The Chemical Composition of Essential Oils from Cinnamomum camphora and Their Insecticidal Activity against the Stored Product Pests

To investigate the chemical composition and insecticidal activity of the essential oils of certain Chinese medicinal herbs and spices, the essential oils were extracted from the stem barks, leaves, and fruits of Cinnamomum camphora (L.) Presl, which were found to possess strong fumigant toxicity against Tribolium castaneum and Lasioderma serricorne adults. The essential oils of the plants were extracted by the method of steam distillation using a Clavenger apparatus. Their composition was determined by gas chromatography/mass spectrometric (GC-MS) analyses (HP-5MS column), and their insecticidal activity was measured by seal-spaced fumigation. D-camphor (51.3%), 1,8-cineole (4.3%), and α-terpineol (3.8%), while D-camphor (28.1%), linalool (22.9%), and 1,8-cineole (5.3%) were the main constituents of its fruits. The essential oils of the C. camphora all showed fumigant and contact toxicity. Other compounds exhibited various levels of bioactivities. The results indicate that the essential oils of C. camphora and its individual compounds can be considered a natural resource for the two stored-product insect management.


Introduction
The red flour beetle (Tribolium castaneum Herbst) and the cigarette beetle (Lasioderma serricorne Fabricius) are worldwide pests of stored products. They are dominant populations in the stored traditional Chinese medicines insect community [1]. The principal method to control these insects is to use synthetic insecticides or fumigants, but that may cause health hazards to warm-blooded animals, pose risk of environmental pollution, and bring development of resistance by insects or pest resurgence [2]. Therefore, an increasing number of scientists have been keen on searching for active natural products to use as botanical insecticides [3,4]. Essential oils continue to be a subject of interest among the international research community, which include lipids, terpenoids, ketones, phenols, and oxygenated derivatives and have been found for their control effects [4][5][6][7][8][9].
During the screening process for new agrochemicals from Chinese medicinal materials and spices, the essential oils of Cinnamomum camphora (L.) Presl (commonly known as camphor tree, camphorwood or camphor laurel) were found to possess fumigant and contact activities against the red floor beetle and the cigarette beetle. C. camphora is a tree native to South China, Taiwan, South Japan, Korea, and Vietnam [10]. It is cultivated as an ornamental plant and as a good material for wood furniture. Additionally, as a source of camphor and camphor oil, this plant is an important source for perfume. In the medical field, this plant can be used to treat muscular strains, inflammation, and rheumatic conditions [11].
In previous research, C. camphora extracts have shown many kinds of bioactivities against T. castaneum, such as the contact activity [12,13], the fumigant activity [14], the repellent activity [12,14], as well as progeny suppression [13,14]. However, those studies only tested the bioactivities of powders/essential oils from C. camphora aerial parts and seldom mentioned the bioactive compounds from them. Moreover, although some studies have analyzed the chemical composition of the different parts from essential oils [15][16][17][18], very little work has been done on the stem barks of this plant. Some species of the Cinnamomum genus have been confirmed to have insecticidal activities on their barks [19,20], whereas there has been no report on the bioactivity of C. camphora. Particularly, we have already analyzed the essential oil of C. camphora leaves (EL, vegetative organs of this plant) and found it to possess strong bioactivities against L. serricorne in our former study [18]. At this time, we bring our attention to its protective and reproductive organs, namely barks and fruits, and we try to find other bioactive compounds from its essential oils as well.
Thus, in this work, we analyzed the chemical composition of the essential oils of stem barks (EB) for the first time and compared the chemical composition between EB, EL, and the essential oils of its fruits (EF). In addition, we investigated the fumigant and contact activities of EB, EL, and EF against T. castaneum and L. serricorne adults, and found bioactive compounds from its essential oils.

Insecticidal Activity
The results of fumigant assays for the essential oils against T. castaneum and L. serricorne adults are presented in Table 2. EB and EL exhibited strong fumigant toxicity with LC 50 values both less than 3.2 mg/L air; meanwhile, EF also showed strong fumigant toxicity against the red flour beetle (LC 50 = 8.5 mg/L air). Moreover, the essential oils showed contact activity against the two stored-product insects, and the results are presented in Table 3. EB and EL were not so effective against T. castaneum in our contact toxicity measure range, and their LD 50 values could not be calculated (LD 50 > 50.0 µg/adult); however, they exhibited contact toxicity against L. serricorne adults with LD 50 values of 7.6 and 21.3 µg/adult, respectively. EF showed contact toxicity against T. castaneum and L. serricorne adults with LD 50 values of 19.0 and 10.1 µg/adult, respectively. The bioactivities of five major individual compounds (D-camphor, linalool, 1,8-cineole, safrole, and α-terpineol) were tested, and the results are listed in Tables 2 and 3 [21]; 5 The mortality of the negative control was 0 for the two insects.
Because we failed to test the bioactivities of safrole, we only compared the fumigant and contact results with the study with similar conditions [21,24]. As the uppermost constituent (29.0%) in EF, safrole showed weak fumigant activity against T. castaneum adults with a LC 50 value of 38.3 mg/L air. However, it had the strongest contact activity (LD 50 = 4.7 µg/adult) among the tested individual compounds against T. castaneum adults. Because EF had fumigant activity with LC 50 values of 8.5 mg/L air, it was not safrole but D-camphor and 1,8-cineole (LC 50 < 2.3 mg/L air and LC 50 = 5.5 mg/L air, respectively) that were the main contributors to its fumigant activity against T. castaneum adults.
On the other hand, some constituents of the essential oils of C. camphora still need safety risk assessment. As the uppermost constituent in EB and EL, the daily maximum human therapeutic dose of D-camphor is about 1.43 mg/kg. This dose is relatively safe, but still lacks information of long-term experiments [42]. Although safrole accounting for 29.0% of the EF showed obvious insecticidal activity, it is carcinogenic. Linalool, based on the volume of use (2011) from the International Fragrance Association, harmed the aquatic compartment [43,44]. Moreover, at very low doses, 1,8-cineole can be used as a flavoring and medicinal ingredient; however, in higher-than-normal doses, it is hazardous for behavior, the respiratory tract, and the nervous system [45]. Although the biological effects of the individual components of essential oils are known, the toxicokinetics and ecotoxicology of their blends and single compounds is still much more difficult to evaluate [20,46]. In general, medicinal herbs are low-risk and relatively well studied experimentally and clinically. However, no experimental data about the safety of the essential oils of the barks leaves and fruits are yet available. Further study should be focused on not only the effectiveness but also the safety of essential oils in order to manage these insects.

Insects
T. castaneum and L. serricorne were reared on wheat flour mixed with yeast (10:1, w/w) in dark incubators at 29 ± 1 • C and 70%-80% relative humidity. Adult insects of mixed sex, about 1 week old, were used for bioassay tests.

Plant Materials and Essential Oils Extraction
Stem barks, leaves, and fruits of C. camphora were all collected in May 2013 from Suzhou City (31.97 • N and 120.49 • E), Jiangsu, China. The samples were identified as Cinnamomum camphora (L.) Presl. The voucher specimens (BNU-dushushan-2013-05-25-005, BNU-dushushan-2013-05-25-006, and BNU-dushushan-2013-05-25-007) were deposited at the Herbarium (BNU) of College of Resources Science and Technology, Beijing Normal University. In this work, essential oils were extracted from barks and fruits (1 kg and 0.745 kg, respectively, of dry matter) with hydrodistillation. A modified Clevenger-type apparatus was used, and each extraction process lasted 6 hours. The essential oils were dehydrated with anhydrous sodium sulfate and stored in airtight containers in a refrigerator at 4 • C.

Chemical Components Determination
GC-MS analysis was run on an Agilent 6890 N gas chromatograph connected to an Agilent 5973 N mass selective detector. They were equipped with a gas chromatography-flame ionization detector (GC-FID) and an HP-5MS (30 m × 0.25 mm × 0.25 µm) capillary column. The essential oil samples were diluted in n-hexane to obtain a 1% solution. The injector temperature was maintained at 250 • C. The volume injected was 1 µL. The flow rate of carrier gas (helium) was 1.0 mL/min with the mass spectrum spectra scanned from 50 to 550 m/z.
The retention indices (RI) were determined from gas chromatograms using a series of n-alkanes (C 5 -C 36 ) under the same operating conditions. Based on RI, the chemical constituents were identified by comparing with n-alkanes as reference. The components of oil were identified by matching their mass spectra with computer libraries, namely Wiley 275 libraries, NIST 05, and RI from other literatures [47].

Fumigant Toxicity
The individual compounds were obtained from TCI Shanghai Development Co., Ltd. (Shanghai, China) (D-camphor, 1,8-cineole) and Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China) (linalool and α-terpineol). The appropriate testing concentrations were determined by range-finding studies. A serial dilution of the essential oils and compounds with five concentrations was prepared in n-hexane. The essential oils and above samples were tested with the method described by Liu and Ho [22]. Ten insects were put inside a glass vial (diameter 2.5 cm, height 5.5 cm, volume 25 mL). On the bottom of the cap, a filter paper (diameter 2 cm) was placed and treated with a 10 µL sample solution. Before placing the cap tightly on the glass vial to form a sealed chamber, the solvent evaporated for 20 s. The negative control was n-hexane. The experiments were performed with five replicates for each treatment. The packets were incubated for 24 h. After that, dead insects were counted, and SPSS V20.0 Probit analysis was used to calculate LC 50 values [48].

Contact Toxicity
The essential oils and individual compounds against the two stored-product insects were measured as described by Liu and Ho [22]. Range-finding studies were run to determine the appropriate testing concentrations. A series of testing samples (five concentrations) was diluted using n-hexane for essential oils and individual compounds. The dorsal thorax of each insect was applied to 0.5 µL aliquots of the dilution. The control group was treated only with n-hexane. Ten treated insects were then transferred to each glass vial and kept in the dark incubator. Each experiment was replicated five times. After 24 h, mortality was recorded, and the LD 50 values were calculated using SPSS V20.0 Probit analysis [48].

Conclusions
The significantly high efficacy of the essential oils of C. camphora and its individual constituents against insects in stored products demonstrated in this study may lead to the development of new natural products. Additionally, further research could concentrate more on the safety, potency, and stability of the essential oils.