Veterinary Parasitology 211 (2015) 223–227
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In vitro anthelmintic activity of Zanthoxylum simulans essential oil against Haemonchus contortus H Qi, W.X. Wang, J.L. Dai, L. Zhu ∗ College of Food and Bioengineering, South China University of Technology, Guangzhou, Guangdong Province, China
a r t i c l e
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Article history: Received 21 October 2014 Received in revised form 27 May 2015 Accepted 30 May 2015 Keywords: Zanthoxylum simulans Essential oil Composition Anthelmintic activity
a b s t r a c t The need for new anthelmintic agents with low impact on the environment is becoming urgent. Phytotherapy is an alternative method to control gastrointestinal nematodes in small ruminants. This study aims to determine the composition of Zanthoxylum simulans essential oil (ZSEO) and evaluate the in vitro ovicidal and larvicidal effects of ZSEO on Haemonchus contortus using egg hatch assay, larval development assay (LDA), and larval migration inhibition assay (LMIA). The chemical composition of ZSEO was determined through gas chromatography and mass spectrometry and 94 compounds were identified from the ZSEO. The major constituents of ZSEO were borneol (18.61%), -elemene (10.87%). ZSEO and borneol both at 40 mg/mL inhibited larval hatching by 100%, with LC50 values of 3.98 and 1.50 mg/mL, respectively. The LC50 value of -elemene was not determined because of its insufficient activity. The results of LDA showed that ZSEO, borneol, and -elemene all at 40 mg/mL inhibited larval development by 99.8%, 100%, and 55.4%, respectively, and exhibited dose-dependent responses with LC50 values of 4.02, 1.99, and 32.17 mg/mL, respectively. The results of LMIA showed that ZSEO, borneol, and -elemene all at 40 mg/mL inhibited larval migration by 74.3%, 97.0%, and 53.2%, respectively. ZSEO presented ovicidal and larvicidal activities in vitro. Therefore, Zanthoxylum may be an alternative source of anthelmintic agents to control gastrointestinal nematodes in sheep. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Helminthosis caused by gastrointestinal nematodes, particularly Haemonchus contortus, is a prevalent and economically significant disease among domesticated animals (Perry and Randolph, 1999; Marie-Magdeleine et al., 2010). Gastrointestinal parasitic disease in small ruminants is characterized by anaemia, haemorrhagic gastroenteritis, hypoproteinaemia, sudden death, and chronic emaciation (Bethony et al., 2006; Botura et al., 2011). Therefore, controlling helminthes in livestock farming is important worldwide. Most anthelmintic drugs are expensive and unavailable to rural subsistence livestock keepers. Furthermore, the large-scale use of anthelmintic drugs has led to the emergence of multiple anthelmintic resistances. The residue of some persistent chemicals in the environment disrupts the ecosystem and poses a threat to human health. Therefore, new treatment approaches are required, among them, the use of medicinal plants have a rapid development because they are less toxic,
∗ Corresponding author. Fax: +86 20 87113849. E-mail address: [email protected] (L. Zhu). http://dx.doi.org/10.1016/j.vetpar.2015.05.029 0304-4017/© 2015 Elsevier B.V. All rights reserved.
biodegradable, and environmentally friendly (Hammond et al., 1997). Zanthoxylum of the family Rutaceae consists of 250 species of deciduous shrubs and trees that are native to warm temperate and subtropical regions of the world. China is home to 45 species and 13 varieties of this genus (Zhang et al., 2014). The fruits of some species such as Z. armatum and Z. bungeanum, with the most popular name being “huajiao” (flower pepper), is a traditional spice with strong spiciness and astringent taste. Many Zanthoxylum species in China are medicinal herbs widely used in traditional Chinese medicine. Essential oils isolated from the genus are effective for the treatment of intestinal parasites, inflammatory diseases, stomach disorders, toothache, diarrhea, and dysentery (Wei et al., 2011). Z. simulans is a prickly shrub that is distributed throughout mainland China and Taiwan (Yang et al., 2002). Z. simulans is an edible material and traditional medicine universally used to kill intestinal parasites as well as treat rheumatoid arthritis and inflammation (Wang et al., 2014). Zanthoxylum contains alkaloids, coumarins, amides, lignans, and flavonoids (Wang et al., 2014; Yang et al., 2002; Chen et al., 1994, 1996). The present study aims to determine the chemical composition of Z. simulans essential oil (ZSEO) and evaluate the in vitro anthelmintic effect of ZSEO and its
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major constituents, borneol and -elemene, against the eggs and infective larvae of H. contortus from sheep. 2. Materials and methods 2.1. Plant materials Fresh aerial parts of Z. simulans were collected from Hunan Province, China, in September 2012 and were identified by Dr. Gong Xun, Kunming Institute of Botany, Chinese Academy of Sciences. The plants were dried under a shade at room temperature. The voucher specimens (No. 0412161) were deposited in Kuming Institute of Botany, Chinese Academy of Sciences. 2.2. Preparation of ZSEO and chemicals Air-dried plant materials of Z. simulans were chopped and subjected to hydrodistillation for 4 h using a clevenger-type apparatus (Kesijia Ltd., Beijing, China) consisting of a 5 L distillation bottle, a 5 mL graduated receiver, and a jacketed-coil condenser. The obtained oils were dried over hydrous sodium sulfate for 24 h, filtered, and then stored at 4 ◦ C in brown sealed glass vials until tested. Borneol and -elemene were purchased from Sigma–Aldrich (St. Louis, USA). To improve the emulsification of ZSEO and compounds in water, 2% Tween 80 solvent was added and mixed the solutions in a vortex shaker until the oil, solvent, and water became a stable emulsion. 2.2.1. Gas chromatography mass spectrometry analysis ZSEO was quantitatively and qualitatively analyzed using an Agilent GC–MS 6890-5975 system equipped with an HP-5 MS fused silica capillary column (30 m × 0.25 mm i.d.; film thickness, 0.25 m). For GC–MS detection, an electron ionization system with 70 eV of ionization energy was used. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The injector and mass transfer line temperatures were set at 250 ◦ C and 280 ◦ C, respectively. The ZSEO solution (1 L) in hexane was injected and then analyzed under the following conditions: initial column temperature at 40 ◦ C for 1 min, increased to 250 ◦ C at a heating ramp rate of 3 ◦ C/min, and then maintained at 250 ◦ C for 20 min. The Kovats indices were calculated for all volatile components using a homologous series of n-alkanes (C8 –C25 ) on the HP-5 MS column. The major ZSEO components were identified by comparison with the standards and verified on the basis of Kovats indices using Wiley (V.7.0) and National Institute of Standards and Technology V.2.0 GC–MS library. The relative concentration of each compound in ZSEO was quantified on the basis of the peak area integrated in the analysis program. 2.2.2. Gas chromatography flame lionization detector analysis An Agilent HP-6890 GC with an HP-5 5% phenylmethylsiloxane capillary column (30 m × 0.25 mm i.d.; film thickness, 0.25 m) and a flame ionization detector were used for GC–FID analysis. Helium at a constant flow rate of 1 mL/min was used as the carrier gas. All other parameters were the same as those described above. Results were expressed as the relative percentage of peak area. 2.3. In vitro anthelmintic assays The anthelmintic bioassay tests of ZSEO and chemicals at different life-cycle stages of H. contortus were performed using egg hatch assay (EHA), larval development assay (LDA), and larval migration inhibition assay (LMIA). For each assay, the four parasitic stages were obtained from donor Small Tail Han sheep which were
experimentally infected with oral administration of a pure aqueous suspension of 10,000 H. contortus third stage larvae (L3). 2.3.1. Egg hatch assay Eggs were collected from the donor sheep and EHA was performed in accordance with the guidelines of World Association for the Advancement of Veterinary Parasitology guidelines (Coles et al., 1992). The parasite eggs were collected from the feces of infected donor sheep, dispersed in water, sifted successively (250, 125, 63, 50, and 30 mm sieves), and then centrifuged at 1000 × g for 15 min. Eggs were washed three times with distilled water. ZSEO and its principal components, namely, borneol and -elemene, were tested for their inhibitory activities against egg hatching. Albendazole (Sigma–Aldrich) was used as a positive control, whereas untreated eggs in PBS or Tween 80 solvent (20 mg/mL) served as negative control. The egg suspension was dispensed in 24-multiwell plates (1 mL per well) with a concentration of 200 eggs/mL and then mixed with the same volume of the sample solution (ZSEO, borneol, or -elemene) that their final concentration prepared at 40, 20, 10, 5, 2.5, and 1.25 mg/mL. Albendazole used as a positive control was dissolved in distilled water to a final concentration of 0.0063 mg/mL. PBS was applied to avoid any nonspecific effects caused by changes in pH (pH 7.2). Five replicates were performed for each test extract. After 48 h of incubation at 27 ◦ C, egg hatching was stopped by adding Lugol’s iodine solution. The number of L1 larvae and unhatched egg per well were then counted under a dissecting microscope at 40× magnification. 2.3.2. Larval development assay LDA was conducted as described by Costa et al. (2008) and Tadesse et al. (2009). Eggs were incubated at 27 ◦ C for 48 h to obtain first-stage larvae. The experiment was conducted in plastic cups of 20 mL. A 1 mL aliquot containing 200 first-stage larvae of H. contortus was mixed with 5 g of sheep feces free of gastrointestinal nematodes and then with different sample treatment solutions. Serial final concentrations (40, 20, 10, 5, 2.5, and 1.25 mg/mL) of the sample solution (ZSEO, borneol, or -elemene) were prepared to a total volume of 7 mL. Albendazole was dissolved in distilled water to a final concentration of 0.0063 mg/mL and used as a positive control. All samples were incubated at room temperature for 6 days. After 6 days, the wall of each cup containing the sample was thoroughly rinsed with 10 mL of water, and the larvae were collected. One drop of Lugol’s iodine solution was added, and all third-stage (L3) larvae were counted under a dissecting microscope at 40× magnification. 2.3.3. Larval migration inhibition assay In vitro experiments were performed to determine the effect of ZSEO on the mobility of ensheathed H. contortus L3 larvae through LMIA as described by Rabel et al. (1994) with modifications. Eggs hatch and larvae develop to L3 after 8 days and were then collected by sedimentation using Baermann devices (Luo et al., 1999). Larvae were stored at 4 ◦ C for 1 month before use. One thousand live L3 larvae dispersed in 1 mL PBS were added to 5 mL centrifuge tubes containing either the negative control (PBS; Tween 80 solvent), the commercial anthelmintic control (levamisole at final concentration 5 mg/mL), or the sample solution (ZSEO, borneol, or -elemene) that their final concentration prepared at 40, 20, 10, 5, 2.5, and 1.25 mg/mL. Incubations were carried out at 28 ◦ C for 3 h. Thereafter, the L3 larvae from each tube were washed with PBS, centrifuged three times at 1400 × g for 10 min. and then transferred to sieves (inserts equipped with a 20 m mesh positioned in a conical tube) (Hernández-Villegas et al., 2011). After 3 h of incubation at room temperature, the number of larvae that migrated through the mesh was counted under a microscope at 40× magnification using a 15% aliquot technique (Hernández-Villegas et al.,
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2011). Migration percentage was calculated as M/T × 100, where T is the total number of L3 larvae deposited on the sieve and M is the number of L3 larvae that successfully migrated through the sieve. Five replicates were run for each ZSEO solution and for the control.
2.4. Statistical analysis Data from EHA, LDA, and LMIA were subjected to Probit analysis by SPSS 13.0 to calculate LC50 , LC90 , and other statistical indices at 95% confidence limits. Chi-square values were calculated using SPSS 13.0 for Windows. Statistical significance was considered at p < 0.05. 3. Results The steam distillation of 4000 g of dried Z. simulans plant material yielded 6.1 mL (0.153% v/w) of yellow oil. The oil sample was analyzed using GC–FID and GC–MS, and the components were identified on the basis of their RI values as well as their mass spectra relative to those reported in literature. The GC–MS analysis indicated 94 components that represent 99.30% of ZSEO (Table 1). Table 2 lists the data on the inhibitory effects of ZSEO, borneol, and -elemene on H. contortus egg hatching. ZSEO for all tested concentrations inhibited egg hatching, with an LC50 value of 3.98 mg/mL (95% CI = 2.89 mg/mL–5.31 mg/mL). Similar to the positive control albendazole, ZSEO at 40 and 20 mg/mL inhibited egg hatching by more than 95% (p > 0.05). With an LC50 value of 1.50 mg/mL (95% CI = 1.15 mg/mL–1.85 mg/mL), borneol exhibited higher ovicidal activity than ZSEO. -elemene did not exhibit a high ovicidal activity, and its LC50 was not determined because more than 40 mg/mL -elemene was needed to inhibit 50% of egg hatching. PBS and Tween 80 solvent showed no effect on the hatching of H. contortus eggs. ZSEO, borneol, and -elemene effectively inhibited the larval development of H. contortus (Table 3). Similar to the positive control albendazole, ZSEO at 40 mg/mL inhibited larval development by more than 99% (p > 0.05). A clear dose-dependent effect was also obtained within the range of the concentrations tested, with an LC50 of 4.02 mg/mL (95% CI = 3.51 mg/mL–4.57 mg/mL). With an LC50 value of 1.99 mg/mL (95% CI = 1.63 mg/mL–2.35 mg/mL), borneol exhibited higher activity than ZSEO for all concentrations. The activity of -elemene was not very significant with LC50 32.17 mg/mL (95% CI = 24.27–47.13 mg/mL). Albendazole was 97.0% effective in inhibiting larval development, whereas the negative control and Tween 80 (20 mg/mL) exhibited 1.4% and 0.8% effectiveness, respectively. Table 4 lists the data on the effects of ZSEO, borneol, and elemene on inhibiting H. contortus larval migration. In the negative control (PBS) and Tween 80 groups, the migration percentage of H. contortus L3 larvae was higher than 83.4%. The migration percentage of the positive control levamisole was 2.2%. The migration percentage of ZSEO at 40 mg/mL was 21.4%. Similar to the positive control, the migration percentage of borneol at 40 and 20 mg/mL was 1.8 and 2.6%. -elemene showed low inhibition for all concentrations tested, its migration percentage was 39.8% at 40 mg/mL. 4. Discussion Helminth infections constitute serious health problems worldwide in both humans and animals. Traditional healers and farmers in different regions of China have used botanical materials isolated from plant leaves, roots, seeds, flowers, and bark as anthelmintic agents (Lv et al., 2012). For centuries, medicinal plants have been used to combat parasitism and are still used for this purpose in
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Table 1 Chemical composition of Zanthoxylum simulans essential oil. RIa 930 978 1008 1030 1059 1089 1132 1072 1078 1096 1099 1105 1117 1120 1123 1130 1135 1140 1143 1148 1160 1168 1170 1178 1190 1196 1206 1217 1220 1242 1257 1265 1335 1351 1375 1384 1388 1390 1428 1437 1439 1449 1460 1486 1487 1490 1503 1514 1556 1549 1562 1578 1587 1598 1625 1639 1651 1659 1689 852 862 982 1017 1033 1062 1094 1095 1118 1158 1164 1082
Componentc
% RAb
Monoterpene hydrocarbons ␣-Thujene -Pinene ␣-Phellandrene Limonene ␥-Terpinene Terpinolene Allo-ocimene Oxygenated monoterpenes cis-Sabinene hydrate cis-Linalool oxide cis-Sabinene hydrate Linalool Thujone endo-Fenchol cis-4-Isopropyl-1-methyl-2-cyclohexen-1-ol trans-2-Pinanol cis-Limonene oxide cis--Terpineol trans-2-Menthenol Camphor Camphene hydrate Isoborneol Borneol Lavandulol Terpinene-4-ol ␣-Terpineol Citronellol Piperitol trans-Carveol exo-2-Hydroxycineole Carvone Geraniol Furomyrcenol Sesquiterpene hydrocarbons ␦-Elemene ␣-Cubebene Isoledene -Bourbonene -Cubebene -Elemene Thujopsene ␥-Elemene Aromadendrene cis--Farnesene Allo aromadendrene Germacrene D Selinene Valencene Bicyclo[4.4.0]dec-1-ene, 2-isopropyl-5-methyl-9-methylenetrans-␥-cadinene Germacrene B Oxygenated sesquiterpenes Elemol Ledol Caryophyllene oxide Viridiflorol Guaiol 1-epi-Cubenol Isospathulenol 9-Aristolen-␣-ol Kongol ␣-Bisabolol Others trans-2-Hexenal trans-2-Hexenol 1-Octen-3-ol p-Cymene Benzyl alcohol trans-2-Octenal 2-Nonanone Nonanal Phenylethyl alcohol Sabina ketone Benzyl acetate Camphenilone
7.60 0.76 0.41 0.25 4.23 1.36 0.37 0.22 34.03 0.19 1.83 0.41 5.54 0.25 0.21 0.24 0.47 0.26 0.25 0.29 1.01 0.26 0.31 18.61 0.36 1.05 0.30 0.27 0.42 0.23 0.41 0.30 0.22 0.34 26.13 0.41 0.59 0.85 0.38 0.45 10.87 0.22 2.89 0.36 0.86 0.99 4.94 1.25 0.18 0.29 0.24 0.36 10.23 0.25 0.47 6.13 0.36 0.38 0.24 0.46 0.3 0.38 1.26 21.31 1.79 3.81 0.34 1.47 0.61 0.18 0.24 0.46 0.65 0.45 0.33 0.49
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Table 1 (Continued) RIa
Componentc
% RAb
1215 1229 1235 1253 1271 1285 1292 1297 1304 1308 1315 1344 1357 1368 1385 1396 1432 1495 1500 1521 1534 1620 1971
Caprylyl acetate Estragole Thymol methyl ether Sabinene hydrate acetate Geranial Bornyl acetate Safrole Thymol 2-Undecanol Carvacrol 2-Methoxy-4-vinylphenol exo-2-Hydroxycineole acetate Eugenol Neryl acetate Geranyl acetate cis-Jasmone ␣-Ionone 2-Tridecanone trans-Methyl isoeugenol Calamenene ␣-Calacorene Dill apiole n-Hexadecanoic acid Total identified (%)
0.24 0.22 0.26 0.46 0.29 0.18 0.31 0.36 1.38 0.28 0.57 0.95 0.65 0.57 0.34 0.67 0.38 0.99 0.26 0.37 0.29 0.19 0.28 99.30
a
Retention index (RI) relative to n-alkanes on the HP-5 MS capillary column. Relative area (peak area relative to the total peak area). c All the compounds were identified using mass spectrum and retention index. Limonene, linalool, borneol, -elemene, germacrene D, caryophyllene oxide, trans2-hexenol were also identified using the method of co-injection with authentic compound. b
Table 2 Effect (mean percent ± SD) of Zanthoxylum simulans essential oil, borneol and elemene on inhibiting hatching of Haemonchus contortus eggs. Concentration (mg/mL)
Essential oils and constituents Essential oil
40.0 20.0 10.0 5.0 2.5 1.25 Albendazole (0.0063) Tween 80 (20) PBS
100 95.6 75.0 51.4 32.6 20.2 100 1.2 1.6
± ± ± ± ± ± ± ± ±
0a 3.20a 7.18b 4.16c 5.22d 2.95e 0a 0.84f 1.14f
-elemene
Borneol 100 98.8 92.2 80.0 62.1 48.6 100 1.2 1.6
± ± ± ± ± ± ± ± ±
0a 1.79a 5.12b 5.83c 5.34d 6.02e 0a 0.84f 1.14f
40.4 25.2 18.8 9.6 6.0 1.2 100 1.2 1.6
± ± ± ± ± ± ± ± ±
7.13b 3.96c 4.32d 2.07e 2.24e 0.84f 0a 0.84f 1.14f
Means with the same letter within the same column are not significantly different at p < 0.05.
Table 3 Effect (mean percent ± SD) of Zanthoxylum simulans essential oil, borneol and elemene on inhibiting Haemonchus contortus larval development. Concentration (mg/mL)
Essential oils and constituents Essential oil
40.0 20.0 10.0 5.0 2.5 1.25 Albendazole (0.0063) Tween 80 (20) PBS
99.8 93.4 84.0 55.4 25.6 18.0 97.0 0.8 1.4
± ± ± ± ± ± ± ± ±
0.44a 1.14b 7.07c 7.93d 5. 23e 3.74f 1.58a,b 0.84g 0.55g
-elemene
Borneol 100.0 98.0 91.2 80.2 52.2 38.2 97.0 0.8 1.4
± ± ± ± ± ± ± ± ±
0a 3.39a 3.42b 2.86c 8.67d 3.90e 1.58a 0.84f 0.55f
55.4 39.2 27.0 16.0 8.4 4.8 97.0 0.8 1.4
± ± ± ± ± ± ± ± ±
5.32b 3.83c 4.18d 1.58e 4.51f 3.42f,g 1.58a 0.84g 0.55g
Means with the same letter within the same column are not significantly different at p < 0.05.
many parts of the world (Anthony et al., 2005). However, commercially available anthelmintic agents in developing countries are unaffordable; in addition, these agents and their secondary metabolites pose a threat to human health (Cabaret, 2008). These
Table 4 Effect (mean percent ± SD) of Zanthoxylum simulans essential oil, borneol and elemene on inhibiting Haemonchus contortus larval migration. Concentration (mg/mL)
Essential oils and constituents Essential oil
40.0 20.0 10.0 5.0 2.5 1.25 Levamisole(5) Tween 80 (20) PBS
21.4 38.2 49.6 59.4 69.2 80.4 2.2 83.4 86.4
± ± ± ± ± ± ± ± ±
-elemene
Borneol b
4.22 3.03c 7.92d 4.87e 3.19f 4.15g 0.83a 4.33g,h 2.97h
1.8 2.6 9.6 23.2 37.2 52.8 2.2 83.4 86.4
± ± ± ± ± ± ± ± ±
a
0.84 1.14a 4.16b 6.38c 9.31d 6.22e 0.83a 4.33f 2.97f
39.6 46.2 60.2 64.6 71.0 78.8 2.2 83.4 86.4
± ± ± ± ± ± ± ± ±
8.62b 4.15b 7.85c 8.62c,d 6.86d,e 7.19e,f 0.83a 4.33f 2.97f
Means with the same letter within the same column are not significantly different at p < 0.05.
issues reinforced the need to derive safe and cheap anthelmintic agents from plants (Fred-Jaiyesimi et al., 2011). Although Zanthoxylum is a known potential source of anthelmintic agents for traditional Chinese medicine, only a few studies have assessed the anthelmintic activity of Zanthoxylum species (Olounlade et al., 2012). Therefore, the present study investigated the effect of ZSEO on the egg hatching and larval motility of H. contortus. To the best of our knowledge, this study is the first to analyze and test ZSEO for its anthelmintic activity. The bio-assays performed to test the in vitro anthelmintic activity of ZSEO were based on established methods. Several in vivo and in vitro techniques have been developed to detect anthelmintic resistance in nematodes (Craven et al., 1999). In vivo tests would be more accurate but with more time consuming, expensive, and lower precision and reproducibility because of the interanimal variation and pharmacodynamics of the drug in the host (Lacey et al., 1990). In vitro tests that use free-living stages of parasitic nematodes are considered the useful methods in examining natural anthelmintics, so both techniques have a role to play and the one will not exclude the other. In the present study, ZSEO efficiently inhibited the egg hatching, larval development, and larval motility of H. contortus. Literature survey reveals that anthelmintic activity against H. contortus in vitro of essential oils from various plant have been well demonstrated. Ribeiro et al. (2014) showed Eucalyptus citriodora essential oils (4 mg/ml concentration) inhibited H. contortus larvae hatching by 97.2% at 4 mg/mL and inhibited larval development by 99.8% at 8 mg/mL. The essential oil from Piper aduncum showed to be effective in inhibiting H. contortus hatchability and the LC90 was calculated as 8.9 mg/mL (Oliveira et al., 2014). Artemisia lancea essential oil at 10 mg/ mL inhibited H. contortus larvae hatching by 99.0%, inhibited larval development by 93.6%, and inhibited larval migration by 77.0% (Zhu et al., 2013a). Eucalyptus staigeriana essential oil inhibited larval hatching by 99.9% at doses of 1.0 mg/mL and inhibited larval development by 98.4% at 8.0 mg/mL (Ribeiro et al., 2013). The maximum effectiveness of Eucalyptus globulus essential oils on H. contortus on eggs hatching was 99.3% in concentration of 21.75 mg/mL and on larvae development was 98.7% in concentration 43.5 mg/mL (Macedo et al., 2009). In view of these findings it is proposed that essential oils may offer an alternative source for the control of gastrointestinal nematodes of sheep and goats. The results of phytochemical tests show that ZSEO contains terpenoids and oxygenated terpenoids that might be involved in the growth inhibition or retardation, maturation damage, reduced reproductive capacity, appetite suppression, and eventual mortality of insects (Júnior, 2003). Previous studies revealed that essential oils exert varying degrees of ovicidal and larvicidal effects against some gastrointestinal nematodes because of their chemical constituents, including linalool (Zhu et al., 2013b), 1,8-cineole (Oliveira
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et al., 2014; Zhu et al., 2013a), camphor (Zhu et al., 2013a), limonene (Ribeiro et al., 2013), anethole, and thymol (Camurc¸a-Vasconcelos et al., 2007). Borneol and -elemene, the two principal constituents of ZSEO, were also evaluated in the present study. The two compounds inhibited the egg hatching, larval development, and larval motility of H. contortus. The inhibitory effect of ZSEO was weaker than that of borneol but stronger than that of -elemene. The current study found that the anthelmintic activity of ZSEO can be partially associated with its major components (borneol and elemene). However, the considerable activity of the essential oil suggests a possible synergistic relationship because of the complex mixture of compounds that can interact with multiple molecular targets in various developmental stages of the parasite (MarieMagdeleine et al., 2009). In conclusion, ZSEO showed promising anthelmintic activities against the eggs and larvae of H. contortus. The traditional use of Z. simulans against intestinal parasites can be supported by the anthelmintic activities of its essential oil against nematodes. Therefore, Zanthoxylum may be an alternative source of anthelmintic agents to control gastrointestinal nematodes in sheep and goats. However, detailed studies are needed to identify and evaluate the active components and elucidate the mechanism of action of Z. simulans. In vivo experiments should also be performed to explore the toxicity and evaluate the effects of Z. simulans. Conflict of interest The authors have no conflict of interest concerning the work reported in this paper. Acknowledgments The authors are grateful for financially supported by the financial support by Fundamental Research Funds for the Central Universities (2013ZZ0081). References Anthony, J.P., Fyfe, L., Smith, H., 2005. Plant active components a resource for antiparasitic agents? Trends Parasitol. 21, 462–468. Bethony, J.M., Loukas, A., Hotez, P.J., Knox, D.P., 2006. Vaccines againstblood-feeding nematodes of humans and livestock. Parasitology 133 (Suppl), S63–S79. Botura, M.B., Silva, G.D., Lima, H.G., Oliveira, J.V.A., Souza, T.S., Santos, J.D.G., Branco, A., Moreira, E.L.T., Almeida, M.A.O., Batatinha, M.J.M., 2011. in vivo anthelminthic activity of an aqueous extract from sisal waste (agave sisalana perr.) against gastro-intestinal nematodes in goats. Vet. Parasitol. 177, 104–110. Cabaret, J., 2008. Pro and cons of targeted selective treatment against digestive-tract strongyles of ruminants. Parasite 15, 506–509. Camurc¸a-Vasconcelos, A.L.F., Bevilaqua, C.M.L., Morais, S.M., Maciel, M.V., Costa, C.T.C., Macedo, I.T.F., Oliveira, L.M.B., Braga, R.R., Silva, R.A., Vieira, L.S., 2007. Anthelmintic activity of croton zehntneri and lippia sidoides essential oils. Vet. Parasitol. 148, 288–294. Chen, I.S., Wu, S.J., Tsai, I.L., 1994. Chemical and bioactive constituents from zanthoxylum simulans. J. Nat. Prod. 57, 1206–1211. Chen, I.S., Wu, S.J., Leo, I.N., Tsai, I.L., Wu, T.S., 1996. Alkaloids from root bark of zanthoxylum simulans. Phytochemistry 42, 217–219. Coles, G.C., Bauer, C., Borgsteede, F., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World association for advancement in veterinary parasitology (waavp) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–43. Costa, C.T.C., Bevilaqua, C.M.L., Camurc¸a-vasconcelos, A.L.F., Maciel, M.V., Morais, S.M., Castor, C.M.S., Braga, R.R., Oliverira, L.M.B., 2008. in vitro ovicidal and larvicidal activity of azadirachta indica extracts on haemonchus contortus. Small Ruminant Res. 74, 284–287.
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In vitro anthelmintic activity of Zanthoxylum simulans essential oil against Haemonchus contortus H Qi, W.X. Wang, J.L. Dai, L. Zhu ∗ College of Food and Bioengineering, South China University of Technology, Guangzhou, Guangdong Province, China
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 27 May 2015 Accepted 30 May 2015 Keywords: Zanthoxylum simulans Essential oil Composition Anthelmintic activity
a b s t r a c t The need for new anthelmintic agents with low impact on the environment is becoming urgent. Phytotherapy is an alternative method to control gastrointestinal nematodes in small ruminants. This study aims to determine the composition of Zanthoxylum simulans essential oil (ZSEO) and evaluate the in vitro ovicidal and larvicidal effects of ZSEO on Haemonchus contortus using egg hatch assay, larval development assay (LDA), and larval migration inhibition assay (LMIA). The chemical composition of ZSEO was determined through gas chromatography and mass spectrometry and 94 compounds were identified from the ZSEO. The major constituents of ZSEO were borneol (18.61%), -elemene (10.87%). ZSEO and borneol both at 40 mg/mL inhibited larval hatching by 100%, with LC50 values of 3.98 and 1.50 mg/mL, respectively. The LC50 value of -elemene was not determined because of its insufficient activity. The results of LDA showed that ZSEO, borneol, and -elemene all at 40 mg/mL inhibited larval development by 99.8%, 100%, and 55.4%, respectively, and exhibited dose-dependent responses with LC50 values of 4.02, 1.99, and 32.17 mg/mL, respectively. The results of LMIA showed that ZSEO, borneol, and -elemene all at 40 mg/mL inhibited larval migration by 74.3%, 97.0%, and 53.2%, respectively. ZSEO presented ovicidal and larvicidal activities in vitro. Therefore, Zanthoxylum may be an alternative source of anthelmintic agents to control gastrointestinal nematodes in sheep. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Helminthosis caused by gastrointestinal nematodes, particularly Haemonchus contortus, is a prevalent and economically significant disease among domesticated animals (Perry and Randolph, 1999; Marie-Magdeleine et al., 2010). Gastrointestinal parasitic disease in small ruminants is characterized by anaemia, haemorrhagic gastroenteritis, hypoproteinaemia, sudden death, and chronic emaciation (Bethony et al., 2006; Botura et al., 2011). Therefore, controlling helminthes in livestock farming is important worldwide. Most anthelmintic drugs are expensive and unavailable to rural subsistence livestock keepers. Furthermore, the large-scale use of anthelmintic drugs has led to the emergence of multiple anthelmintic resistances. The residue of some persistent chemicals in the environment disrupts the ecosystem and poses a threat to human health. Therefore, new treatment approaches are required, among them, the use of medicinal plants have a rapid development because they are less toxic,
∗ Corresponding author. Fax: +86 20 87113849. E-mail address: [email protected] (L. Zhu). http://dx.doi.org/10.1016/j.vetpar.2015.05.029 0304-4017/© 2015 Elsevier B.V. All rights reserved.
biodegradable, and environmentally friendly (Hammond et al., 1997). Zanthoxylum of the family Rutaceae consists of 250 species of deciduous shrubs and trees that are native to warm temperate and subtropical regions of the world. China is home to 45 species and 13 varieties of this genus (Zhang et al., 2014). The fruits of some species such as Z. armatum and Z. bungeanum, with the most popular name being “huajiao” (flower pepper), is a traditional spice with strong spiciness and astringent taste. Many Zanthoxylum species in China are medicinal herbs widely used in traditional Chinese medicine. Essential oils isolated from the genus are effective for the treatment of intestinal parasites, inflammatory diseases, stomach disorders, toothache, diarrhea, and dysentery (Wei et al., 2011). Z. simulans is a prickly shrub that is distributed throughout mainland China and Taiwan (Yang et al., 2002). Z. simulans is an edible material and traditional medicine universally used to kill intestinal parasites as well as treat rheumatoid arthritis and inflammation (Wang et al., 2014). Zanthoxylum contains alkaloids, coumarins, amides, lignans, and flavonoids (Wang et al., 2014; Yang et al., 2002; Chen et al., 1994, 1996). The present study aims to determine the chemical composition of Z. simulans essential oil (ZSEO) and evaluate the in vitro anthelmintic effect of ZSEO and its
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major constituents, borneol and -elemene, against the eggs and infective larvae of H. contortus from sheep. 2. Materials and methods 2.1. Plant materials Fresh aerial parts of Z. simulans were collected from Hunan Province, China, in September 2012 and were identified by Dr. Gong Xun, Kunming Institute of Botany, Chinese Academy of Sciences. The plants were dried under a shade at room temperature. The voucher specimens (No. 0412161) were deposited in Kuming Institute of Botany, Chinese Academy of Sciences. 2.2. Preparation of ZSEO and chemicals Air-dried plant materials of Z. simulans were chopped and subjected to hydrodistillation for 4 h using a clevenger-type apparatus (Kesijia Ltd., Beijing, China) consisting of a 5 L distillation bottle, a 5 mL graduated receiver, and a jacketed-coil condenser. The obtained oils were dried over hydrous sodium sulfate for 24 h, filtered, and then stored at 4 ◦ C in brown sealed glass vials until tested. Borneol and -elemene were purchased from Sigma–Aldrich (St. Louis, USA). To improve the emulsification of ZSEO and compounds in water, 2% Tween 80 solvent was added and mixed the solutions in a vortex shaker until the oil, solvent, and water became a stable emulsion. 2.2.1. Gas chromatography mass spectrometry analysis ZSEO was quantitatively and qualitatively analyzed using an Agilent GC–MS 6890-5975 system equipped with an HP-5 MS fused silica capillary column (30 m × 0.25 mm i.d.; film thickness, 0.25 m). For GC–MS detection, an electron ionization system with 70 eV of ionization energy was used. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The injector and mass transfer line temperatures were set at 250 ◦ C and 280 ◦ C, respectively. The ZSEO solution (1 L) in hexane was injected and then analyzed under the following conditions: initial column temperature at 40 ◦ C for 1 min, increased to 250 ◦ C at a heating ramp rate of 3 ◦ C/min, and then maintained at 250 ◦ C for 20 min. The Kovats indices were calculated for all volatile components using a homologous series of n-alkanes (C8 –C25 ) on the HP-5 MS column. The major ZSEO components were identified by comparison with the standards and verified on the basis of Kovats indices using Wiley (V.7.0) and National Institute of Standards and Technology V.2.0 GC–MS library. The relative concentration of each compound in ZSEO was quantified on the basis of the peak area integrated in the analysis program. 2.2.2. Gas chromatography flame lionization detector analysis An Agilent HP-6890 GC with an HP-5 5% phenylmethylsiloxane capillary column (30 m × 0.25 mm i.d.; film thickness, 0.25 m) and a flame ionization detector were used for GC–FID analysis. Helium at a constant flow rate of 1 mL/min was used as the carrier gas. All other parameters were the same as those described above. Results were expressed as the relative percentage of peak area. 2.3. In vitro anthelmintic assays The anthelmintic bioassay tests of ZSEO and chemicals at different life-cycle stages of H. contortus were performed using egg hatch assay (EHA), larval development assay (LDA), and larval migration inhibition assay (LMIA). For each assay, the four parasitic stages were obtained from donor Small Tail Han sheep which were
experimentally infected with oral administration of a pure aqueous suspension of 10,000 H. contortus third stage larvae (L3). 2.3.1. Egg hatch assay Eggs were collected from the donor sheep and EHA was performed in accordance with the guidelines of World Association for the Advancement of Veterinary Parasitology guidelines (Coles et al., 1992). The parasite eggs were collected from the feces of infected donor sheep, dispersed in water, sifted successively (250, 125, 63, 50, and 30 mm sieves), and then centrifuged at 1000 × g for 15 min. Eggs were washed three times with distilled water. ZSEO and its principal components, namely, borneol and -elemene, were tested for their inhibitory activities against egg hatching. Albendazole (Sigma–Aldrich) was used as a positive control, whereas untreated eggs in PBS or Tween 80 solvent (20 mg/mL) served as negative control. The egg suspension was dispensed in 24-multiwell plates (1 mL per well) with a concentration of 200 eggs/mL and then mixed with the same volume of the sample solution (ZSEO, borneol, or -elemene) that their final concentration prepared at 40, 20, 10, 5, 2.5, and 1.25 mg/mL. Albendazole used as a positive control was dissolved in distilled water to a final concentration of 0.0063 mg/mL. PBS was applied to avoid any nonspecific effects caused by changes in pH (pH 7.2). Five replicates were performed for each test extract. After 48 h of incubation at 27 ◦ C, egg hatching was stopped by adding Lugol’s iodine solution. The number of L1 larvae and unhatched egg per well were then counted under a dissecting microscope at 40× magnification. 2.3.2. Larval development assay LDA was conducted as described by Costa et al. (2008) and Tadesse et al. (2009). Eggs were incubated at 27 ◦ C for 48 h to obtain first-stage larvae. The experiment was conducted in plastic cups of 20 mL. A 1 mL aliquot containing 200 first-stage larvae of H. contortus was mixed with 5 g of sheep feces free of gastrointestinal nematodes and then with different sample treatment solutions. Serial final concentrations (40, 20, 10, 5, 2.5, and 1.25 mg/mL) of the sample solution (ZSEO, borneol, or -elemene) were prepared to a total volume of 7 mL. Albendazole was dissolved in distilled water to a final concentration of 0.0063 mg/mL and used as a positive control. All samples were incubated at room temperature for 6 days. After 6 days, the wall of each cup containing the sample was thoroughly rinsed with 10 mL of water, and the larvae were collected. One drop of Lugol’s iodine solution was added, and all third-stage (L3) larvae were counted under a dissecting microscope at 40× magnification. 2.3.3. Larval migration inhibition assay In vitro experiments were performed to determine the effect of ZSEO on the mobility of ensheathed H. contortus L3 larvae through LMIA as described by Rabel et al. (1994) with modifications. Eggs hatch and larvae develop to L3 after 8 days and were then collected by sedimentation using Baermann devices (Luo et al., 1999). Larvae were stored at 4 ◦ C for 1 month before use. One thousand live L3 larvae dispersed in 1 mL PBS were added to 5 mL centrifuge tubes containing either the negative control (PBS; Tween 80 solvent), the commercial anthelmintic control (levamisole at final concentration 5 mg/mL), or the sample solution (ZSEO, borneol, or -elemene) that their final concentration prepared at 40, 20, 10, 5, 2.5, and 1.25 mg/mL. Incubations were carried out at 28 ◦ C for 3 h. Thereafter, the L3 larvae from each tube were washed with PBS, centrifuged three times at 1400 × g for 10 min. and then transferred to sieves (inserts equipped with a 20 m mesh positioned in a conical tube) (Hernández-Villegas et al., 2011). After 3 h of incubation at room temperature, the number of larvae that migrated through the mesh was counted under a microscope at 40× magnification using a 15% aliquot technique (Hernández-Villegas et al.,
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2011). Migration percentage was calculated as M/T × 100, where T is the total number of L3 larvae deposited on the sieve and M is the number of L3 larvae that successfully migrated through the sieve. Five replicates were run for each ZSEO solution and for the control.
2.4. Statistical analysis Data from EHA, LDA, and LMIA were subjected to Probit analysis by SPSS 13.0 to calculate LC50 , LC90 , and other statistical indices at 95% confidence limits. Chi-square values were calculated using SPSS 13.0 for Windows. Statistical significance was considered at p < 0.05. 3. Results The steam distillation of 4000 g of dried Z. simulans plant material yielded 6.1 mL (0.153% v/w) of yellow oil. The oil sample was analyzed using GC–FID and GC–MS, and the components were identified on the basis of their RI values as well as their mass spectra relative to those reported in literature. The GC–MS analysis indicated 94 components that represent 99.30% of ZSEO (Table 1). Table 2 lists the data on the inhibitory effects of ZSEO, borneol, and -elemene on H. contortus egg hatching. ZSEO for all tested concentrations inhibited egg hatching, with an LC50 value of 3.98 mg/mL (95% CI = 2.89 mg/mL–5.31 mg/mL). Similar to the positive control albendazole, ZSEO at 40 and 20 mg/mL inhibited egg hatching by more than 95% (p > 0.05). With an LC50 value of 1.50 mg/mL (95% CI = 1.15 mg/mL–1.85 mg/mL), borneol exhibited higher ovicidal activity than ZSEO. -elemene did not exhibit a high ovicidal activity, and its LC50 was not determined because more than 40 mg/mL -elemene was needed to inhibit 50% of egg hatching. PBS and Tween 80 solvent showed no effect on the hatching of H. contortus eggs. ZSEO, borneol, and -elemene effectively inhibited the larval development of H. contortus (Table 3). Similar to the positive control albendazole, ZSEO at 40 mg/mL inhibited larval development by more than 99% (p > 0.05). A clear dose-dependent effect was also obtained within the range of the concentrations tested, with an LC50 of 4.02 mg/mL (95% CI = 3.51 mg/mL–4.57 mg/mL). With an LC50 value of 1.99 mg/mL (95% CI = 1.63 mg/mL–2.35 mg/mL), borneol exhibited higher activity than ZSEO for all concentrations. The activity of -elemene was not very significant with LC50 32.17 mg/mL (95% CI = 24.27–47.13 mg/mL). Albendazole was 97.0% effective in inhibiting larval development, whereas the negative control and Tween 80 (20 mg/mL) exhibited 1.4% and 0.8% effectiveness, respectively. Table 4 lists the data on the effects of ZSEO, borneol, and elemene on inhibiting H. contortus larval migration. In the negative control (PBS) and Tween 80 groups, the migration percentage of H. contortus L3 larvae was higher than 83.4%. The migration percentage of the positive control levamisole was 2.2%. The migration percentage of ZSEO at 40 mg/mL was 21.4%. Similar to the positive control, the migration percentage of borneol at 40 and 20 mg/mL was 1.8 and 2.6%. -elemene showed low inhibition for all concentrations tested, its migration percentage was 39.8% at 40 mg/mL. 4. Discussion Helminth infections constitute serious health problems worldwide in both humans and animals. Traditional healers and farmers in different regions of China have used botanical materials isolated from plant leaves, roots, seeds, flowers, and bark as anthelmintic agents (Lv et al., 2012). For centuries, medicinal plants have been used to combat parasitism and are still used for this purpose in
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Table 1 Chemical composition of Zanthoxylum simulans essential oil. RIa 930 978 1008 1030 1059 1089 1132 1072 1078 1096 1099 1105 1117 1120 1123 1130 1135 1140 1143 1148 1160 1168 1170 1178 1190 1196 1206 1217 1220 1242 1257 1265 1335 1351 1375 1384 1388 1390 1428 1437 1439 1449 1460 1486 1487 1490 1503 1514 1556 1549 1562 1578 1587 1598 1625 1639 1651 1659 1689 852 862 982 1017 1033 1062 1094 1095 1118 1158 1164 1082
Componentc
% RAb
Monoterpene hydrocarbons ␣-Thujene -Pinene ␣-Phellandrene Limonene ␥-Terpinene Terpinolene Allo-ocimene Oxygenated monoterpenes cis-Sabinene hydrate cis-Linalool oxide cis-Sabinene hydrate Linalool Thujone endo-Fenchol cis-4-Isopropyl-1-methyl-2-cyclohexen-1-ol trans-2-Pinanol cis-Limonene oxide cis--Terpineol trans-2-Menthenol Camphor Camphene hydrate Isoborneol Borneol Lavandulol Terpinene-4-ol ␣-Terpineol Citronellol Piperitol trans-Carveol exo-2-Hydroxycineole Carvone Geraniol Furomyrcenol Sesquiterpene hydrocarbons ␦-Elemene ␣-Cubebene Isoledene -Bourbonene -Cubebene -Elemene Thujopsene ␥-Elemene Aromadendrene cis--Farnesene Allo aromadendrene Germacrene D Selinene Valencene Bicyclo[4.4.0]dec-1-ene, 2-isopropyl-5-methyl-9-methylenetrans-␥-cadinene Germacrene B Oxygenated sesquiterpenes Elemol Ledol Caryophyllene oxide Viridiflorol Guaiol 1-epi-Cubenol Isospathulenol 9-Aristolen-␣-ol Kongol ␣-Bisabolol Others trans-2-Hexenal trans-2-Hexenol 1-Octen-3-ol p-Cymene Benzyl alcohol trans-2-Octenal 2-Nonanone Nonanal Phenylethyl alcohol Sabina ketone Benzyl acetate Camphenilone
7.60 0.76 0.41 0.25 4.23 1.36 0.37 0.22 34.03 0.19 1.83 0.41 5.54 0.25 0.21 0.24 0.47 0.26 0.25 0.29 1.01 0.26 0.31 18.61 0.36 1.05 0.30 0.27 0.42 0.23 0.41 0.30 0.22 0.34 26.13 0.41 0.59 0.85 0.38 0.45 10.87 0.22 2.89 0.36 0.86 0.99 4.94 1.25 0.18 0.29 0.24 0.36 10.23 0.25 0.47 6.13 0.36 0.38 0.24 0.46 0.3 0.38 1.26 21.31 1.79 3.81 0.34 1.47 0.61 0.18 0.24 0.46 0.65 0.45 0.33 0.49
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Table 1 (Continued) RIa
Componentc
% RAb
1215 1229 1235 1253 1271 1285 1292 1297 1304 1308 1315 1344 1357 1368 1385 1396 1432 1495 1500 1521 1534 1620 1971
Caprylyl acetate Estragole Thymol methyl ether Sabinene hydrate acetate Geranial Bornyl acetate Safrole Thymol 2-Undecanol Carvacrol 2-Methoxy-4-vinylphenol exo-2-Hydroxycineole acetate Eugenol Neryl acetate Geranyl acetate cis-Jasmone ␣-Ionone 2-Tridecanone trans-Methyl isoeugenol Calamenene ␣-Calacorene Dill apiole n-Hexadecanoic acid Total identified (%)
0.24 0.22 0.26 0.46 0.29 0.18 0.31 0.36 1.38 0.28 0.57 0.95 0.65 0.57 0.34 0.67 0.38 0.99 0.26 0.37 0.29 0.19 0.28 99.30
a
Retention index (RI) relative to n-alkanes on the HP-5 MS capillary column. Relative area (peak area relative to the total peak area). c All the compounds were identified using mass spectrum and retention index. Limonene, linalool, borneol, -elemene, germacrene D, caryophyllene oxide, trans2-hexenol were also identified using the method of co-injection with authentic compound. b
Table 2 Effect (mean percent ± SD) of Zanthoxylum simulans essential oil, borneol and elemene on inhibiting hatching of Haemonchus contortus eggs. Concentration (mg/mL)
Essential oils and constituents Essential oil
40.0 20.0 10.0 5.0 2.5 1.25 Albendazole (0.0063) Tween 80 (20) PBS
100 95.6 75.0 51.4 32.6 20.2 100 1.2 1.6
± ± ± ± ± ± ± ± ±
0a 3.20a 7.18b 4.16c 5.22d 2.95e 0a 0.84f 1.14f
-elemene
Borneol 100 98.8 92.2 80.0 62.1 48.6 100 1.2 1.6
± ± ± ± ± ± ± ± ±
0a 1.79a 5.12b 5.83c 5.34d 6.02e 0a 0.84f 1.14f
40.4 25.2 18.8 9.6 6.0 1.2 100 1.2 1.6
± ± ± ± ± ± ± ± ±
7.13b 3.96c 4.32d 2.07e 2.24e 0.84f 0a 0.84f 1.14f
Means with the same letter within the same column are not significantly different at p < 0.05.
Table 3 Effect (mean percent ± SD) of Zanthoxylum simulans essential oil, borneol and elemene on inhibiting Haemonchus contortus larval development. Concentration (mg/mL)
Essential oils and constituents Essential oil
40.0 20.0 10.0 5.0 2.5 1.25 Albendazole (0.0063) Tween 80 (20) PBS
99.8 93.4 84.0 55.4 25.6 18.0 97.0 0.8 1.4
± ± ± ± ± ± ± ± ±
0.44a 1.14b 7.07c 7.93d 5. 23e 3.74f 1.58a,b 0.84g 0.55g
-elemene
Borneol 100.0 98.0 91.2 80.2 52.2 38.2 97.0 0.8 1.4
± ± ± ± ± ± ± ± ±
0a 3.39a 3.42b 2.86c 8.67d 3.90e 1.58a 0.84f 0.55f
55.4 39.2 27.0 16.0 8.4 4.8 97.0 0.8 1.4
± ± ± ± ± ± ± ± ±
5.32b 3.83c 4.18d 1.58e 4.51f 3.42f,g 1.58a 0.84g 0.55g
Means with the same letter within the same column are not significantly different at p < 0.05.
many parts of the world (Anthony et al., 2005). However, commercially available anthelmintic agents in developing countries are unaffordable; in addition, these agents and their secondary metabolites pose a threat to human health (Cabaret, 2008). These
Table 4 Effect (mean percent ± SD) of Zanthoxylum simulans essential oil, borneol and elemene on inhibiting Haemonchus contortus larval migration. Concentration (mg/mL)
Essential oils and constituents Essential oil
40.0 20.0 10.0 5.0 2.5 1.25 Levamisole(5) Tween 80 (20) PBS
21.4 38.2 49.6 59.4 69.2 80.4 2.2 83.4 86.4
± ± ± ± ± ± ± ± ±
-elemene
Borneol b
4.22 3.03c 7.92d 4.87e 3.19f 4.15g 0.83a 4.33g,h 2.97h
1.8 2.6 9.6 23.2 37.2 52.8 2.2 83.4 86.4
± ± ± ± ± ± ± ± ±
a
0.84 1.14a 4.16b 6.38c 9.31d 6.22e 0.83a 4.33f 2.97f
39.6 46.2 60.2 64.6 71.0 78.8 2.2 83.4 86.4
± ± ± ± ± ± ± ± ±
8.62b 4.15b 7.85c 8.62c,d 6.86d,e 7.19e,f 0.83a 4.33f 2.97f
Means with the same letter within the same column are not significantly different at p < 0.05.
issues reinforced the need to derive safe and cheap anthelmintic agents from plants (Fred-Jaiyesimi et al., 2011). Although Zanthoxylum is a known potential source of anthelmintic agents for traditional Chinese medicine, only a few studies have assessed the anthelmintic activity of Zanthoxylum species (Olounlade et al., 2012). Therefore, the present study investigated the effect of ZSEO on the egg hatching and larval motility of H. contortus. To the best of our knowledge, this study is the first to analyze and test ZSEO for its anthelmintic activity. The bio-assays performed to test the in vitro anthelmintic activity of ZSEO were based on established methods. Several in vivo and in vitro techniques have been developed to detect anthelmintic resistance in nematodes (Craven et al., 1999). In vivo tests would be more accurate but with more time consuming, expensive, and lower precision and reproducibility because of the interanimal variation and pharmacodynamics of the drug in the host (Lacey et al., 1990). In vitro tests that use free-living stages of parasitic nematodes are considered the useful methods in examining natural anthelmintics, so both techniques have a role to play and the one will not exclude the other. In the present study, ZSEO efficiently inhibited the egg hatching, larval development, and larval motility of H. contortus. Literature survey reveals that anthelmintic activity against H. contortus in vitro of essential oils from various plant have been well demonstrated. Ribeiro et al. (2014) showed Eucalyptus citriodora essential oils (4 mg/ml concentration) inhibited H. contortus larvae hatching by 97.2% at 4 mg/mL and inhibited larval development by 99.8% at 8 mg/mL. The essential oil from Piper aduncum showed to be effective in inhibiting H. contortus hatchability and the LC90 was calculated as 8.9 mg/mL (Oliveira et al., 2014). Artemisia lancea essential oil at 10 mg/ mL inhibited H. contortus larvae hatching by 99.0%, inhibited larval development by 93.6%, and inhibited larval migration by 77.0% (Zhu et al., 2013a). Eucalyptus staigeriana essential oil inhibited larval hatching by 99.9% at doses of 1.0 mg/mL and inhibited larval development by 98.4% at 8.0 mg/mL (Ribeiro et al., 2013). The maximum effectiveness of Eucalyptus globulus essential oils on H. contortus on eggs hatching was 99.3% in concentration of 21.75 mg/mL and on larvae development was 98.7% in concentration 43.5 mg/mL (Macedo et al., 2009). In view of these findings it is proposed that essential oils may offer an alternative source for the control of gastrointestinal nematodes of sheep and goats. The results of phytochemical tests show that ZSEO contains terpenoids and oxygenated terpenoids that might be involved in the growth inhibition or retardation, maturation damage, reduced reproductive capacity, appetite suppression, and eventual mortality of insects (Júnior, 2003). Previous studies revealed that essential oils exert varying degrees of ovicidal and larvicidal effects against some gastrointestinal nematodes because of their chemical constituents, including linalool (Zhu et al., 2013b), 1,8-cineole (Oliveira
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et al., 2014; Zhu et al., 2013a), camphor (Zhu et al., 2013a), limonene (Ribeiro et al., 2013), anethole, and thymol (Camurc¸a-Vasconcelos et al., 2007). Borneol and -elemene, the two principal constituents of ZSEO, were also evaluated in the present study. The two compounds inhibited the egg hatching, larval development, and larval motility of H. contortus. The inhibitory effect of ZSEO was weaker than that of borneol but stronger than that of -elemene. The current study found that the anthelmintic activity of ZSEO can be partially associated with its major components (borneol and elemene). However, the considerable activity of the essential oil suggests a possible synergistic relationship because of the complex mixture of compounds that can interact with multiple molecular targets in various developmental stages of the parasite (MarieMagdeleine et al., 2009). In conclusion, ZSEO showed promising anthelmintic activities against the eggs and larvae of H. contortus. The traditional use of Z. simulans against intestinal parasites can be supported by the anthelmintic activities of its essential oil against nematodes. Therefore, Zanthoxylum may be an alternative source of anthelmintic agents to control gastrointestinal nematodes in sheep and goats. However, detailed studies are needed to identify and evaluate the active components and elucidate the mechanism of action of Z. simulans. In vivo experiments should also be performed to explore the toxicity and evaluate the effects of Z. simulans. Conflict of interest The authors have no conflict of interest concerning the work reported in this paper. Acknowledgments The authors are grateful for financially supported by the financial support by Fundamental Research Funds for the Central Universities (2013ZZ0081). References Anthony, J.P., Fyfe, L., Smith, H., 2005. Plant active components a resource for antiparasitic agents? Trends Parasitol. 21, 462–468. Bethony, J.M., Loukas, A., Hotez, P.J., Knox, D.P., 2006. Vaccines againstblood-feeding nematodes of humans and livestock. Parasitology 133 (Suppl), S63–S79. Botura, M.B., Silva, G.D., Lima, H.G., Oliveira, J.V.A., Souza, T.S., Santos, J.D.G., Branco, A., Moreira, E.L.T., Almeida, M.A.O., Batatinha, M.J.M., 2011. in vivo anthelminthic activity of an aqueous extract from sisal waste (agave sisalana perr.) against gastro-intestinal nematodes in goats. Vet. Parasitol. 177, 104–110. Cabaret, J., 2008. Pro and cons of targeted selective treatment against digestive-tract strongyles of ruminants. Parasite 15, 506–509. Camurc¸a-Vasconcelos, A.L.F., Bevilaqua, C.M.L., Morais, S.M., Maciel, M.V., Costa, C.T.C., Macedo, I.T.F., Oliveira, L.M.B., Braga, R.R., Silva, R.A., Vieira, L.S., 2007. Anthelmintic activity of croton zehntneri and lippia sidoides essential oils. Vet. Parasitol. 148, 288–294. Chen, I.S., Wu, S.J., Tsai, I.L., 1994. Chemical and bioactive constituents from zanthoxylum simulans. J. Nat. Prod. 57, 1206–1211. Chen, I.S., Wu, S.J., Leo, I.N., Tsai, I.L., Wu, T.S., 1996. Alkaloids from root bark of zanthoxylum simulans. Phytochemistry 42, 217–219. Coles, G.C., Bauer, C., Borgsteede, F., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World association for advancement in veterinary parasitology (waavp) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–43. Costa, C.T.C., Bevilaqua, C.M.L., Camurc¸a-vasconcelos, A.L.F., Maciel, M.V., Morais, S.M., Castor, C.M.S., Braga, R.R., Oliverira, L.M.B., 2008. in vitro ovicidal and larvicidal activity of azadirachta indica extracts on haemonchus contortus. Small Ruminant Res. 74, 284–287.
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