Isolation of Insecticidal Constituents from the Essential Oil of Ageratum houstonianum Mill . against Liposcelis bostrychophila Badonnel

The aim of this research was to determine chemical composition and insecticidal activities of the essential oil of Ageratum houstonianumMill. aerial parts against booklice,Liposcelis bostrychophilaBadonnel, and to isolate any insecticidal constituents from the oil. Essential oil ofA. houstonianumwas obtained by hydrodistillation and analyzed by GC-MS. A total of 35 components in the essential oil were identified.Themajor compounds were precocene II (62.68%), precocene I (13.21%), and β-caryophyllene (7.92%). Based on bioactivity-guided fractionation, precocene II and precocene I were isolated and identified as the active constituents. The essential oil exhibited contact toxicity againstL. bostrychophilawith an LC 50 value of 50.8 μg/cm. Precocene II (LC 50 = 30.4 μg/cm) exhibited stronger acute toxicity than precocene I (LC 50 = 64.0 μg/cm) against the booklice. The essential oil and the two isolated constituents also possessed strong repellent activity against L. bostrychophila. The results indicated that the essential oil and its constituent compounds have potential for development into natural insecticides or repellents for control of insects in stored grains.


Introduction
Botanical pesticides have the advantage of providing novel modes of action against insects that can reduce the risk of cross-resistance as well as offering new leads for the design of target-specific molecules.During the screening program for new agrochemicals from Chinese medicinal herbs and wild plants, essential oil of Ageratum houstonianum (Family: Compositae) aerial parts was found to possess strong insecticidal toxicity against the booklice, Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelidae). A. houstonianum is an annual erect ornamental shrub of 30-70 cm height.It is commonly known as floss flower and native to southeastern Mexico, Central America.Now it is cultured as an ornamental flower and also naturalized as an invasive weed in Anhui, Fujian, Guangdong, Guangxi, Guizhou, Hainan, Hebei, Jiangsu, Nanhai Zhudao, Shandong, Sichuan, Taiwan, Yunnan, and Zhejiang Province, China [1].The whole plant of A. houstonianum is used medicinally in traditional Chinese medicine to clear away heat and toxic materials.People in Central America (Ecuador) use this plant as an antiphlogistic to relieve swelling and pain in the throat [1].

Gas Chromatography-Mass Spectrometry (GC-MS).
Analyses of volatile constituents were determined using an Agilent 5973 GC-MS system operating in the EI mode at 70 eV (equipped with a 30 m HP-5MS column (0.25 mm × 30 m × 0.25 m) and coated with 5% phenylmethylpolysiloxane using a HP-5MS (df = 0.25 m) (Agilent J&W Scientific, USA)).The temperature program used for the analysis was as follows: initial temperature at 60 ∘ C, held for 1 min, ramped at 4 ∘ C/min to 290 ∘ C, and held for 0.5 min.Helium was the carrier gas at 1.0 mL/min; the sample (1 L 1/100, v/v, in acetone) was injected in the split mode (1 : 10).The injector and detector temperatures were performed at 230 ∘ C and 300 ∘ C, respectively.Most constituents were identified by gas chromatography by comparison of their retention indices with those of the literature or with those of authentic compounds available in our laboratories.The retention indices were determined in relation to a homologous series of -alkanes (C 8 -C 24 ) under the same operating conditions.Further identification was made by comparison of their mass spectra with those stored in NIST 05 and Wiley 275 libraries or with mass spectra from the literature [21].
2.5.Insects.L. bostrychophila was obtained from laboratory cultures in the dark in incubators at 28-30 ∘ C and 70-80% r.h. and was reared on a 1 : 1 : 1 mixture, by mass, of milk powder, active yeast, and flour.All containers housing insects and the Petri dishes used in experiments were made escape proof with a coating of polytetrafluoroethylene (Fluon, Blades Biological, UK). Adult insects used in all the experiments were about one week old.
2.6.Contact Toxicity.Contact toxicity of the oil against the booklice was measured as described by Zhao et al. [22].The filter paper with 3.5 cm in diameter (Whatman) was treated with 150 L of the solution (2.0%, 2.4%, 2.9%, 3.5%, 4.2%, and 5.0% in acetone).The treated filter paper after being treated with solid glue (Glue Stick, Jong Ie Nara Co., Ltd., Hong Kong) was placed in a Petri dish (3.5 cm in diameter) and 10 booklice were put on the filter paper.The plastic cover with holes was put and all the Petri dishes were kept in incubators at 27-29C, 70-80% r.h. for 24 h and mortality of insects was observed.Acetone was used as controls and pyrethrum extract was used as a positive control.Pyrethrum extract (25% pyrethrin I and pyrethrin II) was purchased from Fluka Chemie (Buchs, Switzerland).

Repellent Activity.
A commercial repellent, dimethyl phthalate, was purchased from Aladdin-Reagent Company (Shanghai) and used as a positive control.The essential oil and isolated compounds were diluted in acetone to four concentrations (6.4,3.2 1.6, and 0.8 nL/cm 2 ).Filter paper (6 cm in diameter) was cut in half and 150 L of each concentration was applied separately to half of the filter paper as uniformly as possible with a micropipette.The other half (control) was treated with 150 L of absolute acetone.Both the treated half and the control half were then air dried to evaporate the solvent completely (10 sec).A full disc was carefully remade by attaching the tested half to the negative control half with tape.Each remade filter paper after being treated with solid glue was placed in a Petri dish and 20 insects were released in the center of each filter-paper disc and a cover was placed over the Petri dish.Five replicates were used and the experiment was repeated for three times.Counts of the insects present on each strip were made after 2 h and 4 h.The percent repellency of the oil/compounds was then calculated using the formula where Nc was the number of insects present in the negative control half and Nt was the number of insects present in the treated half.The averages were then categorised according to the following scale [23,24] as shown in Table 1.

Data Analysis.
The observed mortality data were corrected for control mortality using Abbott's formula.The results from all replicates in contact toxicity were subjected to Probit analysis using PriProbit Program V1.6.3 to determine LD 50 and LD 90 values [25].The percentage repellency data were subjected to an arcsine square-root transformation before ANOVA and Tukey's tests.

Contact Toxicity.
The essential oil of A. houstonianum aerial parts exhibited contact toxicity against L. bostrychophila with an LC 50 value of 50.8 g/cm 2 (Table 3).
Precocene II (LC 50 = 30.4g/cm 2 ) exhibited stronger acute toxicity than precocene I (LC 50 = 64.0g/cm 2 ) against the booklice.Precocene II shows almost 2 times stronger toxicity than the oil while precocene I exhibits less toxicity than the oil.Thus it seems that contact toxicity of the oil may be mainly attributed to precocene II.However, compared with the positive control, pyrethrum extract (LC 50 = 18.99 g/cm 2 ), A. houstonianum essential oil, and precocene II showed 2.7 and 1.6 times less toxicity to L. bostrychophila, respectively.When compared with the other essential oils in the previous studies using the same bioassay, the essential oil of A. houstonianum exhibited stronger or the same level of acute toxicity against the booklice, for example, essential oils of Acorus calamus [26], Artemisia rupestris and A. frigida [24,27], Curcuma wenyujin [28], Foeniculum vulgare [22], and Valeriana jatamansi [29].

Repellent Activity. The essential oil of A. houstonianum
showed strong repellent activity (Class V) against the booklice at a concentration of 1.6 nL/cm 2 and higher concentration after 4 hr of exposure (Table 4).Moreover, the essential oil of A. houstonianum still exhibited the same level (class IV) of repellent activity against the booklice as commercial repellent, dimethyl phthalate, at a concentration of 0.8 nL/cm 2 after 4 hr of exposure.Precocene I possessed the same strong repellency against the booklice, while precocene II exhibited class IV repellent activity at a concentration of 1.6 nL/cm 2 after 4 hr of exposure.Compared with commercial repellent, dimethyl phthalate, precocene I showed the same level of repellent activity against the booklice, while precocene II exhibited less active at lower concentrations (0.8 nL/cm 2 and 1.6 nL/cm 2 ) (Table 4).This study demonstrates that the essential oil of A. houstonianum had contact toxicity and repellent activity to the booklice.Furthermore, the two isolated constituent compounds, precocene II and precocene I, also exhibited insecticidal and repellent activity against L. bostrychophila.In the previous studies, precocene II and precocene I demonstrated to inhibit the synthesis of juvenile hormone in a number of insects.Consequently, this inhibition can disturb the embryonic development, induce premature metamorphosis, decrease the reproductive potential, and affect the insect behavior including the antifeedant and repellent effect [19,[30][31][32][33][34][35].The above findings suggest that the essential oil and the two isolated constituent compounds show a potential to be developed as possible natural insecticides/repellents for the control of grain storage insects.It seems that this plant is quite safe to human consumption because it has been used as a medicinal herb for clearing away heat and toxic materials [1].However, A. houstonianum is reported to be toxic to grazing animals, causing liver lesions [36].Moreover, no information on toxicity of the essential oil and the isolated constituents to human were available.Thus, to develop a practical application for the essential oil and the isolated constituents as novel natural insecticides, further research into the safety of the essential oil/compounds to humans is needed.Additional studies on the development of formulations are also necessary to improve the efficacy and stability and to reduce cost.

Conclusion
This study indicates that the essential oil of A. houstonianum aerial parts and its isolated constituent compounds have potential for development into natural insecticides and repellents for control of insects in stored grains.

Table 1 :
The scale to be assign repellency of the essential oil and its constituents.

Table 2 :
GC-MS analysis of essential oil of Ageratum houstonianum aerial parts.
* Identification by coinjection of authentic compounds.

Table 4 :
Repellency (PR) after two exposure times for Ageratum houstonianum essential oil and its isolated compounds against Liposcelis bostrychophila.