Bioefficacy of Mentha piperita essential oil against dengue fever mosquito Aedes aegypti L (2025)

Abstract

Objective

To assess the larvicidal and repellent potential of the essential oil extracted from the leaves of peppermint plant, Mentha piperita (M. piperita) against the larval and adult stages of Aedes aegypti (Ae. Aegypti).

Methods

The larvicidal potential of peppermint oil was evaluated against early fourth instar larvae of Ae. aegypti using WHO protocol. The mortality counts were made after 24 and 48 h, and LC₅₀ and LC₉₀ values were calculated. The efficacy of peppermint oil as mosquito repellent was assessed using the human-bait technique. The measured area of one arm of a human volunteer was applied with the oil and the other arm was applied with ethanol. The mosquito bites on both the arms were recorded for 3 min after every 15 min. The experiment continued for 3 h and the percent protection was calculated.

Results

The essential oil extracted from M. piperita possessed excellent larvicidal efficiency against dengue vector. The bioassays showed an LC₅₀ and LC₉₀ value of 111.9 and 295.18 ppm, respectively after 24 h of exposure. The toxicity of the oil increased 11.8% when the larvae were exposed to the oil for 48 h. The remarkable repellent properties of M. piperita essential oil were established against adults Ae. aegypti. The application of oil resulted in 100% protection till 150 min. After next 30 min, only 1-2 bites were recorded as compared with 8-9 bites on the control arm.

Conclusions

The peppermint essential oil is proved to be efficient larvicide and repellent against dengue vector. Further studies are needed to identify the possible role of oil as adulticide, oviposition deterrent and ovicidal agent. The isolation of active ingredient from the oil could help in formulating strategies for mosquito control.

Keywords: Essential oil, Larvicide, Repellent, % protection, Bioefficacy, Mentha piperita, Essential oil, Dengue, Aedes aegypti, Larvicidal potential, Adulticide

1. Introduction

Mosquitoes are a serious threat to public health transmitting several dangerous diseases for over 2 billion people in the tropics[1]. Aedes aegypti (Ae. aegypti), the primary carrier for viruses that cause dengue fever, dengue hemorrhagic fever and yellow fever is widespread over large areas of the tropics and subtropics. Mosquito control and personal protection from mosquito bites are currently the most important measures to control these diseases. Many approaches have been developed and tried to tackle mosquito menace. The use of larvicides and repellents is an obvious practicality and economical means of preventing the transmission of these diseases to humans.

The common approach for the control of mosquito vectors and reducing the transmission of human pathogens is based on the chemical insecticide-based intervention measures[2]. However, in the past, the frequent and repeated use of chemical insecticides has resulted in the worldwide development of insecticide resistance, destabilisation of the ecosystem and toxic effects on human beings and non-target organisms[3]. Thus, there is an urgent need to develop new insecticides for controlling mosquitoes which are more environmentally safe, biodegradable and target-specific against the mosquitoes. In recent years, much effort has, therefore, been focused on plant extracts or phytochemicals as potential sources of mosquito control agents or as lead compounds[4],[5].

Many researchers have reported the effectiveness of plant extracts or essential oils as efficient mosquito larvicides and repellents without posing hazards of toxicity to humans[6]–[10]. Several extracts and compounds from different plant families have been evaluated to show new and promising larvicides[11]–[13]. Further, repellency is known to play an important role in preventing the vector borne diseases by reducing man-vector contact. Ethno-botanical studies show that in some village communities, the use of plant repellents to reduce human vector contact is a common practice[14].

2. Materials and methods

2.1. Mosquito rearing

The present investigations employ the dengue fever mosquito, Ae. aegypti, originated from fields of Delhi and surrounding areas. The colony was maintained in an insectary at (28±1) °C, (80±5)% RH and 14:10 L/D photoperiod[17]. Adults were supplied with freshly soaked deseeded raisins. Periodic blood meals were provided to female mosquitoes for egg maturation by keeping restrained albino rats in the cages. The eggs were collected in a bowl lined with Whatman filter paper and were allowed to hatch in trays filled with de-chlorinated water. Larvae were fed upon a mixture of yeast powder and grinded dog biscuits. The pupae formed were collected and transferred to the cloth cages for adult emergence.

2.2. Larvicidal bioassay

The larvicidal bioassay was performed at (28±1) °C on the Ae. aegypti larvae in accordance with the WHO method for mosquito larvae with slight modifications. The graded series of peppermint oil was prepared using ethanol as the solvent. The early fourth instar larvae of Ae. aegypti, in batches of 15, were taken in plastic bowls containing 99 mL of distilled water and transferred to glass jar containing 100 mL of distilled water and 1 mL of the particular concentration of oil. Four replicates were carried out simultaneously for each dilution making a total of 100 larvae for each concentration. Controls were exposed to the solvent, i.e. ethanol alone.

During the treatment period, no food was offered to the larvae. The larvae were considered moribund if, at the end of 24 h, they showed no sign of swimming movements even after gentle touching with a glass rod. The dead and moribund larvae were recorded after 24 h as larval mortality. The whole set up was kept undisturbed for another 24 h and mortality counts were recorded again after 48 h.

2.3. Statistical analysis of data

The larvicidal tests with more than 20% mortality in controls and pupae formed were discarded and repeated again. If the control mortality ranged between 5%-20%, it was corrected using Abbott's formula.

The data were subjected to regression analysis using computerized SPSS 11.5 Programme. The LC₅₀ and LC₉₀ values with 95% fiducial limits were calculated in each bioassay to measure difference between the test samples. The results obtained with different oils were analyzed using Student's t-test with statistical significance considered for P ≤ 0.05.

2.4. Adult repellency bioassays

The repellency of the peppermint oil against dengue vectors was evaluated using the human-bait technique. For investigation, 25 laboratory-reared, blood-starved, adult female Ae. aegypti that were between 3 and 10 days old were placed into separate laboratory cages (45 cm×45 cm×40 cm). Before each test, the forearm and hand of a human subject were washed with unscented neutral soap, thoroughly rinsed, and allowed to dry 10 min before extract application. An area of (5 cm×5 cm) on each forearm of five human volunteers was marked out with a permanent marker. Approximately 0.1 mL of the oil was applied to the marked area of one forearm of each volunteer while the other forearm was treated with ethanol as control. During the test, the forearm was covered by a paper sleeve leaving the marked area open. The control and treated arms were introduced simultaneously into the cage. An attempt of the mosquito to insert its stylets was considered a bite. The numbers of bites were counted over 3 min, every 15 min, from 1 100 h to 1 400 h.

Protection time was recorded as the time elapsed between repellent application and the observation period immediately preceding that in which a confirmed bite was obtained. If no bites were confirmed at 180 min, tests were discontinued, and protection time was recorded as 180 min. In case, if during the observation period no mosquito landed on the control arm or attempted to bite, the trial was discarded, and the test was repeated with a new batch of mosquitoes to ensure that lack of bites was due to repellence and not because mosquitoes are not predisposed to get a blood meal at the time. The experiments were conducted three times in separate cages, and in each replicate, different volunteers were used to nullify any effect of skin differences on repellency.

The percentage protection was calculated by using the following formula:

3. Results

The investigations revealed that when the early fourth instars of Ae. aegypti were exposed to peppermint oil for 24 h, the LC₅₀ and LC₉₀ values obtained were 111.9 and 295.18 ppm, respectively. The essential oil was proved to be 11.8 % more effective when the exposure of the larvae to essential oil continued for 48 h, exhibiting an LC₅₀ value of 98.66 ppm.

The results obtained proved and established the efficacy of the M. piperita essential oil against the dengue vector larvae. All the treatments resulted in complete mortality of larvae without any pupal or adult emergence. The control or untreated groups did not show any mortality within 48 h. The larvae developed into pupae and then adults within 60-72 h.

The essential oil of M. piperita has exerted promising and remarkable repellent activity against Ae. aegypti adults. The present studies revealed the percentage protection in relation to time (min). Our investigations resulted in 150 min of complete protection time with 0.1 mL of oil applied on the arms of human volunteers. After 150 min, the efficacy of oil decreased a little resulting in non-repellency of 7% of mosquitoes. Consequently, the % protection decreased to 27% in half an hour. First bite of the dengue vector was observed after 165 min of oil application, making the % repellency and protection of 96% and 88.8%, which decreased to be 93.2% and 73.1%, respectively after 180 min. The control treatment did not provide any protection even during the first trial. The number of bites in control treatment ranged only from 3 to 9 in 3 min of exposure period spread over 180 min.

4. Discussion

Essential oils extracted from different plants have been reported to have larvicidal and repellent properties against Ae. aegypti[7],[18]–[20]. Though few reports are available regarding the potential of M. piperita oil as repellent against insects, no work has been carried out regarding its potential as mosquito larvicide agent. The present investigations were performed to assess the potential of essential M. piperita oil as larvicidal and repellent agent against Ae. aegypti.

Our investigations clearly showed that essential oil extracted from M. piperita possesses excellent larvicidal efficiency against dengue vector. The bioassays performed resulted in an LC₅₀ value of 111.9 ppm and LC₉₀ value of 295.18 ppm after 24 h of exposure. It was also observed that the toxicity of the oil increased when the larvae were exposed to the oil for longer duration. Though there are no reports available regarding the potential of peppermint oil as mosquito larvicide, several reports are available on other oils which reveal their efficacy against mosquito larvae. Cheng et al[21] compared the essential oils from the leaves of eight provenances of indigenous cinnamon (Cinnamomum osmophloeum Kaneh.) and reported that the essential oils of cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type had an excellent inhibitory effect against the fourth-instar larvae of Ae. aegypti with LC₅₀ values of 36 mg/L and 44 mg/L; and LC₉₀ values of 79 mg/L and 85 mg/L, respectively. The larvicidal activity of cinnamon and other oils were recorded by Zhu et al[22],[23] against 4th instars of Aedes albopictus, Ae. aegypti, and Culex pipiens pallens. Amer and Mehlhorn[7] reported the larvicidal properties of Lippia citriodora against Ae. aegypti, Anopheles stephensi, and Culex quinquefasciatus. Senthilkumar et al[24] reported larvicidal effect of Blumea mollis essential oil against Culex quinquefasciatus, with LC₅₀ and LC₉₀ values of 52.2 and 108.7 mg/L, respectively.

According to Gleiser and Zygadlo[25], the essential oils of Lippia turbinata and Lippia polystachya exhibit LC₅₀ values of 74.9 and 121 mg/L, respectively against Culex quinquefasciatus. The essential oil of Zanthoxylum armatum was tested against three species of mosquitoes by Tiwari et al[26]. He found that among all the three species Culex quinquefasciatus was the most sensitive with LC₅₀ and LC₉₅ values of 49 and 146 ppm, respectively followed by Ae. aegypti and Anopheles stephensi with LC₅₀ values in the range of 54-58 ppm.

Our investigations also revealed the excellent repellent properties of M. piperita essential oil against adults Ae. aegypti resulting in 100% protection till 150 min. After next 30 min, only 1-2 bites were recorded as compared with 8-9 bites on the control arm. In 2009, Nour et al[27] reported that the essential oils from four basil accessions, Ocimum basilicum, conferred complete repellency against Anopheles mosquito lasting for 1.5 to 2.5 h per one application of 0.1 mL to a human volunteer's arm. The essential oil of Zingiber officinalis showed repellent activity at 4.0 mg/cm² and provided 100% protection up to 120 min against Culex quinquefasciatus[28]. Trachyspermum ammi seed oil could achieve a repellency of 45.0% with repellent dose (RD₅₀) observed as 25.02 mg/mat against Anopheles stephensi adults[29]. Earlier, Tawatsin et al[30] have reported that the essential oils extracted from 18 plant species, belonging to 11 families were more effective against the night-biting mosquitoes (Anopheles dirus, Anopheles albopictus and Culex quinquefasciatus) exhibiting repellency for 4.5-8 h than against Ae. aegypti (repellency 0.3-2.8 h). Essential oil of Cinnamomum zeylanicum, Zingiber officinale, and Rosmarinus officinalis also showed repellent activities against Anopheles stephensi, Ae. aegypti, and Culex quinquefasciatus[31].

There are reports available where essential oils have shown repellent properties in the fields. The essential oils extracted from some Verbenaceae plants have shown repellent and/or insecticidal effects against mosquitoes[8],[32]. Petroleum ether extracts of Vitex agnus castus leaves offered 8 h of bite protection (2 mg/cm²) by different mosquito species in the field[33],[34]. Rural populations of Tamil Nadu, India burn leaves of this plant with grass as fumigant against mosquitoes. Lippia citriodora was reported to have repellent properties against Ae. aegypti[32] and Culex quinquefasciatus[8] and larvicide against Ae. aegypti, Anopheles stephensi, and Culex quinquefasciatus[7].

In conclusion, an attempt has been made to evaluate the possible role of peppermint essential oil to control mosquitoes. As natural products are generally preferred in vector control measures due to their less deleterious effect on non-target organisms, their innate biodegradability and keeping in view the resistance developed by the mosquito larvae against chemical insecticides, it is worthwhile to identify new active compounds from natural products against mosquitoes. Hence, the results reported here open the possibility of further investigations of efficacy of peppermint oil in terms of its adulticidal, oviposition deterrent and ovicidal action against mosquitoes.

Acknowledgments

The authors are highly grateful to University Grants Commission for providing financial assistance to carry out the present investigations. Thanks are extended to Dr. Savithri Singh, Principal, Acharya Narendra Dev College for providing infrastructure and research facilities.

Footnotes

Conflict of interest statement: We declare that we have no conflict of interest.

References

1.Odalo J, Omolo M, Malebo H, Angira J, Njeru P, Ndiege I, et al. Repellency of essential oils of some plants from the Kenyan coast against Anopheles gambiae. Acta Trop. 2005;95:210–218. doi: 10.1016/j.actatropica.2005.06.007. [DOI] [PubMed] [Google Scholar]
2.Paul A, Harrington LC, Scott JG. Evaluation of novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) J Med Entomol. 2006;43:55–60. doi: 10.1603/0022-2585(2006)043[0055:EONIFC]2.0.CO;2. [DOI] [PubMed] [Google Scholar]
3.Jirakanjanakit N, Rongnoparut P, Saengtharatip S, Chareonviriyaphap T, Duchon S, Bellec C, et al. Insecticide susceptible/resistance status in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Thailand during 2003-2005. J Econ Entomol. 2007;100:545–550. doi: 10.1603/0022-0493(2007)100[545:irsias]2.0.co;2. [DOI] [PubMed] [Google Scholar]
4.Ansari MA, Mittal PK, Razdan RK, Sreehari U. Larvicidal and mosquito repellent activities of Pine (Pinus longifolia, Family: Pinaceae) oil. J Vector Borne Dis. 2005;42:95–99. [PubMed] [Google Scholar]
5.Singh RK, Dhiman RC, Mittal PK. Mosquito larvicidal properties of Momordica charantia Linn (Family: Cucurbitaceae) J Vector Borne Dis. 2006;43:88–91. [PubMed] [Google Scholar]
6.Mehlhorn H, Schmahl G, Schmidt J. Extract of the seeds of the plant Vitex agnus castus proven to be highly efficacious as a repellent against ticks, fleas, mosquitoes and biting flies. Parasitol Res. 2005;95:3635. doi: 10.1007/s00436-004-1297-z. [DOI] [PubMed] [Google Scholar]
7.Amer A, Mehlhorn H. Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera: Culicidae) Parasitol Res. 2006a;99:466–472. doi: 10.1007/s00436-006-0182-3. [DOI] [PubMed] [Google Scholar]
8.Amer A, Mehlhorn H. Repellency effect of forty-one essential oils against Aedes, Anopheles and Culex mosquitoes. Parasitol Res. 2006b;99:478–490. doi: 10.1007/s00436-006-0184-1. [DOI] [PubMed] [Google Scholar]
9.Rahuman AA, Bagavan A, Kamaraj C, Vadivelu M, Zahir AA, Elango G, et al. Evaluation of indigenous plant extracts against larvae of Culex quinquefasciatus Say (Diptera: Culicidae) Parasitol Res. 2009a;104:637–643. doi: 10.1007/s00436-008-1240-9. [DOI] [PubMed] [Google Scholar]
10.Rahuman AA, Bagavan A, Kamaraj C, Saravanan E, Zahir AA, Elango G. Efficacy of larvicidal botanical extracts against Culex quinquefasciatus Say (Diptera: Culicidae) Parasitol Res. 2009b;104:1365–1372. doi: 10.1007/s00436-009-1337-9. [DOI] [PubMed] [Google Scholar]
11.Mohan DR, Ramaswamy M. Evaluation of larvicidal activity of the leaf extract of a weed plant, Ageratina adenophora against two important species of mosquitoes, Aedes aegypti and Culex quinquefasciatus. Afr J Biotechnol. 2007;6:631–638. [Google Scholar]
12.Inocent E, Joseph CC, Gikonyo NK, Nkunya MHH, Hassanali A. Mosquito larvicidal constituents from Lantana viburnoides ssp. Viburnoides var. kisi (A. Rich) Verdc (Verbenaceae) J Vector Borne Dis. 2008a;45:240–244. [PubMed] [Google Scholar]
13.Inocent E, Joseph CC, Gikonyo NK, Nkunya MHH, Hassanali A. Growth disruption activities of polar extracts from Kotschya uguensis (Fabaceae) against Anopheles gambiae s.s. larvae. Int J Trop Ins Sci. 2008b;28:220–224. [Google Scholar]
14.Kweka EJ, Mosha F, Lowassa A, Mahande AM, Kitau J, Matowo J, et al. Ethnobotanical study of some of mosquito repellent plants in north-eastern Tanzania. Malar J. 2008;7:152. doi: 10.1186/1475-2875-7-152. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Cappello G, Spezzaferro M, Grossi L, Manzoli L, Marzio L. Peppermint oil (Mintoil®) in the treatment of irritable bowel syndrome: a prospective double blind placebo-controlled randomized trial. Dig Liver Dis. 2007;39:530–536. doi: 10.1016/j.dld.2007.02.006. [DOI] [PubMed] [Google Scholar]
16.Robert IK. Handbook of pesticide toxicology. New York: Academic Press; 2001. p. 823. [Google Scholar]
17.Kumar S, Warikoo R, Wahab N. Larvicidal potential of ethanolic extracts of dried fruits of three species of peppercorns against different instars of an Indian strain of dengue fever mosquito, Aedes aegypti L. (Diptera: Culicidae) Parasitol Res. 2010;107:901–907. doi: 10.1007/s00436-010-1948-1. [DOI] [PubMed] [Google Scholar]
18.Morais SM, Eveline SB, Cavalcanti ESP, Luciana MB, Oliveira CLL, Rodrigues JRB, et al. Larvicidal activity of essential oils from Brazilian Croton species against Aedes aegypti L. J Am Mosq Control Assoc. 2006;22:161–164. doi: 10.2987/8756-971X(2006)22[161:LAOEOF]2.0.CO;2. [DOI] [PubMed] [Google Scholar]
19.Silva WJ, Doria GA, Maia RT, Nunes RS, Carvalho GA, Blank AF, et al. Effects of essential oils on Aedes aegypti larvae: alternatives to environmentally safe insecticides. Bioresour Technol. 2008;99:3251–3255. doi: 10.1016/j.biortech.2007.05.064. [DOI] [PubMed] [Google Scholar]
20.Waliwitya R, Kennedy CJ, Lowenberger CA. Larvicidal and oviposition-altering activity of monoterpenoids, trans-anithole and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae) Pest Mgmt Sci. 2009;65:241–248. doi: 10.1002/ps.1675. [DOI] [PubMed] [Google Scholar]
21.Cheng SS, Liu JY, Tsai KH, Chen WJ, Chang ST. Chemical composition and mosquito larvicidal activity of essential oils from leaves of different Cinnamomum osmophloeum provenances. J Agric Food Chem. 2004;52:4395–4400. doi: 10.1021/jf0497152. [DOI] [PubMed] [Google Scholar]
22.Zhu J, Zeng X, Yanma Liu T, Qian K, Han Y, Xue S, et al. Adult repellency and larvicidal activity of five plant essential oils against mosquitoes. J Am Mosq Control Assoc. 2006;22:515–522. doi: 10.2987/8756-971X(2006)22[515:ARALAO]2.0.CO;2. [DOI] [PubMed] [Google Scholar]
23.Zhu J, Zeng X, O'neal M, Schultz G, Tucker B, Coats J, et al. Mosquito larvicidal activity of botanical-based mosquito repellents. J Am Mosq Control Assoc. 2008;24:161–168. doi: 10.2987/8756-971X(2008)24[161:MLAOBM]2.0.CO;2. [DOI] [PubMed] [Google Scholar]
24.Senthilkumar A, Kannathasan K, Venkatesalu V. Chemical constituents and larvicidal property of Blumea mollis (D.Don) merr. against Culex quinquefasciatus. Parasitol Res. 2008;103:959–962. doi: 10.1007/s00436-008-1085-2. [DOI] [PubMed] [Google Scholar]
25.Gleiser RM, Zygadlo JA. Insecticidal properties of essential oils from Lippia turbinata and Lippia polystachya (Verbenaceae) against Culex quinquefasciatus (Diptera: Culicidae) Parasitol Res. 2007;101:1349–1354. doi: 10.1007/s00436-007-0647-z. [DOI] [PubMed] [Google Scholar]
26.Tiwary M, Naik SN, Tewary DK, Mittal PK, Yadav S. Chemical composition and larvicidal activities of the essential oil of Zanthoxylum armatum DC (Rutaceae) against three mosquito vectors. J Vector Borne Dis. 2007;44:198–204. [PubMed] [Google Scholar]
27.Nour AH, Elhussein SA, Osman NA, Nour AH. Repellent activities of the essential oils of four Sudanese accessions of Basil (Ocimum basilicum L.) against Anopheles mosquito. J Appl Sci. 2009;9:2645–2648. [Google Scholar]
28.Pushpanathan T, Jebanesan A, Govindarajan M. The essential oil of Zingiber officinalis Linn (Zingiberaceae) as a mosquito larvicidal and repellent agent against the filarial vector Culexquinquefasciatus Say (Diptera: Culicidae) Parasitol Res. 2008;102:1289–1291. doi: 10.1007/s00436-008-0907-6. [DOI] [PubMed] [Google Scholar]
29.Pandey SK, Upadhyay S, Tripathi AK. Insecticidal and repellent activities of thymol from the essential oil of Trachyspermum ammi (Linn) Sprague seeds against Anopheles stephensi. Parasitol Res. 2009;105:507–512. doi: 10.1007/s00436-009-1429-6. [DOI] [PubMed] [Google Scholar]
30.Tawatsin A, Asavadachanukorn P, Thavara U, Wongsinkongman P, Bansidhi J, Boonruad T, et al. Repellency of essential oils extracted from plants in Thailand against four mosquito vectors (Diptera: Culicidae) and oviposition deterrent effects against Aedes aegypti (Diptera: Culicidae) Southeast Asian J Trop Med Public Health. 2008;37:915–931. [PubMed] [Google Scholar]
31.Prajapati V, Tripathi AK, Aggarwal KK, Khanuja SPS. Insecticidal, repellent and oviposition-deterrent activity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Bioresour Technol. 2005;96:1749–1757. doi: 10.1016/j.biortech.2005.01.007. [DOI] [PubMed] [Google Scholar]
32.Gillij YG, Gleiser RM, Zygadlo JA. Mosquito repellent activity of essential oils of aromatic plants growing in Argentina. Bioresour Technol. 2008;99:2507–2515. doi: 10.1016/j.biortech.2007.04.066. [DOI] [PubMed] [Google Scholar]
33.Karunamoorthi K, Mulelam A, Wassie F. Laboratory evaluation of traditional insect/mosquito repellent plants against Anopheles arabiensis, the predominant malaria vector in Ethiopia. Parasitol Res. 2008a;103:529–534. doi: 10.1007/s00436-008-1001-9. [DOI] [PubMed] [Google Scholar]
34.Karunamoorthi K, Ramanujam S, Rathinasamy R. Evaluation of leaf extracts of Vitex negundo L. (Family: Verbenaceae) against larvae of Culex tritaeniorhynchus and repellent activity on adult vector mosquitoes. Parasitol Res. 2008b;103:545–550. doi: 10.1007/s00436-008-1005-5. [DOI] [PubMed] [Google Scholar]