Active Component Isolated from Eugenia Caryophyllata Leaves and Its Structural Analogues Show Insecticidal Properties Against Pochazia Shantungensis

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 insecticides, 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.

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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 isolated 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. The insecticidal toxicities of plant-derived were inoculated into the plastic cage. The lid was closed materials are affected by the insect species due to

Table 2 Insecticidal activities of E. caryophyllata oil, isolated eugenol, and eugenol analogues against the nymph of Pochazia shantungensis

a Compound Insect species LC50 (mg/L) 95 % conﬁdence interval

Oil Pochazia shantungensis (nymphs) 352.16 332.26–372.06 Eugenol 124.44 119.72–129.16 Acetyl eugenol 165.11 161.06–169.17 Isoeugenol 83.29 81.22–85.36 Methyl eugenol 126.31 124.32–128.30 Methyl isoeugenol 105.61 103.63–107.59 Negative control (only solvent) – – a Exposed for 72 h

Table 3 Insecticidal activities of E. caryophyllata oil, isolated eugenol, and eugenol analogues against the adults of Pochazia shantungensis

a Compound Insect species LC50 (mg/L) 95 % conﬁdence interval

Oil Pochazia shantungensis (adults) 394.46 392.10–396.82 Eugenol 143.24 138.11–148.29 Acetyl eugenol 170.06 162.21–176.39 Isoeugenol 91.03 89.51–92.55 Methyl eugenol 143.84 136.45–149.95 Methyl isoeugenol 114.48 111.42–117.54 Negative control (only solvent) – – a Exposed for 72 h

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O R1 methoxybenzene skeleton

Compound R1 R2 R3

Eugenol C3H6 CH3 OH

Acetyl eugenol C3H6 C3H5OOH

Isoeugenol C3H6 CH3 OH

Methyl eugenol C3H6 CH3 CH3O

Methyl isoeugenol C3H6 CH3 CH3O

Fig. 1 Structures of eugenol and its structurally related analogues differences in, for example, detoxiﬁcation enzymes and the component was identiﬁed as eugenol (Fig. 1) based on the size and weight of the insect populations (Yang and Lee following: eugenol (4-allyl-2-methoxyphenol) (C10H12O2, 2012). MW: 164); EI-MS (70 eV) m/z 164 [M?]; 1H-NMR

The chemical compositions of E. caryophyllata oil were (600 MHz, CDCl3) dH 6.83–6.85 (d, 2H, J = 12 Hz), analyzed by GC–MS (Table 1). Essential oil of E. 6.68–6.69 (d, 1H, J = 6 Hz), 5.92–5.98 (m, 1H), 5.47 (s, caryophyllata leaves had 12 components, which comprised 1H), 5.04–5.09 (m, 2H), 3.87 (s, 3H), 3.31–3.32 (d, 2H, 13 94.23 % of the total. The relative composition (%) of the J = 6 Hz); C-NMR (600 MHz, CDCl3) dC 146.8, 144.3, volatile constituents of E. caryophyllata oil was a-amorphene 138.2, 132.3, 121.6, 115.6, 114.6, 111.5, 56.2, 40.2. The (1.22 %), b-bourbonene (3.54 %), cadinene (9.52 %), b- 1H-NMR and 13C-NMR spectra of EC62 isolated from E. caryophyllene (6.44 %), a-copaene (9.44 %), a-cubebene caryophyllata matched those of eugenol. (14.48 %), eugenol (18.76 %), germacrene (9.15 %), a-hu- The insecticidal toxicities of eugenol, its structural mulene (3.77 %), methyl eugenol (12.58 %), a-muurolene analogues (isoeugenol, acetyl eugenol, methyl isoeugenol, (3.27 %), and valencene (2.26 %). a-Cubebene, eugenol, and and methyl eugenol), and acetone as a negative control methyl eugenol were the main constituents (45.82 %) of E. were evaluated against the nymph and adults of P. shan- caryophyllata oil. According to Dzamic et al. (2009), the tungensis by leaf-dipping and spray bioassays. Based on volatile components of E. caryophyllata oil are eugenol, a- the LC50 values of eugenol, its structural analogues, and cubebene, methyl eugenol, a-copaene, cadinene, germacrene, negative control against the nymphs of P. shantungensis and b-caryophyllene. In the previous and present studies, the (Table 2), isoeugenol (LC50, 83.29 mg/L) had the most volatile constituents of E. caryophyllata oil were inﬂuenced potent insecticidal activity, followed methyl isoeugenol by the harvest time, handling method, intraspeciﬁc variabil- (LC50, 105.61 mg/L), eugenol (124.44 mg/L), methyl ity, and the extraction method used (Kim et al., 2013). eugenol (126.31 mg/L), and acetyl eugenol (165.11 mg/L). The active constituent of E. caryophyllata oil was iso- In a spray bioassay against the adults of P. shantungensis lated by silica gel chromatography and prep. HPLC, and its (Table 3), isoeugenol (LC50, 91.03 mg/L) showed the structure was determined by some spectroscopic analyses strongest activity, followed by methyl isoeugenol (LC50, (EI-MS, 1H-NMR, and 13C-NMR) and by direct compar- 114.48 mg/L), eugenol (143.24 mg/L), methyl eugenol ison with an authentic standard compound. The active (143.84 mg/L), and acetyl eugenol (170.06 mg/L). The

123 Appl Biol Chem (2016) 59(4):609–614 613 negative control (only acetone) exhibited no activity. These Metcalfa pruinosa to commercially registered insecticides in results indicate that the insecticidal activities of E. Korea. Korean J Pest Sci 15:329–334 Batish DR, Singh HP, Kohli RK, Kaur S (2008) Eucalyptus essential caryophyllata oil against the nymphs and adults of P. oil as a natural pesticide. For Ecol Manag 256:2166–2174 shantungensis were due to eugenol and methyl eugenol. Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Rouabhia M, The insecticidal toxicities of eugenol and its structural Mahdouani K, Bakhrouf A (2007) The chemical composition analogues against the nymphs and adults of P. shantun- and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. 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Korean J Appl Entomol 50:367–372 3 Choi DS, Kim DI, Ko SJ, Kang BR, Lee KS, Park JD, Choi KJ (2012) weak toxicities against sporadic insects. Furthermore, Occurrence ecology of Ricania sp. (Hemiptera: Ricaniidae) and acetyl eugenol, which has an acetyl (C2H3O) group, selection of environmental friendly agricultural materials for exhibited decreased insecticidal activity. Taken together, control. Korean J Appl Entomol 51:141–148 these findings confirm that changing the site of the func- Cuthbertson AGS, Blackburn LF, Northing P, Luo W, Cannon RJC, Walters KF (2009) Leaf dipping as an environmental screening tional group affects insecticidal activity against a sporadic measure to test chemical efficacy against Bemisia tabaci on insect (Pochazia shantungensis). Furthermore, these results poinsettia plants. 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This study aimed to identify alternative, natural insec- Chem Nat Compd 45:259–261 Houghton JT, Ding Y, Griggs DJ, Noguer M, Van Der Linden PJ, ticides and so ameliorate chemical resistance, environ- Xiaosu D, Maskell K, Johnson CA (2001) Climate change 2001: mental toxicity, and detrimental effects on human health the scientific basis. Cambridge University Press, Cambridge (Kim et al. 2013). According to the Sigma-Aldrich MSDS, Kim MG, Yang JY, Lee HS (2013) Acaricidal potentials of active the oral toxicity of eugenol (4-ally-2-methoxyphenol) to properties isolated from Cynanchum paniculatum and acaricidal changes by introducing functional radicals. J Agric Food Chem mouse is low (LD50 value 2000 mg/kg) (Sigma-Aldrich 61:7568–7573 2010). In this regard, eugenol (4-ally-2-methoxyphenol) Kim DY, Kim SH, Ahn HM, Lim SR, Oh JS, Choi SG, Le HJ, Auh and its derivatives may be safe for use against sporadic JH, Choi HK (2015) Differentiation of highbush blueberry insects. Therefore, E. caryophyllata oil and eugenol ana- (Vaccinium corymbosum L.) fruit cultivars by GC–MS-based metabolic profiling. J Korean Soc Appl Biol Chem 58:21–28 logues are promising natural sources of pesticides against Lee HW, Lee HS (2015) Acaricidal potency of active constituent exotic pests. This study is the first to report the insecticidal isolated from Mentha piperita and its structural analogs against toxicity of the active constituent of E. caryophyllata oil pyroglyphid mites. J Korean Soc Appl Biol Chem 58:597–602 against the nymphs and adults of P. shantungensis. Further Shin YH, Moon SR, Yoon CM, Ahn KS, Kim GH (2010) Insecticidal activity of 26 insecticides against eggs and nymphs of Lycorma research should be conducted to reduce the human toxicity delicatula (Hemiptera: Fulgoridae). Korean J Pest Sci of eugenol and its analogues and improve its insecticidal 14:157–163 potency. Sigma-Aldrich (2010) In material safety data sheet (MSDS): toxico- logical information, section 11. Sigma-Aldrich, St Louis Acknowledgments This work was carried out with the support of Song JS, Lee CM, Lee DW (2013) Leaf damage symptom of grape ‘‘Cooperative Research Program for Agriculture Science & Tech- (Vitis vinifera) by tea bagworm (Eumeta minuscula) and control nology Development (Project title: Study on the ecology and devel- efficacies of several insecticides against the Eumeta minuscula. opment of management practice for an exotic and epidemic pest, Korean J Pest Sci 17:27–32 Ricania sp., Project No. PJ0116902016)’’ Rural Development Sung BK, Lee CH, Kim CH, Son JS, Lee HS (2004) Antimite effect Administration, Republic of Korea. of essential oils derived from 24 Rosaceae and Umbelliferae species against stored food mite. Food Sci Biotechnol 13:512–515 White NDG, Leesch JG (1995) Chemical control, in integrated References management of insects in stored products, New York, USA, pp. 287–330 Ahn KS, Lee GS, Lee KH, Song MK, Lim SC, Kim GH (2011) Yang JY, Lee HS (2012) Acaricidal activities of the active component Susceptibility commercially of North American planthopper, of Lycopus lucidus oil and its derivatives against house dust and

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