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Evaluation of benzaldehyde derivatives from Morinda officinalis as anti-mite agents with dual function as acaricide and mite indicator

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus with 12–30% fatality rate. Despite severity of the disease, any medication or treatment for SFTS has not developed yet. One approach to prevent SFTS spreading is to control the arthropod vector carrying SFTS virus. We report that 2–methylbenzaldehyde analogues from M. officinalis have a dual function as acaricide against Dermatophagoides spp. and Haemaphysalis longicornis and indicator (color change) against Dermatophagoides spp. Based on the LD50 values, 2,4,5–trimethylbenzaldehyde (0.21, 0.19 and 0.68 μg/cm3) had the highest fumigant activity against D. farinae, D. pteronyssinus and H. longicornis, followed by 2,3–dimethylbenzaldehyde (0.46, 0.44 and 0.79 μg/cm3), 2,4–dimethylbenzaldehyde (0.66, 0.59 and 0.95 μg/cm3), 2,5–dimethylbenzaldehyde (0.65, 0.68 and 0.88 μg/cm3), 2–methylbenzaldehyde (0.95, 0.87 and 1.28 μg/cm3), 3–methylbenzaldehyde (0.99, 0.93 and 1.38 μg/cm3), 4–methylbenzaldehyde (1.17, 1.15 and 3.67 μg/cm3) and M. officinalis oil (7.05, 7.00 and 19.70 μg/cm3). Furthermore, color alteration of Dermatophagoides spp. was shown to be induced, from colorless to dark brown, by the treatment of 2,3–dihydroxybenzaldehyde. These finding indicated that 2–methylbenzaldehyde analogues could be developed as functional agent associated with the arthropod vector of SFTS virus and allergen.

Introduction

Appearance of new infectious diseases creates serious health threat to the world. Ebola and Severe fever with thrombocytopenia syndrome are the typical example of new infectious diseases, which were recently discovered in West Africa and Northeast Asia1. The SFTS is characterized by fever, leukocytopenia, respiratory problems, gastrointestinal symptoms and thrombocytopenia1. The etiological agent of SFTS is SFTS virus, a novel bunyavirus, which is carried by a tick, Haemaphysalis longicornis. Although the prevalence of SFTS is rare, the disease is a serious disease in which fatality rate range from 12 to 30%2. However, there has not been developed any medication or treatment for SFTS yet. More seriously, the population of H. longicornis is increasing because of global warming2, suggesting that SFTS could threaten the world near future.

House dust mites, such as Dermatophagoides pteronyssinus and Dermatophagoides farinae, play directly as a strong allergen to cause a number of allergic diseases, of which atopic dermatitis and allergic rhinitis are the most prevalent diseases1. Although changes in housing environments with local heating, repeated ventilation and limited use of fitted carpets are a solution to control acari, which are the subclass of ticks and mites, the control of acari by improvement of house conditions has a definite limitation3. Therefore, there is urgent demand for development of an agent controlling acari effectively to prevent allergic diseases and diseases spread by acari including SFTS.

Traditional control methods have been performed using many approaches, such as the following: (1) implementation of exceptional cleaning standards; (2) improvement of habitat of mites and ticks; and (3) use of synthetic acaricides, such as avermectin, γ–benzene hexachloride, chlopyrifos–methyl, DEET, fenitrothion, pirimiphos–methyl, etc4,5,6. In spite of the outstanding effects of these synthetic acaricides against mites and ticks, their repeated treatment has resulted in the occurrence of resistance, environmental impact and side effects in non–target organisms7. For example, Van Leeuwen et al.8 had reported that avermectin in mites acts on glutamate–gated chloride channels and γ-aminobutyric acid (GABA). Resistance of avermectin causes an increase of excretion and decreases of absorption with conjugated compound8. In light of these problems, there is a need for the development of alternatives for the control of mites and ticks, such as more efficient and safer chemicals of natural origin9.

Plants have been found as prospective alternatives to a number of chemicals because of the abundance of materials designated as traditional herbal medicine10,11. Morinda officinalis How (family Rubiaceae), cultivated in subtropical and tropical regions, has been used in traditional herbal medicines and nutrient supplements for more than 2,000 years12,13. To date, various biological activities of M. officinalis roots have been reported, including antiosteoporosis14, antifatigue15, antioxidant16, hypoglycemic17 and antidepressant agents17. In addition, various chemicals have been found in M. officinalis roots, including anthraquinone, carbohydrate, iridoid glucoside, iridoid lactone, rotungenic and β–sitosterol17. These findings indicated that M. officinalis roots should have important pharmacological properties14,15,16,17,18. In this regard, the potential ability of M. officinalis roots to act as a natural acaricide against mites and ticks was assessed in the present study.

Results and Discussion

Essential oil was extracted from M. officinalis roots with a yield of 0.03%. To determine the acaricidal potential, the acaricidal activity of M. officinalis oil was evaluated using the contact and the fumigant bioassays against Dermatophagoides spp. and H. longicornis. Compared with the LD50 values of DEET, the acaricidal activity of M. officinalis oil (7.05, 7.00 and 19.70 μg/cm3) proved to be more toxic than that of DEET (36.50, 34.23 and 52.03 μg/cm3) against D. farinae, D. pteronyssinus and H. longicornis in the fumigant assay, respectively (Table 1). In case of the contact bioassay, the acaricidal effect of M. officinalis oil (5.57, 5.00 and 15.48 μg/cm2) was also more potent than that of DEET (19.64, 14.12 and 47.77 μg/cm2) against D. farinae, D. pteronyssinus and H. longicornis, respectively (Table 1). The negative treatment, injection of acetone alone, did not result in the death of dust mites and ticks in the fumigant and the contact bioassays. The acaricidal potential of M. officinalis oil depends on the species of mites, chemicals and biological conditions19. Thus, M. officinalis oil has the potential ability as a natural acaricide against Dermatophagoides spp. and H. longicornis.

Table 1 Acaricidal activities of M. officinalis oil and acaricide against Dermatophagoides spp. and H. longicornis1

The M. officinalis oil was analyzed by GC–MS and the retention index, retention time and mass spectra of each compound were compared to those reported in the literature20. A list of the constituents from M. officinalis oil is provided in Table 2. The relative composition (%) of the constituents of M. officinalis oil was 1–allyl–4–methoxybenzene (2.42%), 1,2–benzenedicarboxylic acid (4.54%), γ–butyrolactone (2.74%), hexadecanoic acid (6.47%), hexanoic acid (2.42%), 2–methylanthraquinone (8.00%), 2–methylbenzladehyde (31.97%), 8–methylundecene (2.94%), myristaldehyde (6.53%), 1,3,12–nonadecatriene (2.63%), nonanoic acid (4.27%), 9,17–octadecadienal (2.09%), paeonol (11.26%) and γ–stearolactone (2.20%). The constituents of M. officinalis oil were grouped as acids (1,2–benzedicarboxylic acid, hexadecanoic acid, hexanoic acid and nonanoic acid), aldehydes (2–methylbenzaldehyde and myristaldehyde), alkenes (8–methylundecene, 1,3,12–nonadecatriene and 9,17–octadecadienal), benzenes (1–allyl–4–methoxybenzene), lactones (γ–butyrolactone and γ–stearolactone), phenols (paeonol) and quinones (2–methylanthraquinone). According to Yong–tao (2009)21, the volatile components of M. officinalis oil were borneol, diisobutyl phthalate, linoleic acid, 3–methylbenzaldehyde and oleic acid22. In the present and previous studies, the constituents of M. officinalis oil were influenced by the environmental conditions, including the handling method, harvest time, intraspecific variability, storage period and the experimental conditions, which included the method of extraction and the parts of the plant extracted20.

Table 2 Analysis of various constituents from M. officinalis oil identified by GC–MS

To isolate the active constituent of M. officinalis oil, various chromatographic analyses were conducted using column chromatography and liquid chromatography. The MO322 fraction was finally isolated from M. officinalis oil and the structure was identified by spectroscopic analyses including 1H–NMR, 13C–NMR and DEPT–NMR spectra. The isolated MO322 fraction was characterized as 2–methylbenzaldehyde (Figure 1) (C8H8O); EI–MS (70 eV) m/z M+ 120.06; 1H–NMR (CDCl3, 400 MHz) δ 2.48 (s, 3H), 7.26–7.44 (d, J = 7.2 Hz, 1H), 7.45–7.59 (t, J = 5.6 Hz, 1H), 7.61–7.75 (t, J = 5.6 Hz, 1H), 7.77–7.90 (d, J = 5.2 Hz, 1H) and 10.36 (s, 1H); 13C–NMR and DEPT–NMR (CDCl3, 100 MHz) δ 18.6 (CH3), 191.0 (CH), 126.2 (CH), 131.9 (CH), 13.1.9 (CH) 134.4 (C), 134.4 (CH), 139.6 (C). The findings of 2–methylbenzaldehyde were compared with those of a previous study23. The acaricidal activity of 2–methylbenzaldehyde isolated from M. officinalis oil was evaluated using the contact and the fumigant bioassays against Dermatophagoides spp. and H. longicornis and compared with that of DEET. The LD50 values of 2–methylbenzaldehyde in the fumigant bioassay were 0.95, 0.87 and 1.28 μg/cm3 against D. farinae, D. pteronyssinus and H. longicornis, respectively (Table 3). In the contact bioassay, the LD50 values of 2–methylbenzaldehyde were observed to be 0.51, 0.47 and 0.94 μg/cm2 against D. farinae, D. pteronyssinus and H. longicornis, respectively (Table 4). In comparison with the LD50 values of DEET in the fumigant bioassay, 2–methylbenzaldehyde was approximately 38.58, 39.30 and 40.58 times more effective than DEET (36.50, 34.23 and 52.03 μg/cm3) against D. farinae, D. pteronyssinus and H. longicornis (Table 3). In the contact bioassay, it was about 38.48, 30.04 and 50.60 times more toxic than DEET (19.64, 14.12 and 47.77 μg/cm2) against D. farinae, D. pteronyssinus and H. longicornis (Table 4). In this regard, the acaricidal activity of 2–methylbenzaldehyde was much more potent than that of DEET, a commercial acaricide, against D. farinae, D. pteronyssinus and H. longicornis.

Table 3 Acaricidal activities of 2–methylbenzaldehyde analogues and commercial acaricide against Dermatophagoides spp. and H. longicornis, using a fumigant bioassay1
Table 4 Acaricidal activities of 2–methylbenzaldehyde analogues and commercial acaricide against Dermatophagoides spp. and H. longicornis, using a contact bioassay1
Figure 1
figure1

Structure–activity relationships of 2–methylbenzaldehyde analogues.

(a) 2–Methylbenzaldehyde; (b) 3–Methylbenzaldehyde; (c) 4–Methylbenzaldehyde; (d) 2–Hydroxybenzaldehyde; (e) 3–Hydroxybenzaldehyde; (f) 4–Hydroxybenzaldehyde; (g) 2,3–Dimethylbenzladehyde; (h) 2,4–Dimethylbenzladehyde; (i) 2,5–Dimethylbenzladehyde; (j) 2,3–Dihydroxybenzladehyde; (k) 2,4–Dihydroxybenzladehyde; (l) 2,5–Dihydroxybenzladehyde; (m) 2,4,5–Trimethylbenzladehyde; (n) 2,4,5–Trihydroxybenzladehyde.

To establish the structural relationships between 2–methylbenzaldehyde analogues and acaricidal activities against Dermatophagoides spp. and H. longicornis, 2,3–dihydroxybenzaldehyde, 2,5–dihydroxybenzaldehyde, 2,4–dihydroxybenzaldehyde, 2,3–dimethylbenzaldehyde, 2,4–dimethylbenzaldehyde, 2,5–dimethylbenzaldehyde, 2–hydroxybenzaldehyde, 3–hydroxybenzaldehyde, 4–hydroxybenzaldehyde, 3–methylbenzaldehyde, 4–methylbenzaldehyde, 2,4,5–trihydroxybenzaldehyde and 2,4,5–trimethylbenzaldehyde were selected as 2–methylbenzaldehyde analogues for testing (Figure 1). The acaricidal activities of 2–methylbenzaldehyde analogues were evaluated using the contact and the fumigant bioassays against D. farinae, D. pteronyssinus and H. longicornis. In the fumigant bioassay (Table 3), the acaricidal activities of 2,4,5–trimethylbenzaldehyde (0.21, 0.19 and 0.68 μg/cm3) were approximately 178.02, 178.30 and 76.07 times more toxic than those of DEET (36.50, 34.23 and 52.03 μg/cm3) against D. farinae, D. pteronyssinus and H. longicornis, followed by 2,3–dimethylbenzaldehyde (0.46, 0.44 and 0.79 μg/cm3), 2,4–dimethylbenzaldehyde (0.66, 0.59 and 0.95 μg/cm3), 2,5–dimethylbenzaldehyde (0.65, 0.68 and 0.88 μg/cm3), 2–methylbenzaldehyde (0.95, 0.87 and 1.28 μg/cm3), 3–methylbenzaldehyde (0.99, 0.93 and 1.38 μg/cm3) and 4–methylbenzaldehyde (1.17, 1.15 and 3.67 μg/cm3). In the contact bioassay (Table 4), the acaricidal effects of 2,4,5–trimethylbenzaldehyde (0.12, 0.19 and 0.46 μg/cm2) were about 170.99, 73.53 and 103.61 times more active than those of DEET (19.64, 14.12 and 47.77 μg/cm2) against D. farinae, D. pteronyssinus and H. longicornis, followed by 2,3–dimethylbenzaldehyde (0.25, 0.24 and 0.56 μg/cm2), 2,4–dimethylbenzaldehyde (0.32, 0.32 and 0.62 μg/cm2), 2,5–dimethylbenzaldehyde (0.35, 0.36 and 0.74 μg/cm2), 2–methylbenzaldehyde (0.51, 0.47 and 0.94 μg/cm2), 3–methylbenzaldehyde (0.53, 0.50 and 1.02 μg/cm2) and 4–methylbenzaldehyde (0.63, 0.61 and 1.84 μg/cm2). In contrast, 2,3–dihydroxybenzaldehyde, 2,4–dihydroxybenzaldehyde, 2,5–dihydroxybenzaldehyde, 2–hydroxybenzaldehyde, 3–hydroxybenzaldehyde, 4–hydroxybenzaldehyde and 2,4,5–trihydroxybenzaldehyde did not show the acaricidal activity against D. farinae, D. pteronyssinus, or H. longicornis in the contact and the fumigant bioassays. Comparison of the LD50 values from the contact and the fumigant bioassays against D. farinae, D. pteronyssinus and H. longicornis revealed that the acaricidal activities of the structural analogues containing a methyl (CH3) functional group (2,3–dimethylbenzaldehyde, 2,4–dimethylbenzaldehyde, 2,5–dimethylbenzaldehyde, 2–methylbenzaldehyde, 3–methylbenzaldehyde, 4–methylbenzaldehyde and 2,4,5–trimethylbenzaldehyde) were more potent than those of structural analogues containing a hydroxyl (OH) functional group (2,3–dihydroxybenzaldehyde, 2,4–dihydroxybenzaldehyde, 2,5–dihydroxybenzaldehyde, 2–hydroxybenzaldehyde, 3–hydroxybenzaldehyde, 4–hydroxybenzaldehyde, 2,4,5–trihydroxybenzaldehyde). In particular, the number of methyl functional groups influenced the acaricidal activity against D. farinae, D. pteronyssinus and H. longicornis. These observations are similar with the results in the study by Oh et al.18 in which 2′–methylacetophenone, 3′–methylacetophenone and 4′–methylacetophenone were observed to have potent activities while 2′,4′–dihydroxyacetophenone and 2′,6′–dihydroxyacetophenone possessed no activities against D. farinae and D. pteronyssinus. According to Lee and Lee22, the acaricidal effects of 2–methyl–1,4–naphthoquinone, which has a methyl functional group added onto 1,4–naphthoquinone, were more toxic against D. farinae and D. pteronyssinus than those of 2–hydroxy–1,4–naphthoquinone, containing a hydroxyl functional group on 1,4–naphthoquinone.

Color alterations of Dermatophagoides spp. were observed through an optical microscope when before and after treatment with M. officinalis oil and 2–methylbenzaldehyde structural analogues. In particular, Dermatophagoides spp. treated with M. officinalis oil and 2,3–dihydroxybenzaldehyde presented with skin discoloration to a dark brown color in the body, while the untreated mites were colorless (Figure 2). The color change of the mites treated with M. officinalis oil and 2,3–dihydroxybenzaldehyde allowed D. farinae and D. pteronyssinus to be distinguish with the naked eye. According to a previous study, complete elimination is actually impossible due to residual mite excrement and dead mites24. In addition, a vicious cycle of the treatments of the commercial acaricides was continued. These problems have emphasized the demand for the development of new technologies to control the allergens caused by Dermatophagoides spp. In this regard, the mite indicator in this study is valuable because of the color alteration of Dermatophagoides spp. induced by M. officinalis and 2,3–dihydroxybenzaldehyde. For these reasons, a new concept for natural acaricides was found in the present study, involving both the acaricidal activity and the mite indicator against Dermatophagoides spp. The discoloration is likely to be related to the benzene ring metabolisms of the defense mode of action in plants22,25. A similar reaction was previously observed in the whole body of dust mites treated with quinone22. Lee and Lee reported that polyphenol oxidase catalyzed two reactions: hydroxylation and oxidation in the benzene ring metabolisms21. In addition, disease resistance in both insects and mites occurred due to the existence of polyphenol oxidase26.

Figure 2
figure2

Mite indicators against D. farinae and D. pteronyssinus.

(a) untreated mites, (b) mites, treated with 2,3–dihydroxybenzaldehyde.

In the present study, acaricidal effects of M. officinalis oil and 2–methylbenzaldehyde structural analogues were found against Dermatophagoides spp. and H. longicornis. In particular, 2,4,5–trimethylbenzaldehyde had potent activity against Dermatophagoides spp. and H. longicornis. From a point of view, M. officinalis oil and 2–methylbenzaldehyde are the most promising because of the high sensitivity against D. farinae, D. pteronyssinus and H. longicornis. Interestingly, skin color alteration of Dermatophagoides spp. was exhibited, changing from colorless to dark brown through the treatment with 2,3–dihydroxybenzaldehyde. In this regard, 2,3–dihydroxybenzaldehyde could be very useful to observe and remove the allergens.

In conclusion, this work showed that 2–methylbenzaldehyde analogues from M. officinalis oil have a dual function as acaricide and acari indicator, meaning that these compounds could be developed as acaricidal agents. Especially, we believe that 2,3–dihydroxybenzaldehyde have a strong potential as an ideal acaricidal agent to control Dermatophagoides spp. and H. longicornis which are the vector of SFTS virus and allergen. In the registration process, considering the fact that Morinda officinalis is a very cheap plant which can be easily cultivated, the cost would not be a barrier for the commercial development of 2–methylbenzaldehyde isolated from Morinda officinalis.

Methods

Compounds

2,3–Dihydroxybenzaldehyde (97%, Cat No. 189839), 2,4–dihydroxybenzaldehyde (98%, Cat No. 168637), 2,5–dihydroxybenzaldehyde (98%, Cat No. D108200) 2,3–dimethylbenzaldehyde (97%, Cat No. 515353), 2,4–dimethylbenzaldehyde (90%, Cat No. 151041), 2,5–dimethylbenzaldehyde (99%, Cat No. 151068), 2–hydroxybenzaldehyde (98%, Cat No. W300403), 3–hydroxybenzaldehyde (97%, Cat No. H19808), 4–hydroxybenzaldehyde (97%, Cat No. W398403), 3–methylbenzaldehyde (97%, Cat No. T35505), 4–methylbenzaldehyde (97%, Cat No. T35602), 2,4,5–trihydroxybenzaldehyde (99%, Cat No. 526452) and 2,4,5–trimethylbenzaldehyde (99%, Cat No. 493864) were provided from Aldrich (St. Louis, MO, USA). DEET (95%, Cat No. 32570) was obtained from Fluka (Buchs, Switzerland).

Essential oil from M. officinalis

Dried roots of M. officinalis (4 kg) were obtained from a market (Jeonju city, Republic of Korea) and extracted using the steam distillation extraction method27. The essential oil (1.2 g, yield 0.03%) of M. officinalis roots was concentrated by an evaporator (model name: NAJ–100, EYELA, Tokyo, Japan) at 27°C and stored at 4°C to prevent loss of the volatile constituents.

Gas chromatography–mass spectrometer

The volatile components of M. officinalis oil were analyzed by GC–MS (6890 and 5973 series, Agilent, USA) and were separated using DB–5 and HP–Innowax capillary columns (0.25 mm i.d. × 3,000 cm L. × 0.25 μm thickness)7. The initial temperature of the GC column was 50°C, which was gradually increased up to 210°C. The ion source temperature was 230°C. Helium gas as the carrier gas was flowed at a rate of 0.85 mL/min7. Effluents from GC column were introduced into the mass spectrometer. Mass spectra (m/z) were obtained in electron ionization (70 eV) set to scan a range of 50–600 amu for 2 seconds. The volatile components of M. officinalis oil were identified by retention index, retention time and mass spectra and were confirmed by comparison to the published mass spectra data28. The relative composition (%) of volatile components was calculated by the standard of the internal amount.

Isolation and identification

Silica gel (70–230 mesh) was purchased from Merck (Rahway, NJ, USA). M. officinalis oil (12 g) was loaded onto a silica gel column (8 cm i.d. × 70 cm L.) and eluted gradually using a mixed organic solvent (hexane:ethyl acetate, gradient, v/v). The eluted fractions were separated by thin–layer chromatography and a UV lamp (at 264 nm) to obtain eight fractions (MO1–MO8). The acaricidal activities of the eight fractions were evaluated using the fumigant bioassay against Dermatophagoides spp. and H. longicornis at a concentration of 59.0 μg/cm3. Fraction MO3 fraction (1.98 g, yield 16.5%) was found to possess potent activity against Dermatophagoides spp. and H. longicornis and was chromatographed on a silica gel column using a mixed organic solvent (ethyl acetate:hexane = 1:9, v/v) to further separate into five fractions (MO31–MO35). Fraction MO32 (887 mg, yield 44.8%) was identified as the toxic fraction. Next, preparative high performance liquid chromatography (model name: LC–908, JAI Co., Ltd, Tokyo, Japan) was performed. Fraction MO32 was separated into three fractions (MO321–MO323) using a GS column (model name: GS 310, 2 cm i.d. × 50 cm L.) with methanol solvent as the mobile phase at a flow rate of 4.8 mL/min. Finally, the active compound (MO322, 130 mg, yield 14.7%) was isolated. The structure of the compound in MO322 fraction was identified by spectroscopic analyses.

The chemical structure of MO322 fraction isolated from M. officinalis oil was determined using nuclear magnetic resonance (NMR). To confirm the number of carbons (C) and protons (H), 13C–, 1H– and DEPT–NMR (model name: JNM–ECA600 spectrometer, JEOL Ltd., Tokyo, Japan) were implemented at 400 and 100 MHz. The MO322 was melted in CDCl3 and tetramethylsilane was used as the internal standard. In addition, the molecular weight of the MO322 fraction was investigated using EI–MS (model name: JEOL GSX 400 mass spectrometer, JEOL Ltd., Tokyo, Japan) spectra.

Target

The rearing method of D. farinae and D. pteronyssinus modified by Yang and Lee was utilized3. The mites were reared without exposure to any synthetic acaricides. The feed consisted of fry powder and dried yeast (Korea special feed meal Co. Ltd., Jeonju, Republic of Korea) given in a rearing case (15 × 12 × 6 cm) at 25°C and 75% relative humidity (RH) in a dark area. The protein content of the fry powder was maintained above 49%. For experiments, the Dermatophagoides spp. mites were placed in a petri dish (9 cm i.d. × 1.5 cm L.). H. longicornis ticks were collected around Jeonju River and were identified morphologically and genetically. However, the H. longicornis ticks were not maintained in our laboratory.

Acaricidal activity

The acaricidal effects of the oil, 2–methylbenzaldehyde and its analogues were evaluated using the contact and the fumigant bioassays against Dermatophagoides spp. and H. longicornis through the method modified by Yang and Lee5. The experimental concentrations in the study were set to scan a range of 60.0–0.10 μg/cm2. Each sample was dissolved in 50 μL acetone and then applied to filter paper (5.5 cm i.d. × 25 μm thickness, Whatman, UK). Acetone alone was applied as the negative control and DEET was designated as the positive control. The residual solvent of each filter paper was dried under the fume hood for 14 min. In the contact bioassay, each piece was placed on the bottom of a petri dish (6 cm i.d. × 1.5 cm L.). In the case of the fumigant bioassay, a piece of filter paper was put in the lid of a petri dish (6 cm i.d. × 1.5 cm L.) and a thin cotton fabric was inserted to prevent contact of the inoculated mites with the applied filter paper. Each twenty experimental mites (Dermatophagoides spp. and H. longicornis), which included a mixture of males and females, were inoculated in each petri dish and the lids were sealed. All treatments were repeated four times and incubated for 24 h at 25°C in the dark. Mortality was measured by the number of dead ticks and mites, which did not move when prodded with a pin, under an optical microscope (20×, Olympus, Japan).

Color alteration of mites

Color alterations to D. farinae and D. pteronyssinus exposed to 2–methylbenzaldehyde analogues were evaluated using a light microscope (100×), as described by Lee et al25. Color alteration of the mites before and after treatment was compared.

Statistics

Mortalities in all the treatments were measured under an optical microscope (20×, Olympus, Japan). D. farinae and D. pteronyssinus were regarded to be dead when they did not move when touched with a pin. The LD50 values were determined by probit analysis29. Relative toxicity (RT) was calculated by the ratio of the commercial acaricide LD50 value to the LD50 value observed for each compound.

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Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (2013R1A2A2A01067945).

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J.-Y.Y. designed and carried out the experiments, prepared most of the data and wrote the paper; M.-G.K. designed and carried out the experiments, prepared most of the data and wrote the paper; J.-H.P. carried out the experiments for the bioassay and assisted in paper–writing; S.-T.H. wrote the paper; H.-S.L. proposed the key idea of this paper, designed the experiments, carried out color alteration by introduction of functional radicals, managed the research process and wrote the paper.

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Yang, JY., Kim, MG., Park, JH. et al. Evaluation of benzaldehyde derivatives from Morinda officinalis as anti-mite agents with dual function as acaricide and mite indicator. Sci Rep 4, 7149 (2014). https://doi.org/10.1038/srep07149

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