Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess15068 J. Oleo Sci. 64, (8) 861-868 (2015)
Insecticidal Constituents of Essential Oil Derived from Zanthoxylum armatum against Two Stored-Product Insects Cheng-Fang Wang1, 2, Wen-Juan Zhang1, Chun-Xue You1, Shan-Shan Guo1, Zhu-Feng Geng3, Li Fan2＊ , Shu-Shan Du1＊ , Zhi-Wei Deng3 and Yong-Yan Wang1 1
Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Beijing Normal University (NO.19 Xinjiekouwai Street, Beijing 100875, CHINA) 2 China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention ( Xicheng District, Beijing 100088, CHINA) 3 Analytical and Testing Center, Beijing Normal University (NO.19 Xinjiekouwai Street, Beijing 100875, CHINA)
Abstract: In the course of our search for natural bioactive chemicals and investigations on their insecticidal activities from some medicinal plants growing in China, the essential oil derived from the twigs of Zanthoxylum armatum (Rutaceae) was found to possess strong insecticidal activities against two storedproduct insects, Lasioderma serricorne and Tribolium castaneum. A total of 32 constituents of the essential oil were identified by GC and GC-MS analysis, and it revealed (E)-anethole (20.5%), 1,8-cineole (14.0%), 2-tridecanone (12.5%), limonene (9.0%) and piperitone (8.0%) as major components, followed by β phellandrene (6.3%), β -pinene (5.1%) and 4-terpineol (4.4%). From the essential oil, five compounds were isolated and identified as (E)-anethole, 1,8-cineole, 2-tridecanone, limonene and piperitone. The results of insecticidal bioassays showed that the essential oil of Z. armatum exhibited strong fumigant toxicity towards L. serricorne and T. castaneum with LC50 values of 13.83 and 4.28 mg/L air, respectively, and also possessed contact toxicity against two insect species with LD50 values of 18.74 and 32.16 mg/adult, respectively. Among the active compounds, piperitone performed the strongest fumigant toxicity against L. serricorne (LC50 = 1.21 mg/L air) and contact toxicity against T. castaneum (LD50 = 3.16 mg/adult). 1,8-Cineole, limonene and piperitone showed similar fumigant toxicity against T. castaneum with LC50 values of 5.47, 6.21 and 7.12 mg/ L air, respectively. Meanwhile, L. serricorne was the most sensitive to 2-tridecanone (LD50 = 5.74 mg/adult) in the progress of contact toxicity assay. Key words: Z. armatum, essential oil, L. serricorne, T. castaneum, fumigant toxicity, contact toxicity 1 INTRODUCTION The essential oils of natural plants are considered to be more effective than pesticides traditionally used1, 2）, and reduce the pollution to environment and toxicity to mammalian3）. In the course of our search for natural bioactive chemicals and investigations on their insecticidal activities from some medicinal plants growing in China4−6）, the essential oil derived from twigs of Zanthoxylum armatum was found to possess strong insecticidal acDC.（Rutaceae） tivities against Lasioderma serricorne Frbricius and Tribolium castaneum Herbst. Z. armatum, a spiny deciduous arbor, is distributed mainly in southeast and southwest China. It is also found throughout India, Nepal, Japan,
Korea and Vietnam7）. The fruits, seeds and barks of Z. armatum are extensively used as a folk medicine for the prevention of stomach ache, tooth ache, treating cold in the chest and abdomen, preventing snake bites and expelling roundworms8）. Almost all parts of the plant are aromatic and supposed to possess essential oil. An examination of literature shows that a lot of work has been done on essential oils of Z. armatum9−11）. However, most of the essential oils tested in the previous reports were extracted from this plant located in India, Pakistan and Vietnam12−14）, Abbreviations: Z. armatum: Zanthoxylum armatum; L. serricorne: Lasioderma serricorne; T. castaneum: Tribolium castaneum; RI: Retention Index; MS: mass spectrum.
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Correspondence to: Shu-Shan Du, Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Beijing Normal University, NO.19 Xinjiekouwei Street, Beijing 100875, CHINA; Li Fan, China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Xicheng District, Beijing 100088, CHINA E-mail: [email protected]; [email protected] Accepted April 24, 2015 (received for review March 24, 2015)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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work on the essential oil of Z. armatum collected from China is very few15）. In this paper, we report the chemical constituents of essential oil derived from the fresh twigs of Z. armatum in China. It has been reported that the essential oil of Z. armatum is used to control female stable fly, Stomoxys calcitrans and found to possess larvicidal activities against three species of mosquito vectors, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus 16, 17）. The Z. armatum oil also exhibits leech repellent activity and the seed extract shows lousicidal potential against tropical hen louse, Lipeurus lawrensis tropicalis18, 19）. To the best of our knowledge, studies have not been conducted so far to evaluate the insecticidal activities against the stored-product insects. Therefore, we determine the fumigant and contact toxicity of the crude essential oil and its five main components against L. serricorne and T. castaneum adults in this work.
2 EXPERIMENTAL PROCEDURES 2.1 Material 2.1.1 Plants Fresh twigs（5.0 kg）of Z. armatum were collected at October 2011 from Xishuangbanna（21.99°N latitude and 100.83°E longitude）, Yunnan Province, China. A voucher specimen（BNU-CMH-Dushuahan-2011-10-29-003） was deposited at college of resources science & technology, Beijing Normal University. The leaves and twigs were airdried for one week and ground to a powder. 2.1.2 Insects L. serricorne and T. castaneum were obtained from laboratory cultures maintained for the last 3 years in the dark in incubators at 28-30℃ and 70–80％ relative humidiconty. The insects were reared in glass containers （0.5 L） taining wheat flour mixed with yeast（wheat/yeast, 10:1, w/ w）. Adults used in all the experiments were about one week old. 2.2 Isolation of the essential oil and purification of five constituents The powder of Z. armatum twigs was subjected to hydrodistillation using a Clevenger-type apparatus for six hours20）. Anhydrous sodium sulphate was used to remove water after extraction. The essential oil was stored in airtight containers in a refrigerator at 4℃ for subsequent experiments. was chromatographed on The crude essential oil （15 mL） a silica gel（Qingdao Marine Chemical Plant, Shandong province, China）column（45 mm i.d., 500 mm length）by gradient elution with n-hexane first, then with n-hexaneethyl acetate, and last with ethyl acetate. Fractions（200 mL）were collected and concentrated at 35℃, and similar
fractions according to thin layer chromatography（TLC） profiles were combined to obtain 20 fractions. Of these, fractions 3–5, 9–12 were further purified by preparative silica gel column chromatography（PTLC）until obtain the pure compounds for determining structure as limonene（1, 0.85 g）, 1, 8-cineole（2, 1.51 g）,（E）-anethole（3, 2.20 g）, 2-tridecanone（4, 1.02 g）, and piperitone（5, 0.94 g）. The isolated compounds were elucidated based on nuclear magnetic resonance and mass spectrometry. 1 H and 13 C-NMR spectra were recorded on Bruker Avance DRX 500 instruments using CDCl3 as solvent with TMS as internal standard. 2.3 GC-MS and GC-FID analysis The obtained essential oil was packed in amber vial, lightless. A sample of the oil was diluted in n-hexane and subjected to analysis by gas chromatography coupled to flame ionization detector （GC-FID） and gas chromatography coupled to spectrometry（GC-MS）in Beijing Normal University, Beijing, China. GC-MS analysis was running on a （No. MS 200275） Thermo Finnigan Trace DSQ instrument equipped with a flame ionization detector （FID） and a capillary column of HP-5MS（30 m×0.25 mm×0.25 μm）capillary column. The column temperature was programmed at 50℃ for 3 min then increased at 10℃/min until the final temperature of 290℃ was reached, where it was held for 20 min. The injector temperature was maintained at 250℃ and the samples（1 μL, dilute to 1％ with n-hexane）were injected, with a split ratio of 1:50. The carrier gas was helium at flow rate of 1.0 ml/min. Spectra were scanned from 50 to 550 m/z. Most constituents were identified by comparison of their retention indices with those reported in the literatures. The retention indices were determined in relation to a homologous series of n-alkanes（C5 – C36） under the same operating conditions. Further identification was made by comparison of their mass spectra with those stored in NIST 05 and Wiley 275 libraries or with mass spectra from literature21, 22）. Relative percentages of the individual components of the essential oil were obtained by averaging the GC peak area％ reports. 2.4 Isolated Compounds （E）-Anethole（1, Fig. 1）. Slightly yellow oil, C 10H12O. 1 H-NMR（500 MHz, CDCl3）δppm: 7.29（2H, d, J＝8.5 Hz, H-3, 5） , 6.87 （2H, d, J＝8.5 Hz, H-2, 6） , 6.38 （1H, d, J＝15.5 , 1.89（3H, Hz, H-7） , 6.12（1H, m, H-8）, 3.83（3H, s, 1-OCH3） d, J＝6.5 Hz, 8-CH3）. 13C-NMR（125 MHz, CDCl3）δppm: 158.59（C-1）, 138.83（C-4）, 130.35（C-8）, 126.89（C-3, 5）, 123.50（C-7）, 113.91（C-2, C-6）, 55.28（1-OCH3）, 18.43（8CH3）. The 1H and 13C-NMR data were in agreement with the reported data23）. 1,8-Cineole（2, Fig. 1）. Colorless oil, C10H18O. 1H-NMR δppm: 2.03（2H, t, H-2）, 1.68（2H, t, H-6）, （500 MHz, CDCl3） 1.52（4H, m, H-3, 5）, 1.42（1H, m, H-4）, 1.25（6H, s, H-9,
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Insecticidal Constituents of Essential Oil from Zanthoxylum armatum Against Two Stored-Product Insects
Fig. 1 Chemical compounds isolated from the essential oil of Z. armatum twigs. 10）, 1.07（3H, s, H-7）. 13C-NMR（125 MHz, CDCl3）δppm: 100.00（C-8）, 73.61（C-8）, 69.77（C-1）, 32.94（C-4）, 31.51 （C-3, 5）, 28.89（C-2, 6）, 27.58（C-7）, 22.83（C-9, 10）. The 1 H and 13C-NMR data were in agreement with the reported data24）. 2-Tridecanone（ 3, Fig. 1）. Colorless oil, C 13 H 26 O. 1 H-NMR（500 MHz, CDCl3）δppm: 2.43（2H, m, H-3）, 2.15 , 0.90 （3H, s, H-1） , 1.58 （2H, m, H-4） , 1.28 （16H, m, -CH2-） （125 MHz, CDCl3） δppm: 209.43（C （3H, m, H-13）. 13C-NMR ＝O）, 43.84（C-3）, 31.91（C-1）, 29.85（C-4）, 29.61–22.69 （-CH2-）, 14.12（C-13）. The 1H and 13C-NMR data were in agreement with the reported data25）. Limonene （4, Fig. 1） . Slightly yellow oil, C10H16. 1H-NMR （500 MHz, CDCl 3）δppm: 5.43（1H, m, H-6）, 4.74（2H, s, H-9） , 2.05–2.16 （3H, m, H-2, H-4）, 1.93–2.00 （2H, m, H-5） , 1.81–1.85（1H, m, H-3a）, 1.76（3H, s, H-10）, 1.68（3H, s, H-7）, 1.46-1.54（1H, m, H-3b）. 13C-NMR（125 MHz, CDCl3） δppm: 150.25（ C-8）, 133.73（ C-1）, 120.66（ C-6）, 108.37 （C-9）, 41.10（C-4）, 30.82（C-2）, 30.61（C-5）, 27.94（C-3）, 23.46（C-7）, 20.80（C-10）. The 1H and 13C-NMR data were in agreement with the reported data26）. Piperitone（5, Fig. 1）. Colorless oil, C10H16O. 1H-NMR （500 MHz, CDCl 3）δppm: 5.84（1H, s, H-2）, 2.37（1H, m, H-4）, 2.31（ 2H, m, H-6）, 2.01（ 2H, m, H-5）, 1.94（ 3H, s, 1-CH3）, 1.82（1H, m, H-7）, 0.95（3H, d,J＝7.0 Hz, 7-CH3）, . 13C-NMR （125 MHz, CDCl3） 0.85 （3H, d, J＝7.0 Hz, 7-CH3） δppm: 201.30（ C＝O）, 161.08（C-1）, 126.83（C-2）, 51.59 , （C-4）, 30.37（C-6）, 25.81（C-5）, 24.08（C-7）, 22.96（1-CH3） 20.68（7-CH3）, 18.55（7-CH3）. The 1H and 13C-NMR data were in agreement with the reported data27）. 2.5 Insecticidial activity 2.5.1 Fumigant Toxicity The fumigant activity of the essential oil or pure compounds against the two species of insects was measured as described by Liu and Ho28）. Range-finding works were run to assess the appropriate testing concentrations. A serial dilution of the essential oil/compounds（five concentrations）was prepared in n-hexane. A Whatman filter paper （diameter 2.0 cm）was impregnated with 10 μ L dilution and then placed on the underside of the screw cap of a glass vial （diameter 2.5 cm, height 5.5 cm）. The solvent was allowed to evaporate for 20 s before the cap was placed
tightly on the glass vial, each of which contained 10 insects inside to form a sealed chamber. Fluon（ICI America Inc.） was used inside the glass vial to prevent insects from contacting the treated filter paper. n-Hexane was used as a control. Five replicates were carried out for all treatments and controls, and they were incubated under the same conditions for 24 h. The insects were considered dead if appendages did not move when probed with a tiny brush, and the corrected mortality（ ％）＝100× （A-C） （1-C） / , where A＝the mortality of treated with essential oils, C＝ the mortality of control. 2.5.2 Contact Toxicity The contact toxicity of the essential oil or pure compounds against the two species of insects was tested with reference to the method of Liu and Ho28）. Range-finding surveys were run to determine the appropriate testing concentrations. A serial dilution of the essential oil/compounds （five concentrations）was prepared in n-hexane. Aliquots of 0.5 μL of the dilutions were applied topically to the dorsal thorax of the insects. Controls were determined using n-hexane. Ten insects were used for each concentration and control, and the experiment was replicated five times. Mortality was recorded after 24 h of exposure and the corrected mortality（％）＝100× （A-C） （1-C） / , where A ＝the mortality of treated with essential oils, C＝the mortality of control. 2.5.3 Statistical Analysis The corrected mortality represented the average mean of five replicates. The LC50 or LD50 value was analyzed by Probit analysis29）, which was conducted using SPSS 19.0 for Windows 2007. During the Probit analysis, differences at values of p＜0.05 were considered as significant.
3 RESULTS and DISCUSSION 3.1 Chemical compounds of the essential oil The crude essential oil from Z. armatum twigs was pale yellow with a yield of 0.53 ％（v/w）and density of 0.84 g/ mL. Examination of essential oil of Z. armatum by GC-MS revealed the presence of thirty-two components, accounting for 98.2％ of the total oil（Table 1）. The main constituents in the essential oil of Z. armatum were（E）-anethole 863
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C. F. Wang, W. J. Zhang and C. X. You et al.
Table 1 C hemical constituents of the essential oil derived from Z. armatum twigs. Peak no.
RIa
Compounds
％RAb
Identification Methodsc
1
927
α-Thujene
0.1
MS, RI
2
939
α-Pinene
0.7
MS, RI
3
967
β -Thujene
3.8
MS, RI
4
979
β -Pinene
5.1
MS, RI, Co
5
1006
α-Phellandrene
0.2
MS, RI
6
1014
Terpinolene
0.5
MS, RI
7
1019
o-Cymene
0.8
MS, RI
8
1025
Limonene
9.0
MS, RI, Co
9
1029
β -Phellandrene
6.3
MS, RI
10
1032
1,8-Cineole
14.0
MS, RI, Co
11
1037
cis-β -Ocimene
0.2
MS, RI
12
1060
γ-Terpinene
1.0
MS, RI
13
1089
4-Carene
1.5
MS, RI
14
1096
Linalool
0.5
MS, RI, Co
15
1107
Hotrienol
0.2
MS, RI
16
1156
Citronellal
0.2
MS, RI
17
1177
4-Terpineol
4.4
MS, RI, Co
18
1190
Cryptone
0.1
MS, RI
19
1196
α-Terpineol
0.5
MS, RI
20
1251
Piperitone
8.0
MS, RI, Co
21
1332
（E）-Anethole
20.5
MS, RI, Co
22
1382
2-Undecanol
0.4
MS, RI
23
1423
Caryophyllene
1.5
MS, RI
24
1454
α-Caryophyllene
1.0
MS, RI
25
1496
2-Tridecanone
12.5
MS, RI
26
1503
2-Tetradecanol
1.2
MS, RI
27
1519
α-Muurolene
0.3
MS, RI
28
1523
δ-Cadinene
0.8
MS, RI
29
1578
Spathulenol
0.4
MS, RI
30
1583
Caryophyllene oxide
0.5
MS, RI, Co
31
1636
τ-Muurolol
1.0
MS, RI
32
1663
α-Cadinol
1.0
MS, RI
Total identified
98.2
a
Retention index (RI) relative to the homologous series of n-hydrocarbons on the HP-5 MS capillary column. b Relative area (peak area relative to the total peak area). c MS＝mass spectrum, Co＝co-injection with standard compound. （20.5％）, 1,8-cineole（ 14.0％）, 2-tridecanone（ 12.5％）, limonene（ 9.0％）and piperitone（ 8.0％）, followed by β -phellandrene（6.3％）, β -pinene（5.1％）and 4-terpineol （4.4％）. The chemical structures of isolated compounds
from Z. armatum oil are shown in Fig. 1. In the present paper, the chemical composition of the essential oil is comparable to that of the previous reports with some variation in the constituents. For example, Bisht
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J. Oleo Sci. 64, (8) 861-868 (2015)
Insecticidal Constituents of Essential Oil from Zanthoxylum armatum Against Two Stored-Product Insects
reported that 2-undecanone（ 44.6％）, linalool（14.5％）, 2-tridecanene（8.0％） and 1,8-cineole（6.9％） were the major components in the essential oil of Z. armatum leaves from India10）. While Muhammad found linalool was the most dominant constituent30）. The leaf oil of Z. armatum from Vietnam was rich in 1,8-cineole （41.0％） , sabinene （8.4％） , terpinen-4-ol（5.2％）, and linalool（4.5％）14）. Moreover, linalool was reported as the highest level compound of essential oil from Z. armatum pericarps in China, accounting for 71.7％∼75.0％15）. These reports indicate that linalool is a high content composition in Z. armatum. However, in our investigation, linalool was a minor constituent, only presented in 0.5％. The observed differences in chemical composition and content of the essential oil from Z. armatum could be due to harvest time and local, environmental conditions, or may result from herbal parts used and the extraction and analytical methods used. These variations may lead to different biological activities. 3.2 Insecticidial activity The fumigant toxicities of essential oil from Z. armatum twigs and five isolated compounds against L. serricorne and T. castaneum were presented in Table 2. The results showed that the red flour beetle （T. castaneum） treated by the crude essential oil was more sensitive than the cigarette beetle（L. serricorne）with LC50 values of 4.28 and 12.54 mg/L air, respectively. Among the five compounds, piperitone exhibited the strongest fumigant toxicity to the cigarette beetle（LC50＝1.21 mg/L air）, while 1,8-cineole, limonene and piperitone showed similar fumigant toxicity against T. castaneum with LC50 values of 5.47, 6.21 and
7.12 mg/L air, respectively. When compared with the positive control, phosphine （LC50＝9.23×10−3 mg/L air）, MeBr （LC50＝1.75 mg/L air）, piperitone represented 131 times less toxicity to L. serricorne and 1,8-cineole exhibited 3.5 times less toxicity to T. castaneum, respectively（Table 2）. However, the two commercial fumigants are highly toxic to humans and other non-target organisms. The fumigant toxicity of Z. armatum oil and isolated constituents was comparable with some reports recently, for instance, essential oils of Aster ageratoides（ LC 50＝12.14 mg/L）, Citrus wilsonii and its main component γ-Terpinene（LC50 ＝8.18 and 4.09 mg/L, respectively） , and Cayratia japonica （LC50＝15.67 mg/L）against T. castaneum2, 5, 31）; Agastache foeniculum（LC50＝21.57 mg/L）and Litsea cubeba（LC50＝ 22.97 mg/L）against L. serricorne32, 33）. Thus, the essential oil of Z. armatum and its active compounds have potential to be developed as possible natural fumigants for control of L. serricorne and T. castaneum. As the results were shown in Table 3, the essential oil of Z. armatum exhibited contact toxicity against the cigarette beetle and red flour beetle with LD50 values of 18.74 and 32.16 μg/adult, respectively. The most toxic compound to L. serricorne was 2-tridecanone（LD50＝5.74 μg/adult）. While piperitone presented strongest contact toxicity against T. castaneum（LD50＝3.16 μg/adult）, followed by 2-tridecanone with an LD50 value of 5.35 μg/adult. Both the essential oil and its active compounds showed less contact toxicity against the two species of insects comparing with positive control, pyrethrins. When compared with previous reports, the essential oil of Z. armatum showed similar contact toxicity against L. serricorne, e.g. essential oils of
Table 2 Fumigant toxicity of essential oil from Z. armatum twigs and its main components against L. serricorne and T. castaneum adults. Insects
L. serricorne
Treatment
Concentrations (％)
LC50 (mg/L air)
95％ Fiducial limits
Z. armatum
3.95-20.00
13.83
10.73-16.16
18.17
(E)-Anethole
0.99- 5.00
6.31
5.33- 7.19
16.56
1,8- Cineole
0.99- 5.00
5.18
4.63- 5.70
16.79
2-Tridecanone
-
-
-
-
Limonene
1.97-10.00
14.07
12.41-15.76
22.08
Piperitone
0.39- 2.00
1.21
0.75- 1.55
6.69
-
-
Phosphine*
T. castaneum
Chi square (χ2)
-
9.23×10
−3
Z. armatum
1.97-10.00
4.28
1.86- 5.92
26.45
(E)-Anethole
1.97-10.00
9.20
8.03-10.25
17.02
1,8- Cineole
0.99-5.00
5.47
4.73- 6.17
25.30
2-Tridecanone
-
-
-
-
4）
Limonene
0.99- 5.00
6.21
5.38- 7.05
13.34
Piperitone
1.97-10.00
7.12
5.44- 8.38
6.96
MeBr**
-
1.75
-
-
28)
* Data from You et al. ; ** MeBr: Methyl bromide, values from Liu and Ho . 865
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C. F. Wang, W. J. Zhang and C. X. You et al.
Table 3 Contact toxicity of the essential oil from Z. armatum twigs and its main components against L. serricorne and T. castaneum adults. Insects
L. serricorne
T. castaneum
Treatment
Concentrations (％)
LD50 (μg/adult)
95％ Fiducial limits
Chi square (χ2)
Z. armatum
1.97-10.00
18.74
14.99-21.69
13.80
(E)-Anethole
0.99- 5.00
10.32
8.63-11.72
14.03
1,8- Cineole
1.97-10.00
15.58
12.88-18.02
15.18
2-Tridecanone
0.88- 4.44
5.74
5.04- 6.45
9.70
Limonene
0.99- 5.00
13.66
11.63-16.18
11.04
Piperitone
0.63-10.00
22.05
21.07-23.01
7.99
Pyrethrins*
-
0.24
-
-
Z. armatum
5.93-30.00
32.16
27.96-35.88
16.33
(E)-Anethole
1.97-10.00
15.40
13.67-17.11
18.63
1,8- Cineole
1.97-10.00
18.83
17.13-20.69
16.56
2-Tridecanone
0.96- 2.00
5.35
4.94- 5.74
6.46
Limonene
1.97-10.00
14.97
12.88-17.04
20.01
Piperitone
0.59- 2.96
3.16
2.56- 3.65
6.41
Pyrethrins*
-
0.26
-
-
6）
*Data from Wang et al. .
Litsea cubeba（LD50＝27.33 μg/adult）and Cinnamomum camphora（LD 50＝21.25 μg/adult）33, 34）, weaker contact toxicity against T. castaneum, e.g. essential oils of Aster ageratoides（LD50＝8.09 μg/adult）and Amomum tsaoko （LD50＝16.52 μg/adult）2, 6）. In the present work, piperitone showed much different insecticidal activities between the two tested insects. In contrast to contact toxicity test, L. serricorne adults treated with piperitone were more susceptible than T. castaneum adults in the fumigant toxicity test（Table 2 and 3）. This indicates that fumigant toxicity may be a major route of insecticidal activity of piperitone against L. serricorne, while the mode of action in T. castaneum is via contact toxicity. It also has been reported that piperitone possessed insecticidal activity against the third instar larvae of Spodoptera littoralis and the adults of Callosobruchus maculatus35, 36）. Moreover, 2-tridecanone presented strong contact toxicity against L. serricorne and T. castaneum, whereas no fumigant toxicity of 2-tridecanone was measured to the test insects. It is also found that 2-tridecanone has strong contact toxicity to Solenopsis invicta, larvae of Keiferia lycopersicella and Spodoptera exigua37, 38）. In this context, the combination of 2-tridecanone as a contact toxin and piperitone as a fumigant at a certain ratio would develop significantly stronger insecticidal activity than individual compounds.
4 CONCLUSION This paper is the first one to demonstrate botanical effi-
cacy of Z. armatum essential oil towards storage pests. The results suggested that Z. armatum essential oil and its major components could be a promising alternative in the control of stored-product insects. Further investigations should be focused on the safety of essential oil/compounds to humans, the development of formulations and improvement of their efficacy and stability.
ACKNOWLEDGMENT This project was supported by Beijing Municipal Natural Science Foundation（No. 7142093）, National Natural Science Foundation of China（No. 81374069）and the Fundamental Research Funds for the Central Universities.
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Zia-Lu-Haq, M.; Qayum, M.; Khan, H. Behavioral properties of essential oils of Zanthoxylum armatum DC leaves: augmented by chemical profile using GC/GC（2013）. MS. J. Chem. Soc. Pakistan 35, 1595-1600 31）Liu, Z. L.; Yang, K.; Huang, F.; Liu, Q. Z.; Zhou, L.; Du, S. S. Chemical composition and toxicity of the essential oil of Cayratia japonica against two grain storage insects. J. Essent. Oil Res. 24, 237-240 （2012） . 32）Ebadollahi, A.; Safaralizadeh, M. H.; Pourmirza, A. A.; Gheibi, S. A. Toxicity of essential oil of Agastache foeniculum（Pursh）Kuntze to Oryzaephilus surinamensis L. and Lasioderma serricorne F. J. Plant （2010） . Prot. Res. 50, 215-219 33）Yang, K.; Wang, C. F.; You, C. X.; Geng, Z. F.; Sun, R. Q.; Guo, S. S.; Du, S. S.; Liu, Z. L.; Deng, Z. W. Bioactivity of essential oil of Litsea cubeba from China and its main compounds against two stored product insects. J. Asia-Pac. Entomol. 17, 459-466 （2014） . 34）Chen, H. P.; Yang, K.; You, C. X.; Lei, N.; Sun, R. Q.; Geng, Z. F.; Ma, P.; Cai, Q.; Du, S. S.; Deng, Z. W. Chemical constituents and insecticidal activities of the
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Insecticidal Constituents of Essential Oil Derived from Zanthoxylum armatum against Two Stored-Product Insects Cheng-Fang Wang1, 2, Wen-Juan Zhang1, Chun-Xue You1, Shan-Shan Guo1, Zhu-Feng Geng3, Li Fan2＊ , Shu-Shan Du1＊ , Zhi-Wei Deng3 and Yong-Yan Wang1 1
Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Beijing Normal University (NO.19 Xinjiekouwai Street, Beijing 100875, CHINA) 2 China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention ( Xicheng District, Beijing 100088, CHINA) 3 Analytical and Testing Center, Beijing Normal University (NO.19 Xinjiekouwai Street, Beijing 100875, CHINA)
Abstract: In the course of our search for natural bioactive chemicals and investigations on their insecticidal activities from some medicinal plants growing in China, the essential oil derived from the twigs of Zanthoxylum armatum (Rutaceae) was found to possess strong insecticidal activities against two storedproduct insects, Lasioderma serricorne and Tribolium castaneum. A total of 32 constituents of the essential oil were identified by GC and GC-MS analysis, and it revealed (E)-anethole (20.5%), 1,8-cineole (14.0%), 2-tridecanone (12.5%), limonene (9.0%) and piperitone (8.0%) as major components, followed by β phellandrene (6.3%), β -pinene (5.1%) and 4-terpineol (4.4%). From the essential oil, five compounds were isolated and identified as (E)-anethole, 1,8-cineole, 2-tridecanone, limonene and piperitone. The results of insecticidal bioassays showed that the essential oil of Z. armatum exhibited strong fumigant toxicity towards L. serricorne and T. castaneum with LC50 values of 13.83 and 4.28 mg/L air, respectively, and also possessed contact toxicity against two insect species with LD50 values of 18.74 and 32.16 mg/adult, respectively. Among the active compounds, piperitone performed the strongest fumigant toxicity against L. serricorne (LC50 = 1.21 mg/L air) and contact toxicity against T. castaneum (LD50 = 3.16 mg/adult). 1,8-Cineole, limonene and piperitone showed similar fumigant toxicity against T. castaneum with LC50 values of 5.47, 6.21 and 7.12 mg/ L air, respectively. Meanwhile, L. serricorne was the most sensitive to 2-tridecanone (LD50 = 5.74 mg/adult) in the progress of contact toxicity assay. Key words: Z. armatum, essential oil, L. serricorne, T. castaneum, fumigant toxicity, contact toxicity 1 INTRODUCTION The essential oils of natural plants are considered to be more effective than pesticides traditionally used1, 2）, and reduce the pollution to environment and toxicity to mammalian3）. In the course of our search for natural bioactive chemicals and investigations on their insecticidal activities from some medicinal plants growing in China4−6）, the essential oil derived from twigs of Zanthoxylum armatum was found to possess strong insecticidal acDC.（Rutaceae） tivities against Lasioderma serricorne Frbricius and Tribolium castaneum Herbst. Z. armatum, a spiny deciduous arbor, is distributed mainly in southeast and southwest China. It is also found throughout India, Nepal, Japan,
Korea and Vietnam7）. The fruits, seeds and barks of Z. armatum are extensively used as a folk medicine for the prevention of stomach ache, tooth ache, treating cold in the chest and abdomen, preventing snake bites and expelling roundworms8）. Almost all parts of the plant are aromatic and supposed to possess essential oil. An examination of literature shows that a lot of work has been done on essential oils of Z. armatum9−11）. However, most of the essential oils tested in the previous reports were extracted from this plant located in India, Pakistan and Vietnam12−14）, Abbreviations: Z. armatum: Zanthoxylum armatum; L. serricorne: Lasioderma serricorne; T. castaneum: Tribolium castaneum; RI: Retention Index; MS: mass spectrum.
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Correspondence to: Shu-Shan Du, Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Beijing Normal University, NO.19 Xinjiekouwei Street, Beijing 100875, CHINA; Li Fan, China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Xicheng District, Beijing 100088, CHINA E-mail: [email protected]; [email protected] Accepted April 24, 2015 (received for review March 24, 2015)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs
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work on the essential oil of Z. armatum collected from China is very few15）. In this paper, we report the chemical constituents of essential oil derived from the fresh twigs of Z. armatum in China. It has been reported that the essential oil of Z. armatum is used to control female stable fly, Stomoxys calcitrans and found to possess larvicidal activities against three species of mosquito vectors, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus 16, 17）. The Z. armatum oil also exhibits leech repellent activity and the seed extract shows lousicidal potential against tropical hen louse, Lipeurus lawrensis tropicalis18, 19）. To the best of our knowledge, studies have not been conducted so far to evaluate the insecticidal activities against the stored-product insects. Therefore, we determine the fumigant and contact toxicity of the crude essential oil and its five main components against L. serricorne and T. castaneum adults in this work.
2 EXPERIMENTAL PROCEDURES 2.1 Material 2.1.1 Plants Fresh twigs（5.0 kg）of Z. armatum were collected at October 2011 from Xishuangbanna（21.99°N latitude and 100.83°E longitude）, Yunnan Province, China. A voucher specimen（BNU-CMH-Dushuahan-2011-10-29-003） was deposited at college of resources science & technology, Beijing Normal University. The leaves and twigs were airdried for one week and ground to a powder. 2.1.2 Insects L. serricorne and T. castaneum were obtained from laboratory cultures maintained for the last 3 years in the dark in incubators at 28-30℃ and 70–80％ relative humidiconty. The insects were reared in glass containers （0.5 L） taining wheat flour mixed with yeast（wheat/yeast, 10:1, w/ w）. Adults used in all the experiments were about one week old. 2.2 Isolation of the essential oil and purification of five constituents The powder of Z. armatum twigs was subjected to hydrodistillation using a Clevenger-type apparatus for six hours20）. Anhydrous sodium sulphate was used to remove water after extraction. The essential oil was stored in airtight containers in a refrigerator at 4℃ for subsequent experiments. was chromatographed on The crude essential oil （15 mL） a silica gel（Qingdao Marine Chemical Plant, Shandong province, China）column（45 mm i.d., 500 mm length）by gradient elution with n-hexane first, then with n-hexaneethyl acetate, and last with ethyl acetate. Fractions（200 mL）were collected and concentrated at 35℃, and similar
fractions according to thin layer chromatography（TLC） profiles were combined to obtain 20 fractions. Of these, fractions 3–5, 9–12 were further purified by preparative silica gel column chromatography（PTLC）until obtain the pure compounds for determining structure as limonene（1, 0.85 g）, 1, 8-cineole（2, 1.51 g）,（E）-anethole（3, 2.20 g）, 2-tridecanone（4, 1.02 g）, and piperitone（5, 0.94 g）. The isolated compounds were elucidated based on nuclear magnetic resonance and mass spectrometry. 1 H and 13 C-NMR spectra were recorded on Bruker Avance DRX 500 instruments using CDCl3 as solvent with TMS as internal standard. 2.3 GC-MS and GC-FID analysis The obtained essential oil was packed in amber vial, lightless. A sample of the oil was diluted in n-hexane and subjected to analysis by gas chromatography coupled to flame ionization detector （GC-FID） and gas chromatography coupled to spectrometry（GC-MS）in Beijing Normal University, Beijing, China. GC-MS analysis was running on a （No. MS 200275） Thermo Finnigan Trace DSQ instrument equipped with a flame ionization detector （FID） and a capillary column of HP-5MS（30 m×0.25 mm×0.25 μm）capillary column. The column temperature was programmed at 50℃ for 3 min then increased at 10℃/min until the final temperature of 290℃ was reached, where it was held for 20 min. The injector temperature was maintained at 250℃ and the samples（1 μL, dilute to 1％ with n-hexane）were injected, with a split ratio of 1:50. The carrier gas was helium at flow rate of 1.0 ml/min. Spectra were scanned from 50 to 550 m/z. Most constituents were identified by comparison of their retention indices with those reported in the literatures. The retention indices were determined in relation to a homologous series of n-alkanes（C5 – C36） under the same operating conditions. Further identification was made by comparison of their mass spectra with those stored in NIST 05 and Wiley 275 libraries or with mass spectra from literature21, 22）. Relative percentages of the individual components of the essential oil were obtained by averaging the GC peak area％ reports. 2.4 Isolated Compounds （E）-Anethole（1, Fig. 1）. Slightly yellow oil, C 10H12O. 1 H-NMR（500 MHz, CDCl3）δppm: 7.29（2H, d, J＝8.5 Hz, H-3, 5） , 6.87 （2H, d, J＝8.5 Hz, H-2, 6） , 6.38 （1H, d, J＝15.5 , 1.89（3H, Hz, H-7） , 6.12（1H, m, H-8）, 3.83（3H, s, 1-OCH3） d, J＝6.5 Hz, 8-CH3）. 13C-NMR（125 MHz, CDCl3）δppm: 158.59（C-1）, 138.83（C-4）, 130.35（C-8）, 126.89（C-3, 5）, 123.50（C-7）, 113.91（C-2, C-6）, 55.28（1-OCH3）, 18.43（8CH3）. The 1H and 13C-NMR data were in agreement with the reported data23）. 1,8-Cineole（2, Fig. 1）. Colorless oil, C10H18O. 1H-NMR δppm: 2.03（2H, t, H-2）, 1.68（2H, t, H-6）, （500 MHz, CDCl3） 1.52（4H, m, H-3, 5）, 1.42（1H, m, H-4）, 1.25（6H, s, H-9,
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Insecticidal Constituents of Essential Oil from Zanthoxylum armatum Against Two Stored-Product Insects
Fig. 1 Chemical compounds isolated from the essential oil of Z. armatum twigs. 10）, 1.07（3H, s, H-7）. 13C-NMR（125 MHz, CDCl3）δppm: 100.00（C-8）, 73.61（C-8）, 69.77（C-1）, 32.94（C-4）, 31.51 （C-3, 5）, 28.89（C-2, 6）, 27.58（C-7）, 22.83（C-9, 10）. The 1 H and 13C-NMR data were in agreement with the reported data24）. 2-Tridecanone（ 3, Fig. 1）. Colorless oil, C 13 H 26 O. 1 H-NMR（500 MHz, CDCl3）δppm: 2.43（2H, m, H-3）, 2.15 , 0.90 （3H, s, H-1） , 1.58 （2H, m, H-4） , 1.28 （16H, m, -CH2-） （125 MHz, CDCl3） δppm: 209.43（C （3H, m, H-13）. 13C-NMR ＝O）, 43.84（C-3）, 31.91（C-1）, 29.85（C-4）, 29.61–22.69 （-CH2-）, 14.12（C-13）. The 1H and 13C-NMR data were in agreement with the reported data25）. Limonene （4, Fig. 1） . Slightly yellow oil, C10H16. 1H-NMR （500 MHz, CDCl 3）δppm: 5.43（1H, m, H-6）, 4.74（2H, s, H-9） , 2.05–2.16 （3H, m, H-2, H-4）, 1.93–2.00 （2H, m, H-5） , 1.81–1.85（1H, m, H-3a）, 1.76（3H, s, H-10）, 1.68（3H, s, H-7）, 1.46-1.54（1H, m, H-3b）. 13C-NMR（125 MHz, CDCl3） δppm: 150.25（ C-8）, 133.73（ C-1）, 120.66（ C-6）, 108.37 （C-9）, 41.10（C-4）, 30.82（C-2）, 30.61（C-5）, 27.94（C-3）, 23.46（C-7）, 20.80（C-10）. The 1H and 13C-NMR data were in agreement with the reported data26）. Piperitone（5, Fig. 1）. Colorless oil, C10H16O. 1H-NMR （500 MHz, CDCl 3）δppm: 5.84（1H, s, H-2）, 2.37（1H, m, H-4）, 2.31（ 2H, m, H-6）, 2.01（ 2H, m, H-5）, 1.94（ 3H, s, 1-CH3）, 1.82（1H, m, H-7）, 0.95（3H, d,J＝7.0 Hz, 7-CH3）, . 13C-NMR （125 MHz, CDCl3） 0.85 （3H, d, J＝7.0 Hz, 7-CH3） δppm: 201.30（ C＝O）, 161.08（C-1）, 126.83（C-2）, 51.59 , （C-4）, 30.37（C-6）, 25.81（C-5）, 24.08（C-7）, 22.96（1-CH3） 20.68（7-CH3）, 18.55（7-CH3）. The 1H and 13C-NMR data were in agreement with the reported data27）. 2.5 Insecticidial activity 2.5.1 Fumigant Toxicity The fumigant activity of the essential oil or pure compounds against the two species of insects was measured as described by Liu and Ho28）. Range-finding works were run to assess the appropriate testing concentrations. A serial dilution of the essential oil/compounds（five concentrations）was prepared in n-hexane. A Whatman filter paper （diameter 2.0 cm）was impregnated with 10 μ L dilution and then placed on the underside of the screw cap of a glass vial （diameter 2.5 cm, height 5.5 cm）. The solvent was allowed to evaporate for 20 s before the cap was placed
tightly on the glass vial, each of which contained 10 insects inside to form a sealed chamber. Fluon（ICI America Inc.） was used inside the glass vial to prevent insects from contacting the treated filter paper. n-Hexane was used as a control. Five replicates were carried out for all treatments and controls, and they were incubated under the same conditions for 24 h. The insects were considered dead if appendages did not move when probed with a tiny brush, and the corrected mortality（ ％）＝100× （A-C） （1-C） / , where A＝the mortality of treated with essential oils, C＝ the mortality of control. 2.5.2 Contact Toxicity The contact toxicity of the essential oil or pure compounds against the two species of insects was tested with reference to the method of Liu and Ho28）. Range-finding surveys were run to determine the appropriate testing concentrations. A serial dilution of the essential oil/compounds （five concentrations）was prepared in n-hexane. Aliquots of 0.5 μL of the dilutions were applied topically to the dorsal thorax of the insects. Controls were determined using n-hexane. Ten insects were used for each concentration and control, and the experiment was replicated five times. Mortality was recorded after 24 h of exposure and the corrected mortality（％）＝100× （A-C） （1-C） / , where A ＝the mortality of treated with essential oils, C＝the mortality of control. 2.5.3 Statistical Analysis The corrected mortality represented the average mean of five replicates. The LC50 or LD50 value was analyzed by Probit analysis29）, which was conducted using SPSS 19.0 for Windows 2007. During the Probit analysis, differences at values of p＜0.05 were considered as significant.
3 RESULTS and DISCUSSION 3.1 Chemical compounds of the essential oil The crude essential oil from Z. armatum twigs was pale yellow with a yield of 0.53 ％（v/w）and density of 0.84 g/ mL. Examination of essential oil of Z. armatum by GC-MS revealed the presence of thirty-two components, accounting for 98.2％ of the total oil（Table 1）. The main constituents in the essential oil of Z. armatum were（E）-anethole 863
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Table 1 C hemical constituents of the essential oil derived from Z. armatum twigs. Peak no.
RIa
Compounds
％RAb
Identification Methodsc
1
927
α-Thujene
0.1
MS, RI
2
939
α-Pinene
0.7
MS, RI
3
967
β -Thujene
3.8
MS, RI
4
979
β -Pinene
5.1
MS, RI, Co
5
1006
α-Phellandrene
0.2
MS, RI
6
1014
Terpinolene
0.5
MS, RI
7
1019
o-Cymene
0.8
MS, RI
8
1025
Limonene
9.0
MS, RI, Co
9
1029
β -Phellandrene
6.3
MS, RI
10
1032
1,8-Cineole
14.0
MS, RI, Co
11
1037
cis-β -Ocimene
0.2
MS, RI
12
1060
γ-Terpinene
1.0
MS, RI
13
1089
4-Carene
1.5
MS, RI
14
1096
Linalool
0.5
MS, RI, Co
15
1107
Hotrienol
0.2
MS, RI
16
1156
Citronellal
0.2
MS, RI
17
1177
4-Terpineol
4.4
MS, RI, Co
18
1190
Cryptone
0.1
MS, RI
19
1196
α-Terpineol
0.5
MS, RI
20
1251
Piperitone
8.0
MS, RI, Co
21
1332
（E）-Anethole
20.5
MS, RI, Co
22
1382
2-Undecanol
0.4
MS, RI
23
1423
Caryophyllene
1.5
MS, RI
24
1454
α-Caryophyllene
1.0
MS, RI
25
1496
2-Tridecanone
12.5
MS, RI
26
1503
2-Tetradecanol
1.2
MS, RI
27
1519
α-Muurolene
0.3
MS, RI
28
1523
δ-Cadinene
0.8
MS, RI
29
1578
Spathulenol
0.4
MS, RI
30
1583
Caryophyllene oxide
0.5
MS, RI, Co
31
1636
τ-Muurolol
1.0
MS, RI
32
1663
α-Cadinol
1.0
MS, RI
Total identified
98.2
a
Retention index (RI) relative to the homologous series of n-hydrocarbons on the HP-5 MS capillary column. b Relative area (peak area relative to the total peak area). c MS＝mass spectrum, Co＝co-injection with standard compound. （20.5％）, 1,8-cineole（ 14.0％）, 2-tridecanone（ 12.5％）, limonene（ 9.0％）and piperitone（ 8.0％）, followed by β -phellandrene（6.3％）, β -pinene（5.1％）and 4-terpineol （4.4％）. The chemical structures of isolated compounds
from Z. armatum oil are shown in Fig. 1. In the present paper, the chemical composition of the essential oil is comparable to that of the previous reports with some variation in the constituents. For example, Bisht
864
J. Oleo Sci. 64, (8) 861-868 (2015)
Insecticidal Constituents of Essential Oil from Zanthoxylum armatum Against Two Stored-Product Insects
reported that 2-undecanone（ 44.6％）, linalool（14.5％）, 2-tridecanene（8.0％） and 1,8-cineole（6.9％） were the major components in the essential oil of Z. armatum leaves from India10）. While Muhammad found linalool was the most dominant constituent30）. The leaf oil of Z. armatum from Vietnam was rich in 1,8-cineole （41.0％） , sabinene （8.4％） , terpinen-4-ol（5.2％）, and linalool（4.5％）14）. Moreover, linalool was reported as the highest level compound of essential oil from Z. armatum pericarps in China, accounting for 71.7％∼75.0％15）. These reports indicate that linalool is a high content composition in Z. armatum. However, in our investigation, linalool was a minor constituent, only presented in 0.5％. The observed differences in chemical composition and content of the essential oil from Z. armatum could be due to harvest time and local, environmental conditions, or may result from herbal parts used and the extraction and analytical methods used. These variations may lead to different biological activities. 3.2 Insecticidial activity The fumigant toxicities of essential oil from Z. armatum twigs and five isolated compounds against L. serricorne and T. castaneum were presented in Table 2. The results showed that the red flour beetle （T. castaneum） treated by the crude essential oil was more sensitive than the cigarette beetle（L. serricorne）with LC50 values of 4.28 and 12.54 mg/L air, respectively. Among the five compounds, piperitone exhibited the strongest fumigant toxicity to the cigarette beetle（LC50＝1.21 mg/L air）, while 1,8-cineole, limonene and piperitone showed similar fumigant toxicity against T. castaneum with LC50 values of 5.47, 6.21 and
7.12 mg/L air, respectively. When compared with the positive control, phosphine （LC50＝9.23×10−3 mg/L air）, MeBr （LC50＝1.75 mg/L air）, piperitone represented 131 times less toxicity to L. serricorne and 1,8-cineole exhibited 3.5 times less toxicity to T. castaneum, respectively（Table 2）. However, the two commercial fumigants are highly toxic to humans and other non-target organisms. The fumigant toxicity of Z. armatum oil and isolated constituents was comparable with some reports recently, for instance, essential oils of Aster ageratoides（ LC 50＝12.14 mg/L）, Citrus wilsonii and its main component γ-Terpinene（LC50 ＝8.18 and 4.09 mg/L, respectively） , and Cayratia japonica （LC50＝15.67 mg/L）against T. castaneum2, 5, 31）; Agastache foeniculum（LC50＝21.57 mg/L）and Litsea cubeba（LC50＝ 22.97 mg/L）against L. serricorne32, 33）. Thus, the essential oil of Z. armatum and its active compounds have potential to be developed as possible natural fumigants for control of L. serricorne and T. castaneum. As the results were shown in Table 3, the essential oil of Z. armatum exhibited contact toxicity against the cigarette beetle and red flour beetle with LD50 values of 18.74 and 32.16 μg/adult, respectively. The most toxic compound to L. serricorne was 2-tridecanone（LD50＝5.74 μg/adult）. While piperitone presented strongest contact toxicity against T. castaneum（LD50＝3.16 μg/adult）, followed by 2-tridecanone with an LD50 value of 5.35 μg/adult. Both the essential oil and its active compounds showed less contact toxicity against the two species of insects comparing with positive control, pyrethrins. When compared with previous reports, the essential oil of Z. armatum showed similar contact toxicity against L. serricorne, e.g. essential oils of
Table 2 Fumigant toxicity of essential oil from Z. armatum twigs and its main components against L. serricorne and T. castaneum adults. Insects
L. serricorne
Treatment
Concentrations (％)
LC50 (mg/L air)
95％ Fiducial limits
Z. armatum
3.95-20.00
13.83
10.73-16.16
18.17
(E)-Anethole
0.99- 5.00
6.31
5.33- 7.19
16.56
1,8- Cineole
0.99- 5.00
5.18
4.63- 5.70
16.79
2-Tridecanone
-
-
-
-
Limonene
1.97-10.00
14.07
12.41-15.76
22.08
Piperitone
0.39- 2.00
1.21
0.75- 1.55
6.69
-
-
Phosphine*
T. castaneum
Chi square (χ2)
-
9.23×10
−3
Z. armatum
1.97-10.00
4.28
1.86- 5.92
26.45
(E)-Anethole
1.97-10.00
9.20
8.03-10.25
17.02
1,8- Cineole
0.99-5.00
5.47
4.73- 6.17
25.30
2-Tridecanone
-
-
-
-
4）
Limonene
0.99- 5.00
6.21
5.38- 7.05
13.34
Piperitone
1.97-10.00
7.12
5.44- 8.38
6.96
MeBr**
-
1.75
-
-
28)
* Data from You et al. ; ** MeBr: Methyl bromide, values from Liu and Ho . 865
J. Oleo Sci. 64, (8) 861-868 (2015)
C. F. Wang, W. J. Zhang and C. X. You et al.
Table 3 Contact toxicity of the essential oil from Z. armatum twigs and its main components against L. serricorne and T. castaneum adults. Insects
L. serricorne
T. castaneum
Treatment
Concentrations (％)
LD50 (μg/adult)
95％ Fiducial limits
Chi square (χ2)
Z. armatum
1.97-10.00
18.74
14.99-21.69
13.80
(E)-Anethole
0.99- 5.00
10.32
8.63-11.72
14.03
1,8- Cineole
1.97-10.00
15.58
12.88-18.02
15.18
2-Tridecanone
0.88- 4.44
5.74
5.04- 6.45
9.70
Limonene
0.99- 5.00
13.66
11.63-16.18
11.04
Piperitone
0.63-10.00
22.05
21.07-23.01
7.99
Pyrethrins*
-
0.24
-
-
Z. armatum
5.93-30.00
32.16
27.96-35.88
16.33
(E)-Anethole
1.97-10.00
15.40
13.67-17.11
18.63
1,8- Cineole
1.97-10.00
18.83
17.13-20.69
16.56
2-Tridecanone
0.96- 2.00
5.35
4.94- 5.74
6.46
Limonene
1.97-10.00
14.97
12.88-17.04
20.01
Piperitone
0.59- 2.96
3.16
2.56- 3.65
6.41
Pyrethrins*
-
0.26
-
-
6）
*Data from Wang et al. .
Litsea cubeba（LD50＝27.33 μg/adult）and Cinnamomum camphora（LD 50＝21.25 μg/adult）33, 34）, weaker contact toxicity against T. castaneum, e.g. essential oils of Aster ageratoides（LD50＝8.09 μg/adult）and Amomum tsaoko （LD50＝16.52 μg/adult）2, 6）. In the present work, piperitone showed much different insecticidal activities between the two tested insects. In contrast to contact toxicity test, L. serricorne adults treated with piperitone were more susceptible than T. castaneum adults in the fumigant toxicity test（Table 2 and 3）. This indicates that fumigant toxicity may be a major route of insecticidal activity of piperitone against L. serricorne, while the mode of action in T. castaneum is via contact toxicity. It also has been reported that piperitone possessed insecticidal activity against the third instar larvae of Spodoptera littoralis and the adults of Callosobruchus maculatus35, 36）. Moreover, 2-tridecanone presented strong contact toxicity against L. serricorne and T. castaneum, whereas no fumigant toxicity of 2-tridecanone was measured to the test insects. It is also found that 2-tridecanone has strong contact toxicity to Solenopsis invicta, larvae of Keiferia lycopersicella and Spodoptera exigua37, 38）. In this context, the combination of 2-tridecanone as a contact toxin and piperitone as a fumigant at a certain ratio would develop significantly stronger insecticidal activity than individual compounds.
4 CONCLUSION This paper is the first one to demonstrate botanical effi-
cacy of Z. armatum essential oil towards storage pests. The results suggested that Z. armatum essential oil and its major components could be a promising alternative in the control of stored-product insects. Further investigations should be focused on the safety of essential oil/compounds to humans, the development of formulations and improvement of their efficacy and stability.
ACKNOWLEDGMENT This project was supported by Beijing Municipal Natural Science Foundation（No. 7142093）, National Natural Science Foundation of China（No. 81374069）and the Fundamental Research Funds for the Central Universities.
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