Natural Product Research Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Chemical composition of the essential oils isolated from peel of three citrus species and their mosquitocidal activity against Culex pipiens Mohamed E. I. Badawy, Nehad E. M. Taktak & Ahmed F. El-Aswad To cite this article: Mohamed E. I. Badawy, Nehad E. M. Taktak & Ahmed F. El-Aswad (2017): Chemical composition of the essential oils isolated from peel of three citrus species and their mosquitocidal activity against Culex pipiens, Natural Product Research, DOI: 10.1080/14786419.2017.1378216 To link to this article: http://dx.doi.org/10.1080/14786419.2017.1378216
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Date: 11 October 2017, At: 02:07
Natural Product Research, 2017 https://doi.org/10.1080/14786419.2017.1378216
SHORT COMMUNICATION
Chemical composition of the essential oils isolated from peel of three citrus species and their mosquitocidal activity against Culex pipiens Mohamed E. I. Badawya , Nehad E. M. Taktakb and Ahmed F. El-Aswada a
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Faculty of Agriculture, Department of Pesticide Chemistry and Technology, 21545-El-Shatby, Alexandria University, Alexandria, Egypt; bDepartment of Tropical Health, High Institute of Public Health, Alexandria University, Alexandria, Egypt
ABSTRACT
Three essential oils (EOs) were isolated from the peel of citrus fruits Citrus reticulata L., Citrus reticulata chinase Blanco and Citrus sinensis (L.) Osbeck (Family: Rutaceae) and evaluated against Culex pipiens L.1758 (Family: Culicidae). Chemical composition indicated that the EOs were rich in essential phytochemicals including hydrocarbons, monoterpenes and sesquiterpenes. These constituents revealed some variability among the oils displaying interesting chemotypes limonene (35–51%), 1R-α-pinene (1.04–2.5%), γ-terpinene (0.46–5.65%) and sabinene (0.51–5.42%). The toxicity proved that C. sinensis oil had more effect than C. reticulata chinase and C. reticulata oils against larvae (LC50 = 15.35, 16.11 and 32.84 mg/L, respectively). However, C. reticulate was the most active as fumigant against adults (LC50 2.74 μL/L air). The in vivo effect on acetylcholine esterase (AChE), carboxyl esterase (CbE), acid phosphatase (ACP), alkaline phosphatase (ALP), adenosine triphosphatase (ATPase) and glutathione-S-transferase (GST) were also demonstrated. To the best of our knowledge, this is the first report about the chemical composition and mosquitocidal activity of C. reticulata chinase essential oils. Conclusively, the tested essential oils could be used as eco-friendly alternatives in mosquitoes control programme.
ARTICLE HISTORY
Received 24 July 2017 Accepted 29 August 2017 KEYWORDS
Chemical composition; Culex pipiens; essential oils; mosquitocidal activity
Hydro distillation Citrus reticulata L.
Citrus reticulata Chinase
Citrus sinensis L.
GC/MS analysis
Live larvae
Fumigant assay against adults
In-vitro mosquitocidal activity against Culex pipiens
Dead larvae
CONTACT Mohamed E. I. Badawy [email protected], [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1378216. © 2017 Informa UK Limited, trading as Taylor & Francis Group
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M. E. I. BADAWY ET AL.
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1. Introduction Mosquitoes are responsible for the transmission of many disease to humans and animals in the world due to rapid urbanisation and poor water management (Badawy et al. 2016; Reegan et al. 2016). The essential oils (EOs) extracted from some plants are suitable as they are economically and highly active against mosquitoes (Pavela 2015). The EOs were present in a great quantities in Citrus fruit flavedo of the family Rutaceae which includes about 17 species grown throughout the world (Caccioni et al. 1998). In fact, limonene, α-pinene, α-terpinolene, which considered from the active components exist in Citrus EOs, exhibited a wide spectrum of larvicidal activity against mosquitoes, as convinced by many previous studies in other plants (Sanei-Dehkordi et al. 2016; Zahran et al. 2017). Among commercially common species Citrus reticulata (mandrin), Citrus reticulata chinase (tangor) and Citrus sinensis (orange) are considered the important species grown in Egypt. Therefore, the main objective of current study is to identify the chemical composition of three isolated EOs from common Citrus species (C. reticulata, C. reticulata chinase and C. sinensis, Family: Rutaceae) in Egypt by GC/MS and evaluate their larvicidal and adulticidal activity against Culex pipiens L. 1758 (Family: Culicidae). In order to elucidate the mechanism of action of the EOs tested, certain biochemical parameters including target enzymes of acetylcholine esterase (AChE), acid phosphatase (ACP), alkaline phosphatase (ALP) and adenosine triphosphatase (ATPase), and two detoxifying enzymes of carboxyl esterase (CbE) and glutathione-S-transferase (GST) were evaluated in vivo in larvae exposed to 24 and 48 h of the LC50 of each EO.
2. Results and discussion 2.1. Chemical composition of EOs The EOs contain limonene as the major component (43.25% in C. reticulate, 51.49% in C. reticulata chinase and 34.97% in C. sinensis) (Tables S1–S3). α-pinene and myrcene (monoterpenes) are involved in three tested EOs with different amounts. While the C. reticulate oil features with the presence of some hydrocarbon compounds (2,3-dimethyl-octane 4.24%, toluene 4.74% and 1,3-xylene 1.44%) and monoterpenes (α-phellandrene 0.8%, β-pinene 1.24%, 1,8-cineole 0.11%, γ-terpinene 5.65% and geranyl isovalerate 0.15%) (Table S1). These results are similar to the other results proved that limonene was the major component in C. reticulata Blanco EO (46.7–73.38%), while geraniol was completely absent from their study (Chutia et al. 2009; Espina et al. 2011). The major constituents of C. reticulata chinase oil followed by limonene are shown in Table S2 as (R)-m-mentha-6-8-diene (10.81%), sabinene (5.42%), α-myrcene (3.56%), (1R)-α- pinene (2.30%), linalool (2.11%), then citronellol (0.78%). There is no previous studies found related to chemical composition of hybrid mandarin (tangor). On the other hand, C. sinensis oil contains the largest number of identified components (27 components) and distinguished by the presence of some ingredients that did not exist in the other studied oils such as γ-terpinene (1.08%), carene (0.42%), cis-citral (0.69%), 8-hydroxyl geraniol (1.96%), and sesquiterpenes (L-Caryophyllene (0.25%) and valencene (1.62%) (Table S3). Other studies of the chemical composition of C. sinensis consistent with the results of our study with some differences in components percentages (Tao et al. 2009; Khemakhem et al. 2015).
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2.2. Larvicidal activity of essential oils C. sinensis oil was the most active against larvae, even at lower concentrations (LC50 = 15.34 and 12.53 mg/L after 24 and 48 h., respectively) (Table 1). Followed by, the oil of C. reticulata chinase that exhibited LC50 16.11 and 13.22 mg/L, respectively after 24 and 48 h of exposure. Whenever, the C. reticulata oil was the lowest effective against larvae (LC50 = 32.84 and 20.54 mg/L after 24 and 48 h, respectively). Our study is in agreement with that showed the Egyptian C. sinensis oil was more toxic than Greek C. sinensis against Cx. pipiens larvae (LC50 = 51.5 mg/L) (Michaelakis et al. 2009). In addition, Tennyson proved that the orange oil exhibited high larvicidal activity (LC50 = 85.93 mg/L) against Aedes aegypti (Tennyson et al. 2013).
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2.3. Fumigant toxicity of essential oils Among three tested EOs, the highest adulticidal activity was showed by C. sinensis followed by C. reticulata chinase then C. reticulata with LC50 of 2.78, 2.80 and 3.11 μL/L air, respectively (Table 2). The results proved by Zahran et al. showed the C. sinensis oil had LC50 of 3.58 mg/L against Cx. pipiens adults (Zahran et al. 2017). On the contrary, the study demonstrated by Soonwera proved that the repellent activity of C. reticulate oil was higher than C. sinensis oil against Ae. egypti and Cx quinquefasciatus adults (Soonwera 2015). There is no studies reported, the larvicidal and adulticidal activity of C. reticulata chinase against Cx. pipiens until now.
Table 1. Larvicidal activity of EOs isolated from C. reticulata, C. reticulata chinase and C. sinensis against Cx. pipiens.
EOs C. reticulata C. reticulata chinase C. sinensis
Time of exposure (h) 24 48 24 48 24 48
95% Confidence limits (mg/L) LC50a (mg/L) 32.84 20.54 16.11 13.22 15.35 12.54
Lower 27.159 16.032 11.282 10.734 13.545 7.007
Upper 39.304 24.807 20.278 15.462 17.026 16.871
Slopeb ± SE 5.420 ± 0.435 3.459 ± 0.265 2.450 ± 0.224 2.228 ± 0.223 3.255 ± 0.271 2.607 ± 0.250
Interceptc ± SE −8.220 ± 0.667 −4.541 ± 0.379 −2.958 ± 0.322 −2.498 ± 0.319 −3.861 ± 0.372 −2.862 ± 0.345
(χ2)d 16.389 10.189 7.338 6.452 4.532 10.940
a
The concentration causing 50% mortality. Slope of the concentration-mortality regression line ± standard error (SE). c Intercept of the regression line ± SE. d Chi square value. b
Table 2. Fumigant activity of EOs isolated from C. reticulata, C. reticulata chinase and C. sinensis against Cx. pipiens adults after 24 h of exposure. 95% Confidence limits (μL/Lair) EOs C. reticulata C. reticulata chinase C. sinensis a
LC50a (μL/Lair) 3.11 2.80 2.78
Lower 2.957 2.601 2.578
Upper 3.245 2.949 2.934
The concentration causing 50% mortality. Slope of the concentration-mortality regression line ± standard error (SE). c Intercept of the regression line ± SE. d Chi square value. b
Slopeb ± SE 10.969 ± 1.225 10.047 ± 1.455 9.914 ± 1.452
Interceptc ± SE −5.398 ± 0.639 −4.488 ± 0.730 −4.402 ± 0.729
(χ2)d 0.229 0.111 0.117
LC50 (mg/L) 0.00 24 h (32.84) 48 h (20.54) 24 h 16.11 48 h (13.22) 24 h 15.35 48 h (12.54) AChE 1.24a ± 0.001 0.39 g ± 0.140 0.57e ± 0.014 0.50f ± 0.018 0.96c ± 0.013 0.68d ± 0.016 1.10b ± 0.011
ACP 1.07d ± 0.023 2.31b ± 0.002 0.53e ± 0.002 2.85a ± 0.019 1.48c ± 0.051 1.52c ± 0.002 1.01d ± 0.002
ALP 1.15e ± 0.050 2.15a ± 0.010 1.31d ± 0.004 2.07a ± 0.013 1.50c ± 0.090 1.92b ± 0.000 1.53c ± 0.043
ATPase 1.37e ± 0.078 7.29b ± 0.028 5.78d ± 0.124 7.88a ± 0.015 6.23c ± 0.030 7.34b ± 0.130 7.04b ± 0.165
Notes: Different letters in the same column indicate statistically significant differences according to Student-Newman-Keuls (SNK) test (p ≤ 0.05).
C. sinensis
C. reticulata chinase
Untreated larvae C. reticulata
Treatments
Enzyme activity (OD.mg−1 protein.min−1) ± SE CbE 6.50c ± 0.027 8.30a ± 0.050 6.06d ± 0.007 7.04b ± 0.004 4.89e ± 0.026 4.12 g ± 0.041 4.31f ± 0.042
GST 0.025d ± 0.002 0.052a ± 0.002 0.037b ± 0.001 0.040b ± 0.000 0.031c ± 0.001 0.039b ± 0.000 0.031c ± 0.000
Table 3. In-vivo biochemical effects of EOs of C. reticulata, C. reticulata chinase and C. sinensis at LC50 on AChE, ACP, ALP, CbE, GST and ATPase activities in Cx. pipiens larvae.
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NATURAL PRODUCT RESEARCH
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2.4. Biochemical studies Increase of the concentration from 20.5 to 32.8 mg/L of C. reticulata oil led to decrease in AChE activity from 0.57 to 0.39, respectively (Table 3). While the activity was decreased from 0.96 to 0.5, at 13.2 to 16.11 mg/L of C. reticulata chinase oil, respectively. The activity was also decreased from 1.10 to 0.68 at 12.5 and 15.4 mg/L, respectively of C. sinensis. Other treatments caused high inhibition of AChE (68.62, 53.81, and 29.89% at 32.5 mg/L of C. reticulata, 20.50 mg/L of C. reticulata chinase, and 15.40 mg/L of C. sinensis, respectively). It has been reported that the essential oils from aromatic plants, and monoterpenes (α and β-pinene, linalool, thujene, myrcene, geranyl acetate and geranyl formate) act as reversible competitive inhibitors of AChE in different insect species (Picollo et al. 2008; Owokotomo et al. 2015). This suggests the possibility of synergy between predominant bioactive components in three EOs to cause an obvious inhibition in AChE activity irrespective of the minor active compounds (Owokotomo et al. 2015). The EOs of three species caused significant increase of ACP, ALP and ATPase activities at tested concentrations (Table 3). It is expected that alterations on intracellular and external ATP balance will be affected due to the action of the EOs on the cell membrane and the correlation between the intracellular and extracellular (Faleiro 2011). From this point, we can interpret the in vivo activation of ATPase which may be due to the alterations of ATP balance due to the action of the EOs on the cell membrane. EOs decreased the CbE activity with low concentrations range but the activity was increased with high concentrations range (Table 3). Our results are in agreement with the results obtained by Wei et al. who found that Chenopodium ambrosioides oil by either contact or fumigant treatment significantly inhibited CbE (Wei et al. 2015). CbE plays an essential role in resistance mechanism in insects, it is one of important detoxifying enzymes of xenobiotic or insecticides in body insect. The increased activity of CbE could enhance insects’ ability to detoxify the insecticides, leading to the development of resistance (Wei et al. 2015). The results proved that these EOs had activation effects on GST activity (Table 3). GSTs are considered from the important detoxifying enzymes against any xenobiotic compound in insect body (Yan et al. 2009). Certain plant secondary metabolites (monoterpenes) can inhibit GSTs activity, whereas others can activate them (Francis et al. 2005; Caballero et al. 2008). For these reasons, GSTs activity was increased in this study at all used concentrations to protect the Cx. pipiens larvae form damage by secondary metabolites in the tested EOs.
3. Conclusion EOs from peels of Citrus plants (C. reticulata L., C. reticulata chinase and C. sinensis L.) were very effective against Cx. pipiens larvae and adults. The mode of action of these natural products can refer to the inhibition of AChE at all tested concentrations and CbE at low concentrations, but they showed a contrary effect on the ACP, ALP and ATPase enzymes.
Disclosure statement No potential conflict of interest was reported by the authors.
ORCID Mohamed E. I. Badawy
http://orcid.org/0000-0002-6923-5452
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References Badawy ME, Taktak NE, Awad OM, Elfiki SA, Abou El-Ela NE. 2016. Preparation of ecofriendly formulations containing biologically active monoterpenes with their fumigant and residual toxicities against adults of Culex pipiens. J Trop Medic. 2016:8. Article ID 8540830. Caballero C, López-Olguín J, Ruiz M, Ortego F, Castañera P. 2008. Antifeedant activity and effects of terpenoids on detoxication enzymes of the beet armyworm, Spodoptera exigua (Hübner). Spanish J Agric Res. 6:177–184. Caccioni DR, Guizzardi M, Biondi DM, Renda A, Ruberto G. 1998. Relationship between volatile components of citrus fruit essential oils and antimicrobial action on Penicillium digitatum and Penicillium italicum. Int J Food Microbiol. 43:73–79. Chutia M, Bhuyan PD, Pathak M, Sarma T, Boruah P. 2009. Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India. LWT-Food Sci Technol. 42:777–780. Espina L, Somolinos M, Lorán S, Conchello P, García D, Pagán R. 2011. Chemical composition of commercial citrus fruit essential oils and evaluation of their antimicrobial activity acting alone or in combined processes. Food Cont. 22:896–902. Faleiro M. 2011. The mode of antibacterial action of essential oils. Science against microbial pathogens. Comm Cur Res Technol Adv. 2:1143–1156. Francis F, Vanhaelen N, Haubruge E. 2005. Glutathione-S-transferases in the adaptation to plant secondary metabolites in the Myzus persicae aphid. Arch Insect Biochem Physiol. 58:166–174. Khemakhem I, Yaiche C, Ayadi MA, Bouaziz M. 2015. Impact of aromatization by Citrus limetta and Citrus sinensis peels on olive oil quality, chemical composition and heat stability. J Am Oil Chem Soci. 92:701–708. Michaelakis A, Papachristos D, Kimbaris A, Koliopoulos G, Giatropoulos A, Polissiou MG. 2009. Citrus essential oils and four enantiomeric pinenes against Culex pipiens (Diptera: Culicidae). Parasitol Res. 105:769. Owokotomo I, Ekundayo O, Abayomi T, Chukwuka A. 2015. In-vitro anti-cholinesterase activity of essential oil from four tropical medicinal plants. Toxicol Rep. 2:850–857. Pavela R. 2015. Essential oils for the development of eco-friendly mosquito larvicides: A review. Indust Crops Prod. 76:174–187. Picollo M, Toloza A, Cueto GM, Zygadlo J, Zerba E. 2008. Anticholinesterase and pediculicidal activities of monoterpenoids. Fitoterapia. 79:271–278. Reegan AD, Ceasar SA, Paulraj MG, Ignacimuthu S, Al-Dhabi NA. 2016. Current status of genome editing in vector mosquitoes: a review. BioSci Trends. 10:424–432. Sanei-Dehkordi A, Sedaghat MM, Vatandoost H, Abai MR. 2016. Chemical compositions of the peel essential oil of Citrus aurantium and its natural larvicidal activity against the malaria vector Anopheles stephensi (Diptera: Culicidae) in comparison with Citrus paradisi. J Arthropod-Borne Dis. 10:577–585. Soonwera M. 2015. Efficacy of essential oils from Citrus plants against mosquito vectors Aedes aegypti (Linn.) and Culex quinquefasciatus (Say). J Agric Technol. 11:669–681. Tao NG, Liu YJ, Zhang ML. 2009. Chemical composition and antimicrobial activities of essential oil from the peel of bingtang sweet orange (Citrus sinensis Osbeck). Int J Food Sci Technol. 44:1281–1285. Tennyson S, Samraj DA, Jeyasundar D, Chalieu K. 2013. Larvicidal efficacy of plant oils against the dengue vector Aedes aegypti (L.) (Diptera: Culicidae). Middle East J Sci Res. 13:64–68. Wei H, Liu J, Li B, Zhan Z, Chen Y, Tian H, Lin S, Gu X. 2015. The toxicity and physiological effect of essential oil from Chenopodium ambrosioides against the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). Crop Protec. 76:68–74. Yan S, Cui F, Qiao C. 2009. Structure, function and applications of carboxylesterases from insects for insecticide resistance. Protein and Peptide lett. 16:1181–1188. Zahran HE-DM, Abou-Taleb HK, Abdelgaleil SA. 2017. Adulticidal, larvicidal and biochemical properties of essential oils against Culex pipiens L. J Asia-Pacific Entomol. 20:133–139.
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Chemical composition of the essential oils isolated from peel of three citrus species and their mosquitocidal activity against Culex pipiens Mohamed E. I. Badawy, Nehad E. M. Taktak & Ahmed F. El-Aswad To cite this article: Mohamed E. I. Badawy, Nehad E. M. Taktak & Ahmed F. El-Aswad (2017): Chemical composition of the essential oils isolated from peel of three citrus species and their mosquitocidal activity against Culex pipiens, Natural Product Research, DOI: 10.1080/14786419.2017.1378216 To link to this article: http://dx.doi.org/10.1080/14786419.2017.1378216
View supplementary material
Published online: 10 Oct 2017.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 Download by: [University of Liverpool]
Date: 11 October 2017, At: 02:07
Natural Product Research, 2017 https://doi.org/10.1080/14786419.2017.1378216
SHORT COMMUNICATION
Chemical composition of the essential oils isolated from peel of three citrus species and their mosquitocidal activity against Culex pipiens Mohamed E. I. Badawya , Nehad E. M. Taktakb and Ahmed F. El-Aswada a
Downloaded by [University of Liverpool] at 02:07 11 October 2017
Faculty of Agriculture, Department of Pesticide Chemistry and Technology, 21545-El-Shatby, Alexandria University, Alexandria, Egypt; bDepartment of Tropical Health, High Institute of Public Health, Alexandria University, Alexandria, Egypt
ABSTRACT
Three essential oils (EOs) were isolated from the peel of citrus fruits Citrus reticulata L., Citrus reticulata chinase Blanco and Citrus sinensis (L.) Osbeck (Family: Rutaceae) and evaluated against Culex pipiens L.1758 (Family: Culicidae). Chemical composition indicated that the EOs were rich in essential phytochemicals including hydrocarbons, monoterpenes and sesquiterpenes. These constituents revealed some variability among the oils displaying interesting chemotypes limonene (35–51%), 1R-α-pinene (1.04–2.5%), γ-terpinene (0.46–5.65%) and sabinene (0.51–5.42%). The toxicity proved that C. sinensis oil had more effect than C. reticulata chinase and C. reticulata oils against larvae (LC50 = 15.35, 16.11 and 32.84 mg/L, respectively). However, C. reticulate was the most active as fumigant against adults (LC50 2.74 μL/L air). The in vivo effect on acetylcholine esterase (AChE), carboxyl esterase (CbE), acid phosphatase (ACP), alkaline phosphatase (ALP), adenosine triphosphatase (ATPase) and glutathione-S-transferase (GST) were also demonstrated. To the best of our knowledge, this is the first report about the chemical composition and mosquitocidal activity of C. reticulata chinase essential oils. Conclusively, the tested essential oils could be used as eco-friendly alternatives in mosquitoes control programme.
ARTICLE HISTORY
Received 24 July 2017 Accepted 29 August 2017 KEYWORDS
Chemical composition; Culex pipiens; essential oils; mosquitocidal activity
Hydro distillation Citrus reticulata L.
Citrus reticulata Chinase
Citrus sinensis L.
GC/MS analysis
Live larvae
Fumigant assay against adults
In-vitro mosquitocidal activity against Culex pipiens
Dead larvae
CONTACT Mohamed E. I. Badawy [email protected], [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1378216. © 2017 Informa UK Limited, trading as Taylor & Francis Group
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M. E. I. BADAWY ET AL.
Downloaded by [University of Liverpool] at 02:07 11 October 2017
1. Introduction Mosquitoes are responsible for the transmission of many disease to humans and animals in the world due to rapid urbanisation and poor water management (Badawy et al. 2016; Reegan et al. 2016). The essential oils (EOs) extracted from some plants are suitable as they are economically and highly active against mosquitoes (Pavela 2015). The EOs were present in a great quantities in Citrus fruit flavedo of the family Rutaceae which includes about 17 species grown throughout the world (Caccioni et al. 1998). In fact, limonene, α-pinene, α-terpinolene, which considered from the active components exist in Citrus EOs, exhibited a wide spectrum of larvicidal activity against mosquitoes, as convinced by many previous studies in other plants (Sanei-Dehkordi et al. 2016; Zahran et al. 2017). Among commercially common species Citrus reticulata (mandrin), Citrus reticulata chinase (tangor) and Citrus sinensis (orange) are considered the important species grown in Egypt. Therefore, the main objective of current study is to identify the chemical composition of three isolated EOs from common Citrus species (C. reticulata, C. reticulata chinase and C. sinensis, Family: Rutaceae) in Egypt by GC/MS and evaluate their larvicidal and adulticidal activity against Culex pipiens L. 1758 (Family: Culicidae). In order to elucidate the mechanism of action of the EOs tested, certain biochemical parameters including target enzymes of acetylcholine esterase (AChE), acid phosphatase (ACP), alkaline phosphatase (ALP) and adenosine triphosphatase (ATPase), and two detoxifying enzymes of carboxyl esterase (CbE) and glutathione-S-transferase (GST) were evaluated in vivo in larvae exposed to 24 and 48 h of the LC50 of each EO.
2. Results and discussion 2.1. Chemical composition of EOs The EOs contain limonene as the major component (43.25% in C. reticulate, 51.49% in C. reticulata chinase and 34.97% in C. sinensis) (Tables S1–S3). α-pinene and myrcene (monoterpenes) are involved in three tested EOs with different amounts. While the C. reticulate oil features with the presence of some hydrocarbon compounds (2,3-dimethyl-octane 4.24%, toluene 4.74% and 1,3-xylene 1.44%) and monoterpenes (α-phellandrene 0.8%, β-pinene 1.24%, 1,8-cineole 0.11%, γ-terpinene 5.65% and geranyl isovalerate 0.15%) (Table S1). These results are similar to the other results proved that limonene was the major component in C. reticulata Blanco EO (46.7–73.38%), while geraniol was completely absent from their study (Chutia et al. 2009; Espina et al. 2011). The major constituents of C. reticulata chinase oil followed by limonene are shown in Table S2 as (R)-m-mentha-6-8-diene (10.81%), sabinene (5.42%), α-myrcene (3.56%), (1R)-α- pinene (2.30%), linalool (2.11%), then citronellol (0.78%). There is no previous studies found related to chemical composition of hybrid mandarin (tangor). On the other hand, C. sinensis oil contains the largest number of identified components (27 components) and distinguished by the presence of some ingredients that did not exist in the other studied oils such as γ-terpinene (1.08%), carene (0.42%), cis-citral (0.69%), 8-hydroxyl geraniol (1.96%), and sesquiterpenes (L-Caryophyllene (0.25%) and valencene (1.62%) (Table S3). Other studies of the chemical composition of C. sinensis consistent with the results of our study with some differences in components percentages (Tao et al. 2009; Khemakhem et al. 2015).
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2.2. Larvicidal activity of essential oils C. sinensis oil was the most active against larvae, even at lower concentrations (LC50 = 15.34 and 12.53 mg/L after 24 and 48 h., respectively) (Table 1). Followed by, the oil of C. reticulata chinase that exhibited LC50 16.11 and 13.22 mg/L, respectively after 24 and 48 h of exposure. Whenever, the C. reticulata oil was the lowest effective against larvae (LC50 = 32.84 and 20.54 mg/L after 24 and 48 h, respectively). Our study is in agreement with that showed the Egyptian C. sinensis oil was more toxic than Greek C. sinensis against Cx. pipiens larvae (LC50 = 51.5 mg/L) (Michaelakis et al. 2009). In addition, Tennyson proved that the orange oil exhibited high larvicidal activity (LC50 = 85.93 mg/L) against Aedes aegypti (Tennyson et al. 2013).
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2.3. Fumigant toxicity of essential oils Among three tested EOs, the highest adulticidal activity was showed by C. sinensis followed by C. reticulata chinase then C. reticulata with LC50 of 2.78, 2.80 and 3.11 μL/L air, respectively (Table 2). The results proved by Zahran et al. showed the C. sinensis oil had LC50 of 3.58 mg/L against Cx. pipiens adults (Zahran et al. 2017). On the contrary, the study demonstrated by Soonwera proved that the repellent activity of C. reticulate oil was higher than C. sinensis oil against Ae. egypti and Cx quinquefasciatus adults (Soonwera 2015). There is no studies reported, the larvicidal and adulticidal activity of C. reticulata chinase against Cx. pipiens until now.
Table 1. Larvicidal activity of EOs isolated from C. reticulata, C. reticulata chinase and C. sinensis against Cx. pipiens.
EOs C. reticulata C. reticulata chinase C. sinensis
Time of exposure (h) 24 48 24 48 24 48
95% Confidence limits (mg/L) LC50a (mg/L) 32.84 20.54 16.11 13.22 15.35 12.54
Lower 27.159 16.032 11.282 10.734 13.545 7.007
Upper 39.304 24.807 20.278 15.462 17.026 16.871
Slopeb ± SE 5.420 ± 0.435 3.459 ± 0.265 2.450 ± 0.224 2.228 ± 0.223 3.255 ± 0.271 2.607 ± 0.250
Interceptc ± SE −8.220 ± 0.667 −4.541 ± 0.379 −2.958 ± 0.322 −2.498 ± 0.319 −3.861 ± 0.372 −2.862 ± 0.345
(χ2)d 16.389 10.189 7.338 6.452 4.532 10.940
a
The concentration causing 50% mortality. Slope of the concentration-mortality regression line ± standard error (SE). c Intercept of the regression line ± SE. d Chi square value. b
Table 2. Fumigant activity of EOs isolated from C. reticulata, C. reticulata chinase and C. sinensis against Cx. pipiens adults after 24 h of exposure. 95% Confidence limits (μL/Lair) EOs C. reticulata C. reticulata chinase C. sinensis a
LC50a (μL/Lair) 3.11 2.80 2.78
Lower 2.957 2.601 2.578
Upper 3.245 2.949 2.934
The concentration causing 50% mortality. Slope of the concentration-mortality regression line ± standard error (SE). c Intercept of the regression line ± SE. d Chi square value. b
Slopeb ± SE 10.969 ± 1.225 10.047 ± 1.455 9.914 ± 1.452
Interceptc ± SE −5.398 ± 0.639 −4.488 ± 0.730 −4.402 ± 0.729
(χ2)d 0.229 0.111 0.117
LC50 (mg/L) 0.00 24 h (32.84) 48 h (20.54) 24 h 16.11 48 h (13.22) 24 h 15.35 48 h (12.54) AChE 1.24a ± 0.001 0.39 g ± 0.140 0.57e ± 0.014 0.50f ± 0.018 0.96c ± 0.013 0.68d ± 0.016 1.10b ± 0.011
ACP 1.07d ± 0.023 2.31b ± 0.002 0.53e ± 0.002 2.85a ± 0.019 1.48c ± 0.051 1.52c ± 0.002 1.01d ± 0.002
ALP 1.15e ± 0.050 2.15a ± 0.010 1.31d ± 0.004 2.07a ± 0.013 1.50c ± 0.090 1.92b ± 0.000 1.53c ± 0.043
ATPase 1.37e ± 0.078 7.29b ± 0.028 5.78d ± 0.124 7.88a ± 0.015 6.23c ± 0.030 7.34b ± 0.130 7.04b ± 0.165
Notes: Different letters in the same column indicate statistically significant differences according to Student-Newman-Keuls (SNK) test (p ≤ 0.05).
C. sinensis
C. reticulata chinase
Untreated larvae C. reticulata
Treatments
Enzyme activity (OD.mg−1 protein.min−1) ± SE CbE 6.50c ± 0.027 8.30a ± 0.050 6.06d ± 0.007 7.04b ± 0.004 4.89e ± 0.026 4.12 g ± 0.041 4.31f ± 0.042
GST 0.025d ± 0.002 0.052a ± 0.002 0.037b ± 0.001 0.040b ± 0.000 0.031c ± 0.001 0.039b ± 0.000 0.031c ± 0.000
Table 3. In-vivo biochemical effects of EOs of C. reticulata, C. reticulata chinase and C. sinensis at LC50 on AChE, ACP, ALP, CbE, GST and ATPase activities in Cx. pipiens larvae.
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2.4. Biochemical studies Increase of the concentration from 20.5 to 32.8 mg/L of C. reticulata oil led to decrease in AChE activity from 0.57 to 0.39, respectively (Table 3). While the activity was decreased from 0.96 to 0.5, at 13.2 to 16.11 mg/L of C. reticulata chinase oil, respectively. The activity was also decreased from 1.10 to 0.68 at 12.5 and 15.4 mg/L, respectively of C. sinensis. Other treatments caused high inhibition of AChE (68.62, 53.81, and 29.89% at 32.5 mg/L of C. reticulata, 20.50 mg/L of C. reticulata chinase, and 15.40 mg/L of C. sinensis, respectively). It has been reported that the essential oils from aromatic plants, and monoterpenes (α and β-pinene, linalool, thujene, myrcene, geranyl acetate and geranyl formate) act as reversible competitive inhibitors of AChE in different insect species (Picollo et al. 2008; Owokotomo et al. 2015). This suggests the possibility of synergy between predominant bioactive components in three EOs to cause an obvious inhibition in AChE activity irrespective of the minor active compounds (Owokotomo et al. 2015). The EOs of three species caused significant increase of ACP, ALP and ATPase activities at tested concentrations (Table 3). It is expected that alterations on intracellular and external ATP balance will be affected due to the action of the EOs on the cell membrane and the correlation between the intracellular and extracellular (Faleiro 2011). From this point, we can interpret the in vivo activation of ATPase which may be due to the alterations of ATP balance due to the action of the EOs on the cell membrane. EOs decreased the CbE activity with low concentrations range but the activity was increased with high concentrations range (Table 3). Our results are in agreement with the results obtained by Wei et al. who found that Chenopodium ambrosioides oil by either contact or fumigant treatment significantly inhibited CbE (Wei et al. 2015). CbE plays an essential role in resistance mechanism in insects, it is one of important detoxifying enzymes of xenobiotic or insecticides in body insect. The increased activity of CbE could enhance insects’ ability to detoxify the insecticides, leading to the development of resistance (Wei et al. 2015). The results proved that these EOs had activation effects on GST activity (Table 3). GSTs are considered from the important detoxifying enzymes against any xenobiotic compound in insect body (Yan et al. 2009). Certain plant secondary metabolites (monoterpenes) can inhibit GSTs activity, whereas others can activate them (Francis et al. 2005; Caballero et al. 2008). For these reasons, GSTs activity was increased in this study at all used concentrations to protect the Cx. pipiens larvae form damage by secondary metabolites in the tested EOs.
3. Conclusion EOs from peels of Citrus plants (C. reticulata L., C. reticulata chinase and C. sinensis L.) were very effective against Cx. pipiens larvae and adults. The mode of action of these natural products can refer to the inhibition of AChE at all tested concentrations and CbE at low concentrations, but they showed a contrary effect on the ACP, ALP and ATPase enzymes.
Disclosure statement No potential conflict of interest was reported by the authors.
ORCID Mohamed E. I. Badawy
http://orcid.org/0000-0002-6923-5452
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