Chemical Composition and Insecticidal Activity of Essential Oils from Zanthoxylum dissitum Leaves and Roots against Three Species of Storage Pests

This work aimed to investigate chemical composition of essential oils obtained from Zanthoxylum dissitum leaves and roots and their insecticidal activities against several stored product pests, namely the cigarette beetle (Lasioderma serricorne), red flour beetle (Tribolium castaneum) and black carpet beetle (Attagenus piceus). The analysis by GC-MS of the essential oils allowed the identification of 28 and 22 components, respectively. It was found that sesquiterpenoids comprised a fairly high portion of the two essential oils, with percentages of 74.0% and 80.9% in the leaves and roots, respectively. The main constituents identified in the essential oil of Z. dissitum leaves were δ-cadinol (12.8%), caryophyllene (12.7%), β-cubebene (7.9%), 4-terpineol (7.5%) and germacrene D-4-ol (5.7%), while humulene epoxide II (29.4%), caryophyllene oxide (24.0%), diepicedrene-1-oxide (10.7%) and Z,Z,Z-1,5,9,9-tetramethyl-1,4,7-cycloundecatriene (8.7%) were the major components in the essential oil of Z. dissitum roots. The insecticidal activity results indicated that the essential oil of Z. dissitum roots exhibited moderate contact toxicity against three species of storage pests, L. serricorne, T. castaneum and A. piceus, with LD50 values of 13.8, 43.7 and 96.8 µg/adult, respectively.


Introduction
Traditional Chinese Medicines (TCMs) are gaining popularity as a form of complementary and alternative medicine [1]. Over the past decades, research on TCMs has mainly focused on their efficacy [2][3][4][5]. However, studies concerning on the storage and stability of TCMs are insufficient. TCMs mainly include Chinese medicinal herbs and animals parts, which are very vulnerable to insect attack during storage. The antagonistic storage method is one of traditional TCM conservation methods and has been in use for a long time, playing an important role in the protection and utilization of TCM resources. It mainly utilizes some Chinese Medicinal Materials (CMMs) with special volatile odors to prevent another CMM from suffering insect attacks. With the increasing attention of ecological protection and medication security, it is believed that this sustainable control method for stored-product pests would have broad application prospects in the future. In order to develop this traditional method of prevention and control of storage pests, we have established a screening program for new agrochemicals from CMMs [6][7][8][9][10], in which we focus on the volatile substances due to their major role in the antagonistic storage process. During this screening process, the essential oil of Zanthoxylum dissitum Hemsl was found to possess insecticidal activity against three species of stored-product pests, the cigarette beetle (Lasioderma serricorne Frbricius), red flour beetle (Tribolium castaneum Herbst) and black carpet beetle (Attagenus piceus Oilver), which are dominant populations in the stored CMM insect community and also widely distributed pests that destroy stored products worldwide [11][12][13].
Z. dissitum, a climbing woody vine, belongs to the family Rutaceae and is mainly distributed in Guangxi, south of Shanxi and Gansu, Guizhou, and west Sichuan Province [14]. The roots, stems and leaves are used as traditional Chinese medicinal herbs for the treatment of many painful conditions such as sprains, lower back and leg pain, and fractures [15]. Phytochemical studies on Z. dissitum have revealed the presence of coumarins, alkaloids, amides and phytosterols [16][17][18], but to the best of our knowledge there are no reports available on the essential oil of Z. dissitum or its insecticidal activity against stored product insects. Thus, the chemical composition of essential oils derived from Z. dissitum leaves and roots and their insecticidal activities towards three species of stored-product pests are presented in this paper for the first time. It was expected that this work would provide more theoretical background for the conception of useful antagonistic storage methods and comprehensive utilization of TCM resources.

Chemical Composition of the Essential Oils
The GC-FID and GC-MS analysis results for the Z. dissitum essential oils are summarized in Table A1. The Z. dissitum leave and root essential oil yields were 0.9% and 0.3% (v/w), respectively.

Insecticidal Activities
Percent fumigant/contact mortality of each beetle species after 24 h exposure to increasing concentrations of the two essential oils is shown in Table 1.  The essential oils of Z. dissitum leaves and roots exhibited weak fumigant toxicity against L. serricorne adults, T. castaneum adults and A. piceus larvae, with the highest concentration tested (50%) causing less than 50% mortality. In the contact toxicity assays, the highest concentration (50%) of essential oil from roots caused 100% beetle mortality, while the percent mortality caused by 50% concentration of essential oil from leaves was only 33% in L. serricorne adults, 53% in T. castaneum adults and 22% in A. piceus larvae, respectively. The contact toxicity of essential oil from the roots was thus stronger than that of the leaves. In this context, we determined the appropriate testing concentrations of the essential oil of Z. dissitum roots to the three species of insects and calculated the LD50 values by using Probit analysis, with the results listed in Table 2. The three species of storage pests treated by the essential oil of Z. dissitum roots revealed different sensitivity, with LD50 values of 13.8, 43.7 and 96.8 µg/adult, respectively. In Figure 1, it can be seen that the contact toxicity of essential oil from roots towards L. serricorne adults, T. castaneum adults and A. piceus larvae was concentration-dependent.

Chemical Composition of the Essential Oils
As shown in Table A1, the essential oils from two parts of the plant consisted mainly of sesquiterpenoids, which accounted for 74.0% in the leaves and 80.9% in the roots. However, among the chemical components of the two essential oils, there were only nine identical constituents, and the percentage of most of the same components showed great differences. These observed differences in chemical composition and content between the essential oils of leaves and roots could be due to the different effects of environmental factors (such as sunlight, water and soil) on the aerial parts and underground parts, or may result from different metabolic pathways in the plant. These variations of chemical composition may also lead to different biological activities.

Insecticidal Activities
During the fumigant toxicity tests, the essential oils from Z. dissitum leaves and roots showed weak toxicity to L. serricorne adults, T. castaneum adults and A. piceus larvae (Table 1), even though some components of the two essential oils such as caryophyllene oxide, 4-terpineol, estragole and linalool were found to possess fumigant toxicity against T. castaneum adults in previous reports [19][20][21]. This might be due to the high content of sesquiterpenoids and their relative distribution ratio in the essential oils from Z. dissitum leaves and roots. This suggested that the majority of the sesquiterpenoids in the Z. dissitum essential oils demonstrated antagonistic effects in the fumigant toxicity process.
The essential oils of Z. dissitum roots showed strong contact toxicity against L. serricorne adults, T. castaneum adults and A. piceus larvae (Tables 1 and 2). When compared with the positive control pyrethrins, the essential oil demonstrated 57, 168 and 16 times less acute toxicity against the three species of storage pests (Table 2). However, compared with the other essential oils reported in previous studies (Figure 2), the essential oil of Z. dissitum roots exhibited stronger contact toxicity against L. serricorne, e.g., essential oils of Litsea cubeba (LD50 = 27.3 μg/adult) [22], Cinnamomum camphora (LD50 = 21.3 μg/adult) [23], Myristica fragrans (LD50 = 19.3 μg/adult) [7]. The essential oil showed similar contact toxicity to the essential oils of Alpinia blepharocalyx (LD50 = 15.0 μg/adult) and Artemisia stolonifera (LD50 = 12.7 μg/adult) [24,25]. Meanwhile, the essential oil of Z. dissitum roots was found to have weaker toxicity to T. castaneum in comparison with the essential oils of Artemisia stolonifera (LD50 = 8.6 μg/adult) and Liriope muscari (LD50 = 13.4 μg/adult) [25,26].  The leaves and roots were air-dried for one week and ground to a powder. Then the powder was subjected to hydrodistillation for 6 h using a Clevenger-type apparatus. Anhydrous sodium sulfate was used to remove water after extraction. The essential oils were stored at 4 °C for subsequent experiments.

Gas Chromatography and Mass Spectrometry
Gas chromatographic (GC) analysis was performed on an Agilent 6890N instrument equipped with a flame ionization detector and an HP-5MS (30 m × 0.25 mm × 0.25 μm) capillary column. The column temperature was programmed at 50 °C for 3 min then increased at 10 °C/min until the final temperature of 290 °C was reached, where it was held for 20 min. The injector temperature was maintained at 250 °C. The samples (1 μL, dilute to 1% with n-hexane) were injected, with a split ratio of 1:50. Helium was used as carrier gas at a flow rate of 1.0 mL min −1 . Percentage composition of individual components was calculated on the basis of peak area without using correction factors. The chemical constituents of essential oil were identified on an Agilent Technologies 5973N mass spectrometer. Various components were identified by comparison of their retention indices with literature values [27,28], computer matching against the library spectra (NIST 05, Wiley 275 and Adams Library). The retention indices were determined in relation to a homologous series of n-alkanes (C8-C24) under the same operating conditions.

Insects
L. serricorne and T. castaneum were obtained from laboratory cultures maintained in the dark in incubators at 29-30 °C and 70%-80% relative humidity (RH). Both of them were reared on wheat flour mixed with yeast (10:1, w/w) at 12%-13% moisture content. The unsexed adults used in all the experiments were about 1-2 weeks old, and the average weight of per adult insect was 1.9 mg (L. serricorne) and 1.7 mg (T. castaneum), respectively. The larvae of A. piceus were obtained from the warehouse of Traditional Chinese Medicinal Materials in the laboratory and were about 2-3 months old, had an average weight of 6-7 mg. Its cultures condition was the same as L. serricorne and T. castaneum. All containers housing insects used in experiments were made escape proof with a coating of polytetrafluoroethylene (Sino-rich ® , Beijing Sino-rich Tech Co., Ltd., Xuanwu District, Beijing, China).

Fumigant Toxicity
The fumigant toxicity of the essential oils against the three species of insects was measured as described by Liu and Ho [29]. A serial dilution of the essential oils (three concentrations of 50%, 10% and 2%) was prepared in n-hexane. A Whatman filter paper (diameter 2.0 cm) was impregnated with 10 µL 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., London, UK) 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. The corrected mortality of essential oils was calculated according to the formula: corrected mortality (%) = 100 × (A − C)/(1 − C), where A = the mortality of treated with essential oils, C = the mortality of control.

Contact Toxicity
The contact toxicity of the essential oils against the three species of insects was tested with reference to the method of Liu and Ho [29]. The preparation of testing concentrations of the essential oils was same as the fumigant toxicity assay. Aliquots of 0.5 µL of the dilutions were applied topically to the dorsal thorax of the insects. Controls were performed 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. The corrected mortality of essential oils was calculated by the same way with the fumigant toxicity assay. Range-finding surveys were run to determine the appropriate testing concentrations of the essential oil of Z. dissitum roots. A serial dilution (five concentrations) was prepared in n-hexane. The LD50 values were calculated by using Probit analysis [30]. The units were converted according to the formula: LD50 (μg/adult) = LD50 (%) × D (g/mL) × V (µL/adult) × 10 3 , where D = the density of the concentrated essential oil, V = the volume of test solution on per insect.

Conclusions
In this paper, we report the chemical composition and insecticidal activity of Z. dissitum leave and root essential oils for the first time. The results suggest that essential oil of Z. dissitum roots is rich in sesquiterpenoids and has potential application in the study of ecological prevention and control of storage pests. Further investigations should be focused on more detailed biological activity studies of the whole plant and/or different parts, and the safety of the essential oils to humans. * RI, retention index as determined on a HP-5MS column using the homologous series of n-alkanes (C 8 -C 24 ).

Conflicts of Interest
The authors declare no conflict of interest.