Seven herbs against the stored product insect: Toxicity evidence and the active sesquiterpenes from Atractylodes lancea

Ecotoxicology and Environmental Safety 169 (2019) 807–813

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Seven herbs against the stored product insect: Toxicity evidence and the active sesquiterpenes from Atractylodes lancea Shan-shan Guoa, Yang Wanga, Xue Panga, Zhu-feng Genga,b, Ju-qin Caoa,c, Shu-shan Dua,

T

⁎

a Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Faculty of Geographical Science, Beijing Normal University, NO.19 Xinjiekouwai Street, Beijing 100875, China b Analytical and Testing Center, Beijing Normal University, NO.19 Xinjiekouwai Street, Beijing 100875, China c Medical Chemistry Department, School of Basic Medical Sciences, Ningxia Medical University, Xingqing District, Yinchuan 750004, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Herbs Essential oil Atractylodes lancea Tribolium castaneum Toxicity Sesquiterpenes

In this work, the essential oils (EO) were extracted from seven typical Chinese herbs, and their repellent and contact toxicities against Tribolium castaneum adults (red flour beetles) were evaluated. The experimental results showed that the above EOs presented the various levels of repellent and contact toxicities. The EOs extracted from A. lancea and A argyi of the Compositae (Asteraceae) family presented obvious repellent effects (Repellency Percentage > 90% at 3.15 nL/cm2 after 4 h exposure) and strong contact toxicity with LD50 values of 5.78 and 3.09 μg/adult respectively. Based on literature researches and screening results, the EO from A. lancea was analyzed by GC-MS and chosen for further identification of bioactive components. Altogether 59 chemical components were identified and 17 of them were recognized as sesquiterpene compounds, accounting for 57.8% of the total weight of the EO. From the identified sesquiterpenes, three individual compounds (β-eudesmol, hinesol, valencene) were selected for the laboratory bioassays of the toxicity against red flour beetles. It was found that all the three compounds expressed some repellent effects. Although β-eudesmol (31.2%) and hinesol (5.1%) were identified as main constituents and had been considered to be symbolic characteristics of high medicinal value, valencene (0.3%) showed strong repellent property which could be comparable to that of DEET (N, N‑diethyl‑3‑methylbenzamide), a powerful commercial pesticides, and it had best toxicity with LD50 values of 3.25 (μg/adult) in the contact test. This work may provide toxicity evidence of seven common herbs against red flour beetles, add the information for the development and comprehensive utilization of A. lancea, and will contribute to the application of grain preservation.

1. Introduction The stored-product pests usually cause a great deal of economic loss and are harmful to human health, since they eat the stored products, especially food and industrial crops, and release pollutants (defecation), which then make fungi and bacteria easy to breed. The red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), is an important harvested products pest which widely distributes in the world (Herron, 1990; Campbell et al., 2004; Garcìa et al., 2005; Fedina and Lewis, 2007). Although this beetle has indirect contact with human health, it belongs to risk factor as well. The beetle itself is the intermediate host of Pseudanoplocephala crawfordi (Li et al., 1982), and its secretions usually contain some kinds of quinoids which could be responsible for tumor induction (Happ, 1968). T. castaneum is now used as a model organism and it has important research status in genetics, immunology, molecular ecology, toxicology and other research fields ⁎

(An, 2009). The synthetic broad spectrum pesticides have been extensively used to control stored-product pests. No doubt, this method have effectively limited the infestation of the pests in the warehouse. However, the unrestrained use of synthetic broad spectrum pesticides to control pests has been known to cause several long-term side-effects on other living creatures (Theiling and Croft, 1988; Cônsoli et al., 1998; Biondi et al., 2012). It has been expected to develop new security techniques or new types of safe pesticides for the control of stored-product pests. Since ancient time of China, some herbs were burnt in burners or contained in bags (sachets) to emit fragrance, preventing illness and repelling insects, as these herbs could emit the special smell (Lu and Lo, 2015; Yan, 1985). Recently, the resource of herb plants has increasingly attracted the scholarly attention and they might be considered as the safe natural materials to develop new effective insecticides (Adebayo et al., 1999; Gbolade et al., 2000; Isman, 2004, 2006). Our previous researches have

Corresponding author. E-mail address: [email protected] (S.-s. Du).

https://doi.org/10.1016/j.ecoenv.2018.11.095 Received 16 August 2018; Received in revised form 19 November 2018; Accepted 20 November 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

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belongs to the Araceae family. The species was determined and the voucher specimen (labelled as BNU-Dushushan-xxxx-xx-xx) deposited at the Herbarium in the College of Resources Science and Technology, Beijing Normal University. All the collected samples were dried naturally and ground into powders. Then each of the powder samples was extracted by hydrodistillation for 6 h separately. The water left was further removed by adding some of the anhydrous sodium sulphate. The EOs collected were then stored in a refrigerator at 4 °C.

already investigated the behavioral and contact toxicities of some common Chinese herbs on some stored product insects (Zhang et al., 2014, 2015; You et al., 2014a; Wang et al., 2014; Wang, 2015), but the work using this model insect was still lacking. To find more evidence for the toxicity of herbs against red flour beetles, this work evaluated and compared the toxicity of essential oils (EO) of the seven common Chinese herbs, Atractylodes lancea (Thunb.) DC., Artemisia argyi Lévl. et Van., Mentha haplocalyx Briq., Perilla frutescens (L.) Britt. Alpinia officinarum Hance, Amomum tsaoko Crevost et Lemarie and Acorus calamus (Linn.). During our screening, the EO of A. lancea was found to possess relatively high toxicity. A. lancea was a perennial plant widely distributed in China (Institute of Botany, 1975). Based on literature research, the extracts and some constituents of this plant were observed to have insecticidal effects. The diethyl ether extracts of A. lancea rhizomes were confirmed to have fumigant activity against three product insects (Oryzaephilus surinamensis, Sitophilus oryzae and Liposcelis paeta adults) (Lü, He, 2010). After that, from ethanol extracts of the plant, our previous work found the contact and repellent activities of four isolated compounds (atractylodin, atractylodinol, atractylenolides II and III) against the red flour beetles (Chen et al., 2015b). In addition, the experiment showed that some sesquiterpenes isolated from the EO of A. chinensis (relative plant in the same genus) possessed contact toxicity against Drosophila melanogaster adults (Chu et al., 2010). The results implied that the EO of A. lancea might have the similar activity against the stored product insects. The objective of this work is to measure the contact and repellent activity of EOs extracted from seven Chinese common spices against red flour beetles. In this work, repellent and contact activities of EOs extracted from seven spices of plants were evaluated, using T. castaneum adults as the target insect. All the seven plants were identified as typical Chinese herbs, and they widely distributed in China. The results were expected to provide the more experimental evidence for the research of further insect toxicity, the development of new safe pesticides and the use of the natural resource.

2.3. Instrumental analysis GC-MS analysis was performed on a Thermo Finnigan Trace DSQ instrument coupled with a flame ionization detector and an HP-5 MS (30 m × 0.25 mm × 0.25 µm) capillary column. The oven temperature was programmed at 50 °C for 2 min, then increased to the temperature of 150 °C at 2 °C/min and held for 2 min, and then increased to the final temperature of 250 °C at 10 °C/min, and held for 5 min. The injector temperature was set at 250 °C. 1 μL sample (1% solution diluted in nhexane) was injected. Mass spectra were recorded with the mass range m/z 50–550. Relative percentages of the individual components of the EO were calculated from the integration of the peaks in FID chromatograms. Most constituents were identified by comparison of their retention indices with retention index (RI) to a series of n-alkanes (Adams, 2001). Computer matching on NIST 05 and Wiley 275 libraries was used for identification. 2.4. Contact toxicity

T. castaneum were obtained from laboratory cultures maintained for the last 2 years. They were reared in glass containers (0.5 L) containing feed (wheat/yeast, 10:1, w/w) at 12–13% moisture content in the dark incubators at 29 ± 1 °C and 70–80% relative humidity. Adults used in all the experiments were about 7 ± 2 days old regardless of gender.

The contact toxicity against red flour beetles was evaluated with the method (topical application) described by Liu and Ho (1999). The samples include the seven EOs and the individual compounds from the EO of A. argyi (hinesol, β-eudesmol, and valencene). Hinesol and βeudesmol, were obtained from the EO sample of A. lancea by isolation on silica gel column chromatography (Chen et al., 2015b) and were identified by the analysis of MS and NMR spectra. Valencene were purchased from Sigma-Aldrich. The EO samples and the pure compound samples were quantitatively dissolved in n-hexane separately to prepare the stock solutions. After appropriate testing concentrations were determined by a range-finding pretesting, serials of testing solutions with five concentrations were prepared by diluting the stock solution for each of the testing samples. Aliquots of diluent (0.5 μL) were applied topically to the dorsal thorax of the beetles carefully and then transferred to glass vials (10 insects per vial, five replicates per dose). Both treated and control (n-hexane) group insects were kept in dark incubators. Mortality was determined after 24 h of treatment.

2.2. Plant materials and extraction of EO

2.5. Repellent toxicity

The plant samples of the seven herbs were collected in different areas of China. The basic information about samples was listed in Table 1. A. lancea and A. argyi belongs to the Compositae family, M. haplocalyx and P. frutescens belongs to the Labiatae family, A. officinarum and A. tsaoko belongs to the Zingiberaceae family and A. calamus

The repellent activity was tested using the area preference method (Zhang et al., 2011). EO and pure compound samples were diluted in nhexane to prepare serials of testing solutions to five concentrations (78.63, 15.73, 3.15, 0.63 and 0.13 nL/cm2). Filter paper (ϕ 9 cm) was cut into two pieces equally. 500 μL of testing solution was uniformly

2. Materials and methods 2.1. Insects

Table 1 EOs of the seven Chinese herbs. Species

Origin

Used Parts

Dry Weight (kg)

Yields%

Voucher Specimen

A. lancea A. argyi M. haplocalyx P. frutescens A. officinarum A. tsaoko A. calamus*

Anguo, Hebei province (purchased) Jining, Shandong province Jining, Shandong province Beijing Zhanjiang, Guangdong province Yulin, Guangxi Province Dali, Yunnan province

Rhizomes Aerial parts Aerial parts Aerial parts Rhizomes Fruits Rhizomes

0.5 2.0 0.6 9.0 1.9 0.9 3.5

0.8 0.5 1.7 0.1 0.6 0.2 0.9

BNU-Dushushan-2017-11-16-06 BNU-CMH-Dushuahan-2013-10-22-006 BNU-CMH-Dushushan-2013-10-22-101 BNU-CMH-Dushushan-2013-07-09-008 BNU-CMH-Dushushan-2013-06-11-014 BNU-Dushushan-2013-06-09-24 BNU-CMH-Dushuahan-2012-11-25-006

* Data has been reported (Chen et al., 2015a). 808

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Fig. 1. The Bar-diagrams of Repellency percentage (RP) for seven EOs against T. castaneum adults after 2 (A) and 4 h Exposure (B) a. a Means in the same column (2 h and 4 h, respectively) followed by the same letters do not differ significantly (p > 0.05) in ANOVA and Tukey’s tests. RP was subjected to an arcsine square-root transformation before ANOVA and Tukey’s tests. b Data from Chen et al. (2015a). c DEET, N, N‑diethyl‑3‑methylbenzamide, were used as a positive control. Data from Yang et al. (2014).

Table 2 Contact toxicity of seven EOs against red flour beetles. Treatments

LD50 (106 μg/kg)

95% FL (106 μg/ kg)

LD50 (μg/adult)

95% FL (μg/adult)

Slope ± SE

χ2

P

A. lancea A. argyi M. haplocalyx P. frutescens A. officinarum A. tsaokoa A. calamusb Pyrethrinsc

3.40 1.82 10.00 0.82 12.18 9.72 8.47 0.15

3.15–3.69 1.63–2.02 8.76–11.17 0.54–1.04 1.74–21.09 8.01–11.45 6.45–10.24 0.12–0.18

5.78 3.09 17.01 1.39 20.71 16.52 14.40 0.26

5.35–6.28 2.77–3.43 14.90–18.99 0.91–1.76 2.96–35.85 13.62–19.47 10.97–17.41 0.22–0.30

6.67 4.10 4.13 2.57 1.39 1.68 3.05 3.34

6.96 19.95 13.88 20.89 14.22 9.25 14.49 13.11

0.725 0.645 0.930 0.588 0.942 0.971 0.904 0.950

a b c

± ± ± ± ± ± ± ±

0.78 0.45 0.50 0.47 0.41 0.27 0.49 0.32

Data from Wang et al. (2014). Data from Chen et al. (2015a). Data from You et al. (2014b).

painted on a half of the filter paper. The other half was treated with 500 μL of n-hexane (solvent control). The two halves of filter paper were air-dried to remove the solvent (after about 30 s), then were sticked side by side on the bottom of Petri dishes (ϕ 9 cm). With this way, the insects could move freely on the two pieces of the filer paper, while the penetrations of the testing solution between two pieces could be effectively limited. Twenty insects were released in the center of each filter paper disk, and a cover was placed over the Petri dish. For the repellent activity experiment, the number of insects present on each half were recounted after 2 and 4 h. Five tests were done per dose. The above procedure was repeated for three times.

PR (%) = [(Nc-Nt) / (Nc+Nt)] × 100 where Nc is the number of insects present in the negative control half and Nt is the number of insects present in the treated half. The averages were later assigned to different classes (0 to V) using the following scale (percentage repellency). Class, % repellency: 0, 0.01–0.1; I, 0.1–20.0; II, 20.1–40.0; III, 40.1–60.0; IV, 60.1–80.0; and V, 80.1–100.(Liu et al., 2007) Percentage was subjected to an arcsine square-root transformation before variance and Tukey’s tests. Analysis of variance (One-Way ANOVA and GLM Univariate) and Tukey’s test were conducted by using SPSS 20.0 (IBM) for Windows 2007. 3. Results and discussion

2.6. Statistical analysis The EO yields of the seven herbs were listed in Table 1. The EO yields of the seven samples were varied from 0.1% to 1.7%, although the extraction procedure and experimental condition were identical. It was believed that the difference of the EO yields should be related to

For the contact toxicity test, the LD50 values were calculated using Probit analysis, SPSS 20.0 (Sakuma, 1998). The percent repellency (PR) of each sample was then recalculated using the formula: 809

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tsaoko even exhibited the attracting effect under the experimental conditions. Comparing the bar diagrams given in Fig. 1 (A and B), it was found that in the testing rang, the characteristics of the RP values were quite similar for most of the testing samples except those of M. haplocalyx and A. calamus samples at two lower testing doses. At the testing doses of 0.63 nL/cm2, the RP values of M. haplocalyx and A. calamus samples observed after 4 h exposure were much less than those obtained after 2 h exposure. The results of the contact toxic evaluation of the EOs were summarized in Table 2. Some information could be derived from this Table: 1) All the EO samples showed contact toxic effect against the red flour beetle, and the goodness-of-fit test displayed that P values of all the seven samples were larger than 0.05. 2) LD50 value of P. frutescens EO was 1.39 μg/adult, which was much larger than the LD50 value (0.26 μg/adult) of the popular botanical insecticide, pyrethrins. It meant that the contact activity of P. frutescens sample was about 5 times less than that of pyrethrins. 3) The LD50 values of two Compositae plant samples, A. lancea and A. argyi, were 5.78 and 3.09 μg/adult, which were even larger than the LD50 values of P. frutescens EO. However, these two EO samples still expressed the better contact toxic effect under a certain condition. These A. lancea and A. argyi samples got the relatively larger slope values (6.67 and 4.10, Table 2) after the linear regression of the experimental data (y = 6.67x - 23.02; y = 4.10x 15.94, respectively). Lethality of the pests would rapidly rise up, with the testing concentrations of A. lancea sample increased. So that, it could be recognized that the red flour beetles were more sensitive to the variation of the testing concentrations of A. lancea sample. In practice, lethality of the pests treated with A. lancea EO was higher than those treated with A. argyi EO, as the testing concentration was larger than 5.3 μg/adult. The seven EO samples exhibited the various levels of repellent and contact toxicities. Among them, samples of the two Compositae plants (A. argyi and A. lancea) expressed the effective toxic properties against the red flour beetle. A. lancea was one of the plant origins of a traditional Chinese medicine, Rhizoma Atractylodis (Chinese Pharmacopoeia Committee, 2015). It was used for diuretic and analgesic purposes or for treating stomach disorders (Kubo et al., 1983; Nogami et al., 1986). However, there has been no report on the bioactivities of the EO extracted from the rhizomes of A. lancea against stored product insects. Thus, it is necessary to investigate the EO of A. lancea further. Here, the chemical components were determined, and then, some of the identified compounds were selected for the assay of repellent and contact activities against the red flour beetle.

Table 3 Chemical composition of the EO of A. lancea. Compounds

RIa

Content (%)

Identified Methodb

α-Pinene Camphene Sabenene α-Phellandrene o-Cymene (E)-β-Ocimene γ-Terpinene Terpinolene α-Terpineol cis-Sabinol 2-Methoxy-4-methyl-1-(1-methylethyl)benzene Bornyl acetate Linalyl propionate α-Bulnesene Geranyl acetate Helminthogermacrene Isocaryophyllene γ-Elemene α-Caryophyllene Dihydro-cis-α-copaene-8-ol 2-Isopropenyl-4a,8-dimethyl1,2,3,4,4a,5,6,7octahydronaphthalene β-Selinene α-Selinene Valencene β-Sesquiphellandrene Elemol γ-Patchoulene γ-Eudesmol Hinesol β-Eudesmol α-Bisabolol 1,5,5-Trimethyl-6-(2-propenylidene)-1cyclohexene (E)-10-Pentadecen-5-yn-1-ol 4-Biphenylcarboxaldehyde 1,3,3-Trimethyl-1-phthalanol Heneicosane Monoterpenoids Sesquiterpenoids Total

932 954 971 1005 1010 1038 1057 1080 1188 1196 1244

7.3 1.7 0.5 8.2 1.2 0.4 0.2 0.8 1.3 1.0 0.2

MS; MS; MS; MS; MS; MS; MS; MS; MS; MS; MS;

RI RI RI RI RI RI RI RI RI RI RI

1285 1339 1342 1394 1396 1409 1437 1454 1465 1483

0.3 0.2 0.2 0.3 1.2 0.3 0.9 0.2 1.0 0.8

MS; MS; MS; MS; MS; MS; MS; MS; MS; MS;

RI RI RI RI RI RI RI RI RI RI

1489 1492 1497 1517 1551 1620 1621 1637 1648 1681 1701

7.8 0.2 0.3 0.3 2.9 0.1 1.8 5.1 31.2 3.5 1.1

MS; MS; MS; MS; MS; MS; MS; MS; MS; MS; MS;

RI RI RI RI RI RI RI RI RI RI RI

1707 1726 1814 1863

1.4 2.7 0.1 0.1 21.8 57.8 86.9

MS; MS; MS; MS;

RI RI RI RI

a RI, retention index as determined on a HP-5MS column using the homologous series of n-hydrocarbon. b MS, mass spectrum.

the differences of the spices, sampling locations and seasons, used parts or etc. These extracted EOs were used as the testing samples for toxic evaluation against red flour beetles.

3.2. Chemical composition of A. lancea EO The yield of the EO was 0.8% (v/w), with the density of 0.91 g/ml. By the analysis of GC-MS, 59 components were identified from the EO sample of A. lancea and their contents were accounted for 86.9% of the total components present (see Table 3). Seventeen of them accounting for 57.8% of the total weight of the EO were recognized as sesquiterpene compounds. It has been mentioned that sesquiterpenes were considered as the feature chemical components of plants of the Compositae family, and some of them showed the toxic or feeding deterrents to herbivore insects (Fraga, 2004; Wu et al., 2006; Wang et al., 2018). In the EO sample of A. lancea, the main sesquiterpene compounds included β-eudesmol (31.2%), α-phellandrene (8.2%), β-selinene (7.2%), and hinesol (5.1%). This result was some different from the data reported in the published paper (Ji et al., 2001; Guo et al., 2002; Xu et al., 2007; Zhang et al., 2010; Zeng et al., 2012). It was believed that the difference was causes by the location of the collection and the plant physiological status. The contents of β-eudesmol (31.2%) and hinesol (5.1%) were relatively higher in our samples. This phenomenon was well agreed with the view, which predicted that A. lancea and A. chinensis could be classified into one of the groups from Atractylodes

3.1. Toxicity of the EOs The experimental results of their repellent were shown in Fig. 1. By analyzing the results of the repellent tests against red flour beetles after 2 h exposure, it was found that: 1) All the samples showed repellent activities as the testing dose was higher than 15.83 nL/cm2. EOs of A. argyi, A. lancea, M. haplocalyx, P. frutescens and A. calamus exhibited more significant repellent activities, since all values of the repellency percentage (PR) were higher than 90% as the testing doses were higher than 15.83 nL/cm2. 2) The PR values of all the above five samples decreased with the decrease of the testing doses just like those of DEET (a positive control). However, each value at the different doses was better than that of DEET in all the testing range after 2 h treatment. 3) The EO samples of two Zingiberaceae plants (A. officinarum and A. tsaoko) presented relatively weaker repellent activities. As the testing doses were lower than 3.15 nL/cm2, the EO of A. officinarum presented a rather low repellent effect against red flour beetles, while the EO of A. 810

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Fig. 2. Repellency percentage (RP) for the EO and the four individual constituents after 2 (A) and 4 h Exposure (B) a. a Means in the same column (2 h and 4 h, respectively) followed by the same letters do not differ significantly (P > 0.05) in ANOVA and Tukey’s tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukey’s tests. b Data from You et al. (2015). c Data from Yang et al. (2014).

Table 4 Contact toxicity of EO of A. lancea rhizomes and the individual sesquiterpenes. Treatments

LD50 (106 μg/kg)

95% FL (106 μg/ kg)

LD50 (μg/adult)

95% FL (μg/adult)

Slope ± SE

χ2

P

A. lancea Hinesol β-Eudesmol Valencene γ-Elemenea Pyrethrinsb

3.40 – 35.73 1.91 – 0.15

3.15–3.69 – 31.14–43.21 1.63–2.20 – 0.12–0.18

5.78 – 60.74 3.25 – 0.26

5.35–6.28 – 52.93–73.45 2.78–3.74 – 0.22–0.30

6.67 – 3.51 2.65 – 3.34

6.96 – 11.25 17.94 – 13.11

0.725 – 0.981 0.928 – 0.950

a b

± 0.78 ± 0.48 ± 0.30 ± 0.32

Data from Liang et al. (2016). Data from You et al. (2014b). Fig. 3. Structures of the sesquiterpenes. (A) A showed the three bicyclic sesquiterpenes identified from the EO of A. lancea, including spirocyclic bicyclic sesquiterpene and fused bicyclic sesquiterpene (B) B showed three monocyclic sesquiterpenes, including γ-elemene which identified from the EO of A. lancea and two sesquiterpenes isolated from the EO of A. calamus in our previous paper with similar structure.

plants, since both of them contained either β-eudesmol or hinesol as the main constituents (Kitajima et al., 2003).

3.3. Toxicity of the individual compounds The repellent and contact activities against T. castaneum adults were tested for the three individual compounds (β-eudesmol, hinesol and valencene) identified in the EO sample of A. lancea. The experimental 811

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results were presented in Fig. 2 and Table 3 along with DEET and γelemene. γ-Elemene was one of the components identified in the EO, and its toxicity data obtained under the same experimental conditions as this work has been reported by our research group (Liang et al., 2016) From the bar-diagrams given in Fig. 2, it could be seen that all the four identified compounds showed repellent toxicity, especially valencene and γ-elemene exhibited stronger and longer effects. 1) At the first three testing concentrations (78.63, 15.83 and 3.15 nL/cm2), the repellent activities of alencene and γ-elemene were at the same level (P > 0.05) as those of DEET. 2) At the same concentration range, the repellent effect of two main components β-Eudesmol and hinesol was relatively weak, both of them displayed lower repellency level (P < 0.05) than those of DEET. Only at the highest testing dose, the repellent toxicity of β-eudesmol reached Class V. 3). At the highest testing concentration (78.63 nL/cm2), hinesol expressed 60% repellent effect, but the repellent activity of hinesol dropped down rapidly, when the lower doses were adopted. The PR values hinesol were lower than 20% at the last three doses. The four individual compounds showed the different contact activities against red flour beetles (see Table 4). As mentioned above, the EO of red flour beetles exhibited the fair activity (LD50 = 5.78 μg/ adult) against the pests, but the insecticidal effect of its two main components (β-eudesmol and hinesol) was not remarkable. The LD50 value of β-eudesmol was as large as 60.74 μg/adult, and this value of hinesol even could not derive in the testing concentration range. Meanwhile, valencene was one of the minor (0.3%) component in the EO, but it presented obvious toxic activity (LD50 = 3.25 μg/adult). Although γ-elemene had an obvious repellent activity, its contact effect could not be observed. In addition, it was also found that the two fused bicyclic sesquiterpenes, β-eudesmol and valencene possessed contact toxicity against red flour beetles, while the spirocyclic bicyclic sesquiterpenoid (hinesol) and monocyclic sesquiterpene (γ-elemene) were totally ineffective within the test concentration range. The structures of β-eudesmol and valencene were similar, but their contact activities were significantly different. This difference might be caused by the variety of the double bond position on the main structures (Stompor et al., 2015). In the field of traditional Chinese medicine, the white cotton-like crystals (hinesol and β-eudesmol) on rhizoma transection of A. lancea surface were considered to be symbolic characteristics of high quality of the crude drug (Institute of Botany, 1975; Yoshioka et al., 1959). Despite the fact that hinesol and β-eudesmol were the main constituents and have been considered to be symbolic characteristics of high medicinal value, they may be not the contributors in contact and repellent toxicity. Other active compounds in small amounts (such as valencene and γ-elemene) maybe act as actual active material basis. Compositae family was endowed with abundant sesquiterpenes natural products. Hinesol, β-eudesmol and valencene belong to vetispirane, eudesmane- and valencane-types separately. Previous research seldom demonstrated that hinesol or vetispirane possessed any antiinsect effects (Chu et al., 2011). However, the sesquiterpenes with eudesmane skeletons were the noteworthy points in insect toxicological research. β-Eudesmol has been found to exhibit repellent activity against Liposcelis bostrychophila (Chen et al., 2015) and red flour beetles (You et al., 2015). In addition, it was also toxic to fruit flies and some important mosquitoes vectors, such as Drosophila melanogaster (Chu et al., 2011), Culex pipiens (Park and Park, 2012.) Anopheles quadrimaculatus and Aedes aegypti (Ali et al., 2015), as well as leaf-cutting ants (Atta species) and the fire ant, Solenopsis invicta (Marinho et al., 2006). As for valencene, it was found to have toxicity to Anopheles minimus and Coptotermes formosanus (Zhu et al., 2003; Tisgratog et al., 2017). The toxicities of the hinesol and valencene were found in this work for the first time. Some relation could be further found by comparing the structures of γ-elemene with the two active compounds (shyobunone and

isoshyobunone) in our previous paper of A. calamus, one of the seven herbs in this work (Chen et al., 2015a). The previous report has already described that shyobunone and isoshyobunone were a pair of isomers with a double bond located at different positions along the isopropyl side chain, and they were observed to have different contact and repellent against red flour beetles (Fig. 3B). It's worth noting that their molecules both had ketone carbonyl in the ring of the parent nucleus. Compared with shyobunone, the structure of isoshyobunone was more similar to γ-elemene. With ketone carbonyl group, the contact toxicity of isoshyobunone improved significantly with LD50 value of 61.90 μg/ adult, and it showed over 60% repellency at the last three low concentrations (3.15, 0.63 and 0.13 nL/cm2), which was more effective than shyobunone and γ-elemene. The differences might be resulted from the location of the conjugated system as well as the adding of oxygen-containing groups. However, from a cost-benefit standpoint, although these seven EOs or sesquiterpenes possessed equivalent or greater toxicity to the chemical synthetics, both the EOs and its bioactive compounds more expensive than the commercial insecticides at this stage. Their natural abundance, preparation techniques and security are also a bottleneck to their large-scale development. Further research will focus on screening more pesticidal natural products from these common Chinese herbs and conduct safety evaluations. 4. Conclusion The findings confirm that the seven EOs from seven common herbs all have repellent and contact toxicities against T. castaneum adults. Based on literature researches and screening results, the EO from A. lancea rhizomes was chosen for further identification of bioactive components. The repellent and contact toxicities of the active sesquiterpenes were also measured. Although β-eudesmol and hinesol were identified as main constituents and had been considered to be symbolic characteristics of high medicinal value, valencene showed strong repellent property and best toxicity. This work may provide toxicity evidence of seven common herbs against red flour beetles, add the information for the development and comprehensive utilization of A. lancea, and will contribute to the application of grain preservation. Acknowledgments This project was supported by National Key Research and Development Program [grant numbers 2016YFC0500805]. We thank Dr. Q.R. Liu (College of Life Sciences, Beijing Normal University, Beijing, China) and Dr. Q. Cai (College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, Liaoning, China) for the identification of the herbs. References Ali, A., Tabanca, N., Demirci, B., Blythe, E.K., Ali, Z., Baser, K.H.C., Khan, I.A., 2015. Chemical composition and biological activity of four Salvia essential oils and individual compounds against two species of mosquitoes. J. Agric. Food Chem. 63 (2), 447–456. An, F.M., 2009. Toxicological Properties of Acetycholinesterase in Model Insect Tribolium castaneum (Herbst) (Master degree). Southwest University, China, Chongqing. Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography/ quadrupole Mass Spectroscopy. Allured Pub, Corporation, USA. Adebayo, T.A., Gbolade, A.A., Olaifa, J.I., 1999. Comparative study of toxicity of essential oils to larvae of three mosquito species. Niger. J. Nat. Prod. Med. 3, 74–76. Biondi, A., Desneux, N., Siscaro, G., Zappalà, L., 2012. Using organic-certified rather than synthetic pesticides may not be safer for biological control agents: selectivity and side effects of 14 pesticides on the predator Orius laevigatus. Chemosphere 87 (7), 803–812. Campbell, J.F., Arthur, F.H., Mullen, M.A., Steve, L.T., 2004. Insect management in food processing facilities. Adv. Food Nutr. Res. 48, 239–295. Chu, S.S., Jiang, G.H., Liu, Z.L., 2011. Insecticidal compounds from the essential oil of Chinese medicinal herb Atractylodes chinensis. Pest Manag. Sci. 67 (10), 1253–1257. Cônsoli, F.L., Parra, J.R.P., Hassan, S.A., 1998. Side-effects of insecticides used in tomato fields on the egg parasitoid Trichogramma pretiosum Riley (Hym.,

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