Molecules 2013, 18, 5684-5696; doi:10.3390/molecules18055684 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Identification of Insecticidal Constituents of the Essential Oil of Acorus calamus Rhizomes against Liposcelis bostrychophila Badonnel Xin Chao Liu 1, Li Gang Zhou 2, Zhi Long Liu 1,* and Shu Shan Du 3,* 1 Department of Entomology, China Agricultural University, Haidian District, Beijing 100193, China; E-Mail: [email protected] 2 Department of Plant Pathology, China Agricultural University, Haidian District, Beijing 100193, China; E-Mail: [email protected] 3 College of Resources Science and Technology, Beijing Normal University, Haidian District, Beijing 100875, China * Authors to whom correspondence should be addressed; E-Mails: [email protected] (Z.L.L.); [email protected] (S.S.D.); Tel./Fax: +86-10-6273-2800 (Z.L.L.); Tel./Fax: +86-10-6220-8032 (S.S.D.). Received: 20 March 2013; in revised form: 6 May 2013 / Accepted: 13 May 2013 / Published: 15 May 2013 Abstract: The aim of this research was to determine the chemical composition of the essential oil of Acorus calamus rhizomes, its insecticidal activity against the booklouse, (Liposcelis bostrychophila) and to isolate any insecticidal constituents from the essential oil. The essential oil of A. calamus rhizomes was obtained by hydrodistillation and analyzed by GC-FID and GC-MS. A total of 32 components of the essential oil of A. calamus rhizomes was identified and the principal compounds in the essential oil were determined to be α-asarone (50.09%), (E)-methylisoeugenol (14.01%), and methyleugenol (8.59%), followed by β-asarone (3.51%), α-cedrene (3.09%) and camphor (2.42%). Based on bioactivity-guided fractionation, the three active constituents were isolated from the essential oil and identified as methyleugenol, (E)-methylisoeugenol and α-asarone. The essential oil exhibited contact toxicity against L. bostrychophila with an LD50 value of 100.21 µg/cm2 while three constituent compounds, α-asarone, methyleugenol, and 2 2 2 (E)-methylisoeugenol had LD50 values of 125.73 µg/cm , 103.22 µg/cm and 55.32 µg/cm , respectively. Methyleugenol and (E)-methylisoeugenol possessed fumigant toxicity against L. bostrychophila adults with LC50 values of 92.21 μg/L air and 143.43 μg/L air, respectively, while the crude essential oil showed an LC50 value of 392.13 μg/L air. The Molecules 2013, 18 5685 results indicate that the essential oil of A. calamus rhizomes and its constituent compounds have potential for development into natural fumigants/insecticides for control of the booklice. Keywords: Acorus calamus; Liposcelis bostrychophila; fumigant; contact toxicity; essential oil composition 1. Introduction The booklouse, Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae) has a worldwide distribution and has been reported infesting domestic premises, raw material stores, manufacturing factories, and also museums [1]. These are tiny (approximately 1 mm in length), wingless, soft-bodied, light brown insects. Psocids used to be considered as nuisance pests rather than a cause of losses to stored commodities [2]. They were regarded as secondary pests, often overlooked due to their small size and the existence of other more damaging primary pests in cereal grains (e.g., maize weevils Sitophilus zeamais, rice weevils S. oryzae and lesser grain borer, Rhyzopertha dominica) [2]. However, currently, psocids are perhaps the most important category of emerging pests in stored grains and related commodities due to their small size and resistance to chemicals [2,3]. L. bostrychophila exhibits partenogenensis behavior, because all the offspring are female, so the rate of population increase is exponential and infestation could arise from a small indigenous population when favorable conditions occur [2]. Infestations of stored product insects currently are controlled by fumigation or insecticidal treatment of commodities and surfaces [4]. However, many problems are associated with heavy usage of these chemicals, such as the development of resistance, toxic residues in food, workers’ safety, and high cost of procurement [4]. These problems have necessitated a search for alternative eco-friendly insect pest control methods [5]. The use of essential oils or their constituents with low mammalian toxicity can effectively prevent and/or suppress insect pest especially in storage [6]. Investigations in several countries confirm that some plant essential oils not only repel insects, but possess contact and fumigant toxicity against stored product pests as well as exhibiting feeding inhibition or harmful effects on the reproductive system of insects [7]. Essential oils from many plants including medicinal herbs, spices and fruits have been evaluated with success for insecticidal/ repellency activity against stored product insects/mites, in some cases, have been proven more effective than traditionally used organophosphorus pesticides [8–18]. Besides insecticidal and repellent activities, essential oils from different plant sources have exhibited several biological activities, including antibacterial and antifungal [19], larvicidal [20–22], acaricidal [23], and nematicidal [24–26]. As a consequence, this vast arsenal of bioactive compounds has attracted significant and crescent attention of researchers in recent years [7]. During our screening program for new agrochemicals from Chinese medicinal herbs and wild plants, the essential oil of Acorus calamus L. (Acoraceae) rhizomes was found to possess strong insecticidal toxicity against the booklouse (L. bostrychophila). A. calamus (Sweet flag) is a wetland perennial monocot plant, whose scented leaves and rhizomes have been traditionally used medicinally against different ailments like, fever, asthma, bronchitis, cough and mainly for digestive problems such as gas, bloating, colic, and poor digestive function, and Molecules 2013, 18 5686 also used as a sedative, nerve tonic, antimicrobial agent, and expectorant [27,28]. Chemical composition of the essential oils obtained from leaves and rhizomes of A. calamus grown in different countries has been the subject of some studies and a great variation in chemical composition of the essential oils were observed [29–37]. The essential oil of A. calamus has been demonstrated to possess insecticidal activity against many species of insects, e.g., the kelp fly, Coelopa frigida [38], termite, Odontotermes obesus [39], the larger grain borer, Prostephanus truncates [40], the tobacco armyworm, Spodoptera litura [41] and Callosobruchus phaseoli [42]. Moreover, the ethanol extract of A. calamus exhibited strong repellency and contact effect to maize weevil, S. zeamais [43] and a supercritical fluid (CO2) extract of A. calamus possessed strong contact toxicity against German cockroaches, Blattella germanica [44]. However, a literature survey has shown that there is no report on insecticidal of A. calamus essential oil against the booklouse, thus we decided to investigate the chemical constituents and insecticidal activity of the essential oil of A. calamus against L. bostrychophila for the first time and to isolate any active constituent compounds from the essential oil. 2. Results and Discussion 2.1. Essential Oil Chemical Composition The yield of A. calamus essential oil was 1.31% (V/W) and the density of the essential oil was determined to be 0.92 g/mL. GC-MS analysis of the essential oil of A. calamus rhizomes led to the identification and quantification of a total of 32 major components, accounting for 97.52% of the total components present (Table 1). Table 1. Chemical constituents of the essential oil derived from Acorus calamus rhizomes. RI * Compound Composition, % 1 931 α-Pinene 0.17 2 957 Camphene 0.55 3 981 β-Pinene 0.27 4 1030 (D)-Limonene 0.56 5 1032 1,8-Cineol 0.16 6 1094 Linalool 1.07 7 1146 Camphor 2.42 8 1179 Terpinen-4-ol 1.21 9 1191 α-Terpineol 0.66 10 1198 Estragole 0.89 11 1287 Bornyl acetate 0.13 12 1374 Copaene 1.08 13 1400 α-Funebrene 0.23 14 1406 α-Gurjunene 0.69 15 1409 α-Cedrene 3.09 16 1416 α-Bulnesene 0.32 17 1403 Methyleugenol 8.59 18 1422 β-Cedrene 1.52 19 1420 β-Caryophyllene 0.21 20 1454 α-Caryophyllene 0.19 Molecules 2013, 18 5687 Table 1. Cont. RI * Compound Composition, % 21 1500 (E)-Methylisoeugenol 14.01 22 1458 Germacrene D 2.12 23 1504 Cuparene 0.73 24 1537 α-Cadinene 1.35 25 1557 β-Calacorene 0.51 26 1561 Germacrene B 0.26 27 1631 β-Asarone 3.51 28 1654 α-Cadinol 0.27 29 1678 α-Asarone 50.09 30 1685 Acorenone 0.23 31 1686 α-Bisabolol 0.13 32 1775 Acorone 0.11 Total 97.52 Monoterpenoids 7.20 Sesquiterpenoids 13.04 Others 77.28 * RI, retention index as determined on a HP-5MS column using the homologous series of n-hydrocarbons. The principal compounds in the essential oil of A. calamus rhizomes were α-asarone (50.09%), (E)-methylisoeugenol (14.01%), and methyleugenol (8.59%) (Figure 1), followed by β-asarone (3.51%), α-cedrene (3.09%) and camphor (2.42%). Figure 1. Main constituent compounds isolated from the essential oil of A. calamus rhizomes. 8 8 8 12 7 9 7 9 7 9 CH3 O 1 1 1 6 2 6 2 2 10 10 6 10 CH3 CH3 CH3 5 4 5 4 4 O O 5 O 3 3 3 11 11 O O O 11 CH3 CH3 CH3 Methyleugenol (1) (E)-Methylisoeugenol (2) α-Asarone (3) Most of the essential oil components were phenylpropanoids (77.28%) and only 7.20% monoterpenoids and 13.04% sesquiterpenoids were identified (Table 1). The results are quite different from the previous reports. For example, preisocalamenediol (18.0%), acorenone (14.2%), shyobunone (13.3%) and cryptoacorone (7.5%) were major compounds of the essential oil of A. calamus collected from Quebec, Canada [34]. Preisocalamendiol (17.3%), isoshyobunone (13.0%), 1,4-(trans)-1,7- (trans)-acorenone (10.5%), camphor (5.9%) 2,6-diepishyobunone (2.6%) and β-gurjunene (2.5%) were the main components of the essential oil of A.