Appl Biol Chem (2016) 59(4):609–614 Online ISSN 2468-0842 DOI 10.1007/s13765-016-0200-9 Print ISSN 2468-0834 ARTICLE Active component isolated from Eugenia caryophyllata leaves and its structural analogues show insecticidal properties against Pochazia shantungensis Hwa-Won Lee1 . Sang-Guei Lee2 . Hoi-Seon Lee1 Received: 24 March 2016 / Accepted: 15 April 2016 / Published online: 2 May 2016 Ó The Korean Society for Applied Biological Chemistry 2016 Abstract The purpose of this study was to isolate an Introduction active constituent from the essential oil of Eugenia caryophyllata leaves and to evaluate its insecticidal activity During the past 100 years, global warming has resulted in a against the nymph and adults of Pochazia shantungensis. temperature increase of 0.6 °C, with the 1990s the warmest According to some chromatographic methods and spec- decade and 1997 the warmest year since records began troscopic analyses, the active constituent of E. caryophyl- (Houghton et al. 2001). This has affected the habitats of lata leaves was identified as eugenol. Based on the LC50 pests and increased sporadic insects against international values of eugenol and its structural analogues against the trade (Ahn et al. 2011). For example, Lycorma delicatula nymph and adults of P. shantungensis, isoeugenol (LC50, (Shin et al. 2010) and Metcalfa pruinosa (Ahn et al. 2011) 83.29 and 91.03 mg/L) exhibited the highest insecticidal were discovered. Discovery of sporadic insects and their activity, followed by methyl isoeugenol (105.61 and controls are problematic in ecosystem (Shin et al. 2010; 114.48 mg/L), eugenol (124.44 and 143.24 mg/L), methyl Ahn et al. 2011; Song et al. 2013). Particularly, Pochazia eugenol (126.31 and 143.84 mg/L), and acetyl eugenol shantungensis was first identified in Korea in 2010 (Choi (165.11 and 170.06 mg/L). Insecticidal activity against P. et al. 2011). This sporadic insect attacks diverse agricul- shantungensis was dependent on the presence of a func- tural commodities, including fruits, nuts, and berries, and is tional group in 4-ally-2-methoxyphenol. In conclusion, E. a serious pest of fruit trees and forests worldwide (Choi caryophyllata oil and eugenol analogues might be suit- et al. 2012). able alternative synthetic insecticides. To remove various pests (foreign, sporadic, and stored insects), synthetic insecticides such as dinotefuran and Keywords Eugenia caryophyllata Á Eugenol Á Structural imidacloprid are widely used (Duguet and Quan 1990). analogues Á Insecticidal activity Á Pochazia shantungensis However, these chemicals have serious drawbacks, such as genetic resistance in the pests, residual toxicity, and effects on non-target organism on humans and ecosystems (White and Leesch 1995; Lee and Lee 2015). Thus, development of new and safer materials for insect control is required. Plant-derived materials are valuable alternative insecti- cides, because they do not cause side effects and are less toxic (Zettler and Arthur 2000). In particular, essential oils & Hoi-Seon Lee extracted from aromatic plants are potent alternatives to [email protected] synthetic insecticides and do not pose a risk to the envi- 1 Eugenia Department of Bioenvironmental Chemistry, College of ronment or human health (Batish et al. 2008). Agriculture & Life Science, Chonbuk National University, caryophyllata, a member of the Myrtaceae family, is used Jeonju 54896, Republic of Korea as a traditional medication globally (Cho et al. 2004). E. 2 Pest Risk Assessment Division, Animal and Plant Quarantine caryophyllata contains a high eugenol content and exhibits Agency, Gimcheon 39660, Republic of Korea 123 610 Appl Biol Chem (2016) 59(4):609–614 Table 1 Analysis of volatile Compound Mass spectral dataa Retention time (min) Relative (%) constituents derived from E. caryophyllata oil identified by a-Cubebene 41, 91, 105, 161, 120, 204 41:63 14.48 GC–MS Eugenol 55, 77, 103, 149, 164 42:20 18.76 a-Copaene 41, 91, 105, 119, 161, 204 43:22 9.44 b-Bourbonene 81, 123, 161, 204 43:59 3.54 Methyl eugenol 65, 77, 91, 107, 147, 163, 178 45:70 12.58 b-Caryophyllene 107, 133, 148, 176, 204 45:81 6.44 a-Humulene 80, 93, 121, 147, 204 47:87 3.77 Valencene 79, 91, 105, 133, 161, 204 48:31 2.26 Germacrene 41, 79, 91, 105, 119, 161, 204 49:74 9.15 a-Amorphene 79, 93, 119, 161, 204 50:59 1.22 a-Muurolene 41, 93, 105, 161, 204 51:03 3.27 Cadinene 81, 105, 161, 204 52:52 9.52 a Major fragmentation ions, base peak (listed first) and other ions in decreasing order of relative abundance medicinal properties (Yoo et al. 2005). E. caryophyllata ness 9 0.25 mm i.d.) (Kim et al. 2015). The conditions possesses various biological activities, such as antioxidant, were as follows: the initial column temperature was antimicrobial, and insecticidal effects (Chaieb et al. 2007). 501 °C, which was increased to 209 °C; the ion-source However, few studies have reported the insecticidal temperature was 230 °C. The flow rate of helium gas was activities of E. caryophyllata oil and its active component 0.8 mL/min. Mass spectra (m/z) were produced in electron against sporadic insects. Thus, we isolated an active com- ionization (70 eV) mode with a scan range of 50–800 amu ponent from E. caryophyllata and determined the insecti- for 2 s (Cho 2015). The volatile components of E. cidal activities of E. caryophyllata oil, the isolated caryophyllata oil were identified by retention time, reten- constituent, and its structural analogues against the nymphs tion index, and mass spectra and were confirmed by com- and adults of Pochazia shantungensis. parison with an extant mass spectrum library (Table 1). The relative composition (%) of the volatile components was calculated by comparison with internal standards. Materials and methods Isolation and identification Chemicals and material preparation To isolate insecticidal constituent of E. caryophyllata Acetyl eugenol, isoeugenol, methyl eugenol, and methyl leaves, the essential oil of E. caryophyllata leaves (10 g) isoeugenol were used in this study (Sigma-Aldrich, St. was loaded onto a silica gel column (5.8 cm 9 60 cm, Louis, MO, USA). E. caryophyllata leaves (5 kg) were Merck 70–230 mesh, Germany) and sequentially eluted purchased from a regional store in Jeonju, Republic of with n-hexane and mixtures of ethyl acetate (10:1, v:v), Korea. A voucher specimen was authenticated by Jeong- resulting in 30 fractions (EC1–EC30). All fractions were moon Kim (Chonbuk National University, Republic of analyzed by thin-layer chromatography. Similar fractions Korea). E. caryophyllata leaves were powdered and were mixed and assayed. The EC6 fraction exhibited extracted by steam distillation–extraction. The residual insecticidal activity; therefore, this fraction was isolated by solvent of E. caryophyllata oil was removed in an evapo- HPLC (Spectra system P2000, Thermo Separation Prod- rator at 35 °C and stored at 4 °C. ucts, San Jose, CA, USA) and applied to a Porasil column (7.9 mm diameter 9 300 mm, Waters, MA, USA) with hexane:ethyl acetate mixture (7:3, v:v) at 0.4 ml/min, with GC–mass spectrometry detection at 287 nm. Finally, the EC62 fraction (2.3 g) was isolated. The structure of the EC62 fraction was determined The volatile constituents of E. caryophyllata oil were by some spectroscopic analyses. 1H- and 13C-NMR spectra analyzed by GC–Mass (5973 and 6890 series, Agilent, were obtained using a JNM-ECA600 spectrometer (JEOL USA) and were separated using DB-5 and HP-Innowax Ltd, Tokyo, Japan; 1H-600 MHz; 13C-150 MHz), with capillary columns (3000 cm L 9 0.25 lm thick- CDCl3. 123 Appl Biol Chem (2016) 59(4):609–614 611 Leaf-dipping bioassay and sealed using Parafilm. The system was maintained at 20 °C, with a 12:12 L:D cycle, for 72 h. The insecticidal activities of E. caryophyllata oil, the iso- lated constituent, and its structural analogues were assessed Statistical analysis against Pochazia shantungensis. Insecticidal toxicity was bioassayed according to the procedure described by Cuth- Insect mortalities were measured under the naked eyes. The bertson et al. (2009). Various dilutions of separate insec- dead insects were regarded when they did not move, which ticidal compound (1000–50 mg/L) were prepared in were touched with a brush. All treatments were repeated four acetone. One leaf was dipped into each dilution for 5 min times, and the LC50 values were determined by probit analysis. then allowed to air dry, before being placed in sealed petri dishes (60 9 15 cm) for each individual dilution of each insecticide (isolated compound and its derivatives). All Results and discussion sealed petri dishes were incubated at 20 °C, with a 12:12 L:D cycle for 72 h. Distilled water was used as the nega- The insecticidal toxicities of the essential oil extracted tive control. All experiments were replicated four times. from E. caryophyllata leaves were investigated by leaf- dipping and spray bioassays against the nymphs and adults Spray bioassay of P. shantungensis (Tables 2, 3). Based on the LC50 against nymphs of P. shantungensis by the leaf-dipping Insecticidal toxicities of the samples were measured using method, the essential oil of E. caryophyllata (LC50, the spray method against P. shantungensis adults by the 352.16 mg/L) showed insecticidal activity. The insecticidal method described by Choi et al. (2012), with slight modi- toxicity (LC50 value) of E. caryophyllata oil against P. fications. Various concentrations (1000–50 mg/L) of each shantungensis adults by the spray method was 394.46 mg/ sample were liquefied in acetone. The sample was nebu- L. Therefore, E. caryophyllata oil could represent a novel lized into a plastic box (5 9 5 9 10 cm), and 30 insects insecticide.