International Journal of Science and Engineering Applications Volume 4 Issue 5, 2015, ISSN-2319-7560 (Online) Acaricidal Activity of Ethulia conyzoides Extracts and Constituents against Tetranychus urticae Koch A. Mahmoud Dawidar Chem. Dept., Fac. of Sci., Mansoura University Mansoura, Egypt M. Abdel-Mogib Chem. Dept., Fac. of Sci., Mansoura University Mansoura, Egypt M. El Saied. El-Naggar Plant Protection Research Institute, A.R.C., Egypt M. El-Hoseiny Mostafa Plant Protection Research Institute, A.R.C., Egypt Abstract: The acaricidal potential of Ethulia conyzoides L. aerial parts extracts as well as its isolated compounds were investigated against larvae and adults of Tetranychus urticae Koch under laboratory conditions. Chromatographic separation of the extracts gave 3-O-acetyl lupeol, lupan-3-ol, ethuliacoumarin (1) and 7-O-methyl apigenin (Genkwanin), besides a mixture of isoethyliacoumarin A (2), isoethuliacoumarin C (3). The biological evaluation results indicated that ethyl acetate fraction and ethuliacoumarin (1) were the most potent to larvae and adults of Tetranychus urticae. The LC50 values of ethyl acetate fraction were found to be 11.58 and 17.86 ppm, respectively and LC50 values of (1) were found to be 12.72 and 19.22 ppm, respectively after 7 days of treatment. Keywords: Acaricidal activity, Tetranychus urticae Koch, Ethulia conyzoides, 3-O-acetyl lupane, lupan-3-ol, ethuliacoumarin, 7-O-methyl apigenin, isoethyliacoumarin A and isoethuliacoumarin C. 1. INTRODUCTION The two spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a worldwide damaging pest attacking cotton, fruit trees and vegetables in Egypt [1]. Synthetic chemical acaricides has been the major tool in pest control operations. However, the extensive and repeated use of synthetic organic pesticides have led to environmental problems to the human health, non-target organisms and cause some disastrous ecological damage [2]. The move towards green pesticides represents a new trend for discovering natural pesticides. Natural products considered to be an excellent alternative to synthetic pesticides as a mean to reduce negative impacts to human health and the environment [3]. Ethulia conyzoides L., a wild growing Egyptian plant, has for centuries been used in folk medicine as an anti-helminthic for round worms and for abdominal disorders [4, 5], a source of natural antioxidants [6] and antihypertensive agents. It was used to cure headaches and dysmenorrhea [7]. In addition, the methanol extract of the aerial parts of E. conyzoides has antibacterial activity [5]. The molluscicidal activity of E. conyzoides was recorded [4], which was attributed to the presence of ethuliacoumarin A and isoethuliacoumarin A. The previous phytochemical screening revealed that E. conyzoides contains terpenoid 5-methylcoumarins [5, 8, 9, 10, 11, 12], triterpenoids [13, 14], flavonoids [15] and sterols [13]. This article presents the results of phytochemical and acaricidal activity investigation of E. conyzoides. 2. MATERIALS AND METHODS 2.1 General NMR experiments were performed on a Bruker AMX 400 instrument standard pulse sequences operating at 400 MHz in 1H-NMR and 13C-NMR. Chemical shifts are given in δ (ppm) relative to TMS as internal standard material and the coupling constants (J) are in Hz. GC/MS analysis of the volatile fractions were performed on a Varian GC interfaced to Finnegan SSQ 7000 Mass selective Detector (SMD) with ICIS V2.0 data system for MS identification of the GC components, at Agriculture Research Center, Dokki, Cairo. The column used was DB-5 (J&W Scientific, Folosm, CA) cross-linked fused silica capillary column (30 m. long, 0.25 mm. internal diameter) coated with poloy dimethyl-siloxane (0.5 μm. film thickness). The oven temperature was programmed from 50°С for 3 min., at isothermal, then heating by 7°С /min. to 250°С and isothermally for 10 min., at 250°С. Injector temperature was 200°С and the volume injected was 0.5 μl. Transition-line and ion source temperatures were 250°С and 150°С, respectively. The mass spectrometer had a delay of 3 min. to avoid the solvent plead and then scanned from m/z 50 to m/z 300. Ionization energy was set at 70 eV. 2.2 Chemicals Normal CC: column chromatography was performed on silica gel Merck grain size 0.2-0.063 mm; preparative TLC were performed on silica gel International Journal of Science and Engineering Applications Volume 4 Issue 5, 2015, ISSN-2319-7560 (Online) (Kieselgel 60, F 254) of 0.25 mm thickness. Pet. ether (60-80 ̊C), methylene chloride, ethyl acetate and methanol were obtained from Adwic Company. 2.3 Plant material The aerial parts of Ethulia conyzoides L. was collected on canal banks near Mansoura in April, 2009, identified by Prof. Dr. Loutfy Boulos, Professor of Botany, Faculty of Science, Alexandria University, Egypt. 2.4 Extraction and isolation The aerial parts of E. conyzoides (1 Kg) were extracted by soxhlet apparatus using different organic solvents of different polarities composed of pet. ethermethylene chloride- ethyl acetate and methanol successively. In order to obtain four fractions, pet. ether fraction (20.40 g), methylene chloride fraction (9.28 g), ethyl acetate fraction (3.05 g) and methanol fraction (53.72 g), which was further extracted by butanol to give the butanol fraction (2.31 g). E. conyzoides fresh aerial parts (250 g) were processed in order to obtain the volatile oil fraction by means of hydro-distillation technique (0.54 g, 0.22% fresh weight). 2.5 Separation of compounds The pet. ether fraction (9.45g) was defatted using cold methanol to give the defatted pet. ether fraction (9.42 g). The defatted pet ether residue was subjected to column chromatography using silica gel as adsorbent. Elution of the column was performed by using a series of eluents from hexane/ acetone combinations of increasing polarity. The effluents were combined into four sub-fractions according to their TLC patterns. Sub-fraction 1 a yellow orange oily material which was eluted by hexane/ acetone (4: 1) was further purified using preparative TLC eluted by (hexane / ether 3:1) to yield white needle crystals, Rf = 0.69 of 3-O-acetyl lupeol. Pet. ether fraction afforded by GC/MS technique 6 compounds as indicated by table 1. Methylene chloride fraction (9.28 g) was chromatographed over silica gel CC using a series of eluents from pet. ether / ethyl acetate combinations of increasing polarities. The effluents were recombined into eight sub-fractions based on their TLC patterns. The sub-fraction 2 eluted by pet. ether / ethyl acetate (13:7) gave by preparative TLC (silica gel, benzene / ether 4 : 1) lupan-3-ol at Rf = 0.28 and compound (1) at Rf = 0.53. The sub-fraction 3 was purified using preparative TLC (silica gel, CHCl3 / MeOH (39: 1)) to afford a mixture of compounds (2) and (3) at Rf = 0.19. Methylene chloride fraction afforded by GC/MS technique 5 compounds as indicated by table 1. The ethyl acetate fraction (3.05 g) was purified on sephadex-LH-20, which was washed with MeOH, the effluents were combined into five sub fractions based on their TLC patterns. Sub-fraction 5 was further purified using preparative TLC developed in a mixture of benzene / acetone (3:2), where 7-O-methyl apigenin was obtained at Rf = 0.67. The volatile oil fraction (0.54 g, 0.22%) was separated by GC/MS to afford 8 compounds. 2.6 Maintance of spider mite colony Colony of spider mite Tetranychus urticae Koch was reared under laboratory condition (25±2 °C and 60±5 % R.H) at plant protection research institute branch, Dakahlia Governorate. This study colony was isolated from heavily infested castor oil plant leaves. Spider mite colony was reared on castor oil leaves. These leaves were cleaned and placed on moisten cotton wool pad in Petri dishes. This colony was left for one year under the precious conditions in order to get a homogenous and sensitive colony. Spider mite individual were transferred to the leaves by the aid of fine camels hair brush. Breeding leaves were changed twice weekly at summer and once weekly at winter. Adding water was done twice daily to prevent escaping of T. urticae individuals. 2.7 Assessment of acaricidal activity In this respect, laboratory experiments are conducted to evaluate the activity of various tested plant extracts against T. urticae mobile stages (larvae and adult females). The leaf-dip technique was used Gazal et al., (1992) [16]. The indication of mortality was chosen as the failure of mites to respond positively by leg movement followed light brooding with a fine brush. Mortality percentages were determined and corrected by using Abotts, (1925) formula [17] and they are statistically analyzed to estimate LC50, LC90 and slope values according to Finney, (1971) [18]. Toxicity index was computed for different extracts and their isolated compounds by comparing these materials with the most effective extracts or isolated compounds using Sun’s, (1950) equation [19]. Where: A is the most effective compound B is the tested compound 3. RESULTS AND DISCUSSION The chromatographic separation of the plant extracts and the spectral analysis of the separated compounds revealed the identification of the triterpenoids lupyl acetate [20] and was previously isolated from the same plant species [13] and lupeol [21]) that was isolated before from E. conyzoides [13], The monoterperpenoidal 5-methylcoumarins ethuliacoumarin (1) which was isolated previously from the same plant species by Baldaa et al., (1980) and Mahmoud et al., (1998) [8, 9], isoethyliacoumarin A (2) and isoethuliacoumarin C (3) which were isolated previously from the same plant species by (Baldaa et al., 1980) [8], as well as the flavonoid 7-O-methyl apigenin (Genkwanin) [22], which has not been isolated previously from E. conyzoides. The pet ether, methylene chloride and the volatile oil fractions of the aerial parts of E. conyzoides were International Journal of Science and Engineering Applications Volume 4 Issue 5, 2015, ISSN-2319-7560 (Online) analyzed by GC/MS technique. Nineteen compounds which are listed in table 1 were identified. Table 1: Chemical constituents identified in fractions of E. conyzoides aerial parts by GC/MS technique. Rt, min Area % M.F 15.17 0.80 C15H24O Isoaromadendrene epoxide (5) 15.63 0.43 C15H24O Perhydrofarnesyl acetone 17.80 1.17 C18H36O 2,3,5-trimethyl-7HFuro[3,2g][1]benzopyran-7-one 20.76 2.53 C14H12O3 Phytol isomer 21.08 1.50 C20H40O Stigmasterol 35.58 3.74 C29H48O Methylene chloride fraction Benzaldehyde,412.33 methoxy Coniferyl alcohol 16.56 1.90 C8H8O2 0.54 C10H12O3 17.04 1.45 C11H16O3 Neophytadiene 18.02 0.61 C20H38 Phytol 21.05 0.36 C20H40O 11.89 0.43 C15H24 14.43 1.30 C15H24O 15.17 0.47 C15H24O Hexestrol (6) 20.55 0.20 C18H22O2 Acetyl tri-n-butyl citrate 22.73 0.46 C20H34O8 Taraxasterol 25.28 1.36 C30H50O β-Amyrin acetate 27.52 1.52 C32H52O2 Lupeol actate 29.29 2.69 C32H52O2 Component name Pet. ether fraction 4,4-dimethylTetracyclo[6.3.2.0 (2,5).0(1,8)]tridecan-9ol (4) (-)-Loliolide volatile oil fraction Trans-caryophyllene (-)-Caryophyllene oxide Caryophylla4(12),8(13)-dien-5β-ol (Identity) m/z (ret. int. %) 220 (1) [M+], 205 (3) [M- CH3]+, 187 (7) [M- CH3- H2O]+, 163 (10) [C11H15O]+, 136 (100) [C9H12O].+, 91 (37) [C7H7]+, 65 (20) [C5H5]+ 220 (7) [M+], 205 (13) [C15H25]+, 149 (43) [C11H17]+, 134 (96) [C10H14].+, 105 (98) [C8H9] +, 91 (100) [C7H7]+, 77 (67) [C6H5]+, 65 (27) [C5H5]+ 268 (1) [M+], 250 (13) [C18H34]+, 225 (1) [C16H33]+, 109 (35) [C8H13]+, 85 (35) [C6H13]+, 71 (63) [C5H11]+, 58 (100) [C3H6O]+ 228 (1) [M+], 227 (3) [M- H]+, 213 (7) [M- CH3]+, 199 (10) [M- H- CO]+, 185 (100) [M- CH3- CO]+, 175 (100) [M- CH3CO- CO]+, 91 (37) [C7H7]+, 77 (67) [C6H5]+ 297 (1) [M+.], 123 (74) [C9H15]+, 95 (78) [C7H11]+ , 71 (100) [C5H11]+, 55 (63) [C4H7]+. 412 (100) [M+], 394 (13) [M- H2O]+, 300 (43) [C21H32O]+, 255 (54) [C19H27]+, 231 (12) [C16H23O]+, 213 (30) [C16H21]+, 133 (43) [C10H13]+, 55 (70) [C4H7]+ 136 (100) [M+], 135 (100) [M+- H]+, 107 (13) [M+- CO]+, 105 (7) [M+- OCH3]+, 77 (20) [C6H5]+ 180 (74) [M+], 164 (100) [M+-OCH3]+, 137 (100) [C8H9O2]+, 124 (53) [C7H8O2].+, 91 (32) [C7H7]+, 77 (20) [C6H5]+ 196 (26) [M+], 178 (9) [M+- H2O]+, 163 (13) [M+- H2O-CH3]+, 135 (48) [C9H11]+, 111 (100) [C6H7O2]+, 91 (78) [C7H7]+, 65 (9) [C5H9]+. 278 (9) [M+], 123 (65)[C9H15]+, 95 (88) [C17H11]+, 82 (77) [C6H10]+, 71 (4) [C5H11]+, 68 (100) [C5H8]+, 55 (84) [C4H7]+ 297 (4) [M+.], 123 (28) [C9H15]+, 95 (13) [C7H11]+, 71 (100) [C5H11]+, 55 (27) [C4H7]+. 204 (13) [M+], 189 (33) [M-CH3]+, 147 (40) [C11H15]+, 133 (100) [C10H13]+, 91 (86) [C7H7]+, 55 (40) [C4H7]+, 220 (1) [M+], 205 (7) [M-CH3]+, 149 (17) [C11H17]+, 123 (17) [C9H15]+, 109 (50) [C7H9O]+, 93 (86) [C7H9]+, 55 (40) [C4H7]+. 220 (1) [M+], 205 (2) [M-CH3]+, 187 (3) [M-CH3-H2O]+, 136 (100) [C10H16].+, 109 (20) [C7H9O]+, 91 (34) [C7H7]+. 270 (22) [M+], 135 (100) [C9H11O]+, 120 (7) [C8H8O]+, 107 (3) [C7H7O]+, 77 (15) [C6H5]+. 329 (7) [C16H25O7]+, 301 (1) [C15H25O6]+,259(60) [C12H19O6]+, 185 (100) [C9H13O4]+, 157 (100) [C8H13O3]+, 129 (45) [C6H9O3]+. 426 (53) [M+], 411 (27) [M- CH3]+, 393 (7) [M-CH3-H2O]+, 272 (8) [C20H32].+, 207 (78) [C14H23O]+, 135 (97) [C10H15]+, 95 (72)[C7H11]+ , 77 (17) [C6H5]+, 55 (50) [C4H7]+. 468 (99) [M+], 393 (49) [M-CH3-H2O]+, 218 (48) [C16H26]+, 203 (55)[C15H23]+, 189 (81) [C14H21]+. 468 (37) [M+], 453 (17) [M- CH3]+, 393 (10) [M-CH3CH3COOH]+, 203 (37) [C15H23]+, 189 (100) [C14H21]+, 161 (28) [C12H17]+,135 (47) [C10H15]+, 121 (47) [C9H13]+. International Journal of Science and Engineering Applications Volume 4 Issue 5, 2015, ISSN-2319-7560 (Online) The results of acaricidal activity were presented obtained in table (2) that showed the efficiency of E. conyzoides fractions and their isolated compounds against the larvae of T. urticae after 7 days of treatment. The ethyl actate fraction was the most effective at LC50 level followed by butanol fraction, pet. ether fraction, methylene chloride fraction and essential oil extract. The LC50 values of these fractions were 11.58, 21.00, 53.63, 76.47 and 282.23 ppm, respectively. Taking the toxicity index into consideration, it could be also observed that ethyl actate fraction was the most effective fraction against the larvae of T. urticae after 7-days of treatment followed by butanol fraction, pet. ether fraction, methylene chloride fraction and essential oil extract. The susceptibility of the adult females of T. urticae to various plant fractions after 7-days of treatments (table 2) showed that the LC50 values were 17.86, 53.37, 109.73, 175.94 and 334.73 ppm for ethyl acetate fraction, butanol fraction, methylene chloride fraction, pet. ether fraction and essential oil fraction, respectively. The toxicity index of the LC50 values showed that ethyl acetate fraction was the most effective plant fraction against adult females of T. urticae after 7days of treatment, followed by butanol fraction, methylene chloride fraction and pet. ether fraction. Essential oil fraction was the least effect one. Bioassay-guided fractionation led to the isolation and purification of six compounds which were investigated as acaricides. The toxicity of the isolated compounds of E. conyzoides fractions against adult females of T. urticae were presented in table (2). Ethuliacoumarin (1) was the most effective isolated compound against larvae and adult females of T. urticae after 7- days of treatment followed by (isoethyliacoumarin A (2), isoethuliacoumarin C (3)), lupan-3-ol, 3-O-acetyl lupeol and genkwanin. Our results were in agreement with the finding by Kady et al., (1992) who reported that the molluscicidal principles of E. conyzoides were identified as ethuliacoumarin A and isoethuliacoumarin A [4]. International Journal of Science and Engineering Applications Volume 4 Issue 5, 2015, ISSN-2319-7560 (Online) Table 2. Toxicity of plant fractions and isolated compounds against larvae and adult females of T. urticae after 7- days of treatment. Plant extract Pet. ether fraction Methylene Chloride fraction Larvae LC50 (ppm) and confidence limits at 95% LC90 (ppm) and confidence limits at 95% 53.63 1225.41 20.35 93.05 76.47 50.30 105.49 5.69 17.59 4.39 38.37 3-O-acetyl lupeol lupan-3-ol Ethuliacoumarin (1) Isoethyliacoumarin A (2), Isoethuliacoumarin C (3) Genkwanin 116.70 1242.25 68.55 578.12 800.43 282.23 Essential oil fraction 304.99 130.84 21.00 Butanol fraction 7983.04 496.28 11.58 Ethyl acetate fraction 540.97 466.06 245.60 81924.59 6146.57 2315.09 11439.8E+1 Adult females Slope Toxicity index at LC50 value 0.943±0.210 21.59 1.578±0.283 15.14 1.217±0.270 100.00 0.811±0.254 55.14 0.958±0.256 4.10 1.957±0.447 6.31 1.706±0.416 9.27 0.858±0.254 100.00 201.64 100.93 285.45 137.21 60.66 201.32 12.72 3.92 22.24 910.73 628.18 1991.13 774.15 500.10 2160.80 396.98 128.10 21521.78 35.46 8.36 60.72 261.77 171.33 684.08 1.476±0.388 35.87 297.55 69.07 489.80 3714.43 1720.12 459176.86 1.169±0.370 4.27 LC50 (ppm) and confidence limits at 95% LC90 (ppm) and confidence limits at 95% 175.94 2250.52 96.40 273.84 109.73 38.45 212.01 1108.31 20981.68 9095.97 1999.50 14463.1 E+2 17.86 10.26 100.68 25.16 67.54 53.37 31.37 442.08 76.69 334.73 129.12 208.27 532.61 254.23 1373.01 3762.10 1995.07 18461.55 Slope Toxicity index at LC50 value 1.104±0.259 10.15 0.668±0.189 16.28 1.707±0.320 100.00 1.396±0.276 33.46 1.220±0.298 5.34 1.312±0.275 5.51 1.354±0.286 10.55 0.776±0.249 100.00 348.61 184.21 516.21 182.19 87.43 274.70 19.22 7.20 37.31 3307.25 1848.00 11578.63 1609.07 951.62 4858.05 863.57 200.97 39855.5E+1 66.64 92.41 452.89 271.15 1209.23 1.540±0.279 28.84 362.79 195.92 573.89 5745.07 2378.06 57914.33 1.068±0.257 5.30 44.21 International Journal of Science and Engineering Applications Volume 4 Issue 5, 2015, ISSN-2319-7560 (Online) 4. 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