J Arthropod-Borne Dis, December 2018, 12(4): 414–420 X Cai et al.: Insecticidal and … 414 http://jad.tums.ac.ir Published Online: December 25, 2018 Original Article Insecticidal and Acetylcholinesterase Inhibition Activity of Veratrum nigrum Alkaloidal Extract against the German Cockroach (Blattella germanica) Xianghai Cai 1, Qingfeng Li 2, Lei Xiao 3, Hailiang Lu 3, Jian Tang 3, Jianbo Huang 2, *Jianzhong Yuan 2 1State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, the Chinese Academy of Sciences, China 2Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences. Shanghai, China 3Infinitus (China) Company Ltd, Guangzhou, China (Received 30 Mar 2018; accepted 13 Nov 2018) Abstract Background: Veratrum nigrum (Liliaceae) is perennial medicinal plant widely used to treat various conditions. To determine its insecticidal properties against the German cockroach (Blattella germanica), several laboratory tests were carried out. Methods: A 4kg dry sample of V. nigrum root was purchased from the medicinal material market in Yunnan Province in 2015, China. In contact toxicity tests, V. nigrum alkaloidal extract was topically applied to the abdomen of cockroaches using a micro-applicator. In vitro acetylcholinesterase (AChE) activity tests were performed using a modified Ellman method. Results: Veratrum nigrum alkaloidal extract was toxic to male adults and 4th nymphs cockroaches, with median lethal dose (LD50) values of 14.90µg/insect, 14.21µg/insect for adults and 41.45µg/insect, 39.01µg/insect for 4 th nymphs after 24h and 48h exposure, respectively. There was a significant difference between adults and nymphs in terms of tolerance to V. nigrum alkaloidal extract. There was no significant difference in mortalities at 24h and 48h, the lethal effect of V. nigrum alkaloidal extract on German cockroach was quick. AChE activity tests showed that V. nigrum alkaloidal extract had an excellent inhibitory effect on AChE: inhibition in the 4th nymphs and male adults had 50% inhibiting concentra- tion (IC50) values of 3.56mg/ml and 5.78mg/ml respectively. The inhibitory effect of AChE activity was positively cor- related with inhibitory time (0–20min), at a concentration of 1mg/ml, inhibition of nymph and adult AChE activity had 50% inhibiting time (IT50) values of 8.34min and 16.75min, respectively. Conclusion: V. nigrum may be explored as a potential natural insecticide for control of the German cockroach. Keywords: Insecticidal activity, Acetylcholinesterase inhibition, Veratrum nigrum, Medicinal plant, German cockroach Introduction The German cockroach, Blattella germanica Linnaeus is both a serious pest and a mechanical vector for bacteria and other pathogens (1–3). To date, the control of cockroaches in China mainly has relied on two types of insecticides administered as sprays or toxic baits based on synthetic insecticides. Toxic baits may be more effective than spray formulations against Ger- man cockroaches (4). Synthetic insecticides are an effective tool to manage pests but are asso- ciated with negative effects such as insecticide resistance, environmental pollution and human health problems. Over 500 insect species had developed resistance (5). The resistance of the German cockroach to different insecticides is also very serious (6, 7). Therefore, it is essential to develop a new type of environmentally-friend- ly insecticide. Plant extracts are undoubtedly an ideal candidate due to their low mammalian tox- icity, readily biodegradable and low risk to the environment (8). Many medicinal plant extracts have been re- ported to show insecticidal properties against public health pests, such as mosquitoes (9, 10), *Corresponding author: Dr Jianzhong Yuan, E-mail: yuangang188@126.com mailto:yuangang188@126.com J Arthropod-Borne Dis, December 2018, 12(4): 414–420 X Cai et al.: Insecticidal and … 415 http://jad.tums.ac.ir Published Online: December 25, 2018 houseflies (11, 12) and German cockroaches (B. germanica) (13, 14). One such example is Vera- trum nigrum Linnaeus which belongs to the Lil- iaceae family. It is a perennial traditional Chi- nese medical plant and is very prevalent in the wild in China. It has been extensively used for the treatment of hypertension, stroke, excessive phlegm and epilepsy (15, 16). In recent years, it had other properties, such as antifungal (15) and insecticidal activity (17, 18). However, there are few studies on the insecticidal activity of V. nigrum against public health pests such as the B. germanica. Therefore, we investigated the insecticidal activity of V. nigrum against B. ger- manica. Materials and Methods Preparation of V. nigrum alkaloidal extract A 4kg dry sample of V. nigrum root was purchased from the medicinal material market in Yunnan Province in 2015, China. The sam- ple was crushed and extracted three times with 15L methanol at room temperature. The extrac- tion liquid was made up to 1000ml with water after removal in vacuo. The extract was parti- tioned between 1% hydrochloric acid solution and ethyl acetate. Components of the acidic lay- er, adjusted to pH 9–10 with 10% ammonia so- lution, were extracted with ethyl acetate to give an alkaloidal extract (69g). Compounds Acetylthiocholine iodide, 5, 5’-dithiobis (2- nitrobenzoic acid) (DTNB), Triton X-100, and eserine were purchased from Sigma Chemical Company. Positive control, tetramethrin (95%) was purchased from Shanghai Forever Chem- ical Co, Ltd. Insect rearing Blattella germanica strain maintained at 26±1 °C and 60% relative humidity under 16:8h (L:D) cycle in the laboratory for 40yr without exposure to any insecticides. Contact toxicity test A group of 20 male adult or 4th nymph cock- roaches were anesthetized using Ether. A drop- let (1µl) containing five different concentrations of V. nigrum alkaloidal extract (0.375–12%) or tetramethrin (0.375–6%) was topically applied to the abdomen of cockroaches using a micro- applicator (Burkard, UK). A 1µl droplet of ac- etone was applied to the control group. After treatment, cockroaches were transferred to a 200ml glass bottle maintained at a temperature of 261 °C and a humidity of 7510% RH. Mor- tality was assessed 24h and 48h after treatment. All experiments were repeated in triplicate. In vitro acetylcholinesterase (AChE) activity test In vitro acetylcholinesterase (AChE) activity tests were performed as follows: 10 male adults and 4th nymph cockroaches were homogenized using a glass tissue grinder in ice in 20mM phos- phate buffer (pH 7.0) containing 0.5% Triton X-100. The homogenate was then centrifuged at 12000gr for 15min at 4 °C. The supernatant was used as an enzyme source for measuring AChE activity via a modified Ellman method (19). To determine the 50% inhibiting concen- tration (IC50) of the alkaloidal extract, 50µl of the enzyme was added to a test tube containing 50µl of six different concentrations (0.375–12 mg/ml) of the alkaloidal extract and pre-incu- bated at 25 °C for 5min. To determine the 50% inhibiting time (IT50), the above procedure was followed and pre-incubated at 25 °C for six dif- ferent time periods (0.5min, 1min, 5min, 10min, 15min, 20min). After incubation, 1300µl of 20 mM Ellman solution containing 1mM acetylthi- ocholine iodide, 0.23mM 5', 5'-dithiobis (2-ni- trobenzoic acid) and 0.45mM NaHCO3 were added to the test tube which was then incubated at 25 °C for 20min. The reaction was stopped by 100µl 10mM eserine and the absorbance at 412nm was measured. Each assay was repli- cated five times. J Arthropod-Borne Dis, December 2018, 12(4): 414–420 X Cai et al.: Insecticidal and … 416 http://jad.tums.ac.ir Published Online: December 25, 2018 Statistical analysis Mortality corrected using Abbott's formula. All analyses were performed using SPSS 13.0 software (Chicago, IL, USA). LD50, IC50 and IT50 were considered to be significantly different on the basis of non -overlapping of 95% confidence intervals (CLs). Results Contact toxicity test Contact toxicities of V. nigrum alkaloidal extract against male adults and 4th nymphs cock- roaches are shown in Table 1. Veratrum nigrum alkaloidal extract has significant insecticidal ac- tivity against the B. germanica. There was no significant difference in mortality at 24h and 48h (overlapping of 95% CL) at LD50 values of 14.90µg/insect, 14.21µg/insect for adults and 41.45µg/insect, 39.01µg/insect for 4th nymphs after 24h and 48h exposure, respectively. The lethal effect of V. nigrum alkaloidal extract on B. germanica is quick. In vitro acetylcholinesterase (AChE) activity test The results of in vitro acetylcholinesterase (AChE) activity tests are shown in Table 2 and Fig. 1. Veratrum nigrum alkaloidal extract was found to have an excellent inhibitory effect on AChE, showing IC50 values were 3.56mg/ml for nymphs and 5.78mg/ml for adults, respec- tively (Table 2). Inhibition of AChE activity was positively correlated with inhibition time. When the concentration of V. nigrum alkaloidal extract was 1mg/ml, inhibition of AChE activity in both nymphs and adults was at IT50 values of 8.34 min and 16.75min, respectively (Fig. 1). Table 1. Toxicity of Veratrum nigrum alkaloidal extract against male adults and 4th nymphs of Blattella germanica after exposure for 24 and 48h Compounds (µg/insect) Age Exposure time LD50SD 95%fiducial limits SlopeSD Chi square (2) Alkaloidal extract Adult 24 14.901.35 13.40–16.68 3.330.42 1.99 4th Nymph 24 41.454.45 36.02–47.68 2.270.33 3.73 Tetramethrin Adult 24 7.890.88 6.89–9.93 3.390.40 2.89 4th Nymph 24 13.981.26 10.82–18.22 2.790.38 2.43 Alkaloidal extract Adult 48 14.211.40 13.12–15.78 3.230.38 2.25 4th Nymph 48 39.014.03 35.55–46.89 2.650.37 3.68 Tetramethrin Adult 48 7.720.78 6.52–8.90 3.210.37 2.76 4th Nymph 48 13.301.28 10.32–18.00 2.810.33 2.41 Fig. 1. Residual AChE activity in male adults and 4th nymphs of Blattella germanica at 1 mg/ml Veratrum nigrum extract J Arthropod-Borne Dis, December 2018, 12(4): 414–420 X Cai et al.: Insecticidal and … 417 http://jad.tums.ac.ir Published Online: December 25, 2018 Table 2. IC50 values of Veratrum nigrum alkaloidal extract against male adults and 4th nymphs of Blattella germanica AChE activity Age IC50 (mg/ml) (SD) 95% Confidence limit Slope (SD) Chi square (2) Adult 5.780.47 4.78–6.98 1.240.13 1.54 4th nymph 3.560.49 2.36–4.72 1.350.11 1.35 Discussion The Chinese pharmacopoeia includes tradi- tional Chinese medicine plants, shown to have anti-oxidant, free radical scavenging, anti-inflam- matory and anticancer properties (20). In pre- vious studies, V. nigrum extracts had insecticidal effects against Mythimna separata, Aphis crac- civora, Cx. pipiens pallens, Tetranychus cin- nabarinus, and Plutella xylostella (17, 18). Cer- tain Chinese medicinal plant extracts have been reported to be bioactive against B. germanica, such as Pogostemon cablin at a LD50 of 23.45 µg/insect (14), and Chenopodium ambrosioides at a LD50 of 64.47µg/insect (13). Our results showed that V. nigrum alkaloidal extract has significant insecticidal activity against B. germanica. Compared with the control (a chemical insecticide: tetrarmethrin, LD50 of 7.89 µg/insect, 7.72µg/insect for adults and 13.98µg/ insect, 13.30µg/insect for 4th nymphs after 24h and 48h exposure, respectively), V. nigrum al- kaloidal extract showed a little weaker or equiv- alent toxic to the B. germanica. Contact toxicity of V. nigrum alkaloidal extract against male adults was stronger than 4th nymphs B. german- ica. Moreover, the result was similar to insec- ticidal activities of tetramethrin against above both. The tolerance level of the nymphs was sig- nificantly higher than that of adults (non-over- lapping of 95% CL), as also reported by (21-23). The weight difference between males and fe- males may be one reason for the different sus- ceptibility to insecticide (24, 25). In our research, the mean nymph weight (30.18 mg) was light- er than the adult male (54.33mg) but did not appear to be a factor for the increase intolerance. Therefore, there may be other modes of action responsible for this difference, but the mecha- nisms of plant extract toxicity against B. german- ica are poorly reported. The insecticidal target site of the phyto- chemicals against cockroaches has been previ- ously researched, such as the insect GABA re- ceptor was target site for carvacrol, pulegone, and thymol (26). Octopamine receptor was target site for carvacrol, -terpineol and pulegone (27, 28). In addition, the AChE inhibition activity of phytochemicals on B. germanica has been previously studied in vitro (24, 29, 30). In the present study, V. nigrum had an excellent in- hibitory effect on AChE, showing IC50 and IT50 values were 3.56mg/ml，8.34min for nymphs and 5.78mg/ml, 16.75min for adults, respective- ly. AChE in 4th nymph B. germanica was more susceptible to insecticide than in male adult cockroaches, similar to results found by (24, 29). The mechanisms of insecticide resistance in B. germanica have many factors, including target site insensitivity, metabolic detoxification and decreased skin permeability (31, 32). Therefore it is difficult to explain the higher tolerance of nymphs against V. nigrum alkaloidal extract through AChE inhibition alone. In addition, plant extracts have many active ingredients and biological activity is, therefore, the result of a combination of various compo- nents (13, 33). It is necessary to determine the active components of V. nigrum which are cur- rently poorly understood for insecticidal effica- cy. In recent years, 35 active ingredients were identified in V. nigrum alkaloidal extract and the main components were veratramine, followed by veratrosine and jervine (34). This would provide a strong foundation for screening the insecticidal constituents of V. nigrum. Further work focuses J Arthropod-Borne Dis, December 2018, 12(4): 414–420 X Cai et al.: Insecticidal and … 418 http://jad.tums.ac.ir Published Online: December 25, 2018 on insecticidal activities of V. nigrum ingredi- ents against B. germanica. Conclusion Insecticidal activities of phytochemicals de- rived from Chinese medicinal plants have been previously investigated against B. germanica. V. nigrum (the family of Liliaceae) alkaloidal ex- tract have excellent insecticidal activity com- pared to tetramethrin in vivo and inhibitory ef- fect on AChE in vitro. It showed potential for further development as possible natural insec- ticide against B. germanica. 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