CHEM. RES. CHINESE UNIVERSITIES 2010, 26(6), 888—892
Comparison of Constituents and Insecticidal Activities of Essential Oil from Artemisia lavandulaefolia by Steam Distillation and Supercritical-CO2 Fluid Extraction YUAN Hai-bin1,2, SHANG Li-na1, WEI Chun-yan3 and REN Bing-zhong1* 1. Jilin Key Laboratory of Animal Resource Conservation and Utilization, School of Life Sciences, Northeast Normal University, Changchun 130024, P. R. China; 2. College of Agronomy, Jilin Agricultural University, Changchun 130118, P. R. China; 3. Jilin Entry-Exit Inspection and Quarantine Bureau, Changchun 130062, P. R. China Abstract Essential oil was extracted from Artemisia lavandulaefolia DC. by steam distillation(SD) and supercritical-CO2 fluid extraction(SFE), respectively. The constituents of the essentil oils extracted with those two methods were analyzed by gas chromatography-mass spectrometry(GC-MS) and insecticidal activities of the essential oils were evaluated, then the results were compared to assess their biological activity. Thirty-one compounds were identified in the essential oil extracted by SD, and its main components were eucalyptol, α,α,4-trimethyl-3-cyclohexene1-methanol and so on. Twenty-two compounds were identified for the essential oil extracted by SFE, and its main components were cyclodecene, n-hexadecanoic acid and so on. Six chemical compositions were all contained in the essential oils extracted by the two methods, i.e., eucalyptol, α,α,4-trimethyl-3-cyclohexene-1-methanol, caryophyllene, [3aS-(3aα,3bβ,4β,7α,7aS)]-octahydro-7-methyl-3-methylene-4-(1-methylethyl)-1H-cyclopenta[1,3]cyclopropa[1,2]benzene, nerolidol and (–)-Spathulenol. The fumigation toxicity of the essential oil obtained by means of SD to the adults of Sitophilus zeamais is significantly higher than that of the essential oil by means of SFE. The contact toxicity of the essential oil obtained by means of SFE to the adults of S. zeamais is higher than that of the essential oil obtained by means of SD, but the difference is not significant. Keywords Artemisia lavandulaefolia DC.; Essential oil; Supercritical-CO2 fluid extraction; Steam distillation; GC-MS; Insecticidal activity Article ID 1005-9040(2010)-06-888-05
1
Introduction
Wild wormwood(Artemisia lavandulaefolia DC.) is a perennial herb with fragrance belonging to Asteraceae, Artemisia. It is widely distributed around mountains and fields, mainly in the roadsides, forest edges, hillsides, shrub, river and lake lands. The plant can be used as the medicine instead of wormwood (Artemisia argyl Lem)[1], and has the effect of dispelling the cold and damp, warming in the menses, hemostasis and so on. A. lavandulaefolia has been applied in traditional medicines from many cultures for a variety of indications, including cholecystitis, liver ailments, stomatitis, fever, bronchitis and chronic
cervicitis[2,3]. The essential oil of A. lavandulaefolia contains various chemicals[4,5] and has been proved to inhibit the growth of crop pathogens Pyricμlaria grisea and Rhizoctonia solani[6]. Steam distillation(SD) is a traditional method for essential oil extraction. Supercritical-CO2 fluid extraction(SFE) is a new method with increased application in recent years. It is widely used for simple handling and high extraction. The extraction rates and chemical composition vary with the extraction methods[7,8]. The components of essential oil vary, leading to a change of it in biological activity[9]. In this study, the constituents and the insecticidal activities of essentil oils from Artemisia lavandulaefolia by SD and SFE were compared, and
——————————— *Corresponding author. E-mail:
[email protected] Received July 22, 2010; accepted October 14, 2010. Supported by the Technology Development Project of Jilin Province, China(Nos.20090571 and 20090262), the Development and Reform Project of Jilin Province, China(No.20071033), the State Administration of Quality Supervision Project, China(No. 2008IK253), the Jingyue Development Zone Project of Changchun City, China(No.2007C010) and the Scientific Research Fund of Jilin Agricultural University, China(No.2007040).
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the relationship between the insecticidal activity and its constituents was analyzed. This study provides a reference for developing wild wormwood as a new type of plant pesticide resource.
2 2.1
Materials and Methods Materials and Apparatuses
The upper parts of the wild wormwood(Artemisia lavandulaefolia DC.) in the flowering period were collected from Changchun area, dried, crushed and reserved at room temperature for use. The main experimental apparatuses included a 5LHA29508 supercritical fluid extraction apparatus (Nantong Huaan Supercritical Extraction Industrial Company, China) and an Agilent 6890 N-5973 IMSD Gas Chromatography Mass Spectrometer(Agilent, USA). Analytically pure reagents were used. 2.2
Extraction of Essential Oil
2.2.1 Steam Distillation The dried wild wormwood was soaked for 12 h in water, and then distilled for 3 h. The deep green essential oil was obtained. 2.2.2 Supercritical CO2 Extraction The dried wild wormwood was crushed to the particle sizes of 0.3―0.5 mm, and put into the extraction kettle. The optimal conditions were as follows: 20 MPa, 40 °C, 3 h, analytical pressure 10 MPa, analytical temperature 45 °C. The yellow essential oil was obtained. 2.3
Isolation and Identification of Essential Oil
GC conditions were as follows: HP-5 MS quartz capillary column, 30 m×0.32 mm×0.25 μm; injector temperature: 280 °C; carrier gas: helium; carrier gas flow rate: 1 mL/min; programmed temperature(60 °C to maintain the initial 1 min, 25 °C/min rise to 200 °C to maintain 10 min, then 5 °C/min rise to 280 °C, maintained 30 min). 2.4
Determination of Insecticidal Activity
2.4.1 Tested Insects Sitophilus zeamais Motschulsky was rearing in the laboratory[t=25―28 °C; RH=70%―80%; n(D): n(L)=12:12]. 2.4.2 Determination of Fumigation Activity The fumigation activity was evaluated by Flask sealed fumigating method. The insects were put into
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the 30 mL flasks. Essential oil of the wild wormwood was dropped on the filter paper strips(2 cm×7.5 cm) at the doses of 0.5, 1.0, 1.5 and 2.0 μL. After acetone volatile, the filter paper strips were hung on the bottles and the bottles were sealed with the transparent tape. The same amount of acetone was used in the control group. Ten adult insects were included in each bottle, three replicates were used in each treament. The death of the insects was checked after 72 h, and the corrected mortality was calculated. 2.4.3 Determination of Contact Activity Drop method was used. Essential oil was diluted with acetone into four concentrations: 5%, 10%, 15%, and 20%(volume fraction). Acetone was used alone as control. 1 μL of the dilution was dropped on the dorsal surface of adults S. zeamais via a microsyringe, and ten adults per replication were used for control experiment and each concentration. After 24 h, the death of the insects was checked, and corrected mortality was calculated.
3 3.1
Results and Discussion Composition of Essential Oil
The compositions of essential oils extracted by SFE and SD were analyzed by GC-MS. Gas chromatography area normalization method for the determination of the relative content of each component was used. After comparison with the standard spectrum library(NIST05a), thirty-one compounds which have more than 80% similarity were identified for the essential oil extracted by SD(Table 1), and its main components were found to be eucalyptol(10.74%), α,α,4-trimethyl-3-cyclohexene-1-methanol(5.26%), 4carene(4.00%), 4-methyl-1-(1-methylethyl)-bicycle [3.1.0]hexan-3-one(3.63%), caryophyllene oxide (2.60%), caryophyllene(2.16%) and so on. Twentytwo compounds were identified for the essential oil extracted by SFE(Table 1), and its main components were found to be cyclodecene(13.77%), n-hexadecanoic acid(10.41%), (Z,Z,Z)-9,12,15-octadecatrien-1-ol (5.22%), squalene(2.47%), caryophyllene(2.24%), and so on. Six chemical compositions were all included in the essential oils extracted by the two methods, i.e., eucalyptol, α,α,4-trimethyl-3-cyclohexene-1-methanol, caryophyllene, [3aS-(3aα, 3bβ, 4β, 7α, 7aS)]-octahydro-7-methyl-3-methylene-4-(1-methylethyl)-1H-cyclopenta[1,3]cyclopropa[1,2]benzene, nerolidol and (–)-spathulenol.
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Constituents and relative contents of the essential oil of A. lavandulaefolia
Compound
SD
Mass fraction(%) SFE
1 2
4-Methylene-1-(1-methylethyl)-bicyclo[3.1.0]hexane β-Myrcene
0.43 0.70
— —
3
α-Phellandrene
0.34
—
4
1-Methyl-4-(1-methylethyl)-1,3-cyclohexadiene
1.07
—
5
β-Phellandrene
0.45
—
6
3,7-Dimethyl-1,3,6-octatriene
0.28
—
7
Eucalyptol
8
3-Carene
9
10.74
0.37
1.98
—
1-Methyl-4-(1-methylethylidene)-cyclohexene
0.89
—
10
4-Methyl-1-(1-methylethyl)-bicyclo[3.1.0]hexan-3-one
3.63
—
11
Thujone
1.24
—
12
1-Methyl-4-(1-methylethyl)-2-cyclohexen-1-ol
1.26
—
13
α,α,4-Trimethyl-3-cyclohexene-1-methanol
5.26
0.99
14
3-Methyl-6-(1-methylethyl)-2-cyclohexen-1-ol
0.92
—
15
6,6-Dimethyl-bicyclo[3.1.1]hept-2-ene-2-methanol
0.38
—
16
2-Methyl-5-(1-methylethenyl)-2-cyclohexen-1-ol
0.46
—
17
trans-2-Caren-4-ol
0.46
—
18
Acetic acid, 1,7,7-trimethyl-bicyclo[2.2.1]hept-2-yl ester
0.54
—
19
2-Methyl-5-(1-methylethenyl)-2-cyclohexen-1-one
0.20
—
20
3-Methyl-6-(1-methylethyl)-2-cyclohexen-1-one
0.91
—
21
(+)-4-Carene
4.00
—
22
Caryophyllene
2.16
2.24
23
Eugenol
1.62
—
24
1H-Cyclopenta[1,3]cyclopropa[1,2]benzene, octahydro-7-methyl-3-methylene-4-(1-methylethyl)-,
1.63
1.16
[3aS-(3aα,3bβ,4 β,7α,7aS)] 25
β-Humulene
1.07
—
26
r-Elemene
0.63
—
27
Naphthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)
0.30
—
28
Nerolidol
0.24
0.21
29
(–)-Spathulenol
1.99
1.58
30
Caryophyllene oxide
2.60
—
31
α-Cadinol
0.49
—
32
cis-β-Terpineol
—
0.38
33
Borneol
—
0.92
34
1,7,7-Trimethyl-bicyclo[2.2.1]heptan-2-one
—
0.32
35
Copaene
—
0.30
36 37
2,2-Dimethy-l-3-methylene-bicyclo[2.2.1]heptane
—
0.20
10,10-Dimethyl-2,6-dimethylenebicyclo[7.2.0] undecan-5β-ol
—
0.50
38
n-Hexadecanoic acid
—
10.41
39
Phytol
—
1.14
40
Cyclodecene
—
13.77
41
(Z,Z,Z)-9,12,15-Octadecatrien-1-ol
—
5.22
42
(E)-9-Tetradecen-1-ol
—
0.37
43
6,7-Dimethoxy-2H-1-benzopyran-2-one
—
0.67
44
Arteannuin b
—
1.34
45
Eicosanoic acid
—
0.52
46
Azuleno[4,5-b]furan-2,7-dione,3,3a,4,5,9a,9b-hexahydro-3,6,9-trimethyl-, [3R-(3α,3aβ,9aβ,9bα)]-
—
0.65
47
Squalene
—
2.47
3.2
Fumigation Activities Comparison
Table 2
The fumigation activities of essential oils from A. lavandulaefolia by SD and SFE were determined. The corrected mortality after 72 h is list in Table 2. The fumigation activity of essential oil extracted by SD was significant higher than that extracted by SFE (Table 2). At a dose of 1.0 μL, the corrected
Corrected mortality(%) of fumigating activity of A. lavandulaefolia essential oil against S. zeamais after 72 h Morality*(%)
Dose/μL
SD
SFE
0.5 1.0
53.23±0.65b 73.46±1.12 b
26.59±1.19 a 43.02±0.85 a
1.5
84.85±2.10 b
64.58±1.67 a
b
2.0 94.96±0.86 73.41±0.81a * a and b means they have significant difference at 0.05 level.
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mortality of the essential oil extracted by SD was 73.46% while the corrected mortality of the essential oil extracted by SFE was 43.02%. The fumigant activities of the essential oils extracted by both the two methods increased with the dosage increase. 3.3
Contact Activity Comparison
The contact activities of essential oils extracted by SD and SFE were determined, with the corrected mortality after 72 h shown in Table 3. Table 3
Corrected mortality(%) of contact activity of A. lavandulaefolia essential oil against S. zeamais after 72 h
Dose/μL
Mortality*(%) SD
SFE
0.05 0.10
34.83±0.85 a 62.95±1.14 a
47.24±1.75 a 71.91±1.58 a
0.15
a
a
75.29±1.08
83.18±0.67
0.20 84.29±2.15 a 91.03±1.05 a * a means they have no significant difference at 0.05 level.
The contact activity of the essential oil extracted by SFE is higher than that extracted by SD, but the difference is not significant. At a dose of 0.10 μL, the corrected mortality of the essential oil extracted by SFE was 71.91% and the corrected mortality of the essential oil extracted by SD was 62.95%. The contact activities of the essential oils extracted by both the two methods increased with the dosage. 3.4 Relevance Analysis Between Insecticidal Activity and Constituent of Essential Oil The essentil oil of A. lavandulaefolia extracted by SD showed much better fumigant activities than that extracted by SFE against the adults of Sitophilus zeamais(S. zeamais). High content(mass fraction) of terpenes was eucalyptol(10.74%), α,α,4-trimethyl-3-cyclohexene-1methanol(5.26%), 4-carene(4.00%), 4-methyl-1-(1methylethyl)-bicycle[3.1.0]hexan-3-one(3.63%), caryophyllene oxide(2.60%), caryophyllene(2.16%) and 3-carene(1.98%). But carene and caryophyllene oxide were not found in the essentil oil extracted with SFE. Another high content of chemical components of non-terpenes was 4-methyl-1-(1-methylethyl)-bicycle [3.1.0]hexan-3-one(3.63%), and it was also not found in the essentil oil extracted with SFE. These results indicate that carene, caryophyllene oxide and 4-methyl-1-(1-methylethyl)-bicycle[3.1.0]hexan-3-one may be related with the fumigant activities. The contact toxicity of essentil oil from A.
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lavandulaefolia extracted by SFE is higher than that extracted by SD against the adults of S. Zeamais. However, the difference is not significant. High content of terpenes was cyclodecene(13.77%), squalene (2.47%), caryophyllene(2.24%) and arteannuin b (1.34%). Cyclodecene and arteannuin b were not observed in the essentil oil extracted with SD. Two high content chemical components of non-terpenes were n-hexadecanoic acid and (Z,Z,Z)-9,12,15-octadecatrien-1-ol and they were also not found in the essentil oil extracted with SFE either. The results show that cyclodecene, arteannuin b, n-hexadecanoic acid and (Z,Z,Z)-9,12,15-octadecatrien-1-ol may be related with the contact toxicity. 3.5
Discussion
The insecticidal activities and the constituents of essentil oils obtained by means of SD and SFE were apparently different. The fumigation activity of essential oil extracted by SD is significantly higher than that extracted by SFE. The contact activity of the essential oil extracted by SFE is higher than that extracted by SD, but the difference is not significant. The identical compositions in the essentil oils obtained by SD and SFE were eucalyptol, α,α,4trimethyl-3-cyclohexene-1-methanol, caryophyllene, [3aS-(3aα,3bβ,4β,7α,7aS)]-octahydro-7-methyl-3methylene-4-(1-methylethyl)-1H-cyclopenta[1,3]cyclopropa[1,2]benzene, nerolidol and (–)-spathulenol. Among them, eucalyptol and nerolidol have high antibacterial ability[10,11], which indicates that essential oil may have good insecticidal activity. The essential oil of A. lavandulaefolia extracted by SD showed much better fumigant activity than that extracted by SFE against the adults of S. zeamais. Thirty-one compounds were identified for the essential oil extracted by SD, in which eucalyptol(10.74%), α,α,4-trimethyl-3-cyclohexene-1-methanol(5.26%), 4-carene(4.00%), 4-methyl-1-(1-methylethyl)-bicycle[3.1.0]hexan-3-one(3.63%), caryophyllene oxide(2.60%) and caryophyllene(2.16%) were main components. Terpenes were one class of the high content of compounds that accounted for 83.4%. Many studies show that terpenes have repellent activities, antifeedant activities, oviposition deterrentant activety, contact toxicity and fumigant activities
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in plant essentil oil . The results of this experiment are the same with those of the studies. Results from experimental analysis reveal that eucalyptol was the highest concentration among compounds, eucalyptol may result in higher fumigation activity of essential oil. The results of ref.[15] also show that eucalyptol with the highest fumigant activity in essential oil of Vitex negundo on S. zeamais. The essentil oil of A. lavandulaefolia extracted by SFE exhibited much better contact toxicity than that extracted by SD against S. zeamais, but the difference was not significant. Twenty-two compounds were identified for the essential oil extracted by SFE, in which cyclodecene(13.77%), n-hexadecanoic acid(10.41%), (Z,Z,Z)9,12,15-octadecatrien-1-ol(5.22%), squalene(2.47%), caryophyllene(2.24%) were main components. Terpenes accounted for 60% of the compounds. The cyclodecene was the highest concentration among compounds. The contact toxicity may be related to the cyclodecene. Furthermore, arteannuin b(1.34%) was another compound obtained from the essentil oil of A. lavandulaefolia extracted with SFE. Some studies show that A. annua contains artemisinin and A. annua extraction have contact activity against a variety of insects[16―18]. So, arteannuin b may result in higher contact toxicity of essential oil. Shen et al.[19] and Zhang et al.[20] reported that the number of chemical components extracted by SFE was larger than that by SD, which is different from our results. The reason may be the extraction condition and plant materials. Some researches show that 1-phenyl-2,4-pentadiyne, famesene and citronellal were effective insecticide chemical composition in other plant essential oil of Artemisia[8,9]. These compounds were not obtained using these two extraction methods. The reason may
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