# Contact Toxicity and Repellency of the Essential Oil fromBupleurum bicaule Helm against Two Stored Product Insects

Authors: Xiao-Meng Wei; Shan-Shan Guo; Hua Yan; Xian-Long Cheng; Feng Wei; Shu-Shan Du
Journal: Journal of Chemistry
 (2018)

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## Abstract

Essential oils obtained from many plants showed various kinds of insecticidal properties; some of them have been considered as alternative insecticides for pest control. The present study was aimed at determining the chemical composition of the essential oil from the roots ofBupleurum bicauleHelm, as well as evaluating the contact and repellent activities of the oil and four identified compounds againstLasioderma serricorne andLiposcelis bostrychophila adults. The essential oil was extracted by hydrodistillation, and its components were analyzed by gas chromatography-mass spectrometry (GC-MS). 26 components were determined and the main compounds included trans-2-isopropylbicyclo[4.3.0]non-3-en-8-one (25.9%), 4,5-dimethyl-1,2,3,6,7,8,8a,8b-octahydrobiphenylene (23.5%), and 1,4-dimethoxy-2-tert-butylbenzene (4.3%). It was found that the essential oil exhibited contact toxicity againstL. serricorne (LD50 = 11.91 μg/adult), but the contact toxicity againstL. bostrychophilacould not be observed. The essential oil also showed strong repellent activity againstL. serricorne with percent repellency of 100% at 78.63 nl/cm2. Four chemical compounds, 1,4-dimethoxy-2-tert-butylbenzene, bornyl acetate, (2E,4E)-2,4-nonadienal, and β-bisabolene, exhibited various levels of bioactivities. The experimental results indicated that the essential oil ofB. bicauleand its individual compounds could be used in insecticidal and repellent strategies for stored product insects.


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## Body

## 1. Introduction

The economy of agriculture and food industry has been threatened by insects in the process of production, transportation, and storage. It is estimated that about 10–40% of the world’s annual stored product loss was caused by the insects [1]. The cigarette beetle (Lasioderma serricorne Fabricius) and the booklouse (Liposcelis bostrychophila Badonnel) are two major insects in the warehouses of stored foods, grains, and some herbal medicines [2, 3]. Under appropriate environmental conditions, they can reproduce rapidly [4, 5]. The insects not only consume the stored commodities, but also lead to contamination and deterioration of stored products in the short term. A great effort has been focused on the prophylaxis of insects in stored products. Many methods, such as the cold storage, the light traps, and the chemical treatment [5] have been successively used to control the insects. Among them the chemical treatment is the most commonly used method with different chemical synthetic insecticides. However, a series of negative effects such as insecticide residues and environmental damage have been exposed, so that the development of the new environmental friendly, economical, and effective methods [2, 6] is necessary. Antagonistic storage, a traditional method, has been used for Chinese medicinal material conservation for a long history. Our ancestors skillfully used the special odors of some Chinese herbs to repel the insects and to achieve the purpose of protecting another kind of medicinal materials [7]. For instance, Rhizoma Alismatis is easily infected by insects. If it is stored with Cortex Moutan which has a special odor, Rhizoma Alismatis would not be susceptible to insects, and the quality of the two medicinal materials would be maintained for a long time [8]. The antagonistic storage method is mostly related to the special odor reeked off by the stored materials. Thus, the essential oils of the stored materials could be considered as the effective elements in antagonistic storage method. The research on the essential oils would be helpful to reveal the mechanism of the antagonistic storage method. In practice, this kind of research has become a hot topic in the field of controlling stored product insects [9–11].Bupleurum bicauleHelm (Chinese name: Zhuiye Chaihu) is widely distributed in northeast region of China, and its roots have been medicinally used for the treatment of fever, cold, hypochondriac pain, hepatitis, biliary tract infection, irregular menstruation, uterine prolapse, and anal prolapse [12–15]. As is well known, essential oils are abundant in Umbelliferae (Apiaceae) plants [16]. Many of these essential oils obtained from Umbelliferae (Apiaceae) plants have been proven to possess insecticidal activities against certain kinds of insects. For examples, the essential oil ofHeracleum persicum showed fumigant toxicity againstCallosobruchus maculatus [17],Pimpinella anisumessential oil also showed strong fumigant toxicity againstEphestia kuehniella andSitophilus oryzae [18], andFerula asafetidaessential oil possessed strong repellent activity againstTribolium castaneum [19].The phytochemistry ofBupleurumplants has been investigated, and some of the secondary metabolites obtained fromB. salicifolium showed antifeedant activity againstSpodoptera littoralis [20]. ForB. bicaule, the chemical components of the essential oil obtained from this plant have been reported [21]. In this work, the essential oil ofB. bicaule was further investigated, and its insecticidal and repellent activities against the cigarette beetles and booklice adults were evaluated; meanwhile, the same kinds of biological activity tests were carried out with four representative compounds identified in the sample of the essential oil.


## 2. Materials and Methods

### 2.1. Plant Materials and Preparation of the Essential Oil

Roots ofB. bicaulewere collected in Hailar, Inner Mongolia Autonomous Region, China (northern latitude: 9°5′–19°27′; east longitude: 119°30′–120°35′) in August 2016. The species was identified by Dr. Yan H. (National Institutes for Food and Drug Control, China Food and Drug Administration, China). The voucher specimen (NIFDC-20160801-01) was deposited at Herbarium (NIFDC) of National Institutes for Food and Drug Control. After air drying for one week (25–28°C and 13–20% RH), the dried roots (2.4 kg) were ground to powders with a grinding mill. The extraction of essential oil was carried out by hydrodistillation using a modified Clevenger apparatus for 8 h [22]. The distilled oils were dried over anhydrous sodium sulfate and stored in glass vials at 4°C.


### 2.2. GC-MS and GC-FID Analysis

The GC-MS analysis ofB. bicaule essential oil was performed by an Agilent 6890N GC/MS instrument equipped with an Agilent 5973N mass selective detector and a HP-5 MS column (30 m × 0.25 mm × 0.25 μm). The carrier gas was helium, and the flow rate was 1.0 mL/min. Analytical conditions are as follows: column temperature was programmed from 50°C (2 min) to 150°C at 2°C/min and kept isothermal for 2 min, then increased to 250°C at 10°C/min, and held for 5 min. The injector temperature was 250°C. The MS spectra were obtained in the electron-impact mode with ionization energy of 70 eV, and mass range was from m/z 50 to 550.The essential oil was diluted withn-hexane to prepare a GC sample solution with content of 1% (v/v), and the sample solution of 0.1 μL was injected into the instrument. Relative percentages of the individual components were determined by GC-FID via percentage peak area calculations. These components were further identified based on their retention index and by comparison with NIST 05 (Standard Reference Data, Gaithersburg, MD) and Wiley 275 GC-MS databases (Wiley, New York, NY).


### 2.3. Test Insects

The cigarette beetles were originally delivered from Henan University of Technology, China, since 2013, and the species was identified by Dr. Lv J. H. (School of Food Science and Technology, Henan University of Technology, China). The booklice were adopted from China Agricultural University and identified by Dr. Liu Z. L. (China Agricultural University, China). The cigarette beetles were reared on wheat flour mixed with yeast (10 : 1, w/w) at 12-13% moisture content, and the booklice were cultured with the flour mixture, which was prepared with flour, yeast, and milk powder at 10 : 1 : 1 mixed proportion [2]. Insect rearing were carried out in the dark in incubators at 29-30°C and 70–80% RH. All the unsexed insects used in the experiments were one to two weeks old. The escape proof was made of polytetrafluoroethylene (Beijing Sino-Rich Co., Ltd., Beijing, China) coating on all insect containers and experimental Petri dishes.


### 2.4. Bioactivities

#### 2.4.1. Preparation of the Treatment Solutions

The essential oil and four representative compounds found in the oil were selected for bioactivity assays. The chemical compounds (2E,4E)-2,4-nonadienal andβ-bisabolene were purchased from TCI (Shanghai) Development Co., Ltd. (Shanghai, China). 1,4-Dimethoxy-2-tert-butylbenzene was obtained from JW&Y PharmLab (Shanghai), PharmLab Co., Ltd. (Shanghai, China). Bornyl acetate was purchased from Acros Organics Co., Ltd. (Geel, Belgium). N,N-Diethyl-3-methylbenzamide (DEET) was obtained from the National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang, China). Each of the above samples was dissolved in the solvent ofn-hexane, and their stock solutions with the known concentrations were prepared.


#### 2.4.2. Contact Toxicity

In the evaluation of contact toxicity, two kinds of experimental methods were used based on the size of the insects. The contact toxicity against cigarette beetles was carried out with the methods described in the literature [23]. Before the formal test, the appropriate concentration range was determined by preliminary tests. Ultimately, the essential oil and four selected compounds were diluted withn-hexane into five concentrations. Drop 0.5 μL dilutions on the dorsal thorax of each insect using a TopPette (0.5–10 μL, Dragon Laboratory Instruments Co., Ltd., Beijing, China). Ten insects were used for each concentration, and the experiment was replicated five times for each concentration. Then the treated and control insects were transferred to glass vials (2.5 cm in diameter, 5.5 cm in height). After being cultured in an incubator (29-30°C and 70–80% R.H) for 24 h, the mortality of tested insects was observed and recorded, and the LD50 values were calculated by Probit analysis (SPSS 20.0) [24].For booklice, the contact toxicity was tested as described [25]. A filter paper of 5.5 cm in diameter was treated with 300 μL dilutions of each test concentration. The treated filter paper was fixed in a 5.5-cm-diameter Petri dish with solid glue, and 10 test insects were placed in each Petri dish. Five concentrations (inn-hexane) and five replicates of each concentration were used. In the above experiments,n-hexane was used as a negative control. The chemical synthetic insecticide pyrethrins was usually served as a positive control; its mortality was taken from Yang et al.’s data with the same experimental methods [26]. The mortality of insects was recorded after these covered-Petri dishes were placed in an incubator at 29-30°C and 70–80% RH for 24 h. The mortality data was corrected by Abbott’s formula [27]:(1)corrected  mortality%=100×Mc-Mt1-Mc,where Mt is the observed mortality and Mc is the control mortality. With these data, the LD50 values were calculated by Probit analysis (SPSS 20.0).


#### 2.4.3. Repellent Activity

The repellent activity ofB. bicaule essential oil and four components against cigarette beetles and booklice was investigated with the method proposed by Zhang et al. [28]. The experiments were set with five concentrations (cigarette beetles: 78.63, 15.73, 3.15, 0.63, and 0.13 nL/cm2; booklice: 63.17, 12.63, 2.53, 0.51, and 0.10 nL/cm2) and two observation times (2 h and 4 h). For cigarette beetles, the essential oil and four selected compounds were diluted withn-hexane. Filter paper (9 cm in diameter) was symmetrically cut into two pieces, and 500 μL dilution of each concentration was dropped evenly on half of the filter paper, while the other half was treated with 500 μL ofn-hexane as a negative control. After being air-dried for 30 s, both two pieces were stuck to the bottom of the Petri dish (9 cm in diameter) with solid glue abreast. For booklice, due to its tiny body, the diameters of the Petri dish and filter paper were reduced to 5.5 cm. Filter paper was cut into two pieces, and one piece was treated with 150 μL of each solution separately. The other half (negative control) was treated with the same volume ofn-hexane. For each test, 20 insects were placed at the center of the disk and covered quickly with lids. All the above procedures were repeated five times for each concentration, and each experiment was repeated three times.As a commercial insect repellant, DEET was served as positive control. Counts of insects on each half of the paper were recorded separately. The percent repellency (PR) of each test was calculated by the following formula [23]:(2)PR%=Nc-NtNc+Nt×100,where Nc is the number of insects in the negative control half and Nt is the number of insects in the tested half. The percent repellency was transformed to arcsine square root values for analysis of variance (ANOVA), and the effect on the transformed arcsine of the percent repellency of different treatments (the essential oil, the four compounds, and the control) at each concentration and exposure time was analyzed separately. Significant differences in repellence rates among treatments were given by the analysis of Tukey’s test (SPSS 20.0 for Windows 2007).


## 2.1. Plant Materials and Preparation of the Essential Oil

Roots ofB. bicaulewere collected in Hailar, Inner Mongolia Autonomous Region, China (northern latitude: 9°5′–19°27′; east longitude: 119°30′–120°35′) in August 2016. The species was identified by Dr. Yan H. (National Institutes for Food and Drug Control, China Food and Drug Administration, China). The voucher specimen (NIFDC-20160801-01) was deposited at Herbarium (NIFDC) of National Institutes for Food and Drug Control. After air drying for one week (25–28°C and 13–20% RH), the dried roots (2.4 kg) were ground to powders with a grinding mill. The extraction of essential oil was carried out by hydrodistillation using a modified Clevenger apparatus for 8 h [22]. The distilled oils were dried over anhydrous sodium sulfate and stored in glass vials at 4°C.


## 2.2. GC-MS and GC-FID Analysis

The GC-MS analysis ofB. bicaule essential oil was performed by an Agilent 6890N GC/MS instrument equipped with an Agilent 5973N mass selective detector and a HP-5 MS column (30 m × 0.25 mm × 0.25 μm). The carrier gas was helium, and the flow rate was 1.0 mL/min. Analytical conditions are as follows: column temperature was programmed from 50°C (2 min) to 150°C at 2°C/min and kept isothermal for 2 min, then increased to 250°C at 10°C/min, and held for 5 min. The injector temperature was 250°C. The MS spectra were obtained in the electron-impact mode with ionization energy of 70 eV, and mass range was from m/z 50 to 550.The essential oil was diluted withn-hexane to prepare a GC sample solution with content of 1% (v/v), and the sample solution of 0.1 μL was injected into the instrument. Relative percentages of the individual components were determined by GC-FID via percentage peak area calculations. These components were further identified based on their retention index and by comparison with NIST 05 (Standard Reference Data, Gaithersburg, MD) and Wiley 275 GC-MS databases (Wiley, New York, NY).


## 2.3. Test Insects

The cigarette beetles were originally delivered from Henan University of Technology, China, since 2013, and the species was identified by Dr. Lv J. H. (School of Food Science and Technology, Henan University of Technology, China). The booklice were adopted from China Agricultural University and identified by Dr. Liu Z. L. (China Agricultural University, China). The cigarette beetles were reared on wheat flour mixed with yeast (10 : 1, w/w) at 12-13% moisture content, and the booklice were cultured with the flour mixture, which was prepared with flour, yeast, and milk powder at 10 : 1 : 1 mixed proportion [2]. Insect rearing were carried out in the dark in incubators at 29-30°C and 70–80% RH. All the unsexed insects used in the experiments were one to two weeks old. The escape proof was made of polytetrafluoroethylene (Beijing Sino-Rich Co., Ltd., Beijing, China) coating on all insect containers and experimental Petri dishes.


## 2.4. Bioactivities

### 2.4.1. Preparation of the Treatment Solutions

The essential oil and four representative compounds found in the oil were selected for bioactivity assays. The chemical compounds (2E,4E)-2,4-nonadienal andβ-bisabolene were purchased from TCI (Shanghai) Development Co., Ltd. (Shanghai, China). 1,4-Dimethoxy-2-tert-butylbenzene was obtained from JW&Y PharmLab (Shanghai), PharmLab Co., Ltd. (Shanghai, China). Bornyl acetate was purchased from Acros Organics Co., Ltd. (Geel, Belgium). N,N-Diethyl-3-methylbenzamide (DEET) was obtained from the National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang, China). Each of the above samples was dissolved in the solvent ofn-hexane, and their stock solutions with the known concentrations were prepared.


### 2.4.2. Contact Toxicity

In the evaluation of contact toxicity, two kinds of experimental methods were used based on the size of the insects. The contact toxicity against cigarette beetles was carried out with the methods described in the literature [23]. Before the formal test, the appropriate concentration range was determined by preliminary tests. Ultimately, the essential oil and four selected compounds were diluted withn-hexane into five concentrations. Drop 0.5 μL dilutions on the dorsal thorax of each insect using a TopPette (0.5–10 μL, Dragon Laboratory Instruments Co., Ltd., Beijing, China). Ten insects were used for each concentration, and the experiment was replicated five times for each concentration. Then the treated and control insects were transferred to glass vials (2.5 cm in diameter, 5.5 cm in height). After being cultured in an incubator (29-30°C and 70–80% R.H) for 24 h, the mortality of tested insects was observed and recorded, and the LD50 values were calculated by Probit analysis (SPSS 20.0) [24].For booklice, the contact toxicity was tested as described [25]. A filter paper of 5.5 cm in diameter was treated with 300 μL dilutions of each test concentration. The treated filter paper was fixed in a 5.5-cm-diameter Petri dish with solid glue, and 10 test insects were placed in each Petri dish. Five concentrations (inn-hexane) and five replicates of each concentration were used. In the above experiments,n-hexane was used as a negative control. The chemical synthetic insecticide pyrethrins was usually served as a positive control; its mortality was taken from Yang et al.’s data with the same experimental methods [26]. The mortality of insects was recorded after these covered-Petri dishes were placed in an incubator at 29-30°C and 70–80% RH for 24 h. The mortality data was corrected by Abbott’s formula [27]:(1)corrected  mortality%=100×Mc-Mt1-Mc,where Mt is the observed mortality and Mc is the control mortality. With these data, the LD50 values were calculated by Probit analysis (SPSS 20.0).


### 2.4.3. Repellent Activity

The repellent activity ofB. bicaule essential oil and four components against cigarette beetles and booklice was investigated with the method proposed by Zhang et al. [28]. The experiments were set with five concentrations (cigarette beetles: 78.63, 15.73, 3.15, 0.63, and 0.13 nL/cm2; booklice: 63.17, 12.63, 2.53, 0.51, and 0.10 nL/cm2) and two observation times (2 h and 4 h). For cigarette beetles, the essential oil and four selected compounds were diluted withn-hexane. Filter paper (9 cm in diameter) was symmetrically cut into two pieces, and 500 μL dilution of each concentration was dropped evenly on half of the filter paper, while the other half was treated with 500 μL ofn-hexane as a negative control. After being air-dried for 30 s, both two pieces were stuck to the bottom of the Petri dish (9 cm in diameter) with solid glue abreast. For booklice, due to its tiny body, the diameters of the Petri dish and filter paper were reduced to 5.5 cm. Filter paper was cut into two pieces, and one piece was treated with 150 μL of each solution separately. The other half (negative control) was treated with the same volume ofn-hexane. For each test, 20 insects were placed at the center of the disk and covered quickly with lids. All the above procedures were repeated five times for each concentration, and each experiment was repeated three times.As a commercial insect repellant, DEET was served as positive control. Counts of insects on each half of the paper were recorded separately. The percent repellency (PR) of each test was calculated by the following formula [23]:(2)PR%=Nc-NtNc+Nt×100,where Nc is the number of insects in the negative control half and Nt is the number of insects in the tested half. The percent repellency was transformed to arcsine square root values for analysis of variance (ANOVA), and the effect on the transformed arcsine of the percent repellency of different treatments (the essential oil, the four compounds, and the control) at each concentration and exposure time was analyzed separately. Significant differences in repellence rates among treatments were given by the analysis of Tukey’s test (SPSS 20.0 for Windows 2007).


## 2.4.1. Preparation of the Treatment Solutions

The essential oil and four representative compounds found in the oil were selected for bioactivity assays. The chemical compounds (2E,4E)-2,4-nonadienal andβ-bisabolene were purchased from TCI (Shanghai) Development Co., Ltd. (Shanghai, China). 1,4-Dimethoxy-2-tert-butylbenzene was obtained from JW&Y PharmLab (Shanghai), PharmLab Co., Ltd. (Shanghai, China). Bornyl acetate was purchased from Acros Organics Co., Ltd. (Geel, Belgium). N,N-Diethyl-3-methylbenzamide (DEET) was obtained from the National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang, China). Each of the above samples was dissolved in the solvent ofn-hexane, and their stock solutions with the known concentrations were prepared.


## 2.4.2. Contact Toxicity

In the evaluation of contact toxicity, two kinds of experimental methods were used based on the size of the insects. The contact toxicity against cigarette beetles was carried out with the methods described in the literature [23]. Before the formal test, the appropriate concentration range was determined by preliminary tests. Ultimately, the essential oil and four selected compounds were diluted withn-hexane into five concentrations. Drop 0.5 μL dilutions on the dorsal thorax of each insect using a TopPette (0.5–10 μL, Dragon Laboratory Instruments Co., Ltd., Beijing, China). Ten insects were used for each concentration, and the experiment was replicated five times for each concentration. Then the treated and control insects were transferred to glass vials (2.5 cm in diameter, 5.5 cm in height). After being cultured in an incubator (29-30°C and 70–80% R.H) for 24 h, the mortality of tested insects was observed and recorded, and the LD50 values were calculated by Probit analysis (SPSS 20.0) [24].For booklice, the contact toxicity was tested as described [25]. A filter paper of 5.5 cm in diameter was treated with 300 μL dilutions of each test concentration. The treated filter paper was fixed in a 5.5-cm-diameter Petri dish with solid glue, and 10 test insects were placed in each Petri dish. Five concentrations (inn-hexane) and five replicates of each concentration were used. In the above experiments,n-hexane was used as a negative control. The chemical synthetic insecticide pyrethrins was usually served as a positive control; its mortality was taken from Yang et al.’s data with the same experimental methods [26]. The mortality of insects was recorded after these covered-Petri dishes were placed in an incubator at 29-30°C and 70–80% RH for 24 h. The mortality data was corrected by Abbott’s formula [27]:(1)corrected  mortality%=100×Mc-Mt1-Mc,where Mt is the observed mortality and Mc is the control mortality. With these data, the LD50 values were calculated by Probit analysis (SPSS 20.0).


## 2.4.3. Repellent Activity

The repellent activity ofB. bicaule essential oil and four components against cigarette beetles and booklice was investigated with the method proposed by Zhang et al. [28]. The experiments were set with five concentrations (cigarette beetles: 78.63, 15.73, 3.15, 0.63, and 0.13 nL/cm2; booklice: 63.17, 12.63, 2.53, 0.51, and 0.10 nL/cm2) and two observation times (2 h and 4 h). For cigarette beetles, the essential oil and four selected compounds were diluted withn-hexane. Filter paper (9 cm in diameter) was symmetrically cut into two pieces, and 500 μL dilution of each concentration was dropped evenly on half of the filter paper, while the other half was treated with 500 μL ofn-hexane as a negative control. After being air-dried for 30 s, both two pieces were stuck to the bottom of the Petri dish (9 cm in diameter) with solid glue abreast. For booklice, due to its tiny body, the diameters of the Petri dish and filter paper were reduced to 5.5 cm. Filter paper was cut into two pieces, and one piece was treated with 150 μL of each solution separately. The other half (negative control) was treated with the same volume ofn-hexane. For each test, 20 insects were placed at the center of the disk and covered quickly with lids. All the above procedures were repeated five times for each concentration, and each experiment was repeated three times.As a commercial insect repellant, DEET was served as positive control. Counts of insects on each half of the paper were recorded separately. The percent repellency (PR) of each test was calculated by the following formula [23]:(2)PR%=Nc-NtNc+Nt×100,where Nc is the number of insects in the negative control half and Nt is the number of insects in the tested half. The percent repellency was transformed to arcsine square root values for analysis of variance (ANOVA), and the effect on the transformed arcsine of the percent repellency of different treatments (the essential oil, the four compounds, and the control) at each concentration and exposure time was analyzed separately. Significant differences in repellence rates among treatments were given by the analysis of Tukey’s test (SPSS 20.0 for Windows 2007).


## 3. Results and Discussion

### 3.1. Chemical Constituents of the Essential Oil

The yellow essential oil was obtained fromB. bicaulewith a yield of 0.04%(v/w). Its chemical components were analyzed by the GC-MS. 26 compounds were identified and their relative contents in the oil sample were determined. The total amount of all the identified compounds was 85.5% (w/w) of the essential oil. The content of trans-2-isopropylbicyclo[4.3.0]non-3-en-8-one was 25.9%, and the content of 4,5-dimethyl-1,2,3,6,7,8,8a,8b-octahydrobiphenylene was 23.5%. These two compounds could be considered as the major components of the essential oil. The results are summarized in Table 1.Table 1
Chemical composition of the essential oil ofBupleurum bicaule.
Compounds Relative content (%) a R I e x p b R I l i t Identification methodsc Aldehydes (2E,4E)-2,4-Octadienal 0.8 1113 1109 MS; RI (2E,4E)-2,4-Nonadienal 1.2 1214 1217 MS; RI (2E,4E)-2,4-Decadienal 2.8 1317 1316 MS; RI Monoterpenes Bornyl acetate 1.1 1280 1281 MS; RI Trans-2-isopropylbicyclo[4.3.0]non-3-en-8-one 25.9 1765 1765 MS; RI Sesquiterpenes α-Longipinene 0.5 1352 1351 MS; RI β-Elemene 0.8 1391 1393 MS; RI β-Cuvebene 1.5 1390 1388 MS; RI 1,4,7,-Cycloundecatriene, 1,5,9,9-tetramethyl-, Z,Z,Z- 1.9 1456 1454 MS; RI Calarene 2.1 1467 1463 MS; RI γ-Gurjunene 2.0 1479 1472 MS; RI (E,E)-α-Farnesene 0.4 1505 1505 MS; RI β-Bisabolene 2.7 1510 1511 MS; RI Cuparene 2.9 1502 1499 MS; RI (E)-Nerolidol 0.5 1565 1564 MS; RI Spathulenol 0.9 1582 1578 MS; RI Caryophyllene oxide 1.4 1583 1580 MS; RI Humulene oxide II 1.4 1607 1611 MS; RI α-Cadinol 1.4 1653 1654 MS; RI τ-Muurolol 0.6 1662 1660 MS; RI Juniper camphor 0.3 1690 1693 MS; RI Alkynes (6Z)-6-Tridecen-4-yne 2.7 1470 1470 MS; RI Aromatic compounds 1,4-Dimethoxy-2-tert-butylbenzene 4.3 1477 1477 MS; RI Myristicin 1.6 1529 1523 MS; RI 2(1H)Naphthalenone,3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)- 0.3 1772 1773 MS; RI Others 4,5-Dimethyl-1,2,3,6,7,8,8a,8b-octahydrobiphenylene 23.5 1357 1357 MS; RI  Total 85.5 aRetention index (RI) in this experiment relative to the homologous series of n-alkanes (C5–C36) on the HP-5 MS capillary column. bRI in literature. cMass spectrum.With the further analysis of the GS-MS results, it was found that the essential oil contained a plenty of monoterpenes and sesquiterpenes, and their contents accounted for 27.0% and 21.3% of the essential oil, respectively. The other types of chemical components included aldehydes, alkynes, and aromatic compounds. In a published paper [21], Xu and his partners reported that 19 compounds were identified in the root oil ofB. bicaulecollected from Inner Mongolia, China, and all the types of chemical components mentioned above also existed in Xu et al.’s oil sample. The five types of components accounted for about 60% of the essential oils in the two works. However, the significant difference between the results presented in Xu’s paper and our results could be figured out. The compound with the highest content predicted in Xu’s paper was 4-tridecen-6-yne (42.6%), but only its isomer, 6-tridecen-4-yne, was discovered in our sample and its content only accounted for 2.7% of the essential oil. The highest content compound in our sample was trans-2-isopropylbicyclo[4.3.0] non-3-en-8-one (25.9%), and it was not reported in the literature.The other differences were showed in Figure1, the content of aldehydes (12.6%) in Xu’s paper was higher than that of terpenoids (6.2%), and the monoterpenes only accounted for a minor proportion (0.8%). However, in our case, the content of terpenoids was 48.3% (27.0% for monoterpenes and 21.3% for sesquiterpenes), which was much higher than that of aldehyde compounds (4.8%). The above differences might be caused by many variables such as the growth environment of plants, the harvest time, and treatment of samples. These differences might further affect the biological activities.Figure 1
Percentage of different types of compounds in essential oils of this work and literature.It was reported that terpenoids and aromatic compounds exhibited excellent bioactivities against different stored product insect pests [29–32] and aldehydes showed fair contact toxicity and repellent activities [33]. Here, four representative compounds were selected from 26 components identified in our sample as they belong to the chemical types of monoterpenes, sesquiterpenes, aldehydes, or aromatic compounds, respectively. Their bioactivity tests were evaluated as well. They are bornyl acetate, β-bisabolene, (2E,4E)-2,4-nonadienal, and 1,4-dimethoxy-2-tert-butylbenzene. The molecular structures of the four selected compounds are shown in Figure 2.Figure 2
Molecular structures of selected compounds from the essential oil ofBupleurum bicaule.


### 3.2. Contact Toxicity

The results of contact toxicity of the essential oil and selected compounds against cigarette beetles and booklice are listed in Table2. The results showed that the essential oil exhibited fair contact toxicity against cigarette beetles with a LD50 value of 11.91 μg/adult but expressed unobservable toxicity against booklice in our measure range.Table 2
Contact toxicity ofBupleurum bicauleessential oil and its constituents against Lasioderma serricorne(LS) and Liposcelis bostrychophila(LB) adults.
Insects Treatments Concentrations (%) LD50 (µg/adult)/(µg/cm2) 95% FL(µg/adult)/(µg/cm2) Slope ± SE Chi square (χ2) LS Essential oil 1.91–10.00 11.91 7.04–15.66 2.01 ± 0.39 17.50 β-Bisabolene 2.97–15.00 24.99 21.68–29.14 3.86 ± 0.56 3.61 1,4-Dimethoxy-2-tert-butylbenzene 1.97–10.00 23.56 20.03–27.24 3.18 ± 0.55 2.90 Bornyl acetate 0.90–4.40 9.42 8.14–10.87 0.98 ± 0.17 7.53 (2E,4E)-2,4-Nonadienal 0.27–1.30 2.31 2.03–2.61 4.00 ± 0.62 9.44 Pyrethrinsa -- 0.24 0.22–0.30 3.34 ± 0.32 13.11  LB Essential oil 0–5.00 -- -- -- -- β-Bisabolene 0–50.00 -- -- -- -- 1,4-Dimethoxy-2-tert-butylbenzene 0.12–1.00 62.34 55.97–69.66 6.42 ± 0.87 3.93 Bornyl acetate 0.20–0.40 36.67 34.67–38.55 16.73 ± 2.28 7.04 (2E,4E)-2,4-Nonadienal 0.20–0.40 32.23 30.59–33.97 17.08 ± 2.31 9.66 Pyrethrinsa -- 18.72 17.60–19.92 2.98 ± 0.40 10.56 aData from Yang et al. [26].Among the four selected compounds, (2E,4E)-2,4-nonadienal showed the strongest toxicity against cigarette beetles (LD50 = 2.31 μg/adult) and booklice (LD50 = 32.23 μg/cm2). Bornyl acetate also showed certain toxicity against the two insects (LD50 = 9.42 μg/adult and 36.67 μg/cm2, resp.), while the toxicity of 1,4-dimethoxy-2-tert-butylbenzene and β-bisabolene was relatively weak. No toxicity against booklice was observed for β-bisabolene under the testing concentrations.From the results, it could be calculated that the contact toxicity of (2E,4E)-2,4-nonadienal against booklice was only 1.7 times less than that of pyrethrins (LD50 = 18.72 μg/cm2). In You et al.’s work [34], it was reported that the perilla aldehyde isolated from the essential oil of PurplePerillaaerial parts also showed significant contact toxicity against cigarette beetles (LD50 = 3.82 μg/adult). By the structural analysis, it was found that both of (2E,4E)-2,4-nonadienal and perilla aldehyde have aldehyde fragment. The toxic characteristic of these compounds against insects might be related to the presence of an aldehyde group in these compounds. Therefore, the content of (2E,4E)-2,4-nonadienal could be considered as one of the key elements which would affect the contact toxicity of the essential oil against certain kinds of insects.


### 3.3. Repellency

The results of the ANOVAs indicated that the repellent rate against both insects significantly differ with the different treatments. These results are given in Tables3 and 4.Table 3
Percent repellency (PR) of the essential oil fromBupleurum bicaule and its constituents against Lasioderma serricorne adults at 2 h and 4 h after exposure. ∗Comparison of different samples at the same concentration, df = 5.
Times Treatments Concentrations (nL/cm2) 78.63 15.73 3.15 0.63 0.13 2 h Essential oil 100 ± 0c 74 ± 12c 70 ± 12c 66 ± 11b 38 ± 4bc β-Bisabolene 90 ± 6bc 12 ± 9b 34 ± 15bc 10 ± 16a 56 ± 12c 1,4-Dimethoxy-2-tert-butylbenzene 62 ± 11a 40 ± 13bc 34 ± 15bc 6 ± 12a 14 ± 12b Bornyl acetate 74 ± 12ab −28 ± 19a −4 ± 17a −4 ± 9a −36 ± 17a (2E,4E)-2,4-Nonadienal 56 ± 12a 66 ± 20c 4 ± 19ab 16 ± 17ab 60 ± 10c DEET 88 ± 7b 76 ± 14 28 ± 7bc 20 ± 14ab 16 ± 7b F 15.878 20.895 9.823 5.244 39.261 P 0 0 0 0.002 0  4 h Essential oil 100 ± 0c 62 ± 15bcd 24 ± 19ab 28 ± 19bc 12 ± 18b β-Bisabolene 88 ± 12bc 26 ± 17b 24 ± 11ab 14 ± 17abc 34 ± 18bc 1,4-Dimethoxy-2-tert-butylbenzene 46 ± 20a 32 ± 9bc 34 ± 17ab −4 ± 16ab 28 ± 4bc Bornyl acetate 68 ± 13ab −4 ± 12a 14 ± 12ab −8 ± 18ab −18 ± 11a (2E,4E)-2,4-Nonadienal 50 ± 19a 72 ± 9cd 12 ± 18a −14 ± 16a 32 ± 19bc DEET 98 ± 2c 78 ± 9d 58 ± 16b 56 ± 14c 46 ± 7c F 15.428 17.091 2.713 5.753 13.079 P 0 0 0.044 0.001 0 ∗Different letters in the same column (2 h and 4 h, resp.) mean significant differences (P<0.05) in ANOVA and Tukey’s tests. PR was subjected to an arcsine square-root transformation before analysis.Table 4
Percent repellency (PR) of the essential oil fromBupleurum bicaule and its constituents against Liposcelis bostrychophila adults at 2 h and 4 h after exposure. ∗Comparison of different samples at the same concentration, df = 5.
Times Treatments Concentrations (nL/cm2) 63.17 12.63 2.53 0.51 0.10 2 h Essential oil 50 ± 12ab 56 ± 12bc 4 ± 17a 24 ± 19a −6 ± 20a β-Bisabolene 76 ± 15bc 74 ± 15cd 42 ± 19ab 12 ± 18a 18 ± 16a 1,4-Dimethoxy-2-tert-butylbenzene 52 ± 19ab 8 ± 19a 36 ± 13ab 22 ± 18a 16 ± 13a Bornyl acetate 12 ± 14a 20 ± 10ab 32 ± 9ab 10 ± 18a 34 ± 16a (2E,4E)-2,4-Nonadienal 18 ± 20a 74 ± 8cd 24 ± 18a 20 ± 10a 28 ± 14a DEET 100 ± 0c 98 ± 2d 78 ± 10b 66 ± 8a 8 ± 3a F 8.980 12.170 5.299 1.998 2.173 P 0 0 0.002 0.115 0.091  4 h Essential oil 44 ± 13abc 48 ± 15a 16 ± 10b 36 ± 10a −10 ± 16a β-Bisabolene 74 ± 22bc 64 ± 20a −16 ± 15a 48 ± 20a 16 ± 18ab 1,4-Dimethoxy-2-tert-butylbenzene 20 ± 15ab 28 ± 20a 18 ± 16b 30 ± 14a 18 ± 18ab Bornyl acetate 10 ± 19a 24 ± 10a 38 ± 12bc 22 ± 18a 44 ± 18 (2E,4E)-2,4-Nonadienal 28 ± 8ab 36 ± 13a 46 ± 7bc 14 ± 3a 8 ± 20ab DEET 96 ± 3c 82 ± 5a 68 ± 3c 54 ± 6a 22 ± 5ab F 7.308 2.695 10.321 2.014 2.564 P 0 0.045 0 0.113 0.054 ∗Different letters in the same column (2 h and 4 h, resp.) mean significant differences (P<0.05) in ANOVA and Tukey’s tests. PR was subjected to an arcsine square-root transformation before analysis.The essential oil showed strong repellency against cigarette beetles. After 2 h of exposure, the percent repellency (PR) of the essential oil reached 100% at the highest concentration (78.63 nL/cm2). The PR values continuously declined with the decrease of the tested concentrations. The repellent characteristics of the four compounds against the cigarette beetles were variable. At the dose of 78.63 nL/cm2, all the four compounds showed definite repellent activities against cigarette beetles, although their individual efficiency of repellency was not as good as that of the essential oil treatment. The PR values of β-bisabolene, 1,4-dimethoxy-2-tert-butylbenzene, bornyl acetate, and (2E,4E)-2,4-nonadienal were 90%, 62%, 74%, and 56%, respectively, after 2 h of exposure. However, the repellent characteristics of the four compounds were obviously different at lower concentrations. The experimental data were further analyzed by Tukey’ test, and the statistical results (see Table 3) could be used to exam the repellency level of each sample. Testing results of 0.13 nL/cm2 showed that β-bisabolene and (2E,4E)-2,4-nonadienal possessed a higher level of repellency than DEET (P=0.019 and 0.009, resp.) after 2 h of exposure. The repellency of 1,4-dimethoxy-2-tert-butylbenzene was comparable to DEET (P=0.966), while the bornyl acetate showed an insect attractant property.As the results show in Table4, theB. bicaule essential oil showed relatively weak repellency against booklice; the highest repellent rate of the essential oil was only 56% relative to DEET (PR = 98%) at the dose of 12.63 nL/cm2 after 2 h of exposure. However, some of individual compounds showed certain repellent activities. At low concentration (0.10 nL/cm2), the repellency of the four compounds was at the same level as that of DEET (P=0.952, 0.855, 1.000, and 1.000, resp.) after 2 h of exposure. At the highest concentration (63.17 nL/cm2), only β-bisabolene showed an obvious repellent activity with PR value of 76% after 2 h of exposure.The experimental results and data analysis showed that the repellent rates of the essential oil were almost comparable to those of the positive control at all testing concentrations against cigarette beetles. Each of the four compounds exhibited a different level of the repellent activity against cigarette beetles. Accordingly, it could be considered that these compounds would make various degrees of contribution to the strong repellency of the essential oil. Among them,β-bisabolene (a sesquiterpene) might be the most important one which majorly affected the repellent activity against cigarette beetles. It has been reported that the sesquiterpenes showed repellent activities against several insects. In Khani and Heydarian’s work, the essential oil ofTeucrium polium subsp.capitatum (L.), which was rich in sesquiterpene, showed 60% and 52% repellency againstTeucrium castaneum andCallosobruchus maculatus adults [35]. Based on the above findings, it was believed that the essential oil ofB. bicaule and the types of sesquiterpenes compounds would be the exploration direction of natural repellents.


## 3.1. Chemical Constituents of the Essential Oil

The yellow essential oil was obtained fromB. bicaulewith a yield of 0.04%(v/w). Its chemical components were analyzed by the GC-MS. 26 compounds were identified and their relative contents in the oil sample were determined. The total amount of all the identified compounds was 85.5% (w/w) of the essential oil. The content of trans-2-isopropylbicyclo[4.3.0]non-3-en-8-one was 25.9%, and the content of 4,5-dimethyl-1,2,3,6,7,8,8a,8b-octahydrobiphenylene was 23.5%. These two compounds could be considered as the major components of the essential oil. The results are summarized in Table 1.Table 1
Chemical composition of the essential oil ofBupleurum bicaule.
Compounds Relative content (%) a R I e x p b R I l i t Identification methodsc Aldehydes (2E,4E)-2,4-Octadienal 0.8 1113 1109 MS; RI (2E,4E)-2,4-Nonadienal 1.2 1214 1217 MS; RI (2E,4E)-2,4-Decadienal 2.8 1317 1316 MS; RI Monoterpenes Bornyl acetate 1.1 1280 1281 MS; RI Trans-2-isopropylbicyclo[4.3.0]non-3-en-8-one 25.9 1765 1765 MS; RI Sesquiterpenes α-Longipinene 0.5 1352 1351 MS; RI β-Elemene 0.8 1391 1393 MS; RI β-Cuvebene 1.5 1390 1388 MS; RI 1,4,7,-Cycloundecatriene, 1,5,9,9-tetramethyl-, Z,Z,Z- 1.9 1456 1454 MS; RI Calarene 2.1 1467 1463 MS; RI γ-Gurjunene 2.0 1479 1472 MS; RI (E,E)-α-Farnesene 0.4 1505 1505 MS; RI β-Bisabolene 2.7 1510 1511 MS; RI Cuparene 2.9 1502 1499 MS; RI (E)-Nerolidol 0.5 1565 1564 MS; RI Spathulenol 0.9 1582 1578 MS; RI Caryophyllene oxide 1.4 1583 1580 MS; RI Humulene oxide II 1.4 1607 1611 MS; RI α-Cadinol 1.4 1653 1654 MS; RI τ-Muurolol 0.6 1662 1660 MS; RI Juniper camphor 0.3 1690 1693 MS; RI Alkynes (6Z)-6-Tridecen-4-yne 2.7 1470 1470 MS; RI Aromatic compounds 1,4-Dimethoxy-2-tert-butylbenzene 4.3 1477 1477 MS; RI Myristicin 1.6 1529 1523 MS; RI 2(1H)Naphthalenone,3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)- 0.3 1772 1773 MS; RI Others 4,5-Dimethyl-1,2,3,6,7,8,8a,8b-octahydrobiphenylene 23.5 1357 1357 MS; RI  Total 85.5 aRetention index (RI) in this experiment relative to the homologous series of n-alkanes (C5–C36) on the HP-5 MS capillary column. bRI in literature. cMass spectrum.With the further analysis of the GS-MS results, it was found that the essential oil contained a plenty of monoterpenes and sesquiterpenes, and their contents accounted for 27.0% and 21.3% of the essential oil, respectively. The other types of chemical components included aldehydes, alkynes, and aromatic compounds. In a published paper [21], Xu and his partners reported that 19 compounds were identified in the root oil ofB. bicaulecollected from Inner Mongolia, China, and all the types of chemical components mentioned above also existed in Xu et al.’s oil sample. The five types of components accounted for about 60% of the essential oils in the two works. However, the significant difference between the results presented in Xu’s paper and our results could be figured out. The compound with the highest content predicted in Xu’s paper was 4-tridecen-6-yne (42.6%), but only its isomer, 6-tridecen-4-yne, was discovered in our sample and its content only accounted for 2.7% of the essential oil. The highest content compound in our sample was trans-2-isopropylbicyclo[4.3.0] non-3-en-8-one (25.9%), and it was not reported in the literature.The other differences were showed in Figure1, the content of aldehydes (12.6%) in Xu’s paper was higher than that of terpenoids (6.2%), and the monoterpenes only accounted for a minor proportion (0.8%). However, in our case, the content of terpenoids was 48.3% (27.0% for monoterpenes and 21.3% for sesquiterpenes), which was much higher than that of aldehyde compounds (4.8%). The above differences might be caused by many variables such as the growth environment of plants, the harvest time, and treatment of samples. These differences might further affect the biological activities.Figure 1
Percentage of different types of compounds in essential oils of this work and literature.It was reported that terpenoids and aromatic compounds exhibited excellent bioactivities against different stored product insect pests [29–32] and aldehydes showed fair contact toxicity and repellent activities [33]. Here, four representative compounds were selected from 26 components identified in our sample as they belong to the chemical types of monoterpenes, sesquiterpenes, aldehydes, or aromatic compounds, respectively. Their bioactivity tests were evaluated as well. They are bornyl acetate, β-bisabolene, (2E,4E)-2,4-nonadienal, and 1,4-dimethoxy-2-tert-butylbenzene. The molecular structures of the four selected compounds are shown in Figure 2.Figure 2
Molecular structures of selected compounds from the essential oil ofBupleurum bicaule.


## 3.2. Contact Toxicity

The results of contact toxicity of the essential oil and selected compounds against cigarette beetles and booklice are listed in Table2. The results showed that the essential oil exhibited fair contact toxicity against cigarette beetles with a LD50 value of 11.91 μg/adult but expressed unobservable toxicity against booklice in our measure range.Table 2
Contact toxicity ofBupleurum bicauleessential oil and its constituents against Lasioderma serricorne(LS) and Liposcelis bostrychophila(LB) adults.
Insects Treatments Concentrations (%) LD50 (µg/adult)/(µg/cm2) 95% FL(µg/adult)/(µg/cm2) Slope ± SE Chi square (χ2) LS Essential oil 1.91–10.00 11.91 7.04–15.66 2.01 ± 0.39 17.50 β-Bisabolene 2.97–15.00 24.99 21.68–29.14 3.86 ± 0.56 3.61 1,4-Dimethoxy-2-tert-butylbenzene 1.97–10.00 23.56 20.03–27.24 3.18 ± 0.55 2.90 Bornyl acetate 0.90–4.40 9.42 8.14–10.87 0.98 ± 0.17 7.53 (2E,4E)-2,4-Nonadienal 0.27–1.30 2.31 2.03–2.61 4.00 ± 0.62 9.44 Pyrethrinsa -- 0.24 0.22–0.30 3.34 ± 0.32 13.11  LB Essential oil 0–5.00 -- -- -- -- β-Bisabolene 0–50.00 -- -- -- -- 1,4-Dimethoxy-2-tert-butylbenzene 0.12–1.00 62.34 55.97–69.66 6.42 ± 0.87 3.93 Bornyl acetate 0.20–0.40 36.67 34.67–38.55 16.73 ± 2.28 7.04 (2E,4E)-2,4-Nonadienal 0.20–0.40 32.23 30.59–33.97 17.08 ± 2.31 9.66 Pyrethrinsa -- 18.72 17.60–19.92 2.98 ± 0.40 10.56 aData from Yang et al. [26].Among the four selected compounds, (2E,4E)-2,4-nonadienal showed the strongest toxicity against cigarette beetles (LD50 = 2.31 μg/adult) and booklice (LD50 = 32.23 μg/cm2). Bornyl acetate also showed certain toxicity against the two insects (LD50 = 9.42 μg/adult and 36.67 μg/cm2, resp.), while the toxicity of 1,4-dimethoxy-2-tert-butylbenzene and β-bisabolene was relatively weak. No toxicity against booklice was observed for β-bisabolene under the testing concentrations.From the results, it could be calculated that the contact toxicity of (2E,4E)-2,4-nonadienal against booklice was only 1.7 times less than that of pyrethrins (LD50 = 18.72 μg/cm2). In You et al.’s work [34], it was reported that the perilla aldehyde isolated from the essential oil of PurplePerillaaerial parts also showed significant contact toxicity against cigarette beetles (LD50 = 3.82 μg/adult). By the structural analysis, it was found that both of (2E,4E)-2,4-nonadienal and perilla aldehyde have aldehyde fragment. The toxic characteristic of these compounds against insects might be related to the presence of an aldehyde group in these compounds. Therefore, the content of (2E,4E)-2,4-nonadienal could be considered as one of the key elements which would affect the contact toxicity of the essential oil against certain kinds of insects.


## 3.3. Repellency

The results of the ANOVAs indicated that the repellent rate against both insects significantly differ with the different treatments. These results are given in Tables3 and 4.Table 3
Percent repellency (PR) of the essential oil fromBupleurum bicaule and its constituents against Lasioderma serricorne adults at 2 h and 4 h after exposure. ∗Comparison of different samples at the same concentration, df = 5.
Times Treatments Concentrations (nL/cm2) 78.63 15.73 3.15 0.63 0.13 2 h Essential oil 100 ± 0c 74 ± 12c 70 ± 12c 66 ± 11b 38 ± 4bc β-Bisabolene 90 ± 6bc 12 ± 9b 34 ± 15bc 10 ± 16a 56 ± 12c 1,4-Dimethoxy-2-tert-butylbenzene 62 ± 11a 40 ± 13bc 34 ± 15bc 6 ± 12a 14 ± 12b Bornyl acetate 74 ± 12ab −28 ± 19a −4 ± 17a −4 ± 9a −36 ± 17a (2E,4E)-2,4-Nonadienal 56 ± 12a 66 ± 20c 4 ± 19ab 16 ± 17ab 60 ± 10c DEET 88 ± 7b 76 ± 14 28 ± 7bc 20 ± 14ab 16 ± 7b F 15.878 20.895 9.823 5.244 39.261 P 0 0 0 0.002 0  4 h Essential oil 100 ± 0c 62 ± 15bcd 24 ± 19ab 28 ± 19bc 12 ± 18b β-Bisabolene 88 ± 12bc 26 ± 17b 24 ± 11ab 14 ± 17abc 34 ± 18bc 1,4-Dimethoxy-2-tert-butylbenzene 46 ± 20a 32 ± 9bc 34 ± 17ab −4 ± 16ab 28 ± 4bc Bornyl acetate 68 ± 13ab −4 ± 12a 14 ± 12ab −8 ± 18ab −18 ± 11a (2E,4E)-2,4-Nonadienal 50 ± 19a 72 ± 9cd 12 ± 18a −14 ± 16a 32 ± 19bc DEET 98 ± 2c 78 ± 9d 58 ± 16b 56 ± 14c 46 ± 7c F 15.428 17.091 2.713 5.753 13.079 P 0 0 0.044 0.001 0 ∗Different letters in the same column (2 h and 4 h, resp.) mean significant differences (P<0.05) in ANOVA and Tukey’s tests. PR was subjected to an arcsine square-root transformation before analysis.Table 4
Percent repellency (PR) of the essential oil fromBupleurum bicaule and its constituents against Liposcelis bostrychophila adults at 2 h and 4 h after exposure. ∗Comparison of different samples at the same concentration, df = 5.
Times Treatments Concentrations (nL/cm2) 63.17 12.63 2.53 0.51 0.10 2 h Essential oil 50 ± 12ab 56 ± 12bc 4 ± 17a 24 ± 19a −6 ± 20a β-Bisabolene 76 ± 15bc 74 ± 15cd 42 ± 19ab 12 ± 18a 18 ± 16a 1,4-Dimethoxy-2-tert-butylbenzene 52 ± 19ab 8 ± 19a 36 ± 13ab 22 ± 18a 16 ± 13a Bornyl acetate 12 ± 14a 20 ± 10ab 32 ± 9ab 10 ± 18a 34 ± 16a (2E,4E)-2,4-Nonadienal 18 ± 20a 74 ± 8cd 24 ± 18a 20 ± 10a 28 ± 14a DEET 100 ± 0c 98 ± 2d 78 ± 10b 66 ± 8a 8 ± 3a F 8.980 12.170 5.299 1.998 2.173 P 0 0 0.002 0.115 0.091  4 h Essential oil 44 ± 13abc 48 ± 15a 16 ± 10b 36 ± 10a −10 ± 16a β-Bisabolene 74 ± 22bc 64 ± 20a −16 ± 15a 48 ± 20a 16 ± 18ab 1,4-Dimethoxy-2-tert-butylbenzene 20 ± 15ab 28 ± 20a 18 ± 16b 30 ± 14a 18 ± 18ab Bornyl acetate 10 ± 19a 24 ± 10a 38 ± 12bc 22 ± 18a 44 ± 18 (2E,4E)-2,4-Nonadienal 28 ± 8ab 36 ± 13a 46 ± 7bc 14 ± 3a 8 ± 20ab DEET 96 ± 3c 82 ± 5a 68 ± 3c 54 ± 6a 22 ± 5ab F 7.308 2.695 10.321 2.014 2.564 P 0 0.045 0 0.113 0.054 ∗Different letters in the same column (2 h and 4 h, resp.) mean significant differences (P<0.05) in ANOVA and Tukey’s tests. PR was subjected to an arcsine square-root transformation before analysis.The essential oil showed strong repellency against cigarette beetles. After 2 h of exposure, the percent repellency (PR) of the essential oil reached 100% at the highest concentration (78.63 nL/cm2). The PR values continuously declined with the decrease of the tested concentrations. The repellent characteristics of the four compounds against the cigarette beetles were variable. At the dose of 78.63 nL/cm2, all the four compounds showed definite repellent activities against cigarette beetles, although their individual efficiency of repellency was not as good as that of the essential oil treatment. The PR values of β-bisabolene, 1,4-dimethoxy-2-tert-butylbenzene, bornyl acetate, and (2E,4E)-2,4-nonadienal were 90%, 62%, 74%, and 56%, respectively, after 2 h of exposure. However, the repellent characteristics of the four compounds were obviously different at lower concentrations. The experimental data were further analyzed by Tukey’ test, and the statistical results (see Table 3) could be used to exam the repellency level of each sample. Testing results of 0.13 nL/cm2 showed that β-bisabolene and (2E,4E)-2,4-nonadienal possessed a higher level of repellency than DEET (P=0.019 and 0.009, resp.) after 2 h of exposure. The repellency of 1,4-dimethoxy-2-tert-butylbenzene was comparable to DEET (P=0.966), while the bornyl acetate showed an insect attractant property.As the results show in Table4, theB. bicaule essential oil showed relatively weak repellency against booklice; the highest repellent rate of the essential oil was only 56% relative to DEET (PR = 98%) at the dose of 12.63 nL/cm2 after 2 h of exposure. However, some of individual compounds showed certain repellent activities. At low concentration (0.10 nL/cm2), the repellency of the four compounds was at the same level as that of DEET (P=0.952, 0.855, 1.000, and 1.000, resp.) after 2 h of exposure. At the highest concentration (63.17 nL/cm2), only β-bisabolene showed an obvious repellent activity with PR value of 76% after 2 h of exposure.The experimental results and data analysis showed that the repellent rates of the essential oil were almost comparable to those of the positive control at all testing concentrations against cigarette beetles. Each of the four compounds exhibited a different level of the repellent activity against cigarette beetles. Accordingly, it could be considered that these compounds would make various degrees of contribution to the strong repellency of the essential oil. Among them,β-bisabolene (a sesquiterpene) might be the most important one which majorly affected the repellent activity against cigarette beetles. It has been reported that the sesquiterpenes showed repellent activities against several insects. In Khani and Heydarian’s work, the essential oil ofTeucrium polium subsp.capitatum (L.), which was rich in sesquiterpene, showed 60% and 52% repellency againstTeucrium castaneum andCallosobruchus maculatus adults [35]. Based on the above findings, it was believed that the essential oil ofB. bicaule and the types of sesquiterpenes compounds would be the exploration direction of natural repellents.


## 4. Conclusions

In this work, the chemical composition ofB. bicaule essential oil was determined, and its contact and repellent activities against two stored product insects were investigated along with four individual components. A compound (2E,4E)-2,4-nonadienal with striking contact toxicity was found, and the essential oil showed considerable contact and repellent activities, which could be attributed to the existence of different chemical compounds in essential oil. Some of those components might be the material basis of active effect. This work would provide scientific basis for further development and utilization ofB. bicaule in stored product insects control.There must be a certain relationship between the bioactivity and structure of the compounds, which deserves further study and discussion.



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