Mosquito Repellence of Solvent Extracts andActivated Charcoal Obtained from Agro-WasteCoconut Shells

doi:10.21203/rs.3.rs-4076592/v1

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Research Article

Mosquito Repellence of Solvent Extracts andActivated Charcoal Obtained from Agro-WasteCoconut Shells

https://doi.org/10.21203/rs.3.rs-4076592/v1

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Description of the Subject

Burning coconut shells have been used since ages in rural India to ward away mosquitoes; we studied this traditional method in detail in our research using modern techniques such as dry distillation, solvent extraction, chromatography and infrared spectroscopy to decipher its constitution and efficacy.

Objectives

In our research, coconut shells in the form of activated charcoal and chemical extracts obtained from the shell were used to test their mosquito (Aedes aegyptii) repellence and knockdown efficacy using distinct methods.

Methods

Solvent extraction, fractional distillation, column chromatography, Fourier transform infrared spectroscopy, and Peet Grady’s chamber were used as per the WHO guidelines for testing insecticides.

Results

The petroleum ether extract from the solvent extraction technique was found to be most effective, with a 100% mortality rate after 30 mins. The extract mainly contained aromatic compounds, alkanes and ketonic groups.

Conclusion

Activated charcoal obtained from agro-waste coconut shells can be used as an alternative filler in industrially manufactured mosquito coils. The petroleum extract can be used as a great mosquito repellent in contrast to hazardous chemicals already present in the coils.

Agroecology

Biochemical Research Methods

Entomology

Coconut shells

Cocos nucidera

Mosquito repellance

Solvent extracts

Fractional distillation

Mosquitoes are considered pests of humankind in all parts of the world. They serve as a major vector for spreading diseases, not only through pathogens but also through parasites.² Mosquitoes are responsible for the propagation of diseases, specifically malaria, chikungunya, dengue, Japanese encephalitis, West Nile, yellow fever, and Zika, throughout the world.³ Approximately 400,000 deaths are caused per year by malaria.² Although constant developments at generating vaccines for mosquito-borne arboviral diseases are underway, such as inactivated vaccines, viral-vector vaccines, live attenuated vaccines, protein vaccines, or nucleic acid vaccines, approximately half of the world's population is expected to be at risk of arbovirus transmission by 2050.⁴ This raises an alarming need for pest control and eradication or at least control of its propagation vector, i.e., mosquitoes.

Several studies have suggested the use of natural ingredients (including Zanthoxylum limonella, Azadirachta indica, Tinospora rumphii, Citrus grandis, Jatropha curcas, Cymbopogon nardus, and Cocos nucifera) to repel or eradicate mosquito larvae and adults.^2,5–9

In rural India, coconut (Cocos nucifera) coir or shell fibres have been used in bonfires or small flames to ward away mosquitoes; a few references suggest the use of coconut-derived compounds as efficient mosquito repellents.^5–12 This was the very hypothesis for our research, i.e., to find any active component in the traditional ways of repelling mosquitoes and whether it can be used for commercial purposes.

The coconut tree is affectionately called the kalpavriksha—the mythical wish fulfilling tree—by coastal Indians, as each part of the tree can be used for various purposes. In addition to the fruit having nutritious value, research has also shown the use of coconut fibres, kernels, leaves and bark in the production, purification, and isolation of various chemicals: coconut fibres, kernels and bark have been used as industrial fillers in water purification to remove bacteria, organic pollutants, and heavy metals and even in the generation of nanoparticles such as magnetite and palladium. ^12–19 Based on our research and past literature, we assessed the efficiency of coconut shell and its isolated extracts as active ingredients, by part, or as a whole, in terms of mosquito repellent and mosquito-cidal activity. This research is completely novel in its spectrum and bridges the gap between mainstream traditional ways of driving out mosquitoes and the scientific reasons for the similarities based on chemical composition using cutting-edge scientific techniques for extraction and efficacy testing.

Statement of Novelty

By employing modern scientific techniques, our research provides a novel perspective on the traditional practice of burning coconut shells as a mosquito repellent in rural India. Through the application of advanced methods such as dry distillation, solvent extraction, chromatography, and infrared spectroscopy, we delve into the intricate details of the constitution of coconut shells. The innovation lies in the integration of traditional knowledge with contemporary analytical tools, offering a comprehensive understanding of the chemical composition responsible for mosquito repellence. By isolating and characterizing key compounds, our study introduces a fresh and refined approach to age-old practice, opening avenues for the utilization of coconut shell derivatives in mosquito control with enhanced precision and efficacy. To the best of our knowledge, there are no conflicts of scientific interest in the findings of our research.

Statement of Industrial Relevance

Our research holds significant industrial relevance because it provides a sustainable and effective solution for mosquito control derived from agro-waste coconut shells. The identification of activated charcoal and petroleum ether extracts as potent mosquito repellents offers an eco-friendly alternative to conventional insecticides. Activated charcoal sourced from coconut shells has emerged as a promising filler for industrially manufactured mosquito coils, addressing environmental concerns associated with traditional fillers. Furthermore, petroleum ether extract, characterized by its high efficacy and natural composition, is a viable alternative to the hazardous chemicals commonly found in commercial mosquito repellent coils. This breakthrough not only aligns with the growing demand for environmentally friendly pest control solutions but also has the potential to revolutionize the production of mosquito repellent products on an industrial scale.

Preparation of coconut shell charcoal

Approximately 1 kg of Cocos nucifera shells were obtained from local vendors in the Mumbai district. They were peeled, dried and broken into large pieces of approximately 3x3 cm each. The pieces were gradually roasted in an earthen pot apparatus, as shown in Fig. 1, for 8 hrs at high temperatures to remove moisture and organic oils. The organic oils were collected as in (E), which were processed and characterized through solvent extraction and other processes. The shell residue obtained was crushed into fine particles using a mortar and pestle. The particles were filtered through a metal sieve to obtain very fine and uniform particles (diameter of approximately 1 mm).

Activation of charcoal

The charcoal obtained from the above process and wood charcoal (sourced from local vendors) crushed into fine uniform powder were soaked in methanol overnight to separate organic impurities. It was then filtered, dried, and heated at 100℃ in an oven for 3 hrs. This clears off volatile substances and leaves behind pure charcoal carbon.

Solvent extraction

Crude oil was obtained from the dry distillation of the shells (a yield of 1 kg of coconut shells yields approximately 250 cc of crude extract). The obtained crude oil was split for characterization and experimentation. A total of 25 cc of oil was separated by solvent extraction using three solvents with increasing polarities, i.e., petroleum ether (nonpolar), chloroform (polar) and methanol (highly polar). Different extracts obtained using these three solvents were collected and air-dried to evaporate the excess solvent. After complete drying, each extract was named E₁, E₂ or E₃.

Fractional distillation: Part of the crude oil was distilled in a sand bath using a water condenser to obtain distillates D1-D4 at 60°C, 80°C, 90°C and 100°C. These distillates were then characterized further by Fourier transform infrared (FTIR) spectroscopy and silica gel-thin layered chromatography (TLC) to determine their organic composition and purity (supplementary data: D1-D4).

Column chromatography

Part of the dried petroleum ether extract E1 (which showed maximum mortality in the mortality tests) was further characterized using silica gel column chromatography using gradual ratios of the nonpolar polar solvents petroleum ether-chloroform-methanol to obtain 6 fractions, which were further characterized by Fourier transform infrared (FTIR) spectroscopy and silica gel-thin layered chromatography (TLC) to determine their organic composition and purity (supplementary data).

Fourier transform infrared spectroscopy

FTIR, or Fourier transform infrared spectroscopy, is a method employed to acquire the infrared absorption or emission spectra of various substances, including solids, liquids, and gases. An FTIR spectrometer can simultaneously and efficiently capture detailed spectral information across a broad range of infrared wavelengths. This key feature provides a distinct advantage compared to dispersive spectrometers, which record intensity data within a limited range of wavelengths one at a time. The extracts were outsourced at the Haffkine Institute for FTIR, and the results were incorporated into our research.

Preparation of Mosquito Repellent Sticks: To study the correlation between mosquito repellent activity, Jigat (Machilus macrantha: inert adhesive filler) powder, sawdust and coconut or wood charcoal powder were mixed at a ratio of 1:1:1 by weight and kneaded with water to obtain dough. This dough was then rolled onto bamboo sticks (average weight of each stick = 0.5 g) to obtain sticks of uniform length, breadth and weight. They were then air-dried for 24 hrs. The sticks were 11 cm long on average, 0.5 cm wide and 1.5 g in weight. The dried extracts E1-E3 obtained after solvent extraction were dissolved in methanol, and the prepared sticks were dipped in their respective solutions and air-dried to evaporate the methanol.

Mosquito repellence and mortality test: The mosquito repellent tests were performed at the Haffkine Institute, Mumbai, under the WHO guidelines for testing the efficacy of insecticide products on mosquitoes.¹ Tests were performed on 50 non-blood-fed, 2–5-day-old adult female Aedes aegypti mosquitoes in an internal volume of 180 cc in a Peet Grady chamber at 27°C ± 2°C, 80% ± 10% relative humidity (RH) and a photoperiod of 12:12 h (light:dark). The repellent sticks were lit after the mosquitoes were kept captivated in the chamber at T = 0, and the tests were carried out for a total of 30 min from being incensed. Knockdown readings were recorded every 5 min up to T = 30 min, and the mortality of the mosquitoes was calculated by the following formula:

Mortality (%) = No. of mosquitoes knocked out (for 24 hr)/Total number of mosquitoes in the chamber × 100

As shown in Fig. 2, in the coconut charcoal group, the petroleum ether extract had the greatest effect on 100% mosquito mortality within 30 minutes of the test, whereas chloroform and methanol had 37.5% and 0% mortality, respectively. The mortality rates for the wooden charcoal group with extracted petroleum ether extracts were 62.5%, and the mortality rates for the chloroform and methanol subgroups were zero. Among the tested treatments, the crude coconut shell extract with coconut shell charcoal had an average mortality of 62.5%. The mortality rates were greater for the (E1 cc) coconut charcoal sticks dipped in petroleum ether extracts than for their wood charcoal (E1 wc) counterparts. The repellence activity of the three solvent extracts decreased in the order of petroleum ether > chloroform > methanol, with methanol having the least activity. Coconut charcoal enhanced the ability to repel mosquitoes much more efficiently than did the wood charcoal-filled repellent sticks; this effect was observed from chloroform extracts with coconut shell charcoal, which exhibited some repellence activity compared to that of the wooden charcoal counterparts, which exhibited no repellence (similar trends were also observed in the petroleum ether extracts). These findings show that coconut charcoal contributes to the total efficacy of the mosquito repellence activity.

The isolated petroleum ether (E1) extract showed greater repellence than did the crude extract and thus was more refined or free of any inhibitory substances from natural substances. The characterization of the E1 extract revealed the presence of alkanes, ketones and aromatic hydrocarbons, which may be responsible for the mosquito repellence effect (supplementary data). Coconut charcoal shows greater mosquito repellency than its wooden charcoal counterparts.

The coconut shell charcoal sticks were more efficacious than their wood charcoal counterparts, which shows that agro-waste coconut shell charcoal can not only replace but also improve the repellent properties of coconut shell charcoal against mosquitos. This shows that an industrial and commercial replacement of the adsorbent filler, wood charcoal, can be healthily substituted by agro-waste coconut shell charcoal. This will not only improve the quality of the repellant but also put to use an enormous amount of agricultural waste generated from coconut kernels. Since coconut waste is one of the most abundant biomasses found in more than 90 countries globally, with a production of approximately 62.5 million tons of waste per year²⁰, utilizing this large biomass for the benefit of biopesticide production to ward away mosquitoes and avoid mosquito-related biological diseases such as dengue, chikungunya, and malaria seems to be an all-round and wholesome approach for sustainable waste management. The adsorptivity quotient of coconut shell charcoal should be quantified relative to that of its wooden charcoal counterparts at the molecular level, although we weighed the products before and after adsorption and found a comparable quantity with respect to adsorption onto the repellant sticks.

In a further study, the fractions isolated from fractional distillation and column chromatography could be further characterized for their mosquito repellence activities for future prospects to determine and narrow down upon further chemical analyses of the repellent activity from mosquitoes.

Funding and disclosure

This article was supported by Cooperatio NEUR and SVV 12653 of Charles University.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement:

This work was supported by the Cooperatio NEUR and SVV 12653 of Charles University

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The authors declare no competing interests.

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Mosquito Repellence of Solvent Extracts andActivated Charcoal Obtained from Agro-WasteCoconut Shells

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