- freely available
Molecules 2015, 20(12), 21939-21945; doi:10.3390/molecules201219818
Abstract: The essential oil obtained by hydrodistillation from Alpinia kwangsiensis rhizomes was investigated by GC-MS. A total of 31 components representing 92.45% of the oil were identified and the main compounds in the oil were found to be camphor (17.59%), eucalyptol (15.16%), β-pinene (11.15%) and α-pinene (10.50%). These four compounds were subsequently isolated and the essential oil and four isolated compounds exhibited potent insecticidal activity against Lasioderma serricorne adults. During the assay, it was shown that the essential oil exhibited both potential contact (LD50 = of 24.59 μg/adult) and fumigant (LC50 = of 9.91 mg/L air) toxicity against Lasioderma serricorne. The study revealed that the insecticidal activity of the essential oil can be attributed to the synergistic effects of its diverse major components, which indicates that oil of Alpinia kwangsiensis and its isolated compounds have potential to be developed into natural insecticides to control insects in stored grains and traditional Chinese medicinal materials.
The cigarette beetle, Lasioderma serricorne (Fabricius, Coleoptera: Anobiidae) is widely distributed around the world, especially in tropical and subtropical areas . As one of the major insects in stored tobacco products, cereal grains and processed foods, Lasioderma serricorne causes significant losses of grains, foods or traditional Chinese medicinal materials stored in warehouses . Its development and survival are closely affected by the type of food, temperature and humidity, thus, its life cycle can be quite different . Control of Lasioderma serricorne populations around the world is primarily dependent upon continued applications of phosphine . However, its repeated use for decades has led to serious problems, especially in insecticide resistance, disruption of biological control by natural enemies, environmental and human health concerns, rising cost of production and lethal effects on non-target organisms [4,5]. There is therefore an urgent need to find an alternative strategy to control this pest. Due to their abundance and natural biodegradable features, plants have played a significant role in the development of insecticides . In recent years, essential oils have received a great deal of attention as pest control agents. They are typically characterized by low toxicity to human and animals, high volatility, and toxicity to stored grain insect pests . In a previous report , essential oils were reported as applicable to the protection of stored products.
Alpinia, a member of the ginger family (Zingiberaceae), comprises more than 250 species found in Southeast Asia, extending from Japan in the north to Australia in the south and into the Western Pacific . Alpinia kwangsiensis T. L. Wu et Senjen is one of the 50 species of Alpinia found in China. The rhizomes of A. kwangsiensis are used in Chinese traditional medicine for the treatment of abdominal pain, stomach cold vomiting and traumatic injury [10,11]. The chemical constituents of rhizomes of this medicinal herb have been studied . During our mass screening program for new agrochemicals from the wild plants, essential oil of A. kwangsiensis rhizomes was found to possess strong insecticidal activity against the cigarette beetle. According to recent literature, it was shown that there are no reports on the insecticidal activity of the essential oil from A. kwangsiensis rhizomes. Hence, we decided to use the essential oil obtained from A. kwangsiensis rhizome parts and its main chemical constituents to investigate its insecticidal activities against L. serricorne.
2. Results and Discussion
2.1. Chemical Compounds of the Essential Oil
The essential oil was obtained by hydrodistillation from the dried rhizomes of A. kwangsiensis with a yield of 0.16% (v/w) and a density of 0.86 g/mL. The chemical constituents of A. kwangsiensis essential oil are shown in Table 1. The main constituents of A. kwangsiensis essential oil were camphor (17.59%), eucalyptol (15.16%), β-pinene (11.15%) and α-pinene (10.50%) followed by α-terpineol (7.28%), camphene (6.85%) and limonene (5.22%). A total of 31 components were identified in the essential oil of A. kwangsiensis, accounting for 92.45% of the total oil (Table 1).
|Peak No.||Components||RI a||%RA b||Identification Methods c|
|1||Tricyclene||919||0.17 ± 0.07||MS, RI|
|2||3-Thujene||925||0.06 ± 0.02||MS, RI|
|3||Benzaldehyde||928||0.11 ± 0.09||MS, RI|
|4||α-Pinene||932||10.50 ± 0.06||MS, RI, Co|
|5||Camphene||943||6.85 ± 0.04||MS, RI|
|6||β-Pinene||974||11.15 ± 0.03||MS, RI|
|7||α-Phellandrene||999||0.18 ± 0.12||MS, RI|
|8||Benzyl alcohol||1008||0.09 ± 0.04||MS, RI|
|9||β-Terpinene||1013||2.86 ± 0.02||MS, RI|
|10||Eucalyptol||1017||15.16 ± 0.03||MS, RI|
|11||Limonene||1040||5.22 ± 0.02||MS, RI|
|12||γ-Terpinene||1060||0.20 ± 0.05||MS, RI|
|13||Terpinolene||1083||0.26 ± 0.03||MS, RI, Co|
|14||Linalool||1090||1.16 ± 0.02||MS, RI|
|15||Perillene||1094||1.05 ± 0.04||MS, RI|
|16||Campholenic aldehyde||1112||0.71 ± 0.05||MS, RI, Co|
|17||Camphor||1120||17.59 ± 0.06||MS, RI|
|18||Borneol||1159||3.16 ±0.05||MS, RI|
|19||Terpinen-4-ol||1162||1.60 ± 0.12||MS, RI|
|20||Myrtenal||1166||0.38 ± 0.10||MS, RI|
|21||α-Terpineol||1174||7.28 ± 0.02||MS, RI|
|22||Myrtenol||1180||3.25 ± 0.03||MS, RI|
|23||Bornyl acetate||1289||0.58 ± 0.06||MS, RI|
|24||Methyl geranate||1307||0.11 ± 0.10||MS, RI|
|25||Neryl alcohol||1347||0.45 ± 0.03||MS, RI|
|26||α-Caryophyllene||1454||0.39 ± 0.09||MS, RI|
|27||Germacrene D||1480||0.19 ± 0.06||MS, RI|
|28||Valencen||1484||0.32 ± 0.02||MS, RI|
|29||Myristicin||1489||0.84 ± 0.06||MS, RI|
|30||Spathulenol||1578||0.16 ± 0.07||MS, RI|
|31||τ-Cadinol||1617||0.42 ± 0.04||MS, RI|
|Total||92.45 ± 0.06|
a Retention index (RI) relative to the homologous series of n-alkanes on the HP-5 MS capillary column. b Relative area (peak area relative to the total peak area). c MS = mass spectrum, Co = co-injection with standard compound.
However, the principal components of essential oil from A. kwangsiensis rhizomes analyzed in this work differed from those in previous report, for example, in the previous study, cinnamic acid methyl ester was the main isolated and identified component, making up 94.54% of the essential oil from A. kwangsiensis , a fact possibly due to differences in the place of origin and plant parts used.
2.2. Insecticidial Activity
The essential oil of A. kwangsiensis rhizome parts showed contact toxicity against L. serricorne adults with an LD50 value of 24.59 μg/adult (Table 2). Compared with the positive control, pyrethrins, the essential oil showed much less contact toxicity on L. serricorne adults. However, compared with other essential oils in the literature, for example, the essential oil of Litsea cubeba (LD50 = 27.33 μg/adult), the essential oil of A. kwangsiensis possessed stronger contact toxicity against L. serricorne, .
The essential oil of A. kwangsiensis rhizomes also possessed fumigant activity against L. serricorne with an LC50 value of 9.91 mg/L air (Table 2). However, the currently used grain fumigant, phosphine was reported to have fumigant activity against L. serricorne adults with LC50 value of 0.75 mg/L air . The fumigant activity of the essential oil against the L. serricorne was thus 13 magnitudes lower than that of the commercial fumigant phosphine. Compared with other essential oils in previous studies, the essential oil of A. kwangsiensis rhizomes exhibited roughly the same level fumigant toxicity against the cigarette beetles, e.g. essential oils of Pistacia lentiscus (LC50 = 8.44 mg/L air) and Elsholtzia stauntonii (LC50 = 10.99 mg/L air) [6,15], and exhibited stronger fumigant toxicity against L. serricorne than the essential oil of Agastache foeniculum (Lamiaceae) (LC50 = 21.57 mg/L air) . The crude essential oil of L. muscari aerial parts showed pronounced contact toxicity against T. castaneum, L. serricorne and L. bostrychophila, with LD50 values of 13.36, 11.28 μg/adult and 21.37 μg/cm2, respectively (Table 2).
Among the four main compounds, camphor and eucalyptol account for 17.59% and 15.16% of the essential oil and possessed stronger contact (LD50 = 11.30 and 15.58 μg/adult) and fumigant toxicity (LC50 = 2.91 and 5.18 mg/L air) against L. serricorne than that of the essential oil, β-pinene and α-pinene, respectively. What’s more, β-pinene and α-pinene exhibited weaker contact (LD50 = 65.87 and 77.28 μg/adult) and fumigant toxicity (LC50 = 35.69 and 37.57 mg/L air) against L. serricorne than that of the essential oil (Table 2). Thus, the contact and fumigant toxicity of the essential oil might be attributable to a synergistic effect of the sum of the pure components activity.
|Treatments||Fumigant Toxicity||Contact Toxicity|
|LC50 a (μg/mL Air)||95% FL c||Chi Square (χ2)||LD50 b (μg/adult)||95% FL c||Chi Square (χ2)|
The results of this work suggest that among the four main essential oil components, camphor and eucalyptol showed stronger contact and fumigant toxicity against L. serricorne. In previous reports, the four components have been demonstrated to possess insecticidal activities against several stored product insects such as Sitophilus zeamais, Tribolium castaneum, Leptinotarsa decemlineata, and broadbean weevil [18,19,20,21]. The high volatility of these toxic compounds likely delivered fumigant toxicity by vapor action via the respiratory system, but further work is needed to confirm their extract mode of action.
3. Experimental Section
3.1. Plant Material
The fresh rhizomes (2.0 kg) of A. kwangsiensis were harvested from Xishuangbanna (21°08′–22°36′ N latitude and 99°56′–101°50′ E longitude), Yunnan Province, China in August 2013. The plant was identified, and a voucher specimen (BNU-dushushan-2013-08-12-25) was deposited at the Herbarium (BNU) of the College of Resources Science and Technology, Beijing Normal University.
Cultures of the cigarette beetle, L. serricorne, were maintained in the laboratory without exposure to any insecticide before use. They were reared on a sterilized diet (wheat flour/yeast, 10:1, w/w) at 29–30 °C, 70%–80% r.h. in the dark. The unsexed adult beetles used in all the experiments were about 1–2 weeks old. All containers housing insects used in experiments were made escape proof with a coating of polytetrafluoroethylene (Fluon (ICI America Inc., Bridgewater, NJ, USA)).
3.3. Isolation of the Essential Oil and Purification of the Four Constituents
The sample was air-dried and ground to powder using a grinding mill. The powders were subjected to hydrodistillation for 6 h using a modified Clevenger-type apparatus and extracted with n-hexane. Anhydrous sodium sulphate was used to remove water after extraction. The essential oil was stored in airtight containers in a refrigerator at 4 °C for subsequent experiments.
The crude essential oil (9 mL) was chromatographed on a silica gel column (45 mm i.d., 500 mm length, 160 to 200 mesh, Qingdao Marine Chemical Plant, Qingdao, Shandong Province, China) by gradient elution with mixtures of two solvents: petroleum ether and ethyl acetate (the proportion of the two solvents was changed from 100:0–0:100). Each 150 mL of eluate was collected as a fraction and concentrated at 35 °C. According to thin layer chromatography (TLC), fractions with similar profiles were combined to yield 35 fractions. Of these, fractions 3, 9, 13 and 25 were further separated by repeated silica gel column chromatography and PTLC to afford four pure compounds for determining their structure. The isolated compounds were identified based on their nuclear magnetic resonance spectra. 1H- and 13C-NMR spectra were recorded on an AMX500 instrument (Bruker-Biospin, Billerica, MA, USA) using CDCl3 as the solvent with TMS as internal standard. The spectral data of camphor (1.36 g, 96%) matched the data given in ; the spectral data of eucalyptol (0.97 g, 97%) matched that in ; the spectral data were also identical to the published data of β-pinene (0.56 g, 96%) , and α-pinene (0.33 g, 95%) , respectively.
3.4. GC-MS and GC-FID Analysis
The obtained essential oil was packed in lightless amber vials. A sample of the oil was diluted in n-hexane and subjected to analysis by gas chromatography coupled to a flame ionization detector (GC-FID) and gas chromatography coupled to spectrometry (GC-MS) at Beijing Normal University (Beijing, China).
GC-MS analysis was performed on a Trace DSQ instrument (Thermo Finnigan, Lutz, FL, USA) equipped with a flame ionization detector and a HP-5MS (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 literature. The retention indices were determined in relation to a homologous series of n-alkanes (C5–C36) under the same operating conditions. Further identification was made by comparison of their mass spectra with those stored in NIST 05 and Wiley 275 libraries or with mass spectra from reference . Relative percentages of the individual components of the essential oil were obtained by averaging the GC peak area% reports.
3.5. Contact Toxicity
The contact toxicity of the crude essential oil and isolated compounds against L. serricorne adults was measured as described by Liu and Ho . Range-finding studies (96.75–0.22 μg/adult, 1.5 times dilution) were run to determine the appropriate test 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. Ten insects were used for each concentration and control, and the experiment was replicated five times. Both treated and control insects were then transferred to glass vials with culture media and kept in incubators. Mortality was recorded after 24 h and the LD50 values were calculated using Probit analysis  (SPSS V20.0, IBM Inc., Armonk, NY, USA). The positive control, pyrethrins (pyrethrin I and II, 37%) were purchased from Dr. Ehrenstorfer (Augsburg, Germany).
3.6. Fumigant Toxicity
The fumigant activity of the crude essential oil and isolated compounds against L. serricorne adults was tested 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. A Whatman filter paper disc (diameter 2.0 cm) was impregnated with 20 μL dilution and then placed on the underside of the screw cap of a glass vial (diameter 2.5 cm, height 5.5 cm, volume 25 mL). The solvent was allowed to evaporate for 10 s before the cap was placed tightly on the glass vial, each of which contained 10 insects inside to form a sealed chamber. Fluon was used inside the glass vial to prevent insects from contacting the treated filter paper. Preliminary experiments demonstrated that 10 s was sufficient for the evaporation of solvents. n-Hexane was used as a control. Five replicates were carried out for all treatments and controls, and they were incubated under the same conditions as rearing. Mortality was determined after 24 h of treatment, and the LC50 values were calculated using Probit analysis  (IBM SPSS V20.0, IBM Inc., Armonk, NY, USA).
This study demonstrates that the essential oil obtained from A. kwangsiensis rhizomes and its main components (camphor and eucalyptol) exhibit significant insecticidal activity on L. serricorne. These findings also suggest that the bioactivities of the essential oil may be attributed to its bioactive compounds. Considering that the currently used fumigants are synthetic insecticides, the essential oil from A. kwangsiensis rhizomes, camphor and eucalyptol are quite promising leads, and they also show potential for development as possible natural fumigants for the control of stored product insect pests. However, for the practical application of the essential oil and compounds as novel fumigants, further studies on the safety of the essential oil and compounds to humans and on formulation development are needed.
This project was supported by the National Natural Science Foundation of China (No. 81374069), Beijing Municipal Natural Science Foundation (No. 7142093) and Fundamental Research Funds for the Central Universities. The authors thank Liu Q.R. from College of Life Sciences, Beijing Normal University, Beijing 100875, for the identification of the investigated medicinal herb.
Yan Wu, Wen-Juan Zhang, Dong-Ye Huang, Ying Wang and Jian-Yu Wei designed the research. Zhi-Hua Li, Jian-Sheng Sun, Jia-Feng Bai, Zhao-Fu Tian and Ping-Juan Wang performed the research and analyzed the data. Wen-Juan Zhang, Ying Wang and Shu-Shan Du wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
- Ashworth, J.R. The biology of Lasioderma serricorne. J. Stored Prod. Res. 1993, 29, 291–303. [Google Scholar] [CrossRef]
- Kim, S.; Park, C.; Ohh, M.H.; Cho, H.C.; Ahn, Y.J. Contact and fumigant activity of aromatic plant extracts and essential oils against Lasioderma serricorne (Coleoptera: Anobiidae). J. Stored Prod. Res. 2003, 39, 11–19. [Google Scholar] [CrossRef]
- White, N.D.G.; Leesch, J.G. Integrated Management of Insects in Stored Products; Marcel Dekker Inc.: New York, NY, USA, 1995. [Google Scholar]
- Rajendran, S.; Narasimhan, K.S. Phosphine resistance in the cigarette beetle Lasioderma serricorne (Coleoptera: Anobiidae) and overcoming control failures during fumigation of stored tobacco. Int. J. Pest Manag. 1994, 40, 207–210. [Google Scholar] [CrossRef]
- Jovanovic, Z.; Kostic, M.; Popovic, Z. Grain-protective properties of herbal extracts against the bean weevil Acanthoscelides obtectus Say. Ind. Crop. Prod. 2007, 26, 100–104. [Google Scholar] [CrossRef]
- Bachrouch, O.; Jemaa, J.M.B.; Talou, T.; Marzouk, B.; Abderraba, M. Fumigant toxicity of Pistacia lentiscus essential oil against Tribolium castaneum and Lasioderma serricorne. Bull. Insectol. 2010, 63, 129–135. [Google Scholar]
- Batish, D.R.; Sing, P.H.; Kohli, K.R.; Kaur, S. Eucalyptus essential oil as a natural pesticide. For. Ecol. Manag. 2008, 256, 2166–2174. [Google Scholar] [CrossRef]
- Sahaf, B.Z.; Moharramipour, S.; Meshkatalsadat, M.H. Fumigant toxicity of essential oil from Vitex pseudonegundo against Tribolium castaneum (Herbst) and Sitophilus orysae (L.). J. Asia Pac. Entomol. 2008, 11, 175–179. [Google Scholar] [CrossRef]
- Smith, R.M. Alpinia (Zingiberaceae): A proposed new infrageneric classification. Edinburgh J. Bot. 1990, 47, 71–75. [Google Scholar] [CrossRef]
- Medicinal Materials Company in Yunnan Province, List of Traditional Chinese Medicine Resources in Yunnan Province; Science Press: Beijing, China, 1993.
- Guang, Y.H.; Peng, Z.C.; Zhang, Z.L.; Zhang, L.X. Microscopic identification of Dai medicine Alpiniae kwangsiensis Rhizoma and its confused species. J. Chin. Med. Mater. 2014, 37, 411–414. [Google Scholar]
- Na, Z. Chemical constituent analysis of volatile oils from rhizome of Alpinia blepharocalyx and A. kwangsiensis. J. Plant Res. Environ. 2006, 15, 73–74. [Google Scholar]
- Yang, K.; Wang, C.F.; You, C.X.; Geng, Z.F.; Sun, R.Q.; Guo, S.S.; Du, S.S.; Liu, Z.L.; Deng, Z.W. Bioactivity of essential oil of Litsea cubeba from China and its main compounds against two stored product insects. J. Asia Pac. Entomol. 2014, 17, 459–466. [Google Scholar] [CrossRef]
- Naik, J.; Ramesh, B.S.; Gurudutt, K.N. Fumigation studies on cured large cardamom (Amomum subulatum Roxb.) capsules. J. Food Sci. Technol. 2005, 42, 531–533. [Google Scholar]
- Lv, J.H.; Su, X.H.; Zhong, J.J. Fumigant activity of Elsholtzia stauntonii extract against Lasioderma serricorne. S. Afr. J. Sci. 2012, 108, 7–8. [Google Scholar]
- Ebadollahi, A.; Safaralizadeh, M.; Pourmirza, A.; Gheibi, S. Toxicity of essential oil of Agastache foeniculum (Pursh) Kuntze to Oryzaephilus Surinamensis L. and Lasioderma Serricorne F. J. Plant Prot. Res. 2010, 50, 215–219. [Google Scholar] [CrossRef]
- Zhang, W.J.; Yang, K.; You, C.X.; Wang, Y.; Wang, C.F.; Wu, Y.; Geng, Z.F.; Su, Y.; Du, S.S.; Deng, Z.W. Bioactivity of essential oil from Artemisia stolonifera (Maxim.) Komar. and its main compounds against two stored-product insects. J. Oleo Sci. 2015, 64, 299–307. [Google Scholar] [CrossRef] [PubMed]
- Chu, S.S.; Du, S.S.; Liu, Z.L. Fumigant compounds from the essential oil of Chinese Blumea balsamifera leaves against the Maize Weevil (Sitophilus zesmais). J. Chem. 2013, 2013, 1–7. [Google Scholar] [CrossRef]
- Liu, Z.L.; Jiang, G.H.; Zhou, L.; Liu, Q.Z. Analysis of the essential oil of Dipsacus japonicus flowering aerial parts and its insecticidal activity against Sitophilus zeamais and Tribolium castaneum. Z. Naturforsch. C. 2013, 68, 13–18. [Google Scholar] [CrossRef] [PubMed]
- Kordali, S.; Aslan, I.; Calmasur, O.; Cakir, A. Toxicity of essential oils isolated from three Artemisia species and some of their major components to granary weevil Sitophilus granarius (L.) (Coleoptera: Curculinonidae). Ind. Crop. Prod. 2006, 23, 162–170. [Google Scholar] [CrossRef]
- Kordali, S.; Kesdek, M.; Cakir, A. Toxicity of monoterpenes against larvae and adults of Colorado potato beetle Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Ind. Crop. Prod. 2007, 26, 278–297. [Google Scholar] [CrossRef]
- Tanaka, H.; Chou, J.Y.; Mine, M.; Kuroboshi, M. The oxidation of alcohols in N-oxyl-immobilized silica gel/aqueous NaOCl disperse systems. A prominent access to a column-flow system. B. Chem. Soc. Jpn. 2004, 77, 1745–1755. [Google Scholar] [CrossRef]
- Ashnagar, A.; Naseri, N.G.; Bayemani, A. Isolation and determination of the major chemical compounds present in essential oil of the leaves of Myrtus plant grown in Khuzestan Province of Iran. Asian J. Chem. 2009, 21, 4969–4975. [Google Scholar]
- Badjah Hadj Ahmed, A.Y.; Meklati, B.Y.; Waton, H.; Pham, Q.T. Structural studies in the bicyclo [3.1.1] heptane series by 1H- and 13C-NMR. Mag. Reson. Chem. 1992, 30, 807–816. [Google Scholar] [CrossRef]
- Adams, R.P. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. J. Am. Soc. Mass Spectrom. 2001, 16, 1902–1903. [Google Scholar]
- Liu, Z.L.; Ho, S.H. Bioactivity of the essential oil extracted from Evodia rutaecarpa Hook f. et Thomas against the grain storage insects, Sitophilus zeamais Motsch and Tribolium castaneum (Herbst). J. Stored Prod. Res. 1999, 35, 317–328. [Google Scholar] [CrossRef]
- Sakuma, M. Probit analysis of preference data. Appl. Entomol. Zool. 1998, 33, 339–347. [Google Scholar]
- Sample Availability: Not available.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).