View of In Silico Study of Eugenol and trans-Caryophyllene also Clove Oil Fumigant Toxicity on Tribolium castaneum

(1)

http://dx.doi.org/10.11594/jtls.12.03.07

How to cite:

Research Article

In Silico Study of Eugenol and Trans-Caryophyllene also Clove Oil Fumigant Toxicity on Tribolium castaneum

Silvi Ikawati *, Toto Himawan, Abdul Latief Abadi, Hagus Tarno, Alvan Fajarudin

Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Brawijaya, Malang 65145, Indonesia

Article history:

Submission November 2021 Revised November 2021 Accepted July 2022

ABSTRACT

Alternative storage pest control that is more environmentally friendly than the use of synthetic chemical pesticides is to use botanical pesticides from plant essential oils, including clove (Syzygium aromaticum) which contains the main compounds eugenol and trans-caryophyllene. To study the various mechanisms of action of essential oils as botanical insecticides could use in silico approach through molecular docking. This study aims to predict the dominant binding mode(s) of a ligand with a protein of a known three-dimensional structure through docking. Then tested its fumigant activity on Tribolium castaneum. The docking results showed that the trans-caryophyllene and eugenol compounds had a more stable bond strength in the acetylcholinesterase en- zyme T. castaneum than the control compound linalool. In addition, there is a synergy between eugenol and trans-caryophyllene when the two compounds interact with acetylcholinesterase. These results can be used as prediction material that trans-caryo- phyllene and eugenol have potential as protein acetylcholinesterase inhibitors of T.

castaneum. After being tested in the laboratory, clove oil which contains two main compounds, namely eugenol and trans-caryophyllene has the potential to control T.

castaneum with an LC50 value of 5,227 μL/L air.

Keywords: Botanical insecticide, Caryophyllene, Docking, Eugenol, Fumigant, Syzygium aromaticum

*Corresponding author:

E-mail: [email protected]

Introduction

The flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a polyph- agous and cosmopolitan pest in cereal products [1]

and other stored products. The level of the eco- nomic damage of flour beetles to stored products in Nigeria ranges from 14 to 40% [2]. Various control measures against storage pests can be ap- plied individually or in an integrated control program [3].

In the case of mass propagation of stored product pests, synthetic chemical insecticides are often used [4]. Phosphine and methyl bromide are two common fumigant pesticides used for product protection worldwide [5]. For phosphine, The re- sistance to phosphine T. castaneum has been widely reported [6]. Methyl bromide, a broad- spectrum fumigant, has been declared an ozone- depleting substance, so its use has been banned

[5]. Therefore, alternative forms of control that are more environmentally friendly are needed

Alternative pest control is indicated by the presence of products from plants, namely essential oils and their components, where natural compounds from plant sources are believed to have advantages over synthetic pesticides in terms of low toxicity to mammals, rapid degradation, and local availability [7]. Secondary metabolites in the form of plant essential oils play an important role in plant-insect interactions because these compounds can have insecticidal activity against insects [7, 8].

Essential oils generally contain a complex mixture of monoterpenes, phenols, and sesquiterpenes [9, 10]. Unlike pesticides from synthetic chemicals, the bioactivity of essential oils in insect pest control cannot always be attributed to the main ele- ment, in some cases indicating an internal synergy

(2)

JTLS | Journal of Tropical Life Science 340 Volume 12 | Number 3 | September | 2022

eraceae, Apiaceae, Rutaceae, and Poaceae [12].

Plants that are found in Indonesia and have poten- tial as pesticides include cloves (Syzygium aro- maticum). This oil has toxic properties as a food inhibitor, and is a repellent for controlling stored products [13]. Eugenol and -caryophyllene are the main components of clove oil [10, 14, 15]. This component shows the LD50 value variable that is specific to the pest species [16]. So that, clove essential oil has the potential to be developed as a vegetable pesticide. To be able to commercialize it, there are some practical considerations besides chemical considerations, including regulatory re- quirements and costs (in some jurisdictions), the price of the oil commodity used as the active in- gredient, the availability of large volumes of oil, and the chemical consistency of the oil [10]. Es- sential oils have three mechanisms of action as pesticides [17]. The three mechanisms of action are acting on the insect's nervous system; suppres- sion and disturbance of normal growth, development, metamorphosis, and reproduction of insects;

and inhibition of mitochondrial membrane respir- atory enzymes or regulation of oxygen consump- tion and the amount of carbon dioxide released in insects. Essential oil compounds exert their activity on insects through neurotoxic effects involving several mechanisms, most notably through GABA, octopamine synapses, and acetylcholinesterase inhibition [18]. Some of the monoterpenes contained in essential oils are neurotoxic to insects, such as eugenol which has been identified as an important component of essential oils. The mechanism of this neurotoxicity involves nervous system receptors [18]. One of the important enzymes targeted on the nervous system by many insecticides is the enzyme acetylcholinesterase. To study this in the development of botanical insecticides, it is often through in vitro and in vivo testing which takes a relatively long time and is quite ex- pensive. The alternative so that these problems can be overcome is a modeling approach using computational chemical calculation techniques.

The amino acid sequence obtained from the FASTA section was made for a three-dimensional quaternary structure modeling online at www.swissmodel.expasy.org. After all the three- dimensional structure models are ready, the docking process is carried out. The docking process between eugenol and trans-caryophyllene molecules with acetylcholinesterase receptor protein using the PyRx program. The analysis of the docking results includes the interactions that occur. Docking data collection results in the form of binding energy and electrostatic energy. The stages of the in silico study are presented in Figure 2. After the docking data is obtained, the next step is to determine the conformation of the docking result, which has the smallest ki value. To get the value of the inhibition constant (Ki) the Cheng-Prusoff formula is used as follows:

Ki = 𝑒⁽^{𝐷𝐺𝑏𝑖𝑛𝑑}^2.5 ⁾

Where Ki is the constant of inhibition, e is the mathematical constant (2.71828), and DG bind is the binding affinity. Provided DGbind in kJ/mol [19].

Clove oil composition

The chemical composition of clove oil was de- termined by Gas Chromatography-Mass Spec- trometry (GC-MS Shimadzu Model QP-2010).

The compounds were identified by comparing their retention times with known compounds and also comparing their mass spectra with those stored in the Wiley 8 Library (comparison quality

>80%). The number of relative percentages is obtained directly from the peak region of the GC.

GC-MS analysis was performed with a GCMS Shimadzu Model QP-2010 Plus Mass Spectrome- ter equipped with a capillary column (HP-5, 25 m

× 0.25 mm, 0.25 m film thickness). The carrier gas is helium with a flow of 1 mL min-1. The GC oven temperature was held at 50°C for 2 minutes, pro- grammed at 5°C min-1 to 200°C, then held at this

(3)

temperature for 15 minutes. The mass spectrum was recorded at 70 eV. The mass range is from m/z 35-350 amu. The injection block temperature is 250°C.

Rearing of insect test

Insects were obtained from the collection of the Laboratory of Plant Pests, Department of Plant Pests and Diseases, Faculty of Agriculture, Uni- versitas Brawijaya. The feed used for rearing is a composition of 95% wheat flour and 5% instant yeast [20]. The flour used is the blue triangle brand with a total weight of 1 kg of feed. T. castaneum insects were identified using a stereomicroscope to identify the male and female species. After the feed and insects were ready, the feed was placed into a propagation jar (t=12 cm, and d=10) and then mixed evenly, and 100 adult insects were in- fested with a ratio of male and female 1:4 into the feed. Furthermore, the propagation tube was left for seven days for the oviposition process. Then, all imago were removed from the propagation tube. The eggs produced by the imago are awaited until they become pupae. When entering the pupa stage, the male and female pupae were separated based on their genital organs using a stereomicroscope. Next, male and female pupae are placed in separate tubes and left to develop into new imago.

The new adults used in the study were seven to fourteen days old.

Clove oil fumigant toxicity

Testing the activity or fumigant properties of clove oil on adults of T. castaneum was conducted by exposing it to 30 adults (aged 5-7 days) without distinguishing between males and females. The test was carried out in a 250 mL glass flask which was closed using a rubber stopper. Clove oil was dissolved in 1 mL of acetone to obtain the desired concentration (0.8, 1.6, 3.2, and 6.4 μL/L air). For control, only used acetone without clove oil. Each concentration was dripped evenly on a piece of Whatman No 1 filter paper (7 cm x 9 cm), air-dried (10 minutes), and then affixed one side to a rubber cover. The filter paper is installed in such a way that it does not come into contact with the walls of the flask. After that, the flask was stored in an in- cubator at a temperature of 27^oC and 75% Rh.

Each treatment was repeated 5 (five) times. After exposure for 48 hours, insects were transferred to Petri dishes. Insect mortality as soon as possible

can be observed. Insects are considered dead when touched and do not show movement.

Statistical analysis

Clove Oil Fumigant Activities were arranged in a Randomized Block Design. The percentage of mortality due to fumigant activity was analyzed by ANOVA and if there was a significant difference in the treatment, further tests were carried out so that the difference between treatments was known by the DMRT test (0.05). Probit analysis was used to obtain 48 h LC50 and LC90 results. All tests were carried out using the SPSS 23.0 program (SPSS, Inc., Chicago, IL). If there is a death in the control, then the percent death needs to be corrected. If the mortality of insects in control is 20%, then the test must be repeated. The percentage of corrected deaths was calculated based on Abbott's (1925) formula as follows:

𝑃 =X − Y

Y × 100

Where P is the percentage of corrected mortality, X is the percent of live insects in control, and Y is the percent of live insects in the treatment [21].

Results and Discussion

Molecular Interaction of Eugenol and Trans- caryophyllene with Acetylcholinesterase T.

castaneum In Silico

Collection of 3D Structures of Bioactive Compounds

Eugenol (C10H12O2) is a phenylpropanoid formally derived from guaiacol with an allyl chain in the para position with a hydroxy group. It has the role of allergen, plant metabolite, human blood serum metabolite, and sensitizer. It is a phenylpropanoid, monomethoxybenzene, and phenol member. It is derived from guaiacol [22].

Eugenol has a molecular weight of 302,238 g/mol.

The 3-dimensional structure of the eugenol compound was obtained from the compound database, namely PubChem with CID number 3314 (Figure 1).

Trans-caryophyllene (C15H24) is found in many essential oils, especially clove oil. These compounds have roles as non-steroidal anti-in- flammatory drugs, fragrances, metabolites, and insect attractants [23]. Trans-caryophyllene has a molecular weight of 204.35 g/mol. The 3-dimen-

(4)

sional structure of the trans-caryophyllene compound was obtained from the compound database, namely PubChem with CID number 5281515 (Figure 2).

Linalol was chosen as a control compound because it can bind to acetylcholinesterase protein [24]. These compounds have roles as plant metabolites, components of essential oils, antimicrobial agents, and fragrances. It is tertiary alcohol and a monoterpenoid [25]. Linalool has a molecular weight of 154.25 g/mol. The 3- dimensional structure of the linalool compound was obtained from the compound database, namely PubChem with CID number 6549 (Figure 3).

Determination of Target Protein

The receptor protein molecule is the acetyl- cholinesterase enzyme from the insect T. casta- neum downloaded in the form of an amino acid se- quence from www.ncbi.nlm.nih.gov in the Protein

subdatabase with GenBank number: EEZ99262.2.

The amino acid sequence obtained from the FASTA section was then modeled on an online three-dimensional quaternary structure at www.swissmodel.expasy.org. The 3D Acetylcho- linesterase structure of T. castaneum was visual- ized with PyMol software (Figure 4).

Interaction of Acetylcholinesterase with Bioactive Compounds

The interaction of target proteins with bioactive compounds or ligands can be identified through 3 stages: molecular docking, 3D visualization using PyMol, and Protein-Ligand Interac- tion Profiler (PLIP).

The interaction analysis of eugenol and trans- caryophyllene on the acetylcholinesterase enzyme through the molecular docking method was carried out using PyRx 0.8 software. Molecular docking has resulted in 9 conformational models of interactions for each ligand with macromolecules.

Figure 1. The 2D and 3D structures of eugenol compounds [22]

Figure 2. The 2D and 3D structures of trans-caryophyllene compounds [23]

Figure 3. The 2D and 3D structure of Linalool compound [25]

(5)

The docking results obtained are the lowest binding affinity of each compound (Table 1). When there is a fit (fit) in shape and volume between the ligand molecule and the active site or protein binding site, it means that the ligand interaction with the receptor protein occurs [26]. In addition, it is also necessary that the functional groups on the ligand molecule must be inadequate positions of the amino acids that are their partners at the active site or the anchoring site [27]. The match between the ligand molecule and the active site or protein binding site is very specific, such as the described match between the keyhole and the key (lock-and- key) [26]. The active site or the mooring site urges (induces) a conformational change of the ligand [26, 28]. With this conformational change, a cer- tain amount of energy will be released which is known as the Gibbs energy of tethering (∆Gbind) or binding affinity [27]. Binding affinity (∆G) is used as a parameter for the stability of the interaction between compounds and proteins (ligands with receptors). The smaller (negative) value of

∆G indicates that the level of stability is getting better between the ligand and the receptor so that the bond formed will be stronger.

The docking results of trans-caryophyllene

and eugenol compounds showed different results on the binding ability of the acetylcholinesterase enzyme. The binding affinity values of trans-caryophyllene and eugenol with acetylcholi-nesterase of -8.3 and 6.3 kcal/mol were lower than the binding affinity values of linalool with acetylcholinesterase of -5.5 kcal/mol. The lower binding affinity value indicates that the compound can react spon- taneously without using a lot of energy and has a stable bond [29, 30]. So that trans Caryophyllene and eugenol have more stable bond strength than the control compound linalool. The sequence of compounds that best binds to acetylcholinesterase is trans-caryophyllene, eugenol, and then linalool.

In addition, there is a good synergy seen in the combination of Eugenol - transCaryophylene, with a lower binding affinity value than when the eugenol compound interacts with acetylcholinesterase alone.

The 3D visualization with PyMol and Discover- yStudio

This 3D visualization aims to determine the binding site of the compound with acetylcholinesterase protein using PyMol software. The results of the 3D visualization of the compound

a b

Figure 4. The 3D structure of acetylcholinesterase protein is visualized with PyMol software, shown in the form of a cartoon (a) and a surface (b).

Table 1. Docking results, inhibition constants, and LC50 of eugenol and trans-caryophyllene against Acetyl- cholinesterase T. castaneum

Compound Binding Affinity (kcal/mol) Ki (mM) LC50= 2xKi (mM)

Trans-caryophylene -8,3 0,0009 0,0019

Trans-caryophylene - Eugenol -6,2 0,0312 0,0623

Eugenol -6,3 0,0264 0,0527

Eugenol – Trans-caryophylene -8,3 0,0009 0,0019

Linalool -5,5 0,1006 0,2011

(6)

binding to the target protein are shown in Figure 5.

Based on the visualization, it can be seen that trans-caryophyllene and eugenol are located at the same binding position as the control compound linalool. So, it can be said that trans-caryophyllene

and eugenol have the same potential as linalool as protein acetylcholinesterase inhibitors. The linalool compound works by inhibiting the cholinergic system by inhibiting the mechanism of acetylcholinesterase (AchE) [24].

Furthermore, visualization of the molecular

a b

Figure 5. The binding sites of transCaryophyllene (red), eugenol (yellow), linalool (blue) with acetylcholinesterase protein (green) are visualized with PyMOL software in cartoon form (a) and surface (b).

a b

c d

Figure 6. 3D interaction between acetylcholinesterase protein and (A) eugenol, (B) transcaryophyllene - eugenol, (C) transcaryophyllene, and (D) eugenol - transcaryophyllene using PyMol software.

(7)

docking between the two test compounds, namely eugenol, and trans-caryophyllene, was carried out to determine the interaction. This process is carried out by combining macromolecules with one of the ligands using PyMol software, then followed by visualization of acetylcholinesterase- transcaryophyllene with eugenol, and acetylcholinesterase-eugenol with trans-caryophyllene.

The resulting interaction is seen through the docking of the acetylcholinesterase protein with the test compound resulting in an interaction pat- tern that has different characteristics. Following are the interactions of proteins and test compounds in 3D using PyMol software (Figure 6) and interactions in 2D using DiscoveryStudio software

(Figure 7). Interactions that occur between eugenol and transcaryophyllene-eugenol with amino acids include Hydrogen and Pi-Alkyl interactions.

Meanwhile, the interactions that occur between transCaryophyllene and eugenol – transcaryophyllene with amino acids are Pi-Sigma and Pi- Alkyl.

Visualization with Protein-Ligand Interaction Profiler (PLIP). This 3D visualization aims to determine the amino acid residues and the bond dis- tance between the compound and the acetylcholinesterase protein. The tool used is the PLIP web- server (https://projects.biotec.tu-dresden.de/plip- web/plip). The visualization results can be seen in Figures 8-12. The binding site of trans-caryo-

a b

c d

Figure 7. 2D interaction between acetylcholinesterase protein and (a) eugenol, (b) transcaryophyllene - eugenol, (c) transcaryophyllene, and (d) eugenol - transcaryophyllene using the DiscoveryStudio software.

(8)

JTLS | Journal of Tropical Life Science 346 Volume 12 | Number 3 | September | 2022 Figure 8. Visualization of trans-caryophyllene with acetylcholinesterase using PLIP

Figure 9. Visualization results of eugenol with acetylcholinesterase using PLIP

(9)

phyllene in acetylcholinesterase protein involves 114 amino acids TYR, 126 TRP, 351 PHE, and 392 PHE which are present in hydrophobic bonds (Figure 8). Meanwhile, the binding site for eugenol involved 126 TRP amino acids, 391 TYR on hydrophobic bonds, and 171 GLY, and 258 GLU on hydrogen bonds (Figure 9). The binding site of linalool involves 126 TRP amino acids, 391 TYR on hydrophobic bonds, and 171 GLY on hydrogen bonds (Figure 10).

The similarity of binding sites between trans- Caryophyllene and eugenol with the control compound linalool can be seen in the similarity of amino acid residues. Trans-Caryophyllene has the same bond compared to linalool at 126 TRP, while eugenol also has the same bond as linalool at 126 TRP, 391 TYR, and 171 GLY. This similarity can be used as a prediction material that trans-caryophyllene and eugenol have potential as inhibitors of acetylcholinesterase proteins such as linalool.

The analysis of the interaction of eugenol and trans-caryophyllene on the acetylcholi-nesterase enzyme through the molecular docking method (adjusting two molecules together, namely protein and ligand in 3D and using computational chemi-

cal calculations) and their visualization showed that trans-caryophyllene and eugenol have poten- tial as protein inhibitors of T. castaneum acetyl- cholinesterase.

Clove Oil Composition

From the results of the GC-MS analysis, it was found that there are two main components contained in clove oil, namely eugenol and trans- caryophyllene (Table 2). The largest component was eugenol and trans-caryophyllene with 89 and 10% percentages, respectively.

The content of eugenol and trans-caryophyllene in this study is following the results of previous studies. Eugenol (85.39%) and transcaryophyllene (9.22%) were identified as two major components of clove oil [31]. clove oil and its main effective composition eugenol show many beneficial advantages [32].

Clove Oil Fumigant Activity

In this study, clove oil fumigant activity was evaluated against T. castaneum imago using the filter paper method (Figure 11). The results showed that the clove oil concentration signifi- Figure 10. Visualization of linalool with acetylcholinesterase using PLIP

(10)

Parameter Value

LC50 (μL/L air) 5,227

LC90 (μL/L air) 87,157

Intercept (±SE) -0,753 (1.433)

Slope (±SE) 1,049 (1,060)

Chi² 0,612

df 1

cantly affected the mortality of T. castaneum after 24 hours of exposure (F=26,860, df=3, P=0.000).

Here, concentration is the main effect, while corrected mortality is the dependent variable. Clove oil at the lowest to the highest concentration resulted in increased mortality until, at the highest concentration, mortality reached 54%. So, from these results, it can be estimated the value of LC50

through probit analysis (Table 3).

The LC50 and LC90 values of clove oil fumi- gants against T. castaneum were 3,920 and 65,368 L/L water, respectively. From the LC50 results, it can be concluded that clove oil has fairly good fu- migant toxicity against T. castaneum. Clove oil has contact toxicity against storage pest T. casta- neum [33] and Cryptolestes ferrugineus [34]as well as fumigant toxicity against Lasioderma ser- ricorne [35]. The slope of the response curve for the clove oil concentration is quite high, namely 1.08. The slope value of the high concentration- response curve indicates that small variations in essential oil concentrations lead to large variations in mortality [36]. This study demonstrated the potential of clove oil and its components against the pest T. castaneum through an in silico test. How- ever, further research can be carried out by testing each of its components in the laboratory to com- plete the data in this study.

Conclusion

In this study, docking results showed that the trans-caryophyllene and eugenol compounds had a more stable bond strength in the acetylcholi- nesterase enzyme T. castaneum than the control

potential to control T. castaneum with an LC50

value of 5,227 μL/L air.

Acknowledgment

Authors gratefully acknowledge BPPM, Faculty of Agriculture, the University of Brawijaya by PNBP Research Grant for financial support.

References

1. Good NE (1936) The flour beetles of the genus Tribolium. USDA Technical Bulletin 5 27–28.

2. Ajayi FA, Rahman SA (2006) Susceptibility of some staple processed meals to red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae).

Pakistan J Biol Sci. DOI: 10.3923/pjbs.2006.1744.1748 3. Lo PK (1986) Recognition and control of insect pests in

stored grains. Post-harvest prevention of paddy/rice loss Taiwan Council of Agriculture, Asian Productivity Organization,Taipei 171–186.

4. Gasch T (2014) The use of semiochemicals for stored product protection.

5. Rajendran S, Sriranjini V (2008) Plant products as fumigants for stored-product insect control. Journal of Stored Products Research 44 (2): 126–135. DOI:

10.1016/j.jspr.2007.08.003.

6. Devi MA, Sahoo D, Singh TB, Rajashekar Y (2019) Toxicity, repellency and chemical composition of essential oils from Cymbopogon species against red flour beetle Tribolium castaneum Herbst (Coleoptera:

Tenebrionidae). J Consum Prot Food Saf. DOI:

10.1007/s00003-019-01264-y.

7. Ebadollahi A, Safaralizadeh M, Pourmirza A (2010) Fumigant Toxicity of Lavandula Stoechas L. Oil Against Three Insect Pests Attacking Stored Products. Journal of Plant Protection Research 50 (1): 56–60. DOI:

10.2478/v10045-010-0010-8.

8. Lashgari A, Mashayekhi S, Javadzadeh M, Marzban R (2014) Effect of Mentha piperita and Cuminum cyminum essential oil on Tribolium castaneum and Sitophilus oryzae. Archives Of Phytopathology And Plant Protection 47 (3): 324–329. DOI:

10.1080/03235408.2013.809230.

9. Hummelbrunner LA, Isman MB (2001) Acute, Sublethal, Antifeedant, and Synergistic Effects of Monoterpenoid Essential Oil Compounds on the Tobacco Cutworm, Spodoptera litura (Lep., Noctuidae). Journal of Agricultural and Food Chemistry 49 (2): 715–720. DOI:

10.1021/jf000749t.

10. Isman MB (2016) Pesticides based on plant essential oils:

Phytochemical and practical considerations. ACS Symp

(11)

Ser. DOI: 10.1021/bk-2016-1218.ch002.

11. Bernards MA (2010) Plant natural products: a primer The present review is one in the special series of reviews on animal–plant interactions. Canadian Journal of Zoology 88 (7): 601–614. DOI: 10.1139/Z10-035.

12. Tripathi AK, Upadhyay S, Bhuiyan M, Bhattacharya PR (2009) A review on prospects of essential oils as biopesticide in insect-pest management. Kyōtoshi Maizō

Bunkazai Kenkyūjo.

13. Plata-Rueda A, Campos JM, da Silva Rolim G et al.

(2018) Terpenoid constituents of cinnamon and clove essential oils cause toxic effects and behavior repellency response on granary weevil, Sitophilus granarius.

Ecotoxicology and Environmental Safety 156 263–270.

DOI: 10.1016/j.ecoenv.2018.03.033.

14. Jirovetz L, Buchbauer G, Stoilova I et al. (2006) Chemical Composition and Antioxidant Properties of Clove Leaf Essential Oil. Journal of Agricultural and Food Chemistry 54 (17): 6303–6307. DOI:

10.1021/jf060608c.

15. Park I-K, Shin S-C (2005) Fumigant Activity of Plant Essential Oils and Components from Garlic ( Allium sativum ) and Clove Bud ( Eugenia caryophyllata ) Oils against the Japanese Termite ( Reticulitermes speratus Kolbe). Journal of Agricultural and Food Chemistry 53 (11): 4388–4392. DOI: 10.1021/jf050393r.

16. Koul O, Walia S, Dhaliwal GS (2008) Essential Oils as Green Pesticides: Potential and Constraints. Biopestic Int 4 (1): 63–84.

17. Liao M, Xiao J-J, Zhou L-J et al. (2016) Insecticidal Activity of Melaleuca alternifolia Essential Oil and RNA- Seq Analysis of Sitophilus zeamais Transcriptome in Response to Oil Fumigation. PLOS ONE 11 (12):

e0167748. DOI: 10.1371/journal.pone.0167748.

18. Regnault-Roger C, Vincent C, Arnason JT (2012) Essential Oils in Insect Control: Low-Risk Products in a High-Stakes World. Annual Review of Entomology 57 (1): 405–424. DOI: 10.1146/annurev-ento-120710- 100554.

19. Yung-Chi C, Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. DOI:

10.1016/0006-2952(73)90196-2.

20. Campolo O, Verdone M, Laudani F et al. (2013) Response of four stored products insects to a structural heat treatment in a flour mill. Journal of Stored Products Research 54 54–58. DOI: 10.1016/J.JSPR.2013.05.001.

21. Abbott WS (1925) A method for computing the effectiveness of an insecticide. J. Econ. Entomol.

22. PubChem. 2020a. Eugenol.

https://pubchem.ncbi.nlm.nih.gov/compound/Eugenol 23. PubChem. 2020b. beta-Caryophyllene.

https://pubchem.ncbi.nlm.nih.gov/compound/beta-

Caryophyllene

24. Pavela R, Benelli G (2016) Essential Oils as Ecofriendly Biopesticides? Challenges and Constraints. Trends Plant Sci. DOI: 10.1016/j.tplants.2016.10.005

25. Motiejunas D, Wade RC (2006) Structural, energetic, and dynamic aspects of ligand-receptor interactions. Compr Med Chem II. DOI: 10.1016/b0-08-045044-x/00250-9 26. Sugita M, Hamano M, Kasahara K et al. (2020) New

Protocol for Predicting the Ligand-Binding Site and Mode Based on the 3D-RISM/KH Theory. Journal of Chemical Theory and Computation 16 (4): 2864–2876.

DOI: 10.1021/ACS.JCTC.9B01069.

27. PubChem. 2020c. Linalool.

https://pubchem.ncbi.nlm.nih.gov/compound/Linalool 28. Foloppe N, Chen I-J (2009) Conformational Sampling

and Energetics of Drug-Like Molecules. Curr Med Chem.

DOI: 10.2174/092986709789057680.

29. Richards S, Gibbs RA, Weinstock GM et al. (2008) The genome of the model beetle and pest Tribolium castaneum. Nature 452 (7190): 949–955. DOI:

10.1038/nature06784.

30. Vangone A, Bonvin AMJJ (2015) Contacts-based prediction of binding affinity in protein–protein complexes. Elife. DOI: 10.7554/eLife.07454.

31. Ikawati S, Himawan T, Abadi AL, Tarno H (2020) Thermostability, photostability, and toxicity of clove oil nanoparticles against Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae). Biodiversitas Journal of Biological Diversity 21 (10): 4764–4771. DOI:

10.13057/BIODIV/D211041.

32. Hu Q, Zhou M, wei S (2018) Progress on the Antimicrobial Activity Research of Clove Oil and Eugenol in the Food Antisepsis Field. Journal of Food Science 83 (6): 1476–1483. DOI: 10.1111/1750- 3841.14180.

33. Ikawati S, Himawan T, Abadi AL, Tarno H (2021) Toxicity nanoinsecticide based on clove essential oil against Tribolium castaneum (Herbst). Journal of Pesticide Science 46 (2): 222–228. DOI:

10.1584/JPESTICS.D20-059.

34. Ikawati S, Himawan T, Abadi AL, Tarno H (2021) Characterization of clove oil nanoparticles and their insecticidal activity against cryptolestes ferrugineus (Stephens) (coleoptera: Laemophloeidae). Agrivita 43 (1): 43–55. DOI: 10.17503/AGRIVITA.V1I1.2532.

35. Ikawati S, Dhuha MS, Himawan T (2017) Bioactivity of Citrus Hystrix D.C. Leaf Extract Against Cigarette Beetle Lasioderma Serricorne (F.). Journal of Tropical Life Science 7 (3): 189–196.

36. Pinheiro PF, Queiroz VT de, Rondelli VM et al. (2013) Insecticidal activity of citronella grass essential oil on Frankliniella schultzei and Myzus persicae. Ciência e Agrotecnologia. DOI: 10.1590/s1413- 70542013000200004.

(12)

This page is intentionally left blank.