832
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Contact Toxicity and Repellency of the Essential Oil from Mentha haplocalyx Br iq . against Lasioderma serricorne by Wen-Juan Zhang a ), Kai Yang a ), Chun-Xue You a ), Cheng-Fang Wang* a ) b ), Zhu-Feng Geng c ), Yang Su d ), Ying Wang a ), Shu-Shan Du* a ), and Zhi-Wei Deng c ) a
) Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Beijing Normal University, No. 19 Xinjiekouwai Street, Beijing 100875, P. R. China (phone/fax: þ 86-10-62208022; e-mail: [email protected]) b ) 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, P. R. China (phone/fax: þ 86-10-62208022; e-mail: [email protected]) c ) Analytical and Testing Center, Beijing Normal University, No. 19 Xinjiekouwai Street, Beijing 100875, P. R. China d ) Tibet Judicial Police Hospital, No. 10 Zhaji Road, Chengguan District, Lhasa 850000, Tibet Autonomous Region, P. R. China
The chemical composition of the essential oil obtained by hydrodistillation from the aerial parts of Mentha haplocalyx was investigated by GC-FID and GC/MS analyses. In sum, 23 components, representing 92.88% of the total oil composition, were identified, and the main compounds were found to be menthol (59.71%), menthyl acetate (7.83%), limonene (6.98%), and menthone (4.44%). By bioassayguided fractionation (contact toxicity), three compounds were obtained from the essential oil and identified as menthol, menthyl acetate, and limonene. The essential oil and the three isolated compounds exhibited potent contact toxicity against Lasioderma serricorne adults, with LD50 values of 16.5, 7.91, 5.96, and 13.7 mg/adult, respectively. Moreover, the oil and its isolated compounds also exhibited strong repellency against L. serricorne adults. At the lower concentrations tested and at 2 h after exposure, menthol showed even significantly stronger repellency than the positive control DEET. The study revealed that the bioactivity properties of the essential oil can be attributed to the synergistic effects of its diverse major and minor components, which indicates that the M. haplocalyx oil and its isolated compounds have potential for the development as natural insecticides and/or repellents to control insects in stored grains and traditional Chinese medicinal materials.
Introduction. – Antagonistic storage, i.e., storage of traditional Chinese medicinal materials with insect-repellent volatile odors together with medicinal materials vulnerable to insects, has been used as one of the traditional conservation methods to prevent insect harm. With the improvement of the sense of environmental protection and medication security, this method is believed to have broad prospects of application in the future. To inherit and further develop this traditional method of prevention and control of stored-grain insects, Mentha haplocalyx essential oil was investigated as potential insect repellent and Lasioderma serricorne adults as target insects. It was expected that this study would provide some theoretical bases for the conception of antagonistic storage. The cigarette beetle (L. serricorne), which is widely distributed, can not only cause significant losses because of the consumption of grains, but also elevate temperature Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
833
and moisture conditions that lead to an accelerated growth of molds, including toxigenic species [1] [2]. Currently, recommended pest-control methods for durable stored products rely heavily on the use of synthetic insecticides or fumigants, which may cause possible health hazards to warm-blooded animals, risks of environmental pollution, development of resistance by insects, and pest resurgence [3]. These problems have necessitated the search for alternative, ecologically safe insect-pest control methods [4]. The essential oils and their constituents isolated from many plants, including medicinal herbs, spices, and fruits, have been evaluated successfully for their insecticidal or repellent activity against stored-product insects and have even proven to be more effective than traditionally-used pesticides, in some cases [5 – 11]. Moreover, 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 [4] [12]. Essential oils from many plants including medicinal herbs, spices, and fruits have been evaluated with success for their insecticidal/repellent activity against stored-product insects/mites. In some cases, they have been proven more effective than organophosphorus pesticides that were traditionally used [6] [13] [14]. During a screening program for new agrochemicals from local wild plants and Chinese medicinal herbs, the essential oil isolated from M. haplocalyx aerial parts and some of its major compounds have been found to possess insecticidal and repellent activity against L. serricorne. M. haplocalyx, which is widely distributed in the Chinese provinces Jiangsu, Anhui, Jiangxi, and Zhejiang, is not only used as popular vegetable in China, but also for the treatment of nerve-center, breath, procreation, and digestive-system disorders [15]. However, there is no report on the insecticidal and repellent activities of this essential oil. Hence, the chemical composition and the insecticidal and repellent activities against L. serricorne of the M. haplocalyx essential oil was investigated, and three main essential-oil constituents were isolated. Results and Discussion. – Chemical Composition of the Essential Oil. The yield and density of the essential oil isolated from M. haplocalyx aerial parts were 1.67% (v/w) and 0.86 g/ml, respectively. The composition of the oil sample was characterized by GCFID and GC/MS analyses, and the components were identified based on the comparison of their retention indices (RIs) and mass spectra with those reported in the literature [5] [6] [9] [16]. In sum, 23 components, representing 92.88% of the total oil composition, were identified (Table 1). The main oil constituents were menthol (59.71%), menthyl acetate (7.83%), limonene (6.98%), and menthone (4.44%). The chemical composition of the essential oil isolated from the aerial parts of M. haplocalyx presented here was not the same as those reported in previous studies. For example, the GC/MS results for the essential-oil composition of M. haplocalyx collected in four different regions of China (Yunnan, Guangxi, Jiangsu, and Hebei Province) were quite different [17]. The oil sample from Yunnan had eucalyptol (16.96%), limonene (18.45%), and pinene (22.00%) as the main constituents, a composition very similar to that of the sample from Hebei, which showed the same main components, but in different proportions, with eucalyptol (12.45%), pinene (23.00%), and limonene (33.33%). However, the oil sample from Guangxi had carvone (25.46%), limonene (28.75%), and eucalyptol (16.96%) as main compounds, while that
834
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 1. Chemical Composition of the Essential Oil Isolated from Mentha haplocalyx Aerial Parts Peak No.
Compound name and class
Chemical formula
RI a )
Relative content [%]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
o-Xylene Car-3-ene a-Phellandrene Caryophyllene Terpinene Limonene 2-Isopropyltoluene Isolimonene Camphor Isopulegol Menthone Menthol a-Terpineol Carvone Menthyl acetate Isopulegol acetate g-Elemene a-Bourbonene Elemene Farnesene Germacrene D 3,3-Dimethylhexane Diisooctyl phthalate
C8H10 C10H16 C10H16 C15H24 C10H16 C10H16 C10H14 C10H16 C10H16O C10H18O C10H18O C10H20O C10H18O C10H14O C12H22O2 C12H20O2 C15H24 C15H24 C15H24 C15H24 C15H24 C8H18 C24H38O4
811 1003 1005 1042 1059 1060 1104 1140 1143 1146 1156 1169 1188 1242 1260 1287 1340 1385 1390 1438 1479 1742 2135
2.70 0.87 0.34 1.00 0.91 6.98 0.11 0.10 0.10 0.56 4.44 59.71 0.19 4.85 7.83 0.14 0.15 0.13 0.10 0.08 1.32 0.09 0.18
Monoterpenoids Sesquiterpenoids Esters Others Total identified
79.16 1.78 8.15 2.79 92.88
a ) RI: Retention index determined on a HP-5MS column relative to a homologous series of n-alkanes (C5 – C36 ).
from Jiangsu was dominated by pinene (11.10%), limonene (42.30%), and pulegone (25.50%). Conclusively, the composition of the M. haplocalyx volatile oils was highly dependent on the regional habitat conditions, which might result in different biological activities of the oils. Contact Toxicity. As shown in Table 2, the essential oil obtained by hydrodistillation from M. haplocalyx aerial parts and three isolated main compounds, i.e., menthol, menthyl acetate, and limonene, exhibited strong contact toxicity against L. serricorne adults. Menthyl acetate, menthol, and limonene showed stronger contact toxicity against L. serricorne adults than the essential oil (LD50 values of 5.96, 7.91, and 13.7, resp., vs. 16.5 mg/adult). Among the three isolated compounds, menthyl acetate showed the strongest contact toxicity, although it was almost 25 times lower than that of pyrethrins used as positive control. However, compared with that of essential oils and essential-oil components reported in the literature, menthyl acetate showed the highest level of contact toxicity
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
835
Table 2. Contact Toxicity of the Essential Oil from Mentha haplocalyx Aerial Parts and Its Main Constituents against Lasioderma serricorne Adults Treatment
LD50 [mg/adult]
95% Fiducial limits
Slope SE
c2
Essential oil Menthol Menthyl acetate Limonene a ) Pyrethrins a )
16.5 7.91 5.96 13.7 0.24
14.93 – 18.23 5.22 – 9.89 4.18 – 7.84 11.63 – 16.18 0.16 – 0.35
0.22 0.03 1.89 0.36 1.06 0.17 3.21 0.54 1.31 0.20
4.25 7.94 9.57 11.04 17.36
a
) Data from Yang et al. [17].
against L. serricorne adults. Indeed, Yang et al. [18] reported LD50 values of 27.3, 65.6, 76.8, and 12.7 mg/adult for the Litsea cubeba essential oil, b-pinene, a-pinene, and linalool against L. serricorne adults. Data from Du et al. [19] showed that, among the compounds isolated from Myristica fragrans essential oil, elemicin exhibited the strongest contact toxicity against L. serricorne, with an LD50 value of 9.8 mg/adult. The other isolated compounds had lower contact toxicity, i.e., methyleugenol, eugenol, methylisoeugenol, myristicin, and safrole showed LD50 values of 12.8, 13.2, 21.3, 20.5, and 14.6 mg/adult, respectively. In another study [20], the essential oil of Artemisia argyi and its isolated compounds including eucalyptol, camphor, and b-caryophyllene also showed contact toxicity against L. serricorne adults with LD50 values of 6.42, 15.6, 11.3, and 35.5 mg/adult, respectively. Hence, these results suggest that menthyl acetate has potential for development as a novel natural insecticide for stored products. Repellent Activity. The results of the repellency assays for the essential oil and the three isolated constituents menthol, menthyl acetate, and limonene against L. serricorne adults are shown in the Figure, and the scale used to categorize the repellency is presented in Table 3. As can be seen, the repellent activity was not only dependent on the tested concentrations, but also on the duration of exposure. Among the tested oil and compounds, menthol tended to exhibit the strongest repellency at both 2 and 4 h after treatment, especially at the lower tested concentrations of 0.31 and 0.06 nl/cm2. At these lower concentrations and at 2 h after exposure, menthol showed even significantly stronger repellency than the positive control DEET. At three tested concentrations (7.86, 0.31, and 0.06 nl/cm2 ), menthol exhibited higher levels of repellency at 4 h after exposure (Class V, IV, and IV, resp.; Fig., b) than at 2 h after exposure (Class IV, III, and III, resp.; Fig., a.). This suggests that menthol not only exhibited a strong repellent activity, but also showed persistent efficacy. On the other hand, the other two compounds, menthyl acetate and limonene, tended to exhibit a Table 3. Scale Used to Categorize the Repellency of the Mentha haplocalyx Essential Oil and Its Main Constituents Class
Repellency [%]
Class
Repellency [%]
0 I II
> 0.01 – < 0.1 0.1 – 20.0 20.1 – 40.0
III IV V
40.1 – 60.0 60.1 – 80.0 80.1 – 100.0
836
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Figure. Percentage repellency (PR) of the Mentha haplocalyx essential oil and its three isolated main compounds against Lasioderma serricorne at a) 2 h and b) 4 h after exposure. Means with same letters above the bars do not differ significantly (P > 0.05) in ANOVA and TukeyÏs tests. The PR was subjected to an arcsine square-root transformation before ANOVA and TukeyÏs tests.
lower repellent activity than the positive control DEET. Nevertheless, they all contributed to the repellent activity of the total essential oil. The observed differences in repellency might be attributed to the different volatility of the oil and its isolated compounds and to different mechanisms of action on L. serricorne adults. However, there is not sufficient evidence to confirm these hypotheses at present. Hence, further investigations need to be conducted in the future. Conclusions. – The results suggest that M. haplocalyx essential oil and its major constituents might have potential use as ecological prevention and control of storage pests. Since the natural resources of M. haplocalyx are abundant, further investigations that focus on more detailed biological activity studies should be conducted, to elucidate the bioactivity mechanism of the tested essential oil for various applications.
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
837
This project was supported by the National Natural Science Foundation of China (No. 81374069), the Beijing Municipality Natural Science Foundation (No. 7142093), and the Fundamental Research Funds for the Central Universities. Experimental Part Plant Material. The aerial parts (0.6 kg) of Mentha haplocalyx were collected in October 2013 in Jining City (35.238 N and 116.338 E), Shandong Province, P. R. China. The plant was identified by Dr. Q. R. Liu, (College of Life Sciences, Beijing Normal University, Beijing, China) and a voucher specimen (BNU-CMH-Dushushan-2013-10-22-101) has been deposited with the Herbarium (BNU) of the College of Resources Science and Technology, Beijing Normal University. The aerial parts were air-dried for one week and ground to powder. Essential-Oil Extraction. The ground powder of M. haplocalyx aerial parts was subjected to hydrodistillation using a modified Clevenger-type apparatus for 6 h. The essential oil was extracted with hexane, dried (anh. Na2SO4 ), and stored in an airtight container in a refrigerator at 48. GC-FID and GC/MS Analyses of the Essential Oil. The GC-FID and GC/MS analyses were performed with a Thermo Finnigan Trace DSQ GC/MS instrument equipped with a flame ionization detector (FID) and a HP-5MS cap. column (30 m 0.25 mm i.d., filmthicknes 0.25 mm). The column temp. was programmed isothermal at 508 for 2 min, then rising from 50 to 1508 at 28/min, held isothermal at 1508 for 2 min, rising from 150 to 2508 at 108/min, and finally held isothermal at 2508 for 5 min; injector temp., 2508; carrier gas, He (1.0 ml/min); injection vol., 0.1 ml (1% soln. dil. in hexane); m/z range, 50 – 550 amu. Most oil constituents were identified by comparing their retention indices (RIs), determined rel. to the retention times (tR ) of a series of n-alkanes (C5 – C36 ), with those reported in the literature [5] [6] [9] [16]. Further identification of the isolated constitutes was made by comparing their mass spectra with those given in the NIST 05 and Wiley 275 mass-spectral libraries or in the literature [16]. The relative contents of the individual essential-oil components were obtained by averaging the GC peakarea-percentage reports. Bioassay-Directed Fractionation and Isolation of Limonene, Menthyl Acetate, and Menthol. The crude essential oil (10 ml) was chromatographed on a silica-gel column (SiO2 ; Qingdao Marine Chemical Plant, Shandong Province, P. R. China; 30 mm i.d., 500 mm length) by gradient elution with hexane first, then with hexane/AcOEt, and finally with AcOEt, to obtain 25 fractions. Based on the contact toxicity, Frs. 3, 8, and 16 were chosen for further fractionation. Using prep. thin-layer chromatography (PTLC), three purified compounds identified as limonene (6 1); 0.39 g), menthyl acetate (11; 0.47 g), and menthol (12; 3.34 g) were obtained. The identification of the isolated compounds was performed by NMR analysis (Bruker Avance DRX 500 instrument; in CDCl3 ; d in ppm rel. to Me4Si as internal standard) and comparison of the NMR data with the literature: limonene (colorless oil) [21], menthyl acetate (colorless oil) [22], and menthol (colorless crystals) [22]. Tested Insect Species. Lasioderma serricorne adults were obtained from laboratory cultures maintained for the last two years in dark in incubators at 29 18 and 70 – 80% rel. humidity. The insects were reared in glass containers (0.5 l) containing wheat flour and traditional Chinese medicinal materials at 12 – 13% moisture content mixed with yeast (wheat feed/yeast, 10 : 1, (w/w)). Adults used in all the experiments were ca. 7 2 d old, regardless of gender. Contact Toxicity. The contact toxicity of the essential oil and isolated compounds against L. serricorne adults was evaluated as described by Liu and Ho [23]. Range-finding studies were run to determine the appropriate testing concentrations. A serial dilution of the essential oil and its three isolated compounds (five concentrations) was prepared in hexane. Aliquots of 0.5 ml of the dilutions were applied topically to the dorsal thorax of the L. serricorne adults. Controls were determined using hexane. Both treated and control insects were then transferred to glass vials (ten insects per vial, five replicates per dose) with culture media and kept in incubators. The mortality was recorded after 24 h of treatment, 1)
Italic numbers in parentheses refer to the peak numbers in Table 1.
838
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
and the LD50 values were calculated using Probit analysis [24]. Positive control pyrethrins (pyrethrin 1, 24%; pyrethrin 2, 13%; cinnerin 1, 2%; cinnerin 2, 2%; jasmolin 1, 1%; jasmolin 2, 1%) were purchased from Dr. Ehrenstorfer GmbH (Germany). Repellency Test. The repellent activity of the essential oil and the pure compounds towards L. serricorne adults was tested using the area preference method [11]. The essential oil and the compounds were diluted in hexane to different concentrations (39.32, 7.86, 1.57, 0.31, and 0.06 nl/cm2 ), and hexane was used as control. Filter paper (9 cm in diameter) was cut in half, and 500 ml of treatment soln. was placed on one half of the filter paper and allowed to dry for 30 s. The other half (control) was treated with 500 ml of 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 half of the filter paper were made after 2 and 4 h. The percent repellency (PR) of each volatile oil/compound was then calculated using Eqn. 1: PR [%] ¼ [(Nc ¢ Nt )/(Nc þ Nt )] · 100
(1)
where Nc is the number of insects present in the negative-control half and Nt is the number of insects present in the treated half. Analysis of variance (one-way ANOVA and GLM univariate) and TukeyÏs test were conducted by using SPSS 20.0 for Windows 2007. PR Values were subjected to arcsine square-root transformation before variance and TukeyÏs tests. The average PR were then assigned to different classes (0 – V) according to the scale presented in Table 3 [25]. A commercial repellent, DEET (N,N-diethyl-3-methylbenzamide), was purchased from Dr. Ehrenstorfer GmbH and used as positive control. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
J. R. Ashworth, J. Stored Prod. Res. 1993, 29, 291. N. Magan, R. Hope, V. Cairns, D. Aldred, Eur. J. Plant Pathol. 2003, 109, 723. J. L. Zettler, F. H. Arthur, Crop Prot. 2000, 19, 577. M. B. Isman, Annu. Rev. Entomol. 2006, 51, 45. S. S. Chu, Q. Z. Liu, G. H. Jiang, Z. L. Liu, J. Serb. Chem. Soc. 2013, 78, 209. R. Fang, C. H. Jiang, X. Y. Wang, H. M. Zhang, Z. L. Liu, S. S. Du, Z. W. Deng, Molecules 2010, 15, 9391. Y. Liang, J. L. Li, S. Xu, N. N. Zhao, L. G. Zhou, J. Cheng, Z. L. Liu, J. Econ. Entomol. 2013, 106, 513. Z. L. Liu, S. S. Du, e-J. Chem. 2011, 8, 1937. Z. L. Liu, N. N. Zhao, C. M. Liu, L. Zhou, S. S. Du, Molecules 2012, 17, 12049. J. Lu, X. Su, J. Zhong, S. Afr. J. Sci. 2012, 108, 103. J. S. Zhang, N. N. Zhao, Q. Z. Liu, Z. L. Liu, S. S. Du, L. G. Zhou, Z. W. Deng, J. Agric. Food. Chem. 2011, 59, 9910. M. B. Isman, Pest Manag. Sci. 2008, 64, 8. S. S. Chu, Q. R. Liu, Z. L. Liu, Biochem. Syst. Ecol. 2010, 38, 489. S. Rajendran, V. Srianjini, J. Stored Prod. Res. 2008, 44, 126. C. Y. Liang, W. L. Li, H. Q. Zhang, B. R. Ren, Chin. Wild Plant Resour. 2003, 22, 9. R. P. Adams, ÐIdentification of Essential Oil Components by Gas Chromatography/Quadrupole Mass SpectroscopyÏ, Allured Publishing Corporation, Carol Stream, IL, 2001. X. H. Guo, D. G. Wan, M. L. Chen, X. Wang, L. Q. Huang, Nat. Prod. Res. Dev. 2011, 23, 1139. K. Yang, C. F. Wang, C. X. You, Z. F. Geng, R. Q. Sun, S. S. Guo, S. S. Du, Z. L. Liu, Z. W. Deng, J. Asia-Pacific Entomol. 2014, 17, 459. S.-S. Du, K. Yang, C. F. Wang, C. X. You, Z. F. Geng, S. S. Guo, Z. W. Deng, Z. L. Liu, Chem. Biodiversity 2014, 11, 1449. W. J. Zhang, C. X. You, K. Yang, R. Chen, Y. Wang, Y. Wu, Z. F. Geng, H. P. Chen, H. Y. Jiang, Y. Su, N. Lei, P. Ma, S. S. Du, Z. W. Deng, J. Oleo Sci. 2014, 63, 829.
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
839
[21] C. Pouchert, J. A. Behnke, in ÐAldrich Ô Library of 13C and 1H FT-NMR SpectraÏ, Aldrich Chemical Co., Milwaukee, WI, USA, 1993, Vol. 1, 70C, 71A, 71B. [22] D. A. Lanfranchi, M. C. Blanc, M. Vellutini, P. Bradesi, J. Casanova, F. Tomi, Magn. Reson. Chem. 2008, 46, 1188. [23] Z. L. Liu, S. H. Ho, J. Stored Prod. Res. 1999, 35, 317. [24] M. Sakuma, Appl. Entomol. Zool. 1998, 33, 339. [25] F. A. Talukder, P. E. Howse, J. Stored Prod. Res. 1995, 31, 55. Received July 7, 2014
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Contact Toxicity and Repellency of the Essential Oil from Mentha haplocalyx Br iq . against Lasioderma serricorne by Wen-Juan Zhang a ), Kai Yang a ), Chun-Xue You a ), Cheng-Fang Wang* a ) b ), Zhu-Feng Geng c ), Yang Su d ), Ying Wang a ), Shu-Shan Du* a ), and Zhi-Wei Deng c ) a
) Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Beijing Normal University, No. 19 Xinjiekouwai Street, Beijing 100875, P. R. China (phone/fax: þ 86-10-62208022; e-mail: [email protected]) b ) 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, P. R. China (phone/fax: þ 86-10-62208022; e-mail: [email protected]) c ) Analytical and Testing Center, Beijing Normal University, No. 19 Xinjiekouwai Street, Beijing 100875, P. R. China d ) Tibet Judicial Police Hospital, No. 10 Zhaji Road, Chengguan District, Lhasa 850000, Tibet Autonomous Region, P. R. China
The chemical composition of the essential oil obtained by hydrodistillation from the aerial parts of Mentha haplocalyx was investigated by GC-FID and GC/MS analyses. In sum, 23 components, representing 92.88% of the total oil composition, were identified, and the main compounds were found to be menthol (59.71%), menthyl acetate (7.83%), limonene (6.98%), and menthone (4.44%). By bioassayguided fractionation (contact toxicity), three compounds were obtained from the essential oil and identified as menthol, menthyl acetate, and limonene. The essential oil and the three isolated compounds exhibited potent contact toxicity against Lasioderma serricorne adults, with LD50 values of 16.5, 7.91, 5.96, and 13.7 mg/adult, respectively. Moreover, the oil and its isolated compounds also exhibited strong repellency against L. serricorne adults. At the lower concentrations tested and at 2 h after exposure, menthol showed even significantly stronger repellency than the positive control DEET. The study revealed that the bioactivity properties of the essential oil can be attributed to the synergistic effects of its diverse major and minor components, which indicates that the M. haplocalyx oil and its isolated compounds have potential for the development as natural insecticides and/or repellents to control insects in stored grains and traditional Chinese medicinal materials.
Introduction. – Antagonistic storage, i.e., storage of traditional Chinese medicinal materials with insect-repellent volatile odors together with medicinal materials vulnerable to insects, has been used as one of the traditional conservation methods to prevent insect harm. With the improvement of the sense of environmental protection and medication security, this method is believed to have broad prospects of application in the future. To inherit and further develop this traditional method of prevention and control of stored-grain insects, Mentha haplocalyx essential oil was investigated as potential insect repellent and Lasioderma serricorne adults as target insects. It was expected that this study would provide some theoretical bases for the conception of antagonistic storage. The cigarette beetle (L. serricorne), which is widely distributed, can not only cause significant losses because of the consumption of grains, but also elevate temperature Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
833
and moisture conditions that lead to an accelerated growth of molds, including toxigenic species [1] [2]. Currently, recommended pest-control methods for durable stored products rely heavily on the use of synthetic insecticides or fumigants, which may cause possible health hazards to warm-blooded animals, risks of environmental pollution, development of resistance by insects, and pest resurgence [3]. These problems have necessitated the search for alternative, ecologically safe insect-pest control methods [4]. The essential oils and their constituents isolated from many plants, including medicinal herbs, spices, and fruits, have been evaluated successfully for their insecticidal or repellent activity against stored-product insects and have even proven to be more effective than traditionally-used pesticides, in some cases [5 – 11]. Moreover, 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 [4] [12]. Essential oils from many plants including medicinal herbs, spices, and fruits have been evaluated with success for their insecticidal/repellent activity against stored-product insects/mites. In some cases, they have been proven more effective than organophosphorus pesticides that were traditionally used [6] [13] [14]. During a screening program for new agrochemicals from local wild plants and Chinese medicinal herbs, the essential oil isolated from M. haplocalyx aerial parts and some of its major compounds have been found to possess insecticidal and repellent activity against L. serricorne. M. haplocalyx, which is widely distributed in the Chinese provinces Jiangsu, Anhui, Jiangxi, and Zhejiang, is not only used as popular vegetable in China, but also for the treatment of nerve-center, breath, procreation, and digestive-system disorders [15]. However, there is no report on the insecticidal and repellent activities of this essential oil. Hence, the chemical composition and the insecticidal and repellent activities against L. serricorne of the M. haplocalyx essential oil was investigated, and three main essential-oil constituents were isolated. Results and Discussion. – Chemical Composition of the Essential Oil. The yield and density of the essential oil isolated from M. haplocalyx aerial parts were 1.67% (v/w) and 0.86 g/ml, respectively. The composition of the oil sample was characterized by GCFID and GC/MS analyses, and the components were identified based on the comparison of their retention indices (RIs) and mass spectra with those reported in the literature [5] [6] [9] [16]. In sum, 23 components, representing 92.88% of the total oil composition, were identified (Table 1). The main oil constituents were menthol (59.71%), menthyl acetate (7.83%), limonene (6.98%), and menthone (4.44%). The chemical composition of the essential oil isolated from the aerial parts of M. haplocalyx presented here was not the same as those reported in previous studies. For example, the GC/MS results for the essential-oil composition of M. haplocalyx collected in four different regions of China (Yunnan, Guangxi, Jiangsu, and Hebei Province) were quite different [17]. The oil sample from Yunnan had eucalyptol (16.96%), limonene (18.45%), and pinene (22.00%) as the main constituents, a composition very similar to that of the sample from Hebei, which showed the same main components, but in different proportions, with eucalyptol (12.45%), pinene (23.00%), and limonene (33.33%). However, the oil sample from Guangxi had carvone (25.46%), limonene (28.75%), and eucalyptol (16.96%) as main compounds, while that
834
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 1. Chemical Composition of the Essential Oil Isolated from Mentha haplocalyx Aerial Parts Peak No.
Compound name and class
Chemical formula
RI a )
Relative content [%]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
o-Xylene Car-3-ene a-Phellandrene Caryophyllene Terpinene Limonene 2-Isopropyltoluene Isolimonene Camphor Isopulegol Menthone Menthol a-Terpineol Carvone Menthyl acetate Isopulegol acetate g-Elemene a-Bourbonene Elemene Farnesene Germacrene D 3,3-Dimethylhexane Diisooctyl phthalate
C8H10 C10H16 C10H16 C15H24 C10H16 C10H16 C10H14 C10H16 C10H16O C10H18O C10H18O C10H20O C10H18O C10H14O C12H22O2 C12H20O2 C15H24 C15H24 C15H24 C15H24 C15H24 C8H18 C24H38O4
811 1003 1005 1042 1059 1060 1104 1140 1143 1146 1156 1169 1188 1242 1260 1287 1340 1385 1390 1438 1479 1742 2135
2.70 0.87 0.34 1.00 0.91 6.98 0.11 0.10 0.10 0.56 4.44 59.71 0.19 4.85 7.83 0.14 0.15 0.13 0.10 0.08 1.32 0.09 0.18
Monoterpenoids Sesquiterpenoids Esters Others Total identified
79.16 1.78 8.15 2.79 92.88
a ) RI: Retention index determined on a HP-5MS column relative to a homologous series of n-alkanes (C5 – C36 ).
from Jiangsu was dominated by pinene (11.10%), limonene (42.30%), and pulegone (25.50%). Conclusively, the composition of the M. haplocalyx volatile oils was highly dependent on the regional habitat conditions, which might result in different biological activities of the oils. Contact Toxicity. As shown in Table 2, the essential oil obtained by hydrodistillation from M. haplocalyx aerial parts and three isolated main compounds, i.e., menthol, menthyl acetate, and limonene, exhibited strong contact toxicity against L. serricorne adults. Menthyl acetate, menthol, and limonene showed stronger contact toxicity against L. serricorne adults than the essential oil (LD50 values of 5.96, 7.91, and 13.7, resp., vs. 16.5 mg/adult). Among the three isolated compounds, menthyl acetate showed the strongest contact toxicity, although it was almost 25 times lower than that of pyrethrins used as positive control. However, compared with that of essential oils and essential-oil components reported in the literature, menthyl acetate showed the highest level of contact toxicity
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
835
Table 2. Contact Toxicity of the Essential Oil from Mentha haplocalyx Aerial Parts and Its Main Constituents against Lasioderma serricorne Adults Treatment
LD50 [mg/adult]
95% Fiducial limits
Slope SE
c2
Essential oil Menthol Menthyl acetate Limonene a ) Pyrethrins a )
16.5 7.91 5.96 13.7 0.24
14.93 – 18.23 5.22 – 9.89 4.18 – 7.84 11.63 – 16.18 0.16 – 0.35
0.22 0.03 1.89 0.36 1.06 0.17 3.21 0.54 1.31 0.20
4.25 7.94 9.57 11.04 17.36
a
) Data from Yang et al. [17].
against L. serricorne adults. Indeed, Yang et al. [18] reported LD50 values of 27.3, 65.6, 76.8, and 12.7 mg/adult for the Litsea cubeba essential oil, b-pinene, a-pinene, and linalool against L. serricorne adults. Data from Du et al. [19] showed that, among the compounds isolated from Myristica fragrans essential oil, elemicin exhibited the strongest contact toxicity against L. serricorne, with an LD50 value of 9.8 mg/adult. The other isolated compounds had lower contact toxicity, i.e., methyleugenol, eugenol, methylisoeugenol, myristicin, and safrole showed LD50 values of 12.8, 13.2, 21.3, 20.5, and 14.6 mg/adult, respectively. In another study [20], the essential oil of Artemisia argyi and its isolated compounds including eucalyptol, camphor, and b-caryophyllene also showed contact toxicity against L. serricorne adults with LD50 values of 6.42, 15.6, 11.3, and 35.5 mg/adult, respectively. Hence, these results suggest that menthyl acetate has potential for development as a novel natural insecticide for stored products. Repellent Activity. The results of the repellency assays for the essential oil and the three isolated constituents menthol, menthyl acetate, and limonene against L. serricorne adults are shown in the Figure, and the scale used to categorize the repellency is presented in Table 3. As can be seen, the repellent activity was not only dependent on the tested concentrations, but also on the duration of exposure. Among the tested oil and compounds, menthol tended to exhibit the strongest repellency at both 2 and 4 h after treatment, especially at the lower tested concentrations of 0.31 and 0.06 nl/cm2. At these lower concentrations and at 2 h after exposure, menthol showed even significantly stronger repellency than the positive control DEET. At three tested concentrations (7.86, 0.31, and 0.06 nl/cm2 ), menthol exhibited higher levels of repellency at 4 h after exposure (Class V, IV, and IV, resp.; Fig., b) than at 2 h after exposure (Class IV, III, and III, resp.; Fig., a.). This suggests that menthol not only exhibited a strong repellent activity, but also showed persistent efficacy. On the other hand, the other two compounds, menthyl acetate and limonene, tended to exhibit a Table 3. Scale Used to Categorize the Repellency of the Mentha haplocalyx Essential Oil and Its Main Constituents Class
Repellency [%]
Class
Repellency [%]
0 I II
> 0.01 – < 0.1 0.1 – 20.0 20.1 – 40.0
III IV V
40.1 – 60.0 60.1 – 80.0 80.1 – 100.0
836
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Figure. Percentage repellency (PR) of the Mentha haplocalyx essential oil and its three isolated main compounds against Lasioderma serricorne at a) 2 h and b) 4 h after exposure. Means with same letters above the bars do not differ significantly (P > 0.05) in ANOVA and TukeyÏs tests. The PR was subjected to an arcsine square-root transformation before ANOVA and TukeyÏs tests.
lower repellent activity than the positive control DEET. Nevertheless, they all contributed to the repellent activity of the total essential oil. The observed differences in repellency might be attributed to the different volatility of the oil and its isolated compounds and to different mechanisms of action on L. serricorne adults. However, there is not sufficient evidence to confirm these hypotheses at present. Hence, further investigations need to be conducted in the future. Conclusions. – The results suggest that M. haplocalyx essential oil and its major constituents might have potential use as ecological prevention and control of storage pests. Since the natural resources of M. haplocalyx are abundant, further investigations that focus on more detailed biological activity studies should be conducted, to elucidate the bioactivity mechanism of the tested essential oil for various applications.
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
837
This project was supported by the National Natural Science Foundation of China (No. 81374069), the Beijing Municipality Natural Science Foundation (No. 7142093), and the Fundamental Research Funds for the Central Universities. Experimental Part Plant Material. The aerial parts (0.6 kg) of Mentha haplocalyx were collected in October 2013 in Jining City (35.238 N and 116.338 E), Shandong Province, P. R. China. The plant was identified by Dr. Q. R. Liu, (College of Life Sciences, Beijing Normal University, Beijing, China) and a voucher specimen (BNU-CMH-Dushushan-2013-10-22-101) has been deposited with the Herbarium (BNU) of the College of Resources Science and Technology, Beijing Normal University. The aerial parts were air-dried for one week and ground to powder. Essential-Oil Extraction. The ground powder of M. haplocalyx aerial parts was subjected to hydrodistillation using a modified Clevenger-type apparatus for 6 h. The essential oil was extracted with hexane, dried (anh. Na2SO4 ), and stored in an airtight container in a refrigerator at 48. GC-FID and GC/MS Analyses of the Essential Oil. The GC-FID and GC/MS analyses were performed with a Thermo Finnigan Trace DSQ GC/MS instrument equipped with a flame ionization detector (FID) and a HP-5MS cap. column (30 m 0.25 mm i.d., filmthicknes 0.25 mm). The column temp. was programmed isothermal at 508 for 2 min, then rising from 50 to 1508 at 28/min, held isothermal at 1508 for 2 min, rising from 150 to 2508 at 108/min, and finally held isothermal at 2508 for 5 min; injector temp., 2508; carrier gas, He (1.0 ml/min); injection vol., 0.1 ml (1% soln. dil. in hexane); m/z range, 50 – 550 amu. Most oil constituents were identified by comparing their retention indices (RIs), determined rel. to the retention times (tR ) of a series of n-alkanes (C5 – C36 ), with those reported in the literature [5] [6] [9] [16]. Further identification of the isolated constitutes was made by comparing their mass spectra with those given in the NIST 05 and Wiley 275 mass-spectral libraries or in the literature [16]. The relative contents of the individual essential-oil components were obtained by averaging the GC peakarea-percentage reports. Bioassay-Directed Fractionation and Isolation of Limonene, Menthyl Acetate, and Menthol. The crude essential oil (10 ml) was chromatographed on a silica-gel column (SiO2 ; Qingdao Marine Chemical Plant, Shandong Province, P. R. China; 30 mm i.d., 500 mm length) by gradient elution with hexane first, then with hexane/AcOEt, and finally with AcOEt, to obtain 25 fractions. Based on the contact toxicity, Frs. 3, 8, and 16 were chosen for further fractionation. Using prep. thin-layer chromatography (PTLC), three purified compounds identified as limonene (6 1); 0.39 g), menthyl acetate (11; 0.47 g), and menthol (12; 3.34 g) were obtained. The identification of the isolated compounds was performed by NMR analysis (Bruker Avance DRX 500 instrument; in CDCl3 ; d in ppm rel. to Me4Si as internal standard) and comparison of the NMR data with the literature: limonene (colorless oil) [21], menthyl acetate (colorless oil) [22], and menthol (colorless crystals) [22]. Tested Insect Species. Lasioderma serricorne adults were obtained from laboratory cultures maintained for the last two years in dark in incubators at 29 18 and 70 – 80% rel. humidity. The insects were reared in glass containers (0.5 l) containing wheat flour and traditional Chinese medicinal materials at 12 – 13% moisture content mixed with yeast (wheat feed/yeast, 10 : 1, (w/w)). Adults used in all the experiments were ca. 7 2 d old, regardless of gender. Contact Toxicity. The contact toxicity of the essential oil and isolated compounds against L. serricorne adults was evaluated as described by Liu and Ho [23]. Range-finding studies were run to determine the appropriate testing concentrations. A serial dilution of the essential oil and its three isolated compounds (five concentrations) was prepared in hexane. Aliquots of 0.5 ml of the dilutions were applied topically to the dorsal thorax of the L. serricorne adults. Controls were determined using hexane. Both treated and control insects were then transferred to glass vials (ten insects per vial, five replicates per dose) with culture media and kept in incubators. The mortality was recorded after 24 h of treatment, 1)
Italic numbers in parentheses refer to the peak numbers in Table 1.
838
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
and the LD50 values were calculated using Probit analysis [24]. Positive control pyrethrins (pyrethrin 1, 24%; pyrethrin 2, 13%; cinnerin 1, 2%; cinnerin 2, 2%; jasmolin 1, 1%; jasmolin 2, 1%) were purchased from Dr. Ehrenstorfer GmbH (Germany). Repellency Test. The repellent activity of the essential oil and the pure compounds towards L. serricorne adults was tested using the area preference method [11]. The essential oil and the compounds were diluted in hexane to different concentrations (39.32, 7.86, 1.57, 0.31, and 0.06 nl/cm2 ), and hexane was used as control. Filter paper (9 cm in diameter) was cut in half, and 500 ml of treatment soln. was placed on one half of the filter paper and allowed to dry for 30 s. The other half (control) was treated with 500 ml of 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 half of the filter paper were made after 2 and 4 h. The percent repellency (PR) of each volatile oil/compound was then calculated using Eqn. 1: PR [%] ¼ [(Nc ¢ Nt )/(Nc þ Nt )] · 100
(1)
where Nc is the number of insects present in the negative-control half and Nt is the number of insects present in the treated half. Analysis of variance (one-way ANOVA and GLM univariate) and TukeyÏs test were conducted by using SPSS 20.0 for Windows 2007. PR Values were subjected to arcsine square-root transformation before variance and TukeyÏs tests. The average PR were then assigned to different classes (0 – V) according to the scale presented in Table 3 [25]. A commercial repellent, DEET (N,N-diethyl-3-methylbenzamide), was purchased from Dr. Ehrenstorfer GmbH and used as positive control. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
J. R. Ashworth, J. Stored Prod. Res. 1993, 29, 291. N. Magan, R. Hope, V. Cairns, D. Aldred, Eur. J. Plant Pathol. 2003, 109, 723. J. L. Zettler, F. H. Arthur, Crop Prot. 2000, 19, 577. M. B. Isman, Annu. Rev. Entomol. 2006, 51, 45. S. S. Chu, Q. Z. Liu, G. H. Jiang, Z. L. Liu, J. Serb. Chem. Soc. 2013, 78, 209. R. Fang, C. H. Jiang, X. Y. Wang, H. M. Zhang, Z. L. Liu, S. S. Du, Z. W. Deng, Molecules 2010, 15, 9391. Y. Liang, J. L. Li, S. Xu, N. N. Zhao, L. G. Zhou, J. Cheng, Z. L. Liu, J. Econ. Entomol. 2013, 106, 513. Z. L. Liu, S. S. Du, e-J. Chem. 2011, 8, 1937. Z. L. Liu, N. N. Zhao, C. M. Liu, L. Zhou, S. S. Du, Molecules 2012, 17, 12049. J. Lu, X. Su, J. Zhong, S. Afr. J. Sci. 2012, 108, 103. J. S. Zhang, N. N. Zhao, Q. Z. Liu, Z. L. Liu, S. S. Du, L. G. Zhou, Z. W. Deng, J. Agric. Food. Chem. 2011, 59, 9910. M. B. Isman, Pest Manag. Sci. 2008, 64, 8. S. S. Chu, Q. R. Liu, Z. L. Liu, Biochem. Syst. Ecol. 2010, 38, 489. S. Rajendran, V. Srianjini, J. Stored Prod. Res. 2008, 44, 126. C. Y. Liang, W. L. Li, H. Q. Zhang, B. R. Ren, Chin. Wild Plant Resour. 2003, 22, 9. R. P. Adams, ÐIdentification of Essential Oil Components by Gas Chromatography/Quadrupole Mass SpectroscopyÏ, Allured Publishing Corporation, Carol Stream, IL, 2001. X. H. Guo, D. G. Wan, M. L. Chen, X. Wang, L. Q. Huang, Nat. Prod. Res. Dev. 2011, 23, 1139. K. Yang, C. F. Wang, C. X. You, Z. F. Geng, R. Q. Sun, S. S. Guo, S. S. Du, Z. L. Liu, Z. W. Deng, J. Asia-Pacific Entomol. 2014, 17, 459. S.-S. Du, K. Yang, C. F. Wang, C. X. You, Z. F. Geng, S. S. Guo, Z. W. Deng, Z. L. Liu, Chem. Biodiversity 2014, 11, 1449. W. J. Zhang, C. X. You, K. Yang, R. Chen, Y. Wang, Y. Wu, Z. F. Geng, H. P. Chen, H. Y. Jiang, Y. Su, N. Lei, P. Ma, S. S. Du, Z. W. Deng, J. Oleo Sci. 2014, 63, 829.
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
839
[21] C. Pouchert, J. A. Behnke, in ÐAldrich Ô Library of 13C and 1H FT-NMR SpectraÏ, Aldrich Chemical Co., Milwaukee, WI, USA, 1993, Vol. 1, 70C, 71A, 71B. [22] D. A. Lanfranchi, M. C. Blanc, M. Vellutini, P. Bradesi, J. Casanova, F. Tomi, Magn. Reson. Chem. 2008, 46, 1188. [23] Z. L. Liu, S. H. Ho, J. Stored Prod. Res. 1999, 35, 317. [24] M. Sakuma, Appl. Entomol. Zool. 1998, 33, 339. [25] F. A. Talukder, P. E. Howse, J. Stored Prod. Res. 1995, 31, 55. Received July 7, 2014
