Repellant and insecticidal activities of shyobunone and isoshyobunone derived from the essential oil of Acorus calamus rhizomes
Hai-Ping Chen1, Kai Yang2, Li-Shi Zheng1, Chun-Xue You2, Qian Cai1, Cheng-Fang Wang3
1 College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, Liaoning, China
2 Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, College of Resources Science and Technology, Beijing Normal University, NO.19 Xinjiekouwai Street, Beijing 100875, China
3 China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Xicheng District, Beijing 100088, China
|Date of Submission||30-Jul-2014|
|Date of Acceptance||15-Sep-2014|
|Date of Web Publication||16-Sep-2015|
China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Xicheng District, Beijing 100088
Source of Support: This project was supported by the National Natural
Science Foundation of China (No. 81374069) and Beijing Municipal Natural
Science Foundation (No. 7142093), Conflict of Interest: None
| Abstract|| |
Context: It was found that the essential oil of Acorus calamus rhizomes showed insecticidal activity. Aim: The aim of this study was to determine the chemical composition of the essential oil from A. calamus rhizomes, evaluate insecticidal and repellant activity against Lasioderma serricorne (LS) and Tribolium castaneum (TC), and to isolate any insecticidal constituents from the essential oil. Materials and Methods: Essential oil from A. calamus was obtained by hydrodistillation and analyzed by gas chromatography (GC) flame ionization detector and GC-mass spectrometry. The insecticidal and repellant activity of the essential oil and isolated compounds was tested using a variety of methods. Results: The main components of the essential oil were identified to be isoshyobunone (15.56%), β-asarone (10.03%), bicyclo[6.1.0]non-1-ene (9.67%), shyobunone (9.60%) and methylisoeugenol (6.69%). Among them, the two active constituents were isolated and identified as shyobunone and isoshyobunone. The essential oil showed contact toxicity against LS and TC with LD 50 values of 14.40 and 32.55 μg/adult, respectively. The isolated compounds, shyobunone and isoshyobunone also exhibited strong contact toxicity against LS adults with LD 50 values of 20.24 and 24.19 μg/adult, respectively, while the LD 50 value of isoshyobunone was 61.90 μg/adult for TC adults. The essential oil, shyobunone and isoshyobunone were strongly repellent (98%, 90% and 94%, respectively, at 78.63 nL/cm 2 , after 2 h treatment) against TC. Conclusion: The essential oil, shyobunone and isoshyobunone possessed insecticidal and repellant activity against LS and TC.
Keywords: Acorus calamus, contact toxicity, Lasioderma serricorne, repellency, Tribolium castaneum
|How to cite this article:|
Chen HP, Yang K, Zheng LS, You CX, Cai Q, Wang CF. Repellant and insecticidal activities of shyobunone and isoshyobunone derived from the essential oil of Acorus calamus rhizomes. Phcog Mag 2015;11:675-81
|How to cite this URL:|
Chen HP, Yang K, Zheng LS, You CX, Cai Q, Wang CF. Repellant and insecticidal activities of shyobunone and isoshyobunone derived from the essential oil of Acorus calamus rhizomes. Phcog Mag [serial online] 2015 [cited 2021 May 6];11:675-81. Available from: http://www.phcog.com/text.asp?2015/11/44/675/165543
| Introduction|| |
The red flour beetle, Tribolium castaneum (TC) Herbst and the cigarette beetle, Lasioderma serricorne (LS) Fabricius are the most widespread and destructive primary insect pests of stored cereals.  The infestations of stored product insects currently not only cause significant losses due to the consumption of grains but also result in the rise of temperature and moisture which lead to an accelerated growth of molds, including toxigenic species.  Control of stored product insects relies heavily on the use of synthetic insecticides and fumigants, that has led to problems such as disturbances of the environment, increasing costs of application, pest resurgence, pest resistance to pesticides and lethal effects on nontarget organisms in addition to direct toxicity to users.  These problems have necessitated a search for alternative eco-friendly insect pest control methods.  Botanical pesticides have the advantage of providing novel modes of action against insects that can reduce the risk of cross-resistance as well as offering new leads for design of target-specific molecules. , The use of essential oils or their constituents with low mammalian toxicity can effectively prevent and/or suppress insect pest especially in storage.  Essential oils from many plants have been evaluated with success for insecticidal/repellency activity against stored-product insects/mites, in some cases, have been proven more effective than traditionally used organophosphorus pesticides. ,, During our screening program for new agrochemicals from local wild plants and Chinese medicinal herbs, the essential oil from Acorus calamus rhizomes has been found to possess contact and repellent activities towards LS and TC.
Acorus calamus (Linn.), a member of the family Araceae, is a perennial and semiaquatic plant with creeping rhizomes. Commonly known as sweet flag, A. calamus wildly grows along swampy and marshy areas in the northern temperate and subtropical regions of Asia, North America and Europe.  A. calamus is well known for its beneficial and medicinal properties in Indian medical system. Pharmacological studies have revealed that the plant possesses a wide range of therapeutic activities, including behavior-modifying, anticonvulsant, acetyl cholinesterase inhibitory, , antispasmodic, antidepressant, anxiolytic, ,, anti-diabetic,  hypolipidemic,  antidiarrheal,  bronchodilatory,  anti-inflammatory,  cytoprotective  and analgesic properties.  In addition, the essential oil of A. calamus has been demonstrated to possess repellency activity against the maize weevil, S. zeamais and insecticidal activity against many species of insects, e.g., the larger grain borer, Prostephanus truncates,  the tobacco armyworm, Spodoptera litura and the booklouse, Liposcelis bostrychophila.  However, a literature survey has shown that there is no report on contact/repellency of A. calamus essential oil against the red flour beetle and the cigarette beetle, thus we decided to investigate the chemical constituents and contact/repellency activity of the essential oil of A. calamus against TC and LS for the first time and to isolate any biologically active compounds from its essential oil.
| Materials and methods|| |
Rhizomes (3.5 kg) of A. calamus were collected in September 2012 in Dali City (35.23°N and 116.33°E), Yunnan province of China. The rhizomes were air-dried for one week and ground to a powder. The plant was identified by Dr. Liu, Q.R. (College of Life Sciences, Beijing Normal University, Beijing, China) and a voucher specimen (BNU-CMH-Dushuahan-2012-11-25-006) was deposited at the Herbarium (BNU) of College of Life Sciences, Beijing Normal University.
Cigarette beetles (LS) and red ﬂour beetles (TC) were obtained from laboratory cultures maintained for the last 2 years in dark in incubators at 29°C ± 1°C and 70-80% relative humidity. The insects were reared in glass containers (0.5 L) containing wheat flour at 12-13% moisture content mixed with yeast (10:1, w/w). Adults used in all the experiments were about 7 ± 2 days old regardless of gender.
Extraction and composition of essential oil
The ground powder of A. calamus rhizomes was subjected to hydrodistillation using a modified Clevenger-type apparatus for 6 h and extracted with n-hexane. Anhydrous sodium sulphate was used to remove water after extraction. The essential oil was stored in airtight container in a refrigerator at 4°C.
Gas chromatography-mass spectrometry (GC-MS) analysis was performed on a Thermo Finnigan Trace DSQ instrument equipped with a flame ionization detector and an HP-5 MS (30 m × 0.25 mm × 0.25 μm) capillary column. The column temperature was programmed at 50°C for 2 min, then increased at 2°C/min to the temperature of 150°C and held for 2 min, and then increased at 10°C/min until the final temperature of 250°C was reached, where it was held for 5 min. The injector temperature was maintained at 250°C and the volume injected was 0.1 mL of 1% solution (diluted in n-hexane). The carrier gas was helium at flow rate of 1.0 mL/min. Spectra were scanned from 50 to 550 m/z. Most constituents were identified by comparison of their retention indices with those reported in the literatures. The retention indices were determined in relation to a homologous series of n-alkanes (C 5 -C 36 ) under the same operating conditions. GC retention time and their mass spectra that stored in NIST 05 and Wiley 275 libraries or from literature were used for identify the essential oil components.  Relative percentages of the individual components of the essential oil were obtained by averaging the GC peak area% reports.
Purification and characterization of two constitunent compounds
The crude essential oil (5 ml) was chromatographed on a silica gel (Qingdao Marine Chemical Plant, Shandong province, China) column (30 mm i.d., 500 mm length) by gradient elution with n-hexane first, then with n-hexane-ethyl acetate, and last with ethyl acetate to obtain 22 fractions. Based on contact toxicity/repellent test, fraction 3 and 15 were chosen for further fractionation. With PTLC, two purified compounds were obtained and they were analysised by various NMR techniques including 1 H NMR and 13 C NMR. Combining all the NMR spetra data, the two isolated compounds were finally recognized as shyobunone ( 1 , 0.24 g) , and isoshyobunone ( 2 , 0.35 g). , NMR experiments were performed on Bruker Avance DRX 500 instrument using CDCl 3 as solvent with TMS as internal standard.
Contact toxic activity test
The contact toxicity of the essential oil/pure compounds against LS and TC adults was measured as described by Liu and Ho.  Range-finding studies were run to determine the appropriate testing concentrations. A serial dilution of the essential oil/compounds (five concentrations) was prepared in n-hexane. Aliquots of 0.5 μL of the dilutions were applied topically to the dorsal thorax of the insects. Controls were determined using n-hexane. Five replicates were carried out for all treatments and controls. Both treated and control insects were then transferred to glass vials (10 insects/vial) with culture media and kept in incubators. Mortality was recorded after 24 h and the LD 50 values were calculated using Probit analysis.  Positive control, pyrethrins (pyrethrin I and II, 37%) were purchased from Dr Ehrenstorfer GmbH.
The repellent activity of the essential oil/pure compounds to TC adults was tested using the area preference method.  The essential oil/compounds was diluted in n-hexane to different concentrations (78.63, 15.73, 3.15, 0.63 and 0.13 nL/cm 2 ), and n-hexane was used as the control. Filter paper (9 cm in diameter) was cut in half. 500 μL of treatment solution was placed on one half of the filter paper and allowed to dry for 30s. The other half was treated with 500 μL of n-hexane. The treated side was then joined to the control side by tape and placed in glass petri dishes (9 cm in diameter). Twenty insects were released in the center of each filter paper disk, and a cover was placed over the Petri dish. Five replicates were used. Counts of the insects present on each strip were made after 2 and 4 h. The percent repellency (PR) of each volatile oil/compound was then calculated using the formula:
PR (%) = ([Nc − Nt]/[Nc + Nt]) ×100
Where N c is the number of insects present in the negative control half and N t is the number of insects present in the treated half. Analysis of variance (One-Way ANOVA and GLM Univariate) and Tukey's test were conducted by using SPSS 20.0 (IBM, Armonk, NY) for Windows 2007. Percentage mortality data were subjected to arcsine square-root transformation before analysis of variance. A commercial repellent, N, N-diethyl-3-methylbenzamide (DEET), was purchased from the National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang 110021, China) and used as a positive control.
| Results|| |
Chemical compounds of the essential oil
The yield of A. calamus rhizomes essential oil was 1.00% (v/w) and the density of the essential oil was determined to be 0.93 g/ml. GC-MS analysis of the essential oil of A. calamus rhizomes led to the identification and quantification of a total of 56 major components, accounting for 89.21% of the total components present [Table 1].
|Table 1: Chemical components of the essential oil of A. calamus rhizomes |
Click here to view
The essential oil of A. calamus rhizomes showed strong contact toxicity against LS and TC adults with LD 50 values of 14.40 and 32.55 μg/adult, respectively [Table 2]. Compared with the positive control pyrethrins (37% pyrethrin I and pyrethrin II), the crude essential oil demonstrated 60 and 125 times less toxicity against the two insect species because the pyrethrins had acute contact toxicity to LS and TC adult with LD 50 values of 0.24 μg/adult and 0.26 μg/adult, respectively. The isolated compounds, shyobunone and isoshyobunone also exhibited strong contact toxicity against LS adults with LD 50 values of 20.24 and 24.19 μg/adult, respectively [Table 2], while the LD 50 value of isoshyobunone, was 61.90 μg/adult for TC adults.
|Table 2: Contact toxicity of essential oil of Acorus calamus rhizomes and its main components against LS and TC adults |
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The results of repellency assays for the essential oil and isolated compounds against TC adults are presented in [Figure 1] and [Figure 2]. However, the crude essential oil showed no obvious repellency against LS adults because the essential oil at dose of 78.63 and 15.73 nL/cm 2 has weak repellency (56% and 26%, respectively) to LS after 2 h treatment. A. calamus rhizomes oil at dose of 78.63 nL/cm 2 showed 98% and 98% repellency against TC adults at 2 and 4 h after exposure, respectively. The repellent responses of TC adults to the essential oil at dose of 15.73 nL/cm 2 (P = 0.291) and 3.15 nL/cm 2 (P = 0.103) were the same level compared to that at the highest concentration treatment. Shyobunone and isoshyobunone also showed obvious repellency (>80%) at dose of 78.63 and 15.73 nL/cm 2 after 4 h treatment. However, compared with shyobunone, isoshyobunone produced stronger repellency (100% and 92%, respectively, at 15.73 nL/cm 2 , after 2 and 4 h treatment). At the lowest concentration (0.13 nL/cm 2 ), isoshyobunone still showed repellency (64%) against TC adults at 2 h after exposure.
|Figure 1: Percentage repellency (PR) of the essential oil from Acorus calamus rhizomes and its constituents against Tribolium castaneum at 4 h after exposurea. aMeans in the same column followed by the same letters do not differ significantly (P > 0.05) in ANOVA and Tukey's tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukey's tests. bPositive control|
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|Figure 2: Constituent compounds isolated from the essential oil of Acorus calamus rhizomes|
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| Discussion|| |
The main constituents of A. calamus rhizomes essential oil were isoshyobunone (15.56%), β-asarone (10.03%), bicyclo[6.1.0]non-1-ene (9.67%), shyobunone (9.60%) and methylisoeugenol (6.69%). The results were different from the previous reports. These differences might have been due to harvest time and local, climatic and seasonal factors as well as storage duration of medicinal herbs. , For example, α-asarone (50.09%), (E)-methylisoeugenol (14.01%), methyleugenol (8.59%), β-asarone (3.51%), α-cedrene (3.09%) and camphor (2.42%) were the main components of the essential oil of A. calamus rhizomes obtained from China.  However, the essential oil of A. calamus rhizomes collected from Italy contained acorenone (21.6%), (Z)-sesquilavandulol (13.0%), shyobunone (7.0%), α-asarone (5.1%) and dehydroxyisocalamendiol (3.5%)  while the essential oil of A. calamus collected from Quebec, Canada contained preisocalamenediol (18.0%), acorenone (14.2%), shyobunone (13.3%) and cryptoacorone (7.5%).  The essential oil of A. calamus contained various chemical constituents, and the proportion of each chemical constituent of the oil particularly β-asarone varied in different genotypes and corresponds to the ploidy level.  It is reported that the tetraploids have higher (70-96%) β-asarone content, than the triploids (5-19%), and almost negligible in diploid genotypes. , The above discussions suggest that further studies on plant cultivation and essential oil standardization would be expected because chemical composition of the essential oil varies greatly among the plant population.
To our knowledge, this is the first report regarding to insecticidal action of shyobunone and isoshyobunone against stored-grain insects, as exemplified here with LS and TC. Shyobunone showed more toxicity against LS and much less toxicity against TC than isoshyobunone [Table 2]. However, all the two isolated constituent compounds possessed less activity against LS adult than the crude essential oil [Table 2], suggesting that there may be some other stronger active compounds in small amounts in the essential oil or may be some synergistic action between the various compounds. In addition, we have an interesting discovery in this work. Shyobunone (1) and isoshyobunone (2) have the same molecular formula (C 15 H 24 O). They are a pair of isomers with a double bond located at different positions along the isopropyl side chain [Figure 3], but their contact toxicity is very different. Differences in the biological activities of geometric isomers were reported in coleopteran pests of stored products and in a yellow fever vector mosquito. In previous research, similar phenomena were also observed. cis-Asarone is toxic in addition to having strong antifeedant activity, whereas the trans isomer acts only as an antifeedant with no appreciable toxicity.  Park et al.  reported that the insecticidal activity against Sitophilus oryzae (L.), Callosobruchus chinensis (L.), and LS (F.) was more evident in (Z)-asarone than that in (E)-asarone. In addition, (Z)-9-octadecenoic acid was a more potent repellent agent than (E)-9-octadecenoic acid against Aedes aegypti (L.) adult females.  The tiny structural difference of these compounds may account for the significant differences in their insecticidal action. This action includes insect mortality and sublethal effects on behavior, depending on insect and mode of application.
|Figure 3: Percentage repellency (PR) of the essential oil from Acorus calamus rhizomes and its constituents against Tribolium castaneum at 2 h after exposurea. aMeans in the same column followed by the same letters do not differ significantly (P > 0.05) in ANOVA and Tukey's tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukey's tests. bPositive control|
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Many essential oils and their constituents have been evaluated for repellency against insects.  For example, Zhang et al. reported that geraniol and citronellol exhibited stronger repellency against TC adults than DEET, whereas limonene and citronella showed the same level of repellency against TC adults compared with DEET.  The origanum oil, linalool and p-cymene at dose of 0.03 mg/cm 2 showed 98%, 83% and 85% repellency (after 2 h treatment) against TC adults, respectively.  However, in this paper, we report the repellency action of shyobunone and isoshyobunone for the first time. In this study, compared with the positive control, DEET, essential oil (P = 0.051), isoshyobunone (P = 0.721) exhibited the same level of repellency against TC adults, while shyobunone demonstrated less repllency than isoshyobunone [Figure 1].
| Conclusion|| |
The above discussions suggest that the essential oil and its four compounds show the potential to be developed as natural insecticides and repellents against stored-products insects. However, for the practical application of the essential oil and the four compounds as novel insecticides/repellents, further studies on the safety of the essential oil and its four compounds toward human beings and on the development of formulations are necessary to improve the efficacy and stability, and to reduce cost.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]