Volume 8, No.2, Agustus 2015 63 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
Volume 8, No.2, Agustus 2015 64 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).
Volume 8, No.2, Agustus 2015 65 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
Volume 8, No.2, Agustus 2015 66 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
Volume 8, No.2, Agustus 2015 67 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
Volume 8, No.2, Agustus 2015 68 Table 1. Result of phytochemical screening of soursop seeds (Annona muricata
L.) in powder and extract
No Secondary Metabolites Soursop seed powder Ethanol 70% Extract Ethyl acetate Extract N-heksan Extract
1 Alkaloids - - - -
2 Flavonoids - - - -
3 Saponins + + - -
4 Kuinon - - - -
5 Tannin - - - -
6 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.
Concentra-tion (ppm)
% Mortality
Type Solvent Control
n-hexane Ethyl acetate Ethanol 70% Negative (Solvent)
Positive (Temephos
1 ppm)
750 93,35 95 100 0 100
500 78,35 88,35 100 0 100
250 50 73,35 98,35 0 100
125 20 65 58,35 0 100
50 15 40 45 0 100
LC50
(ppm)
198,610 74,798 67,042 - -
Linear regression
a = -115,7371 b = 70,9888 r = 0,9654
a = -34,9054 b = 45,5671 r = 0,9926
a = -43,2775 b = 52,5234 r = 0,9302
Volume 8, No.2, Agustus 2015 69 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.
Volume 8, No.2, Agustus 2015 70 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.
Volume 8, No.2, Agustus 2015 71 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.
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