Toxicities of active constituent isolated from Thymus vulgaris flowers and its structural derivatives against Tribolium castaneum (Herbst)

Insecticidal activity of 2-isopropyl-5-methylphenol isolated from Thymus vulgaris oil and its derivatives against Tribolium castaneum adults and larvae were evaluated using contact and fumigant bioassays. To elucidate the structure–activity relationship, 2-isopropyl-5-methylphenol and its derivatives were determined. Based on the LD50 values, 2-isopropylphenol was most toxic to T. castaneum adults and larvae, followed by 4-isopropylphenol, 2-methylphenol, 3-methylphenol, and phenol in contact and fumigant bioassays. These results indicate that the introduction of a functional group such as methyl and isopropyl group into the phenol skeleton has an important influence on contact and fumigant toxicities. Furthermore, 2-isopropyl-5-methylphenol and its derivatives could be used as an alternative to control stored-grain insects.


Introduction
Tribolium castaneum is the destructive primary storedgrain insect of stored grains and cause serious damage to quantitative and qualitative losses of grain products (Padin et al. 2002). Insect control in stored grains is dependent in large measure on the use of synthetic insecticide. However, the use of synthetic fumigants and insecticides for decades to control stored-grain insects have leaded to a several undesirable effects, including health and environmental problems, insect toxicity and resistance to non-target organisms (Donahay et al. 1992;Sousa et al. 2009). Therefore, there is an urgent need to develop new types of safe, environmental, convenient and low-cost alternatives. For this reason, various studies have been concentrating their efforts on the search for plant materials as an alternative to synthetic fumigants and insecticides to control stored-grain insects (Huang et al. 1997).
Major constituents and essential oils derived from plants are significant natural sources of insect growth regulators, acute fumigants, fungicides, and insecticides against diverse insect species and they are developed as ecologically potential pesticides. To the best of our knowledge, nothing has been reported in journals about the insecticidal toxicities of the essential oils derived from Thymus vulgaris flowers against T. castaneum adults and larvae. The present study describes the insecticidal toxicities of active constituent isolated from T. vulgaris flowers and its derivatives using the contact and fumigant bioassays against T. castaneum adults and larvae.

Stored grain insect and bioassay
Tribolium castaneum adults and larvae were obtained from National Academy of Agricultural Science, RDA (Korea). They were reared wheat flour in plastic boxes (W 9 L 9 H, 30 9 30 9 20 cm). The laboratory cultures maintained in a 15-h light/9-h dark photoperiod at 71 ± 5 % relative humidity (RH) and 28 ± 1°C. For the fumigant and contact bioassays, about 2-3 weeks old adults and 12-to 16-day-old larvae were tested in this study. To investigate the insecticidal activities of T. vulgaris oil, the active compound, and its structurally related derivatives against T. castaneum adults and larvae, the fumigant and contact methods modified by Jeon et al. (2015) were used. To evaluate fumigant toxicity against T. castaneum adults and larvae, filter paper (5 cm diameter) was impregnated with acetone solutions (100 lL) at various concentrations (0.60-0.010 mg/cm 3 ) of each sample. To evaluate contact toxicity at various concentrations (1.0-0.01 mg/cm 2 ) for the contact toxicity were liquefied in acetone (100 lL) and applied to filter paper. Each piece of filter paper for the fumigant bioassay was attached to the lid of Petri dish (5.5 9 1.5 cm) after the treated and negative control pieces of filter paper were dried for 11 min in air. Twenty adults and larvae were added to each Petri dish which covered with a 100 mesh cloth to avoid contact with the treated sample. Each piece of filter paper for the toxicity bioassay was added to the bottom of a Petri dish after being dried in air. The larvae and adults were added to the Petri dish, and lid was closed. The treatment and control Petri dishes were placed in incubators (27 ± 2°C; 60 ± 5 % RH; photoperiod of 12 h light/12 h dark) for 48 h. All treatments were replicated five times. After 48 h of treatment, the mortality and lethal dose (LD 50 ) values of adults and larvae were determined.

Statistical analysis
Mortality was corrected for negative control mortality using Abbott's formula. The LD 50 values of the test sample were calculated by probit analysis and transformed to arcsine square-root values for analysis of variance (Finney 1971).

Results and discussion
To study the contact and fumigant toxicities of the essential oil extracted from T. vulgaris flowers, the dosage mortality responses to T. castaneum adults and larvae were determined (Table 1). Significant differences were observed in contact and fumigant toxicities against T. castaneum adults and larvae. The LD 50 value of T. vulgaris oil against T. castaneum adults and larvae were 0.141 and 0.117 mg/cm 3 in the fumigant bioassay, respectively. In the contact bioassay, the LD 50 value of T. vulgaris oil against T. castaneum adults and larvae was 0.236 and 0.158 mg/cm 2 , respectively. Tribolium castaneum adults and larvae were about 1.7 and 1.4 times more susceptible to the fumigant (1) (3) R 1 =OH; R 2 =CH 3 ; R 3 , R 4 , R 5 , R 6 =H (4) R 2 =OH; R 2 =H; R 3 = CH 3 ; R 4 , R 5 , R 6 =H (5) R 3 =OH; R 2 = CH(CH 3 ) 2 ; R 3 , R 4 , R 5 , R 6 =H (6) R 4 =OH; R 2 =H; R 3 =H; R 4 = CH(CH 3 ) 2 ; R 5 , R 6 =H The contact and fumigant toxicities were determined for 2-isopropyl-5-methylphenol and its derivatives against T. castaneum adults and larvae and compared with famous insecticide, dichlorvos, which was the positive control ( Fig. 1; Tables 2 and 3). The LD 50 values of 2-isopropyl-5methylphenol in the fumigant toxicity were 0.088 and 0.110 mg/cm 3 against T. castaneum larvae and adults ( Table 2). The LD 50 values of 2-isopropyl-5-methylphenol in the contact toxicity were 0.118 and 0.105 mg/cm 2 . Tribolium castaneum larvae were about 1.3 times more susceptible to fumigant action of 2-isopropyl-5-methylphenol. However, there were no significant differences were observed between contact and fumigant toxicity against T. castaneum adults. The fumigant toxicities of five derivatives were compared with that of dichlorvos against T. castaneum adults and larvae (Table 2). Based on the LD 50 values, 2-isopropylphenol (0.073 and 0.104 mg/cm 3 ) was most toxic to T. castaneum larvae and adult, followed by 4-isopropylphenol (0.079 and 0.106 mg/cm 3 ), 2-methylphenol (0.101 and 0.117 mg/cm 3 ), 3-methylphenol (0.116 and times more susceptible to fumigant action compared to adults. The contact toxicities of five derivatives were compared with that of dichlovos against T. castaneum larvae and adults (Table 3). Based on the LD 50 values, 2-isopropylphenol (0.049 and 0.081 mg/cm 2 ) was most toxic to T. castaneum larvae and adults, followed by 4-isopropylphenol (0.109 and 0.96 mg/cm 3 ), 2-methylphenol (0.113 and 107 mg/cm 3 ), 3-methylphenol (0.131 and 0.115 mg/cm 3 ), and phenol ([0.210 mg/cm 3 ). For five derivatives except for 2-isopropylphenol, the adults of T. castaneum were more susceptible to contact action compared to larvae. The contact and fumigant toxicities of five derivatives and how toxicity varies with structure were investigated. Phenol, which is the skeleton of 2-isopropyl-5-methylphenol exhibited no contact and fumigant toxicities against T. castaneum adults and larvae. However, introduction of functional group, such as isopropyl and methyl, in phenol ring significantly increased their contact and fumigant toxicity against T. castaneum adults and larvae. 2-Isopropylphenol, conjugated with isopropyl group, exhibited the highest contact and fumigant toxicities against T. castaneum adults and larvae. In addition, 4-isopropylphenol (conjugated with isopropyl group) much more effective against T. castaneum adults in the contact and fumigant bioassay than 2-methylphenol (conjugated with methyl group) and 3-methylphenol (conjugated with methyl group). These results indicate that the contact and fumigant mode of action of these compounds may be largely attributable to isopropyl functional group in phenol ring. According to previous studies, the toxicity and efficacy of the monoterpenes against stored-grain insects influenced by the bioassay method (Prates et al. 1998;Park et al. 2003). For example, Abdelgaleil et al. (2009) reported that treatment of T. castaneum with 1,8-cineole and myrcene was the most toxic fumigant against T. castaneum, but the two compounds were not active as a contact toxicant. Lee et al. (1997) reported that the insecticidal susceptibilities were influenced by structural characteristics of monoterpenoids such as degree of saturation, shape, and types of functional groups. Furthermore, Samarasekera et al. (2008) reported that the structural variation in the molecule is key factor in enhancing the mosquitocidal activity. Similar studies have been reported by Lee et al. (2008), introduction of functional group, such as ally, benzyl, isopropyl, and vinyl cinnamates had potent insecticidal activities against stored-grain insect, Sitophilus oryzae. Our results confirm the importance of the isopropyl groups in the insecticidal mode of action of phenol skeleton, as reported by others (Jeon et al. 2015). These observations raise the possibility that the presence of isopropyl functional group augments toxicity. As to questions on possible toxicity of T. vulgaris oil, the fact that it is consumed by humans, as herb food flavoring is indicative of its non-toxicity to humans. Furthermore, Sigma-Aldrich acute toxicity database indicated that the mammalian toxicity of 2-isopropyl-5-methylphenol isolated from Ruta graveolens oil and its structural analogs are relatively low mammalian toxicity (Sigma-Aldrich 2010, USA). So it may be given preference on synthetic pesticides for managing stored-grain insects.