J Arthropod-Borne Dis, 2012, 6(2): 90–97 WX Zhu et al.: Evaluation of Essential Oil … 90 Original Article Evaluation of Essential Oil and its Three Main Active Ingredients of Chinese Chenopodium ambrosioides (Family: Chenopodiaceae) against Blattella germanica Wei Xiang Zhu, Kun Zhao, Sha Sha Chu, *Zhi Long Liu Department of Entomology, China Agricultural University, Beijing, China (Received 9 Oct 2010; accepted 18 Sep 2011) Abstract Background: The efficacy of essential oil of Chenopodium ambrosioides flowering aerial parts and its three main active ingredients was evaluated against Blattella germanica male adults. Methods: Composition of essential oil was determined by GC-MS. Topical application bioassay was used to evalu- ate contact toxicity of essential oil and three main components. Fumigant toxicity of essential oil and its main com- ponents was measured using a sealed space method. Results: Twenty-two components were identified in the essential oil and the main components were (Z)-ascaridole (29.7%), isoascaridole (13.0%), ρ-cymene (12.7%) and piperitone (5.0%). The essential oil and (Z)-ascaridole, isoascaridole and -cymene possessed fumigant toxicity against male German cockroaches with LC50 values of 4.13, 0.55, 2.07 and 6.92 mg/L air, respectively. Topical application bioassay showed that all the three compounds were toxic to male German cockroaches and (Z)-ascaridole was the strongest with a LD50 value of 22.02 g/adult while the crude oil with a LD50 value of 67.46 g/adult. Conclusion: The essential oil from Chinese C. ambrosioides and its three main active ingredients may be explored as natural potential insecticides in the control of cockroaches. Keywords: Blattella germanica, Chenopodium ambrosioides, Essential oil, Fumigant, Contact toxicity Introduction German cockroach, Blattella germanica (L) is an important pest of homes, restaurants, and commercial food processing facilities worldwide. They are a major public health concern in hospitals, kitchens, and food man- ufacturing plants because they are able to car- ry a variety of bacteria and other pathogenic organisms. They are the mechanical vectors to a few pathogens that can cause disease such as food poisoning, typhoid, pneumonia and asthma (Brenner 1995). Body parts, cast skins, and feces of cockroaches are human aller- gens, 2nd in importance only to the house dust mites. Currently, control of cockroach pop ulations primarily depends on continued ap- plications of residual insecticides, such as pro- poxur, acephate, dimethyl 2, 2-dichlorovinyl phosphate (dichlorvos, DDVP), and pyreth- roids and stomach poisons, such as hydrame- thylnon and sulfluramid. However, the repeated application of these insecticides may possess undesirable side effects, such as the disruption of natural biological control systems, and the development of resistance (Chang and Ahn 2001). There are also serious concerns about human health. These problems have highlight- ed the need for the development of new types of selective cockroach-control alternatives. There has been growing interest in the *Corresponding author: Dr Zhi Long Liu, E-mail: zhilongliu@cau.edu.cn J Arthropod-Borne Dis, 2012, 6(2): 90–97 WX Zhu et al.: Evaluation of Essential Oil … 91 use of plant oils for protection of agricul- tural products and control of public health insects because they are often of low mam- malian toxicity, readily biodegradable and pose low danger to the environment if used in small amounts (Rajendran and Srianjini, 2008). During our screening program for new agrochemicals from Chinese medicinal herbs, Chenopodium ambrosioides L (Family: Che- n- opodiaceae) was found to possess strong in- secticidal activity against German cockroaches (Fig. 1). Chenopodium ambrosioides is an ar- omatic herb that grows in Central and South America and now distributed throughout the tropical parts of the world (Duke et al. 2002). It has also been employed by empiri- cal herbalists and healers against intestinal parasites (especially small tapeworms and round worms) throughout Latin America, as well as in the West Indies (Quinlan et al. 2002). The plant is also distributed in the southern provinces of China. The aerial parts of this plant have been used as condiment, tradi- tional purgative for intestinal worms and acesodyne and in the Chinese traditional med- icine. This herb can expel wind, treat rheu- matism (Jiangsu New Medical College 1977). Essential oil of C. ambrosioides has been shown to possess insecticidal and repellent activities against several stored product in- sects (Su 1991, Tapondjou et al. 2002) and medical important insect pests (Toloza et al. 2006, Gillij et al. 2008). In the present study, insecticidal activity C. ambrosioides essential oil and three main active ingredients against the German cock- roaches were investigated. Materials and Methods Test insects Male adults (5–10 days old) were collect- ed from a synchronously reared laboratory colony of insecticide-susceptible German cock- roaches. Cockroaches were supplied ad libi- tum with Purina No 5012 Rat Chow (Labor- atory Animal Centre, Chinese Academy of Medicinal Sciences, Beijing 100021), and water was provided in glass tubes with cot- ton stoppers. All colonies were kept in plas- tic tanks at room temperature. Plants Fresh aerial parts (15 kg of leaves, stems and flowers) of C. ambrosioides were har- vested in August 2008 from Fuzhou (26.08 North latitude and 119.28 East longitude), Fujian Province (Fuzhou 350013), PR Chi- na. The aerial parts were air-dried for one week and ground to a powder. The species was identified and the voucher specimens (CMH-TuJingJie-FuJian-2008-08) were de- posited at the Department of Entomology, China Agricultural University, Beijing 100094. Extraction of essential oil The ground powder of C. ambrosioides aerial parts was subjected to hydrodistilla- tion using a modified Clevenger-type appa- ratus (XWD-C-1000, Shanghai XinWangDe Laboratory Equipment Co, China) for 6 h and extracted with n-hexane. Anhydrous so- dium sulphate was used to remove water af- ter extraction. Essential oil was stored in an airtight container in a refrigerator at 4 C. Gas chromatography and mass spectrom- etry Gas chromatographic analysis was per- formed on the Agilent 6890N while the es- sential oil was identified on a mass spec- trometer Agilent Technologies 5973N. They were equipped with a flame ionization de- tector and capillary column with HP-5MS (30m× 0.25mm× 0.25μm). The GC settings were as follows: the initial oven temperature was held at 60 C for 1 min and ramped at 10 C min−1 to 180 C for 1 min, and then ramped at 20 C min−1 to 280 C for 15 min. The injector temperature was maintained at 270 C. The samples (1 μl) were injected neat, with a split ratio of 1: 10. The carrier J Arthropod-Borne Dis, 2012, 6(2): 90–97 WX Zhu et al.: Evaluation of Essential Oil … 92 gas was helium at flow rate of 1.0 ml min−1. Spectra were scanned from 20 to 550 m/z at 2 scans s-1. Most constituents were identified by gas chromatography by comparison of their retention indices with those of the liter- ature or with those of authentic compounds available in our laboratories. The retention in- dices were determined in relation to a ho- mologous series of n-alkanes (C8–C24) under the same operating conditions. Further iden- tification was made by comparison of their mass spectra on both columns with those stored in NIST 05 and Wiley 275 libraries or with mass spectra from literature (Adams 2001). Component relative percentages were calculated based on GC peak areas without using correction factors. (Z)-Ascaridole and isoascaridole were iso- lated by using bioassay-directed fractiona- tion on repeated silica column from the es- sential oil of C. ambrosioides and confirmed by MS, 1H-NMR and 13C-HNM data. -Cy- mene (98%) was purchased from Aladdin Reagent (China) Co., Ltd, Shanghai 201206, PRChina. Positive control, pyrethrum extract (25% pyrethrine I and pyrethrine II) was purchased from Fluka Chemie. Fumigant toxicity A serial dilution of C. ambrosioides es- sential oil (20–1.3%, 5 concentrations) and pure compounds (2.5–0.3% for (Z)-ascaridole, 20–0.6% for another two compounds, 5 con- centrations) was prepared in n-hexane. A Whatman filter paper (diameter 6.0 cm) were each impregnated with 50 μl dilution, and then placed on the underside of the screw cap of a glass vial (diameter 7.2 cm, height 19.0 cm, volume 750 ml). The solvent was allowed to evaporate for 30 s before the cap was placed tightly on the glass vial, each of which contained 10 male cockroaches inside to form a sealed chamber. Fluon (ICI Amer- ica Inc) was used inside glass vial to prevent insects from contacting the treated filter pa- per. Preliminary experiments demonstrated that 30 s were sufficient for the evaporation of solvents. n- Hexane was used as controls. Five replicates were carried out for all treat- ments and controls, and they were incubated at 24–26 C, 75% RH, 12:12 LD photo- period for 24 h and then mortality was rec- orded. Mortality was defined as inability to move when placed on the dorsal side and ina- bility to respond to prodding. Results from all replicates were subjected to probit analysis using the PriProbit Program V1.6.3 to de- termine LC50 values (Sakuma 1998). Topical application bioassay Groups of ten adult male cockroaches were anaesthetized with carbon dioxide for 15 seconds before treatment. A serial dilu- tion of the essential oil (7.0–1.3%, 5 concen- trations) and pure compounds (10–0.6%, 6 concentrations) was prepared in acetone. Al- iquots of 2 µ l of the solution were dispensed from an Arnold Automatic Micro-applicator (Burkard, Ricksmanworth, England) and ap- plied to the dorsal thorax of individual in- sects. Controls were determined using ace- tone. Both treated and control cockroaches were then transferred to glass vials (10 in- sects/ vial) and kept in incubators (24–26 C, 75% RH, 12:12 LD photoperiod). Mor- tality of cockroaches was observed at 24 h post-treatment. Five replicates were carried out for all treatments and controls. Results from all replicates were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LD50 values (Sakuma 1998). Results The results of GC-MS of C. ambrosioides essential oil are presented in Table 1. A total of 22 active ingredients were identified in the essential oil, accounting for 88.6% of the total oil (Table 1). The main components were (Z)-ascaridole (29.7%), isoascaridole (13.0%), and ρ-cymene (12.7%) followed by piperitone (5.0%), isothymol (4.9%), and 3, 4-epoxy-- menthan-2-one (4.1%). J Arthropod-Borne Dis, 2012, 6(2): 90–97 WX Zhu et al.: Evaluation of Essential Oil … 93 (Z)-Ascaridole, isoascaridole and -cy- mene possessed fumigant toxicity against male German cockroaches with LC50 values of 0.55, 2.07 and 6.92 mg/L air, respectively while the crude essential oil with a LC50 val- ue of 4.13 mg/L air (Table 2). (Z)-Ascaridole also showed the strongest contact toxicity to German cockroaches with a LD50 value of 22.02 g/adult (based on the LD50 values, with no overlap in 95% fiducial limits) by using topical application bioassay while the crude essential oil with a LD50 value of 64.47 g/adult (Table 3). Isoascaridole and -cymene also had weak contact toxicity against Ger- man cockroaches with LD50 values of 96.28 and 119.90 g/adult, respectively. Table 1. Chemical constituents of essential oil derived from Chenopodium ambrosioides Compound RI* Chemical Formula Relative Area (%) -Pinene 931 C10 H16 1.3 -Pinene 981 C10 H16 0.3 δ-4-Carene 1002 C10 H16 1.9 -Terpinene 1017 C10 H16 1.1 -Cymene 1024 C10 H14 12.7 ,α-Dimenthylstyrene 1118 C10 H12 0.7 trans--Mentha-2,8-dienol 1126 C11 H18 O2 0.6 trans--2,8-Menthadien-1-ol 1139 C10 H16 O 0.6 2-Ethylcyclohexanone 1158 C8 H14 O 0.9 ,-4-Trimethylbenzyl alcohol 1182 C10 H14 O 2.8 cis-Piperitol 1196 C10 H16 0.5 (Z)-Ascaridole 1245 C10 H16 O2 29.7 Piperitone 1250 C10 H16 5.0 3,4-Epoxy--menthan-2-one 1276 C10 H16 O2 4.1 Thymol 1292 C10 H14 O 1.1 Carvacrol 1298 C10 H14 O 4.9 Isoascaridole 1295 C10 H16 O2 13.0 Precocene II 1368 C13 H16 O3 1.5 Caryophyllene oxide 1584 C15H24O 2.2 Geranyl tiglate 1700 C15 H24 0.8 Hexahydrofarnesyl acetone 1842 C18 H36 O 1.7 Phytol 2119 C20 H40 O 1.2 Total 88.6 *RI, retention index as determined on a HP-5MS column using the homologous series of n-hydrocarbons; Table 2. Fumigant toxicity of essential oil and components from Chenopodium ambrosioides against male cock- roach adults Compounds LC50 (mg/L air) 95% fiducial limits Slope±SE Chi square (χ 2 ) Ascaridole 0.55 0.47–0.63 7.25±0.69 10.15 -Cymene 6.92 6.11–7.85 7.06±0.45 8.23 Isoascaridole 2.07 1.78–2.43 6.17±0.49 9.46 Crude oil 4.13 3.62–4.74 5.06±0.40 11.21 DDVP 0.01* - - - *Data from Jang et al. (2005) J Arthropod-Borne Dis, 2012, 6(2): 90–97 WX Zhu et al.: Evaluation of Essential Oil … 94 Table 3. Contact toxicity of essential oil and components from Chenopodium ambrosioides against male cockroach adults Compounds LD50 (g/adult) 95% fiducial limits Slope±SE Chi square (χ 2 ) Ascaridole 22.02 19.92–24.43 6.12±0.73 9.48 -Cymene 119.90 102.13–143.51 5.23±0.47 13.40 Isoascaridole 96.28 78.04–117.93 3.44±0.35 14.67 Crude oil 64.47 59.21–70.46 4.09±0.43 7.40 Pyrethrum extract 1.70 1.16–3.78 4.23±0.47 6.80 Fig. 1. Flowering Chenopodium ambrosioides (Orginal) RT: 0.00 - 28.05 SM: 7G 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Tim e (m in) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R el at iv e A bu nd an ce 7.85 8.764.63 15.26 17.69 13.44 26.7024.2810.42 18.93 NL: 5.77E7 TIC MS 3-03 Fig. 2. GC-MC graph of Chenopodium ambrosioides essential oil profiles J Arthropod-Borne Dis, 2012, 6(2): 90–97 WX Zhu et al.: Evaluation of Essential Oil … 95 Discussion Among the three isolated compounds, (Z)-ascaridole was proved to be the most ac- tive (fumigant) compound because it showed 7 times more toxic to the German cockroaches compared with the crude essential oil (Table 2). Isoascaridole also demonstrated stronger fumigant toxicity against German cockroaches than the crude essential oil (based on the LC50 values, with no overlap in 95% fiducial limits). However, ρ-cymene had less toxic to German cockroaches than the crude essential oil. In the previous report (Jang et al. 2005), 41 naturally occurring monoterpenoids was eval- uated for fumigant toxicity against German cockroaches and verbenone was most toxic to the German cockroaches with a LD50 value of 11.5 mg/L air. In the present study, (Z)- ascaridole was 20 times more toxic to the German cockroaches compared to ver- benone. However, fumigant toxicity bioas- say demonstrated that all the three isolated compounds and the crude essential oil were less toxic to the cockroaches compared to the commercial insecticide, DDVP because of DDVP with a LC50 value of 0.007 mg/L air. The three isolated compounds exhibited contact toxicity against the German cock- roaches (Table 3). However, compared with the control (pyrethrum extracts, LD50= 1.70 μg/adult), all the three compounds and the essential oil showed less toxic to the German cockroaches in the topical application bio- assay. The above findings suggested that the C. ambrosioides essential oil and the three components especially (Z)-ascaridole may pos- sess potential to be developed as novel natu- ral insecticides, especially fumigants in the control of cockroaches. In the previous studies, several essential oils have been evaluated and demonstrated to possess insecticidal, antiffedant, and re- pellent activities against cockroaches, such as mint oil (Appel et al. 2001), catnip essen- tial oil (Peterson et al. 2002), majoram oil (Jang et al. 2005), garlic and thyme oil (Tunaz et al. 2009), citrus oils (Yoon et al. 2009), and essential oils derived from star anise Illicium verum (Chang and Ahn 2001), American pep- pertree Schinus molle (Ferrero et al. 2007), and nutmeg Myristic1a fragrans (Jung et al. 2007). Moreover, naturally occurring mono- terpenoids, components of essential oils were also evaluated for insecticidal and repellent activities against cockroaches (Ngoh et al. 1998, Jang et al. 2005) and cineole, l-fen- chone, limonene, linalool, menthone, pule- gone, and thujone at 50 g/ml air (14 h ex- posure) caused 100% mortality of male adult German cockroaches (Lee et al. 2003). The aerial parts of this plant have been used as traditional purgative for intestinal worms in Chinese medicine (Jiangsu New Medical College 1977). However, the essen- tial oil of C. ambrosioides is an irritant to the mucous membrane of the gastrointestinal tract, kidney and liver (Gadano et al. 2006). Overdoses of this oil have caused death in men and rats (Monzote et al. 2006). Intake of 10 mg/kg of the oil has been known to cause cardiac disturbances, convulsions, res- piratory disturbances, sleepiness, vomiting and weakness and even death. Moreover, ascaridole is toxic and has a pungent, not very pleasant flavor; in pure form, it is an explosive sensi- tive to shock (Potawale et al. 2008). For the practical use of ascaridole and the crude es- sential oil as novel natural fumigants/ insec- ticides, further studies are necessary on the safety of these materials to human, and on the development of formulations to improve efficacy and stability, and to cut cost as well. The main constituents of the essential oil were (Z)-ascaridole, isoascaridole, ρ-cymene and piperitone (Table1). However, there were great variations in chemical composi- tion of the essential oils of C. ambrosioides. For example, α-terpinyl acetate (73.9%) and ρ-cymene are major constituents of C. am- J Arthropod-Borne Dis, 2012, 6(2): 90–97 WX Zhu et al.: Evaluation of Essential Oil … 96 brosioides essential oil from Mexico and con- tent of ascaridole is only 2% (Pino et al. 2003). In another report, limonene (32.5%), trans- pinocarveol (26.7%) and geranial (5.0%) were main components of C. ambrosioides essen- tial oil from Mexico (Sagrero-Nieves and Bart- ley, 1995). The main components of C. am- brosioides essential oil derived from Brazil were (Z)-ascaridole (61.4%) and (E)-ascaridole (18.6%) (Jardim et al. 2008). However, the C. ambrosioides essential oil from Nigeria con- tained α-terpinene (56.0%), α-terpinyl acetate (15.7%) and ρ-cymene (15.5%) and no asca- ridole was detected in the oil (Muhayimana et al. 1998). The essential oil from India con- tained α-terpinene (47.4%), ρ-cymene (25.8%) and ascaridole (14.8%) (Singh et al. 2008). However, (Z)-ascaridole (29.7%), isoascaridole (13.0%), and ρ-cymene (12.7%) are the three main components of the essential oil from the Chinese C. ambrosioides (Table 1). For the practical use of the crude essen- tial oil of Chinese C. ambrosioides as a new natural insecticide, standardization of the essential oil is needed. 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