INTRODUCTION:

The species Aedes aegypti and Aedes albopictus are mosquitoes that could transmit dengue virus. These mosquitoes are also vectors of Zika, chikungunya, and yellow fever viruses¹. Mosquitoes are attracted to chemical compounds emitted by mammals². The number of dengue cases reported to WHO increased in 2020, dengue affected several countries, despite a risk of infection existing in 129 countries³, 70% of the actual burden is in Asia. In 2022, as of August 24, 2 597 067 cases and 2 065 deaths had been reported⁴. Dengue is widespread throughout the tropics, with local variations in risk influenced by climate parameters. Outbreaks occur in the rainy season⁵. The virus has four different types, infection with one type usually gives lifelong immunity to that type, but only short-term immunity to the others⁶.

The Aedes mosquito breeds in clear water⁷. Aedes aegypti is a daytime feeder, its peak biting periods are early in the morning and in the evening before sunset⁸. Female Aedes aegypti feed multiple times when each egg-laying period, leading to clusters of infected individuals⁹.

Personal protection from mosquito bites: using window screens, repellents, coils, and vaporizers¹⁰. The persistent synthetic chemicals, and non-biodegradable such as N, N-Diethyl-3-methylbenzamide (DEET), DEPA, DMP, DEBA, and allethrin are utilized in most commercial mosquito repellent formulations. A mosquito repellent is a substance applied to skin or other surfaces that discourages mosquitoes. It makes the surface unpleasant and unattractive to mosquitoes to reduce the human mosquito contact¹¹. DEET has a strong smell, creating oily, and burning sensations, particularly when applied at higher doses. Additionally, emerging problems related to the development of side effects, toxicity to non-target organisms, resistance, and ecological concerns¹². Therefore, alternative active compounds are required. Mosquito-repellent ingredients from plants developed into an essential oil using palmarosa. Palmarosa is one of the aromatic plants found in India. Besides India, palmarosa is grown in Indonesia¹³. Palmarosa oil contains the main components that have the potential as mosquito repellents, there are geraniol, geranyl acetate, and linalool. The application of 1ml of palmarosa oil provided 98.7% protection indoors and 96.52% outdoors during observation from Anopheles sundaicus bites¹⁴.

MATERIALS AND METHODS:

Materials:

Palmarosa essential oil (PEO) is extracted by steam distillation of the palmarosa plant, which was collected from Rumah Atsiri, Tawangmangu, Central Java, Indonesia. Ethanol, diethyl ether, and potassium hydroxide were purchased from Sigma-Aldrich. Aedes aegypti was obtained from the Research and Development Center for Vector Health and Disease Reservoirs in Yogyakarta (BBTKLPP).

Quality evaluation of essential oil palmarosa:

a) Organoleptic:

The physical properties of PEO were observed without changing the identity. Physical properties include smell, taste, and color. The determination of color was carried out by taking a sample of 10mL into a test tube, leaning it on a whiteboard, and observing directly¹⁵.

b) Determination of Relative Density:

The pycnometer is filled with distilled water at a temperature of 25±0.2ºC, closed, and weighed. The pycnometer is emptied, washed, and dried. The pycnometer was filled with 1mL of PEO at a temperature of 25±0.2ºC, closed, and weighed, calculating the specific density of PEO¹⁶.

c) Determination of Refractive Index:

Dripped oil on the prism at a temperature of 20°C, then adjusted the slides, a clear dark and bright outline was obtained¹⁵.

d) Determination of Solubility in Alcohol:

A total of 1mL of PEO, 70% ethanol is added drop by drop at 20°C, and each addition is shaken until the solution is as clear as possible, if the solution is not clear, compare it with the turbidity in 70% ethanol¹⁷.

e) Determination of Acid Value:

PEO (0.5mg) dissolved in 10ml of ethanol and 2-3 drops of PP, then titrated with a standard 0.1N potassium hydroxide solution¹⁵.

f) Determination of Esters Value:

25ml of 0.5 N KOH in alcohol, then reflux for 1h. After that, add 10ml of distilled water. Add a few drops of PP and titrate it against 0.5 N HCl¹⁵.

g) Determination of Optical Rotation:

2 dm-long polarimeter tube was read at 20°C using D-line polarized sodium light. Different concentrations of oil solutions were prepared in ethanol¹⁸.

h) Determination of Iron:

Determination of iron (Fe) was carried out using dry destruction analysis, by weighing a sample of 0.5grams in a porcelain dish heated at a temperature of 800°C for 2hours using a furnace and storing it in the furnace for one hour. After the cold sample was added 5ml of concentrated HNO₃ and heated until dissolved and cooled. After that, 10ml of distilled water was added, filtered with Whatman paper, and then analyzed by an AAS (Atomic Absorption Spectrophotometer)¹⁹.

Chemical identification:

Identification of essential oil component of palmarosa oil was carried out using Gas Chromatography under the following conditions: carrier gas He; Rastex RTX-5MS column; column length 30m; ID 0.25mm; EI (Electron impact) ionizing type; gas velocity 80.0ml/min; temperature rise 10°C/min; initial temperature 60°C; final temperature 200°C; temperature of detector 250°C; temperature of injector 200°C; column pressure 36.2 kPa; the start time at 0 minutes; and the end at 15 minutes.

Methods of Evaluation of the Mosquito Repellent Activity:

The female mosquitoes (aged 3-7 days) were fasted for 24 hours, then 25 mosquitoes were placed in the compartment 35 x 35 x 35 cm. Test on probandus arm first was washed and dried with a soft towel or tissue. The probandus arms inserted into the barrel test containing Aedes aegypti mosquitoes. Each probandus arm was smeared with PEO in concentrations of 12.5%; 25%; 50%; 75%; 100%. The positive control used repellant commercial with 12.5% DEET levels, and the negative control was the arm without treatment. Observations were made for 5 minutes every 1 hour and carried out for 6 hours, repeated three times. Then count the number of mosquitoes that perch and/or bite.

Repellent activity = (C – T) /C × 100%,

where C indicates the number of mosquitoes perched and/or biting on control and T indicates the number of mosquitoes perched and/or biting on arms smeared with PEO²⁰.

Table 2: Chemical composition of essential oil palmarosa

S. No	Compounds	Retention Time (min)	Composition (%)	Molecular Formula	Molecular Mass
1	Limonene	5.197	0.08±0.01	C₁₀H₁₆	136
2	Ocimene	5.352	0.32±0.02	C₁₀H₁₆	136
3	Linalool	6.125	1.55±0.06	C₁₀H₁₈O	154
4	Geraniol	8.501	83.75±2.07	C₁₀H₁₈O	154
5	Geranial	8.645	0.24±0.02	C₁₀H₁₆O	152
6	Nerol	9.031	0.06±0.01	C₁₀H₁₈O	154
7	Geranyl Acetate	10.143	9.82±2.08	C₁₂H₂₀O₂	196
8	Trans Caryophyllene	10.839	1.17±0.05	C₁₅H₂₄	204
9	Farnesol	14.660	0.48±0.04	C₁₅H₂₆O	222
10	Geranyl hexanoate	14.828	0.99±0.16	C₁₆H₂₈O₂	252

Table 3: Efficacy of PEO in repelling Aedes aegypti based on human bait collection

Sample	Repellent activity (%)
Sample	1 h	2 h	3 h	4 h	5 h	6 h
Positive control (DEET 12.5%)	95.50±3.37	94.59±2.21	89.19±4.41	72.07±1.27	58.56±8.92	18.92±7.96
Concentration 12.5%	96.40±1.27	92.79±3.37	90.29±3.37	84.68±1.27	66.67±3.37	58.65±4.41
Concentration 25 %	97.30±2.21	97.30±2.21	89.19±5.84	86.49±7.96	78.38±2.21	60.36±3.37
Concentration 50 %	97.30±0.00	97.30±2.21	95.5±1.27	88.29±2.55	81.98±1.27	62.16±2.21
Concentration 75 %	98.20±1.27	98.20±1.28	97.30±2.21	91.89±3.82	85.59±3.37	80.18±4.59
Concentration 100 %	99.10±1.27	99.10±1.27	98.20±2.55	97.30±3.82	94.59±4.41	89.19±2.21

Evaluation of the Mosquito Repellent Activity:

The repellent activity by evaluating the protection period against the bait of Aedes aegypti within one hour to six hours. The repellent activity was carried out to determine the ability of protection.

Figure 2: Repellent activity of different concentrations of PEO against Aedes aegypti mosquitoes at different time intervals.

Observation time was carried out for 6 hours by testing 6 concentrations of essential oils (12.5%; 25%; 50%; 75%; 100%). negative control using probands hands without treatment. positive control using mosquito repellent lotion with DEET 12.5%. The results of the repellent research are declared effective if they have a resistance to mosquito bites of >80% and are declared ineffective if the resistance is <80%.

DISCUSSION:

Physicochemical properties PEO are useful methods in determining the quality of it. It was determined according to the standard of PEO (ISO 4727:2021). The refractive index of PEO is closely related to the constituent components of essential oils, if there are more long-chain components, the density of the essential oil medium would increase, which causes the refractive index value to be greater. In addition, the refractive index is influenced by the presence of water, the greater the water content. Essential oils generally contain fragrant chiral compounds, so that the property of rotating the plane of polarization to the right (dextrorotatory) or left (levorotatory) when placed in a light²¹. The value of optical rotation is determined by the type and concentration of the compound, temperature, and the distance traveled by the light through the compound. The degree of rotation and its direction are used to determine the essential oil purity criteria.

The solubility of essential oils is determined by the components contained in the oil. Essential oils generally contain oxygenated terpenes so they are more soluble in ethanol than their solubility in unoxygenated terpenes. If the unoxygenated terpene content is higher, the solubility will decrease and it will be more difficult to dissolve, this is because the unoxygenated terpene compound is non polar. It can be concluded that the higher the solubility of the essential oil in ethanol, the better the quality of the essential oil. The value of the acid number is less than five then it is relatively not easily rancid and has a longer shelf life, while the value of the acid number is more than five it will cause irritation to the skin, the smaller the value of the acid number, the better the quality of the essential oil. The quality of PEO based on ISO 4727:2021 has met the parameters.

In general, pure essential oils can be subdivided into two distinct groups of chemical constituents; the hydrocarbons which are made up almost exclusively of terpenes and the oxygenated compounds²². The insecticidal activity is due to the presence of secondary metabolites²³. Aromatic plants contain a variety of volatile molecules²⁴. The combined GC/MS has emerged as one of the important analyses to give effective resolution of individual components in the complex mixture especially essential oils²⁵. Volatile secondary metabolites such as terpenes and terpenoids are similar to other research revealed that the major components of PEO were geraniol (83.75±2.07%), geranyl acetate (9.82±2.08%), linalool (1.55±0.06%), and trans caryophyllene (1.17±0.05%). The chemical profiles of a variety of other palmarosa have been published, and are comparable to our results. Nevertheless, Minoba et al., PEO from Nepal with geraniol (61.4%), geranyl acetate (22.6%) and linalool (4.9%)¹³. Jnanesha, et al., extracted the leaf oil of Palmarosa from India, with geraniol (67.9-76.1%), geranyl acetate (11,4-21,7%), linalool (1,2-2,3%), and trans caryophyllene (0,2-1,1%) being the main compounds²¹. We suggest that such divergence might also be linked to the differences in the location of the trees and the distillation technique. Other factors may include the time of harvest, age of the plant, and method of distillation²⁶.

PEO exhibited good repellent activity against Aedes aegypti mosquitoes (Table 3). The mosquitoes start landing and start biting in 1 h until 6 h. Concentrations of 75% and 100% essential oil showed >80% repellent activity up to 6h. The highest concentration of 100% showed the highest repellent activity against the mosquitoes which decreased as the time increased. The female Aedes Aegypti mosquitoes aged 3-7 days were used, this reason because adult mosquitoes need food to breed in this case. Mosquitoes feed in the form of blood from the arm of the proband used in this study. Moreover, the major component of PEO oil is geraniol elicits a spick response of the antenna of the Aedes aegypti female mosquito. The experimental results of the repellent activity showed that the greater the concentration of essential oil in the repellent, the greater the protection against Aedes aegypti mosquitoes. PEO with a concentration of 12.5% had a greater protection than 12.5% DEET against Aedes aegypti mosquitoes. Essential oil-based repellents are considered safer and more accessible than synthetic repellents and may have a lower probability of resistance development in mosquitoes. In the present study, all of the concentrations of PEO exhibited repellent activity against Aedes aegypti. These results may be candidates for the formulation of safe and effective mosquito repellents. One of the mosquito repellent applications that could be made candles with the aroma of PEO²⁷.

CONCLUSION:

This study will facilitate the identification quality and repellent activity of palmarosa essential oil (PEO). evaluation methods and their range of value can be utilized by traders of raw materials to avoid adulteration. The result of GC-MS has chemical constituents, there are geraniol, geranyl acetate, linalool, and trans caryophyllene. The quality of PEO based on ISO 4727:2021 has met the parameters. It would improve the confidence of both producer & consumer as well.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Rumah Atsiri, Tawangmangu, Indonesia and BPTTLK Yogyakarta for their kind support during preparing sample and all other lab studies.

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Received on 26.10.2022 Modified on 11.05.2023

Research J. Pharm. and Tech 2023; 16(12):5990-5995.

DOI: 10.52711/0974-360X.2023.00972

S. No.	Parameters	Results	Standard (ISO 4727:2021)
1.	Organoleptic test	Color: pale yellow Odor: rose – like with grassy note	Color: pale yellow or yellow Odor: rose – like with grassy note
2.	Relative density (25ºC)	0.893±0.001 g/cm³	0.880-0.894
3.	Refractive Index (nD25)	1.471	1.471-1.478
4.	Miscibility in ethanol 70% (volume fraction) at 20ºC	The sample is soluble in 70% ethanol in a ratio of 1:1 part by volume	The solubility in a volume fraction of 70 % ethanol shall be one volume in not more than two volumes to give a clear solution
5.	Ester value	23.48±0.97	-
6.	Acid value	1.352±0.048	-
7.	Optical rotation	-0.037+0.018°	between -1◦ and 3◦
8.	Iron Fe	5.966±0.070 mg/kg	-