Acute and sub-lethal toxicity of eight essential oils of commercial interest against the
1
filariasis mosquito Culex quinquefasciatus and the housefly Musca domestica
2 3
Giovanni Benelli a,b*, Roman Pavela c, Cristiano Giordani d, Luca Casettari e, Giulia Curzi f, 4
Loredana Cappellacci g, Riccardo Petrelli g, Filippo Maggi g 5
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a Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80,
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56124 Pisa, Italy 8
b The BioRobotics Institute, Sant’Anna School of Advanced Studies, viale Rinaldo Piaggio
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34, 56025 Pontedera, Pisa, Italy 10
c Crop Research Institute, Drnovska 507, 161 06, Prague 6, Czech Republic
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d Instituto de Física, Universidad de Antioquia, Medellín AA 1226, Colombia
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e Department of Biomolecular Sciences, University of Urbino, Urbino, Italy
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f Pharma & Food Consulting srl, Camerino, Italy
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g School of Pharmacy, University of Camerino, via Sant’Agostino 1, 62032, Camerino, Italy
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* Corresponding author. Tel.: +39-0502216141. Fax: +39-0502216087. E-mail address: 17
benelli.giovanni@gmail.com; giovanni.benelli@santannapisa.it (G. Benelli). 18
19 20
*Manuscript
Abstract
21 22
The massive use of synthetic insecticides led to negative effects on the environment 23
and human health. Therefore, researchers looked at natural products as effective alternatives 24
to conventional pesticides. Here, commercially valuable essential oils (EOs) were selected 25
from mint (Mentha x piperita, Mentha spicata), basil (Ocimum basilicum), helichrysum 26
(Helichrysum italicum), yarrow (Achillea ligustica), geranium (Pelargonium odoratissimum), 27
cinnamon (Cinnamomum verum) and ginger grass (Lippia alba). The chemical composition 28
of these EOs assayed was analyzed by GC-MS. Then, we investigated their insecticidal 29
potential in acute and sub-lethal toxicity assays against mosquito vectors of filariasis (Culex 30
quinquefasciatus) and house flies (Musca domestica). Against C. quinquefasciatus 4th instar
31
larvae, the most toxic EO was C. verum (LC50 = 40.7 µl L−1), followed by L. alba (LC50 =
32
59.6 µl L−1), while against M. domestica adults, the most toxic EOs were C. verum and H. 33
italicum (LD50 = 42 µg adult−1). The exposure of mosquito larvae to a sub-lethal concentration
34
(LC30=25 mg L−1) led to a reduction of adult emergence and fertility. Besides, adult flies that
35
survived after exposure to a sub-lethal dose of C. verum EO (LD20=10 µg adult−1) showed a
36
marked decrease in male and female longevity, as well as to a reduction in fecundity, fertility, 37
and natality. Overall, C. verum and H. italicum EOs showed a highly promising insecticidal 38
potential on two key insect vectors and pests. The relatively low prices of the selected EOs, 39
their availability on the market and the noteworthy global production of the bulky materials, 40
make them as ideal candidate ingredients to be used in insecticidal formulations. 41
42
Keywords: Achillea ligustica; Cinnamomum verum; Helichrysum italicum; Lippia alba;
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Ocimum basilicum; Pelargonium odoratissimum
44 45
1. Introduction
46 47
The massive use of synthetic insecticides until the second half of 20th century 48
produced negative effects on the environment and human health, and forced the agrochemical 49
companies to reduce and/or avoid the use of a substantial number of detrimental substances 50
from their chemical arsenals. At the same time, researchers looked at natural products as 51
effective alternatives to conventional insecticides (Pavela and Benelli, 2016). As a matter of 52
fact, it has been estimated that the market of plant-borne insecticides will reach the 7 % of the 53
global pesticide market by 2025 (Isman, 2015) and that of synthetic pesticides is expected to 54
decline by 1.5 % per year (Thakore, 2006). Among natural products, plant essential oils (EOs) 55
are complex mixtures (even above hundreds of constituents) of small, volatile and lipophilic 56
compounds produced by several aromatic species. They gained commercial importance 57
because of their massive use in flavors and fragrances, foodstuffs, beverages, cosmetics and 58
pharmaceutics (Lubbe and Verpoorte, 2011). Currently, 300 EOs are used worldwide on an 59
industrial scale (CBI, 2009). They are obtained from several botanical families among which 60
Asteraceae, Lamiaceae, Lauraceae, Geraniaceae and Verbenaceae are currently recognized as 61
the most important ones (Benelli 2015a; Pavela, 2016). 62
Recently, the European Food Safety Authority (EFSA) is simplifying the regulatory 63
path for some botanicals, including EOs, by evaluating them as ‘low-risk active substances’ 64
(LRASs) as reported in the EC Regulation No. 1107/2009. Important strengths favoring the 65
application of EO-based insecticides are the availability of bulky materials from which some 66
EOs are obtained, the high yields of EOs obtainable from cheap plant sources, the relatively 67
ease of preparation by distillation and chemical characterization by gas chromatography 68
coupled with mass spectrometry (GC-MS) (Rubiolo et al., 2010), and their acknowledged 69
safety to humans and environment (they are Generally Recognized as Safe, GRAS) (Isman et 70
al., 2011). Actually, the restricted regulatory requirements are limiting the diffusion and 71
marketing of EOs-based insecticides (Pavela and Benelli, 2016). 72
In the present work, we investigated the insecticidal effects of an EOs panel of 73
commercial interest against two insects of high economic importance, the filariasis mosquito 74
vector Culex quinquefasciatus Say (Diptera: Culicidae), which is also an important vector of 75
St. Louis encephalitis and West Nile virus (Benelli, 2015b; Vadivalagan et al., 2017), and the 76
housefly Musca domestica L. (Diptera: Muscidae), which transmit pathogens of public health 77
relevance causing more than one hundred diseases (WHO, 1991). The needing of novel and 78
effective green pesticides to control these two insects is pressing (Benelli and Mehlhorn, 79
2016; Benelli et al., 2016; Benelli and Romano, 2017), since both developed resistance to a 80
rather wide number of synthetic pesticides currently marketed (Hardstone et al., 2014; 81
Naqqash et al., 2016; Benelli and Beier, 2017). 82
For the purpose, commercially valuable EOs were selected from mint (Mentha x 83
piperita L., Mentha spicata L.), basil (Ocimum basilicum L.), helichrysum (Helichrysum
84
italicum (Roth) G. Don), yarrow (Achillea ligustica All.), geranium (Pelargonium
85
odoratissimum (L.) L’Hér.), cinnamon (Cinnamomum verum J. Presl) and ginger grass
86
(Lippia alba (Mill.) N.E.Br. ex Britton & P. Wilson) (Fig. 1). The commercial values of these 87
EOs have been estimated as 155-230, 80-100, 40-45, 30-45 and 18-20 €/kg for C. verum, P. 88
odoratissimum, O. basilicum, M. x piperita amd M. spicata, respectively (Lubbe and
89
Verpoorte, 2011). The relatively low prices, their availability on the market and the 90
noteworthy global production of the bulky materials, make them as ideal candidate 91
ingredients to be used in insecticidal formulations, also in organic farms. 92
Mint EOs, like those coming from M. x piperita and M. spicata, are dominated by 93
oxygenated monoterpenes such as menthol, carvone, pulegone and piperitone. They are used 94
against skin irritations and sunburn, as well as antipyretic, anti-inflammatory, and nasal 95
decongestant. Other applications consist in their incorporation in perfumes and as flavoring 96
agents in foodstuffs (Kumar et al. 2011). Basil EO is recognized as antioxidant, anesthetic, 97
anti-inflammatory, antimicrobial and antiproliferative agent. These properties are ascribable 98
to the presence of methyl chavicol and linalool (Rodrigues et al., 2017; Varga et al., 2017). 99
The economic interest around the helichrysum EO is linked to the abundance of the 100
monoterpenoid neryl acetate which makes it an ideal ingredient of glamorous perfumes and 101
personal care products (Appendino et al., 2015). Helichrysum EO is also effective as wound 102
healing agent and against several skin disorders (Schnaubelt, 1999). Ligurian yarrow EO has 103
exhibited important inhibitory properties against oral pathogens (Cecchini et al., 2012), while 104
apple-scented geranium EO obtained from P. odoratissimum is used in perfumery and 105
cosmetics, as well as in aromatherapy and the food industry (Lis-Balchin and Roth, 2000). 106
Frequently it is used to replace the very expensive EO from Rosa x damascena Herrm., which 107
account up to $7500/kg (Blerot et al., 2016). Cinnamon EO is widely used on an industrial 108
level, e.g., to prepare pharmaceutics, seasonings, cosmetics, food and beverages (Li et al., 109
2013). The major component of this oil is the aromatic cinnamaldehyde, which is responsible 110
for important biological effects, namely anti-inflammatory (Chao et al., 2005), antioxidant 111
(Murcia et al., 2004) and antibacterial ones (Chang et al., 2001). The ginger grass EO 112
obtained from L. alba is valuable for the pharmaceutical industry due to the diverse biological 113
activities exhibited, namely antioxidant, antimicrobial, anti-inflammatory, sedative, 114
antigenotoxic, immunomodulatory and antiproliferative (García et al., 2017). Its composition 115
is quite variable, being characterized by different chemotypes according to genetic factors and 116
geographic origin (Hennebelle et al., 2008). Among these, the carvone/limonene chemotype is 117
considered as the most important in South America (da Silva Lima et al., 2016; U.N., 2005). 118
The effects of EOs against insect pests and vectors can be classified into two main 119
groups, namely behavioral (e.g., repellent, anti-feedant, inhibition of oviposition) and 120
physiological ones (e.g., acute and sub-lethal toxicity, inhibition of development and growth) 121
(Isman, 2018). In the present work, after analyzing the chemical composition of the above 122
mentioned EOs by gas chromatography-mass spectrometry (GC-MS), we assayed them for 123
acute toxicity against 4th instar larvae of C. quinquefasciatus and adults of M. domestica. 124
Once the most effective EO was determined, we evaluated the effects of its sub-lethal 125
concentrations on egg emergence, longevity and fertility in both targeted insects. 126
127
2. Materials and Methods
128 129
2.1. Essential oils 130
131
C. verum, M. crispa, O. basilicum and M. piperita EOs were kindly provided by
132
Phalada Agro Research Foundations Pvt Ltd. (http://phaladaagro.com), Bangalore, India. The 133
EO of L. alba was purchased from Centro de Investigación de Excelencia – CENIVAM 134
(http://cenivam.uis.edu.co/cenivam/), Bucaramanga, Santander, Colombia. EOs of H. italicum 135
and P. odoratissimum were kindly furnished by APPO (Associazione Produttori Piante 136
Officinali delle Marche, http://www.ladistilleriaappo.it), Ancona, Italy. A. ligustica EO was 137
obtained from the aerial parts collected from a population cultivated in the Botanical Garden 138
of the University of Camerino in June 2016 (Cecchini et al., 2012; Maggi et al., 2009). For 139
this sample, a voucher specimen was archived in the Herbarium Camerinensis of the School 140
of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy, under the 141
codex CAME 13420. 142
143
2.2. Chemical analysis of the EOs by GC-MS 144
Chemical analysis of EOs was performed by an Agilent 6890N gas chromatograph 146
coupled to a 5973N mass spectrometer and equipped with a HP-5 MS (5% 147
phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 μm film thickness; J & W Scientific, 148
Folsom) capillary column. The analytical conditions used were as follows: oven programmed 149
for 5 min at 60°C then 4°C min−1 up to 220°C, then 11°C min−1 up to 280°C, held for 15 min; 150
temperatures of injector and detector were set to 280 °C; He was used as the carrier gas with a 151
flow rate of 1 mL min−1; split ratio was1:50; mass spectra were acquired in full scan in the 152
range of 29–400 m z−1 using electroimpact (EI, 70 eV) mode. For each EO, a dilution in
n-153
hexane (1:100) was prepared and 2 µL of the solution was injected into the GC-MS system. 154
For data analysis, the MSD ChemStation software (Agilent, Version G1701DA D.01.00) and 155
NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library v. 2.0 156
were used. The identification of peaks was made by means of co-injection with standards 157
available in our laboratory, together with correspondence of retention indices (according to 158
Van den Dool and Kratz formula) and mass spectra with respect to those occurring in 159
ADAMS, NIST 08 and FFNSC2 libraries (Adams, 2007, NIST 08, 2008, FFNSC2, 2012). 160
Semi-quantification of essential oil components was made by peak area normalization 161
considering the same response factor for all volatile components. Values (%) were the mean 162 of 3 chromatographic analyses. 163 164 2.3. Insects 165 166
C. quinquefasciatus was reared in the laboratory colony of Crop Research Institute,
167
Czech Republic. The larvae were fed on dog biscuits and yeast powder in the ratio 3:1. Adults 168
were provided with a sucrose solution (10% w:v) and for blood feeding were a 1-week-old 169
chick. Early 4th instar C. quinquefasciatus larvae were used in the study.
Adult houseflies (Musca domestica L.) were tested here. They were obtained from a 171
laboratory colony of Crop Research Institute, Prague. Larvae were reared in the mixture of 172
sterilized bran with milk powder and water. Adults were fed ad libitum with sugar water 173
(10% w:v) and to milk powder. 174
All tested insects were maintained at 25°C ± 1°C, with 50–70% RH, and 16:8 175
photoperiod (L:D). All experiments were performed under the same conditions. 176
177
2.4. Larvicidal toxicity on mosquitoes 178
179
Mosquito larvicidal trials were carried out according to WHO (1996) standard 180
procedures, with slight modifications (Pavela, 2015b). The essential oils were diluted in 181
dimethyl sulfoxide to prepare a serial dilution of the test dosage. For experimental treatment, 182
1 mL of serial dilution was added to 224 mL of distilled water in a 500-mL glass bowl and 183
shaken gently to produce a homogeneous test solution. The larvae of C. quinquefasciatus 184
were transferred in water into a bowl of the prepared test solution (25 larvae/beaker). Four 185
duplicate trials were carried out for every sample concentration, and for each trial, a negative 186
control was included using distilled water containing the same amount of dimethyl sulfoxide 187
as the test sample. A different series of concentrations (resulting from the previous screening) 188
was used for each essential oil to obtain mortality ranging between 10% and 90%. At least 5 189
concentrations were selected for the calculation of lethal doses. Mortality was determined 190
after 24 h of exposure, during which no food was given to the larvae. 191
192
2.5. Adulticidal toxicity on house flies 193
Acute toxicity (measured as mortality after 24 h) was determined by topical 195
application on M. domestica adults (3–6 days old). A micro-electric applicator was used to 196
deliver 1 μL doses of different dosages of EOs mixed with acetone, to the pronota of CO2
-197
anesthetized flies. Initial screening to approximate the active dose range determined a range 198
of doses that were used to establish the lethal doses. Certified acetone was used as the control 199
treatment. A minimum of 5 concentrations were replicated at least for 4 times (80 flies per 200
single replication) in the final bioassays. All treated flies from each replicate were placed in a 201
plastic box (10 cm diameter x 12 cm high) and had free access to water. Mortality was 202
assessed 24 h after the treatment was performed. Flies that did not respond to mechanical 203
stimulation were considered dead. 204
205
2.6. Sub-lethal effects on mosquitoes 206
207
At the beginning of the fourth instar, C. quinquefasciatus larvae were moved into a 208
plastic container (20 x 20 x 20 cm) with 3 L of drinking water. After acclimatization (after 209
approximately 1 h), a dose of the EO found as most effective in acute toxicity assays (C. 210
verum) was mixed into the water, corresponding to the calculated LC30 (25 mg L−1). The EO
211
was emulsified using DMSO; water with an adequate DMSO content was used as control. 100 212
larvae were stored in each container. After 12 h of exposure, the larvae were transferred into 213
clean water, where they were left until the incubation of adults. The larvae were fed on dog 214
biscuits and yeast powder in a 3:1 ratio. Larvae mortality was determined after 24 and 48 h of 215
exposure; total mortality, percentage of incubated adults, and their sex were determined. 216
The survived adults were used for determination the effect of C. verum EO on fertility. 217
The mosquitoes were kept on breeding cages (25 x 25 x 30 cm). As food sugar water was 218
given and to lay their eggs, a plastic container with water was placed in the middle of each 219
cage. Untreated mosquitoes were used as control. The number of laid eggs per single female 220
mosquito was noted each day. To evaluate emergence and for calculation of potential number 221
of larvae on all survive female, 100 freshly-laid eggs were analyzed. The eggs were placed 222
into plastic dishes (diameter of 12cm and height of 6 cm) filled with water. The number of 223
emerged larvae were noted. 224
All tests were replicated 3 times, with the conditions of the experiment as described in 225
“Insects” paragraph. 226
227
2.7. Sub-lethal effects on house flies 228
229
The following method was used to evaluate the sub-lethal effects of C. verum EO on 230
longevity, fecundity and natality of M. domestica. Housefly adults (1-2 days old) were 231
divided onto their sex. The C. verum oil was applied topically on fly adults at its LD20 dose
232
(10 μg fly−1). The EO application was done following the same method described in the
233
paragraph “Adulticidal toxicity on house flies”. To shed light on the effects of C. verum EO 234
on longevity and fecundity, M. domestica flies were kept on breeding cages (25 x 25 x 30 235
cm). Into each cage there, 20 male and 20 female flies were placed. As food were given sugar 236
water and powdered milk and to lay their eggs, a Petri dish was placed in the middle of each 237
cage, arranged with cotton which was soaked in sweet milk. Untreated males and females 238
were used as control. 239
The mortality of both sexes and the number of laid eggs per single female fly were 240
supervised each day. The ratio of laid eggs per female fly was regulated by the number of 241
female flies present in each assay. To estimate fly aging post-treatment, a period, within 242
which 50% and 99% mortality (LT50 and LT90) of the fly adults was seen, was set, with use of
243
the linear regression analysis. 244
To evaluate natality, we considered 100 freshly-laid eggs, i.e. on each day within the 245
entire period of 10 days (total n=1000) in which the test was performed. The eggs were then 246
placed into plastic dishes (diameter of 12 cm and height of 6 cm) where feedings were 247
arranged. The, the number of emerged larvae were evaluated. 248
All tests was replicated 3 times, with the conditions of the experiment as described in 249 “Insects” paragraph. 250 251 2.8. Statistical analysis 252 253
Experimental tests showing > 20 % of control mortality was discharged and repeated. 254
When the control mortality ranged from 1–20%, the observed mortality was corrected by the 255
Abbott's formula (Abbott, 1925) the LC50, LC90 regression equation, and a 95% confidence
256
limit were calculated by probit analysis (Finney, 1971). 257
Lethal time was calculated as time (in days) to indigent of 50% or 99% natural 258
mortality of adult M. domestica. 95% Confidence intervals (CI) were not adjusted for multiple 259
inferences. Essential oil activity was considered significantly different when the 95% CI fail 260
to overlap. 261
Data about average number of eggs, mortality, fertility and natality were statistically 262
evaluated by using a two-fold F-test (* p=0.05; ** p=0.01). Mortality (%) was sujected to 263
angular transformation (y = arcsen x √%) before ANOVA. 264
265
3. Results and Discussion
266 267
3.1. Chemical composition of the eight essential oils 268
The chemical compositions of the commercial EOs assayed for insecticidal activity are 270
reported in Table. 1, whereas the GC-MS chromatograms of the most active ones are depicted 271
in Fig. 2. A total of sixty-six volatile components was identified in the EO of M. x piperita, 272
corresponding to 99.8% of the total composition. This EO was dominated by oxygenated 273
monoterpenes (81.0%) such as menthol (26.0%), menthone (20.7%), iso-menthone (11.6%), 274
menthyl acetate (9.5%) and neo-menthol (5.3%) (Fig. 3). Monoterpene hydrocarbons gave a 275
minor contribution (12.3%), with limonene as the most representative compound (7.1%). This 276
composition is in line with peppermint batches of industrial interest (Fejér et al., 2017). 277
A total of fifty-five compounds was identified in the EO of M. spicata, accounting for 278
99.4% of the total EO. Also in this case, the major fraction of EO was given by oxygenated 279
monoterpenes (66.7%), but monoterpene hydrocarbons were here more abundant (27.8%) 280
compared with peppermint. Carvone (58.2%) and limonene (22.5%) (Fig. 3) strongly 281
characterized the M. spicata EO. This chemical profile was consistent with those reported in 282
literature (Chrysargyris et al., 2017). 283
The EO of O. basilicum was composed of thirty-seven components, accounting for 284
99.8% of the total composition. The EO was strongly characterized by only two components, 285
namely methyl chavicol (77.9%) and linalool (15.6%) (Fig. 3). Based on these results we can 286
assign this basil EO sample to the high-methyl chavicol chemotype as proposed by Varga et 287
al., (2017). 288
A total of fifty-four components was identified in the EO of H. italicum, accounting 289
for 95.2% of the overall composition. The EO was characterized by oxygenated monoterpenes 290
(57.8%) such as the ester neryl acetate (45.4) which is responsible for the characteristic floral 291
scent (Fig. 3). Other noteworthy components in this fraction were neryl propanoate (5.0%) 292
and linalool (3.9%). In addition, the sesquiterpene hydrocarbons (27.8%) gave an important 293
contribution to the overall EO composition. Among them, -curcumene (9.0%) and ar-294
curcumene (7.6%) were the most abundant compounds. Finally, oxygenated sesquiterpenes 295
(6.2%) and monoterpene hydrocarbons (2.3%) were poor, being mainly represented by 296
rosifoliol (3.7%) and -pinene (2.3%), respectively. The overall chemical profile of this EO 297
was in line with those found in other spontaneous and cultivated populations of H. italicum 298
used on an industrial level (Morone-Fortunato et al., 2010; Melito et al., 2016; Leonardi et al., 299
2013). 300
A total of one hundred and thirty-two volatile components was identified in the EO 301
from the aerial parts of A. ligustica, corresponding to 88.5% of the total composition. This EO 302
was characterized by oxygenated monoterpenes (29.1%), sesquiterpene hydrocarbons 303
(23.7%), oxygenated sesquiterpenes (22.3%) and monoterpene hydrocarbons (12.3%), with 304
linalool (10.8%), germacrene D (11.8%), viridiflorol (12.6%) and -pinene (6.0%) as the 305
most representative compounds, respectively (Fig. 3). Other components occurring in 306
noteworthy levels (> 2%) were terpinen-4-ol (6.0%), 1,8-cineole (4.4%) and (E)-307
caryophyllene (2.1%). This profile was consistent with that previously reported by us 308
(Cecchini et al., 2012; Maggi et al., 2009) and showed some differences with respect to 309
batches of different geographic origin (Tuberoso et al., 2005; Badeer et al., 2007). 310
A total of ninety-two compounds was identified in the EO of P. odoratissimum, 311
accounting for 98.5% of the total EO. The chemical composition of this EO was dominated 312
by oxygenated monoterpenes (71.5%) such as citronellol (30.1%), iso-menthone (16.2%) and 313
citronellyl formate (9.1%) (Fig. 3). Minor contributions were given by sesquiterpene 314
hydrocarbons (14.6%) and monoterpene hydrocarbons (7.5%), with 6,9-guaiediene (5.7%) 315
and -pinene (2.9%) as the most representative compounds, respectively. The chemical 316
profile of this EO showed some similarities with that studied by Matusinsky et al. (2015) who 317
found citronellol (24.9%), geraniol (13.0%), citronellyl formate (7.7%) and iso-menthone 318
(4.7%) as the major compounds. On the other hand, deep differences emerged when 319
compared with investigations of Balchin and Roth (2000) and Andrade et al. (2011) who 320
found methyl eugenol (31.2-79.8%) and iso-menthone (4.6-16.9%), and methyl eugenol 321
(96.8%) as the main volatile components, respectively. 322
A total of thirty-nine components was identified in the EO from the bark of C. verum, 323
accounting for 99.1% of the total composition. They were almost entirely represented by 324
aromatic compounds (97.8%), with (E)-cinnamaldehyde (82.7%) and (E)-o-methoxy 325
cinnamaldehyde (10.1%) as the most representative compounds (Fig. 3). The remaining 326
components were all present in scant amounts (≤0.8%). Interestingly, this EO was almost 327
devoid of eugenol (occurring at trace levels), which is often found as a major volatile 328
component of the cinnamon bark (Azeredo et al., 2014; Yap et al., 2015; Jumbo et al., 2014; 329
Ju et al., 2018). This chemical profile was in line with those reported in literature for 330
cinnamon bark EO (Li et al., 2013; Sienkiewicz et al., 2014). 331
A total of ninety-three compounds was identified in the EO of L. alba, corresponding 332
to 99.2% of the total composition. The EO was characterized by oxygenated monoterpenes 333
(42.8%), monoterpene hydrocarbons (32.9%) and sesquiterpene hydrocarbons (21.9%), with 334
carvone (35.2%), limonene (32.0%) and germacrene D (14.8%) as the major components, 335
respectively (Fig. 3). The remaining constituents were all present in scant amounts, with 336
piperitenone (2.1%), -bourbonene (1.8%), piperitone (1.1%) and -elemene (1.0%) as the 337
most abundant ones. According to Hennebelle et al. (2008), L. alba EOs show a broad 338
chemical polymorphism, with eight different chemotypes and the EO investigated in this 339
study belonged to the limonene/carvone chemotype. 340
341
3.2. Acute and sub-lethal toxicity of the essential oils against mosquitoes and flies 342
Results of the acute toxicity experiments on C. quinquefasciatus and M. domestica of 344
the eight tested EOs were provided in Tables 2 and 3, respectively. Against 4th instar larvae of 345
C. quinquefasciatus mosquitoes, the most toxic EO was C. verum (LC50 = 40.7 µl L−1),
346
followed by L. alba (LC50 = 59.6 µl L−1), O. basilicum (LC50 = 68.6 µl L−1), M. spicata (LC50
347
= 88.2 µl L−1) and A. ligustica (LC50 = 89.5 µl L−1), while the other three tested EOs showed
348
LC50 values higher than 100 µl L−1 (Table 2).
349
Furthermore, against adults of M. domestica flies, the most toxic EOs were C. verum 350
and H. italicum (LD50 = 42 µg adult−1), followed by P. odoratissimum (LD50 = 54 µg adult−1),
351
Mentha x piperita (LD50 = 59 µg adult−1), O. basilicum (LD50 = 70 µg adult−1) and M. spicata
352
(LD50 = 86 µl L−1), while the other two tested EOs showed LD50 values higher than 100
353
µg/adult(Table 3). 354
Since C. verum showed the best toxic potential against both targeted insect species, we 355
selected it for sub-lethal tests. Table 4 showed the emergence and fertility of C. 356
quinquefasciatus adults that survived to a sub-lethal dose (LC30=25 mg/l) of C. verum EO
357
administered to 4th instar larvae. The exposure to a sub-lethal concentration of the selected EO 358
led to a significant larval mortality as well as reduction in adult emergence. The fertility of 359
adults, in terms of both eggs/female and emergence from eggs was also reduced (Table 4). In 360
addition, Table 5 summarized the longevity and fertility of M. domestica adults that survived 361
post-exposure to a sub-lethal dose (LD20=10 µg adult−1) of C. verum EO. The EO LD20
362
treatment on flies led to a marked decrease in male and female longevity, as well as to a 363
significant reduction in fecundity, fertility and natality (Table 5). 364
As far as we know, this is the first report on the effect of lethal and sub-lethal doses or 365
concentrations of the EO from C. verum on insects. As demonstrated by our tests, although 366
the efficacy of lower than lethal doses or concentrations of this EO does not cause acute 367
toxicity, it results in subsequent significant reduction of longevity, fecundity and fertility of 368
M. domestica and C. quinquefasciatus adults. If compared with the great amount of data
369
available about acute toxicity of EOs against insects (Benelli 2015b; Pavela, 2015a), studies 370
testing their sub-lethal toxicity are limited. In agreement with our data, a sub-lethal dose of 371
the EO from Thymus vulgaris L. was previously found to reduce not only the fecundity of the 372
treated M. domestica adults, but also the vitality of larvae hatched from eggs laid by the 373
treated females, resulting in almost 100% mortality of the larvae (Pavela, 2007). A similar 374
negative effect of the thyme EO was also found when applied in lethal concentrations against 375
C. quinquefasciatus larvae (Pavela, 2009), where even a short-term exposure caused
376
subsequent almost 90% mortality of the larvae. The overall significant reduction of larval 377
natality and of the fecundity of subsequently hatched adults of C. quinquefasciatus was also 378
observed for furanochromene isolated from the seeds of Ammi visnaga (Pavela et al., 2016). 379
Thus, as repeatedly demonstrated, even sub-lethal doses or concentrations of some secondary 380
plant metabolites may have a significant negative impact on the fertility and vitality of the 381
subsequent generation. This finding is very important as potential botanical insecticides 382
developed based on these EOs may result in a significant reduction of the overall frequency of 383
incidence of the target insect species even when applied in lower doses or concentrations, 384
thereby significantly reducing the risk of disease transmission. 385
Although the effects of several EOs against M. domestica adults were studied in the 386
past (Pavela, 2008; Zhang et al., 2017), only sporadic information about the efficacy of the 387
EO from C. verum is available. The effects of EOs against mosquito larvae have been studied 388
to a much higher extent (Pavela, 2015a). Currently, EOs showing LC90 < 100 µl L-1 in tests
389
are considered as highly promising for the development of botanical larvicides. In our case, 390
this efficacy was reached only by the EO from C. verum, with LC90 estimated as 64 µl L-1.
391
Other authors have also observed a very good larvicidal efficacy of EOs obtained from 392
Cinnamomum genus. For example, Cheng et al., (2004) estimated LD90 as 79 µl L-1 on larvae
393
of Aedes aegypti L. for the EO from leaves of Cinnamomum osmophloeum Kaneh. 394
Overall, the relevant insecticidal activity exhibited by the cinnamon EO seems to be 395
linked to the major compound, i.e. cinnamaldehyde (Fig. 3). This aromatic aldehyde has 396
shown a wide spectrum of efficacy, being toxic on several arthropod vectors (Jeon et al., 397
2017; Tak and Isman, 2017). At cellular level, cinnamaldehyde is able to inhibit enzymes 398
involved in cytokinesis, as well as to reduce the ATPase activity of cell membranes (Gill and 399
Holley, 2006). Furthermore, it causes loss of membrane integrity and membrane 400
depolarization and decrease cell respiration (Bouhdid et al., 2010). Cinnamon EO is 401
recognized as safe by the United States Food and Drug Administration (FDA) (Jeon et al., 402
2017). Thus, its use as ingredient in botanical insecticides is highly recommended. 403
404
4. Conclusions
405 406
Overall, based on our findings, the C. verum and H. italicum EOs showed a highly 407
promising insecticidal potential on two key insect pests and vectors. The relatively low prices 408
of the selected EOs, their availability on the market and the noteworthy global production of 409
the bulky materials, make them as ideal candidate ingredients to be used in insecticidal 410
formulations. 411
Considering that we found a significant effect of sub-lethal doses or concentrations of 412
the EO from C. verum on the target insect species, further tests will be needed to ascertain the 413
effect of this EO on non-target organisms, although as already demonstrated several times, 414
EOs are relatively friendly to non-target organisms including humans (Pavela, 2016; Pavela 415
and Benelli, 2016), aquatic plankton (Pavela, 2014) or fishes (Pavela and Govindarajan, 416
2017). Given that as previously found, synergistic and antagonistic relationships play an 417
important role in insecticidal efficacy of EOs (Pavela, 2015b; Benelli et al., 2017a,b), it will 418
be important to study mutual synergistic relationships between the major compound – 419
cinnamaldehyde and other minor compounds to understand these relationships and to try to 420
experimentally increase the insecticidal efficacy. 421
Besides, further optimization and standardization of their insecticidal activity through 422
microencapsulation (Pavela, 2016) and formulation on green-coated nanomaterials (Benelli 423 2016, 2018) are ongoing. 424 425 Acknowledgments 426 427
R. Pavela would like to thank the Ministry of Agriculture of the Czech Republic for 428
financial support about botanical pesticide and basic substances research (Project No. 429
RO0417). F. Maggi thanks the University of Camerino (Fondo di Ateneo per la Ricerca, FAR 430
2014/2015, FPI 000044) for financial support. 431
432
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T ab le 1. Ch em ica l c om po siti on o f th e e ig ht ess en ti al oil s a ss ay ed fo r in se cti cid al ac ti vit y. Co m p o n en t a RI b RI li t c La m ia ce a e As te ra ce ae G er a n ia ce ae La u ra ce ae Ve rb en ac ea e ID e M. x p ip erita M. sp ica ta O. b a sil icu m H . it a li cu m A. li gu stica P. o d o ra ti ss im u m C. v eru m L. a lb a 4-m eth yl-pe ntan ol 838 830 0. 1 b, c (2 E) -he xe na l 849 846 T rf b, c (3 E) -he xe nol 850 844 0. 1 0. 1 b, c (3 Z )-he xe nol 850 850 T r b, c n-he xa nol 863 863 T r b, c 2-m eth yl bu ty l a ce tat e 876 875 T r b, c 1-no ne ne 888 888 T r b, c st yre ne 897 897 0. 1 b, c 2, 5-dieth yl-tetra hy dro fu ra n 899 896 0. 1 0. 1 b, c iso bu ty l iso bu ty ra te 912 911 T r T r b, c 2-bu ten oic ac id , 3 -m eth yl-, e th yl este r 920 924 T r b, c α-th ujen e 921 924 0. 1 T r T r b, c α-pin en e 926 932 1. 5 0. 9 2. 0 0. 5 3.0 0. 1 T r a, b,c eth yl ti glate 934 929 T r b, c ca m ph en e 939 946 T r 0. 1 0. 1 a, b,c th uja -2, 4( 10) -d ien e 945 953 T r b, c be nz ald eh yd e 954 952 T r 0. 7 T r a, b,c sa bin en e 966 969 0. 7 0. 6 0. 8 T r a, b,c β-pin en e 969 974 1. 7 1 6. 0 T r a, b,c 1-oc ten -3 -ol 976 974 T r T r a, b,c 6-m eth yl- 5-he pten -2 -one 985 981 0. 1 b, c 3-oc tan on e 986 979 T r b, c m yrc en e 989 988 0. 8 1. 6 T r 0. 3 0. 4 a, b,c 3-oc tan ol 998 988 0. 8 0. 4 T r b, c α-ph ell an dre ne 100 2 100 2 T r 1. 2 a, b,c iso bu ty l 2 -m eth ylb uty ra te 100 4 100 4 T r b, c (3 Z) -h ex en yl ac etat e 100 9 100 4 T r b, c -te rp in en e 101 4 101 4 0. 1 0. 9 b, c 1, 4-cin eo le 101 6 101 2 T r b, c Table
2-m eth ylb uty l iso bu ty ra te 101 8 102 1 T r b, c p -m eth yl-an iso le 101 8 101 5 0. 2 b, c p-c ym en e 102 3 102 0 0. 2 0. 6 tr 1. 6 1. 2 Tr T r a, b,c li m on en e 102 5 102 4 7. 1 22. 5 0. 1 0. 3 0. 7 Tr 32. 0 a, b,c β-ph ell an dre ne 102 5 102 5 0. 2 b, c 1, 8-cin eo le 102 6 102 6 0. 5 0. 3 4. 4 0. 3 a, b,c (Z )-β -o cim en e 103 8 103 2 T r 0. 8 T r a, b,c sa li cy lald eh yd e 104 6 104 4 0. 1 b, c (E )-β -o cim en e 104 7 104 4 T r 0. 1 0. 2 a, b,c iso bu ty l a ng elate 104 9 104 5 T r b, c γ-t erp in en e 105 6 105 4 0. 2 1. 8 0. 1 T r a, b,c cis -sa bin en e h yd ra te 106 3 106 5 0. 3 0. 1 b, c cis -li na lo ol ox id e 107 0 106 7 0. 1 0. 1 b, c ace to ph en on e 107 0 107 3 Tr b, c n-o ctan ol 107 2 106 3 0. 1 T r b, c ter pin olen e 108 5 108 6 0. 1 0. 1 0. 4 T r a, b,c p-cy m en en e 108 6 108 9 T r b, c tra n s-li na lo ol ox id e 108 7 108 4 0. 1 b, c 6-cam ph en on e 109 0 109 5 T r b, c o-g ua iac ol 109 1 108 9 Tr b, c iso bu ty l ti glate 109 4 108 8 T r b, c tra ns -sa bin en e h yd ra te 109 5 109 8 0. 2 b, c lin alo ol 110 0 109 5 0. 2 15. 6 3. 9 10. 8 1. 3 0. 2 a, b,c cis -th ujo ne 110 2 110 1 0. 1 a, b,c n-n ona na l 110 5 110 0 T r b, c 2-m eth yl bu ty l-2 -m eth yl bu ty ra te 110 5 110 0 0. 2 b, c ho tri en ol 110 5 110 3 0. 1 b, c iso pe nty l iso va lera te 110 8 110 2 T r b, c cis -ro se o xid e 110 9 110 6 3. 3 b, c en d o-fe nc ho l 110 9 111 4 T r b, c 2-m eth yl bu ty l iso va lera te 111 0 110 3 0. 2 b, c ph en yl eth yl alco ho l 111 2 110 7 0. 7 b, c but an oic ac id , 3 -m eth yl-, 3-m eth yl- 3-bu ten yl este r 111 5 111 5 T r b, c tra ns -p in en e h yd ra te 111 6 111 9 T r b, c
cis -p -m en th -2 -en -1 -ol 111 7 111 8 0. 2 b, c tra n s-p-m en th a-2, 8-die n- 1-ol 111 7 111 9 0. 1 b, c α-ca m ph olen al 112 3 112 2 T r b, c tra ns -ro se o xid e 112 5 112 2 1. 1 b, c 3-oc tan ol ac etate 112 7 112 0 0. 1 b, c lim on a k eto ne 113 0 113 7 0. 4 b, c cis -li m on en e o xid e 113 0 113 2 T r b, c cis -p -m en th a-2, 8-die n- 1-ol 113 2 113 3 T r b, c tra ns -p in oc arv eo l 113 2 113 5 1. 1 a, b,c tra ns -li m on en e o xid e 113 5 113 7 T r b, c tra ns -p -m en th -2 -en -1 -ol 113 6 113 6 0. 2 b, c ca m ph or 113 9 114 1 0. 1 0. 1 0. 2 T r a, b,c ne o-iso pu leg ol 114 1 114 4 1. 8 0. 1 b, c (3 Z)-he xe ny l iso bu tan oa te 114 6 114 2 T r b, c m en th on e 115 0 114 8 20. 7 0. 6 0. 2 1.0 b, c iso am yl ti glate 115 0 114 8 0. 1 T r b, c 3-m eth yl- 2-bu ten yl 3-m eth yl-bu tan oa te 115 1 114 7 T r b, c ne ro l o xid e 115 4 115 4 0. 1 b, c cit ro ne ll al 115 4 114 8 0. 1 T r a, b,c pin oc arv on e 115 6 116 0 1. 0 b, c bor ne ol 116 0 116 5 0. 3 0. 3 a, b,c iso -m en th on e 116 1 115 8 11. 6 0. 2 0. 1 16. 2 b, c ne o-m en th ol 116 3 116 1 5. 3 b, c -terp in eo l 116 3 116 2 0. 1 0. 1 b, c hy dro cin na m ald eh yd e 116 4 116 3 0. 4 b, c cis -p in oc am ph on e 116 8 117 2 0. 1 b, c m en th ol 116 9 116 7 26 .0 2. 1 1. 2 a, b,c ter pin en -4 -ol 117 2 117 4 0. 3 1 6. 0 a, b,c iso -m en th ol 118 0 117 9 0. 7 0. 1 b, c p-cym en -8 -ol 118 3 117 9 T r b, c tra n s-p-m en th a-1( 7), 8-dien -2 -ol 118 5 118 7 T r b, c ne o-iso -m en th ol 118 6 118 4 0. 1 b, c α-te rp in eo l 118 7 118 6 0. 6 0. 3 0. 1 1. 7 0. 2 T r a, b,c
m yrten al 119 1 119 5 0. 2 a, b,c my rten ol 119 1 119 4 0. 4 a, b,c n eo -d ih yd ro c arv eo l 119 2 119 3 0. 1 b, c cis -d ih yd ro c arv on e 119 3 119 1 1. 7 0. 2 b, c tra n s-d ih yd ro c arv on e 119 9 120 0 0. 2 0. 9 b, c m eth yl ch av ico l 120 1 119 5 0. 1 77. 9 b, c tra ns -p ip erit ol 120 4 120 7 0. 1 b, c oc tan ol ac etate 121 5 121 1 0. 1 b, c (Z )-c in na m ald eh yd e 121 8 121 9 0. 4 b, c iso -d ih yd ro c arv eo l 121 8 121 2 0. 2 b, c tra n s-c arv eo l 122 4 121 5 0. 3 0. 3 b, c ne ro l 122 8 122 7 2. 5 a, b,c hy dro cin na m yl alco ho l 123 2 122 7 0. 1 b, c cit ro ne llo l 123 3 122 3 30. 1 a, b,c ne o iso -d ih yd ro c arv eo l 123 4 122 6 0. 5 b, c pu le go ne 123 5 123 3 1. 7 b, c cis -c arv eo l 123 6 122 6 0. 2 b, c cis -3 -he xe ny l-iso va lera te 123 8 123 8 1.0 b, c ne ral 124 0 123 5 0. 2 Tr 0. 2 a, b,c ca rv on e 124 0 123 9 58. 2 Tr 35. 2 a, b,c o-a nisa ld eh yd e 124 3 124 2 0. 8 b, c he xy l iso va lera te 124 4 124 3 0. 1 Tr b, c cis -m yrtan ol 124 7 125 0 Tr b, c pip erit on e 125 4 124 9 1. 4 0. 4 0. 2 1. 1 b, c cis -m yrtan ol 125 5 125 0 0. 1 b, c cis -p ip erit on e e po xid e 125 5 125 0 0. 3 b, c ge ra nio l 125 8 124 9 1. 9 0. 1 a, b,c cis -c hr ysa nth en yl ac etate 125 9 126 1 0. 3 b, c 2-ph en yl eth yl ac etate 126 0 125 8 T r b, c cis -c arv on e o xid e 126 4 125 9 0. 1 b, c tra n s-a sc arid ol gly co l 126 5 126 6 Tr b, c pe ril la ald eh yd e 126 9 126 9 Tr b, c iso pip erit en on e 127 0 127 1 tr b, c ge ra nial 127 2 126 4 0. 4 0. 2 0. 1 a, b,c n eo -m en th yl ac etate 127 4 127 1 0. 1 b, c n-d ecan ol 127 5 126 6 0. 2 b, c
(E )-c in na m ald eh yd e 127 5 127 0 82. 7 a, b,c tra ns -c arv on e o xid e 127 6 127 3 0. 1 0. 1 b, c cit ro ne ll yl fo rm ate 127 7 127 1 9. 1 b, c ne ry l f or m ate 128 1 128 0 0. 1 b, c bor ny l a ce ta te 128 2 128 7 0. 6 a, b,c hex yl -a ng elate 128 8 127 5 0. 1 b, c th ymo l 128 9 128 9 Tr 0. 1 0. 2 a, b,c m en th yl ac eta te 129 3 129 4 9. 5 0. 2 b, c tra ns -p in oc arv yl ac etate 129 6 129 8 0. 5 b, c n-t rid ec an e 130 0 130 0 Tr a, b,c ca rv ac ro l 130 2 129 8 Tr 0. 5 a, b,c ge ra ny l f orm ate 130 3 129 8 0. 7 b, c iso -m en th yl ac etate 130 4 130 4 0. 2 b, c (E )-c in na m yl alco ho l 130 4 130 4 T r b, c ne o iso -is op uleg yl ac etate 130 9 131 2 0. 1 b, c non yl ac etate 131 4 131 3 0. 1 b, c my rten yl ac eta te 132 2 132 4 Tr b, c (3 Z)-he xe ny l ti glate 132 6 131 9 T r b, c iso -d ih yd ro c arv eo l ac etate 132 7 132 6 0. 2 b, c δ-ele m en e 133 1 133 5 Tr T r b, c tra n s-c arv yl ac etate 133 7 133 9 Tr b, c pip erit en on e 133 7 134 0 2. 1 a, b,c hy dro cin na m ic ac id 134 1 134 7 T r b, c α-cube be ne 134 5 134 5 0. 1 T r a, b,c cit ro ne ll yl ac etate 135 5 135 0 0. 3 a, b,c eu gen ol 135 5 135 6 0. 2 T r T r a, b,c cy clo sa ti ve ne 136 0 136 9 0. 1 b, c cis -c arv yl ac etat e 136 1 136 5 0. 2 b, c pip erit en on e o xid e 136 2 136 6 0. 5 b, c α-ylan ge ne 136 4 137 3 0. 4 b, c ne ry l a ce tate 136 5 135 9 45. 4 a, b,c α-copa ene 136 8 137 4 0. 3 0. 5 0. 2 0. 1 a, b,c β-bour bo ne ne 137 7 138 7 0. 3 1. 7 Tr 1.0 1. 8 b, c tra n s-m yrtan ol ac etate 138 0 138 5 0. 1 b, c β-cu beb en e 138 4 138 7 0. 2 b, c
ge ra ny l a ce tate 138 6 137 9 0. 1 b, c β-ele m en e 138 6 138 9 0. 1 0. 1 0. 1 1. 0 a, b,c iso -it ali ce ne 139 0 140 1 0. 1 b, c be nz yl iso va lera te 139 2 139 5 0. 1 b, c ph en yl eth yl iso bu tan oa te 139 3 139 3 0. 1 b, c ita li ce ne 139 5 140 5 3. 4 b, c (Z )-jas m on e 139 5 139 2 0. 1 b, c n-t etrad ec an e 140 0 140 0 Tr tr a, b,c α-cis -b erg am oten e 141 0 141 1 2. 5 b, c (E )-c ar yo ph yll en e 141 0 141 7 1. 5 1. 1 0. 3 2. 1 1. 3 0. 3 a, b,c β-ylan ge ne 141 0 142 0 0. 3 b, c de cy l a ce tate 141 3 140 7 0. 2 b, c β-co paen e 142 0 143 0 0. 1 0. 1 0. 3 b, c co um arin 142 9 143 2 0. 7 a, b,c α-tra n s-b erg am oten e 143 1 143 2 0. 7 1.0 Tr tr b, c α-gu aien e 143 2 143 7 0. 4 b, c β-gu rju ne ne 143 5 143 1 0. 2 b, c aro m ad en dre ne 143 5 143 9 Tr a, b,c 6, 9-gu aiad ie ne 143 7 144 2 0. 3 5. 7 b, c (E )-c in na m ic ac id 143 7 143 5 0. 42 b, c 4, 6,9 -tri m eth yl-de c- 8- e-3, 5-dio ne 143 9 0. 8 b (Z )-β -fa rn ese ne 144 0 144 0 0. 2 b, c cis -m uu ro la -3, 5-dien e 144 1 144 8 0. 4 b, c oc ty l iso va lera te 144 1 144 0 0. 1 b, c α-hu m ulen e 144 3 145 2 0. 1 0. 1 0. 1 0. 2 0. 1 a, b,c cit ro ne ll yl pro pa no ate 144 5 144 4 1. 1 b, c (E )-c in na m yl ac eta te 144 5 144 6 0. 3 b, c a ll o-a ro m ad en dre ne 145 1 145 8 0. 6 0. 3 0. 5 b, c ne ry l p ro pa no ate 145 6 145 2 5.0 b, c cis -c ad in a-1(6 ), 4-die ne 145 6 146 1 0. 8 0. 3 0. 2 b, c α-ac ora dien e 145 8 146 4 0. 3 b, c (E )-β -fa rn ese ne 145 9 145 4 0. 5 0. 1 0. 4 0. 6 a, b,c de hy dro -se sq uicin eo le 146 6 146 9 Tr b, c β-ac ora dien e 146 8 146 9 0. 1 b, c α-am orp he ne 147 3 148 3 0.6 b, c
γ-c urc um en e 147 6 148 1 9.0 0. 3 b, c ge rm ac re ne D 147 6 148 4 1. 1 0. 4 11. 8 1. 8 0. 1 14. 8 b, c ge ra ny l p ro pa no ate 147 6 147 6 0. 5 b, c ar -c urc um en e 148 1 147 9 7. 6 0. 7 0. 1 T r b, c tra ns -m uu ro la -4( 14) ,5 -dien e 148 3 149 3 Tr T r b, c ph en yl eth yl 3-m eth yl bu tan oa te 148 4 0. 1 b δ-se li ne ne 148 6 149 2 1. 4 b, c vir id if lo re ne 148 6 149 6 1.0 b, c bic yc lo ge rm ac re ne 148 8 150 0 0. 3 0. 7 0. 6 b, c α-zin gib ere ne 149 2 149 3 0. 1 0. 3 b, c α-m uu ro len e 149 4 150 0 T r 0. 3 0. 3 T r 0. 2 b, c iso da uc en e 149 5 150 0 0. 3 b, c δ -am orp he ne 150 0 151 1 0. 1 Tr b, c n-p en ta de ca ne 150 0 150 0 T r a, b,c β-bisa bo len e 150 6 150 5 0. 3 T r 0. 1 b, c (E ,E )-α -fa rn ese ne 150 8 150 5 1. 9 a, b,c cu beb ol 150 7 151 4 0. 4 b, c β-cu rc um en e 150 8 151 4 0. 3 b, c ge ra ny l iso bu tan oa te 151 5 151 4 0. 1 b, c tra ns -c alam en en e 151 7 152 1 0. 1 T r b, c δ-ca din en e 151 7 152 2 0. 1 0. 1 1. 6 1. 1 T r 0. 1 b, c β-se sq uip he ll an dre ne 151 9 152 1 0. 4 b, c tra n s-c ad in a-1, 4-dien e 152 4 153 3 0. 1 b, c it ali ce ne e th er 152 5 153 6 0. 2 b, c (E )-γ -b isa bo le ne 152 7 152 9 0. 1 b, c cit ro ne ll yl bu tan oa te 152 9 153 0 0. 8 b, c (E )-o-m eth ox y cin na m ald eh yd e 153 0 152 9 10. 1 b, c α-ca lac ore ne 153 5 154 4 0. 1 0. 2 b, c fu ro pe larg on e A 153 7 153 8 0. 1 b, c (E )-α -b isa bo le ne 154 0 154 0 1. 4 0. 1 0. 1 0. 1 b, c ge rm ac re ne B 154 6 155 9 T r b, c β-ca lac ore ne 155 5 156 4 Tr b, c pa lu stro l 155 6 156 7 0. 4 b, c ge ra ny l b utan oa te 156 2 156 2 0. 8 b, c
(E )-p-m eth ox y-cin na m ald eh yd e 156 2 156 2 0. 3 b, c (E )-n ero lid ol 156 4 156 3 T r Tr T r a, b, c sp ath ule no l 156 6 157 7 0. 2 0. 1 0. 1 0. 4 b, c ca ry op hy ll en e o xid e 157 1 158 2 0. 1 0. 1 0. 3 0. 9 0. 3 T r 0. 1 a, b,c ar -tu rm ero l 157 7 158 2 0. 2 b, c glo bul ol 158 1 159 0 0. 1 b, c 2-ph en yl eth yl ti glate 158 2 158 4 1. 9 b, c viri dif lo ro l 158 3 159 2 12. 6 a, b, c sa lv ial -4 (1 4) -en -1 -on e 158 3 159 4 T r b, c ne ry l iso va ler ate 158 5 158 2 0. 8 b, c gu aio l 159 1 160 0 0. 8 b, c a ll o-ced ro l 159 2 158 9 0. 7 b, c glo bu lo l 159 2 159 0 0. 7 b, c co pab or neo l 159 3 159 2 0. 5 b, c ro sif oli ol 159 7 160 0 3. 7 b, c ge ra ny l iso va lera te 160 3 160 6 0. 1 b, c 1, 10 -di -ep i-cu ben ol 160 6 161 8 0. 1 b, c 10 -ep i--e ud esm ol 160 8 162 2 0. 1 1. 0 1. 3 b, c ere m oli ge no l 161 9 162 9 T r b, c 1-ep i-cu ben ol 161 9 162 7 0. 9 0. 1 b, c m uu ro la -4 ,1 0(1 4) -d ien - 1- β-ol 162 0 163 0 0. 9 b, c cu ben ol 162 3 164 5 0. 3 b, c (3 Z)-he xe ny lp he ny l ac etate 163 0 163 2 0. 1 b, c ep i-α -m uu ro lo l 163 3 164 0 0. 5 T r b, c ep i-α -c ad in ol 163 4 163 8 0. 3 b, c β-eu de sm ol 163 9 164 9 0. 3 0. 1 b, c α-m uu ro lo l 163 9 164 4 0. 1 T r b, c α-eu de sm ol 164 3 165 2 0. 3 0. 1 b, c α-ca din ol 164 6 165 2 0. 5 0. 2 0. 1 b, c cis -c ala m en en -10 -ol 165 2 166 0 0. 4 b, c tra ns -c alam en en -10 -ol 166 0 166 8 0. 8 b, c bul ne so l 155 9 167 0 0. 2 b, c ep i-β -e ud esm ol 156 5 0. 1 b (E )-c it ro ne ll yl ti glate 166 6 166 6 0. 6 b, c
ca da len e 166 9 167 5 T r b, c eu de sm a-4 (1 5), 7-dien -1β -ol 167 6 168 7 0. 3 b, c ep i-α -b isa bo lo l 167 8 168 3 T r 0. 7 b, c α-bisa bo lo l 168 0 168 5 0. 1 a, b,c n-h ep ta de ca ne 169 9 170 0 T r a, b,c ge ra ny l ti glate 170 1 169 6 0. 1 0. 6 b, c n-p en ta de ca na l 171 3 171 5 T r b, c ch am az ulen e 171 6 171 5 0. 3 b, c (6 R ,7 R )-b isa bo lo ne 174 1 174 0 0. 2 b, c ge ra nio l h ex an oa te 175 4 175 5 0. 1 b, c be nz yl be nz oa te 175 8 175 9 Tr 0. 1 b, c he xa hy dro fa rn es yl ac eto ne 184 4 184 5 Tr T r b, c phe ne thy l-b en zo ate 184 6 184 4 0. 1 b, c n-o ctad ec an ol 206 4 207 0 Tr b, c 13 -h ex ylo xa cy clo tri de c-10 -en -2 -on e 205 5 0. 6 b phy to l 211 8 211 6 0. 1 a, b,c phe ne th yl cin na m ate 217 0 215 8 Tr b, c n-tri co sa ne 230 0 230 0 Tr a, b,c n-p en tac osa ne 250 0 250 0 Tr a, b,c n-h ep tac osa ne 270 0 270 0 Tr a, b,c n-n on ac osa ne 290 0 290 0 Tr a, b,c T otal id en ti fied (% ) 99. 8 99. 4 99. 8 95. 2 88. 5 98. 5 99. 1 99. 2 G ro up ed c om po un ds (% ) M on oterp en e hy dro ca rb on s 12. 3 27. 8 0. 2 2. 3 12. 3 7. 5 0. 1 32. 9 Ox yg en ated m on oterp en es 81. 0 66. 7 18. 3 57. 8 29. 1 71. 5 Tr 42. 8 S esq uit erp en e hy dro ca rb on s 3. 5 3. 9 2. 7 27. 8 23. 7 14. 6 0. 4 21. 9 Ox yg en ated se sq uit erp en es 0. 1 0. 1 6. 2 22. 3 2. 6 0. 1 1. 3 A ro m ati cs 0. 1 0. 1 78. 3 0. 2 0. 2 2. 1 97. 8 Oth ers 2. 9 0. 7 0. 1 0. 9 0. 3 0. 2 0. 6 0. 3 a Co m po un ds are li ste d i n o rd er of th eir elu ti on fro m a HP -5M S c ol um n. b L in ea r re ten ti on in de x o n H P -5 M S c olu m n, ex pe rime ntally d eter m in ed u sin g h om olo go us se ries o f C 8 -C 30 a lk an es.
