formed pink color indicates the presence of tannins katekuat. Subsequently the precipitate was filtered, the filtrate saturated with sodium acetate powder, add a few drops of solution of iron (III) chloride 1 %, blue ink color showed the presence of tannins galat (Fransworth NR, 1966). Alkaloids The 2.12 g of soursop seed powder or 0,67 g of n-hexane extract and ethyl acetate extract; 0,15g of ethanol extract was inserted in a porcelain bowl and then added 5 mL of ammonia 30% crushed and then added 20 mL chloroform and crushed again, then filtered. The filtrate obtained was added HCl 1 N as much as 5 ml and then separated into 2 sections namely A and B. The filtrate A coupled with Mayer reagent, filtrate B coupled with Dragrendroff reagent. Reagent Meyer gives a white


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precipitate, and Dragendorff reagent give an red brick precipitate (Fransworth NR, 1966). Larvasidal activity test Larvae maintenance Mosquito eggs were incubated in a plastic container (tray) with size of 20 x 15 x 10 cm3 which containing distilled water. The eggs hatched within 24 hours of becoming the first instar larvae, then the 2nd day will have change to be instar II stage of development. At this stage larvae was fed with chicken liver, then after 1-2 days will be changed again to the third instar. Implementation of experimental test larvicidal activity Larvicidal activity test was conducted using ”Pesticide Efficacy Testing Standards Household and Vector Control”. Carefully weighed approximately 100 mg extract and then dissolved in 100 mL of solvent. This solution was a main liquor (1000 ppm). The 18.750 ml main liquor were pipettled to 12,500 mL; 6,250 mL; 3.125 mL; 1,250 mL respectively and inserted into plastic cups of 25 mL to obtain a concentration of 750 ppm, 500 ppm, 250 ppm, 125 ppm, 50 ppm, then completely evaporated. Each concentration was made in 3 plastic cups (triplo), then into individual plastic cups partially added to 25 mL of distilled water homogenized, and included 20 third instar larvae of A. aegypti. Observations were carried out after 24 hours of exposure to the test solution and counted the number of dead larvae. Negative controls used solvent without the extract while positive controls carried out on Temephos 1 ppm. Data analysis Test data were analyzed systematically using probit analysis method. Probit analysis was used to determine the percentage of larval mortality or LC50 of A. aegypti L. uses Epa Probit Analysis Program Used For Calculating LC/EC Values Version 1.5 was employed in this research. Result and Discussion Phytochemical screening The phytochemical screening with Farnsworth method was conducted using sour sop seed powder and soursop seeds extract. Both in powder and extract of sour sop seed having metabolite compound such as saponin, triterpenoid, and cumarin. The result of phytochemical test is shown in Table 1.


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Table 1. Result of phytochemical screening of soursop seeds (Annona muricata L.) in powder and extract No

Secondary Metabolites

1 2 3 4 5 6

Soursop Ethanol Ethyl Nseed 70% acetate heksan powder Extract Extract Extract + + - /+ -/+ -/+ -/+

Alkaloids Flavonoids Saponins Kuinon Tannin Steroids / triterpenoids 7 Volatile oil 8 Coumarin + + Notes : + = positive reaction − = negative reaction

+

-

Table 1 indicated the results of the qualitative identification of secondary metabolite content of the soursop seed powder (Annona muricata L.) containing of saponins, triterpenoids, and coumarin. Screening used n-hexane, ethyl acetat and 70% ethanol extracts demonstrates chemical group constituents of triterpenoids, triterpenoids and coumarine, and saponin, triterpenoids, and coumarine consecutively. Larvicidal activity test Larvicidal activity test results showed in Table 2. Table 2. The average percentage mortality of larvae of Ae. aegyptiL. extract after exposure to n-hexane, ethyl acetate and ethanol 70% soursop seeds on a 24-hour observation. % Mortality Concentration (ppm)

750 500 250 125 50 LC50 (ppm) Linear regression

n-hexane

93,35 78,35 50 20 15 198,610

Type Solvent Ethyl acetate Ethanol 70%

95 88,35 73,35 65 40 74,798

a=a = -34,9054 115,7371 b = 45,5671 b = 70,9888 r = 0,9926 r = 0,9654


100 100 98,35 58,35 45 67,042 a = -43,2775 b = 52,5234 r = 0,9302

Control Negative Positive (Solvent) (Temephos 1 ppm) 0 100 0 100 0 100 0 100 0 100 -

-

-

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The results showed that the larvae of A. aegypti exposed with seed extract of soursop (without skin) for 24 hours had the following LC50 n-hexane extract of soursop seeds 198.610 ppm, ethyl acetate 74.798 ppm and 70% ethanol extract 67.042 ppm LC50. This indicate that the 70% ethanol extract of soursop seed result the highest activity as larvicides. It can be interpreted that the seed extract of soursop (without skin) also demonstrated larvicidal activity. This is caused by secondary metabolites contained in the soursop seed in the type of saponin, coumarin and triterepenoid (Riswanto, 2009). Saponins are be able to diffuse into the cuticle layer of larvae to damage cell membranes and toxic compounds penetrate into the larvae. Saponins have a strong bitter taste and cause irritation of the stomach. Larvae digestion tract, particularly the midgut (midgut) is the major site of absorption of nutrients and digestive secretion enzymes. Saponin absorption into the intestine larvae can inhibit the action of digestive enzymes and cause distruction to the cells in the digestion tract (Susilowati et al., 2009). Triterpenoids also thought to function as antifeedant on the larvae appetite. This led to the loss of energy and development of larvae is hampered (Nopitasari, 2013). In addition, coumarin is also reported as larvicides. It is potentially able to change the detoxification ability to reversibly or irreversibly to inhibit the cytochrome P450 enzyme (Venugopala et al., 2014). Another ability of secondary metabolites in seed soursop is that sugar apple seeds potentially shows larvicides against mosquito larvae A. aegypti L. Mortality of larvae on seed extract of soursop seeds (Annona muricata L.) is also resulted from the effects of the component compounds acetogenin toxic squamosin compound. After the larvae exposed to the extract, the compound cross into the body of A. aegypti through physical contact and killing of the larva. Prijono (1994) in Ward et al. (2005) states that the absorption of toxic insecticides contact occurs largely in the cuticle. Active compounds will penetrate into the insect's body through the part that is covered by a thin cuticle, such as membrane between segments. The ability of the compound asetogenin stomach poison works by absorption of chemical constituents in soursop seed extract into the wall of larvae and able to inhibit oxidative chain to inhibit the cell respiration of A. aegypti because by stopping of breathing process. Squamocin compounds in the seeds of soursop are thought to diffuse from the thin cuticle layer to spread throughout the body of A. Aegypti through hemolimfa flow (Wardhana, 2005). Mortality of A. aegypti larvae showed the following symptoms signs: larvae do not move when touched, larvae bodies is white pale, elongated and stiff. The differences between normal and died of A. aegypti larvae is shown on Figure 1. .


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A

B

Figure 1. The normal (A) and the died (B) of the third instar larvae of A. aegypti die

Concentration of sour sop seed extract (ppm) Figure 2. Average of percentage of mortality of soursop seed extract on a 24 hour observation Figure 2 shows that the higher the concentration of soursop seed extract, the higher the death rate of Ae. aegypti L. The solvent n-hexane, ethyl acetate and 70% ethanol and distilled water as a negative control test demonstrated the same activity against larvae of A. aegypti. This indicates that the solvent does not affect the mortality of larvae. Temephos as a positive control, in which the larvicidal activity at a concentration of 1 ppm trials, have demonstrated 100% mortality against larvae of Ae. aegypti L.


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Conclusion The results of phytochemical screening of the seeds of soursop (Annona muricata L.) obtained by the content of secondary metabolites. The test with the larvicidal activity concluded that the 70% ethanol extract of the seeds of the soursop has the highest activity against A. aegypti L. with LC50 values of 97, 462 ppm. Suggestion Ethanol 70% extract of the seeds of soursop (Annona muricata L.) has a good chance to be used as biological insecticides to control mosquito larvae that are environmentally friendly. References Departemen Pertanian Indonesia, 2012. Metode Standar Pengujian Efikasi Pestisida Rumah Tangga dan Pengendalian Vektor. Direktorat Pupuk dan Pestisida dan Direktorat Jenderal Prasarana dan Sarana Pertanian Kementrian Pertanian. p: 20-23 Farnsworth NR, 1966. Biological and phytochemical screening of plants. Journal of pharmaceutica, Sci. 55(3): 225-276. Hadi dan Upik Kesumawati, 2005. Penyakit Tular Vektor: Demam Berdarah Dengue. Bogor: Fakultas Kedokteran Hewan IPB; Kaihena M, Vika L, Maria N, 2012. Efektivitas ekstrak etanol daun sirih (Piper betle L) terhadap mortalitas larva nyamuk Anopheles sp dan Culex. Molucca Medica, Volume 4, Nomor 1, hlm. 88-105. Mulyawati AP, Hayati EK, Nashihuddin A, Tukimin, 2010. Uji efektivitas dan identifikasi senyawa ekstrak biji sirsak (Annona muricata Linn.) yang besifat bioaktif insektisida nabati terhadap hama thrips. Alchemy, 2(1): 104-1575. Nopitasari, 2013. Uji aktivitas ekstrak biji langsat (Lansium domesticum Cor.) sebagai larvasida Aedes aegypti. (Skripsi) Universitas Tanjungpura, p 12-14 Palgunadi BU, Asih Rahayu, 2011. Aedes aegypti sebagai vektor penyakit demam berdarah dengue. Surabaya: Universitas Wijaya Kusuma. diakses dari Demam Berdarah Dengue. Universitas Wijaya Kusuma Surabaya. http://elib.fk.uwks.ac.id/asset/archieve/jurnal/vol2.no1.Januari2011/AE DES%20AEGYPTI%20SEBAGAI%20VEKTOR%20PENYAKIT%20 DEMAM%20BERDARAH%20DENGUE.pdf. Diakses tanggal 13 Januari 2015. Riswanto S, 2009. Uji efektivitas pestisida nabati terhadap hama Spodoptera litura (Lepidoptera: Noctuidae) pada tanaman tembakau (Nicotiaana tabaccum L.). (Skripsi) Medan: Universitas Sumatera Utara, p 20-23 Rosmayanti K, 2014. Uji Efektivitas Ekstrak Biji Sirsak (Annona muricata L ) sebagai Larvasida Pada Larva Aedes segypti Instar III/IV (skripsi). Jakarta: Fakultas Kedokteran Dan Ilmu Kesehatan Universitas Islam Negeri Syarif Hidayatullah; p 1-3, 16-17, 37-40


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Susanti PD, Danang B, Dini S, Susilawati. 2013. Penggunaan ekstrak kulit kayu gemor (Nothaphoebe coriacea K.) sebagai larvasida hayati terhadap tingkat mortalitas jentik nyamuk Aedes aegypti serta dampaknya pada kualitas air hujan. 9:117– 122. Susilowati D, Rahayu MP, Prastiwi R. Efek Penolak Serangga dan Larvasida Ekstrak Daun Jeruk Purut Citrus hystrix terhadap Aedes aegypti. Jurnal Biomedika. 2009; 2 (1): 56–65. Taslimah, 2014. Uji efikasi biji srikaya (Annona squamosa L.) sebagai bioinsektisida dalam upaya integrated vector management terhadap Aedes aegypti (skripsi). Jakarta: Fakultas Kedokteran Dan Ilmu Kesehatan Universitas Islam Negeri Syarif Hidayatullah; p.1-6, 23-28,76-81 Venugopala KN, Raquel MG, Kabange K, Bandar EA, Mahesh VA, Bharti O, 2014. Evaluation of halogenated coumarins for antimosquito properties. Hindawi Publishing Corporation The Scientific World Journal, Vol. 2014 Article ID 189824, 6 p. Wardhana AH, Amir H, Muharsini S, dan Yuningsih, Veteriner BP, 2006. Uji keefektifan biji sirsak (Annona muricata) dan akar tuba (Derris edliptica) terhadap larva Chrysomya bezziana secara in vitro. 1013-1017 Available at: http://peternakan.litbang.deptan.go.id/publikasi/semnas/pro06-152.pdf. Diakses tanggal 21 Februari 2015. Wardhana A.H, Amir H, dan J.Manurung, Veteriner BP, 2005. uji efikasi ekstrak heksan daging biji srikaya (Annona squamosa L) terhadap pertumbuhan larva lalat Chrysomya bezziana secara In Vitro. Seminar Nasional Teknologi Peternakan dan Veteriner; 170-177. Wardhana A.H, Amir H, dan J.Manurung, Veteriner BP, 2005. Efektifitas ekstrak biji srikaya ( Annona squamosa L) dengan pelarut air, metanol dan heksan terhadap mortalitas larva caplak Boophilus microplus secara In Vitro, Jurnal Ilmu ternak dan Veteriner, 10(2):134–142. World Health Organization, 2010. Dengue: Guidelines for Diagnosis, Trearment, Prevention and Control, New edition. Swiss. p 5.


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IDENTIFICATION OF SOURSOP SEEDS (Annona muricata L.)EXTRACT AS A CANDIDATE AGAINST THE Aedes aegypti L. MUSQUITO DBD VECTOR CONTROL

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