Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Bioactivity Evaluation of cajuputi (: ) Crude Extracts against Aedes Mosquito Azlinda Abu Bakar1,2

1Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains , 16150 Kubang Kerian, Kelantan 2Department of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur

ABSTRACT Melaleuca cajuputi crude extract in four different solvents viz dichloromethane, ethyl acetate, hexane, and methanol were evaluated for their insecticidal properties against Aedes aegypti and Aedes albopictus mosquito. Bioassay against larva and adult mosquito was done following World Health Organization’s guidelines. Late 3rd and/or early 4th instar of Aedes larvae were assayed for different concentrations ranging from 10 to 120 mg/L of M. cajuputi crude extract. Larvicidal effects were observed in dichloromethane, hexane, and methanol. Dichloromethane gave the highest of mean mortality, against Ae. aegypti (12.6 ± 0.98) and Ae. albopictus (10.2 ± 0.37) with LC50 of 104.8 mg/L and 106 mg/L, respectively. The adulticidal bioassay was tested against 3 - 5 days old of female mosquitoes with the range concentrations from 0.04 to 6.21 mg/cm2. Amongst solvents used, extracts of dichloromethane and hexane showed effects against the adult mosquito. 2 Extract in hexane gave 100% mortality against both Aedes with LC50 of 0.015 mg/cm (Ae. aegypti) and 0.022 mg/cm2 (Ae. albopictus). In conclusion, the extract of M. cajuputi could be exploited in the development of potential -based products in controlling dengue Aedes vectors, particularly in the ARTICLE INFO adult mosquito. Article history: Received: 4 February 2020 Keywords: Aedes sp., bioactivity, crude extracts, Accepted: 27 April 2020 Published: 28 August 2020 Melaleuca cajuputi, solvents polarity

E-mail address: Current affiliation [email protected] School of Biological Sciences, Universiti Sains Malaysia, 11800 Gelugor, Pulau Pinang ISSN: 1511-3701 © Universiti Putra Malaysia Press e-ISSN 2231-8542 Azlinda Abu Bakar

INTRODUCTION screening of various plant extracts in In Asia, the mosquito Aedes aegypti is a Malaysia, had shown that Melaleuca was primary vector for dengue and chikungunya the most effective when tested against (Sam & Abu Bakar, 2006). Meanwhile, its Aedes spp. larva in the laboratory (Bakar related taxon Aedes albopictus has been et al., 2018). Nevertheless, evaluation of recognized as a secondary vector for dengue the essential oils of the same plant had also and serves as a vector competence in the shown its potential insecticidal effects as maintenance of dengue, and chikungunya well (Abu Bakar et al., 2012; Bakar et al., virus in areas where Ae. aegypti is less 2019). According to Lowe’s report (as cited abundant or absent (Effler et al., 2005; in De Monte et al., 2014, p. 63), there are Grard et al., 2014; Li et al., 2012; Wong et many different extractive techniques and al., 2013). The global widespread of vector approaches working together with various mosquitoes such as Aedes in certain tropical methodologies and solvents to improve and sub-tropical areas causes a major the recovery and, the pharmacological outbreak of dengue and other mosquito- profile of their extract products. However, borne disease-related illness (Rezza, 2014). as a result of the differences among the Utilization of insecticides in the strategy extractive processes and methods, there for disease outbreak control is undeniably is a discrepancy in the qualitative and effective, due to cost-effective, immediate quantitative composition of the extracts action, and high efficiency against a obtained from the same plant. broad range of vectors. Unfortunately, the It is known that various solvents of effectiveness is threatened by negative and different polarities would extract different harmful side effects on human, non-target phenolic compounds from with a high animals, and the environment have become degree of accuracy (Wong & Kitts, 2006). apparent. Nevertheless, the development Furthermore, previous studies have shown of resistance among vector populations that solvents with a high polarity such as (Hamid et al., 2018) is the biggest threat methanol displayed high effectiveness to the program’s efficacy. Thus, the urge as antioxidants (Altemimi et al., 2017). and interest in searching less hazardous The objective of this present study was to alternatives of vector/pest control from plant evaluate the in vitro bioactivity of Melaleuca resources are therefore being renewed and cajuputi plant extracts derived from four continues today. different solvents polarity viz. hexane (non- To date, many potential plant species polar), dichloromethane (moderately polar), with known insecticidal properties and ethyl acetate (polar), and methanol (polar) phytochemicals which are rich with against larvae and adult of dengue vector biodegradable active compounds are being mosquitoes, Ae. aegypti and Ae. albopictus. screened and evaluated (Sharma et al., 2006). For instance, a study on the bioactivity

304 Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020) The Effect ofM. cajuputi Extracts against Aedes

MATERIALS AND METHODS dichloromethane, and methanol with the Collection of Plant Specimens ratio of 1g sample to 10 mL solvent in a 10L plastic container. The samples were shaken The of M. cajuputi were collected and mixed vigorously and left to sit for 72 from Port Dickson, Negeri Sembilan area, hrs. The extracts were filtered through glass 2° 31′ 21.1440″ N, 101° 47′ 46.6620″ E in funnel with filter paper Whatman No.1. Malaysia. Port Dickson is located 120 km The extract was concentrated using rotary towards the south from Kuala Lumpur 3° evaporator type EYELA (N-1001S-WD, 8’ 27.0708’’ N and 101° 41’ 35.5452’’ E. Japan) at 45°C for eight hrs. The residue The voucher specimen was sent to Forest obtained was kept in an amber glass vial Research Institute of Malaysia (FRIM) in to be used for subsequent bioassay testing. Kepong, Selangor for species confirmation and specimen deposited at the herbarium. Larval Bioassay

Preparation of Mosquito The larval bioassay was following the standard guidelines (World Health Laboratory strain mosquito was obtained Organization [WHO], 2005). Five (5) from Vector Control Research Unit, School different concentrations of extracts were of Biological Sciences, Universiti Sains prepared at 10, 50, 80, 100, and 120 mg/L. Malaysia, Pulau Pinang in the form of A 10 mL stock solution was prepared at a eggs. The mosquito colony was cultured concentration of 100,000 mg/L (100,000 and reared in the laboratory under the ppm) and kept in a refrigerator at 4 - 5 °C. optimized condition: relative humidity (RH) Five replicates of 20 late third instar larvae 80% ± 5% and room temperature 28.5 °C were used in each bioassay. The numbers ± 2 °C. During the maintenance period, of dead larvae were counted after 24 hrs. mosquitoes’ larvae were provided with of exposure. Positive and negative control prepared powdered food which contains solutions were prepared by mixing 1mL cat’s biscuit, powder milk, grounded (dried) solvent in 199mL of distilled water and cow liver, yeast, and vitamin B complex. 2 mL of acetone in 198 mL of distilled The mosquito colony was continuously water respectively. During the observation, maintained throughout the study period. food was not supplied to the larvae. The

lethal concentrations (LC50 and LC90) were Preparation of the Crude Extracts calculated by probit analysis (Finney, 1971). The freshly collected leaves of M. cajuputi were dried at room temperature (29 - 31°C) Adulticidal Bioassay for 5 - 7 days. The dried leaves were Bioassay of adulticide was performed as grounded mechanically using a household described in the WHO (2016) guideline. blender. The grounded leaves were extracted Five (5) different concentrations of 2.0 mL with solvents viz: hexane, ethyl acetate, plant extracts of 0.04, 0.08, 0.12, 2.48, and

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6.21 mg/cm2 were applied homogeneously ANOVA. Results with the value of p ≤ 0.05 at the filter papers Whatman No 1 (12 x 15 were reported to be statistically significant. cm). The control paper was treated with All data were analyzed and calculated using 2.0 mL acetone. The impregnated papers SPSS statistics software. were dried at room temperature for 24 hrs and kept (4 - 5°C) in an aluminum foil. RESULTS Four replicates of twenty-five female (3 - 5 The insecticidal bioefficacy of the M. days old, blood starved) mosquitoes were cajuputi crude extracts of dichloromethane, aspirated from the mosquito cage into a ethyl acetate, hexane, and methanol were plastic holding tube. The mosquitoes were tested at 10 mg/L, 50 mg/L, 80 mg/L, 100 allowed to acclimatize in the tube for 1 mg/L, and 120 mg/L against dengue vectors, hr. and later were exposed to the treated Ae. aegypti and Ae. albopictus. Table 1 impregnated filter paper for 1 hr. At the end summarizes the bioactivity against larvae of the 1 hr. exposure period, the mosquitoes and adults’ stage of Aedes vectors. From the were transferred back to the holding tube results obtained, dichloromethane, hexane, and kept for mortality observation for 24 hrs. and methanol showed some larvicidal A pad of cotton wool soaked in 10% sugar effects when tested against Aedes larvae. water was placed on the mesh-screen. The Meanwhile, adulticidal effects were only number of moribund and dead mosquitoes observed in dichloromethane and methanol. was recorded at intervals of 1, 5, 10, 15, 20, The bioassay test against larvae and adults 25, 30, 45-, and 60-minutes post-exposure. Aedes mosquito showed a significant Any knocked down mosquitoes, were increase in the mortality percentage (%) considered moribund and counted as dead. A with the increase of concentration. mosquito was classified as dead or knocked In Table 2, the paired t-test and the one- down if it is immobile or unable to stand or way analysis of variance (ANOVA) were take off. analyzed in mean mortality of Aedes sp. larvae, and solvents used. Statistical analysis Statistical Analysis of one-way ANOVA revealed no significant Percentage mortality that lies between 5% difference between and within groups to 20% will be corrected using Abbott’s among solvents and Aedes sp. Meanwhile formula (Abbot, 1925). Larvicidal and paired t-test between solvents and Aedes sp. adulticidal effects were reported in median showed a significant difference (p ≤ 0.05) in lethal concentration (LC50) with a 95% hexane (p = 0.04) and methanol (p=0.003) confidence interval subjected to a log probit solvent respectively. The highest larvicidal analysis test. The comparative effectiveness activity was observed in dichloromethane of crude extracts among different types of against Aedes sp. with the LC50 values of solvents and Aedes mean mortality were 104.8 mg/L and 106.0 mg/L for Ae. aegypti analyzed using paired t-test and one-way and Ae. albopictus at 24 hrs. respectively

306 Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020) The Effect ofM. cajuputi Extracts against Aedes

Table 1 Bioactivity of Melaleuca cajuputi crude extracts against Ae. aegypti and Ae. albopictus Solvents Larvicidal Adulticidal Dichloromethane √ √ Ethyl acetate - - Hexane √ √ Methanol √ -

Note. √ toxic effects

Table 2 Mean mortality (± SE) of M. cajuputi crude extracts against larvae of Ae. aegypti and Ae. albopictus Aedes sp. Dose *Mean mortality ± SE (mg/L) Dichloromethane1 Hexane2 Methanol3 aAe. aegypti 10 1.20 ± 0.20 0 1.40 ± 0.25 50 3.40 ± 0.25 1.20 ± 0.20 1.60 ± 0.25 80 6.80 ± 0.37 4.20 ± 0.37 2.40 ± 0.40 100 9.80 ± 0.66 5.40 ± 0.51 2.80 ± 0.20 120 12.6 ± 0.98 6.60 ± 0.51 3.00 ± 0.32 Control** 0 0 0 bAe. albopictus 10 0 0 0 50 0 0 0 80 7.40 ± 0.25 0.60 ± 0.25 0 100 10.0 ± 0.32 1.00 ± 0.32 1.60 ± 0.25 120 10.2 ± 0.37 2.00 ± 0.32 2.00 ± 0.00 Control** 0 0 0

Note. *Mean value of five replicates Control** = acetone 0.1% 1, a,b No significant difference 1, 2, 3, a No significant difference 2, a, b Significant differencep < 0.05 1, 2, 3, b No significant difference (p = 0.04)

(Table 3). Figures 1 and 2 show the highest albopictus are presented in Table 4. There percentage mortality values of 63% in Ae. was a significant difference between hexane aegypti and 51% in Ae. albopictus at 120 and dichloromethane in mean mortality of mg/L of dichloromethane extract in M. Aedes spp. with the p-values of 0.007 for cajuputi. Ae. aegypti and 0.003 for Ae. albopictus. The results of the adulticidal activity However, no significant difference was of hexane and dichloromethane extracts observed in mean mortality between Aedes of M. cajuputi against Ae. aegypti and Ae. spp. in each solvent used, hexane and

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Table 3 LC values of M. cajuputi crude extracts against Aedes sp. larvae Ae. aegypti Ae. albopictus

2 2 Solvents LC50 95% df χ LC50 95% df χ (mg/L) Confidence (mg/L) Confidence Limit Limit LCL UCL LCL UCL Dichloromethane 104.8 94.9 117.8 2 0.43 106.0 N/A N/A 2 15.9

Hexane 164.4 132.1 260.3 2 0.71 429.0 N/A N/A 2 1.60

Methanol 349.5 N/A N/A 2 0.19 N/A N/A N/A N/A N/A Note. LCL = lower confidence limit; UCL = upper confidence limit; df = degree of freedom

80 70 63 60 50 50

40 34 33 27 30 21 17 15 20 12 14 6 7 8 Mortality (%) 10 6 0 0 -10 10 50 80 100 120 -20 Hexane Dose Concentration (mg/L) Dichloromethane Methanol Figure 1. Percentage mortality of M. cajuputi crude extracts against Ae. aegypti larvae

70 60 50 51 50 37 40 30 20 8 10 10 Mortality (%) 10 3 5 0 0 0 0 0 0 0 0 0 50 80 100 120 -10 -20 Hexane Dose Concentration (mg/L) Dichloromethane Methanol Figure 2. Percentage mortality of M. cajuputi crude extracts against Ae. albopictus larvae

308 Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020) The Effect ofM. cajuputi Extracts against Aedes dichloromethane. The probit analysis of extract against Ae. albopictus showed

95% confidence limits LC50 (UCL-LCL) minimal larvicidal effects after 24 hr. was also calculated and presented in Table 5. of exposure at various concentrations. The chi-square values were not significantly Meanwhile, observation in adulticidal different at p ≤ 0.05. Among the solvents assays using dichloromethane, ethyl used, hexane gives lower LC50 of 0.015 mg/ acetate, hexane, and methanol showed cm2 (0.005-0.025) and 0.022 mg/cm2 (0.009- nonconformity with the results of the 0.003) in Ae. aegypti and Ae. albopictus, larvicidal assays. Of these, dichloromethane respectively. and hexane extracts of M. cajuputi showed In this study, the results showed that adulticidal effects against Ae. aegypti and bioactivity of M. cajuputi crude extracts Ae. albopictus. However, hexane extract was against Aedes spp. was varied according the most effective against Ae. aegypti and to the species, stage of life, and solvents Ae. albopictus adult’s mosquito. From Table used (Tables 1, 2, and 4). The extract of 4, more than 50% mortality was observed in M. cajuputi in dichloromethane possessed Ae. aegypti (74%) and Ae. albopictus (69%) moderate effects against Ae. aegypti at lowest concentration of 0.04 mg/cm2 and larvae. On the other hand, the sensitivity 100% mortality when exposed at higher of dichloromethane, hexane, and methanol concentrations of 2.48 and 6.21 mg/cm2.

Table 4 Adulticidal effects ofM. cajuputi crude extracts against Aedes aegypti and Aedes albopictus Solvents Dose 1 Ae. aegypti 2Ae. albopictus 2 Used (mg/cm ) Mortality *Mean mortality Mortality *Mean mortality (%) ± SD (%) ± SD aHexane 0.04 74 18.50 ± 1.29 69 17.25 ± 1.26 N=100 0.08 77 19.25 ± 0.96 71 17.75 ± 1.89 0.12 79 19.75 ± 0.96 89 22.25 ± 3.30 2.48 100 25.00 ± 0.00 100 25.00 ± 0.00 6.21 100 25.00 ± 0.00 100 25.00 ± 0.00 **Control 0 0 0 0 bDichloromethane 0.04 31 7.75 ± 1.15 54 13.50 ± 3.00 N=100 0.08 50 12.50 ± 3.00 56 14.00 ± 2.16 0.12 55 13.75 ± 0.96 57 16.75 ± 2.50 2.48 80 20.00 ± 1.83 80 20.00 ± 0.00 6.21 87 19.25 ± 6.29 81 20.20 ± 0.5 **Control 0 0 0 0

Note. *Mean value of four replicates **Control=acetone 0.1% 1, 2, a No significant differencep > 0.05 1, a, b Significant differencep ≤ 0.05 (p = 0.007) 1, 2, b No significant differencep > 0.05 2, a, b Significant differencep ≤ 0.05 (p = 0.003)

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Table 5 LC values of M. cajuputi crude extracts against Aedes sp. adults Ae. aegypti Ae. albopictus

2 2 Solvents LC50 95% df χ LC50 95% df χ (mg/L) Confidence (mg/L) Confidence Limit Limit LCL UCL LCL UCL Dichloromethane 0.116 0.071 0.176 3 3.73 0.030 0.007 0.069 3 0.83

Hexane 0.015 0.005 0.025 3 4.57 0.022 0.009 0.033 3 5.86

Note. LCL = lower confidence limit; UCL = upper confidence limit; df = degree of freedom

DISCUSSION years ago (Walling, 1957). Most chemical Many researchers have reported the potential reactions that are carried out in laboratories of plant extracts for controlling mosquito- or the industry are in the form of solutions. borne diseases (Ghosh et al., 2012; Kamaraj Hence the proper and appropriate solvent et al., 2010; Rehimi et al., 2011). Up to date, selection as one of the reaction parameters is there are now more than 2,000 potential important for a good and successful reaction plant species that have been evaluated for (Reichardt, 2005). According to Rawani et their insecticidal properties worldwide al. (2010), phytochemicals found in plants (Maiza et al., 2013; Roark, 1947; Shaalan may play an important role (bioactivities) in et al., 2005; Sukumar et al., 1991). In some vector control if applied appropriately. The developing countries, pesticidal plants phytochemicals in plants can be obtained offer unique and challenging opportunities from the whole plant or specific parts of for the exploration and development of the plant with different solvents such as their botanical sources. Furthermore, one petroleum ether, benzene, chlorophyll, of the most important factors affecting the methanol, and acetone. benefits and efficiency of bioactivity from Ghosh et al. (2012) showed that the plant materials and their consequent health extraction of active biochemical from plants is the extraction solvents used (Ngo et al., depended upon the polarity of the solvents. 2017). Thus, the selection of solvents used Polar solvents will extract polar molecules depends on the purpose either as of choice and non-polar solvents extract non-polar for yielding high content or for specific molecules. In this study, hexane (polarity extraction of phytochemical compounds index of 0.1), dichloromethane (polarity that would be useful for its medicinal and/ index of 3.1), ethyl acetate (polarity index or insecticidal properties. of 4.4), and methanol (polarity index The polarity effect depends on the of 5.1) (Corradini et al., 1998; Harris, reactivity and selectivity of radical 2015) had been used to investigate the chemistry that has been identified over 50 insecticidal properties of M. cajuputi extract

310 Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020) The Effect ofM. cajuputi Extracts against Aedes against Ae. aegypti and Ae. albopictus. in different solvents used can be carried out From the results obtained, hexane and to characterize the insecticidal properties in dichloromethane solvents that had lower different research settings. It can be used as a to moderate polarity index were observed solution of variants efficacy in a situation of to give moderate effects against Aedes chemical instability of whole or unprocessed mosquito. These findings agree with the plant products. previous study by Ghosh et al. (2012), which described the efficacy of solvents polarity ACKNOWLEDGEMENTS in the bioassays. Biochemical extracted The author is thankful to the Universiti Sains using moderate polarity index solvents Malaysia and Ministry of Higher Education showed good results in a few bioassays. for the scholarship program. The gratitude This study revealed the bioactivity variance is also extended to Dr. Sallehudin Sulaiman of M. cajuputi crude extracts when tested and Dr. Rasadah Mat Ali for their guidance against larvae and adults of Aedes sp. Even and advice during the research, and for though larvicidal effects were observed in providing the laboratory facility at the Forest dichloromethane, hexane, and methanol Research Institute Malaysia. This research extracts of M. cajuputi, the effectiveness did not receive any specific grant from was slightly weak. However, the adulticidal funding agencies in the public, commercial, activity showed good effects against Ae. or not-for-profit sectors. aegypti and Ae. albopictus. REFERENCES CONCLUSION Abbott, W. S. (1925). A method of computing the The bioactivity of crude plant extracts is effectiveness of an insecticide. Journal of characterized by a mixture of complex Economic Entomology, 18(2), 265-266. active compounds. Thus, plants containing Abu Bakar, A., Sulaiman, S., Omar, B., & Mat Ali, beneficial phytochemicals may supplement R. (2012). Evaluation of Melaleuca cajuputi and would be useful plant-based insecticides (Family: Myrtaceae) essential oil in aerosol spray for future development. Variations of cans against dengue vectors in low cost housing flats. Journal of Arthropod Borne Diseases, an insecticidal potential of M. cajuputi 5(2), 28-35. crude extract varied with the different solvents used in the extraction process. Altemimi, A., Lakhssassi, N., Baharlouei, A., Watson, D. G., & Lightfoot, D. A. (2017). Due to the variation’s efficacy in the Phytochemicals: Extraction, isolation, and larvicidal and adulticidal effects against identification of bioactive compounds from Ae. aegypti and Ae. albopictus mosquitoes. plant extracts. Plants, 6(4), 42. doi: 10.3390/ More work is still needed to confirm its plants6040042 effectiveness, especially in the field. A Bakar, A. A., Sulaiman, S., Omar, B., & Ali, R. M. study on the phytochemicals of an active (2018). Screening of five plant extracts for compound of the M. cajuputi crude extract larvicidal efficacy against larvae ofAedes aegypti

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