Quality Evaluation and Effectiveness of Palmarosa (Cymbopogon martinii var. motia) Essential oils as repellents against Aedes aegypti

 

Nastiti Utami*, Novena Yety Lindawati, Tiara Karunia Kristy

Department of Pharmacy, Sekolah Tinggi Ilmu Kesehatan Nasional, Sukoharjo, Central Java, 57552, Indonesia.

*Corresponding Author E-mail: nastiti.utami@stikesnas.ac.id

 

ABSTRACT:

Dengue Hemorrhagic Fever is one of the health problems caused by the dengue virus. Activities that may break the chain of transmission by killing mosquitoes directly or avoiding their bites by using repellents. Natural repellents are harmless to the environment and humans, one of which is the essential oil of palmarosa. This study was conducted to evaluate the quality of palmarosa essential oil (PEO) and determine its repulsion of PEO against the Aedes aegypti mosquito. Testing the repulsion of PEO with concentrations of 100%, 75%, 50%, 25%, and 12.5% against the Aedes aegypti This research was conducted experimentally; the arm was exposed for 6 hours and observed every hour for 5 minutes. The results showed PEO that from water vapor distillation was clear, slightly yellowish, smelled rose-like with grassy, ​​refractive index 1.471, the sample was soluble in 70% ethanol with a ratio of 1:1 by volume, relative density 0.893±0.034, ester value 23.48±0.97, acid value 1.352±0.048, optical rotation 0.037±0.018, Iron Fe 5.966±0.070mg/kg. PEO contains geraniol, geranyl acetate, and linalool which have potential as repellents. The results of the repellent activity showed that the greater the concentration of essential oil in the repellent, the greater the protection against Aedes aegypti. The concentration of PEO (12.5%) had greater protection than 12.5% DEET against bites of Aedes aegypti within one hour to six hours.

 

KEYWORDS: Dengue, Cymbopogon martinii, essential oil, mosquito, repellent.

 

 


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 1: Quality evaluation of essential oil palmarosa

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

-

 


Ethics:

The study protocol was approved by The Health Research Ethics Committee Dr. Moewardi General Hospital, Indonesia.

 

RESULT:

Quality evaluation of essential oil palmarosa:

Quality evaluation of PEO requires methods in determining the quality of essential oils was determined according to the Standard of PEO (ISO 4727:2021). The result of organoleptic PEO was yellow color and odor like rose – like with grassy (Table 1).

 

The quality of PEO showed a refractive index of 1.471, optical rotation of -0.037+0.018°, and specific density of 0.893±0.001g/cm3. The solubility test in ethanol shows whether the sample is an oil or not. Dissolve easily in ethanol, so that the more polar compounds contained in the oil. From the results of the research that has been obtained, it was found that in a 1:1 ratio, 1ml of PEO was dissolved in 1ml of 70% ethanol. Ethanol contains a hydroxyl group (OH), therefore essential oils can dissolve in ethanol.

 

The acid value indicates the free acid content in PEO, the greater the acid value could affect the quality PEO, namely the compound is able to change the distinctive odor of PEO. The results of the acid number test obtained are 1.352±0.048. The presence of Fe (iron) content can cause turbidity of PEO and cause the color of the PEO to become dark and cloudy, therefore the presence of Fe compounds in PEO should be as low as possible. Determination of Fe levels were determined using AAS analysis and the results were 5.966±0.070 mg/kg.

 

Chemical composition of essential oil palmarosa

The chemical compound of PEO is analyzed by GC/MS. The GC/MS total ion chromatogram of the PEO was obtained results are seen in (Table. 2). GC-MS analyses have the presence of 10 compounds (Figure. 1). The majority of volatile oils extracted from PEO contain 83.75±2.07% geraniol.

 

Figure 1: GC–MS chromatogram of PEO; 1 (Limonene); 2 (Ocimene); 3 (Linalool); 4 (Geraniol); 5 (Geranial); 6 (Nerol); 7 (Geranyl Acetate); 8 (Trans Caryophyllene); 9 (Farnesol); 10 (Geranyl hexanoate)

 

The table 2 shows that palmarosa oil is composed of 10 compounds, with 4 main compositions, starting with the most dominant being geraniol, geranyl acetate, linalool, and trans caryophyllene.


 

 

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 (%)

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

Accepted on 06.11.2023           © RJPT All right reserved

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

DOI: 10.52711/0974-360X.2023.00972