Trends Sci. 2025; 22(12): 10379

Application of Eucheuma Cottonii Carrageenan as an Emulsifier in the Making of Mosquito Repellent Lotion from Clove Leaf Essential Oil (Syzigium Aromaticum)

Nurhayati¹, Hasnah Natsir^1,*, Indah Raya¹, Paulina Taba¹,

Abdul Wahid Wahab¹, Herlina Rasyid¹ and Rizal Irfandi²

¹Department of Chemistry, Faculty of Mathematics and Natural Sciences, Hasanuddin University,

South Sulawesi 90245, Indonesia

²Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Makassar,

Jalan Daeng Tata Raya Makassar 90244, Indonesia

(^*Corresponding author’s e-mail: [email protected])

Received: 16 April 2025, Revised: 24 May 2025, Accepted: 5 June 2025, Published: 20 August 2025

Abstract

Dengue fever in Indonesia continues to increase every year. One form of prevention is the utilisation of clove leaf essential oil (Syzigium aromaticum) as an anti-mosquito lotion. The abundance of Eucheuma cottonii in Indonesia has the potential to increase the production of carrageenan which can function as an emulsifier for lotions. The objectives of this research to analyze the characteristics and effectiveness of anti-mosquito lotion from clove leaf essential oil with the addition of carrageenan based on red algae E. cottonii. The method used was carrageenan extraction with 0.1 N KOH solvent, antimicrobial test with disc diffusion method and antimosquito effectiveness test. Analysis of carrageenan using FTIR showed the presence of galactose 4-sulfate functional group which identified kappa type in the spectrum of 846 - 927 cm^–1. Microstructure characteristics of carrageenan using SEM 2000× magnification showed a rough surface and irregular clumps. Characterization of clove leaf essential oil (Syzigium aromaticum) using GC-MS instrument showed the main content of the compound 4.allyl-2-methoxyphenol with an area of 78.89% which includes eugenol compounds. The optimal anti-mosquito lotion was F2 with 4% carrageenan and 15% clove leaves assential oil. Formula 2 has a level of liking in the sensory test in the neutral category, but has good effectiveness with the support of several characteristics that meet quality standards including a pH value of 7.1, a viscosity value of 5,481.8 cP, a moisture content of 4.91% and microbial inhibition of E. coli and S. aureus at a good concentration of 40%.

Keywords: Anti-mosquito lotion, Carrageenan, Clove leaf essential oil, Eucheuma cottonii

Introduction

Indonesia is an archipelago with a tropical climate, so it has a lot of biodiversity on land and sea. Seaweed is an abundant and important marine resource for the national economy, one of which Eucheuma species produces agar and carrageenan. Indonesia imports 1,380 tonnes or about 70% of domestic carrageenan needs, while local production has not met the growing demand [1]. Seaweed from red algae is more widely cultivated, because it has higher biological activity, so the content of primary ans secondary metabolite compounds is also

higher than other types of algae [2].

In addition to abundant marine natural resources, Indonesia also has abundant terrestrial natural resources. One of its superior products is the clove plant, which produces essential oil. Clove contain eugenol (70% - 96%) with many advantages, such as anticancer, antibacterial, antifungal, antioxidant, anti-inflammatory and anti-insect. Eugenol is neurotoxic which can disrupt the nervous system of mosquitoes and its punget aroma can provide better protection against mosquito bites. These advantages can be utilised as a natural and safe anti-mosquito product for humans. Various studies on anti-mosquito lotions from essential oils of various plants rely solely on aroma to repel mosquitoes, such as citronella-containing lemongrass [3] and evodiamine-containing zodia leaves [4].

Currently, health problems, especially dengue fever, are still a big problem. Since the beginning of 2024, more than 14 million cases of dengue fever and more than 10,000 deaths due to dengue fever worldwide [5]. In Indonesia, in 2024 dengue cases will be reported to reach 88,593 with a death toll of 621 people [6]. Dengue fever is an infectious disease caused by the dengue virus which is transmitted through the bite of the Aedes aegypti or Aedes albopictus mosquito [7].

One way to prevent dengue fever is to eliminate or reduce the population of vector mosquitoes such as Aedes sp. The main vector control activities in Indonesia are carried out on mosquito larvae and adult mosquitoes. Control of adult mosquitoes is done by fogging to break the chain of transmission from infected mosquitoes to humans. Specifically for mosquito larvae, mosquito nest eradication is carried out by draining, covering and burying programs chemically with insecticides, biologically using natural enemies such as predators, bacteria, and other methods such as using mosquito nets, using mosquito repellents and installing wire mesh [6,8]. Individual self-protection from the risk of DHF virus transmission can be done in various ways including using repellents, and wearing clothes that reduce mosquito bites.

People generally use mosquito coils, mosquito sprays, and electric mosquito repellents that are derived from chemicals and have negative impacts on the human body such as shortness of breath, and also cause air pollution [9]. Most of the mosquito repellents available today contain diethyltoluamide (DEET) chemicals as active ingredients. DEET compounds are easily absorbed through the skin and enter the bloodstream, affecting the nervous system. In particular, DEET causes seizures and even death in some human individuals. The dangers and warnings are a clear enough reason that alternative safe active ingredients are needed in the formulation of mosquito repellent to facilitate its use, mosquito repellent will be made in the preparation of mosquito repellent lotion.

Based on the above problems, in this study, an anti-mosquito lotion was made from clove leaf essential oil, using carrageenan from red algae (E. cottonii) as an emulsifier.

Materials and methods

Preparation of red algae (E. cottonii)

The red algae obtained were soaked with clean water for 30 min, then washed thoroughly to remove dirt and attached objects such as sand and gravel. Furthermore, it is drained and cut with a size of 2 - 4 cm.

Extraction of carrageenan from red algae (E. cottonii)

Prepared red algae were then weighed as much as 160 g. Furthermore, it was extracted using 0.1 N KOH solvent with a ratio of solvent and raw material of 5:1 for 60 min at a temperature of 70 - 80 °C. The extracted E. cottonii filtrate was washed with water and filtered. Then dried in the oven at 60 °C, and mashed using a hammer mill and sieved using 100 mesh sieving.

Extraction of clove leaf essenttial oil

Weighed 5 kg of washed and dried clove leaves, distilled for 7 h, then separated the oil and water.

Lotion formulation

The formulation of anti-mosquito lotion refers to the research conducted by Dewi et al. [10] modified. The modification is done by replacing the active ingredient extract with clove leaf essential oil extract (Syzigium aromaticum) and adding an emulsifier in the form of carrageenan extracted from red algae (E. cottonii). This test was carried out 3 times. The formulation of anti-mosquito lotion can be seen in Table 1.

Table 1 Anti-mosquito lotion formula with carrageenan.

Description: From 100%, 60 mL of lotion preparation was made.

Sensory test of anti-mosquito lotion

The sensory test used hedonic scale with 15 panelists and was processed using SPSS V. 25 with Kruskal-Wallis test and Mann-Whitney test as the next step. The parameters observed were appearance, color, aroma, and texture. Observations with a hedonic scale worth 1 to 5: 1) Very dislike, 2) Dislike, 3) Neutral, 4) Like, 5) Very like [11].

Characteristic test of anti-mosquito lotion

Characteristic tests on anti-mosquito lotions consist of pH measurements, viscosity [12], specific gravity and water content.

Antimicrobial test against E. coli and S. aureus bacteria

Testing the antibacterial activity of the lotion was done by disc diffusion method using paper discs with a diameter of 5 mm. Paper discs were dipped into the sample at each concentration of 20%, 30% and 40%, then placed on NA media that had been inoculated with test bacteria (E. coli and S. aureus). Then incubated at 37 ºC for 2×24 h. Positive control was 0.1% chloramphenicol solution and negative control was aquades [13].

Effectiveness test of anti-mosquito lotion

The effectiveness test was conducted by introducing 20 test mosquitoes into each of the 5 test cages (control, F0, F1, F2 and F3). Then the left hand was smeared with antimosquito lotion evenly and the right hand was not smeared (as a comparison). Replication was done twice. The observation time was 20 min with an interval of 5 min [19].

Characterization of clove leaf essential oil extract (Syzigium aromaticum) using Gas Chromatography-Mass Spectrometry (GC-MS)

Essential oil from clove leaves is then analyzed using GC-MS with RTx-5MS column conditions with a length of 30 meters (0.25 mm), column oven temperature of 70 °C, injection temperature of 300 °C, water control mode used in the form of pressure with the pressure used of 13.7 kPa, total flow of 50 mL/min, and column flow of 0.5 mL/min [14].

Characterization of E. cottonii carrageenan using Fourier Transform Infrared (FTIR)

Characterization of carrageenan structure using FTIR spectrophotometry was carried out by mixing 1 mg of fucoidan powder with 100 mg of KBr, crushed in a mortar until homogeneous to obtain a pellet. The pellet was put into a simple pan to record the infrared spectrum at a wavelength of 4,000 - 500 cm^–1.

Characterization of E. cottonii carrageenan using Scanning Electron Microscope (SEM)

As much as 200 mg of carrageenan powder was printed on carbon tape, dried and then coated with gold. Placed on the device and observed the surface of the sample directly with 10 - 1500× magnification, dept of field 4 - 0.4 mm and resolution of 1 - 10 nm.

Results and discussion

Sensory test of anti-mosquito lotion

Extract of Clove Leaf Essential Oil Sensory testing was conducted by 15 panelists by measuring the level of appearance, color, aroma, and texture using a hedonic scale. Data were analyzed using Kruskal-Wallis test followed by Mann-Whitney test using SPSS 25.

Table 2 Average value of sensory test of anti-mosquito lotion preparations of clove leaf essential oil extracts.

Description: 1 = Strongly dislike, 2 = Dislike, 3 = Neutral, 4 = Like, 5 = Strongly like a, b, c, d = Similar letter notation means no significant difference at the Mann-Whitney test level (p > 0.05).

Figure 1 Distribution of panelists’ level of liking for the preparation of anti-mosquito lotion.

The panelist liking test was conducted to determine the level of panelist preference for the lotion preparation that had been made. The appearance of F0 tends to be preferred because it has the characteristics of lotions in general that are not given the addition of clove leaf essential oil extract and E. cottonii carrageenan. Based on the level of panelists’ preference for color, panelists preferred F0 compared to other formulas. This is because the color of F0 is white and the other formulas are brown. The color change that occurs is due to the addition of clove leaf essential oil and E. cottonii carrageenan. The white color tends to have a greater appeal because the majority of lotions spread in the community are white so that the level of acceptance of panelists for lotions other than white is still low. The clove leaf essential oil lotion preparation has a distinctive aroma, namely the distinctive aroma of clove leaf essential oil. This is due to the eugenol content that dominates clove essential oil. The aroma of cloves is still not a special attraction for lotion preparations because of its distinctive aroma characteristics that tend to be sharp and spicy. Several studieshave found good percentage for lotion preparations. Commonly used percentage are around 5% - 15%, depending on the type of essential oil and the intended use. Generally, higher concentrations are applied for more than just moisturizing the skin, such as antiinflammatory and antimosquito. The texture liking level of the lotion tended to favor F0 by the panelists because of its smooth and soft texture. When compared to the other formulas which tended to be rough according to the variation in concentration of E. cottonii carrageenan addition and slightly oily with the addition of clove leaf essential oil.

Based on the sensory test, the ideal antimosquito lotion formula is F2 with 15% clove leaf essential oil concentration which is good for warding off mosquitoes. Some other plant essential oils with a percentage of about 15% can provide 50% protection for 6 h. Regarding antimosquito lotion products on the market, the percentage of essential oil used is not specified. However, some studies can serve as a reference for the percentage that is effective and safe for the skin ranging from 5% - 20%.

Characteristic test of anti-mosquito lotion

Characteristic of anti-mosquito lotion can be seen in Table 3.

Table 3 Characteristic of anti-mosquito lotion.

Description: ^a = SNI 16-4399-1996; ^b = Herbal Pharmacopoeia Edition II.

Measuring the pH of clove essential oil extract anti-mosquito lotion aims to ensure the safety of the user’s skin. A pH value that is too acidic can irritate the skin while a pH value that is too alkaline can dry the skin [15]. Based on the SNI 16-4399-1996 standard, the ideal pH value for lotions is between 4.5 - 8.0. Based on the pH measurement data of the lotion using a pH meter, from F0 to F3, the pH decreased. This is in accordance with research conducted by Dewi et al. [10], which states that the higher the clove essential oil content, the smaller the pH obtained. Referring to SNI standards, all formulas meet the requirements to be applied to the skin with a pH value of 6.98 - 7.74.

The viscosity test of anti-mosquito lotion aims to determine the viscosity level of the lotion so that it can have good spreadability on the skin. According to Dewi et al. [10], the presence of thickeners in lotion formulations is useful to prevent particle separation from emulsions so as to maintain lotion stability. Based on the results of the table above, all formulas meet the quality requirements of anti-mosquito lotion preparations. According to Dewi et al. [10], the higher the concentration of clove oil added, the lower the viscosity, due to the consistency of clove oil which is more liquid than the lotion preparation base. Good viscosity is indicated by the fact that the higher the viscosity value, the harder particles movement will tend to move, so the lotion will be more stable.

Based on Table 3 all lotion formulas meet the SNI 16-4399-1996 standard and exceed the specific gravity of water. According to Rowe et al. [16], the specific gravity of lotion tends to be greater than the specific gravity of water depending on the increase in emulgator concentration in the preparation, the increase in specific gravity of lotion preparation is directly proportional to the concentration of emulgator (carrageenan E. cottonii).

The anti-mosquito lotion moisture content test is one of the tests to measure the quality of lotion preparations that are effective and safe to use. Too high moisture content can cause rapid growth of microorganisms. The right moisture content will make the lotion have good adhesion and effectiveness in its function as an anti-mosquito lotion. There is no reference in SNI standards or other standards, but based on Herbal Pharmacopoeia Edition II [17], a good moisture content to maintain the quality of simplisia is ≤ 10%. The following is the water content of each formula. Based on the table above, all formulas have a moisture content of < 10% so that the quality of the lotion can be maintained properly. This is also in accordance with the formulation of each lotion produced in the addition of distilled water.

Antimicrobial test against the growth of E. coli and S. aureus bacteria

Antimicrobial testing of anti-mosquito lotions uses the Agar Diffusion Disk method. According to Rivera et al. [18], the Agar Diffusion Disk method is based on the diffusion of a certain amount of antimicrobial agent contained in a paper disk placed on the surface of an agar medium that has been previously seeded with a standardized microbial inoculum. As the sample diffuses, it will form a zone of inhibition, the concentration of which will be sufficient to inhibit the growth of the inoculated microorganism from growing. The larger the diameter of the inhibition zone produced and exceeds that of the positive control, the greater the potential in inhibiting the growth of the inoculated microorganisms. In the antimicrobial testing of this lotion, the test bacteria used were E. coli and S. aureus. The positive control used chloramphenicol 0.1% and the negative control used DMSO 0.1%.

Figure 2 Lotion antimicrobial test.

Table 4 Diameter of the clear zone of the anti-mosquito lotion dosage formula.

Based on the observation results (Figure 2), clear zone diameter data was obtained for each anti-mosquito lotion formula. The diameter of the clear zone in each anti-mosquito lotion preparation formula with 2 test bacteria showed significant potential inhibition of microorganism growth as the concentration of the lotion preparation formula increased (Table 4). Although still not exceeding the inhibitory ability of the positive control (chloramphenicol), each formula can increase its inhibitory power with increasing concentration. So it can be concluded that the higher the concentration of the lotion preparation formula, the higher the antimicrobial ability. Antimicrobial testing of clove leaf mosquito repellent lotion is conducted to increase the usability of the lotion as askin protector from pathogenic microorganisms. In addition, the presence of antimicrobial activity in the lotion may indicate that the lotion is more effective as a mosquito repellent due to the lethal effect of the same antimicrobial active compound in the antimosquito effectiveness test [19].

Effectiveness test of anti-mosquito lotion

The anti-mosquito lotion effectiveness test was conducted in cages containing mosquitoes that had never sucked blood with a total of 5 cages. The test used 4 lotion formulas and 1 comparison (no lotion). The results of the lotion effectiveness test can be seen in Table 5.

Table 5 Effectiveness test of anti-mosquito lotion.

Description:

F0 = Without the addition of clove leaf essential oil extract.

F1 = Addition of clove leaf essential oil extract by 10%.

F2 = Addition of 15% clove leaf essential oil extract.

F3 = Addition of clove leaf essential oil extract by 20%.

Based on the lotion effectiveness test, it shows that the higher the concentration of clove leaf essential oil extract added to the lotion formula, the more effective it is as an anti-mosquito lotion. This is due to the sharp aroma of clove leaf essential oil that can prevent mosquitoes from sticking to the skin. The eugenol content in clove leaves has neurotoxic properties that can cause insects to become inactive. Neurotoxic works in the process of suppressing the nervous system of insects which can be characterized by the insect’s body which when touched feels soft and limp. In a study conducted by Juniyanti et al. [20], showed that the effectiveness of clove leaf extract lotion was good at a concentration of 3% with a repulsion percentage of 85.5% of 50 test mosquitoes. This was also stated by Ndalu [21], which resulted in an average value of 1.3 mosquitoes from 25 test mosquitoes.

GC-MS analysis of clove leaf essential oil (Syzigium aromaticum)

Analysis of the constituent compound components of clove leaf essential oil extract (Syzigium aromaticum) using GC-MS instrument. The results obtained in the form of spectra and chromatograms. Mass spectra analysis is based on the base peak, similarity index (SI) and fragmentation pattern of the spectrum compared to the spectrum of the Wiley 229 LIB library. The analysis results can be seen in Figure 3 and The interpretation results can be seen in Table 6.

Figure 3 GC-MS results of clove leaf essential oil.

Table 6 Interpretation of GC chromatogram of clove leaf essential oil.

In the GC chromatogram data in Table 6, there are 3 peaks that have a larger area than other peaks, namely peaks number 2, 4, and 7. The compound at peak number 2 with a retention time of 13.7 min and S1 of 95 is similar to the compound 4-allyl-2-methoxyphenol with tthe molecular formula C₁₀H₁₂O₂. The compound at peak number 4 with a retention time of 14.425 min and S1 of 97 is similar to the compound caryophyllene with tthe molecular formula C₁₅H₂₄. The compound at peak number 7 with a retention time of 16.6 min and S1 of 97 is similar to the compound (-)-5-oxatricyclo[8.2.0.0(4,6)]dodecane,12-trimethyl-9-methylene with tthe molecular formula C₁₅H₂₄O_. Among the 3 compounds, the most is the compound 4.allyl-2-methoxyphenol with an area of 78.89% which is included in the eugenol compound. The same thing was reported by Sanga et al. [22], the most compound of clove essential oil is eugenol (59.38%) with a retention time of 17.946; and Budiman et al. [23] eugenol is 87.24%.

Characterization of carrageenan using FTIR

The wavelength absorption in Table 7 shows a broad absorption at a wavelength of 3,479 cm^–1 indicating an -OH group, and a sharp and narrow absorption at a wavelength of 1,070 cm^–1 indicating a galactose glycosidic bond. In addition, there are sharp and narrow absorptions at wavelengths of 927 and 846 cm^–1 that identify the presence of 3,6- anhydrogalactose group and galactose-4-sulfate group. The presence of 3,6- anhydrogalactose group shows the characteristics of kappa carrageenan. This is in line with research by Amin et al. [24], that the characteristics of carrageenan extracted from E. cottonii belong to the kappa type with an absorption region of 848.53 cm^–1 showing galactose-4-sulfate bonds and 928.56 cm^–1 showing 3,6-anhydrogalactose bonds. The characteristic of kappa carrageenan is the presence of D-galactose-4-phosphate and 3,6-anhydro-D-galactose groups [25].

Figure 4 Identification results of E. cottonii carrageenan and standard carrageenan.

Table 7 infrared absorption data of E. cottonii carrageenan.

Characterization of carrageenan E. cottonii using SEM

SEM analysis is useful to determine the microstructure (including porosity and crack shape) of solid objects [26].

Figure 5 Structure of E. cottonii carrageenan magnification (a) 300× and (b) 2000×.

Based on Figure 5, the microstructure characteristics of carrageenan using SEM magnification of 300× shows a rough surface and irregular clumps, while at 2000× magnification shows pores on the surface that look more clearly there are irregular clumps. This is due to the strong reaction between carrageenan and KOH which can increase the ionic strength of the carrageenan polymer chain and form a more stable gel. This is in accordance with research by Bhernama [27], who compared the structure of carrageenan with KOH and NaOH. The results obtained, carrageenan with KOH solvent has an uneven surface structure with gel lumps, while with NaOH solvent the surface tends to be flat and smooth. According to Winarno [28], this is because the solvents potassium hydroxide and sodium hydroxide have a major influence on the extraction of seaweed E. cottonii into carrageenan, which K⁺ ions and Na⁺ have a different role in the strength of the gel. K⁺ ions produce a better surface structure than Na⁺ ions. KOH solvent significantly affects the viscosity of carrageenan through molecular degradation and sulfate reduction, but its strong ionic interactions improve functional properties such as gel strength, making it a versatile choice in various applications such as antimosquito lotions [29].

Conclusions

Characterization of carrageenan from red algae (E. cottonii) using FTIR indicated that it is a type of kappa carrageenan with the characteristics of the 3,6- anhydrogalactose functional group at a wavelength of 927 cm^–1. The results of analysis using SEM instrument showed a rough surface and irregular clumps. Characterization of clove leaf essential oil (Syzigium aromaticum) using GC-MS showed the presence of the main component contained in clove leaf essential oil, namely 4.allyl-2-methoxyphenol. Based on the tests to determine the conformity of the lotion with the predetermined quality standards, the optimal percentage of carrageenan in making anti-mosquito lotion is F2 with 4% carrageenan. Formula 2 has a level of liking in the sensory test in the neutral category, but has good effectiveness with the support of several characteristics that meet quality standards including a pH value of 7.1, a viscosity value of 5,481.8 cP, a moisture content of 4.91% and microbial inhibition of E. coli and S. aureus at a good concentration of 40%.

Acknowledgements

The greatest appreciation goes to the research team who have fully contributed to the completion of this research, and thanks to hasanuddin university for providing equipment that supports this research as well as the panelists who have participated in completing the necessary data.

Declaration of Generative AI in Scientific Writing

The author acknowledge the use of generative tools (e.g., QuillBot) in the preparation of this manuscript, specifically for language editing and grammar correction. No content generation or data interpretation was performed by AI. The authors take full responsibility for the content and conclusions of this work.

CRediT Author Statement

Nurhayati: Conceptualization, Methodology, Software, Investigation, Funding Acquisition, Resources, and Writing – original draf.

Hasnah Natsir: Conceptualization, Methodology, Recources, Validation, Formal analysis, Data Curation, and Project Administration..

Indah Raya: Conceptualization, Methodology, Formal analysis, Validation, and Data Curation.

Paulina Taba: Data curation, Formal Analysis, Validation, and Supervision.

Abdul Wahib Wahab: Validation, Formal analysis, Data Curation, Writing-Reviewing and Editing.

Herlina Rasyid: Formal analysis, Data curation, Validation, Software, and Visualization.

Rizal Irfandi : Data Curation, Software, and Visualization.

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