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Chemical Constituents and Activities of the Essential Oil from Myristica fragrans against Cigarette Beetle Lasioderma serricorne by Shu-Shan Du* a ), Kai Yang a ), Cheng-Fang Wang a ) b ), Chun-Xue You a ), Zhu-Feng Geng c ), ShanShan Guo a ), Zhi-Wei Deng c ), and Zhi-Long Liu* d ) a
) State Key Laboratory of Earth Surface Processes and Resource Ecology, Beijing Normal University, Beijing 100875, P. R. China (phone: þ 86-10-62208022; e-mail: [email protected]) b ) Key Laboratory of Radiological Protection and Nuclear Emergency, Chinese Center for Disease Control and Prevention, Beijing 100088, P. R. China c ) Analytical and Testing Center, Beijing Normal University, Haidian District, Beijing 100875, P. R. China d ) Department of Entomology, China Agricultural University, Haidian District, Beijing 100193, P. R. China (e-mail: [email protected])
Essential oil extracted from nutmeg seeds (Myristica fragrans Houtt.) by hydrodistillation was subjected to GC/MS and GC analysis. A total of 27 constituents were identified, of which eugenol (19.9%), methylisoeugenol (16.8%), methyleugenol (16.7%), sabinene (11.8%), and terpinen-4-ol (8.5%) were the major components. The essential oil was tested against Lasioderma serricorne for insecticidal and repellent activity, the LD50 value at the end of 24 h exposure period was 19.3 mg/adult. Six active compounds were isolated by bioassay-guided fractionation. They were identified as eugenol (1), methyleugenol (2), methylisoeugenol (3), elemicin (4), myristicin (5), and safrole (6). Among these isolates, 4 showed the strongest contact toxicity against L. serricorne adults with an LD50 value of 9.8 mg/ adult. Repellency of crude oil and active compounds were also determined. Compounds 1, 2, 4, and 5 were strongly repellent against the cigarette beetle and exhibited the same level of repellency compared with the positive control, DEET. The results indicate that the essential oil of M. fragrans and its active constituents have potential for development as natural insecticides and repellents to control L. serricorne.
Introduction. – The cigarette beetle, Lasioderma serricorne (Fabricius), is a widespread and destructive pest that has a wide range of feeding habits, including tobacco, tea, cereal grains, beans, cocoa beans, and animal and plant specimens [1] [2]. Control of L. serricorne population around the world primarily relies on the use of synthetic pesticides [3]. Although effective, their extensive use for decades has disrupted biological control by natural enemies and led to outbreaks of insect species, development of resistance to the chemical, and undesirable effects on non-target organisms, in addition to direct toxicity to users [4] [5]. Therefore, viable alternatives to synthetic chemical insecticides are required. Many plant essential oils, which have been shown to possess a broad spectrum of pest-control properties, have been widely investigated for their larvicidal, toxic, repellent, growth-inhibiting, ovicidal, antifeedant, and antioviposition effects [5 – 7]. In our study, essential oil from Myristica fragrans Houtt. was found to possess significant insecticidal and repellent activities against L. serricorne adults. 2014 Verlag Helvetica Chimica Acta AG, Zrich
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M. fragrans is a famous tropical spice and medicinal plant. The dried nutmegs have long been used in traditional Chinese medicine for the treatment of a wide range of ailments, including chronic diarrhea, abdominal pain, indigestion, and loss of appetite, and vomiting [8]. The chemical composition of essential oil from M. fragrans and its biological properties, such as bactericidal, antioxidant, anticonvulsant, and cytotoxic activities, have been widely exploited [9 – 12]. However, very little information is available with respect to managing stored-product pest, the cigarette beetle, with M. fragrans despite its excellent pharmacological actions. In this study, we assessed contact toxicity and repellent activity of essential oil from M. fragrans against L. serricorne adults, and obtained six alkylbenzene compounds based on bioassay-guided isolation. Further, we determined its chemical composition, as well as the biological activities of the isolated compounds towards the cigarette beetle. Results and Discussion. – Chemical Composition of Essential Oil. Results of the GC/MS analysis of the essential oil from M. fragrans, in a yield of 2.4%, are compiled in Table 1. A total of 27 components in the crude oil were identified, accounting for 98.1% of the total oil (Table 1). Eugenol (1; 19.9%), methylisoeugenol (3; 16.8%), methyleugenol (2; 16.7%), sabinene (11.8%), and terpinen-4-ol (8.5%) were the major components. The chemical compound classes were monoterpenoids (35.6%), sesquiterpenoids (0.4%), and alkylbenzenes (59.0%). The chemical composition of the nutmeg oil in this work differed from that reported in other studies. For example, GC/ MS analysis on essential oil from Indian M. fragrans resulted in the identification of 49 components; sabinene (20.2%), terpinen-4-ol (12.1%), safrole (6; 10.3%), a-pinene (9.7%), b-phellandrene (6.6%), and g-terpinene (5.9%) were the major constituents [13]. Terpinen-4-ol (15.0%), sabinene (13.1%), and g-terpinene (11.2%) were found to be the major constituents of the essential oil of Brazilian M. fragrans [9]. The main volatile components of M. fragrans collected from Andaman Nicobar Island were sabinene (41.7%), a-pinene (9.4%), b-pinene (7.3%), terpinen-4-ol (5.8%), limonene (3.7%), and myristicin (5; 2.7%) [14]. The essential oil isolated from Nigerian M. fragrans was found to contain sabinene (49.1%), a-pinene (13.2%), a-phellandrene (6.7%), and terpinen-4-ol (6.4%) as major constituents [15]. The above results revealed that monoterpene components were given priority to the nutmeg essential oil, whereas, alkylbenzenes were found as the principle components in this work. The essential oil also showed wide variation in the yield of the major constituents, which probably occurred as a result of various factors that can affect the composition of the essential oils, such as genetic factors, growing location, the regional climate, herbal parts used, or the extraction and analytical methods used. Isolated Compounds. Based on bioassay-guided fractionation (contact toxicity), six bioactive compounds were isolated and identified as eugenol (1), methyleugenol (2), methylisoeugenol (3), elemicin (4), myristicin (5), and safrole (6) by their spectroscopic data and comparison with literature data. Their chemical structures are given in the Figure. Insecticidal Activity. The contact toxicities of crude oil and its active constituents are collected in Table 2. The essential oil derived from M. fragrans exhibited strong contact toxicity against L. serricorne with an LD50 value of 19.3 mg/adult. Among the six bioactive compounds, elemicin had the strongest contact toxicity to the cigarette beetle
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Table 1. Chemical Constituents of the Essential Oil Isolated from M. fragrans Compound
RI a )
Chemical formula
Relative content [%]
(1R )-a-Pinene Sabinene b-Pinene a-Myrcene a-Phellandrene Car-3-ene Isoterpinolene o-Cymene g-Terpinene ( Z )-a-Terpineol Terpinolene ( Z )-4-Thujanol Linalool b-Terpinyl acetate ( E )-p-Menth-2-en-1-ol ( Z )-1-Methyl-4-(1-methylethyl)cyclohex-2-en-1-ol Terpinen-4-ol a-Terpineol ( Z )-Piperitol Safrole (6) Eugenol (1) Methyleugenol (2) Methylisoeugenol (3) Myristicin (5) a-Cubebene Elemicin (4) Caryophyllene
1097 1133 1135 1152 1163 1169 1176 1184 1222 1231 1255 1266 1272 1281 1294 1315 1361 1377 1399 1496 1582 1596 1691 1712 1828 1851 1883
C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H14 C10H16 C10H18O C10H16 C10H18O C10H18O C12H20O2 C10H18O C10H18O C10H18O C10H18O C10H18O C10H10O2 C10H12O2 C11H14O2 C11H14O2 C11H12O3 C15H24 C12H16O3 C15H24
2.6 11.8 3.1 0.8 0.5 0.8 1.4 0.8 2.5 0.2 1.1 0.2 0.2 3.1 0.4 0.2 8.5 0.7 0.1 1.6 19.9 16.7 16.8 2.3 0.2 1.7 0.2
Monoterpenoids Sesquiterpenoids Alkylbenzenes Others Total identified
35.6 0.4 59.0 3.1 98.1
a ) RI, Retention index as determined on a HP-5MS column using the homologous series of n-alkanes (C10 – C36 ).
Figure. Structures of compounds isolated from the essential oil of M. fragrans seeds
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Table 2. Contact Toxicity of the Essential Oil from M. fragrans and Its Constituents against L. serricorne Treatment
LD50 [mg/adult]
95% Fiducial limits
Slope SE
c2
M. fragrans Eugenol (1) Methyleugenol (2) Methylisoeugenol (3) Elemicin (4) Myristicin (5) Safrole (6) Pyrethrins a )
19.3 13.2 12.8 21.3 9.8 20.5 14.6 0.2
17.1 – 21.7 11.9 – 14.8 11.2 – 14.6 18.9 – 24.0 8.6 – 11.1 17.5 – 23.5 12.3 – 16.9 0.2 – 0.4
3.6 0.4 3.9 0.4 3.1 0.4 3.5 0.4 3.3 0.4 2.9 0.4 2.4 0.3 3.3 0.3
19.1 20.0 10.1 12.0 17.5 16.8 12.9 17.4
a
) Positive control.
(LD50 ¼ 9.8 mg/adult). L. serricorne adults showed the strongest tolerance to methylisoeugenol with an LD50 value of 21.3 mg/adult. Thus, elemicin possessed two times more toxicity than methylisoeugenol. However, compared with pyrethrins (positive control), elemicin showed 40 times less toxicity against L. serricorne adults. Eugenol (1), methyleugenol (2), and safrole (6) exhibited similar toxicities towards the target insects, while myristicin (5; LD50 20.5 mg/adult), analog of 6, was less toxic than 6 (LD50 14.6 mg/adult). This was also reported by Jung et al. [16], who found myristicin (5) less toxic than safrole (6) against adult female Blattella germanica. Their conclusion is in agreement with ours on L. serricorne adults. The major structural difference between 5 and 6 is the presence of a MeO group in the skeleton of safrole, which might be a key factor influencing the activity. Repellent Activity. The results of repellency assays for the essential oil and isolated compounds against L. serricorne adults are presented in Tables 3 and 4. At 78.63 nl cm 2, the nutmeg oil showed 66 and 62% repellency against the cigarette beetle at 2 and 4 h after exposure, respectively. Its activity was similar at 15.73 nl cm 2 treatment, but decreased rapidly at 0.13 nl cm 2. Among the six constituents of the crude essential oil, eugenol (1) exerted strong repellency (86 and 88%, at 78.63 nl cm 2, after 2 and 4 h Table 3. Percentage Repellency ( PR ) of the Essential Oil from M. fragrans and Its Constituents against L. serricorne at 2 h after Exposure a ) Treatment
78.63 nl cm 2
15.73 nl cm 2
3.15 nl cm 2
0.63 nl cm 2
M. fragrans Eugenol (1) Methyleugenol (2) Methylisoeugenol (3) Elemicin (4) Myristicin (5) Safrole (6) DEET b )
66 16 ac ( IV) 86 14 bd ( V) 92 7 bd ( V ) 56 12 c ( III ) 82 9 abd ( V) 70 8 ace ( IV) 66 11 ac ( IV) 88 7 de ( V)
60 6 ab ( III) 70 12 ac ( IV ) 76 9 ac ( IV ) 48 7 b ( III ) 70 12 a ( IV ) 72 9 ac ( IV ) 84 9 c ( V) 76 14 ac ( IV )
16 5 ae ( I ) 20 6 a ( II ) 48 7 bdf ( III) 56 7 b ( III) 2 11 c ( I ) 8 16 ad ( I ) 6 14 a ( I ) 46 11 c () 56 14 bcd ( III) 4 9 d () 64 13 bc (IV ) 36 14 c () 43 12 df ( III ) 16 9 a ( I ) 28 7 ef ( II ) 20 14 a ( II )
0.13 nl cm 2 20 10 a () 10 13 a () 22 16 a () 36 15 b ( II ) 30 6 b ( II ) 18 11 a () 4 9 a () 16 7 b ( I )
a ) Means in the same column, followed by the same letters, do not differ significantly ( P < 0.05) in ANOVA and Tukeys tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukeys tests. b ) Positive control, N,N-diethyl-3-methylbenzamide.
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Table 4. Percentage Repellency ( PR ) of the Essential Oil from M. fragrans and Its Constituents against L. serricorne at 4 h after Exposure a ) Treatment
78.63 nl cm 2
15.73 nl cm 2
3.15 nl cm 2
0.63 nl cm 2
0.13 nl cm 2
M. fragrans Eugenol (1) Methyleugenol (2) Methylisoeugenol (3) Elemicin (4) Myristicin (5) Safrole (6) DEET b )
62 17 a ( IV ) 88 9 ab ( V) 82 11 ab ( V) 44 12 a ( III ) 86 9 ab ( V) 80 13 ab ( IV ) 64 9 a ( IV ) 98 4 b ( V )
52 13 ad ( III) 14 9 a (I ) 42 7 ad ( III) 2 11 a ( I ) 72 15 ab ( IV ) 78 11 b ( IV ) 64 12 a ( IV) 24 14 bd ( II ) 88 9 b (V ) 32 7 c () 4 15 b () 32 11 c () 28 15 c ( II ) 16 7 c () 12 11 b () 14 9 b ( I ) 76 11 bf ( IV) 62 16 be (IV ) 16 14 c ( I ) 32 12 bd ( II ) 58 8 aef ( III ) 30 5 d ( II ) 8 11 b () 20 13 c () 44 16 cde ( III) 58 15 e ( III) 16 7 cd ( I ) 38 13 d ( II ) 78 9 bf ( IV ) 58 16 e ( III) 56 14 a ( III) 46 7 d ( III)
a ) Means in the same column, followed by the same letters, do not differ significantly ( P < 0.05) in ANOVA and Tukeys tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukeys tests. b ) Positive control, N,N-diethyl-3-methylbenzamide.
treatment, resp.). At the assay concentration of 0.63 nl cm 2, 1 still showed repellency (64%) against the cigarette beetle at 4 h after exposure (Table 3). Methyleugenol (2), elemicin (4), and myristicin (5) also showed strong repellency ( > 70%) at 78.63 and 15.73 nl cm 2 after 2-h treatment. However, their repellency decreased at 0.13 nl cm 2. Compared with the positive control (DEET (N,N-diethyl-3-methylbenzamide)), 1, 2, 4, and 5 exhibited the same level of repellency against L. serricorne adults. At the highest concentration, 78.63 nl cm 2, weak repellency was exerted by methylisoeugenol (3) and safrole (4) at 4 h after exposure. Moreover, there was no obvious doseeffect relationship between the compounds and their % repellency (PR) values. Conclusions. – The presented results establish that the application of natural plant products possessing highly biologically active compounds in terms of contact toxicity and repellent effect against L. serricorne adults can be a potential method in integrative control management of this pest. The nutmeg seed used as traditional Chinese medicine and spice is considered to be of low toxicity to human body. However, further investigations need to be also conducted to evaluate the cost and efficacy of the essential oil on a wide range of insect pests. This project was funded by the State Key Laboratory of Earth Surface Processes and Resource Ecology (2013-ZY-11) and the National Nature Science Foundation of China (No. 81374069). The authors are grateful to Dr. H.-B. Yin, College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, P. R. China, for identification of the plant.
Experimental Part 1
13
General. H- and C-NMR spectra: Bruker Avance DRX 500 instrument; in CDCl3 ; d in ppm rel. to Me4Si as internal standard, J in Hz. EI-MS: ThermoQuest Trace 2000 mass spectrometer; at 70 eV (probe); in m/z (rel. %). GC/MS Analysis of Essential Oil. GC/MS Analysis was performed on a Thermo Finnigan Trace DSQ instrument equipped with a flame ionization detector and an HP-5 MS (30 m 0.25 mm, 0.25 mm) cap. column. The column temp. was programmed at 508 for 2 min, then increased at 28/min to 1508, held for 2 min, and then increased at 108/min until the final temp. of 2508 was reached, which was held for 5 min.
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The injector temp. was maintained at 2508. The samples (1 ml) were diluted to 1% with hexane. The carrier gas was He at a flow rate of 1.0 ml min 1. The spectra were scanned from m/z 50 to 550. Most constituents were identified by GC by comparison of their retention indices (RIs) with those in the literature or with those of authentic compounds available in our laboratories. The RI values were determined in relation to a homologous series of n-alkanes (C10 – C36 ) under the same operating conditions. Further identification was made by comparison of their MS spectra with those stored in NIST 05 and Wiley 275 libraries or with MS spectra from the literature [17]. Component relative percentages were calculated based on GC peak areas without using correction factors. Plant Material and Essential-Oil Extraction. Dried seed kernels of M. fragrans (1 kg) were purchased from Anguo Chinese Medicinal Herbs Market (Anguo 071200, Baoding, Hebei Province, P. R. China). The nutmegs were ground to a powder using a grinding mill (Retsch, Germany). A voucher specimen (CMH-M. fragrans-2011-10-010) of the plant was deposited with the College of Resources Science and Technology, Beijing Normal University. The nutmegs were submitted to hydrodistillation for 6 h using a Clevenger-type apparatus and extracted with hexane. Na2SO4 was used to remove H2O after extraction. Isolation of Active Compounds. The crude essential oil (5 ml) was chromatographed on a SiO2 column (Qingdao Marine Chemical Plant, Shandong Province, P. R. China; 4 mm i.d., 500 mm length) by gradient elution with hexane first, then with hexane/AcOEt, and finally with AcOEt to obtain 20 fractions. Based on contact toxicity, Frs. 2, 8, and 12 were chosen for further fractionation. They were separated on repeated CC (SiO2 ) and prep. TLC to afford six pure compounds determined as eugenol (1; 0.75 g), methyleugenol (2; 0.68 g), methylisoeugenol (3; 0.53 g), elemicin (4; 0.14 g), myristicin (5; 0.11 g), and safrole (6; 0.15 g). The structures of the isolated compounds were elucidated based on their NMR spectra. Eugenol ( ¼ 2-Methoxy-4-(prop-2-en-1-yl)phenol; 1). Colorless oil. 1H-NMR (500 MHz) 1): 6.87 (d, J ¼ 8.5, HC(5)); 6.72 (d, J ¼ 8.5, HC(6)); 6.71 (s, HC(2)); 5.95 – 6.01 (m, HC(8)); 5.52 (s, OH); 5.07 – 5.12 (m, CH2(9)); 3.90 (s, MeO); 3.35 (d, J ¼ 6.5, CH2(7)). 13C-NMR (125 MHz): 146.4 (C(3)); 143.9 (C(4)); 137.8 (C(8)); 131.9 (C(1)); 121.2 (C(6)); 115.5 (C(5)); 114.3 (C(2)); 111.1 (C(9)); 55.9 (MeO); 39.9 (C(7)). EI-MS: 164 (100), 150 (36), 147 (5), 131 (27), 103 (29), 77 (28), 55 (13), 39 (7). The spectral data were in agreement with those reported in [18] [19]. Methyleugenol ( ¼ 1,2-Dimethoxy-4-( prop-2-en-1-yl)benzene; 2) . Colorless oil. 1H-NMR (500 MHz): 6.83 (d, J ¼ 8.0, HC(5)); 6.75 (d, J ¼ 8.0, HC(6)); 6.74 (s, HC(2)); 5.95 – 6.03 (m, HC(8)); 5.11 (dd, J ¼ 15.0, 5.0, 1 H of CH2(9)); 5.08 (d, J ¼ 5.0, 1 H of CH2(9)); 3.90 (s, MeOC(3)); 3.89 (s, MeOC(4)); 3.36 (d, J ¼ 6.5, CH2(7)). 13C-NMR (125 MHz): 148.9 (C(3)); 147.4 (C(4)); 137.7 (C(8)); 132.7 (C(1)); 120.4 (C(6)); 115.6 (C(5)); 111.9 (C(2)); 111.3 (C(9)); 56.0 (MeOC(3)); 55.8 (MeOC(4)); 39.8 (C(7)). EI-MS: 178 (100), 163 (33), 147 (32), 131 (11), 107 (28), 103 (39), 91 (50), 77 (26), 58 (9), 43 (27). The spectral data were in agreement with those reported in [20]. Methylisoeugenol ( ¼ 1,2-Dimethoxy-4-[(1E)-prop-1-en-1-yl]benzene; 3). Colorless oil. 1H-NMR (500 MHz): 6.92 (d, J ¼ 1.5, HC(2)); 6.88 (dd, J ¼ 8.5, 1.5, HC(6)); 6.82 (d, J ¼ 8.5, HC(5)); 6.36 (d, J ¼ 15.5, CH2(7)); 6.10 – 6.17 (m, HC(8)); 3.92 (s, MeOC(3)); 3.89 (s, MeOC(4)); 1.89 (d, J ¼ 7.0, Me(9)). 13 C-NMR (125 MHz): 149.0 (C(3)); 148.2 (C(4)); 131.2 (C(8)); 130.6 (C(1)); 123.8 (C(6)); 118.7 (C(5)); 111.3 (C(2)); 108.6 (C(7)); 55.9 (MeOC(3)); 55.8 (MeOC(4)); 18.4 (C(9)). EI-MS: 178 (100), 163 (48), 147 (13), 135 (10), 115 (12), 107 (35), 91 (28), 77 (12), 51 (3). The spectral data were in agreement with those reported in [21]. Elemicin ( ¼ 1,2,3-Trimethoxy-5-(prop-2-en-1-yl)benzene; 4). Colorless oil. 1H-NMR (500 MHz): 6.58 (s, HC(2), HC(6)), 5.94 – 6.02 (m, HC(8)); 5.12 (dd, J ¼ 15.6, 5.0, 1 H of CH2(9)); 5.05 (d, J ¼ 5.0, 1 H of CH2(9)); 3.89 (s, MeOC(3), MeOC(5)); 3.85 (s, MeOC(4)); 3.35(d, J ¼ 6.5, CH2(7)). 13 C-NMR (125 MHz): 153.2 (C(3), C(5)); 137.2 (C(1), C(4)); 135.8 (C(8)); 116.0 (C(9)); 105.4 (C(2), C(6)); 60.9 (MeOC(4)); 56.1 (MeOC(3), MeOC(5)); 40.5 (C(7)). EI-MS: 208 (100), 193 (63), 177 (13), 165 (10), 150 (11), 133 (19), 118 (13), 105 (14), 91 (17), 77 (19), 65 (8), 43 (15). The spectral data were in agreement with those reported in [20]. 1)
For signal assignments of NMR spectra, cf. the atom numbering in Fig. 1.
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Myristicin ( ¼ 4-Methoxy-6-( prop-2-en-1-yl)-1,3-benzodioxole; 5) . Colorless oil. 1H-NMR (500 MHz): 6.41 (s, HC(6)); 6.38 (s, HC(2)); 5.96 (s, OCH2O); 5.91 – 5.95 (m, HC(8)); 5.11 (dd, J ¼ 15.0, 4.0, 1 H of CH2(9)); 5.09 (d, J ¼ 4.0, 1 H of CH2(9)); 3.91 (s, MeO); 3.31 (d, J ¼ 6.5, CH2(7)). 13 C-NMR (125 MHz): 148.9 (C(5)); 143.5 (C(3)); 137.4 (C(8)); 134.6 (C(1)); 133.5 (C(4)); 115.8 (C(9)); 107.8 (C(6)); 102.7 (C(2)); 101.2 (OCH2O); 56.6 (MeO); 40.2 (C(7)). EI-MS: 192 (100), 165 (24), 147 (13), 119 (21), 91 (29), 65 (16), 39 (7). The spectral data were in agreement with those reported in [22] [23]. Safrole ( ¼ 5-(Prop-2-en-1-yl)-1,3-benzodioxole; 6). Colorless oil. 1H-NMR (500 MHz): 6.77 (d, J ¼ 7.5, HC(5)); 6.77 (s, HC(2)); 6.66 (d, J ¼ 7.5, HC(6)); 5.96 (s, OCH2O); 5.91 – 5.95 (m, HC(8)); 5.08 (dd, J ¼ 15.6, 5.0, 1 H of CH2(9)); 5.05 (d, J ¼ 5.0, 1 H of CH2(9)); 3.33 (d, J ¼ 6.5, CH2(7)). 13C-NMR (125 MHz): 137.6 (C(3)); 133.9 (C(4)); 121.3 (C(8)); 116.0 (C(1)); 115.7 (C(6)); 109.1 (C(5)); 108.2 (C(2), C(9)); 100.8 (OCH2O); 39.9 (C(7)). EI-MS: 162 (100), 131 (41), 104 (43), 77 (27), 51 (16), 39 (6). The spectral data were in agreement with those reported in [24]. Insects. L. serricorne was obtained from laboratory cultures maintained for the last two years in the dark in incubators at 28 – 308 and 70 – 80% rel. humidity. The insects were reared in glass containers (0.5 l) containing wheat flour at 12 – 13% moisture content mixed with yeast (wheatfeed/yeast 10 : 1 (w/w)). Adults used in all the experiments were ca. 5 – 9 d old. Contact Toxicity. The contact toxicity of the crude essential oil and isolated compounds against L. serricorne adults was evaluated as described by Liu and Ho [25]. Range-finding studies were run to determine the appropriate testing concentrations. A serial dilution of the essential oil/compounds (five concentrations) was prepared in hexane. Aliquots of 0.5 ml of the dilutions were applied topically to the dorsal thorax of the insects. Controls were determined using hexane. Ten insects were used for each concentration and control, and the experiment was replicated five times. Both treated and control insects were then transferred to glass vials and kept in incubators. Mortality was recorded after 24 h, and the LD50 values were calculated using Probit analysis [26]. Positive control (pyrethrins I and II; 37%) was purchased from Dr. Ehrenstorfer GmbH. Repellency Test. The repellent activity to L. serricorne adults was tested using the area preference method [27]. Petri dishes of 9 cm in diameter were used to confine cigarette beetles during the experiment. The crude essential oil and the isolated compounds were diluted in hexane to different concentrations (78.63, 15.73, 3.15, 0.63, and 0.13 nl cm 2 ), and hexane was used as control. Filter paper of 9 cm in diameter was cut in half, and 500 ml of each concentration was applied separately to half of the filter paper as uniformly as possible with a micropipette. The other half (control) was treated with 500 ml of hexane. Both the treated and control half were then air-dried to evaporate the solvent completely (30 s). A full disk was carefully remade by attaching the tested half to the negative control half with tape. Care was taken so that the attachment did not prevent free movement of insects from the one half to the other, but the distance between the filter paper halves remained sufficient to prevent seepage of test samples from one half to the other. Each reassembled filter paper after treatment with solid glue was placed in a Petri dish with the seam oriented in one of four randomly selected different directions to avoid any insecticidal stimuli affecting the distribution of insects. Twenty insects were released in the center of each filter paper disk, and a cover was placed over the Petri dish. Five replicates were used, and the experiment was repeated three times. Counts of the insects present on each strip were made after 2 and 4 h. The PR value of each volatile oil/compound was then calculated using Eqn. 1: PR [%] ¼ [(Nc Nt )/(Nc þ Nt )] · 100
(1)
where Nc is the number of insects present in the negative control half and Nt is the number of insects present in the treated half. Analysis of variance (ANOVA) and Tukeys test were conducted by using SPSS 13.0 for Windows 2007. Percentage was subjected to an arcsine square-root transformation before ANOVA and Tukeys tests. A commercial repellent, DEET, was purchased from the National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang 110021, P. R. China) and used as positive control. The averages were then categorized according to the scale in Table 5 [28].
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Table 5. The Scale Used to Categorize Repellency of the Essential Oil and Its Compounds Class
Percent repulsion [%]
0 I II III IV V
0.01 – 0.1 0.1 – 20 20.1 – 40 40.1 – 60 60.1 – 80 80.1 – 100
REFERENCES [1] R. T. Arbogast, P. E. Kendra, R. W. Mankin, R. C. McDonald, J. Stored Prod. Res. 2002, 38, 349. [2] M. P. Blanc, N. Lugon-Moulin, C. Panighini, H. Pijnenburg, L. Rossi, Bull. Entomol. Res. 2006, 96, 111. [3] M. G. Jahromi, A. A. Pourmirza, M. H. Safaralizadeh, Afr. J. Biotechnol. 2012, 11, 280. [4] J. L. Zettler, F. H. Arthur, Crop Prot. 2000, 19, 577. [5] M. B. Isman, Annu. Rev. Entomol. 2006, 51, 45. [6] M. B. Isman, Crop Prot. 2000, 19, 603. [7] S. Rajendran, V. Srianjini, J. Stored Prod. Res. 2008, 44, 126. [8] Z. M. Yuan, J. Wang, T. Z. Jia, Chin. Tradit. Pat. Med. 2007, 29, 442. [9] R. K. Lima, M. G. Cardoso, M. A. Andrade, P. L. Guimaraes, L. R. Batista, D. L. Nelson, J. Am. Oil Chem. Soc. 2012, 89, 523. [10] V. M. M. Valente, G. N. Jham, O. D. Dhingra, I. Ghiviriga, J. Food Safety 2011, 31, 197. [11] A. Wahab, R. U. Haq, A. Ahmed, R. A. Khan, M. Raza, Phytother. Res. 2009, 23, 153. [12] A. Piras, A. Rosa, B. Marongiu, A. Atzeri, M. A. Dessi, D. Falconieri, S. Porcedda, J. Food Sci. 2012, 77, 448. [13] G. Singh, P. Marimuthu, C. S. Heluani, C. Catalan, J. Food Sci. 2006, 70, 141. [14] M. Pal, M. Srivastava, D. K. Soni, A. Kumar, S. K. Tewari, Int. J. Pharm. Life Sci. 2011, 10, 1115. [15] I. A. Ogunwande, N. O. Olawore, K. A. Adeleke, O. Ekundayo, J. Essent. Oil Bear. Pl. 2003, 6, 21. [16] W. C. Jung, Y. S. Jang, T. T. Hieu, C. K. Lee, Y. J. Ahn, J. Med. Entomol. 2007, 44, 524. [17] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy, Allured Publishing Corporation, Carol Stream, IL, 2001. [18] S. Mohottalage, R. Tabacchi, P. M. Guerin, Flavour Fragrance J. 2007, 22, 130. [19] E. M. Elgendy, S. A. Khayyat, Russ. J. Org. Chem. 2008, 44, 823. [20] M. Miyazawa, G. Kohno, Nat. Prod. Res. 2005, 19, 29. [21] X. C. Liu, L. G. Zhou, Z. L. Liu, S. S. Du, Molecules 2013, 18, 5684. [22] C. M. Passreiter, Y. Akhtar, M. B. Isman, Z. Naturforsch., C 2005, 60, 411. [23] A. G. Gonzalez, J. Bermejo Barrera, J. G. Diaz, L. Arancibia Lopez, P. P. De Paz, Syst. Ecol. 1988, 16, 641. [24] S. S. Chu, C. F. Wang, S. S. Du, S. L. Liu, Z. L. Liu, J. Insect Sci. 2011, 11, 152. [25] Z. L. Liu, S. H. Ho, J. Stored Prod. Res. 1999, 35, 317. [26] M. Sakuma, Appl. Entomol. Zool. 1998, 33, 339. [27] J. S. Zhang, N. N. Zhao, Q. Z. Liu, Z. L. Liu, S. S. Du, L. G. Zhou, Z. W. Deng, J. Agric. Food Chem. 2011, 59, 9910. [28] F. A. Talukder, P. E. Howse, J. Stored Prod. Res. 1995, 31, 55. Received April 1, 2014
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Chemical Constituents and Activities of the Essential Oil from Myristica fragrans against Cigarette Beetle Lasioderma serricorne by Shu-Shan Du* a ), Kai Yang a ), Cheng-Fang Wang a ) b ), Chun-Xue You a ), Zhu-Feng Geng c ), ShanShan Guo a ), Zhi-Wei Deng c ), and Zhi-Long Liu* d ) a
) State Key Laboratory of Earth Surface Processes and Resource Ecology, Beijing Normal University, Beijing 100875, P. R. China (phone: þ 86-10-62208022; e-mail: [email protected]) b ) Key Laboratory of Radiological Protection and Nuclear Emergency, Chinese Center for Disease Control and Prevention, Beijing 100088, P. R. China c ) Analytical and Testing Center, Beijing Normal University, Haidian District, Beijing 100875, P. R. China d ) Department of Entomology, China Agricultural University, Haidian District, Beijing 100193, P. R. China (e-mail: [email protected])
Essential oil extracted from nutmeg seeds (Myristica fragrans Houtt.) by hydrodistillation was subjected to GC/MS and GC analysis. A total of 27 constituents were identified, of which eugenol (19.9%), methylisoeugenol (16.8%), methyleugenol (16.7%), sabinene (11.8%), and terpinen-4-ol (8.5%) were the major components. The essential oil was tested against Lasioderma serricorne for insecticidal and repellent activity, the LD50 value at the end of 24 h exposure period was 19.3 mg/adult. Six active compounds were isolated by bioassay-guided fractionation. They were identified as eugenol (1), methyleugenol (2), methylisoeugenol (3), elemicin (4), myristicin (5), and safrole (6). Among these isolates, 4 showed the strongest contact toxicity against L. serricorne adults with an LD50 value of 9.8 mg/ adult. Repellency of crude oil and active compounds were also determined. Compounds 1, 2, 4, and 5 were strongly repellent against the cigarette beetle and exhibited the same level of repellency compared with the positive control, DEET. The results indicate that the essential oil of M. fragrans and its active constituents have potential for development as natural insecticides and repellents to control L. serricorne.
Introduction. – The cigarette beetle, Lasioderma serricorne (Fabricius), is a widespread and destructive pest that has a wide range of feeding habits, including tobacco, tea, cereal grains, beans, cocoa beans, and animal and plant specimens [1] [2]. Control of L. serricorne population around the world primarily relies on the use of synthetic pesticides [3]. Although effective, their extensive use for decades has disrupted biological control by natural enemies and led to outbreaks of insect species, development of resistance to the chemical, and undesirable effects on non-target organisms, in addition to direct toxicity to users [4] [5]. Therefore, viable alternatives to synthetic chemical insecticides are required. Many plant essential oils, which have been shown to possess a broad spectrum of pest-control properties, have been widely investigated for their larvicidal, toxic, repellent, growth-inhibiting, ovicidal, antifeedant, and antioviposition effects [5 – 7]. In our study, essential oil from Myristica fragrans Houtt. was found to possess significant insecticidal and repellent activities against L. serricorne adults. 2014 Verlag Helvetica Chimica Acta AG, Zrich
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M. fragrans is a famous tropical spice and medicinal plant. The dried nutmegs have long been used in traditional Chinese medicine for the treatment of a wide range of ailments, including chronic diarrhea, abdominal pain, indigestion, and loss of appetite, and vomiting [8]. The chemical composition of essential oil from M. fragrans and its biological properties, such as bactericidal, antioxidant, anticonvulsant, and cytotoxic activities, have been widely exploited [9 – 12]. However, very little information is available with respect to managing stored-product pest, the cigarette beetle, with M. fragrans despite its excellent pharmacological actions. In this study, we assessed contact toxicity and repellent activity of essential oil from M. fragrans against L. serricorne adults, and obtained six alkylbenzene compounds based on bioassay-guided isolation. Further, we determined its chemical composition, as well as the biological activities of the isolated compounds towards the cigarette beetle. Results and Discussion. – Chemical Composition of Essential Oil. Results of the GC/MS analysis of the essential oil from M. fragrans, in a yield of 2.4%, are compiled in Table 1. A total of 27 components in the crude oil were identified, accounting for 98.1% of the total oil (Table 1). Eugenol (1; 19.9%), methylisoeugenol (3; 16.8%), methyleugenol (2; 16.7%), sabinene (11.8%), and terpinen-4-ol (8.5%) were the major components. The chemical compound classes were monoterpenoids (35.6%), sesquiterpenoids (0.4%), and alkylbenzenes (59.0%). The chemical composition of the nutmeg oil in this work differed from that reported in other studies. For example, GC/ MS analysis on essential oil from Indian M. fragrans resulted in the identification of 49 components; sabinene (20.2%), terpinen-4-ol (12.1%), safrole (6; 10.3%), a-pinene (9.7%), b-phellandrene (6.6%), and g-terpinene (5.9%) were the major constituents [13]. Terpinen-4-ol (15.0%), sabinene (13.1%), and g-terpinene (11.2%) were found to be the major constituents of the essential oil of Brazilian M. fragrans [9]. The main volatile components of M. fragrans collected from Andaman Nicobar Island were sabinene (41.7%), a-pinene (9.4%), b-pinene (7.3%), terpinen-4-ol (5.8%), limonene (3.7%), and myristicin (5; 2.7%) [14]. The essential oil isolated from Nigerian M. fragrans was found to contain sabinene (49.1%), a-pinene (13.2%), a-phellandrene (6.7%), and terpinen-4-ol (6.4%) as major constituents [15]. The above results revealed that monoterpene components were given priority to the nutmeg essential oil, whereas, alkylbenzenes were found as the principle components in this work. The essential oil also showed wide variation in the yield of the major constituents, which probably occurred as a result of various factors that can affect the composition of the essential oils, such as genetic factors, growing location, the regional climate, herbal parts used, or the extraction and analytical methods used. Isolated Compounds. Based on bioassay-guided fractionation (contact toxicity), six bioactive compounds were isolated and identified as eugenol (1), methyleugenol (2), methylisoeugenol (3), elemicin (4), myristicin (5), and safrole (6) by their spectroscopic data and comparison with literature data. Their chemical structures are given in the Figure. Insecticidal Activity. The contact toxicities of crude oil and its active constituents are collected in Table 2. The essential oil derived from M. fragrans exhibited strong contact toxicity against L. serricorne with an LD50 value of 19.3 mg/adult. Among the six bioactive compounds, elemicin had the strongest contact toxicity to the cigarette beetle
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Table 1. Chemical Constituents of the Essential Oil Isolated from M. fragrans Compound
RI a )
Chemical formula
Relative content [%]
(1R )-a-Pinene Sabinene b-Pinene a-Myrcene a-Phellandrene Car-3-ene Isoterpinolene o-Cymene g-Terpinene ( Z )-a-Terpineol Terpinolene ( Z )-4-Thujanol Linalool b-Terpinyl acetate ( E )-p-Menth-2-en-1-ol ( Z )-1-Methyl-4-(1-methylethyl)cyclohex-2-en-1-ol Terpinen-4-ol a-Terpineol ( Z )-Piperitol Safrole (6) Eugenol (1) Methyleugenol (2) Methylisoeugenol (3) Myristicin (5) a-Cubebene Elemicin (4) Caryophyllene
1097 1133 1135 1152 1163 1169 1176 1184 1222 1231 1255 1266 1272 1281 1294 1315 1361 1377 1399 1496 1582 1596 1691 1712 1828 1851 1883
C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H14 C10H16 C10H18O C10H16 C10H18O C10H18O C12H20O2 C10H18O C10H18O C10H18O C10H18O C10H18O C10H10O2 C10H12O2 C11H14O2 C11H14O2 C11H12O3 C15H24 C12H16O3 C15H24
2.6 11.8 3.1 0.8 0.5 0.8 1.4 0.8 2.5 0.2 1.1 0.2 0.2 3.1 0.4 0.2 8.5 0.7 0.1 1.6 19.9 16.7 16.8 2.3 0.2 1.7 0.2
Monoterpenoids Sesquiterpenoids Alkylbenzenes Others Total identified
35.6 0.4 59.0 3.1 98.1
a ) RI, Retention index as determined on a HP-5MS column using the homologous series of n-alkanes (C10 – C36 ).
Figure. Structures of compounds isolated from the essential oil of M. fragrans seeds
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Table 2. Contact Toxicity of the Essential Oil from M. fragrans and Its Constituents against L. serricorne Treatment
LD50 [mg/adult]
95% Fiducial limits
Slope SE
c2
M. fragrans Eugenol (1) Methyleugenol (2) Methylisoeugenol (3) Elemicin (4) Myristicin (5) Safrole (6) Pyrethrins a )
19.3 13.2 12.8 21.3 9.8 20.5 14.6 0.2
17.1 – 21.7 11.9 – 14.8 11.2 – 14.6 18.9 – 24.0 8.6 – 11.1 17.5 – 23.5 12.3 – 16.9 0.2 – 0.4
3.6 0.4 3.9 0.4 3.1 0.4 3.5 0.4 3.3 0.4 2.9 0.4 2.4 0.3 3.3 0.3
19.1 20.0 10.1 12.0 17.5 16.8 12.9 17.4
a
) Positive control.
(LD50 ¼ 9.8 mg/adult). L. serricorne adults showed the strongest tolerance to methylisoeugenol with an LD50 value of 21.3 mg/adult. Thus, elemicin possessed two times more toxicity than methylisoeugenol. However, compared with pyrethrins (positive control), elemicin showed 40 times less toxicity against L. serricorne adults. Eugenol (1), methyleugenol (2), and safrole (6) exhibited similar toxicities towards the target insects, while myristicin (5; LD50 20.5 mg/adult), analog of 6, was less toxic than 6 (LD50 14.6 mg/adult). This was also reported by Jung et al. [16], who found myristicin (5) less toxic than safrole (6) against adult female Blattella germanica. Their conclusion is in agreement with ours on L. serricorne adults. The major structural difference between 5 and 6 is the presence of a MeO group in the skeleton of safrole, which might be a key factor influencing the activity. Repellent Activity. The results of repellency assays for the essential oil and isolated compounds against L. serricorne adults are presented in Tables 3 and 4. At 78.63 nl cm 2, the nutmeg oil showed 66 and 62% repellency against the cigarette beetle at 2 and 4 h after exposure, respectively. Its activity was similar at 15.73 nl cm 2 treatment, but decreased rapidly at 0.13 nl cm 2. Among the six constituents of the crude essential oil, eugenol (1) exerted strong repellency (86 and 88%, at 78.63 nl cm 2, after 2 and 4 h Table 3. Percentage Repellency ( PR ) of the Essential Oil from M. fragrans and Its Constituents against L. serricorne at 2 h after Exposure a ) Treatment
78.63 nl cm 2
15.73 nl cm 2
3.15 nl cm 2
0.63 nl cm 2
M. fragrans Eugenol (1) Methyleugenol (2) Methylisoeugenol (3) Elemicin (4) Myristicin (5) Safrole (6) DEET b )
66 16 ac ( IV) 86 14 bd ( V) 92 7 bd ( V ) 56 12 c ( III ) 82 9 abd ( V) 70 8 ace ( IV) 66 11 ac ( IV) 88 7 de ( V)
60 6 ab ( III) 70 12 ac ( IV ) 76 9 ac ( IV ) 48 7 b ( III ) 70 12 a ( IV ) 72 9 ac ( IV ) 84 9 c ( V) 76 14 ac ( IV )
16 5 ae ( I ) 20 6 a ( II ) 48 7 bdf ( III) 56 7 b ( III) 2 11 c ( I ) 8 16 ad ( I ) 6 14 a ( I ) 46 11 c () 56 14 bcd ( III) 4 9 d () 64 13 bc (IV ) 36 14 c () 43 12 df ( III ) 16 9 a ( I ) 28 7 ef ( II ) 20 14 a ( II )
0.13 nl cm 2 20 10 a () 10 13 a () 22 16 a () 36 15 b ( II ) 30 6 b ( II ) 18 11 a () 4 9 a () 16 7 b ( I )
a ) Means in the same column, followed by the same letters, do not differ significantly ( P < 0.05) in ANOVA and Tukeys tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukeys tests. b ) Positive control, N,N-diethyl-3-methylbenzamide.
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Table 4. Percentage Repellency ( PR ) of the Essential Oil from M. fragrans and Its Constituents against L. serricorne at 4 h after Exposure a ) Treatment
78.63 nl cm 2
15.73 nl cm 2
3.15 nl cm 2
0.63 nl cm 2
0.13 nl cm 2
M. fragrans Eugenol (1) Methyleugenol (2) Methylisoeugenol (3) Elemicin (4) Myristicin (5) Safrole (6) DEET b )
62 17 a ( IV ) 88 9 ab ( V) 82 11 ab ( V) 44 12 a ( III ) 86 9 ab ( V) 80 13 ab ( IV ) 64 9 a ( IV ) 98 4 b ( V )
52 13 ad ( III) 14 9 a (I ) 42 7 ad ( III) 2 11 a ( I ) 72 15 ab ( IV ) 78 11 b ( IV ) 64 12 a ( IV) 24 14 bd ( II ) 88 9 b (V ) 32 7 c () 4 15 b () 32 11 c () 28 15 c ( II ) 16 7 c () 12 11 b () 14 9 b ( I ) 76 11 bf ( IV) 62 16 be (IV ) 16 14 c ( I ) 32 12 bd ( II ) 58 8 aef ( III ) 30 5 d ( II ) 8 11 b () 20 13 c () 44 16 cde ( III) 58 15 e ( III) 16 7 cd ( I ) 38 13 d ( II ) 78 9 bf ( IV ) 58 16 e ( III) 56 14 a ( III) 46 7 d ( III)
a ) Means in the same column, followed by the same letters, do not differ significantly ( P < 0.05) in ANOVA and Tukeys tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukeys tests. b ) Positive control, N,N-diethyl-3-methylbenzamide.
treatment, resp.). At the assay concentration of 0.63 nl cm 2, 1 still showed repellency (64%) against the cigarette beetle at 4 h after exposure (Table 3). Methyleugenol (2), elemicin (4), and myristicin (5) also showed strong repellency ( > 70%) at 78.63 and 15.73 nl cm 2 after 2-h treatment. However, their repellency decreased at 0.13 nl cm 2. Compared with the positive control (DEET (N,N-diethyl-3-methylbenzamide)), 1, 2, 4, and 5 exhibited the same level of repellency against L. serricorne adults. At the highest concentration, 78.63 nl cm 2, weak repellency was exerted by methylisoeugenol (3) and safrole (4) at 4 h after exposure. Moreover, there was no obvious doseeffect relationship between the compounds and their % repellency (PR) values. Conclusions. – The presented results establish that the application of natural plant products possessing highly biologically active compounds in terms of contact toxicity and repellent effect against L. serricorne adults can be a potential method in integrative control management of this pest. The nutmeg seed used as traditional Chinese medicine and spice is considered to be of low toxicity to human body. However, further investigations need to be also conducted to evaluate the cost and efficacy of the essential oil on a wide range of insect pests. This project was funded by the State Key Laboratory of Earth Surface Processes and Resource Ecology (2013-ZY-11) and the National Nature Science Foundation of China (No. 81374069). The authors are grateful to Dr. H.-B. Yin, College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, P. R. China, for identification of the plant.
Experimental Part 1
13
General. H- and C-NMR spectra: Bruker Avance DRX 500 instrument; in CDCl3 ; d in ppm rel. to Me4Si as internal standard, J in Hz. EI-MS: ThermoQuest Trace 2000 mass spectrometer; at 70 eV (probe); in m/z (rel. %). GC/MS Analysis of Essential Oil. GC/MS Analysis was performed on a Thermo Finnigan Trace DSQ instrument equipped with a flame ionization detector and an HP-5 MS (30 m 0.25 mm, 0.25 mm) cap. column. The column temp. was programmed at 508 for 2 min, then increased at 28/min to 1508, held for 2 min, and then increased at 108/min until the final temp. of 2508 was reached, which was held for 5 min.
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The injector temp. was maintained at 2508. The samples (1 ml) were diluted to 1% with hexane. The carrier gas was He at a flow rate of 1.0 ml min 1. The spectra were scanned from m/z 50 to 550. Most constituents were identified by GC by comparison of their retention indices (RIs) with those in the literature or with those of authentic compounds available in our laboratories. The RI values were determined in relation to a homologous series of n-alkanes (C10 – C36 ) under the same operating conditions. Further identification was made by comparison of their MS spectra with those stored in NIST 05 and Wiley 275 libraries or with MS spectra from the literature [17]. Component relative percentages were calculated based on GC peak areas without using correction factors. Plant Material and Essential-Oil Extraction. Dried seed kernels of M. fragrans (1 kg) were purchased from Anguo Chinese Medicinal Herbs Market (Anguo 071200, Baoding, Hebei Province, P. R. China). The nutmegs were ground to a powder using a grinding mill (Retsch, Germany). A voucher specimen (CMH-M. fragrans-2011-10-010) of the plant was deposited with the College of Resources Science and Technology, Beijing Normal University. The nutmegs were submitted to hydrodistillation for 6 h using a Clevenger-type apparatus and extracted with hexane. Na2SO4 was used to remove H2O after extraction. Isolation of Active Compounds. The crude essential oil (5 ml) was chromatographed on a SiO2 column (Qingdao Marine Chemical Plant, Shandong Province, P. R. China; 4 mm i.d., 500 mm length) by gradient elution with hexane first, then with hexane/AcOEt, and finally with AcOEt to obtain 20 fractions. Based on contact toxicity, Frs. 2, 8, and 12 were chosen for further fractionation. They were separated on repeated CC (SiO2 ) and prep. TLC to afford six pure compounds determined as eugenol (1; 0.75 g), methyleugenol (2; 0.68 g), methylisoeugenol (3; 0.53 g), elemicin (4; 0.14 g), myristicin (5; 0.11 g), and safrole (6; 0.15 g). The structures of the isolated compounds were elucidated based on their NMR spectra. Eugenol ( ¼ 2-Methoxy-4-(prop-2-en-1-yl)phenol; 1). Colorless oil. 1H-NMR (500 MHz) 1): 6.87 (d, J ¼ 8.5, HC(5)); 6.72 (d, J ¼ 8.5, HC(6)); 6.71 (s, HC(2)); 5.95 – 6.01 (m, HC(8)); 5.52 (s, OH); 5.07 – 5.12 (m, CH2(9)); 3.90 (s, MeO); 3.35 (d, J ¼ 6.5, CH2(7)). 13C-NMR (125 MHz): 146.4 (C(3)); 143.9 (C(4)); 137.8 (C(8)); 131.9 (C(1)); 121.2 (C(6)); 115.5 (C(5)); 114.3 (C(2)); 111.1 (C(9)); 55.9 (MeO); 39.9 (C(7)). EI-MS: 164 (100), 150 (36), 147 (5), 131 (27), 103 (29), 77 (28), 55 (13), 39 (7). The spectral data were in agreement with those reported in [18] [19]. Methyleugenol ( ¼ 1,2-Dimethoxy-4-( prop-2-en-1-yl)benzene; 2) . Colorless oil. 1H-NMR (500 MHz): 6.83 (d, J ¼ 8.0, HC(5)); 6.75 (d, J ¼ 8.0, HC(6)); 6.74 (s, HC(2)); 5.95 – 6.03 (m, HC(8)); 5.11 (dd, J ¼ 15.0, 5.0, 1 H of CH2(9)); 5.08 (d, J ¼ 5.0, 1 H of CH2(9)); 3.90 (s, MeOC(3)); 3.89 (s, MeOC(4)); 3.36 (d, J ¼ 6.5, CH2(7)). 13C-NMR (125 MHz): 148.9 (C(3)); 147.4 (C(4)); 137.7 (C(8)); 132.7 (C(1)); 120.4 (C(6)); 115.6 (C(5)); 111.9 (C(2)); 111.3 (C(9)); 56.0 (MeOC(3)); 55.8 (MeOC(4)); 39.8 (C(7)). EI-MS: 178 (100), 163 (33), 147 (32), 131 (11), 107 (28), 103 (39), 91 (50), 77 (26), 58 (9), 43 (27). The spectral data were in agreement with those reported in [20]. Methylisoeugenol ( ¼ 1,2-Dimethoxy-4-[(1E)-prop-1-en-1-yl]benzene; 3). Colorless oil. 1H-NMR (500 MHz): 6.92 (d, J ¼ 1.5, HC(2)); 6.88 (dd, J ¼ 8.5, 1.5, HC(6)); 6.82 (d, J ¼ 8.5, HC(5)); 6.36 (d, J ¼ 15.5, CH2(7)); 6.10 – 6.17 (m, HC(8)); 3.92 (s, MeOC(3)); 3.89 (s, MeOC(4)); 1.89 (d, J ¼ 7.0, Me(9)). 13 C-NMR (125 MHz): 149.0 (C(3)); 148.2 (C(4)); 131.2 (C(8)); 130.6 (C(1)); 123.8 (C(6)); 118.7 (C(5)); 111.3 (C(2)); 108.6 (C(7)); 55.9 (MeOC(3)); 55.8 (MeOC(4)); 18.4 (C(9)). EI-MS: 178 (100), 163 (48), 147 (13), 135 (10), 115 (12), 107 (35), 91 (28), 77 (12), 51 (3). The spectral data were in agreement with those reported in [21]. Elemicin ( ¼ 1,2,3-Trimethoxy-5-(prop-2-en-1-yl)benzene; 4). Colorless oil. 1H-NMR (500 MHz): 6.58 (s, HC(2), HC(6)), 5.94 – 6.02 (m, HC(8)); 5.12 (dd, J ¼ 15.6, 5.0, 1 H of CH2(9)); 5.05 (d, J ¼ 5.0, 1 H of CH2(9)); 3.89 (s, MeOC(3), MeOC(5)); 3.85 (s, MeOC(4)); 3.35(d, J ¼ 6.5, CH2(7)). 13 C-NMR (125 MHz): 153.2 (C(3), C(5)); 137.2 (C(1), C(4)); 135.8 (C(8)); 116.0 (C(9)); 105.4 (C(2), C(6)); 60.9 (MeOC(4)); 56.1 (MeOC(3), MeOC(5)); 40.5 (C(7)). EI-MS: 208 (100), 193 (63), 177 (13), 165 (10), 150 (11), 133 (19), 118 (13), 105 (14), 91 (17), 77 (19), 65 (8), 43 (15). The spectral data were in agreement with those reported in [20]. 1)
For signal assignments of NMR spectra, cf. the atom numbering in Fig. 1.
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Myristicin ( ¼ 4-Methoxy-6-( prop-2-en-1-yl)-1,3-benzodioxole; 5) . Colorless oil. 1H-NMR (500 MHz): 6.41 (s, HC(6)); 6.38 (s, HC(2)); 5.96 (s, OCH2O); 5.91 – 5.95 (m, HC(8)); 5.11 (dd, J ¼ 15.0, 4.0, 1 H of CH2(9)); 5.09 (d, J ¼ 4.0, 1 H of CH2(9)); 3.91 (s, MeO); 3.31 (d, J ¼ 6.5, CH2(7)). 13 C-NMR (125 MHz): 148.9 (C(5)); 143.5 (C(3)); 137.4 (C(8)); 134.6 (C(1)); 133.5 (C(4)); 115.8 (C(9)); 107.8 (C(6)); 102.7 (C(2)); 101.2 (OCH2O); 56.6 (MeO); 40.2 (C(7)). EI-MS: 192 (100), 165 (24), 147 (13), 119 (21), 91 (29), 65 (16), 39 (7). The spectral data were in agreement with those reported in [22] [23]. Safrole ( ¼ 5-(Prop-2-en-1-yl)-1,3-benzodioxole; 6). Colorless oil. 1H-NMR (500 MHz): 6.77 (d, J ¼ 7.5, HC(5)); 6.77 (s, HC(2)); 6.66 (d, J ¼ 7.5, HC(6)); 5.96 (s, OCH2O); 5.91 – 5.95 (m, HC(8)); 5.08 (dd, J ¼ 15.6, 5.0, 1 H of CH2(9)); 5.05 (d, J ¼ 5.0, 1 H of CH2(9)); 3.33 (d, J ¼ 6.5, CH2(7)). 13C-NMR (125 MHz): 137.6 (C(3)); 133.9 (C(4)); 121.3 (C(8)); 116.0 (C(1)); 115.7 (C(6)); 109.1 (C(5)); 108.2 (C(2), C(9)); 100.8 (OCH2O); 39.9 (C(7)). EI-MS: 162 (100), 131 (41), 104 (43), 77 (27), 51 (16), 39 (6). The spectral data were in agreement with those reported in [24]. Insects. L. serricorne was obtained from laboratory cultures maintained for the last two years in the dark in incubators at 28 – 308 and 70 – 80% rel. humidity. The insects were reared in glass containers (0.5 l) containing wheat flour at 12 – 13% moisture content mixed with yeast (wheatfeed/yeast 10 : 1 (w/w)). Adults used in all the experiments were ca. 5 – 9 d old. Contact Toxicity. The contact toxicity of the crude essential oil and isolated compounds against L. serricorne adults was evaluated as described by Liu and Ho [25]. Range-finding studies were run to determine the appropriate testing concentrations. A serial dilution of the essential oil/compounds (five concentrations) was prepared in hexane. Aliquots of 0.5 ml of the dilutions were applied topically to the dorsal thorax of the insects. Controls were determined using hexane. Ten insects were used for each concentration and control, and the experiment was replicated five times. Both treated and control insects were then transferred to glass vials and kept in incubators. Mortality was recorded after 24 h, and the LD50 values were calculated using Probit analysis [26]. Positive control (pyrethrins I and II; 37%) was purchased from Dr. Ehrenstorfer GmbH. Repellency Test. The repellent activity to L. serricorne adults was tested using the area preference method [27]. Petri dishes of 9 cm in diameter were used to confine cigarette beetles during the experiment. The crude essential oil and the isolated compounds were diluted in hexane to different concentrations (78.63, 15.73, 3.15, 0.63, and 0.13 nl cm 2 ), and hexane was used as control. Filter paper of 9 cm in diameter was cut in half, and 500 ml of each concentration was applied separately to half of the filter paper as uniformly as possible with a micropipette. The other half (control) was treated with 500 ml of hexane. Both the treated and control half were then air-dried to evaporate the solvent completely (30 s). A full disk was carefully remade by attaching the tested half to the negative control half with tape. Care was taken so that the attachment did not prevent free movement of insects from the one half to the other, but the distance between the filter paper halves remained sufficient to prevent seepage of test samples from one half to the other. Each reassembled filter paper after treatment with solid glue was placed in a Petri dish with the seam oriented in one of four randomly selected different directions to avoid any insecticidal stimuli affecting the distribution of insects. Twenty insects were released in the center of each filter paper disk, and a cover was placed over the Petri dish. Five replicates were used, and the experiment was repeated three times. Counts of the insects present on each strip were made after 2 and 4 h. The PR value of each volatile oil/compound was then calculated using Eqn. 1: PR [%] ¼ [(Nc Nt )/(Nc þ Nt )] · 100
(1)
where Nc is the number of insects present in the negative control half and Nt is the number of insects present in the treated half. Analysis of variance (ANOVA) and Tukeys test were conducted by using SPSS 13.0 for Windows 2007. Percentage was subjected to an arcsine square-root transformation before ANOVA and Tukeys tests. A commercial repellent, DEET, was purchased from the National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang 110021, P. R. China) and used as positive control. The averages were then categorized according to the scale in Table 5 [28].
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Table 5. The Scale Used to Categorize Repellency of the Essential Oil and Its Compounds Class
Percent repulsion [%]
0 I II III IV V
0.01 – 0.1 0.1 – 20 20.1 – 40 40.1 – 60 60.1 – 80 80.1 – 100
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