Research Article | Open Access
Fumigant Compounds from the Essential Oil of Chinese Blumea balsamifera Leaves against the Maize Weevil (Sitophilus zeamais)
Essential oil of Chinese medicinal herb, Blumea balsamifera leaves, was found to possess fumigant toxicity against the maize weevils, Sitophilus zeamais. The main components of the essential oil of B. balsamifera were 1,8-cineole (20.98%), borneol (11.99%), β-caryophyllene (10.38%), camphor (8.06%), 4-terpineol (6.49%), α-terpineol (5.91%), and caryophyllene oxide (5.35%). Bioactivity-guided chromatographic separation of the essential oil on repeated silica gel columns led to isolate five constituent compounds, namely, 1,8-cineole, borneol, camphor, α-terpineol, and 4-terpineol. 1,8-Cineole, 4-terpineol, and α-terpineol showed pronounced fumigant toxicity against S. zeamais adults (LC50 = 2.96 mg/L, 4.79 mg/L, and 7.45 mg/L air, resp.) and were more toxic than camphor (LC50 = 21.64 mg/L air) and borneol (LC50 = 21.67 mg/L air). The crude essential oil also possessed strong fumigant toxicity against S. zeamais adults (LC50 = 10.71 mg/L air).
Fumigants play a very important role in insect pest elimination in stored products not only because of their ability to kill a broad spectrum of pests but also because of their easy penetration into the commodity while leaving minimal residues . Currently, phosphine and methyl bromide (MeBr) are the two common fumigants used for stored-product protection all over the world. Due to insect resistance to phosphine and MeBr as an ozone-depleting compound, there is a global interest in looking for new fumigants [1, 2] and plant essential oils and their components have been shown to possess potential to be developed as new fumigants. Plant essential oils and their components may have the advantage over conventional fumigants in terms of low mammalian toxicity, rapid degradation, and local availability [3, 4]. Essential oils derived from more than 75 plant species have been studied for fumigant toxicity against stored product insects .
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 design of target-specific molecules . During the screening program for new agrochemicals from Chinese medicinal herbs, the essential oil of Blumea balsamifera DC. (Family: Asteraceae) leaves was found to possess strong fumigant toxicity to the maize weevil, Sitophilus zeamais (Motsch). The maize weevil (S. zeamais) is one of the most widespread and destructive primary insect pests of stored cereals . Infestations not only cause significant losses due to the consumption of grains; they also result in elevated temperature and moisture conditions that lead to an accelerated growth of molds, including toxigenic species. B. balsamifera is a perennial evergreen shrub native of Southeast Asia but is distributed throughout tropical Asia. It is a small tree that can grow up to 4 m in height and imparts a strong camphorous odor around it. Use of B. balsamifera leaves has been recommended as traditional Chinese medicine in the treatment of various diseases . It has anti-inflammatory, anticatarrhal and expectorant properties which render it useful in the treatment of both upper and lower respiratory tracts like sinusitis, influenza, and asthmatic bronchitis. Several studies on the chemical constituents of B. balsamifera have been reported, and a number of flavonoids, sesquiterpenoids, and triterpenoids, have been isolated from this plant [8–17]. Chemical composition of the essential oil of B. balsamifera leaves has been studied [18–22]. However, the essential oil of B. balsamifera leaves was not evaluated to have insecticidal activity against grain storage insects and the bioactive (fumigant) constituent compounds of the essential oil have not been isolated and identified from this Chinese medicinal herb. In this paper, we report the isolation and identification of five active compounds contained in the essential oil of B. balsamifera leaves against the maize weevil by bioassay-guided fractionation.
1H nuclear magnetic resonance (NMR) spectra were recorded on Bruker ACF300 (300 MHz (1H)) and AMX500 (500 MHz (1H)) instruments using deuterochloroform (CDCl3) as the solvent with tetramethylsilane (TMS) as the internal standard. Electron impact ionone mass spectra (EIMS) were determined on a Micromass VG7035 mass spectrometer at 70 eV (probe). The crude essential oil (20 mL) was chromatographed on a silica gel (Merck 9385, 1,000 g) column (85 mm i.d., 850 mm length) by gradient elution with a mixture of solvents (n-hexane, n-hexane-ethyl acetate, and acetone). Fractions of 500 mL were collected and concentrated at 40°C, and similar fractions according to TLC profiles were combined to yield 28 fractions. Each fraction was tested with fumigant toxicity bioassay (see the following) to identify the bioactive fractions (fractions 5, 9, 11, 14, and 16). Fractions that possessed fumigant toxicity, with similar TLC profiles, were pooled and further purified by preparative silica gel column chromatography (PTLC) until obtaining three pure compounds for determining structure (Figure 1). The spectral data of 1,8-cineole (1) (2.7 g) matched with the previous reports [23, 24]. The data of camphor (2) (1.6 g) and borneol (3) (1.2 g) matched with the previous reports [23, 25, 26]. The spectral data were identical to the published data of 4-terpineol (4) (1.1 g) and α-terpineol (5) (0.9 g) (Table 3) [23, 27, 28].
2.1. Chinese Medicinal Herb and Hydrodistillation
Fresh leaves of B. balsamifera were collected from the suburb of Nanning City (22.8°N, 108.3°E, Guangxi Zhuang Autonomous Region, China) at August 2008. The plant was identified by Dr. QR Liu (College of Life Sciences, Beijing Normal University, China), and a voucher specimen (CMH-Dafengai-Guangxi-2008-08) was deposited in the Department of Entomology, China Agricultural University. To obtain volatile essential oil, the air-dried samples were first ground to a powder then soaked in water at a ratio of 1 : 4 (w/v) for 1 h, prior to hydrodistillation using a round bottom container over a period of 6 h. The volatile essential oil was collected in a specific receiver, measured, dried over anhydrous sulfate, weighed, and stored in airtight containers.
2.2. Analysis of the Essential Oil
Components of the essential oil of B. balsamifera leaves were separated and identified by gas chromatography-mass spectrometry (GC-MS) Agilent 6890N gas chromatography hooked to Agilent 5973N mass selective detector. They equipped with a flame ionization detector and capillary column with HP-5MS (). The GC settings were as follows: the initial oven temperature was held at 60°C for 1 min and ramped at 10°C min−1 to 180°C for 1 min, and then ramped at 20°C min−1 to 280°C for 15 min. The injector temperature was maintained at 270°C. The samples (1 μL) were injected neat, with a split ratio of 1 : 10. The carrier gas was helium at flow rate of 1.0 mL min−1. Spectra were scanned from 20 to 550 m/z at 2 scans s−1. Most constituents were identified by gas chromatography by comparison of their retention indices with those of the literature [18–22] or with those of authentic compounds available in our laboratories. The retention indices were determined in relation to a homologous series of n-alkanes (C8–C24) under the same operating conditions. Further identification was made by comparison of their mass spectra on both columns with those stored in NIST 05 and Wiley 275 libraries or with mass spectra from literature . Component relative percentages were calculated based on GC peak areas without using correction factors.
2.3. Fumigant Toxicity 
The maize weevils were obtained from laboratory cultures maintained for the last 15 years in the dark in incubators at 29–30°C and 65–75% r.h. They were reared on whole wheat at 12–13% moisture content. The unsexed adults used in the experiments were 2–4 weeks posteclosion. A Whatman filter paper (CAT number 1001020, diameter 2.0 cm) was placed on the underside of the screw cap of a glass vial (diameter 2.5 cm, height 5.5 cm, volume 24 mL). Ten microliters of an appropriate concentration of compounds/oil (1.0%–40.0%, 5 concentrations) were added to the filter paper. The solvent was allowed to evaporate for 30 seconds before the cap was placed tightly on the glass vial (with 10 insects). n-Hexane was used as controls. The vials were upright and the Fluon (ICI America Inc) coating restricted the insects to the lower portion of the vial to prevent them from the treated filter paper. Six replicates were used in all treatments and controls, and they were incubated at 29–30°C and 65–75% RH for 24 hrs. The insects were then transferred to clean vials with some culture media and kept in an incubator for determination of end-point mortality, which was reached after one week. The insects were considered dead if appendages did not move when probed with a camel brush. The observed mortality data were corrected for control mortality using Abbott’s formula. Results from all replicates were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC50 and LC90 values .
3. Results and Discussion
3.1. Chemical Constituents of the Essential Oil
Hydrodistillation of dried leaves of B. balsamifera yielded 0.88% essential oil (v/w), and the density of the essential oil was determined as 0.87%. The results of GC-MS of B. balsamifera essential oil are presented in Table 1. A total of 27 components were identified in the essential oil of B. balsamifera, accounting for 99.23% of the total oil (Table 1). The main components are 1,8-cineole (20.98%), borneol (11.99%), β-caryophyllene (10.38%), camphor (8.06%), 4-terpineol (6.49%), α-terpineol (5.91%), and caryophyllene oxide (5.35%). Monoterpenoids represented 12 of the 27 compounds, corresponding to 70.47% of the whole oil while 13 of the 27 constituents were sesquiterpenoids (27.42% of the crude essential oil). The chemical composition of B. balsamifera essential oil was different from that reported in other studies. For example, borneol (33.2%), caryophyllene (8.2%), ledol (7.1%), tetracyclo[6,3,2,0,(2.5).0(1,8) tridecan-9-ol, 4,4-dimethyl (5.2%), phytol (4.6%), caryophyllene oxide (4.1%), guaiol (3.4%), thujopsene-13 (4.4%), dimethoxydurene (3.6%), and γ-eudesmol (3.2%) were the dominant components in the essential oil of B. balsamifera leaves collected from Bangladesh (Chittagong, 22.20°N, 91.50°E) . However, borneol (57.7%), caryophyllene (7.6%), and camphor (5.0%) were the three main components of the essential oil of B. balsamifera from Guizhou, China (Luodian Country, 25.43°N, 106.75°E) , while bornel (52.4%) and camphor (17.7%) were the two components of the essential oil from Yunnan, China (Puer City, 22.48°N, 100.58°E) . There were great geographic variations in chemical composition of essential oils of B. balsamifera harvested in three provinces of Vietnam . The major components of the essential oils were borneol (57.8%), caryophyllene (8.3%), δ-cadinol (8.0%), and caryophyllene oxide (3.1%) (Ha Giang, 22.80°N, 104.98°E); borneol (50.6%), camphor (18.7%), caryophyllene (10.1%), δ-cadinol (3.1%), patchoulene (3.0%), and veridiflorol (2.0%) (Hanoi, 21.02°N, 105.51°E); camphor (70.1%), caryophyllene (10.5%), borneol (5.7%), and carvacrol (5.7%) (Dak Lak, 22.80°N, 104.98°E). The above findings suggest that further studies on plant cultivation and essential oil standardization are necessary because chemical composition of the essential oil varies greatly with the plant population.
|RI: retention index as determined on a HP-5MS column using the homologous series of -hydrocarbons.|
RA: relative area (peak area relative to total peak area).
3.2. Fumigant Toxicity of Isolated Constituent Compounds against the Maize Weevils
1,8-Cineole (1) (LC50 = 2.96 mg/L air), 4-terpineol (4) (LC50 = 4.79 mg/L air), and α-terpineol (5) (LC50 = 7.45 mg/L air) showed stronger fumigant toxicity than the crude essential oil (LC50 = 10.71 mg/L air) against S. zeamais (Table 2). It indicates that fumigant toxicity of the essential oil may be attributed to the three constituent compounds. In the previous studies, 1,8-cineole was found to exhibit fumigant toxicity against red flour beetles, Tribolium castaneum adult with LC50 = 41 μL/L air , 15.3 μL/L air , and 1.52 mg/L air . It also possesses fumigant toxicity against several other stored product insects and cockroaches as well as mosquitoes, for example, the rice weevil (S. oryzae) (LC50 = 22.8 μL/L air ), the lesser grain borer (Rhyzopertha dominica) (LC50 = 9.5 μL/L air ). However, compared with the current used fumigant (MeBr, LC50 = 0.67 mg/L air), the three compounds and the crude essential oil exhibited only 4–16 times less toxic to the maize weevils. The three constituent compounds and the crude essential oil were more toxic to S. zeamais than another two isolated compounds, camphor (2) (LC50 = 21.67 mg/L air) and borneol (3) (LC50 = 29.64 mg/L air) (Figure 2). Compared with the other essential oils in the previous studies that were tested using a similar bioassay, B. balsamifera essential oil exhibited stronger fumigant toxicity against S. zeamais adults, for example, essential oils of Schizonepeta multifida , Murraya exotica , and several essential oils from Genus Artemisia [36–38]. Moreover, the two other active constituent compounds, 4-terpineol and α-terpineol, have been found to possess strong fumigant toxicity against several grain storage insects, such as S. granarius, S. oryzae, T. castaneum, T. confusum, and R. dominica [31, 32, 39–43]. The two other active constituents, camphor and borneol, were also demonstrated fumigant toxicity against grain storage insects [31, 41, 44, 45].
|MeBr: Methyl bromide; values from Liu and Ho .|
Considering the currently used fumigants are synthetic insecticides, fumigant activity of the crude essential oil of B. balsamifera leaves and the three isolated active compounds are quite promising and they show potential to be developed as possible natural fumigants for control of stored product insects with low toxicity to humans. Although dried leaves of B. balsamifera were used as a common Chinese medicinal herb , there are no toxicity data for this herb and the three isolated compounds and available on human consumption. For the practical use of the three compounds and crude essential oil as novel botanical insecticides, further studies are necessary on the safety of these materials to human, on phytotoxicity to crop seeds, and on the development of formulations to improve efficacy and stability, and to cut cost as well. The isolated three active compounds exhibited fumigant toxicity against several grain storage insects [31, 32, 39–43]. However, little has been done on mechanisms of action of these three monoterpenes. In addition, further testing is necessary to evaluate the spectrum of fumigant activity against other stored-product insects (as well as other life stages of these stored-product insects, especially eggs).
This work was funded by the Hi-Tech Research and Development of China 2011AA10A202 and 2006AA10A209. The authors are grateful to Dr. SL Liu, Department of Biology, GuangXi Traditional Chinese Medical University, for his collection of the medicinal herb. They are grateful to Dr. QR Liu, College of Life Sciences, Beijing Normal University, Beijing, China, for identification of Chinese medicinal herb.
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