Chemical composition and insecticidal property of Myrsine stolonifera (Koidz.) walker (Family: Myrsinaceae) on Musca domestica (Diptera: Muscidae)

Accepted Manuscript Title: Chemical composition and insecticidal property of Myrsine stolonifera (Koidz.) Walker (Family: Myrsinaceae) on Musca domestica (Diptera: Muscidae) Authors: Xue Gui Wang, Qian Li, Su Rong Jiang, Pei Li, Ji Zhi Yang PII: DOI: Reference:

S0001-706X(16)30554-X http://dx.doi.org/doi:10.1016/j.actatropica.2017.02.026 ACTROP 4219

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Acta Tropica

Received date: Revised date: Accepted date:

30-7-2016 5-2-2017 16-2-2017

Please cite this article as: Wang, Xue Gui, Li, Qian, Jiang, Su Rong, Li, Pei, Yang, Ji Zhi, Chemical composition and insecticidal property of Myrsine stolonifera (Koidz.) Walker (Family: Myrsinaceae) on Musca domestica (Diptera: Muscidae).Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.02.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chemical composition and insecticidal property of Myrsine stolonifera (Koidz.) Walker (Family: Myrsinaceae) on Musca domestica (Diptera: Muscidae)

Xue Gui Wang1* ,Qian Li 1, Su Rong Jiang, Pei Li Li, Ji Zhi Yang Biorational Pesticide Research Lab, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, People's Republic of China

* Corresponding author, XG Wang ;E-mail: [email protected]. 1

These authors contributed equally to the work.

Abstract Musca domestica is one of the most important pests of human health, and has developed strong resistance to many chemicals used for its control. One important approach for creating new pesticides is the exploration of novel compounds from plants. During a wide screening of plants with insecticidal properties that grow in southern China, we found that the methanolic extracts of Myrsine stolonifera had insecticidal activity against the adults of M. domestica. However, the insecticidal constituents and mechanisms of the M. stolonifera extracts remain unclear. The insecticidal components of the methanolic extracts of M. stolonifera were isolated with activity-guided fractionation. From the spectra of nuclear magnetic resonance (NMR) and mass spectrometry (MS), the compounds were identified as syringing (1), 2,6-dimethoxy-4hydroxyphenol-1-O-β-D-glu (2) , kaempferol-3-O-glu-rha-glu (3), and quercetin-3-O-glu-rha-glu (4). This study is the first to report the spectral data for compounds 3 and 4, and their LC50 values were 0.52 mg/g sugar and 0.36 mg/g sugar 24 h after treatment of the adults of M. domestica, respectively. Compounds 3 and 4 (LC25) also inhibited the activities of the enzymes carboxylesterase, glutathione S-transferase, mixed function oxidase, and acetylcholine esterase of adult M. domestica, particularly mixed function oxidase and acetylcholine esterase. The cytotoxic effects of compounds 3 and 4 on cell proliferation, mitochondrial membrane potentials (MMP) and

reactive oxygen species (ROS) were demonstrated on SL-1 cells. From the extracts of M. stolonifera, quercetin-3-O-glu-rha-glu and kaempferol-3-O-glu-rha-glu have displayed comparable toxicities to rotenone on M. domestica and also exhibited cytotoxic effects on SL-1 cells; therefore, the extracts of M. stolonifera and their compounds have potential as botanical insecticides to control M. domestica. Keywords:

Myrsine

stolonifera;

Musca

domestica;

Activity-guided

fractionation;

Kaempferol-3-O-glu- rha-glu; Quercetin-3-O-glu-rha-glu; Cytotoxic effect

1. Introduction Musca domestica L. (Diptera: Muscidae) is an important global health pest that carries pathogenic bacteria, including Shigella sp., Vibrio cholerae, Escherichia coli, Staphylococcus aureus, and Salmonella sp. and disseminates many infectious diseases, including typhoid fever, cholera, and bacillary dysentery cholera, among others (Mansour et al., 2011; Kumar et al., 2014). For most of the chemical insecticides applied to control M. domestica, include pyrethroids, organophosphates, and carbamates (Scott et al., 2009; Khan et al., 2013; Abbas et al., 2014; Højland et al., 2014), the housefly has developed resistance because of high selection pressure. Therefore, the task is urgent to develop effective, low toxic insecticides with minimal environmental issues (i.e., residues) to control the housefly. Natural compounds and their derivatives, which are often less toxic, biodegrade more easily, have shorter intervals between applications are more difficult to develop resistance, and exhibit a variety of biological effects as botanical insecticides (Jun et al., 2007; Ruiu et al., 2008). From some of the early reports, many crude extracts and compounds isolated from plants have strong insecticidal effects on the larvae and adults of M. domestica. Wang et al. (2011) isolated and identified four active compounds from Ficus sarmentosa var. henryi and found that 7-hydroxyl-coumarin has strong insecticidal activities with a lethal concentration (LC50) value of 72.13 μg/g sugar for adult of M. domestica at 48 h after treatment. Mansour et al. (2011) found that the larvicidal LC50 values for M. domestica are < 100 μg/mL for Piper nigrum (50.1 μg/mL), Azadirachta indica (76.9 μg/mL), Conyza aegyptiaca (77.0 μg/mL) and Cichorium intybus (96.8 μg/mL). Contact and fumigant activities of Eucalyptus globules oil to the larvae and pupae of M. domestica were also demonstrated (Kumar et al., 2012). Pavela (2013) found that plumbagin

(purchased from the chemical company Sigma–Aldrich in the Czech Republic) is sufficiently acutely toxic and sublethal doses cause significant reductions in the longevity, fecundity, and fertility of the housefly. Thus, for alternatives to chemical pesticides, the screening and isolation of compounds with insecticidal properties from plants is one of the areas of focus for new pesticide development. Myrsine stolonifera (Koidz.) Walker (Family: Myrsinaceae) (Zhang and Wu, 1979) is a shrub widely distributed in south-western China, including in the provinces of Sichuan, Yunnan, and Guizhou. The shrub provides a traditional Chinese medicine used to reduce fever and promote diuresis. During the wide screening for plants with insecticidal compounds in southern China, we found that the methanolic extract of M. stolonifera has insecticidal activities against the adults of M. domestica (Wang et al., 2010), while we are not aware of previous reports on the insecticidal constituents of M. stolonifera. Therefore, the objective of the present study was to isolate these compounds, to identify their chemical structures, and to determine the insecticidal activities on M. domestica and cytotoxicity to SL-1 cells. 2. Material and methods 2.1. Plant materials The roots, stems, and leaves of M. stolonifera were collected from the Jin fo Mountain Natural Reserve, Chongqing Municipality, China, in September 2013. An authenticated voucher specimen (No. CN 20) is identified by associate Prof. Hu Chao, Sichuan Agricultural University (major: Plant taxonomy) and deposited at our laboratory. 2.2. Chemicals The chemicals used in the study, including the reduced form of triphosphopyridine nucleotide (NADPH), phenyl methane sulfonyl fluoride (PMSF), reduced glutathione (GSH), 1-chloro-2, 4-dinitrobenzene (CDNB), p-nitrophenol, p-nitroanisole (ρ-NA), and eserine (product No.: E8375) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China. Ethylene diaminetetraacetic acid (EDTA) was acquired from Sinapharm Chemical Reagent Co. Ltd., Shanghai, China. Bovine serum albumin (BSA), alpha-naphthyl acetate (α-NA), acetylthiocholine iodide (ATCI), fast blue B salt, and 95% technical rotenone, a commercial botanical insecticide from Derris trifoliata Lour., were obtained from Beijing Solarbio Science and Technology Co. Ltd.,

Beijing, China. 2.3. Insect rearing The larvae of M. domestica were reared with a mixed feed contained wheat bran plus yeast plus milk powder plus water with the weight proportions of 250: 3.2: 8: 500. Adults were feed by sugar and water, and laid egg in feed of larvae. The insects were fed at 25-28 °C. When the adults of M. domestica appeared, they were kept in culture for 48 h before use (Wang et al., 2011). 2.4. Insect line and cell culture The culture of the SL-1 cell line was derived from ovarian cells of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), which was obtained from the Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, Laboratory of Insect Toxicology, South China Agricultural University (Guangzhou, China). The culture of cell line is followed the methods of Wen et al. (2013) and Liu et al. (2015). 2.5. Extraction, isolation and identification of insecticidal ingredients Bioactivity-guided fractionation was held to explore the insecticidal active ingredient to M. domestica adults as target insects (Wang et al., 2011; Mansour et al., 2014). The air-dried roots (8.3 kg), stems (10 kg), and leaves (7.5 kg) of M. stolonifera were ground and extracted three times with methanol (50 L each) at room temperature, followed by combining and concentrating at 55 °C in vacuo by a circulating water vacuum pump to obtain the methanol extract of 1328g, 1680g and 1250g, respectively. The methanol extracts of roots, stems, and leaves were assayed for insecticidal activity with the stomach poisoning method (Wang et al., 2011). The root- and stem-methanol extracts were combined as rhizomes because of the similar constituents. The leaf methanol extract and rhizomes were subjected to D101 macroporous adsorption resin column chromatography (CC) eluted with ethyl alcohol-water mixtures of increasing polarity (95:5 to 5:95 by volume) to separate 5 fractions: 30%, 50%, 70%, 95% ethanol, and water-soluble fractions, respectively. These fractions were assayed for insecticidal activities against M. domestica, and the 95% ethanol fraction of the rhizomes exhibited better activity. The stronger active fractions were subjected to silica gel column chromatography eluted with chloroform-methanol mixtures of increasing polarity (95:5 to 60:40 by volume) to separate 7 fractions, F1-F7. These fractions were assayed for insecticidal activity against M. domestica, and F1, F4, and F7 fractions exhibited activity. The F1 fraction (12.0 g) was further

separated by silica gel CC using chloroform-methanol (95:5), followed by Sephadex LH-20 CC using methanol to produce compound 1 (10.0 mg). The F4 fraction (17.6 g) was further separated by silica gel CC using chloroform-methanol (92:8), followed by Sephadex LH-20 CC using methanol to yield compound 2 (4.01 mg). The F7 fraction (23.4 g) was further separated by chloroform-methanol (4:1) and Sephadex LH-20 CC using methanol to separate compounds 3 (29.54 mg) and 4 (23.09 mg). The chemical ingredients from fractions isolated with bioactivity-guided fractionation were identified by the interpretation of the spectroscopic data (1H-NMR,

13

C-NMR and MS) and by a

comparison with the data reported by others. The 1H (500 MHz), 13C (125 MHz), and 2D NMR spectra were recorded on a Bruker DRX-500 instrument using TMS as the internal standard. A Thermo Fisher LCQ Fleet mass spectrometer was used to collect ESI-MS. HR-ESI-MS was conducted on a Bruker Bio-TOF-IIIQ mass spectrometer. 2.6. Insecticidal activities of extractions, fractions and compounds 1-4 against M. domestica 0.05g root-, stem-, and leaf methanol fractions were accurately weighted into 10 mL marked test tube and diluted with acetone for the 10 mg/mL concentration. 1 g sucrose were accurately weighted into a finger tube (25 mm × 75 mm), followed by moving 1 mL solution (10 mg/mL) into the tube for a toxic mixing of 5 mg/g sugar, with only acetone as a negative control. Twenty M. domestica imagos were taken into each finger tube as a replication and three replications were set for each treatment after acetone vaporized absolutely. The mortality and the corrected mortality were determined at 24, 36, and 48 h after treatment. The fractions from the rhizomes and leaves eluted with D101 macroporous adsorption resin CC by 30%, 50%, 70%, 95% ethanol, and water (Table 2) and the F1-F7 fractions (Table 3) from 95% ethanol fraction of the rhizomes (100% corrected mortality at 48 h after treatment) , and finally compounds 1-4 (Table 5) were also assayed with the same method, while the concentrates of 30%-, 50%-, 70%-, 95%-, water-soluble fractions (2 mg/g sugar), F1-F7 (2 mg/g sugar) and compounds 1-4 (0.5 mg/g sugar) were different. The toxicities of the two stronger insecticidal activities from compounds 1-4 were assayed with same method. A series of concentrations of 1000, 500, 250, 125, and 62.5 μg/g sugar was used to assay the stronger insecticidal compounds against M. domestica adults, while rotenone was set for the positive control (Delaney and Wilkins, 1995), with the concentrations of 500, 250, 125, 62.5 and 31.25 μg/g sugar. The mortality and corrected mortality were determined at 12, 24, 36, 48 h

after treatment. Each treatment had three replications. The corrected mortalities were converted into probit values, and the concentrations were switched common logarithm, and finally established the toxic regressive equations between probit values (Y) and common logarithm(X). The LC50 and LC95 (lethal concentrations) values, with 95% confidence intervals (CIs), the slopes of the regressions and the chi-square tests of the toxicity bioassays at 12, 24, 36 48 h after treatment were calculated with POLO2.0 (Leora Software, www.leorasoftware. com) (Wang et al., 2015). 2.7. Preparation of samples and enzyme assays Six hundred adults of M. domestica were treated with the LC25 concentration at 24 h of the two stronger insecticidal compounds, with rotenone and acetone acted as positive and negative controls, respectively. Each assay had three replications and each replication was fifty adults. Randomly screened out 5 active adults for each replication and sampled in each enzyme assay after 12 and 24 h treatment, respectively. Mixed function oxidase (MFO) was determined from the internal organs of adults (Hansen and Hodgson, 1971), acetylcholine esterase (AChE) from the heads of adults (Gorun et al., 1978), and carboxylesterase (CarE) and glutathione S-transferase (GSTs) from the whole bodies of adults (Habig, 1981; Zhang et al., 2007). In each independent replication, 5 adults were homogenized with 1 mL of homogenization buffer (0.1 mol/L sodium phosphate buffer, pH 7.0 for the CarE assay; 0.1 mol/L sodium phosphate buffer, pH 6.5 for the GSTs assay; 0.1 mol/L sodium phosphate buffer, pH 7.8, containing 0.1 mmol/L EDTA, 0.1 mmol/L DTT, and 0.1 mmol/L PMSF, for the MFO assay; and 0.1 mol/L sodium phosphate buffer, pH 7.5 for the AChE assay). The homogenate was centrifuged at 12,000 rpm for 15 min at 4°C, and the clear supernatant was collected and used as the enzyme source for the analyses of the activity of the enzymes. All enzyme assays were held on UV 2000-Spectrophotometer (Unic [Shang Hai] Instrument Co., LTD). The CarE activity was determined with two naphthyl-substituted substrates as described by Zhang et al. (2007). For each reaction, 1.8 mL of phosphate buffer (0.04 mol/L, pH 7.0) that contained the final concentrations of 3 × 10-4 mol/L substrate and 3 × 10-4 mol/L eserine with 0.5 mL of enzyme was incubated at 30 °C for 15 min. To stop the reaction, 0.9 mL of fast blue B salt solution was added (mixture of 1% fast blue B salt and 5 parts 5% SDS). The optical density (OD) was displayed with changes of absorbance at 600 nm for the production of α-naphthol. The method described by Habig (1981) was adapted to determine the activity of GSTs using CDNB as the substrate. The reaction solution contained 790 μL of sodium phosphate buffer (0.1

mol/L, pH 6.5), 30 μL of CDNB (15 mmol/L), 30 μL of GSH (30 mmol/L), and 50 μL of enzyme stock solution. The optical density at 340 nm was recorded at intervals of 10 s for 2 min at 27 °C. The activity of MFO was assayed using β-NA as the substrate (Hansen and Hodgson, 1971). The reaction solution contained 1 mL of enzyme stock solution, 1 mL of β-NA (4 mmol/L), 0.2 mL of NADPH (0.5 mmol/L), and 0.8 mL of sodium phosphate buffer (0.1 mol/L, pH 7.8). The incubation was conducted in a water-bath (37°C, 30 min), and then, 1 mL of HCl (1 mol/L) was added. The OD values were recorded at 400 nm. The activity of AChE was determined using a method by Gorun et al. (1978) with slight modifications. The reaction solution contained 0.1 mL of sodium phosphate buffer (0.1 mol/L, pH7.5), 50 μL of ATCI (0.75 mmol/L), and 50 μL of enzyme stock solution. The incubation was conducted in a water bath (30 °C, 15 min), and then, 1.8 mL of DTNB-phosphoric acid-ethanol was added. The OD values were recorded at 412 nm at intervals of 10 s for 2 min. 2.8. Cytotoxicity against the SL-1 cell line The inhibition of cell proliferation was detected with the MTT assay following the descriptions of Mosmann (1983) and the half maximal inhibitory concentrations (IC50) was got though the regression equations between the inhibitions of cell proliferation and a series of concentrations of tested compounds (Wen et al., 2013). The effects of the two stronger insecticidal compounds with IC50 values on the reactive oxygen species (ROS), and cell mitochondrial membrane potentials (MMP) (ΔΨm) of SL-1 cells were measured using the DCFHDA method (Michal et al., 2004; Wang et al., 2011) and Rhodamine-123 as the dye, following the methods of Han et al. (2009) and Wang et al. (2011), respectively. 2.9. Statistical analyses The data obtained from the enzyme assays for AChE, GSTs, CarE, and MFO were compared using analysis of variance (Duncan’s multiple range test were used for the multiple comparisons, P<0.05) with the SPSS v.17.0 statistical software package (IBM, www.ibm.com) for the Microsoft Windows 7 operating system (www.microsoft. com). 3. Results and discussion 3.1. Insecticidal activities of methanol extracts, ethanol fractions and fractions F1-F7 from the 95% ethanol fraction of the rhizomes against M. domestica adults

The insecticidal activities of root, stem, and leaf methanol extracts (5 mg/g sugar) against the adults of M. domestica are provided in Table 1. The methanol extracts from roots and stems had the highest insecticidal activity and caused 48.15% and 55.19% corrected mortality 48 h after treatment, respectively, followed by the leaf extract (38.15%). The effect on mortality was significant for the stem extract (P<0.001). The insecticidal activities of the fractions (2 mg/g sugar) eluted by 100% water, 30%, 50%, 70%, and 95% ethanol from the methanolic extracts of leaves and the rhizomes were tested. The 95% ethanol fraction of the rhizomes had the strongest activity against M. domestica adults (100% corrected mortality) 48 h after treatment, which was followed by the 30% ethanol, 70% ethanol and water extract fractions (92.59%, 88.89% and 81.48% corrected mortality, respectively). Although the insecticidal activity of the fractions from the leaf methanol extracts was < 75% corrected mortality, the effects of both leaf and rhizome extracts on mortality were significant (P<0.05; Table 2). The insecticidal activities of the fractions 1-7 (2 mg/g sugar) from the 95% ethanol fraction of rhizome are shown in Table 3. The strongest activity was exhibited by Fr. 7 (100% corrected mortality), followed by Fr. 1 (89.63%), Fr. 4 (72.22%), and Fr. 5 (68.89%), and these corrected mortalities were significantly higher than those of the other treatments (P<0.01) 48 h after treatment. 3.2. Spectroscopic analyses of the active compounds Four known compounds (Fig. 1), syringin (1) (Wu et al., 1999), 2, 6-dimethoxy-4hydroxyphenol-1-O-β-D-glucopyranoside (2) (Saijo et al., 1989; Ishirnaru et al., 1990), kaempferol 3-O-glu-rha-glu (3), and quercetin-3-O-glu-rha-glu (4) were isolated with bioactivity-guided fractionation. Based on the search of SciFinder, compounds 1-4 were the first time to be isolated from M. stolonifera. Moreover, the spectral data for kaempferol-3-O-glu-rha-glu (3) and quercetin3-O-glu-rha-glu (4) are first reported in this study. MS and 1H-NMR and 13C-NMR data for the four compounds are as follows. Compound 1: ESI-MS m/z: 395.1[M+Na]+,407.9 [M+Cl]—; 1H-NMR (500 MHz, CD3OD) δ: 6.77 (2H, s, H-3, 5), 6.57 (1H, d, J = 15.9 Hz, H-7), 6.35 (1H, dt, J = 15.9,5.6 Hz, H-8), 4.91 (1H, d, J = 7.6 Hz, H-1'),4.24 (2H, dd, J = 5.5,1.0 Hz, H-9) 3.88 (6H, s, 2,6-OCH3), and 3.80-3.25 (sugar moiety).

13

C-NMR (125 MHz, CD3OD) δ: 154.2 (C-2, 6), 135.6 (C-1), 135.1 (C-4), 131.1 (C-8),

130.0 (C-7),105.4 (C-3, 5), 105.2 (C-1'), 78.3 (C-3'), 77.6 (C-5'), 75.7 (C- 2'), 71.3 (C-4'), 63.5 (C-6'), 62.5 (C-9), and 57.1 (2,6-OCH3). Compound 2: ESI-MS m/z: 331.1 [M-H ]—, 355.1 [M+Na]+; 1H-NMR (500 MHz, C5D5N) δ: 11.37 (1H, s, 4-OH), 6.56 (2H, s, H-3, 5), 5.60 (1H, d, J = 6.9 Hz, H-1'), 4.42 (1H, dd, J = 11.5, 2.2Hz,H-6'a), 4.35 (1H, brd, J = 4.8Hz,H-6'b), 4.32-4.34 (3H, m, H-2',H-3',H-4'), 3.92 (1H, m, H-5'), and 3.71 (6H, s, OCH3).13C-NMR (125 MHz, C5D5N) δ: 155.7 (C-4), 154.3 (C-2, 6), 129.2 (C-1), 105.6 (C-1'), 94.9 (C-3, 5), 78.3 (C-5'), 78.1 (C-3'), 75.9 (C-2'), 71.4 (C-4'), 62.5 (C-6'), and 56.2 (2,6-OCH3). Compound 3: ESI-MS m/z: 779.3 [M+Na]+,755.4[M-H ]—,791.4 [M+Cl]—;1H-NMR (500 MHz, CD3OD) and13C-NMR (125 MHz, CD3OD)data are displayed in Table 4. Compound 4: ESI-MS m/z: 795.2 [M+Na]+, 771.5[M-H ]—; 1H-NMR (500 MHz, CD3OD) and13C-NMR (125 MHz, CD3OD) data are displayed in Table 4. 3.3. Insecticidal activities of compounds 1-4 against M. domestica adult The insecticidal activities of compounds 1-4 (500 µg/mg sugar) against M. domestica adults are listed in Table 5. The activity of compound 4 was the strongest during the experiment period and reached 92.86% corrected mortality at 48 h after treatment, which was equalled to that of rotenone (100%) (P<0.05), followed by compound 3 (82.14%), compound 1 (64.29%) and compound 2 (60.72%). The toxicities of two stronger activities compounds 3 and 4 on the M. domestica adults are displayed in Table 6. Based on the LC50 values, the toxicity of rotenone was slightly stronger than that of compound 4 (confidence intervals overlapped), but was significant with that of compound 3 (confidence intervals did not overlap) at 12 h after treatment. From 24 to 36 h, the toxicities of rotenone increased rapidly and were significantly different from those of the two tested compounds (confidence intervals did not overlap). The toxicity of compound 4 was equal to that of rotenone (confidence intervals overlapped) and significant with that of compound 3 (confidence intervals did not overlap) at 48 h after treatment. Cis et al. (2006) isolated syringin from Rhaponticum pulchrum and found significant anti-feeding activities against the larvae of Sitophilus granaries L. (Coleoptera: Curculionidae) and Tribolium confusum Jac.du Val. (Coleoptera: Tenebrionoidea). This study is the first to report the insecticidal activity of compounds 1-4 on M. domestica, and the stomach insecticidal activity of compound 4 on adult M. domestica was very strong, even if the

effects are slightly weaker than those of rotenone (P<0.05). 3.5. Inhibition of enzyme activities The CarE activities of M. domestica adults were inhibited slightly by compounds 3, 4 and not significantly different from that of the negative control at 12 h after treatment (0.96 μmol/mg protein. 30 min; P>0.05). Nevertheless, the inhibition by rotenone (0.34 μmol/mg protein.30 min), was clearly stronger than by the negative control. The inhibition of CarE activity by compounds 4 and rotenone increased to 0.57 and 0.17 μmol/mg protein.30 min, respectively, 24 h post-treatment, and were significantly higher than that by the negative control (P<0.05; Fig. 2A). The inhibition of GSTs activity by rotenone was the highest (0.57 OD/mg protein.min) 12 h after treatment, followed by the inhibitions by compounds 3 and 4, which were not significant with that by the negative control (0.93 OD/mg protein.min) (P>0.05). Almost all of treatments after 24 h, including those with compounds 3 and 4, the inhibition of GSTs activity decreased and was not significantly different from that of negative control (1.01 OD/mg protein.min; P>0.05), whereas the inhibition by rotenone increased to 0.45 OD/mg protein.min and was significantly different from those of the other treatments (P<0.05; Fig. 2B). The activities of MFO in M. domestica adults decreased significantly with compounds 3, 4 and the rotenone, and the activities were significantly different from those of the negative control (P< 0.05) from 12 to 24 h (Fig. 2C). Cytochrome P450 monooxygenases (P450s) is the key element of MFO system, which has been proved to be related to detoxification and is functionally associated with insect resistance to pesticides, metabolism of insecticides in insects, and so on (Silva et al., 2012). Based on our results, compounds 3 and 4 have showed good inhibition on the MFO activity in M. domestica adults. Whether the tested two compounds did inhibit the P450s activities of M. domestica or not is still unclear. It’s necessary to hold a longer experimental time to detect the MFO activity and detoxification metabolism for compounds 3 and 4 on in our further investigation. AChE is a key enzyme that ensures the normal conduction of nerve impulses at the synapse, which is a well-known target of organophosphates and carbamates, and some natural insecticidal compounds from plants also inhibit AChE activity and cause the death of insects (Yu, 2008). Based on our results, rotenone showed the strongest inhibition of AChE activity (5.14 OD/mg protein.min), which was significantly different from compound 4 (P<0.05) but not from compound 3 (P>0.05). The inhibition of AChE activity was only slightly decreased by the rotenone and compounds 3, 4 at

24 h after treatment, but the effect of rotenone (6.25 OD/mg protein.min) still remained stronger than those of other treatments (P<0.05; Fig. 2D). Anderson and Coats (2012) reported that carvacrol and nootkatone, two terpenoid compounds extracted from the heartwood of Chamaecyparis nootkatensis (D.Don) Spach, both are toxic against M. domestica, Aedes aegypti, Dermacentor variabilis, Periplaneta americana. Carvacrol was observed to cause slight inhibition of the AChE in M. domestica, D. variabilis and P. americana, but it did not inhibit the AChE of mosquito. Nootkatone did not inhibit the AChE in any of the four arthropod models tested. López et al. (2015) reported an increase in AChE activity by approximately 15-35% following treatment with 40 μM geraniol, camphor, γ-terpinene, and linalool, whereas the activity decreased (60–40%) with 5 mM carvone, γ-terpinene, and fenchone. In our study, the AChE activity of M. domestica adult was clearly inhibited by compounds 3 and 4, with similar neurotoxic symptoms, including overexcitation, a continuous vibration of the wings, progressive convulsions and ultimately death. We guessed that the two compounds had their primary effect on the transference of nerve impulses and the effects of the two compounds on the electrophysiology of M. domestica adult were worthy of further research. 3.6. Cytotoxicity against the SL-1 cell line Currently, the cytotoxic effects of compounds are extensive investigation in the development of new pesticides (Choi et al., 2008; Ikeda et al., 2009). Wang et al. (2011) found strong inhibitory activity of quercetin, isolated from F. sarmentosa var. henryi, against BTI-Tn-5B1-4 cells with the MTT assay and reactive oxygen species (ROS). According to our results (Fig. 3A), rotenone, the positive control, had the strongest inhibitory activity against cell proliferation of the SL-1 cell line (42.83% inhibition rate), which was significantly different from those of compound 3 (16.90% inhibition rate) and compound 4 (16.12% inhibition rate) (P<0.05), whereas the effects of the two tested compounds were not significantly different from one another (P>0.05). The effect of rotenone (28.32 Rhodamine-123 fluorescent intensity values) on the MMP of the SL-1 cell was the strongest and was significantly different from those of compound 3 (24.48) and compound 4 (23.20); the negative control (14.69) was the lowest and significant with other treatments (Fig. 3B). Meanwhile, compounds 3 and 4 displayed strong effects on the ROS (23.99 and 22.91 DFC fluorescent intensity values, respectively), although the effects were less than that of rotenone (27.04; P<0.05). The effect of the negative control on the ROS (14.43) was the weakest and was

significantly different from those of the other treatments (P<0.05; Fig. 3C). The insecticide cytotoxicology, as a new branch of toxicology, has rapidly developed in recent years. Many important models, including S2, BmN, SL-1, Sf21 and Sf9 cell lines have been introduced to research the mechanisms of necrosis, apoptosis or autophagy induced by synthetic or biogenic pesticides and virus infections (Zhong et al., 2016). Huang et al. (2011) found that the proliferation of Sl-1 cells was clearly inhibited after 48 h of treatment with azadirachtin A. The up-regulation of p53 was also observed before apoptosis and cell cycle arrest occurred. Shao et al. (2016) reported that azadirachtin A significantly inhibits cell proliferation by inducing autophagic and apoptotic cell death in S. litura cultured cell line (SL-1 cell). Their findings also indicated that azadirachtin induced apoptosis through autophagy. 3β-O-(6'-O-methyl-β-D-glucuronopyranosyl) oleana-11, 13(18)-dien-28-oic acid, one novel triterpenoid glycoside isolated from the stem bark of Aralia armata (Wall.) Seem was demonstrated the more obvious proliferation inhibition activities on Sl-1 cell than the positive-control rotenone. 3β-hydroxyoleana-11, 13(18)-diene-28, 30-dioic acid (1) and 3-oxooleana-11,13(18)-diene-28, 30-dioic acid, other two new triterpenoids from A. armata, also displayed the effects on morphology of Sl-1 cell and resulted in cell blebbing and vacuole forming (Miao et al., 2016). Other similar effects of plant secondary metabolites on SL-1, such as momordicin I and II, also have been demonstrated (Liu et al., 2015). In our research, the inhibition rates of cell proliferation for compounds 3 and 4 were significantly lower than that of rotenone (P<0.05), but the two compounds displayed significant effects on the MMP (ΔΨm) and the ROS of the SL-1 cells, even rough the effects were slightly lower than those of rotenone. 4. Conclusion The present results indicate that the extractions, fractions, compounds from M. stolonifera had strong insecticidal activities. Meanwhile, the active compounds 3 and 4 also displayed the strong inhibition activities on the metabolism enzymes of MFO and the key enzymes of normal conduction of nerve impulses, AChE. The cytotoxicities of the two compounds against the SL-1 were also demonstrated. Therefore, the compounds extracted and isolated from M. stolonifera had insecticidal activities and might be useful in the effective management of M. domestica. Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (31101493).

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HO OH

H3CO

H3CO

OCH3

HO

O

O

HO

O

HO HO

OCH3 O

HO HO

OH

2,6-dimethoxy-4-hydroxyphenol-1-O-β-D-glu

OH

Compound 2

syringin Compound 1

OH

OH

OH

HO

O HO

O OH O

O O

O O

O HO

HO

O

OH OH

OH

O OH

O

HO

O

OH

O

O O

HO

OH OH OH

kaempferol-3-O-glu-rha-glu

quercetin-3-O-glu-rha-glu

Compound 3

Compound 4 Fig 1. Chemical structures of compounds 1-4

OH OH

OH O

OH OH

HO

HO

A

1.0

a ab

.8

ab

ab

b .6 b

.4

c .2 0.0 2.0

C

a 1.5 a 1.0 b .5

b

b

b

b

b

0.0 12

GSTs activity (mOD.mg protein-1.min-1)

a

AChE activity (mOD.mg protein-1.min-1)

CarE activity (mol.mg protein-1.30min-1) MFO activity (mol.mg protein-1.30min-1)

1.2

1.4

B

a

1.2 a

a

1.0 ab

a

ab

.8

b

.6

b

.4 .2 0.0

20

D

a a

15 b 10

b

bc

b c

c 5

0 12

24

24

Treatment time (h) negative control kaempferol-3-O-glu-rha-glu quercetin-3-O-glu-rha-glu rotenone

Fig 2. Effects of kaempferol-3-O-glu-rha-gl and quercetin-3-O-glu-rha-glu on the activities of the enzymes

CarE(A), GSTs (B), MFO (C), and AChE (D) of adults of M. domestica LC25 values of kaempferol-3-O-glu-rha-gl, quercetin-3-O-glu-rha-glu and rotenone were 0.123 mg/g sugar, 0.089 mg/g sugar, 0.029 mg/g. sugar at 24h after treatment according to the results of Table 6, respectively. CarE, GSTs, MFO, and AChE activities were determined from three replications ± SE. Different letters on the columns indicate significant differences from ANOVA, followed by Duncan’s Multiple Comparison tests (P<0.05). The F values of CarE, GSTs, MFO, and AChE activities at 12 h and 24 h were 56.83, 21.34, 57.46, 42.44 and 42.94, 29.85, 38.95, 36.52, respectively.

30 20

b

b

10 0 Compd 3 Compd 4 Rotenone

35 30

35 B b

25

a b

20 15

c

10 5

DCF fluorescent intensity

40

a

A

Rhodamine123 fluorescent intensity

Inhibition rate(%)

50

30

C

a b

25

c

20 15

d

10 5

0 NC

Compd 3Compd 4Rotenone

0 NC

Compd 3Compd 4Rotenone

Fig 3. Effects of compounds 3 and 4 on the inhibition rate (A), mitochondrial membrane potential (B), and

reactive oxygen species (C) of SL-1 cells. The inhibition rate and the fluorescent intensities of Rhodamine-123 and DCF were the means of three replications ± SE. The columns with a different letter are significantly different at the 5% level (Duncan’s; P<0.05). The concentrations of kaempferol-3-O-glu-rha-gl, quercetin-3-O-glu-rha-glu and rotenone in the determination of the inhibition rate were 20 µg/mL, and for the mitochondrial membrane potential and reactive oxygen species, the concentrations were the IC50 values of 65.58 µg/mL, 62.08 µg/mL and 26.85 µg/mLfor kaempferol-3-O-glu-rha-gl, quercetin-3-O-glu-rha-glu and rotenone, respectively. The negative control was 1% DMSO. The abbreviations of NC, Compd 3, and Compd 4 stand for negative control, kaempferol-3-O-glu-rha-gl, and quercetin-3-O-glu-rha-glu, respectively. The F values of the inhibition rate, fluorescent intensities of Rhodamine-123 and DCF were 620.16, 282.85 and 160.78, respectively.

Table 1 Insecticidal activities of the methanol extracts against adult M. domestica Corrected mortality（%）(±SE) Methanol extract

N 24 h

36 h

48 h

Root

30

0.00±0.00 a1)

24.07±3.03 b

48.15±2.19 ab

Stem

30

10.00±2.15 a

44.81±2.89 a

55.19±2.67 a

Leave

30

0.00±0.00 a

17.41±2.37 b

38.15±1.45 b

30

0a

0c

0c

F3,8=1

F3,8=13.95

F3,8=38.18

P=0.441

P<0.01

P<0.001

Negative control

Data are expressed as the means ± SEs of three replicates. Mean values with different letters within the same column are significantly different (P< 0.05; ANOVA), which also applies to the following tables. The mortalities of the negative control were 0.0%, 3.33%, and 3.33% at 24, 36, and 48 h after treatment, respectively.

Table 2 Insecticidal activities of the ethanol fractions isolated from the different methanol extractions against M. domestica Corrected mortality（%）(±SE) Fraction

Rhizomes

Leaves

N 12 h

24 h

36 h

48 h

100%water

30

0.00±0.00 b

28.55±1.77 bc

64.27±3.67 a

81.48±4.61 ab

30% ethanol

30

3.33±0.33 b

53.55±3.09 ab

85.71±4.78 a

92.59±5.19 ab

50% ethanol

30

23.33±1.41 a

46.41±2.59 ab

71.42±4.18 a

77.78±4.56 ab

70% ethanol

30

20.00±1.69 a

57.13±3.30 a

82.14±4.64 a

88.89±4.98 ab

95% ethanol

30

23.33±1.64 a

57.13±3.30 a

85.71±4.78 a

100.00±0.00 a

100%water

30

0.00±0.00 b

17.83±1.94 cd

24.97±1.39 b

25.93±1.48 c

30% ethanol

30

0.00±0.00 b

3.54±0.55 d

24.97±1.73 b

40.74±2.29 c

50% ethanol

30

16.67±1.18 a

42.84±2.4 ab

67.85±3.88 a

70.37±4.00 b

70% ethanol

30

16.67±1.36 a

49.98±2.98 ab

60.70±3.39 a

72.13±3.93 b

95% ethanol

30

3.33±0.33 b

3.54±0.55 d

10.68±0.66 b

25.93±1.48 c

acetone

30

0b

0d

0b

0d

-

-

F10, 22=1.70

F10, 22=8.50

F10, 22=14.72

F10, 22=18.77

-

-

P=0.143

P<0.001

P<0.001

P<0.001

Negative control

The mortalities of the negative control were 0.0%, 6.67%, 6.67%, and 10.00% at 12, 24, 36, and 48 h after treatment, respectively.

Table 3 Insecticidal activities of the fractions 1-7 from the 95% ethanol fraction from the rhizomes against adult M. domestica Corrected mortality（%）(±SE) Fraction

N 12 h

24 h

36 h

48 h

Fr. 1

30

3.33±3.33 b

16.67±3.33 a

26.67±3.33 b

51.48±2.55 bc

Fr. 2

30

0.00±0.00 b

13.33±3.33 ab

20.00±0.00 b

24.07±3.03 e

Fr. 3

30

6.67±3.33 ab

16.67±3.33 a

26.67±3.33 b

44.44±2.56 cd

Fr. 4

30

6.67±3.33 ab

16.67±3.33 a

23.33±3.33 b

51.85±1.85 bc

Fr. 5

30

13.33±3.33 a

13.33±3.33 ab

53.33±3.33 a

55.19±2.89 b

Fr. 6

30

0.00±0.00 b

6.67±3.33 c

20.00±0.00 b

37.77±2.22 d

Fr. 7

30

6.67±3.33 ab

20.00±0.00 a

56.67±3.33 a

89.63±0.37 a

Negative control

30

0b

0c

0c

0f

F7,16=3.17

F7,16=5.12

F7,16=49.37

F7,16=69.12

P= 0.026

P=0.003

P<0.001

P<0.001

The mortalities of the negative control were 0.0%, 0.0%, 0.0%, and 3.33% at 12, 24, 36, and 48 h after treatment, respectively.

Table 4 Spectral data for kaempferol-3-O-glu-rha-glu (compound 3) and quercetin-3-O-glu-rha-glu (compound 4) Compound 3, CD3OD No.

δH (mult, J = Hz)

δC

Compound 4, CD3OD HMBC

δH (mult, J = Hz)

δC

2

159.4

159.3

3

134.8

134.9

4

179.6

179.6

5

163.0

163.0

6

6.22 (1H, d, 2.5)

10

6.22 (1H, d, 2.0)

165.8

7 8

100.0

6.42 (1H, brs)

100.0 165.8

95.0

6.42 (1H, d, 2.0)

94.9

9

158.5

158.5

10

105.7

105.7

1'

122.9

123.2

2'

8.05 (2H, s, H-2', 6')

132.4

3'

6.93 (2H, d, 7.5, H-3',5')

116.2

7.68 (1H, d, 2.0) 1'

117.9 145.9

161.5

4'

HMBC

149.8

5'

6.93

116.2

6.91 (1H, d, 8.4)

116.2

6′

8.05

132.4

7.56 (1H, dd, 2.0, 8.4)

123.3

Glu-1"

5.37 (1H, d, 7.5)

101.2

5.30 (1H, d, 7.6)

101.2

2"

3.36 (2H, m, Glu-3", 5")

77.9

3.36 (1H, m)

77.9

3"

3.77 (1H, t, 8.5)

82.0

3.80 (1H, m)

82.5

2", 4"

4"

3.29 (2H, m, Glu-4", Glu-4'")

71.4

3.33 (1H, m)

71.3

5"

5"

3.36

76.9

3.34 (1H, m)

77.0

6"

3.33 (2H, m, Glu-6"a, Glu -3'")

68.2

3.38 (1H, m)

68.2

3.82 (1H, dd, 5.5, 11.0)

3

3.81 (1H, brd, 11.0)

Glu-1'"

4.79 (1H, d, 6.5)

104.5

4.79 (1H, d, 7.5)

104.7

2'"

3.45 (1H, m)

75.4

3.44 (1H, m)

75.4

3'"

3.33

78.3

3.44 (1H, d, 3.5)

78.2

4'"

3.29

71.3

3.45 (1H, m)

71.1

5'"

3.63 (2H, t, 9.0, Glu-5", Rha-2)

77.8

3.61 (1H, m)

77.9

6'"

3.73 (1H, dd, 5.0, 12.0)

62.6

3.74 (1H, dd, 5.0, 12.0)

62.4

3.83 (1H, m)

2", 3'"

4'"

3.84 (1H, m)

Rha-1

4.50 (1H, s)

102.2

4.51 (1H, s)

102.2

2

3.63

72.3

3.62 (1H, m)

72.1

3

3.51 (1H, m)

72.3

3.52 (1H, m)

72.3

Rha-2

4

3.27 (1H, m)

73.9

3.28 (1H, t, 9.5)

73.9

Rha-3

5

3.44 (1H, m)

69.7

3.43(1H,m)

69.7

6

1.11 (3H, d, 6.0)

17.9

1.12(3H,d,6.2)

17.9

δ The data of compound 3 were determined through the HSQC spectrum and HMBC spectrum. δ The data of compound 4 were determined through the HSQC spectrum and results of compound 3.

6"

Table 5 Insecticidal activities of compounds isolated from M. stolonifera against adult M. domestica Corrected mortality（%）(±SE) Compound

N 12 h

24 h

36 h

48 h

Syringin

30

30.00±5.77 bc

43.33±3.33 c

54.19±2.97 c

64.29±3.57 c

2,6-dimethoxy-4-hydroxyphenol-1-O-β-D-glu

30

26.67±3.33 d

43.33±6.67 c

47.17±5.56 c

60.72±3.57 c

Kaempferol-3-O-glu-rha-glu

30

40.00±5.77 bc

53.33±3.33 c

68.35±5.76 b

82.14±3.57 b

Quercetin-3-O-glu-rha-glu

30

43.33±3.33 b

66.67±3.33 b

75.37±3.20 b

92.86±3.57 a

Rotenone

30

60.00±5.77 a

83.33±3.33 a

100.00±0.00 a

100.00±0.00 a

Negative control

30

0e

0d

0d

0d

F5,12=19.86

F5,12=53.63

F5,12=81.82

F5,12=153.42

P<0.001

P<0.001

P<0.001

P<0.001

The mortalities of the negative control were 0.0%, 0.0%, 3.33%, and 6.67% at 12, 24, 36, and 48 h after treatment, respectively.

Table 6 The toxicities of kaempferol-3-O-glu-rha-glu (compound 3) and quercetin-3-O-glu-rha-glu (compound 4) to adult M. domestica Compound

Kaempferol-3O-glu-rha-glu

Quercetin-3-Oglu-rha-glu

N

Treatment LC50 (mg/g sugar) LC95 (mg/g sugar) time (h) 95% CI a 95% CI a

Chi-square

P*

150

12

0.90 (0.55-2.68)

13.88 (3.97-3.57*102)

1.39±0.32

4.58(13)

<0.05

150

24

0.52 (0.37-0.90)

4.02 (1.89-17.43)

1.83±0.24

15.96(13)

<0.05

150

36

0.46 (0.30-0.98)

11.05 (3.19-257.20)

1.18±0.27

1.82(13)

<0.05

150

48

0.40 (0.31-0.63)

2.14 (1.15-7.60)

2.27±0.31

18.43(13)

<0.05

150

12

0.76 (0.45-2.49)

19.51 (4.55-113.12)

1.17±0.29

2.26(13)

< 0.05

150

24

0.36 (0.23-0.71)

10.22 (2.92-64.65)

1.13±0.26

1.67(13)

<0.01

150

36

0.19 (0.11-0.31)

5.80 (1.84-32.92)

1.10±0.27

1.72(13)

<0.05

150

48

0.11 (0.049-0.17)

3.14 (1.17-14.91)

1.12±0.27

3.74(13)

<0.05

150

12

0.33 （0.19-1.06）

10.72 （2.31-91.42）

1.09±0.28

1.35(13)

<0.01

150

24

0.12 （0.071-0.19）

3.19 (1.01-67.53)

1.14±0.27

2.57(13)

<0.01

150

36

0.048 （0.023-0.073）

1.04 (0.45-8.16)

1.23±0.28

4.03(13)

<0.05

150

48

0.033 （0.016-0.048）

0.34 (0.20-1.13)

1.61±0.34

3.46(13)

<0.05

Rotenone

a LC 50

Slope ± SE

values are significantly different when the 95% CIs do not overlap. *Goodness-of-fit test is significant at P< 0.05.