Chemical Composition and Antimicrobial Activity of the Essential Oil from Ambrosia trifida L.

Abstract

The essential oil obtained by steam distillation of dried aerial parts of Ambrosia trifida L. from Northeast China was analyzed by GC and GC–MS. The essential oil yield based on dried plant material was 0.12% and thirty-five compounds (corresponding to 86.7% of the total weight) were identified. The main components were: bornyl acetate (15.5%), borneol (8.5%), caryophyllene oxide (8.3%), α-pinene (8.0%), germacrene D (6.3%), β-caryophyllene (4.6%), trans-carveol (2.9%), β-myrcene (2.6%), camphor (2.4%) and limonene (3.2%). A. trifida essential oil demonstrated bactericidal and fungicidal activity against six bacterial strains and two fungal strains, using the agar diffusion method.

Introduction

The genus Ambrosia (Asteraceae) is classified as part of the tribe Heliantheae. It comprises some 35-40 species, mostly found in the Americas. A. trifida L. (great or giant ragweed) and A. artemisiifolia L. (common ragweed) are two common species found in China, where A. trifida occurs mainly in Northeast China. A. trifida is American in origin and in ecological terms it is spreading worldwide as a pioneer species [1]. It invades cultivated fields and reduces crop productivity [2,3]. This plant is wild and sometimes cultivated by the North American Indians for food and medicine. It is used as a tea in the treatment of pneumonia, fevers, nausea, intestinal cramps, diarrhoea and mucous discharges and menstrual disorders [4]. A. trifida leaves are very astringent, emetic and febrifugal and are applied to insect bites and various skin complaints. The pollen is harvested commercially and manufactured into pharmaceutical preparations for the treatment of allergies to the plant, but ingesting or touching the pollen of A. trifida can cause allergic reactions and hay fever in some people [5].

Many kinds of metabolites including sesquiterpene lactones, phenolics, ambrosin, isabelin, and psilostachyin have been isolated and identified from A. artemisiifolia [6,7] and more recently, the chemical composition and antimicrobial activity of essential oil from A. artemisiifolia have been reported [8]. However, there are only a few investigations on phytochemistry of A. trifida. Several sesquiterpenes and thiarubrines were isolated and identified from A. trifida tissues [6,9]. The volatile chemicals from A. trifida leaves and their allelopathic potential on other plant species were investigated in our previous paper [10]. This study concerns chemical composition and antimicrobial activity of the essential oil from A. trifida.

Results and Discussion

The essential oil obtained by steam distillation of aerial parts of A. trifida collected in Northeast China was isolated with a yield of 0.12% (based on dried plant material). The isolated oil was a yellowish liquid with a strong aromatic fragrance. Table 1 shows its chemical composition. Thirty-five compounds were identified by comparison of their retention indexes and the mass spectra of each GC component with those of standards and with reported data. Terpenes and their derivatives predominated, with the most abundant one being bornyl acetate (15.5%), followed by borneol (8.5%), caryophyllene oxide (8.3%), α-pinene (8.0%), germacrene D (6.3%), β-caryophyllene (4.6%), trans-carveol (2.9%), β-myrcene (2.6%), camphor (2.4%) and limonene (2.2%), respectively These main components comprised more than 86 % of the essential oil. Although most of these compounds are well documented as essential oil components in various plant species [11], to our knowledge this is the first report of their occurrence in the essential oil of A. trifida.

Interestingly, there were significant differences between the main components of the essential oil of A. trifida L. and those previously determined in A. artemisiifolia L. [8], which belongs to the same genus. Thus, terpene alcohols such as spathulenol, longipinanol, isospathuienol, α-eudesmol and γ-epi-eudesmol are quantitatively abundant in A. artemisiifolia oil, whilst they were only present in much smaller quantities in A. trifida oil (Table 2).

Table 1. Chemical composition of the essential oil from A. trifida from Northeast China

Peak Number	Compound	Retention index	Relative amount (%)
1	α-pinene	935	8.0
2	β-myrcene	992	2.6
3	limonene	1031	2.2
4	α-terpinolene	1087	1.8
5	camphor	1143	2.4
6	cis-β-terpineol	1145	0.6
7	borneol	1170	8.5
8	trans-carveol	1217	2.9
9	bornyl acetate	1275	15.5
10	α-cubebene	1350	1.5
11	α-terpinyl acetate	1354	0.6
12	isoledene	1376	1.1
13	β-caryophyllene	1420	4.6
14	β-farnesene	1450	1.8
15	germacrene D	1480	6.3
16	(E)-methylisoeugenol	1500	1.4
17	γ-cadinene	1520	0.8
18	δ-cadinene	1530	0.7
19	longipinanol	1565	1.1
20	caryophyllene oxide	1581	8.3
21	spathulenol	1583	0.6
22	globulol	1586	0.4
23	carotol	1594	0.6
24	cubenol	1630	0.4
25	isospathulenol	1638	0.8
26	β-cedren-9-α-ol	1645	1.9
27	α-eudesmol	1654	0.7
28	γ-eudesmol acetate	1780	1.0
29	hexahydrofarnesyl acetone	1844	1.5
30	2-methyl-nonadecane	1890	0.9
31	isophytol	1945	0.5
32	manoyl oxide	2000	1.1
33	heneicosane	2100	0.6
34	docosane	2200	0.5
35	abieta-8,11,13-trien-7-one	2312	0.5
-	total isolate	-	86.7
-	unknown	-	13.3

Compounds were listed in order of elution. Retention indices were calculated from retention times relative to those of n-alkanes (C5 to C26) on the non-polar HP-5 column.

Table 1. Chemical composition of the essential oil from A. trifida from Northeast China

Peak Number	Compound	Retention index	Relative amount (%)
1	α-pinene	935	8.0
2	β-myrcene	992	2.6
3	limonene	1031	2.2
4	α-terpinolene	1087	1.8
5	camphor	1143	2.4
6	cis-β-terpineol	1145	0.6
7	borneol	1170	8.5
8	trans-carveol	1217	2.9
9	bornyl acetate	1275	15.5
10	α-cubebene	1350	1.5
11	α-terpinyl acetate	1354	0.6
12	isoledene	1376	1.1
13	β-caryophyllene	1420	4.6
14	β-farnesene	1450	1.8
15	germacrene D	1480	6.3
16	(E)-methylisoeugenol	1500	1.4
17	γ-cadinene	1520	0.8
18	δ-cadinene	1530	0.7
19	longipinanol	1565	1.1
20	caryophyllene oxide	1581	8.3
21	spathulenol	1583	0.6
22	globulol	1586	0.4
23	carotol	1594	0.6
24	cubenol	1630	0.4
25	isospathulenol	1638	0.8
26	β-cedren-9-α-ol	1645	1.9
27	α-eudesmol	1654	0.7
28	γ-eudesmol acetate	1780	1.0
29	hexahydrofarnesyl acetone	1844	1.5
30	2-methyl-nonadecane	1890	0.9
31	isophytol	1945	0.5
32	manoyl oxide	2000	1.1
33	heneicosane	2100	0.6
34	docosane	2200	0.5
35	abieta-8,11,13-trien-7-one	2312	0.5
-	total isolate	-	86.7
-	unknown	-	13.3

Compounds were listed in order of elution. Retention indices were calculated from retention times relative to those of n-alkanes (C5 to C26) on the non-polar HP-5 column.

Table 2. Main composition of the essential oils from A. trifida and A. artemisiifolia

A. trifida (Relative amount, %)	A. artemisiifolia [8] (Relative amount, %)
bornyl acetate (15.5%)	germacrene D (24.1%),
borneol (8.5%)	limonene (16.8%)
caryophyllene oxide (8.3%)	α-pinene (8.0%)
α-pinene (8.0%)	β-myrcene (7.4%)
germacrene D (6.3%)	borneol (2.9%)
β-caryophyllene (4.6%)	spathulenol (1.6%)
trans-carveol (2.9%)	longipinanol (1.6%)
β-myrcene (2.6%)	isospathulenol (1.5%)
camphor (2.4%)	α-eudesmol(1.4%)
limonene (2.2%)	γ-epi-eudesmol (1.3%)

Table 2. Main composition of the essential oils from A. trifida and A. artemisiifolia

A. trifida (Relative amount, %)	A. artemisiifolia [8] (Relative amount, %)
bornyl acetate (15.5%)	germacrene D (24.1%),
borneol (8.5%)	limonene (16.8%)
caryophyllene oxide (8.3%)	α-pinene (8.0%)
α-pinene (8.0%)	β-myrcene (7.4%)
germacrene D (6.3%)	borneol (2.9%)
β-caryophyllene (4.6%)	spathulenol (1.6%)
trans-carveol (2.9%)	longipinanol (1.6%)
β-myrcene (2.6%)	isospathulenol (1.5%)
camphor (2.4%)	α-eudesmol(1.4%)
limonene (2.2%)	γ-epi-eudesmol (1.3%)

Results of the antimicrobial activity tests of the A. trifida essential oil against bacteria and fungi are given in Table 3. Dilute solutions (2% or 4%) of the oil demonstrated bactericidal and fungicidal activity against all microorganisms tested. Particularly significant were the inhibition zone diameters observed for 4% essential oil solution against Staphylococcus aureus and Candida albicans, and for 2% essential oil solution against Klebsiella pneumoniae, while Bacillus subtilis, Pseudomonas aeruginosa and Asperigillus niger were less sensitive to the oil.

Table 3. Antimicrobial activity of the essential oil from A. trifida from Northeast China

Microorganisms	Diameters of inhibition zones (mm)
	2% oil solution	4% oil solution	Control
Gram (+) bacteria
Bacillus subtilis	14.0	12.5	9.0
Staphylococcus aureus	22.5	25.5	10.0
Enterococcus faecalis	20.0	18.5	9.5
Gram (-) bacteria
Escherichia coli	20.0	17.0	10.5
Pseudomonas aeruginosa	14.5	16.5	9.0
Klebsiella pneumoniae	23.5	20.5	11.0
Fungi
Asperigillus niger	16.0	14.5	11.0
Candida albicans	19.0	23.5	10.5

Data are average diameters of inhibition zones from two independent determinations.

Table 3. Antimicrobial activity of the essential oil from A. trifida from Northeast China

Microorganisms	Diameters of inhibition zones (mm)
	2% oil solution	4% oil solution	Control
Gram (+) bacteria
Bacillus subtilis	14.0	12.5	9.0
Staphylococcus aureus	22.5	25.5	10.0
Enterococcus faecalis	20.0	18.5	9.5
Gram (-) bacteria
Escherichia coli	20.0	17.0	10.5
Pseudomonas aeruginosa	14.5	16.5	9.0
Klebsiella pneumoniae	23.5	20.5	11.0
Fungi
Asperigillus niger	16.0	14.5	11.0
Candida albicans	19.0	23.5	10.5

Data are average diameters of inhibition zones from two independent determinations.

Conclusions

Our GC and GC-MS study of the essential oil from A. trifida from Northeast China led to the identification of 35 compounds, representing 86.7% of the total mass. The major components were terpenes and their derivatives, and the most prominent one was bornyl acetate (15.5%). The antimicrobial activity results presented here demonstrate that this plant essential oil has a commercial potential.

Experimental

Plant Material and Isolation of the Essential Oil

Flowering aerial parts of A. trifida were collected from the Shenyang Experimental Station of Ecology, Chinese Academy of Sciences (Northeast China, N 41º31´, E 123º24´) in August 2005. Harvested plant material was air-dried in a shaded area at ambient temperature. A voucher specimen was deposited in Institute of Applied Ecology, Chinese Academy of Sciences, China. The essential oil was obtained by steam distillation in a Clevenger-type apparatus, according to the literature [12]. Isolated oil was dried over a layer of anhydrous sodium sulphate and submitted to chemical and microbiological analysis.

Essential Oil Analysis

The oil was analyzed by capillary GC and GC-MS. Oil (25 μL) was diluted in dichloromethane (2 mL) before injection and 1 μL of this solution was directly used for analysis. GC analysis of the oil was performed on a Hewlett-Packard 5890A gas chromatograph equipped with a split/splitless injector (250 ºC, split ratio 1:30) and a FID operated at 250 ºC. A HP-5 fused silica capillary column (25 m × 0.32 mm i.d., 0.52 μm film thickness) was used. The operating conditions were as follows: 5 min at 50°C initial hold, then from 50-280°C at 2.5 °C/min.; injector temperature, 250 °C; detector temperature, 280 °C; carrier gas, H₂ at 1 mL/min. Retention indices were determined with C5 to C26 alkane standards as reference. Relative amounts of individual components are based on peak areas obtained without FID response factor correction. Identification of the components was assigned by comparison of their retention indices and confirmed by GC-MS [13,14]. GC–MS analyses were performed on a Hewlett-Packard 5890/5970A system, equipped with a HP-5 MS capillary column (30 m ×0.25 mm i.d., film thickness 0.25 μm). Helium was used as carrier gas, the inlet pressure was 200 kPa, the linear velocity 1 mL/min (70 ºC), split flow 10mL/min. Temperature programme: 40–260 °C at a rate of 4 °C/min; injector temperature, 250 °C; detector temperature, 260 °C. The electron energy was 70 eV. Mass spectra were obtained by automatic scanning of the mass range m/z 45 to 629 amu. at 2 scan/s. Chromatographic peaks were checked for homogeneity with the aid of the mass chromatograms of the characteristic fragment ions reported in the NIST 98 and WILEY 138 databases.

Antimicrobial Activity

The essential oil diluted in absolute ethanol was each tested for antimicrobial activity by the agar diffusion method [15] at concentrations of 4% and 2% (v/v). Two fungal strains (Candida albicans and Asperigillus niger) and six bacterial strains (Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 29665) and Escherichia coli (ATCC 25922)) were used. All innocula were subcultures from stock cultures into nutrient broth (10 mL) and incubated at 37 °C. After 24 h the broth culture was subcultured into fresh broth (10 mL) and incubated for a further 18 h under the same conditions. The resulting broth culture was then used as the inoculum for the agar diffusion test. The tests were carried out by pouring agar into Petri dishes to form 4 mm thick layers and adding dense inocula of the tested microorganisms (about 106 microorganisms/mL) in order to obtain semiconfluent growth. One drop of 4% and one of 2% essential oil solution in absolute ethanol were poured onto the agar, prepared as required. Reference samples were produced according to WHO standards [16]. Sensitivity of the bacteria was tested on Mueller-Hinton agar and Saburaud dextrose agar was used to test oil activity against fungi. Incubation lasted 18 h at 37 °C. Reading of results was carried out by measuring the diameters of the zones of inhibition and clear growth (in mm) and comparing to absolute ethanol as control.