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Insecticidal activity of camphene, zerumbone and α-humulene from Cheilocostus speciosus rhizome essential oil against the Old-World bollworm, Helicoverpa armigera
Ecotoxicology and Environmental Safety 148 (2018) 781–786
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Insecticidal activity of camphene, zerumbone and α-humulene from Cheilocostus speciosus rhizome essential oil against the Old-World bollworm, Helicoverpa armigera ⁎
T
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Giovanni Benellia,b, , Marimuthu Govindarajanc,d, , Mohan Rajeswaryc, Baskaralingam Vaseeharane, Sami A. Alyahyaf, Naiyf S. Alharbig, Shine Kadaikunnang, Jamal M. Khaledg, Filippo Maggih a
Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy The BioRobotics Institute, Scuola Superiore Sant’Anna, Viale Rinaldo Piaggio 34, 56025 Pontedera, Pisa, Italy Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar 608002, Tamil Nadu, India d Department of Zoology, Government College for Women, Kumbakonam 612001, Tamil Nadu, India e Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Science Block, 6th floor, Burma Colony, Karaikudi 630003, Tamil Nadu, India f National Center for Biotechnology, King Abdulaziz City for Science and Technology, Riyadh 11442, Saudi Arabia g Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia h School of Pharmacy, University of Camerino, Via Sant’Agostino 1, 62032 Camerino, Italy b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Acute toxicity Cheilocostus speciosus Essential oil Green pesticides Insecticide resistance Larvicide
The fast-growing resistance development to several synthetic and microbial insecticides currently marketed highlighted the pressing need to develop novel and eco-friendly pesticides. Among the latter, botanical ones are attracting high research interest due to their multiple mechanisms of action and reduced toxicity on non-target vertebrates. Helicoverpa armigera (Lepidoptera: Noctuidae) is a key polyphagous insect pest showing insecticide resistance to several synthetic molecules used for its control. Therefore, here we focused on the rhizome essential oil extracted from an overlooked Asian plant species, Cheilocostus speciosus (J. Konig) C. Specht (Costaceae), as a source of compounds showing ingestion toxicity against H. armigera third instar larvae, as well as ovicidal toxicity. In acute larvicidal assays conducted after 24 h, the C. speciosus essential oil achieved a LC50 value of 207.45 µg/ml. GC and GC-MS analyses highlighted the presence of zerumbone (38.6%), α-humulene (14.5%) and camphene (9.3%) as the major compounds of the oil. Ingestion toxicity tests carried out testing these pure molecules showed LC50 values of 10.64, 17.16 and 20.86 µg/ml, for camphene, zerumbone and α-humulene, respectively. Moreover, EC50 values calculated on H. armigera eggs were 35.39, 59.51 and 77.10 µg/ml for camphene, zerumbone and α-humulene, respectively. Overall, this study represents the first report on the toxicity of C. speciosus essential oil against insect pests of agricultural and medical veterinary importance, highlighting that camphene, zerumbone and α-humulene have a promising potential as eco-friendly botanical insecticides.
1. Introduction
several alternative hosts and is characterized by high mobility and fecundity (Tay et al., 2013). Most importantly, the Old-World bollworm developed resistance to a number of insecticidal products used for control operations (Ramasubramaniam and Regupathy, 2004; Srinivas et al., 2004; Young et al., 2005). To avoid frequent and massive overuse of synthetic pesticides with relevant non-target effects on human health and the environment, current criteria of Integrated Pest Management (IPM) research
Helicoverpa armigera Hubner (Lepidoptera: Noctuidae), commonly known as the Old-World bollworm, is a polyphagous insect causing high losses to various crops, including cotton, maize, sorghum, legumes and several horticultural crops (Michael and Donald, 1996; Sharma, 2001; Sundararajan and Kumuthakalavalli, 2001; Talekar et al., 2006; Kriticos et al., 2015). It is worthy to note that H. armigera can survive on
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Corresponding author at: Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy. Corresponding author at: Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar 608002, Tamil Nadu, India. E-mail addresses: [email protected], [email protected] (G. Benelli), [email protected] (M. Govindarajan).
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https://doi.org/10.1016/j.ecoenv.2017.11.044 Received 26 October 2017; Received in revised form 14 November 2017; Accepted 17 November 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 148 (2018) 781–786
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from peak area normalization, without using correction factors. GC-MS was done on an Agilent 6890 GC with 5973 N mass selective detector and an HP–5 (5% phynylmethylpolysiloxane) capillary column. Oven temperature was from 50 to 280 °C, at the rate of 4 °C min−1 and held at 280 °C for 5 min. The inlet and interface temperatures were 250 and 280 °C, respectively. Carrier He was at 1.0 ml min−1 (constant flow). The sample (0.2 μl) was injected with a split ratio of 20:1. EI-MS was carried at 70 eV. Ion source and quadrupole temperatures were 230 and 150 °C, respectively. Compounds’ identification was performed by comparison of calculated RI (van Den Dool and Krats, 1963) using a mixture of n-alkanes (C8-C30) (Supelco, Bellefonte, CA) with those of literature, as well as by MS computer matching with WILEY, ADAMS and NIST 08 databases.
supported the development of novel and eco-friendly pesticides of herbal or microbial origin (Tewary et al., 2005; Miresmaili and Isman, 2014; Pavela and Benelli, 2016). Among them, plant essential oils have been recently proposed as noteworthy candidates, since a significant number of molecules contained in these oils are effective at low doses, cheap and exert toxicity through multiple mechanisms of action (Benelli, 2015; Pavela, 2015). Recently, several herbal preparations from various plant species have been tested on H. armigera, to study their toxic and antifeedant potential. Good examples are Eucalyptus camaldulensis Dehnh., Tylophora indica (Burm. F.) Merr. (Kathuria and Kaushik, 2005), Entandrophragma candollei Harms (Koul et al., 2003), Gnidia glauca (Fresen.) Gilg, Toddalia asiatica (L.) Lam. (Sundararajan and Kumuthakalavalli, 2001), Capsicum annum L. (Tamhane et al., 2005), Atalantia monophylla DC. (Baskar et al., 2009), Solanum pseudocapsicum L. (Jeyasankar et al., 2012), Caesalpinia crista L. (Nathala and Dhingra, 2006) and Melia dubia Cav. (syn. of M. azedarach L.) (Koul et al., 2000). Cheilocostus speciosus (J. Konig) C. Specht, popularly known as crape ginger, belongs to the family of Costaceae, which is strictly correlated with the Zingiberaceae family (Specht and Stevenson, 2006). This plant is native to Southeast Asia, with special reference to Greater Sunda Islands (Indonesia), and is widely cultivated for horticultural purposes (Specht and Stevenson, 2006). The leaves of C. speciosus have an acrid taste while the rhizomes are cooked and eaten (Lim, 2014). In India, C. speciosus can be found cultivated in gardens, since local people used it to prepare herbal remedies as well as ornamental. On the other hand, wild populations of C. speciosus are now rare (Mathur and Joshi, 2013). According to Ayurvedic medicine, the bitter rhizomes have astringent, aphrodisiac, purgative, depurative, febrifuge, expectorant and tonic properties; they are also useful to treat burning wounds, leprosy, skin diseases, diabetes, asthma, bronchitis and anemia (Chowdhury and Das, 2015; Pattanayak et al., 2013; Vishnuprasad et al., 2013; Sathasivampillai et al., 2017). Besides, the root is used to treat intestinal worms (Mathur and Joshi, 2013). The phytochemical composition of C. speciosus essential oil has been poorly investigated (Rao, 1971; Saraf, 2010), whereas its toxicity against insect pests remains unknown. In this research, we focused our attention on the rhizome essential oil extracted from the overlooked Asian plant species, C. speciosus, as a source of compounds showing ingestion toxicity against H. armigera third instar larvae. The C. speciosus oil was studied by GC and GC-MS analyses. Acute toxicity assays were conducted to evaluate the larvicidal and ovicidal toxicity of the C. speciosus on H. armigera. Furthermore, the major oil constituents, namely camphene, zerumbone and α-humulene, were tested against H. armigera eggs and third instar larvae, calculating their EC50/LC50 and EC90/LC90 values, respectively.
2.3. Insect rearing H. armigera insects were reared individually in plastic vials on castor leaves. The rearing was carried out in laboratory with 12:12 h (L:D) photoperiod, 28 ± 2 °C and 75 ± 5% R.H. Eggs as well as healthy and uniform sized (41.6 ± 0.65 mg/larva) third instar larvae were used for the toxicity experiments. 2.4. Ingestion toxicity assays on larvae For the evaluation of larvicidal activity, the C. speciosus essential oil and its three major components were selected. They were camphene, zerumbone and α-humulene. The three pure compounds were purchased by Sigma-Aldrich India. Here, the leaf-dipping method by Park et al. (2002) was employed, with some modifications. H. armigera larvae were individually transferred in plastic cages (29 cm × 8 cm) containing treated or control castor leaves. The petioles of the leaves were covered with wet cotton plugs. Based on preliminary assays, the C. speciosus essential oil was tested at 100–500 µg/ml, while camphene, zerumbone and α-humulene were evaluated at concentrations from 5 to 50 µg/ml. C. speciosus essential oil, camphene, zerumbone and α-humulene-free leaves served as control, they were treated with distilled water only. In each replicate, 20 pre-starved (4 h) 3rd instar larvae of H. armigera were tested. 5 replicates were done for each dose. After 24 h, the number of dead larvae was noted. Larval mortality (%) was calculated using Abbott's formula (1925). 2.5. Ovicidal activity A total of 700 eggs was divided into seven lots (n=100 each), which were dipped in 150–900 µg/ml of C. speciosus essential oil or 25–150 µg/ml of three major compounds. Then, the eggs hatched in controls and treatments were noted and the ovicidal activity (%) was estimated using Abbott's formula (1925). For each tested concentration, 5 replicates were done. The hatch rates were evaluated 120 h posttreatment.
2. Materials and methods 2.1. C. speciosus collection and oil distillation Rhizomes of C. speciosus were harvested from Munnar mountains (India, 10°05′21″N 77°03′35″E, 1700 m a.s.l.). C. speciosus was identified at Annamalai University (ID: AU ZOO 963). C. speciosus oil was obtained by hydrodistillation of fresh rhizomes (1 kg) in a Clevenger apparatus (4 h). The C. speciosus oil layer was separated, dried using Na2SO4 and stored in a dark vial at 8 °C for subsequent tests.
2.6. Statistical analysis Ovicidal data were analyzed using ANOVA followed by Tukey's HSD test (P < 0.05). Larval mortality data were subjected to probit analysis; we estimated LC50, LC90, their 95% CI, related parameters and chisquare values. As software, we used the Statistical Package of Social Science (SPSS) version 13.0 for Windows. P = 0.05 was used as threshold to establish significance levels.
2.2. GC and GC-MS analysis GC analysis was done on a Varian GC with a FID and BPI (100% dimethyl polysiloxane) capillary column. Carrier He was at 1.0 ml min−1 and 8 psi inlet pressure. Temperature was from 60 to 220 °C, at 5 °C min−1, with a hold time of 6 min. Injector and detector temperatures were 250 and 300 °C, respectively. The oil sample (0.2 μl) was injected (1:20 split ratio), and quantitative values were obtained
3. Results 3.1. Essential oil extraction and chemical composition The yield of C. speciosus rhizome oil was 1.9 ml/kg fresh weight. 782
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Table 1 Chemical composition of the Cheilocostus speciosus essential oil. Major constituents are given in bold. Compound Monoterpene hydrocarbons α-Pinene Camphene Myrcene α-Phellandrene δ−3-Carene p-Cymene Limonene Terpinolene Oxygenated monoterpenes 1,8-Cineole Linalool Camphor Borneol α-Terpineol Sesquiterpene hydrocarbons (E)-Caryophyllene α-Humulene δ-Amorphene Oxygenated sesquiterpenes Caryophyllene oxide Humulene epoxide II β-Eudesmol Zerumbone Total identified (%)
Retention Index
Literature Retention Indexa
928 945 987 1005 1008 1021 1025 1083
932 946 988 1003 1008 1020 1024 1086
1029 1102 1143 1167 1190
1026 1095 1145 1169 1186
1419 1453 1500
1417 1454 1498
1574 1601 1645 1736
1583 1608 1656 1734
Composition (%) 20.1 1.7 9.3 1.2 1.6 2.1 1.6 1.1 1.5 9.9 3.2 1.7 1.9 1.8 1.3 19.0 3.4 14.5 1.1 46.8 2.6 3.8 1.8 38.6 95.8
Mode of identificationb
RI, MS RI, MS, Std RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, RI, RI, RI, RI,
MS MS MS MS MS
RI, MS RI, MS, Std RI, MS RI, MS RI, MS RI, MS RI, MS, Std
MS = Mass spectra. Std = authentic standard. a RI values taken from Adams (2007). b RI = Retention index. Fig. 1. The three major constituents of Cheilocostus speciosus essential oil: (a) camphene, (b) zerumbone, and (c) α-humulene.
3.3. Ovicidal activity
Table 1 shows the constituents of the essential oil in order of their elution from a HP-5 column and their percentage composition. A total of twenty molecules (i.e., 95.8% of the C. speciosus essential oil) were identified. The C. speciosus oil was dominated by sesquiterpenes (65.8%) with oxygenated compounds (46.8%) more abundant than hydrocarbons (19.0%). Zerumbone (38.6%) and α-humulene (14.5%) were the main representative compounds of these groups, respectively (Fig. 1). Monoterpenes gave a minor contribution (30.0%), with hydrocarbons (20.1%) more abundant than oxygen-containing compounds (9.9%). Camphene (9.3%) was by far the major compound of the monoterpene fraction (Fig. 1). The abundance of remaining seventeen compounds ranged from 0.8% to 3.6%.
The ovicidal results achieved testing the C. speciosus essential oil and its three major compounds against H. armigera eggs are given in Table 3. As a general trend, the ovicidal activity was concentrationdependent. Among the pure compounds tested, camphene showed the best ovicidal activity, if compared to the other compounds. EC50 values calculated on H. armigera eggs were 35.39, 59.51 and 77.10 µg/ml for camphene, zerumbone and α-humulene, respectively (Table 3). 4. Discussion The Old-World bollworm, H. armigera – selected as target insect in this study – is a polyphagous pest of high economic importance showing insecticide resistance to a number of synthetic molecules used for its control (Kranthi et al., 2002; Torres-Vila et al., 2002; Srinivas et al., 2004; Young et al., 2005). More generally, the fast-growing resistance development to several synthetic and microbial insecticides currently marketed in many insect pests highlighted the pressing need to develop novel and eco-friendly pesticides (Bass et al., 2014; Naqqash et al., 2016; Ranson and Lissenden, 2016). Thus, herbal formulations and plant metabolites are attracting high research interest due to their ability to trigger toxicity through various
3.2. Ingestion toxicity of the oil and selected pure constituents The larvicidal results of C. speciosus oil and three major compounds from C. speciosus against H. armigera third instar larvae are given in Table 2. The C. speciosus essential oil achieved LC50 and LC90 values of 207.45 µg/ml and 403.88 µg/ml, respectively. Among the three compounds, the most potent larvicidal one was camphene, with a LC50 of 10.64 µg/ml and LC90 of 20.29 µg/ml, while zerumbone LC50 was 17.16 µg/ml and the LC90 was 32.20 µg/ml; α-humulene had a LC50 value of 20.86 µg/ml while its LC90 value was 41.72 µg/ml. 783
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Table 2 Larvicidal activity of the essential oil of Costus speciosus and its major chemical compounds against third instar larvae of Helicoverpa armigera. Treatment
Concentration (µg/ml)
Mortality (%) ± SDa
LC50 (µg/ml) (95% LCL-UCL)
LC90 (µg/ml) (95% LCL-UCL)
Slope
Regression equation
χ2 (d.f.)
Essential oil
100 200 300 400 500 5 10 15 20 25 8 16 24 32 40 10 20 30 40 50
28.4 ± 0.8 46.2 ± 1.2 67.3 ± 0.8 88.6 ± 0.6 100.0 ± 0.0 26.3 ± 1.2 44.8 ± 0.6 68.2 ± 0.6 87.3 ± 0.8 100.0 ± 0.0 24.9 ± 0.8 45.3 ± 0.8 67.2 ± 1.2 88.4 ± 0.6 100.0 ± 0.0 27.6 ± 1.2 48.2 ± 0.6 66.4 ± 0.8 87.3 ± 0.6 98.5 ± 0.6
207.45 (184.45–227.89)
403.88 (374.80–442.02)
3.01
y=10.42+0.186x
5.580 (4) n.s.
10.64 (9.52–11.64)
20.29 (18.85–22.16)
2.74
y=8.35+3.798x
4.866 (4) n.s.
17.16 (15.43–18.73)
32.20 (29.96–35.12)
2.56
y=7.17+2.416x
4.579 (4) n.s.
20.86 (18.42–23.00)
41.72 (38.65–45.78)
3.51
y=11.33+1.809x
2.843 (4) n.s.
Camphene
Zerumbone
α-Humulene
SD = standard deviation. d.f. = degrees of freedom. n.s. = not significant (P > 0.05). a Values are means ± SD of 5 replicates. Table 3 Ovicidal activity of the essential oil of Costus speciosus and its major chemical compounds on eggs of Helicoverpa armigera. Treatment
Essential oil Camphene Zerumbone α-Humulene
Egg hatchability (%) Control
150 µg/ml
300 µg/ml
100 ± 0.0a Control 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
89.4 ± 1.2b 25 µg/ml 55.1 ± 0.8 b 78.4 ± 1.4 b 87.2 ± 0.8b
71.6 ± 1.0 50 µg/ml 38.3 ± 1.2 59.6 ± 1.0 67.7 ± 1.2
c
c c c
450 µg/ml
600 µg/ml
750 µg/ml
900 µg/ml
59.3 ± 0.8d 75 µg/ml 21.7 ± 0.8 d 38.4 ± 1.2 d 56.3 ± 1.0d
38.2 ± 1.2e 100 µg/ml NH 20.2 ± 0.8e 35.2 ± 1.2e
19.2 ± 1.4 f 125 µg/ml NH NH 17.2 ± 1.2 f
NH 150 µg/ml NH NH NH
EC50 (µg/ml)
95% LCL-UCL
χ2 (d.f.=6)
485.92
412.15–557.10
10.154 n.s.
35.39 59.51 77.10
11.54–48.48 46.67–70.21 64.63–88.71
12.120 n.s. 9.933 n.s. 9.550 n.s.
Within each row, different letters indicate significant differences (ANOVA, Tukey's HSD, P < 0.05). NH = no hatchability. n.s. = not significant (P > 0.05). a Values are mean ± SD of 5 replicates.
flavoring and appetizer as well as a remedy for pains, inflammations, diarrhea and worm infestations (Yob et al., 2011). This compound exhibited in vivo antinociceptive, antiinflammatory and anticancer effects (Yob et al., 2011). The bioactivity of zerumbone depends on its capacity to act as Michael acceptors, which make it capable of trapping thiols by covalent coupling (Appendino et al., 2015). Interestingly, the zerumbone-rich hexane extract from Z. zerumbet was proven to be highly toxic to Aedes aegypti (L.) and Culex quinquefasciatus Say larvae (Mahardika et al., 2017). α-Humulene is one the main compounds responsible for aroma of hops and hemp, two members of the Cannabaceae family with insecticidal essential oils (Bedini et al., 2016; Benelli et al., 2017a), as well as for that of shampoo ginger (Yob et al., 2011). This sesquiterpene showed toxic effects against Anopheles stephensi Liston, A. aegypti and C. quinquefasciatus larval instars. In addition, it exerted repellency towards adults of the same mosquito vectors and oviposition deterrence as well (da Silva et al., 2015). This compound resulted selective towards mosquitoes, since its toxicity on non-target aquatic species, such as Diplonychus indicus Venkatesan & Rao and Gambusia affinis Baird and Girard, was quite low (Govindarajan et al., 2016). α-Humulene together with zerumbone are the main volatile components of Z. zerumbet essential oil, which was highly effective against pyrethroid-resistant strains of A. aegypti (Sutthanont et al., 2010). Interestingly, when occurring in mixture with other terpenes, αhumulene is able to stimulate oviposition in females of H. armigera
mechanisms of action at the same time, reduced toxicity on non-target vertebrates and – in some cases – large availability and low costs for farmers living in developing countries (Benelli and Mehlhorn 2016; Pavela and Benelli, 2016; Stevenson et al., 2017). In the ingestion toxicity assays conducted here, the toxicity of C. speciosus rhizome essential oil against H. armigera larvae and eggs was moderate, with a LC50 value of 207.45 µg/ml and an EC50 value of 485.92 µg/ml, respectively. On the other hand, the insecticidal efficacy of the three compounds characterizing the essential oil was highly promising, with extremely low LC50 values, 10.64, 17.16 and 20.86 µg/ ml for camphene, zerumbone and α-humulene, respectively. Zerumbone, α-humulene and camphene are terpenes endowed with high lipophilicity that allows them to be incorporated into the larval body of insect where they exert toxic effects. To the best of our knowledge, their mechanisms of action on larvae of H. armigera are unknown to date. Overall, terpenoids can exert insecticidal activity via different ways, e.g., by affecting the cholinergic, octopaminergic and GABAergic systems or by neutralizing the detoxicative enzymes of insects (Pavela and Benelli, 2016). Zerumbone and α-humulene are biogenetically related sesquiterpenes belonging to the humulane group (Appendino et al., 2015; Sut et al., 2017). Zerumbone is the main bioactive component obtained from rhizomes of shampoo ginger (Zingiber zerumbet (L.) Smith), a perennial herb occurring in several tropical countries where it is used as
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Trends Parasitol. 32, 187–196. Rao, B.G., Narasimha, V., Joseph, P.L., 1971. Activity of some essential oil towards phytopathogenic fungi. Riechstoffe Aromen Koerper-pflegemit 21,405-410. Saraf, A.E., 2010. Phytochemical and antimicrobial studies of medicinal plant Costus speciosus (Koen.). Eur. J. Chem. 7, S405–S413. Sathasivampillai, S.V., Rajamanoharan, P.R.S., Munday, M., Heinrich, M., 2017. Plants used to treat diabetes in Sri Lankan Siddha Medicine – An ethnopharmacological review of historical and modern sources. J. Ethnopharmacol. 198, 531–599. Sharma, H.C., 2001. Cotton Bollworm/legume Pod Borer, Helicoverpa armigera (Hübner) (Noctuidae: Lepidoptera): Biology and Management. Crop protection Compendium. ICRISAT, Patancheru, India. Specht, C.D., Stevenson, D.W., 2006. A new phylogeny-based generic classification of Costaceae (Zingiberales). Taxon 55, 153–163. Srinivas, R., Udikeri, S.S., Jayalakshmi, S.K., Sreeramulu, K., 2004. Identification of factors responsible for insecticide resistance in Helicoverpa armigera. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 137, 261–269. Stevenson, P.C., Isman, M.B., Belmain, S.R., 2017. Pesticidal plants in Africa: a global vision of new biological control products from local uses. Ind. Crops Prod. http://dx. doi.org/10.1016/j.indcrop.2017.08.034ucts.
(Jallow et al., 1999). Camphene is a volatile monoterpene hydrocarbon occurring in many essential oils, for instance that of Wedelia prostrata (Hook. et Arn.) Hemsl. and is effective against 4th instar larvae of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), showing LC50 values below 10 ppm (Benelli et al., 2017b) Interestingly, chlorination of camphene was used to manufacture insecticial compounds, such as Toxaphene, which has been used for long time to combat insect pests (Coelhan and Parlar, 1996) until its ban after the insurgence of resistance (El-Sebae et al., 1993). Overall, to the best of our information, this study is the first report on the C. speciosus essential oil against insect pests of agricultural and medical veterinary importance, highlighting that blends of camphene, zerumbone and α-humulene have a promising potential for the development of eco-friendly and effective botanical insecticides, including microencapsulation (Pavela, 2016) and nanoformulations (Benelli, 2016a,b, 2018). Further studies are needed on the mode of action of these terpenes on different target insects, as well as their toxicity of these compounds in blend on non-target species, such as parasitoids and predators of moth pests, as well as earthworms. Acknowledgements The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group No. RG-1438- 074. The authors express their sincere thanks to Professor and Head of the Department of Zoology, Annamalai University for the laboratory provisions granted. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ mass Spectrometry. Carol. Stream, Allured., IL, USA. Appendino, G., Minassi, A., Collado, J.A., Pollastro, F., Chianese, G., Taglialatela-Scafati, O., Ayyari, M., Garcia, V., Muñoz, E., 2015. The thia-Michael reactivity of zerumbone and related cross-conjugated dienones: disentangling stoichiometry, regiochemistry, and addition mode with an NMR-spectroscopy-based cysteamine assay. Eur. J. Org. Chem. 17, 3721–3726. Baskar, K., Kingsley, S., Vendan, S.E., Paulraj, M.G., Duraipandiyan, V., Ignacimuthu, S., 2009. Antifeedant, larvicidal and pupicidal activities of Atalantia monophylla (L) Correa against Helicoverpa armigera Hubner (Lepidoptera: Noctuidae). Chemosphere 75, 355–359. Bass, C., Puinean, A.M., Zimmer, C.T., Denholm, I., Field, L.M., Foster, S.P., Gutbrod, O., Nauen, R., Slater, R., Williamson, M.S., 2014. The evolution of insecticide resistance in the peach potato aphid, Myzus persicae. Insect Biochem. Mol. Biol. 51, 41–51. Bedini, S., Flamini, G., Cosci, F., Ascrizzi, R., Benelli, G., Conti, B., 2016. Cannabis sativa and Humulus lupulus essential oils as novel control tools against the invasive mosquito Aedes albopictus and fresh water snail Physella acuta. Ind. Crops Prod. 85, 318–323. Benelli, G., Govindarajan, M., AlSalhi, M.S., Devanesan, S., Maggi, F., 2017b. High toxicity of camphene and γ-elemene from Wedelia prostrata essential oil against larvae of Spodoptera litura (Lepidoptera: noctuidae). Environ. Sci. Pollut. Res. http://dx.doi. org/10.1007/s11356-017-9490-7. Benelli, G., Mehlhorn, H., 2016. Declining malaria, rising of dengue and Zika virus: insights for mosquito vector control. Parasitol. Res. 115, 1747–1754. Benelli, G., Pavela, R., Lupidi, G., Nabissi, M., Petrelli, R., Ngahang Kamte, S.L., Cappellacci, L., Fiorini, D., Sut, S., Dall’Acqua, S., Maggi, F., 2017a. The crop-residue of fiber hemp cv. Futura 75: from a waste product to a source of botanical insecticides. Environ. Sci. Poll. Res. http://dx.doi.org/10.1007/s11356-017-0635-5. Benelli, G., 2015. Plant-borne ovicides in the fight against mosquito vectors of medical and veterinary importance: a systematic review. Parasitol. Res. 114, 3201–3212. Benelli, G., 2016a. Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol. Res. 115, 23–34. Benelli, G., 2018. Gold nanoparticles – against parasites and insect vectors. Acta Trop. 178, 73–80. Benelli, G., 2016b. Green synthesized nanoparticles in the fight against mosquito-borne diseases and cancer—a brief review. Green synthesized nanoparticles in the fight against mosquito-borne diseases and cancer—a brief review 95, 58–68. Chowdhury, A., Das, A.P., 2015. Ethnopharmacological survey of wetland plants used by local ethnic people in sub- Himalayan terai and duars of West Bengal, India. Am. J. Ethnomed. 2, 122–135. Coelhan, M., Parlar, H., 1996. The nomenclature of chlorinated bornanes and camphenes relevant to toxaphene. Chemosphere 32, 217–228. da Silva, R.C.S., Milet-Pinheiro, P., da Silva, P.C.B., da Silva, A.G., da Silva, M.V., do
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Tewary, D.K., Bhardwaj, A., Shanker, A., 2005. Pesticidal activities in five medicinal plants collected from mid hills of western Himalayas. Ind. Crops Prod. 22, 241–247. Torres-Vila, L.M., Rodrıguez-Molina, M.C., Lacasa-Plasencia, A., Bielza-Lino, P., 2002. Insecticide resistance of Helicoverpa armigera to endosulfan, carbamates and organophosphates: the Spanish case. Crop Prot. 21, 1003–1013. van Den Dool, H., Krats, P.D., 1963. A generalization of the retention index system including linear temperature programmed gas—liquid partition chromatography. J. Chromatogr. A 11, 463–471. Vishnuprasad, C.N., Pradeep, N.S., Cho, Y.W., Gangadharan, G.G., Han, S.S., 2013. Fumigation in Ayurveda: potential strategy for drug discovery and drug delivery. J. Ethnopharmacol. 149, 409–415. Yob, N.J., Jofrry, S.M., Affandi, M.M.R.M.M., The, L.K., Salleh, M.Z., Zakaria, Z.A., 2011. Zingiber zerumbet (L.) Smith: a review of its ethnomedicinal, chemical, and pharmacological uses. Evid. Based Complem. Alternat. Med. http://dx.doi.org/10.1155/ 2011/543216. Young, S.J., Gunning, R.V., Moores, G.D., 2005. The effect of piperonyl butoxide on pyrethroid‐resistance‐associated esterases in Helicoverpa armigera (Hübner) (Lepidoptera: noctuidae). Pest Manag. Sci. 61, 397–401.
Sundararajan, G., Kumuthakalavalli, R., 2001. Antifeedant activity of aqueous extract of Gnidia glauca Gilg. and Toddalia asiatica Lam. on the gram pod borer, Helicoverpa armigera (Hubner). J. Environ. Biol. 22, 11–14. Sut, S., Maggi, F., Nicoletti, M., Baldan, V., Dall, A.S., 2017. New drugs from old natural compounds: scarcely investigated sesquiterpenes as new possible therapeutic agents. Curr. Med. Chem. http://dx.doi.org/10.2174/0929867324666170404150351. Sutthanont, N., Choochote, W., Tuetun, B., Junkum, A., Jitpakdi, A., Chaithong, U., Riyong, D., Pitasawat, B., 2010. Chemical composition and larvicidal activity of edible plant-derived essential oils against the pyrethroid-susceptible and -resistant strains of Aedes aegypti (Diptera: culicidae). J. Vector Ecol. 35, 106–115. Talekar, N.S., Opena, R.T., Hanson, P., 2006. Helicoverpa armigera management: a review of AVRDC's research on host plant resistance in tomato. Crop Prot. 5, 461–467. Tamhane, V.A., Chougule, N.P., Giri, A.P., Dixit, A.R., Sainani, M.N., Gupta, V.S., 2005. In vivo and in vitro effect of Capsicum annum proteinase inhibitors on Helicoverpa armigera gut proteinases. Biochim. Biophys. Acta 1722, 156–167. Tay, W.T., Soria, M.F., Walsh, T., Thomazoni, D., Silvie, P., Behere, G.T., Anderson, C., Downes, S., 2013. A brave new world for an old world pest: Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil. PLoS One 8, e80134.
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Insecticidal activity of camphene, zerumbone and α-humulene from Cheilocostus speciosus rhizome essential oil against the Old-World bollworm, Helicoverpa armigera ⁎
T
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Giovanni Benellia,b, , Marimuthu Govindarajanc,d, , Mohan Rajeswaryc, Baskaralingam Vaseeharane, Sami A. Alyahyaf, Naiyf S. Alharbig, Shine Kadaikunnang, Jamal M. Khaledg, Filippo Maggih a
Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy The BioRobotics Institute, Scuola Superiore Sant’Anna, Viale Rinaldo Piaggio 34, 56025 Pontedera, Pisa, Italy Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar 608002, Tamil Nadu, India d Department of Zoology, Government College for Women, Kumbakonam 612001, Tamil Nadu, India e Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Science Block, 6th floor, Burma Colony, Karaikudi 630003, Tamil Nadu, India f National Center for Biotechnology, King Abdulaziz City for Science and Technology, Riyadh 11442, Saudi Arabia g Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia h School of Pharmacy, University of Camerino, Via Sant’Agostino 1, 62032 Camerino, Italy b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Acute toxicity Cheilocostus speciosus Essential oil Green pesticides Insecticide resistance Larvicide
The fast-growing resistance development to several synthetic and microbial insecticides currently marketed highlighted the pressing need to develop novel and eco-friendly pesticides. Among the latter, botanical ones are attracting high research interest due to their multiple mechanisms of action and reduced toxicity on non-target vertebrates. Helicoverpa armigera (Lepidoptera: Noctuidae) is a key polyphagous insect pest showing insecticide resistance to several synthetic molecules used for its control. Therefore, here we focused on the rhizome essential oil extracted from an overlooked Asian plant species, Cheilocostus speciosus (J. Konig) C. Specht (Costaceae), as a source of compounds showing ingestion toxicity against H. armigera third instar larvae, as well as ovicidal toxicity. In acute larvicidal assays conducted after 24 h, the C. speciosus essential oil achieved a LC50 value of 207.45 µg/ml. GC and GC-MS analyses highlighted the presence of zerumbone (38.6%), α-humulene (14.5%) and camphene (9.3%) as the major compounds of the oil. Ingestion toxicity tests carried out testing these pure molecules showed LC50 values of 10.64, 17.16 and 20.86 µg/ml, for camphene, zerumbone and α-humulene, respectively. Moreover, EC50 values calculated on H. armigera eggs were 35.39, 59.51 and 77.10 µg/ml for camphene, zerumbone and α-humulene, respectively. Overall, this study represents the first report on the toxicity of C. speciosus essential oil against insect pests of agricultural and medical veterinary importance, highlighting that camphene, zerumbone and α-humulene have a promising potential as eco-friendly botanical insecticides.
1. Introduction
several alternative hosts and is characterized by high mobility and fecundity (Tay et al., 2013). Most importantly, the Old-World bollworm developed resistance to a number of insecticidal products used for control operations (Ramasubramaniam and Regupathy, 2004; Srinivas et al., 2004; Young et al., 2005). To avoid frequent and massive overuse of synthetic pesticides with relevant non-target effects on human health and the environment, current criteria of Integrated Pest Management (IPM) research
Helicoverpa armigera Hubner (Lepidoptera: Noctuidae), commonly known as the Old-World bollworm, is a polyphagous insect causing high losses to various crops, including cotton, maize, sorghum, legumes and several horticultural crops (Michael and Donald, 1996; Sharma, 2001; Sundararajan and Kumuthakalavalli, 2001; Talekar et al., 2006; Kriticos et al., 2015). It is worthy to note that H. armigera can survive on
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Corresponding author at: Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy. Corresponding author at: Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar 608002, Tamil Nadu, India. E-mail addresses: [email protected], [email protected] (G. Benelli), [email protected] (M. Govindarajan).
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https://doi.org/10.1016/j.ecoenv.2017.11.044 Received 26 October 2017; Received in revised form 14 November 2017; Accepted 17 November 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
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from peak area normalization, without using correction factors. GC-MS was done on an Agilent 6890 GC with 5973 N mass selective detector and an HP–5 (5% phynylmethylpolysiloxane) capillary column. Oven temperature was from 50 to 280 °C, at the rate of 4 °C min−1 and held at 280 °C for 5 min. The inlet and interface temperatures were 250 and 280 °C, respectively. Carrier He was at 1.0 ml min−1 (constant flow). The sample (0.2 μl) was injected with a split ratio of 20:1. EI-MS was carried at 70 eV. Ion source and quadrupole temperatures were 230 and 150 °C, respectively. Compounds’ identification was performed by comparison of calculated RI (van Den Dool and Krats, 1963) using a mixture of n-alkanes (C8-C30) (Supelco, Bellefonte, CA) with those of literature, as well as by MS computer matching with WILEY, ADAMS and NIST 08 databases.
supported the development of novel and eco-friendly pesticides of herbal or microbial origin (Tewary et al., 2005; Miresmaili and Isman, 2014; Pavela and Benelli, 2016). Among them, plant essential oils have been recently proposed as noteworthy candidates, since a significant number of molecules contained in these oils are effective at low doses, cheap and exert toxicity through multiple mechanisms of action (Benelli, 2015; Pavela, 2015). Recently, several herbal preparations from various plant species have been tested on H. armigera, to study their toxic and antifeedant potential. Good examples are Eucalyptus camaldulensis Dehnh., Tylophora indica (Burm. F.) Merr. (Kathuria and Kaushik, 2005), Entandrophragma candollei Harms (Koul et al., 2003), Gnidia glauca (Fresen.) Gilg, Toddalia asiatica (L.) Lam. (Sundararajan and Kumuthakalavalli, 2001), Capsicum annum L. (Tamhane et al., 2005), Atalantia monophylla DC. (Baskar et al., 2009), Solanum pseudocapsicum L. (Jeyasankar et al., 2012), Caesalpinia crista L. (Nathala and Dhingra, 2006) and Melia dubia Cav. (syn. of M. azedarach L.) (Koul et al., 2000). Cheilocostus speciosus (J. Konig) C. Specht, popularly known as crape ginger, belongs to the family of Costaceae, which is strictly correlated with the Zingiberaceae family (Specht and Stevenson, 2006). This plant is native to Southeast Asia, with special reference to Greater Sunda Islands (Indonesia), and is widely cultivated for horticultural purposes (Specht and Stevenson, 2006). The leaves of C. speciosus have an acrid taste while the rhizomes are cooked and eaten (Lim, 2014). In India, C. speciosus can be found cultivated in gardens, since local people used it to prepare herbal remedies as well as ornamental. On the other hand, wild populations of C. speciosus are now rare (Mathur and Joshi, 2013). According to Ayurvedic medicine, the bitter rhizomes have astringent, aphrodisiac, purgative, depurative, febrifuge, expectorant and tonic properties; they are also useful to treat burning wounds, leprosy, skin diseases, diabetes, asthma, bronchitis and anemia (Chowdhury and Das, 2015; Pattanayak et al., 2013; Vishnuprasad et al., 2013; Sathasivampillai et al., 2017). Besides, the root is used to treat intestinal worms (Mathur and Joshi, 2013). The phytochemical composition of C. speciosus essential oil has been poorly investigated (Rao, 1971; Saraf, 2010), whereas its toxicity against insect pests remains unknown. In this research, we focused our attention on the rhizome essential oil extracted from the overlooked Asian plant species, C. speciosus, as a source of compounds showing ingestion toxicity against H. armigera third instar larvae. The C. speciosus oil was studied by GC and GC-MS analyses. Acute toxicity assays were conducted to evaluate the larvicidal and ovicidal toxicity of the C. speciosus on H. armigera. Furthermore, the major oil constituents, namely camphene, zerumbone and α-humulene, were tested against H. armigera eggs and third instar larvae, calculating their EC50/LC50 and EC90/LC90 values, respectively.
2.3. Insect rearing H. armigera insects were reared individually in plastic vials on castor leaves. The rearing was carried out in laboratory with 12:12 h (L:D) photoperiod, 28 ± 2 °C and 75 ± 5% R.H. Eggs as well as healthy and uniform sized (41.6 ± 0.65 mg/larva) third instar larvae were used for the toxicity experiments. 2.4. Ingestion toxicity assays on larvae For the evaluation of larvicidal activity, the C. speciosus essential oil and its three major components were selected. They were camphene, zerumbone and α-humulene. The three pure compounds were purchased by Sigma-Aldrich India. Here, the leaf-dipping method by Park et al. (2002) was employed, with some modifications. H. armigera larvae were individually transferred in plastic cages (29 cm × 8 cm) containing treated or control castor leaves. The petioles of the leaves were covered with wet cotton plugs. Based on preliminary assays, the C. speciosus essential oil was tested at 100–500 µg/ml, while camphene, zerumbone and α-humulene were evaluated at concentrations from 5 to 50 µg/ml. C. speciosus essential oil, camphene, zerumbone and α-humulene-free leaves served as control, they were treated with distilled water only. In each replicate, 20 pre-starved (4 h) 3rd instar larvae of H. armigera were tested. 5 replicates were done for each dose. After 24 h, the number of dead larvae was noted. Larval mortality (%) was calculated using Abbott's formula (1925). 2.5. Ovicidal activity A total of 700 eggs was divided into seven lots (n=100 each), which were dipped in 150–900 µg/ml of C. speciosus essential oil or 25–150 µg/ml of three major compounds. Then, the eggs hatched in controls and treatments were noted and the ovicidal activity (%) was estimated using Abbott's formula (1925). For each tested concentration, 5 replicates were done. The hatch rates were evaluated 120 h posttreatment.
2. Materials and methods 2.1. C. speciosus collection and oil distillation Rhizomes of C. speciosus were harvested from Munnar mountains (India, 10°05′21″N 77°03′35″E, 1700 m a.s.l.). C. speciosus was identified at Annamalai University (ID: AU ZOO 963). C. speciosus oil was obtained by hydrodistillation of fresh rhizomes (1 kg) in a Clevenger apparatus (4 h). The C. speciosus oil layer was separated, dried using Na2SO4 and stored in a dark vial at 8 °C for subsequent tests.
2.6. Statistical analysis Ovicidal data were analyzed using ANOVA followed by Tukey's HSD test (P < 0.05). Larval mortality data were subjected to probit analysis; we estimated LC50, LC90, their 95% CI, related parameters and chisquare values. As software, we used the Statistical Package of Social Science (SPSS) version 13.0 for Windows. P = 0.05 was used as threshold to establish significance levels.
2.2. GC and GC-MS analysis GC analysis was done on a Varian GC with a FID and BPI (100% dimethyl polysiloxane) capillary column. Carrier He was at 1.0 ml min−1 and 8 psi inlet pressure. Temperature was from 60 to 220 °C, at 5 °C min−1, with a hold time of 6 min. Injector and detector temperatures were 250 and 300 °C, respectively. The oil sample (0.2 μl) was injected (1:20 split ratio), and quantitative values were obtained
3. Results 3.1. Essential oil extraction and chemical composition The yield of C. speciosus rhizome oil was 1.9 ml/kg fresh weight. 782
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Table 1 Chemical composition of the Cheilocostus speciosus essential oil. Major constituents are given in bold. Compound Monoterpene hydrocarbons α-Pinene Camphene Myrcene α-Phellandrene δ−3-Carene p-Cymene Limonene Terpinolene Oxygenated monoterpenes 1,8-Cineole Linalool Camphor Borneol α-Terpineol Sesquiterpene hydrocarbons (E)-Caryophyllene α-Humulene δ-Amorphene Oxygenated sesquiterpenes Caryophyllene oxide Humulene epoxide II β-Eudesmol Zerumbone Total identified (%)
Retention Index
Literature Retention Indexa
928 945 987 1005 1008 1021 1025 1083
932 946 988 1003 1008 1020 1024 1086
1029 1102 1143 1167 1190
1026 1095 1145 1169 1186
1419 1453 1500
1417 1454 1498
1574 1601 1645 1736
1583 1608 1656 1734
Composition (%) 20.1 1.7 9.3 1.2 1.6 2.1 1.6 1.1 1.5 9.9 3.2 1.7 1.9 1.8 1.3 19.0 3.4 14.5 1.1 46.8 2.6 3.8 1.8 38.6 95.8
Mode of identificationb
RI, MS RI, MS, Std RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, RI, RI, RI, RI,
MS MS MS MS MS
RI, MS RI, MS, Std RI, MS RI, MS RI, MS RI, MS RI, MS, Std
MS = Mass spectra. Std = authentic standard. a RI values taken from Adams (2007). b RI = Retention index. Fig. 1. The three major constituents of Cheilocostus speciosus essential oil: (a) camphene, (b) zerumbone, and (c) α-humulene.
3.3. Ovicidal activity
Table 1 shows the constituents of the essential oil in order of their elution from a HP-5 column and their percentage composition. A total of twenty molecules (i.e., 95.8% of the C. speciosus essential oil) were identified. The C. speciosus oil was dominated by sesquiterpenes (65.8%) with oxygenated compounds (46.8%) more abundant than hydrocarbons (19.0%). Zerumbone (38.6%) and α-humulene (14.5%) were the main representative compounds of these groups, respectively (Fig. 1). Monoterpenes gave a minor contribution (30.0%), with hydrocarbons (20.1%) more abundant than oxygen-containing compounds (9.9%). Camphene (9.3%) was by far the major compound of the monoterpene fraction (Fig. 1). The abundance of remaining seventeen compounds ranged from 0.8% to 3.6%.
The ovicidal results achieved testing the C. speciosus essential oil and its three major compounds against H. armigera eggs are given in Table 3. As a general trend, the ovicidal activity was concentrationdependent. Among the pure compounds tested, camphene showed the best ovicidal activity, if compared to the other compounds. EC50 values calculated on H. armigera eggs were 35.39, 59.51 and 77.10 µg/ml for camphene, zerumbone and α-humulene, respectively (Table 3). 4. Discussion The Old-World bollworm, H. armigera – selected as target insect in this study – is a polyphagous pest of high economic importance showing insecticide resistance to a number of synthetic molecules used for its control (Kranthi et al., 2002; Torres-Vila et al., 2002; Srinivas et al., 2004; Young et al., 2005). More generally, the fast-growing resistance development to several synthetic and microbial insecticides currently marketed in many insect pests highlighted the pressing need to develop novel and eco-friendly pesticides (Bass et al., 2014; Naqqash et al., 2016; Ranson and Lissenden, 2016). Thus, herbal formulations and plant metabolites are attracting high research interest due to their ability to trigger toxicity through various
3.2. Ingestion toxicity of the oil and selected pure constituents The larvicidal results of C. speciosus oil and three major compounds from C. speciosus against H. armigera third instar larvae are given in Table 2. The C. speciosus essential oil achieved LC50 and LC90 values of 207.45 µg/ml and 403.88 µg/ml, respectively. Among the three compounds, the most potent larvicidal one was camphene, with a LC50 of 10.64 µg/ml and LC90 of 20.29 µg/ml, while zerumbone LC50 was 17.16 µg/ml and the LC90 was 32.20 µg/ml; α-humulene had a LC50 value of 20.86 µg/ml while its LC90 value was 41.72 µg/ml. 783
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Table 2 Larvicidal activity of the essential oil of Costus speciosus and its major chemical compounds against third instar larvae of Helicoverpa armigera. Treatment
Concentration (µg/ml)
Mortality (%) ± SDa
LC50 (µg/ml) (95% LCL-UCL)
LC90 (µg/ml) (95% LCL-UCL)
Slope
Regression equation
χ2 (d.f.)
Essential oil
100 200 300 400 500 5 10 15 20 25 8 16 24 32 40 10 20 30 40 50
28.4 ± 0.8 46.2 ± 1.2 67.3 ± 0.8 88.6 ± 0.6 100.0 ± 0.0 26.3 ± 1.2 44.8 ± 0.6 68.2 ± 0.6 87.3 ± 0.8 100.0 ± 0.0 24.9 ± 0.8 45.3 ± 0.8 67.2 ± 1.2 88.4 ± 0.6 100.0 ± 0.0 27.6 ± 1.2 48.2 ± 0.6 66.4 ± 0.8 87.3 ± 0.6 98.5 ± 0.6
207.45 (184.45–227.89)
403.88 (374.80–442.02)
3.01
y=10.42+0.186x
5.580 (4) n.s.
10.64 (9.52–11.64)
20.29 (18.85–22.16)
2.74
y=8.35+3.798x
4.866 (4) n.s.
17.16 (15.43–18.73)
32.20 (29.96–35.12)
2.56
y=7.17+2.416x
4.579 (4) n.s.
20.86 (18.42–23.00)
41.72 (38.65–45.78)
3.51
y=11.33+1.809x
2.843 (4) n.s.
Camphene
Zerumbone
α-Humulene
SD = standard deviation. d.f. = degrees of freedom. n.s. = not significant (P > 0.05). a Values are means ± SD of 5 replicates. Table 3 Ovicidal activity of the essential oil of Costus speciosus and its major chemical compounds on eggs of Helicoverpa armigera. Treatment
Essential oil Camphene Zerumbone α-Humulene
Egg hatchability (%) Control
150 µg/ml
300 µg/ml
100 ± 0.0a Control 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
89.4 ± 1.2b 25 µg/ml 55.1 ± 0.8 b 78.4 ± 1.4 b 87.2 ± 0.8b
71.6 ± 1.0 50 µg/ml 38.3 ± 1.2 59.6 ± 1.0 67.7 ± 1.2
c
c c c
450 µg/ml
600 µg/ml
750 µg/ml
900 µg/ml
59.3 ± 0.8d 75 µg/ml 21.7 ± 0.8 d 38.4 ± 1.2 d 56.3 ± 1.0d
38.2 ± 1.2e 100 µg/ml NH 20.2 ± 0.8e 35.2 ± 1.2e
19.2 ± 1.4 f 125 µg/ml NH NH 17.2 ± 1.2 f
NH 150 µg/ml NH NH NH
EC50 (µg/ml)
95% LCL-UCL
χ2 (d.f.=6)
485.92
412.15–557.10
10.154 n.s.
35.39 59.51 77.10
11.54–48.48 46.67–70.21 64.63–88.71
12.120 n.s. 9.933 n.s. 9.550 n.s.
Within each row, different letters indicate significant differences (ANOVA, Tukey's HSD, P < 0.05). NH = no hatchability. n.s. = not significant (P > 0.05). a Values are mean ± SD of 5 replicates.
flavoring and appetizer as well as a remedy for pains, inflammations, diarrhea and worm infestations (Yob et al., 2011). This compound exhibited in vivo antinociceptive, antiinflammatory and anticancer effects (Yob et al., 2011). The bioactivity of zerumbone depends on its capacity to act as Michael acceptors, which make it capable of trapping thiols by covalent coupling (Appendino et al., 2015). Interestingly, the zerumbone-rich hexane extract from Z. zerumbet was proven to be highly toxic to Aedes aegypti (L.) and Culex quinquefasciatus Say larvae (Mahardika et al., 2017). α-Humulene is one the main compounds responsible for aroma of hops and hemp, two members of the Cannabaceae family with insecticidal essential oils (Bedini et al., 2016; Benelli et al., 2017a), as well as for that of shampoo ginger (Yob et al., 2011). This sesquiterpene showed toxic effects against Anopheles stephensi Liston, A. aegypti and C. quinquefasciatus larval instars. In addition, it exerted repellency towards adults of the same mosquito vectors and oviposition deterrence as well (da Silva et al., 2015). This compound resulted selective towards mosquitoes, since its toxicity on non-target aquatic species, such as Diplonychus indicus Venkatesan & Rao and Gambusia affinis Baird and Girard, was quite low (Govindarajan et al., 2016). α-Humulene together with zerumbone are the main volatile components of Z. zerumbet essential oil, which was highly effective against pyrethroid-resistant strains of A. aegypti (Sutthanont et al., 2010). Interestingly, when occurring in mixture with other terpenes, αhumulene is able to stimulate oviposition in females of H. armigera
mechanisms of action at the same time, reduced toxicity on non-target vertebrates and – in some cases – large availability and low costs for farmers living in developing countries (Benelli and Mehlhorn 2016; Pavela and Benelli, 2016; Stevenson et al., 2017). In the ingestion toxicity assays conducted here, the toxicity of C. speciosus rhizome essential oil against H. armigera larvae and eggs was moderate, with a LC50 value of 207.45 µg/ml and an EC50 value of 485.92 µg/ml, respectively. On the other hand, the insecticidal efficacy of the three compounds characterizing the essential oil was highly promising, with extremely low LC50 values, 10.64, 17.16 and 20.86 µg/ ml for camphene, zerumbone and α-humulene, respectively. Zerumbone, α-humulene and camphene are terpenes endowed with high lipophilicity that allows them to be incorporated into the larval body of insect where they exert toxic effects. To the best of our knowledge, their mechanisms of action on larvae of H. armigera are unknown to date. Overall, terpenoids can exert insecticidal activity via different ways, e.g., by affecting the cholinergic, octopaminergic and GABAergic systems or by neutralizing the detoxicative enzymes of insects (Pavela and Benelli, 2016). Zerumbone and α-humulene are biogenetically related sesquiterpenes belonging to the humulane group (Appendino et al., 2015; Sut et al., 2017). Zerumbone is the main bioactive component obtained from rhizomes of shampoo ginger (Zingiber zerumbet (L.) Smith), a perennial herb occurring in several tropical countries where it is used as
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(Jallow et al., 1999). Camphene is a volatile monoterpene hydrocarbon occurring in many essential oils, for instance that of Wedelia prostrata (Hook. et Arn.) Hemsl. and is effective against 4th instar larvae of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), showing LC50 values below 10 ppm (Benelli et al., 2017b) Interestingly, chlorination of camphene was used to manufacture insecticial compounds, such as Toxaphene, which has been used for long time to combat insect pests (Coelhan and Parlar, 1996) until its ban after the insurgence of resistance (El-Sebae et al., 1993). Overall, to the best of our information, this study is the first report on the C. speciosus essential oil against insect pests of agricultural and medical veterinary importance, highlighting that blends of camphene, zerumbone and α-humulene have a promising potential for the development of eco-friendly and effective botanical insecticides, including microencapsulation (Pavela, 2016) and nanoformulations (Benelli, 2016a,b, 2018). Further studies are needed on the mode of action of these terpenes on different target insects, as well as their toxicity of these compounds in blend on non-target species, such as parasitoids and predators of moth pests, as well as earthworms. Acknowledgements The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group No. RG-1438- 074. The authors express their sincere thanks to Professor and Head of the Department of Zoology, Annamalai University for the laboratory provisions granted. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ mass Spectrometry. Carol. Stream, Allured., IL, USA. Appendino, G., Minassi, A., Collado, J.A., Pollastro, F., Chianese, G., Taglialatela-Scafati, O., Ayyari, M., Garcia, V., Muñoz, E., 2015. The thia-Michael reactivity of zerumbone and related cross-conjugated dienones: disentangling stoichiometry, regiochemistry, and addition mode with an NMR-spectroscopy-based cysteamine assay. Eur. J. Org. Chem. 17, 3721–3726. Baskar, K., Kingsley, S., Vendan, S.E., Paulraj, M.G., Duraipandiyan, V., Ignacimuthu, S., 2009. Antifeedant, larvicidal and pupicidal activities of Atalantia monophylla (L) Correa against Helicoverpa armigera Hubner (Lepidoptera: Noctuidae). Chemosphere 75, 355–359. Bass, C., Puinean, A.M., Zimmer, C.T., Denholm, I., Field, L.M., Foster, S.P., Gutbrod, O., Nauen, R., Slater, R., Williamson, M.S., 2014. The evolution of insecticide resistance in the peach potato aphid, Myzus persicae. Insect Biochem. Mol. Biol. 51, 41–51. Bedini, S., Flamini, G., Cosci, F., Ascrizzi, R., Benelli, G., Conti, B., 2016. Cannabis sativa and Humulus lupulus essential oils as novel control tools against the invasive mosquito Aedes albopictus and fresh water snail Physella acuta. Ind. Crops Prod. 85, 318–323. Benelli, G., Govindarajan, M., AlSalhi, M.S., Devanesan, S., Maggi, F., 2017b. High toxicity of camphene and γ-elemene from Wedelia prostrata essential oil against larvae of Spodoptera litura (Lepidoptera: noctuidae). Environ. Sci. Pollut. Res. http://dx.doi. org/10.1007/s11356-017-9490-7. Benelli, G., Mehlhorn, H., 2016. Declining malaria, rising of dengue and Zika virus: insights for mosquito vector control. Parasitol. Res. 115, 1747–1754. Benelli, G., Pavela, R., Lupidi, G., Nabissi, M., Petrelli, R., Ngahang Kamte, S.L., Cappellacci, L., Fiorini, D., Sut, S., Dall’Acqua, S., Maggi, F., 2017a. The crop-residue of fiber hemp cv. Futura 75: from a waste product to a source of botanical insecticides. Environ. Sci. Poll. Res. http://dx.doi.org/10.1007/s11356-017-0635-5. Benelli, G., 2015. Plant-borne ovicides in the fight against mosquito vectors of medical and veterinary importance: a systematic review. Parasitol. Res. 114, 3201–3212. Benelli, G., 2016a. Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol. Res. 115, 23–34. Benelli, G., 2018. Gold nanoparticles – against parasites and insect vectors. Acta Trop. 178, 73–80. Benelli, G., 2016b. Green synthesized nanoparticles in the fight against mosquito-borne diseases and cancer—a brief review. Green synthesized nanoparticles in the fight against mosquito-borne diseases and cancer—a brief review 95, 58–68. Chowdhury, A., Das, A.P., 2015. Ethnopharmacological survey of wetland plants used by local ethnic people in sub- Himalayan terai and duars of West Bengal, India. Am. J. Ethnomed. 2, 122–135. Coelhan, M., Parlar, H., 1996. The nomenclature of chlorinated bornanes and camphenes relevant to toxaphene. Chemosphere 32, 217–228. da Silva, R.C.S., Milet-Pinheiro, P., da Silva, P.C.B., da Silva, A.G., da Silva, M.V., do
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