Acaricidal activity of 9-oxo-10,11-dehydroageraphorone extracted from Eupatorium adenophorum in vitro

Experimental Parasitology 140 (2014) 8–11

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Acaricidal activity of 9-oxo-10,11-dehydroageraphorone extracted from Eupatorium adenophorum in vitro Fei Liao a,1, Yanchun Hu a,⇑,1, Hui Tan a, Lei Wu a, Yunfei Wang a, Yue Huang a, Quan Mo a, Yahui Wei b a b

Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, China Key Laboratory of Resource Biology and Biotechnology in Western China, School of Life Science, Northwest University, Xi’an 710069, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 We proved that the euptox A have

high effectively acaricidal activity to S. scabiei and P. cuniculi.  We have determined the LT50 and LC50 against P. cuniculi and S. scabiei.  The acaricidal activity of euptox A is better than the ivermectin.  Low-dose and safety.

a r t i c l e

i n f o

Article history: Received 8 July 2013 Received in revised form 24 August 2013 Accepted 19 February 2014 Available online 11 March 2014 Keywords: 9-Oxo-10,11-dehydroageraphorone Acaricidal activity Sarcoptes scabiei Psoroptes cuniculi

a b s t r a c t The acaricidal activity of the 9-oxo-10,11-dehydroageraphorone (euptox A), a cadenine sesquiterpene from Eupatorium adenophorum (E. adenophorum) against Sarcoptes scabiei and Psoroptes cuniculi was tested in vitro. A complementary log–log (CLL) model was used to analyze the data of the toxicity tests in vitro. The results showed euptox A had strong toxicity against mites, killing all S. scabiei at 3 and 4 mg/ml (m/v) concentration, while 4 mg/ml euptox A was also found to kill all P. cuniculi within a 4 h period. Similarly, 2, 3 and 4 mg/ml concentration of euptox A had strong toxicity against S. scabiei, with median lethal time (LT50) values at 0.687, 0.526, 0.326 h, respectively. 3 mg/ml and 4 mg/ml showed strong acaricidal action against P. cuniculi; the LT50 values were 0.693 and 0.493 h, respectively. The median lethal concentration (LC50) values were 1.068 mg/ml for Scabies mite and 0.902 mg/ml for P. cuniculi in 2 h. The results indicate that euptox A has strong acaricidal activity and may exploit as novel drugs for the effective control of animal acariasis. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Animal acariasis, one of important veterinary skin diseases, may reduce the productivity and the quality of animal products, even lead to death (Shang et al., 2013). At present, chemical drugs have been widely used to treat and control the psoroptic and sarcoptic ⇑ Corresponding author. Fax: +86 835 2885302. E-mail address: [email protected] (Y. Hu). Both authors contributed equally to this work and should be considered as first authors. 1

http://dx.doi.org/10.1016/j.exppara.2014.02.009 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.

mange in veterinary clinic, and obtained the relative good treatment effectiveness, including ivermectin, and abamectin, etc. But the chemical control could increase resistance in target species, toxicity and environmental hazards (Borges et al., 2013). Euptox A, a cadenine sesquiterpene, is the main toxin extracted from Eupatorium adenophorum (He et al., 2008; Seawright et al., 1998). Euptox A takes a large proportion of E. adenophorum toxins (Bohlmann and Gupta, 1981; Kaushal et al., 2001), can cause not only the allergic bronchial pneumonia of horses which is characterized by pulmonary interstitial fibrosis, emphysema, alveolar epithelisation and reduced tolerance to exercise (O’sullivan, 1985;

F. Liao et al. / Experimental Parasitology 140 (2014) 8–11

Oelrichs et al., 1995), but the contact dermatitis of other domestics animals like cattle and goats (Bai et al., 2011). Besides, according to some studies, for mice, lesions occur in the liver. The hepatic injury in these animals is characterised by multiple areas of focal necrosis of the parenchyma associated with degeneration and loss of the epithelium lining the small bile ducts (Bhardwaj et al., 2001; Katoch et al., 2000). A large number of reports have indicated that the secondary metabolites synthesized and accumulated in E. adenophorum have wide biological activities. For example, there are reports of chronic respiratory disease and exercise intolerance in horses in Australia due to ingestion of Ea (O’sullivan, 1985); ethanol extract form leaves of E. adenophorum were anti-Inflammatory potential (Chakravarty et al., 2011), acaricidal activity (Nong et al., 2013; Seddiek et al., 2013), antioxidant activity(Kundu et al., 2013), and other extract form E. adenophorum torium had toxic activity against Oncomelania hupensis, the intermediate host snail of Schistosoma japonicum (Zou et al., 2009), Tinea (Romero-Cerecero et al., 2012) and Aphis gossypii (Sun et al., 2004). The acetone extract of E. adenophorum had strong toxicity against Cabbage aphids and Brevicoryne brassicae (Xu et al., 2009). But what is the specific material properties has not been reported. Euptox A is the main toxin extracted from E. adenophorum. The purpose of this study is to evaluate the possible acaricidal activity of euptox A against the Sarcoptes scabiei and Psoroptes cuniculi. We found that the euptox A has strong toxicity against S. scabiei and P. cuniculi in vitro. 2. Materials and methods 2.1. Extraction and purification of euptox A E. adenophorum was collected from Xichang City of Sichuan Province, Southwest China in July, 2012. Fifty grams milled leaves were mixed with 100 ml water. Mixture contained euptox A, coumarin, gallotannic acid and volatile oil was ultrasonic extracted by carbinol and hexyl acetate for 30 min at 40 °C. In order to separate euptox A from the extraction, sample was purified by Silica Column Chromatography method and Silica Gel Thin-layer Chromatography and then used to analyze the existence of euptox A in the final extraction (He et al., 2006). According to the HPLC determination result, the purity of the toxin we had extracted was over 96%. 2.2. Mites

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distilled water and glycerin (Fichi et al., 2007) acted as an untreated control, and 2% ivermectin as the positive control. The viability of mites was checked regularly by stimulation, with a needle, and mites were recorded as dead if no reaction was shown (Pasay et al., 2010). As for S. scabiei incubation conditions remained the same. 2.4. Statistical analyses All computations were done by employing the statistical software (SPSS, version 20.0) (Zhang et al., 2012). The significance of differences in mean mite mortality between different concentrations was calculated by probability method. The median lethal time value (LT50) and the median lethal concentration value (LC50) were calculated by the complementary log–log (CLL) model (Qiu et al., 2012). 3. Results There were considerable differences in acaricidal activity between the differences concentration of euptox A. A high concentration group (4% euptox A) caused 100% mortality in test mites (Figs. 1 and 2). However, in the groups of positive control group of ivermectin, mites remained alive in the sample after the 4 h period. The reason probably is that the test mites P. cuniculi and S. scabiei produced resistance to the ivermectin. Mortalities for the mites with the 5 concentration groups are shown in Tables 1 and Table 2. Toxicity was evaluated using a CLL model, Pearson’s Chi-square test and the Hosmer–Lemeshow goodness-of-fit statistic indicated that the data fitted the CLL model (P > 0.05). The lethal concentration and lethal time values of the euptox A for the P. cuniculi and S. scabiei are shown in Tables 3 and Table 4. The euptox A demonstrated stronger toxicity against mites, and its activity was affected by concentration and time. The data confirms that euptox A at concentrations of 2, 3 and 4 mg/ml was highly toxic to S. scabiei and P. cuniculi. But at concentrations of 2, 3 and 4 mg/ml was stronger toxic to S. scabiei than P. cuniculi. The LC50 values were 0.68 mg/ ml for S. scabiei and 1.212 mg/ml for P. cuniculi within 2 h. 4. Discussion In this study, euptox A extracted from E. adenophorum has strong acaricidal activity. euptox A belongs to a cadenine sesquiterpene. A large number of reports indicated that the cadenine sesquiterpene has wide biological activities such as antitumor activity

S. scabiei and P. cuniculi were isolated from scabs and ear cerumen which were collected from the infested legs and ears of naturally infected rabbits. Scabs and ear cerumen were placed in Petri dishes and incubated at 35 °C for 30 min in an incubator. The adult mites were then collected for testing. After collection of materials, the rabbits were treated immediately. Institutional ethical and animal care guidelines were adhered to during the sampling exercise and all procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. 2.3. Assay of acaricidal activity in vitro The euptox A was diluted from the concentration of 0.5–4 mg/ml (0.5, 1, 2, 3, 4 mg/ml) in 10% glycerin. The 0.5 ml sample was added to Petri dishes (10 cm diameter, 2 cm high) with filter paper chips to absorb the liquid. For P. cuniculi, 10 specimens were placed on the filter paper in Petri dishes to be tested and incubated at 25 °C under 75% relative humidity (Nong et al., 2013). Six replicates were performed for each concentration of euptox A. Six Petri dishes containing 0.5 ml

Fig. 1. Dead Sarcoptes scabiei, observed by microscopy following treatment with euptox A after 4 h.

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F. Liao et al. / Experimental Parasitology 140 (2014) 8–11 Table 3 The probit regression analysis of toxicity (LT50) of the euptox A against mites in vitro.

Fig. 2. Dead Psoroptes cuniculi, observed by microscopy following treatment with euptox A after 4 h.

(Kikuchi et al., 2012), antigerminative activity (Amand et al., 2012), neurotrophic activity (Trzoss et al., 2013), larvicidal activity (Montenegro et al., 2013), antiprotozoal activity (Wube et al., 2010), and so on. The data demonstrates that the euptox A from E. adenophorum has a strong toxic effect against S. scabiei and P. cuniculi. Toxicity was showed to be time- and concentrationdependent, with the extract displaying similar effects to the alcohol extract from E. adenophorum against S. scabiei and P. cuniculi, with the LC50 values were 0.22 g/ml for Scabies mite and 0.64 g/ml for P. cuniculi in 1 h (Nong et al., 2013), as well as those of neem (Azadirachta indica) oil against the larvae of the rabbit mite S. scabiei in vitro (Du et al., 2008). The reason probably is that the principal active component of the alcohol extract is euptox A because that the euptox A is soluble in organic solvents, such as alcohol, methanol, acetone (Bai et al., 2011). In the current study, the euptox A showed better acaricidal activity (LC50, 0.902 mg/

Concentration

Regression line

LT50 (h) (95%FL)

Pearson Chi-square

Psoroptes cuniculi 0.5 mg/ml 1 mg/ml 2 mg/ml 3 mg/ml 4 mg/ml

y = 2.280x – 1.335 y = 2.096x – 0.548 y = 2.100x – 0.176 y = 1.809x + 0.288 y = 2.440x + 0.821

3.852 1.825 1.212 0.693 0.461

(3.164 – 5.421) (1.439 – 2.190) (0.828 – 1.512) (0.276 – 1.013) (0.130 – 0.725)

9.153 12.775 17.115 10.366 12.832

Sarcoptes scabiei 0.5 mg/ml 1 mg/ml 2 mg/ml 3 mg/ml 4 mg/ml

y = 1.423x – 1.032 y = 1.131x – 0.370 y = 1.036x + 0.169 y = 1.729x + 0.482 y = 1.940x + 0.945

5.311(3.681 – 15.766) 2.124 (1.347 – 3.152) 0.687 (0.038 – 1.195) 0.526 (0.134 – 0.852) 0.326 (0.028 – 0.628)

10.403 11.531 12.208 23.097 17.763

Table 4 The probit regression analysis of toxicity (LC50) of the euptox A against mites in vitro. Time (h)

LC50 (mg/ml) (95%FL)

Pearson Chi-square

Psoroptes cuniculi 1h y = 2.411x – 0.745 2h y = 1.707x – 0.077 3h y = 2.049x + 0.351 4h y = 2.103x + 0.601

Regression line

2.037 0.902 0.674 0.518

(1.750 (0.661 (0.493 (0.353

– – – –

2.392) 1.128) 0.840) 0.664)

17.550 18.805 16.847 16.859

Sarcoptes scabiei 1h y = 2.198x – 0.392 2h y = 1.963x – 0.056 3h y = 1.885x + 0.189 4h y = 2.590x + 0.414

1.507 1.068 0.794 0.692

(1.265 (0.846 (0.587 (0.548

– – – –

1.775) 1.288) 0.985) 0.826)

16.010 19.385 2.343 18.963

ml) than the alcohol extract from E. adenophorum (LC50, 0.22 g/ ml), whilst the petroleum ether extract of neem oil comparatively weak bioactivity (LC50, 500.0 ll/ml) (Deng et al., 2012). Previous study, the compound has been shown to exhibit toxicity to larvae

Table 1 The acaricidal activity of the euptox A against Psoroptes cuniculi in vitro. Concentration

0.5 mg/ml 1 mg/ml 2 mg/ml 3 mg/ml 4 mg/ml Positive control Untreated control

Time (h) 1 h Mean mortality(%) ± SD

2 h Mean mortality(%) ± SD

3 h Mean mortality(%) ± SD

4 h Mean mortality(%) ± SD

8.33 ± 7.52D 25.0 ± 0.4c 40.0 ± 8.94C 65.0 ± 10.4B 81.7 ± 9.83A 38.3 ± 13.2C 0.00 ± 0.00D

28.0 ± 7.71D 61.0 ± 14.7BC 71.3 ± 12.1B 73.3 ± 8.16B 90.0 ± 13.6A 46.7 ± 8.37c 0.00 ± 0.00E

38.3 ± 7.52E 68.3 ± 7.52cD 80.0 ± 14.1BC 86.7 ± 5.16aB 98.3 ± 4.08A 58.3 ± 11.6D 1.66 ± 4.08F

51.6 ± 11.7E 71.6 ± 7.52cD 83.3 ± 10.3bC 95.0 ± 5.47AB 100 ± 0.00A 65.0 ± 8.36D 3.33 ± 5.16F

Note: The difference between data with the different capital letter within a column is very significant (P < 0.01), and the difference between data with the different small letters within a column is significant (P < 0.05).

Table 2 The acaricidal activity of the euptox A against Sarcoptes scabiei in vitro. Concentration

0.5 mg/ml 1 mg/ml 2 mg/ml 3 mg/ml 4 mg/ml Positive control Untreated control

Time (h) 1 h Mean mortality(%)±SD

2 h Mean mortality(%)±SD

3 h Mean mortality(%)±SD

4 h Mean mortality(%)±SD

15.0 ± 10.4D 35.0 ± 10.4C 60.0 ± 12.6b 71.7 ± 7.52aB 85.0 ± 10.4A 40.0 ± 8.94C 0.00 ± 0.00E

26.7 ± 10.3C 50.0 ± 14.1b 63.3 ± 10.3B 81.7 ± 11.7A 90.0 ± 8.94A 48.3 ± 11.6b 0.00 ± 0.00D

38.3 ± 9.83E 56.6 ± 10.3D 71.7 ± 11.6bC 83.3 ± 12.1aB 96.7 ± 5.16A 65.0 ± 10.4CD 1.67 ± 4.08F

41.7 ± 7.52D 61.7 ± 11.7C 83.3 ± 7.52B 100 ± 0.00A 100 ± 0.00A 70.0 ± 14.1BC 1.67 ± 4.08E

Note: The difference between data with the different capital letter within a column is very significant (P < 0.01), and the difference between data with the different small letters within a column is significant (P < 0.05).

F. Liao et al. / Experimental Parasitology 140 (2014) 8–11

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