Larvicidal activity of Persea americana Mill. against Aedes aegypti

Larvicidal activity of Persea americana Mill. against Aedes aegypti

Asian Pac J Trop Med 2014; 7(Suppl 1): S167-S170 S167 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal ...

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Asian Pac J Trop Med 2014; 7(Suppl 1): S167-S170

S167

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm

doi: 10.1016/S1995-7645(14)60225-X

Document heading

Larvicidal

activity of Persea americana Mill. against Aedes aegypti 1,2*

Rosalinda C. Torres

, Alicia G. Garbo1, Rikkamae Zinca Marie L. Walde2

Industrial Technology Development Institute, Department of Science and Technology, General Santos Ave., Bicutan, Taguig City 1631, Philippines

1

National Research Council of the Philippines, Department of Science and Technology, General Santos Ave., Bicutan, Taguig City 1631, Philippines

2

ARTICLE INFO

ABSTRACT

Article history: Received 23 Jun 2014 Received in revised form 6 Jul 2014 Accepted 19 Jul 2014 Available online 20 Aug 2014

Objective: To evaluate the toxicity of the ethanol and hexane extracts of the different parts of Persea americana Mill. (P. americana) toward third and fourth instars larvae of Aedes aegypti (Ae. aegypti) and to characterize the ethanol extract by qualitative phytochemical analysis. Methods: The seeds, peels and pulp of P. americana were processed for crude extraction using 95% ethanol and n-hexane. Crude extracts were bio-assayed for larvicidal activity against Ae. aegypti following the World Health Organization standard bioassay method. The mortality was observed at 24 h and 48 h after treatment and data were subjected to probit analysis to determine lethal concentrations (LC50 and LC90). The ethanol extract was characterized by phytochemical analysis. Results: Both the hexane and ethanol extracts from the different parts of P. americana exhibited evidence of larvicidal toxicity. The hexane extract from the seeds exhibited the highest toxicity with LC50 and LC90 values of 9.82 mg/L and 22.19 mg/L, respectively, while the ethanol seed extract exhibited LC50 of 16.48 mg/L and LC90 45.77 mg/L, respectively. This was closely followed by the ethanol extract of the peels with an LC50 of 10.35 mg/L and LC90 of 26.29 mg/L. The pulp extracted with ethanol also yielded great larvicidal toxicity with LC50 of 21.32 mg/L and LC90 of 59.45 mg/L. Results of the phytochemical analysis of the ethanol seed extract indicated presence of alkaloids, tannins, saponins, unsaturated steroids and triterpenoids, flavonoids (leucoanthocyanins), fats and oils. Conclusions: Both the hexane and ethanol extracts of P. americana showed promising potential as an alternative source of a more sustainable, non-toxic and environmentally friendly solution for the control of dengue vector, Ae. aegypti.

Keywords:

Persea americana Dengue vector Aedes aegypti Larvicidal bioassay Phytochemical analysis

1. Introduction M osquito-borne diseases contribute significantly to disease burden, death, poverty, and social debility all over the world, particularly in tropical countries[1]. Among these diseases, dengue remains the most serious vectorborne disease in the Philippines. Dengue is a severe, often fatal, most rapidly emerging febrile disease transmitted by female Aedes aegypti (Ae. aegypti) mosquitoes. It is an acute infection that kills much faster than AIDS. In the Philippines, dengue fever and dengue hemorrhagic fever are widespread in all its regions according to World Health Organization. Treatment is mainly supportive as there is no specific therapy available to date. These are the reasons *Corresponding author: Rosalinda C. Torres, Ph.D., Chemicals and Energy Division, Industrial Technology Development Institute, Department of Science and Technology, General Santos Ave., Bicutan, Taguig City, Metro Manila 1631, Philippines. Tel: (632) 837-20-71 loc. 2226 E-mail: [email protected] Foundation Project: Supported by the National Research Council of the Philippines of the Department of Science and Technology, Taguig City, Metro Manila 1631, Philippines.

behind the efforts of many institution and research bodies to find a solution in controlling the vector of dengue, Ae. aegypti. Chemical control is said to be an effective way used generally in daily life[2], but alarms environmental and human advocates since widespread use of synthetic insecticides has led to many negative consequences[3]. This results in the increasing attention and consideration to natural products [4]. G reen larvicides are now being considered because plants consist of several bioactive components that are of low toxicity and biodegradable. Unlike the conventional insecticide which is based on single active ingredient, green insecticides comprise botanical blends of bioactive chemical compounds which act concertedly on both behavioural and physiological processes[5]. The decades of research about interactions between plants and insects revealed the potential use of plants in fundamental pest control programs[6]. Persea americana M ill. ( P. americana ) from the L auraceae family is commonly known as avocado and alligator pear. It is a much more branched, medium-sized tree with shiny evergreen, elliptic leaves and delicious

Rosalinda C. Torres et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S167-S170

fruit[7]. Originally, it was only native to humid tropical areas of Mexico until it was later cultivated to other regions of Latin America as well as to USA and Europe. Today, the fruits are extensively cultivated worldwide on large scale in various subtropical countries such as the Philippines[8]. O ur main focus is now on the research study of P. americana as a potential vector control measure against Ae. aegypti that is acceptable to the populace, cost effective and more importantly, safe for the environment. The use of natural alternatives such as plant extracts can provide the best option as environmentally safe natural larvicides. Therefore, the objectives of the study were to evaluate the toxicity of the ethanol and hexane extracts of the different parts of P. americana Mill. against third and fourth instars larvae of Ae. aegypti and to characterize the ethanol extract by qualitative phytochemical analysis. 2. Materials and methods 2.1. Botanical material T he fruits of P. americana were collected in seven different localities in the Philippines including Davao, M indoro, B atangas, N egros O riental, I loilo, P alawan, and Cavite. The seeds, peels and pulp of the plant were processed for crude extraction and larvicidal bioassay. Specimen labels were placed in each plant. Herbarium specimens of the plants collected were prepared and sent to the National Museum of the Philippines for necessary identification and authentication.

2.2. Sample preparation The seeds, peels and pulp of P. americana were separated and processed individually. Both fresh and dried plant materials were used in the study. These were ground with either Waring blender or Wiley mill. About 300 g of plant materials were macerated with 900 m L of ethanol and n-hexane for 48 h. Then, the mixture was filtered with a

coarse filter paper and the filtrate was concentrated under vacuum at 55 °C to a syrupy consistency. This was further evaporated in a water bath at 55 °C until completely dried. 2.3. Rearing of Ae. aegypti larvae

The eggs of Ae. aegypti were reared in the Entomology L aboratory of the I ndustrial T echnology D evelopment Institute, Department of Science and Technology, Taguig City, Metro Manila 1631, Philippines. The egg rafts were kept

in the basin containing tap water that served as the culture medium at (28±2) °C and a photoperiod of 12 h light followed by 12 h dark (12L: 12D). Appropriate amount of brewer’s yeast were added to enhance the growth of the larvae. The third and fourth instars larvae were used in the study. 2.4. Larvicidal bioassay against third and fourth instars larvae of Ae. aegypti The larvicidal bioassay was conducted following the World

Health Organization method[9]. Batches of 20 third or fourth

instars larvae were transferred by means of droppers to small disposable test vessels, each containing 100 mL of water. The appropriate volume of dilution was added to 100 mL water in the cups to obtain the desired target dosage, starting with the lowest concentration. The test containers were held at (28±2) °C and a photoperiod of 12 h light followed by 12 h dark (12L: 12D). Four or more replicates were set up for each concentration and an equal number of controls were set up simultaneously with tap water, to which 1 mL ethanol (or the organic solvent used) was added. Each test was run three times on different days. The results were recorded where the LC50 and LC90 values and slope and heterogeneity analysis were also noted. 2.5. Data analysis Data from all replicates were pooled for analysis. LC50 and LC90 values were calculated from a log dosage-probit mortality regression line using computer software programs. S tandard deviation of the means of LC 50 values were calculated and recorded. A test series is valid if the relative standard deviation (or coefficient of variation) is less than 25% or if confidence limits of LC50 overlap (significant level at P<0.05).

2.6. Phytochemical analysis The qualitative phytochemical analysis of the ethanol extract of P. americana seeds was performed according to the method of Guevara[10].

3. Results Figures 1 to 4 show the lethal concentration expressed in LC50 and LC90 values of crude ethanol and hexane extracts

of the seeds, peels and pulp of P. americana against third and fourth instars larvae of Ae. aegypti. Figure 1 shows the lethal concentration of the ethanol seed extracts collected at different plant sites in the Philippines. Results showed that the ethanol extract from Batangas yielded the most potent toxicity effect with LC50 and LC90 values of 16.48 mg/ L and 45.77 mg/L, respectively. This was followed closely by the ethanol extract from Davao with an LC50 of 18.21 mg/ L and LC90 of 41.38 mg/L, and the ethanol extract from Negros Oriental with an LC50 value of 20.61 mg/L and LC90 of 44.39 mg/L, respectively. 251

Lethal concentration (mg/L)

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201

151

101 51

1

Batangas LC50

Davao Negros Oriental Mindoro Sources LC90

Palawan

Iloilo

Figure 1. Comparison of lethal concentration LC50 and LC90 of the ethanol extracts of P. americana seeds from different sources.

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36 31 26 21 16 11 6 1

101 81 61 41 21 1

Davao LC50

LC90

Sources

Palawan

Figure 4. Comparison of lethal concentration LC50 and LC90 of the hexane extracts of P. americana peels from two sources.

LC50

LC90

Palawan Sources

Cavite

Table 1 shows the results of the qualitative phytochemical analysis of the ethanol extract of P. americana seeds. T he presence of different phytochemicals in the seeds of P. americana such as alkaloids, saponins, flavonoids (leucoanthocyanins), unsaturated steroids and triterpenoids, tannins, fats and oils may be attributed to its potent toxicities against the dengue vector, Ae. aegypti. Other parts of P. americana such as peels and pulp were also extracted and tested for larvicidal activity. As shown in Figure 3, the peels from Mindoro yielded the most potent toxicity with LC50 of 10 .35 mg/L and LC90 of 26 .29 mg/L, respectively. 101 81 61 41 21 1

LC50

Mindoro

LC90

Davao Sources

Batangas

Figure 3. Comparison of lethal concentration LC50 and LC90 of the ethanol extracts of P. americana peels from different sources.

As illustrated in Figure 4, the hexane extract of the P. americana peels from Davao exhibited higher toxicity than that from Palawan with an LC50 and LC90 values that ranged

As shown in Figure 5, the P. americana pulp from Davao extracted with ethanol also had great larvicidal toxicity with LC50 of 21.32 mg/L and LC90 of 59.45 mg/L. The pulp from Mindoro extracted with hexane had an LC50 and LC90 values that ranged from 32.77 mg/L to 46.09 mg/L (average=39.43 mg/L) and from 78.49 mg/L to 97.84 mg/L (average=88.17 mg/ L), respectively. Lethal concentration (mg/L)

Davao

Figure 2. Comparison of lethal concentration LC50 and LC90 of the hexane extracts of P. americana seeds from different sources.

Lethal concentration (mg/L)

from 19.87 mg/L to 22.80 mg/L (average=21.34 mg/L) and from mg/L to 44.19 mg/L (average=43.02 mg/L), respectively.

41.84 Lethal concentration (mg/L)

Lethal concentration (mg/L)

A s shown in F igure 2 , the hexane extract from the seeds of P. americana collected from the 3 sites, namely, Davao, Palawan and Cavite exhibited very potent lethal concentrations. The hexane extract from Davao yielded the highest toxicity with LC50 and LC90 values of 9.82 mg/ L and 22.19 mg/L, respectively. This was followed by the one collected in Palawan with LC50 of 13.39 mg/L and LC90 of 21.53 mg/L.

101 81 61 41 21 1

LC50

Davao (ethanol extract) LC90

Mindoro (hexane extract)

Figure 5. Comparison of lethal concentration LC50 and LC90 of the extracts of P. americana pulp from two sources.

4. Discussion P eople have used various plant parts, products and secondary metabolites of plant origin in pest control since early historical times[5]. That is why several studies have been dedicated on testing natural products against mosquitoes specifically, Ae. aegypti, which produced varying results [11-13] . S everal P hilippine plants have been found to have various medicinal properties such as larvicidal properties[14]. In the present study, hexane and ethanol extracts of the seeds, peels and pulp of P. americana were subjected

Table 1 Result of the qualitative phytochemical analysis of the ethanol extract of P. americana seeds. Qualitative tests Mayer’s test Dragendorff’s test Dragendorff’s and Mayer’s test Froth test Na2CO3 test Keller Killani test Bate-Smith &Metcalf test Willstatter “Cyanidin” test FeCl3 test Liebermann-Burchard test Borntrager’s test Spot test

Sources

Results Formation of slightly turbid solution (+) Formation of slightly turbid solution (+) Formation of slightly turbid solution (+) Formation of honeycomb froth greater than 2 cm (+) Non-formation of dense and stable froth (-) Formation of reddish-brown colored interface (+) Formation of strong red colored solution (+) Formation of red color (+) Formation of blue-black colored solution (+) Formation of red colored solution (+) Non-formation of red colored solution (-) Formation of greasy/oily appearance (+)

Indication Presence of alkaloids Presence of alkaloids Absence of quaternary bases and/or amineoxides Presence of saponins Absence of free fatty acids Presence of 2-deoxysugars Presence of leucoanthocyanins Presence of γ-benzopyrone nucleus (flavonoids) Presence of hydrolyzable tannins Presence of unsaturated steroid/triterpenoids Absence of anthraquinones Presence of fats and oils

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to larvicidal toxicity assay. All the plant parts that were studied showed great toxicities against Ae. aegypti, especially the hexane extract from the seeds collected in Davao with LC50 of 9.82 mg/L. One literature reported that the hexane extract of P. americana seeds showed an LC50 8.87 mg/L, similar to the lethal concentration (9.82 mg/L) previously reported in the present study[15]. The extracts were considered to be bioactive since they showed lethal concentrations (LC50 and LC90) extremely lower than 1 000 mg/ L[11]. Based from the results, P. americana from different sources yielded varied results which can be attributed to type of soil, the climate of the source and the season in which the plant was collected. The solvent used can also contribute to the variation since it has been shown that the extraction of active biochemical from plants depends upon the polarity of the solvents used[5]. P hytochemical analysis of the ethanol extract of P. americana seeds showed presence of alkaloids, tannins, saponins, unsaturated steroids and triterpenoids, 2-deoxysugars, flavonoids (leucoanthocyanins), fats and oils. It is said that several groups of phytochemicals such as alkaloids, steroids, terpenoids, essential oils and phenolics from different plants have been reported previously for their insecticidal activities[16]. P lants comprise a vast untapped pool of bioactive phytochemicals that may be widely used in pest control programs instead of the conventional synthetic insectides. The remarkable toxicity effects exhibited by the seeds, peels and pulp of P. americana against the third and fourth instars larvae of Ae. aegypti indicate its potential use as green or natural larvicides for the control of dengue vector. Further study on the plant should therefore be fully explored. It is worthwhile to study extensively the larvicidal property by isolating and identifying the active components that cause larval mortality and then use them in field trials in order to assess their full potential as an alternative to chemical larvicides. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements The National Research Council of the Philippines of the Department of Science and Technology, Taguig City, Metro Manila 1631, Philippines is gratefully acknowledged for providing the research grant, and the Industrial Technology Development Institute of the Department of Science and Technology for the unwavering support in the continuous

implementation of this project. References

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