Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3792

Full Length Research Paper

Chemical composition and larvicidal activity of Citrus limonia Osbeck bark essential oil

Paulo Roberto Barros Gomes
  • Paulo Roberto Barros Gomes
  • Federal Institute of Education, Science and Technology of Pará, Paragominas - PA, Brazil.
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Dionney Andrade de Sousa
  • Dionney Andrade de Sousa
  • Department of Chemical Technology, Federal University of Maranhão, 65080-805, São Luís, MA, Brazil.
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Gustavo Oliveira Everton
  • Gustavo Oliveira Everton
  • Department of Chemical Technology, Federal University of Maranhão, 65080-805, São Luís, MA, Brazil.
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Maria Alves Fontenele
  • Maria Alves Fontenele
  • Coordination of Food Engineering, Federal University of Maranhão, 65915-240, Imperatriz, MA, Brazil.
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Jucilane Novais Lopes e Marques
  • Jucilane Novais Lopes e Marques
  • Federal Institute of Education, Science and Technology of Pará, Paragominas - PA, Brazil.
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Adriana Crispim de Freitas
  • Adriana Crispim de Freitas
  • Coordination of Food Engineering, Federal University of Maranhão, 65915-240, Imperatriz, MA, Brazil.
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Virlane Kelly Lima Hunaldo
  • Virlane Kelly Lima Hunaldo
  • Coordination of Food Engineering, Federal University of Maranhão, 65915-240, Imperatriz, MA, Brazil.
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Hilton Costa Louzeiro
  • Hilton Costa Louzeiro
  • Department of Pharmacy, Federal University of Maranhão, 65080-805, São Luís, MA, Brazil.
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Maria do Livramento de Paula
  • Maria do Livramento de Paula
  • Coordination of Licenciatura in Natural Sciences, Federal University of Maranhão, Pinheiro, MA, Brazil.
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Jonas Batista Reis
  • Jonas Batista Reis
  • Department of Chemical Technology, Federal University of Maranhão, 65080-805, São Luís, MA, Brazil.
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Andréa Vasconcelo Melo
  • Andréa Vasconcelo Melo
  • Department of Chemical Technology, Federal University of Maranhão, 65080-805, São Luís, MA, Brazil.
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Helson Souza de Lima
  • Helson Souza de Lima
  • Biodiversity and Biotechnology, Federal University of Maranhão, São Luís - Ma, Brazil.
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Victor Elias Mouchrek Filho
  • Victor Elias Mouchrek Filho
  • Department of Chemical Technology, Federal University of Maranhão, 65080-805, São Luís, MA, Brazil.
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  •  Received: 30 March 2020
  •  Accepted: 05 June 2020
  •  Published: 31 July 2020


Aedes aegypti mosquito arouses the interest of public authorities, as it is a vector for four diseases (dengue, zika, chikungunya, and yellow fever) and one of the ways to combat it is through insecticides. In this study, the main constituent, the predominant class of essential oil extracted from the husks of Citrus limonia Osbeck was identified and evaluated to know if it has biological activity against larvae in the third stage of Ae.aegypti. gas chromatography coupled with mass spectrometry (CG-MS); larvicidal activity as described by the World Health Organization (WHO) was evaluated and the lethal concentration (LC50) from the Probit model was calculated. The results show that the oil consists mainly of limonene, beta-Pinene, meta-Cymene, beta.-Phellandrene and alpha-Pinene, in which the predominant class was monoterpenes and the lethal concentration, CL50, was 67.18 µg.mL-1. Therefore, the oil has potential larvicidal activity.

Key words: Volatile compounds, monoterpenes, aedes aegypti, limonene, natural insecticide.



Aedes aegypti draws the public attention authorities. The interest in that mosquito is based on the principle that it is necessary to combat it, due to  the  diseases  it  transmits (dengue, zika, chicungunya and yellow fever) and the outbreaks and deaths that those diseases have caused in recent  years  (Bhatt  et  al.,  2013).  In  this   manner,   to understand better the dimension of the problem, between 2013 and 2015 in Brazil, two million people were diagnosed with dengue and one million were infected with the zika virus, thus resulting in the largest outbreak reported in that country (Brasil, 2017; Cardoso et al., 2015). In conseqeunce to the fast spread of these diseases, especially the Zika virus, the World Health Organization (WHO) decreed in February 2016 emergency of Global Public Health when the relationship of that disease with the cases of microcephaly registered in Brazil was verified (Brasil, 2017). So, faced with this scenery, the authorities sought to minimize the number of cases through public awareness policies, showing the risks, symptoms and measures that prevent the mosquito's proliferation, and in the development of vaccines; until now there is only for yellow fever (Brasil, 2020; Rothman, 2004). While the development of vaccines for other diseases, such as dengue, zika and chicungunya, are still in the testing phase (Pang and Loh, 2017; Tripp and Ross, 2016), combating the vector mosquito remains the most effective control, be it in the larval or adult phase.

In the literature, there are reports of combating the Ae.aegypti mosquito using two methods: Predation and chemical insecticides. In the predation method, fish species (Trichogaster trichopteros and Astyanax fasciatus) were used to prey on larvae in a certain period of time (Cavalcanti et al., 2007). Chemical insecticides, on the other hand, generally use compounds based on carbamates, pteroids and organophosphates to kill larvae or adult insects at low concentrations, which is recommended by WHO (Govindarajan and Benelli, 2016). It is a fact that between the two methods the most effective are the insecticides, as these act on both larvae and adults. However, studies show that the intensive use of these compounds provoked attacks on non-target organisms and increased the resistance of the mosquito population (da Rocha Voris et al., 2018), which limits their use and encourages the search for other insecticides preferably, of plant origin that are effective and have less impact on the environment.

Among the larvicides of plant origin, the most studied are those based on essential oils. As these compounds are formed by complex structures, it is believed that one of its components, be it the majority or the minority, is responsible for biological activity. Among the compounds that have proven larvicidal activity, either alone or as the main constituent of an essential oil, limonene stands out (Cavalcanti et al., 2004; Estevam et al., 2016; Giatropoulos et al., 2012; Gomes et al., 2019; Millezi, 2014; Santos et al., 2011). In citrus species, this component is present around 80% (Ladaniya, 2008) so that a large part of the plants have larvicidal activity.

Among the various species of citrus plants, whose limonene is the main component, there is Citrus limonia Osbeck, known as pink lemon or china lemon; this species of plant has  been  widely  cultivated  in  orchards and nurseries, due to the early maturation of its fruits and the economic values ​​at the beginning of the harvest (Reda et al., 2005). Studies of the chemical composition revealed that the leaves and peels of the C. limonia Osbeck fruit consist, respectively, of 40 and 82% of limonene (Cavalcanti et al., 2004; Estevam et al., 2016; Millezi, 2014). In addition, this species of plant has some known biological activities which are: Trypanocidal, antibacterial, leishmanicidal (Estevam et al., 2016; Millezi, 2014) and larvicidal (Cavalcanti et al., 2004). It was emphasize that the larvicidal activity of the essential oil was demonstrated in the aerial parts (leaves and branches) of this plant, in which the low lethality against Ae.aegypti larvae in the 3rd stage was proven (Cavalcanti et al., 2004). Although the essential oil extracted from the leaves has a low larvicidal activity, until now there are no studies of this activity for the fruit peels. Thus, according to the view talked here before, in this study the main constituent was identified, the predominant class of essential oil was extracted from the husks of Citrus limonia Osbeck and assessed if it has biological activity against larvae in the third stage of Ae.aegypti.



Obtaining and extracting essential oil

Fruit collection was carried out in a rural area of ​​São Brás dos Macacos, municipality of São José de Ribamar-MA, Brazil, in January 2017, geographic coordinates latitude 02° 35'51, 8''S and Longitude 44° 09 '33, 3''W, and certified by the Laboratory of Botanical Studies with number 11.170. After collection, the shells were removed with a stylus.

The essential oil was extracted by hydrodistillation and the average yield was calculated from the density and weight measurements of the crude material. For extraction, 400 g of the samples was weighed and mix in 4000 mL of distilled water in a 1:10 ratio. Then, mixture was placed in a 1000 mL round-bottom flask and attached it to the Clevenger extractor under 100°C heating in an electric blanket for 4 h. After that time, the extracted oil was collected and dried by percolation in an anhydrous sodium sulfate solution. These operations were perform in triplicates and store the samples in amber glass ampoules under refrigeration to avoid possible losses of volatile constituents. A density pycnometer was used to measure density.

Chromatographic GC/MS analysis

In this manner, the components of essential oil by gas chromatography was identify coupled to mass spectrometry (GC/MS) in a gas chromatograph of the Shimadzu brand, coupled to a mass spectrometer of the model QP2020AS, using helium as carrier gas with flow in the 2.5 mL column .min-1; injector temperature: 280°C, split 1:50; BPX 5% phenylpolysilphenylene-siloxane capillary column (30 m × 0.25 mm × 0.25 mm) and oven temperature programming from 60 to 280ºC. In the Mass Spectrometer, the temperature was 280°C and the recorded spectra were 35 to 550 m/z. We injected aliquots of 1 µL (automatic injector  CP - 8410) of the samples diluted in the proportion of 20 µL in 1.5 mL of hexane. The oil components were identified by comparing their retention index with data obtained from authentic substances existing in NIST14 reference libraries.

Capture and creation of Ae. aegypti

The larvae were obtained through ovitraps in the period from January to February 2019. Ovitraps are prepared by the addition of water and two eucatex straws in polyethylene buckets with a capacity of 500 mL, where eggs are expected to be deposited by mosquito females. After hatching, the larvae in the 3rd stage were kept at room temperature 25 ± 2ºC and relative humidity of 70 to 80%, being fed with dog food.

Larvicidal bioassay

To carry out this experiment, the methodology described by the World Health Organization (World Health Organization, 2005) was followed. In the preliminary stage, ten larvae was transferred in the 3rd stage to disposable cups and expelled to three concentrations of oil (10, 50 and 100 µgmL-1) in 24 h to check whether or not there was activity. After confirming this, five solutions of essential oil (50 to 130 µgmL-1) dissolved in Dimethyl sulfoxide (DMSO) 0.1% was prepared. For each concentration tested, the negative control (DMSO 0.1%) was used with all tests performed in quintuplicates.

Statistical analysis

The average mortality data were submitted to Probit (Finney and Tattersfield, 1952) analysis for the calculation of LC50 and other statistics with 95% reliable limits, upper confidence limit, lower limit and chi-square calculated using the Medcalc 19.2 software with level of significance p <0.05.


Oil extraction and chromatographic GC/MS analysis

The oil extracted by the hydrodistillation technique gave an average yield and density, respectively, of 2.54% (w/w) and 0.842 gmL-1. In the chromatographic analysis by GC/MS, 18 compounds were identified (Table 1), where the five largest were, respectively, Limonene, β-pinene, m-Cymene, β-phellandrene and α-pinene and the class predominant was that of monoterpenes with 93.97%.



Larvicidal activity

In this study, it was observed that the essential oil extracted from the peels of C. limonia Osbeck has larvicidal activity (Table 2) CL50 67.18 µg mL-1, in an exposure time of 24 h, according to the criteria described by Cheng et al. (2003) that consider the activity when the lethal concentration (LC50) is less than 100 µg mL-1.





Diversity of species used

Forty two species distributed across 29 families and 35 genera were mentioned by traditional healers. These medicinal plant species are used to treat 17 ailments; 24 in humans, 6 in livestock and 12 in both human and livestock. The Lamiaceae were most presented with four species, followed by the Anacardiaceae, Boraginaceae, and Solanaceae families with three species each. Asteraceae, Cucurbitaceae, Cuppresaceae, Euphorbiaceae and Rutaceae each with two species and the rest representing one species. Whereas, Clutia abyssinica, Lactucainermis and Clerodendrum myricoides were the most frequently mentioned plant species, followed by  Lagenaria siceraria and   Stephania abyssinica. Shrubs constituted (45.24%) followed by trees (33.33%), herbs (16.67%) and climbers (4.76%) of the used species. Fifty nine percent of the medicinal plants were collected from the wild and the remaining ones from home gardens.

Informant consensus factor

Based on the used citations of the key informants, plants species were clustered into nine different categories (Table 1) to calculate the ICF values. The ICF values range between 0.81 (for lung infection and cough) and 0.98 (for gonorrhea and sexual transmitted disease). Thus, all clusters had an ICF value greater than 0.5 showing that all of them could be considered for validation in support of its traditional use






Thus, the diseases transmitted by the Ae. aegypti mosquito arouse the interest of public authorities who are looking for ways to control them from combating the vector, whether in larvae or mosquitoes. However, one of the main means used in this control, larvicides or chemical   insecticides,   has   the  main  disadvantage  of resistance to mosquitoes and damage to the environment. On the other hand, some essential oil-based larvicides have shown good results in controlling the vector mosquito. In this study, the main constituent, the predominant class of essential oil extracted from the husks of C. limonia Osbeck was identified and whether it has biological activity against larvae in the third stage of Ae. aegypti was evaluated. Here, it was shown that the essential oil is mostly made up of limonene, in which the monoterpernos class prevailed, and it was demonstrated that the oil has larvicidal activity against Ae. aegypti, and can be a potential substitute for chemical larvicides.

In the first finding, the oil was extracted by hydrodistillation and subjected to chemical analysis to identify the main component and the predominant class. From this analysis, it was found that the essential oil C. limonia Osbeck is made up mostly of limonene in which the monoterpernos class predominated. However, the amounts of limonene identified in this study differ from the amounts described in previous studies, where the values ​​range from 33 to 82% (Cavalcanti et al., 2004; Estevam et al., 2016; Millezi, 2014). Although the result for the amount of limonene is within the expected value for citrus species (Ladaniya, 2008), the differences in the composition of the components identified in the oil are explained by some factors, such as collection period, extraction time, temperature, intensity of solar radiation (Gobbo-Neto and Lopes, 2007), seasonality (Silva et al., 2019), the age and development of plants (Gobbo-Neto and Lopes, 2007) among others.

In the second finding, it was demonstrated that the essential oil C. limonia Osbeck has larvicidal activity from the comparison of the result obtained in the LC50 with the criterion established by Cheng (2003), since there is no defined standard to evaluate larvicidal efficacy of an oil. Thus, according to these authors, an essential oil is active when the LC50 is less than or equal to 100 µg mL-1.

The results obtained in this study also confirm the action of different types of monoterpenes with larvicidal activity. In previous studies, it is reported that isolated hydrocarbon monoterpenes (limonene and alpha-pinene) have greater larvicidal activity than oxygenated monoterpenes (carvone, terpinen-4-ol and α-terpineol) (Lucia et al., 2013; Santos et al.,  2011),  as  well  as  it  is also reported that these classes of compounds in essential oil have larvicidal activity (Dias and Moraes, 2014). In this study, these compounds were identified, but it turns out that the presence of the oxygenated monoterpenes in the essential oil reduced the potency of the hydrocarbon monoterpenes so that the essential oil had a low larvicidal activity, thus confirming the synergistic effect of other compounds. The evidence that the limonene present in greater quantity in the essential oil extracted from citrus peels has larvicidal activity is reported in studies with Citrus limon (CL50 15.48 µg mL-1) (Gomes et al., 2019), C. sinensis ( CL50 28.68 µg mL-1) and C. paradisi (CL50 37.03 µg mL-1) (Giatropoulos et al., 2012) in which it has been shown that the most effective to date is C. limon.

These findings should be interpreted with caution, as the identification of the compounds by the GC/MS technique only shows that it is there, but does not indicate the exact quantity of each component. Regarding the larvicidal efficacy of essential oil, further studies are needed to qualify it as a substitute for the chemical larvicide. However compared to other citrus essential oils described in the literature, C. limonia Osbeck oil would not be indicated as the first option, due to the low larvicidal activity.

In summary, the demonstration of the chemical composition and larvicidal activity shows the potential of the essential oil C. limonia Osbeck, as well as opening the opportunity to investigate the mechanism of action and the development of products that help combat the transmission of diseases by the vector mosquito.




To sum up, the essential oil from Citrus limonia Osbeck peels was extracted by hydrodistillation, the chemical analysis and larvicidal activity was determined against Ae. aegypti. Chemical analysis by gas chromatography coupled to the mass spectrum showed the presence of eighteen compounds where the five largest were, respectively, limonene, beta-Pinene, meta-cymene, beta.-Phellandrene and alpha.-Pinene and the predominant class was of  monoterpenes.  The  essential oil has low biological activity against larvae in the 3rd stage of Ae. aegypti when compared to other citruses described in the literature. Therefore, due to these characteristics, essential oil has potential biological activity and can replace synthetic larvicides.



The authors declare that they have no conflict of interests.



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