Volatile composition and antimicrobial activity of the essential oil of Artemisia absinthium growing in Western Ghats region of North West Karnataka, India

Abstract

Context: Artemisia absinthium L. (Asteraceae) is an aromatic, herbaceous, perennial plant commonly known as wormwood. Artemisia absinthium is traditionally used as an anthelmintic, antiseptic, antispasmodic and for bacillary dysentery, cancers and neurodegenerative diseases.

Objective: The essential oil composition of the leaves of A. absinthium growing in the Western Ghats region of North West Karnataka, India, is investigated for the first time in this region and the oil was screened for antimicrobial properties.

Materials and methods: The chemical composition of the hydro-distilled essential oil obtained from the leaves of A. absinthium was analyzed by GC-FID and GC/MS. The oil was tested against five Gram positive and, eight Gram negative bacteria and three fungi by the tube-dilution method at a concentration range of 5000–9 µg/mL.

Results: Results demonstrated that the leave oil was found to be rich in oxygenated monoterpenes (39.7% and 41.1%). The major compounds were borneol (18.7% and16.7%), methyl hinokiate (11.9% and 12.9%), isobornyl acetate (4.0% and 4.7%), β-gurjunene (3.8% and 4.4%) and caryophyllene oxide (3.7% and 4.3%), among 64 identified compounds, comprising 91.7% and 90.1% of the total oil. The organism Micrococcus luteus was found more susceptible to the oil with an MIC value of 25 ± 4 µg/mL, followed by Micrococcus flavus, Bacillus subtilis, Penicillium chrysogenum and Aspergillus fumigatus with MIC values of 58 ± 8, 65 ± 8, 84 ± 15 and 91 ± 13 µg/mL, respectively.

Discussion and conclusion: The oil showing antimicrobial activity against bacteria and fungi validate the traditional use of the plant as an antiseptic.

• Borneol
• essential oil composition
• GC
• GC/MS
• methyl hinokiate

Introduction

Artemisia absinthium L. (Asteraceae) is an aromatic, herbaceous, perennial plant with a hard woody rhizome; it is commonly known as wormwood and distributed in Europe and Asia (Tan et al., 1998). Artemisia absinthium is bitter aromatic, used to stimulate the appetite, for gastrointestinal complaints, as a carminative, choleretic and in spasmodic disorders of the intestines and biliary tract (Wichtl, 1994). It is commonly used in food industry in the preparation of aperitifs, bitters and spirits (Basta et al., 2007; Mishra et al., 1999). The plant has been traditionally used as anthelmintic, antiseptic, antispasmodic, febrifuge, stomachic, cardiac stimulant, for the restoration of declining mental function and inflammation of the liver, and to improve memory (Guarrera, 2005; Howes et al., 2003; Kaul, 1997; Wake et al., 2000). In traditional Chinese medicine this plant is used for treating acute bacillary dysentery, cancers and neurodegenerative diseases (Gilani & Janbaz, 1995; Muto et al., 2003; Parekh et al., 2009; Zhang et al., 2005). It is a medicinal plant having diverse pharmacological properties such as anthelmintic (Tariq et al., 2009), antipyretic (Khattak et al., 1985), antitumor (Chemesova et al., 1987), anti-inflammatory (Lee et al., 2004), antidepressant (Mahmoudi et al., 2009), antimicrobial (Caner et al., 2008; Juteau et al., 2003), antioxidant (Brunet et al., 2005; Gilani & Janbaz, 1995; Mahmoudi et al., 2009), antiplasmodial (Ramazani et al., 2010), antileishmanial activities (Tariku et al., 2011), cognitive enhancement (Wake et al., 2000), antiprotozoal potential (Valdes et al., 2008), intoxicating (Meschler & Howlett, 1999), neurotoxic (Donald, 1981) and acaricidal properties (Chiasson et al., 2001).

Essential oil of this plant from different countries has been summarized in Table 1. A literature review revealed that the essential oil of A. absinthium has been the subject of limited investigation in India. In light of the above mentioned information, the aim of the present study was (i) to investigate the essential oil composition of A. absinthium from Western Ghats region of North West Karnataka, India, using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis; (ii) to evaluate their antimicrobial activity.

Table 1. Previously reported major constituents of essential oil of A. absinthium from different countries.

Country	Major constituents
Belgium	Sabinene (9.3%), myrcene (5.4%), sabinyl acetate (18.6%) (Orav et al., 2006)
Canada	Myrcene (10.8%), trans-thujone (10.1%) and trans-sabinyl acetate (26.4%) (Lopes-Lutz et al., 2008)
Cuba	Bornyl acetate (23.0%) (Pino et al., 1997)
Egypt	α-Phellandrene (50.5%) and terpinen-4-ol (12.0%) (Aboutabl et al., 1998)
Estonia	Epoxyocimenes (59.7%) (Orav et al., 2006)
Ethiopia	Camphor (27.4%), dehydrocostus lactone (41.8%) (Tariku et al., 2011)
France	cis-Chrysanthenol (69.0%) (Carnat et al., 1992); cis-epoxyocimene (49.7%) and cis-chrysanthenyl acetate (36.7%) (Juteau et al., 2003)
Germany	1,8-Cineol (3.4%), curcumeme structures (8.9%), neryl-3-methyl butanoate (3.8%) (Orav et al., 2006)
Greece	Caryophyllene oxide (23.3%), p-cymene (16.8%), 1,8-cineole (18.9%) and (Z)-lanceol acetate (7.3%) (Basta et al., 2007); β-thujone (38.7%) (Orav et al., 2006)
Hungry	Sabinene (18.1%), myrcene (17.7%) (Orav et al., 2006)
India	β-Phellandrene (10.0%), thujone (9.22%), α-himachalene (7.0%) and β-caryophyllene (5.0%) (Kaul et al., 1979)
Iran	β-Pinene (34.0%), p-cymene (14.6%), α-pinene (8.3%), α-thujone (6.9%) (Rahimizadeh et al., 2001); thujone (5.1%) (Sefidkon et al., 2003); β-pinene (23.8%) and trans-thujone (18.6%) (Rezaeinodehi & Khangholi, 2008)
Italy	Epoxyocimenes (23.1-56.6%) (Orav et al., 2006)
Latvia	trans-Verbenol (9.2%), sabinyl acetate (23.6%), curcumene structures (9.0%) (Orav et al., 2006)
Lithuania	trans-Verbenol (11.7%), sabinyl acetate (13.7%), curcumene structures (6.3%) (Orav et al., 2006); thujones (cis + trans, 10.2–36.3%), trans-sabinyl acetate (9.8-39.2%), myrcene (5.1-9.2%), β-pinene (5.4–10.4%), linalool (4.7%), trans-sabinol (6.4%) and 1,8-cineole (5.2-7.1%) (Judzentiene et al., 2009)
Morocco	Thujone (Derwich et al., 2009)
Russia	Epoxyocimenes (22.1%), sabinene (9.3%) (Orav et al., 2006); myrcene (35.0%), α-pinene (6.0%) and nerol (3.0%) (Goraev et al., 1962)
Scotland	Sabinene (30.1%), myrcene (18.0%) (Orav et al., 2006)
Serbia and Montenegro	β-Thujone (19.8–63.4%), cis-β-epoxyocimene (10.7%), trans-sabinyl acetate (8.8–15.5%), linalyl 3-methylbutanoate (7.5–12.5%), geranyl 3-methylbutanoate (4.0–12.9%) (Blagojevica et al., 2006)
Spain	cis-Epoxyocimene (76.3–39.9%), cis-chrysanthenyl acetate (33.4%) (Arino et al., 1999a,b); 1,8-cineole (18.0%), carvone (18.5%), thymol (10.8%) and carvacrol (9.7%), β-(6.2%) (Orav et al., 2006)
Turkey	Chamazulene (17.8%), nuciferyl butyrate (8.2%) and propionate (5.1%) (Kordali et al., 2005)
USA	trans-Thujone (33.1%) and cis-sabinyl acetate (32.8%) (Tucker et al., 1993)

Materials and methods

Plant material

The leaves of A. absinthium were collected in March 2011, at a height of 800 m from the medicinal garden of Regional Medical Research Centre (ICMR) Belgaum (N 15.88668; E 74.52353), Karnataka, India. The plant was identified by Dr. Harsha Hegde, Research Scientist, Regional Medical Research Centre (ICMR), Belgaum (voucher specimen No. RMRC-539).

Isolation of essential oil

The fresh plant material (500 g) was subjected to hydro-distillation using Clevenger-type apparatus for 5 h. The oil was collected and dried over anhydrous sodium sulfate and stored in sealed vials at −4 °C until analysis. The oil yield was 0.03% v/w.

Gas chromatography

The GC analysis of the oil was carried out on Varian 450 gas chromatograph equipped with FID, using stationary phase CP Sil-8-CB (30 m × 0.25 mm i.d., 0.25 µm film thickness) and BP 21 (60 m × 0.25 mm i.d., 0.25 µm film thickness) columns. Nitrogen was a carrier gas at 1.0 mL/min flow rate. Temperature programming was set to 60–220 °C at 3 °C/min, the injector and detector temperatures were 230 and 250 °C, respectively. The injection volume was 1.0 µL of 1% solution diluted in n-hexane; split ratio was 1:50.

Gas chromatography–mass spectrometry

The GC-MS analysis of the oil was carried out on Thermo Scientific Trace Ultra GC (Thermo Fisher Scientific Austria, Vienna, Austria) interfaced with a Thermo Scientific ITQ 1100 Mass Spectrometer (Thermo Fisher Scientific Austria) fitted with TG-5 (30 m × 0.25 mm i.d., 0.25 µm film thickness) and BP 21 (60 m × 0.25 mm i.d., 0.25 µm film thickness) columns. The oven temperature was programmed from 60 to 220 °C at 3 °C/min using helium as a carrier gas at 1.0 mL/min. The injector temperature was 230 °C, injection size was 0.1 µL of 1% solution prepared in n-hexane; split ratio was 1:50. MS were taken at 70 eV with mass scan range of 40–450 amu.

Identification of the components

Identification of constituents were done on the basis of Retention Index (RI, determined with reference to homologous series of n-alkanes C₈–C₂₈, under identical experimental conditions), MS library search (NIST 08 MS Library (Version 2.0 f; Thermo Fisher Scientific Austria) and WILEY MS 9^th Edition (Thermo Fisher Scientific Austria)), and by comparing with the MS literature data (Adams, 2007). The relative amounts of individual components were calculated based on the GC peak area (FID response) without using a correction factor.

Antimicrobial activity

The in vitro antimicrobial activity of the essential oil of A. absinthium was evaluated by the tube dilution method (Joshi et al., 2010; Murthy et al., 2006) and represented as minimum inhibitory concentration (MIC).

Microbial strains

The microorganisms were obtained from the National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune. The microorganisms were Staphylococcus aureus (NCIM 2079), Staphylococcus epidermidis (NCIM 2493), Micrococcus flavus (NCIM 2379), Micrococcus luteus (NCIM 2103), Bacillus subtilis (NCIM 2063) as Gram positive bacteria; Escherichia coli (NCIM 2574), Klebsiella pneumoniae (NCIM 2957), Serratia marcescens (NCIM 2078), Proteus vulgaris (NCIM 2813), Proteus mirabilis (NCIM 2241), Pseudomonas aeruginosa (NCIM5029), Salmonella typhimurium (NCIM 2501), Enterobacter aerogenes (NCIM 2694) as Gram negative bacteria; Aspergillus niger (NCIM 620), Aspergillus fumigatus (NCIM 902) and Penicillium chrysogenum (NCIM 733) (fungi).

Preparation of test sample

The essential oil of A. absinthium was dissolved in 10% dimethyl sulphoxide (DMSO), which is reported to be non-toxic to microorganisms at this percentage (Joshi, 2010). Erythromycin (Alembic Ltd., Solan, Himachal Pradesh, India), amikacin (Iskon Remedies, Sirmour, Himachal Pradesh, India) and amphotericin B (Chandra Bhagat Pharma Pvt. Ltd., Ankleshwar, India) were used as the positive reference standards for Gram positive bacteria, Gram negative bacteria and fungi, respectively.

Preparation of inocula

The inocula of microbial strains were prepared from 18 h-old culture and suspensions were adjusted to 0.5 McFarland standard turbidity (∼10⁷ for bacteria (1 to 2 × 10⁸CFU/mL for E. coli) and ∼10³CFU/mL (McFarland, 1987).

Tube-dilution assay

Tube-dilution method was used to determine the MIC of the essential oil of A. absinthium dissolved in 10% DMSO. The final concentration of the oil was 5000 µg/mL. Serial two-fold dilutions were prepared from the stock solution to give concentration ranging from 5000--9 µg/mL against bacterial and fungal strains. Erythromycin, amikacin and amphotericin B were dissolved in sterile distilled water and two-fold dilutions were prepared (1000–2 µg/mL). One milliliter of each concentration was mixed with 1.0 mL of sterile peptone water (10⁴CFU/mL for bacteria and 10³CFU/mL for fungal concentration, obtained from a McFarland turbidity standard no. 0.5). Solvent control was prepared with DMSO (10%) and a blank control was prepared from the virgin media. Tubes were incubated for 24 and 48 h at 37 °C for bacteria and fungi, respectively. Assays were performed in replicates and the mean value of six experiments was recorded (n = 6) with SEM. MICs were determined as the lowest concentration that inhibits the visible microbial growth (Joshi, 2010; Murthy et al., 2006).

Results and discussion

The results of the GC and GC/MS analyses of the oil from the leaves of A. absinthium are summarized in Table 2. Sixty-four constituents were identified, which constitute 91.7% and 90.1% of the total oil. The major constituents were borneol (18.7% and 16.7%) and methyl hinokiate (11.9% and 12.9%). Other minor constituents were isobornyl acetate (4.0% and 4.7%), β-gurjunene (3.8% and 4.4%), caryophyllene oxide (3.7% and 4.3%) and (E)-β-ocimene (3.5% and 2.9%). The oil was found to be rich in oxygenated monoterpenes (39.7% and 41.1%), followed by oxygenated sesquiterpenes (23.5% and 24.6%), monoterpene hydrocarbons (12.3% and 10.2%), sesquiterpene hydrocarbons (11.3% and 10.7%) and phenyl derivatives (3.9% and 3.5%). Artemisia absinthium plants produced several chemotypes of essential oils in different countries (Aboutabl et al., 1998; Arino et al., 1999a,b; Basta et al., 2007; Carnat et al., 1992; Goraev et al., 1962; Judzentiene et al., 2009; Juteau et al., 2003; Kordali et al., 2005; Pino et al., 1997; Orav et al., 2006; Rahimizadeh et al., 2001; Sefidkon et al., 2003). The main chemotype of wormwood essential oils was thujone (Blagojevica et al., 2006; Derwich et al., 2009; Kaul et al., 1979; Lopes-Lutz et al., 2008; Rezaeinodehi & Khangholi, 2008; Tucker et al., 1993). In this report thujone was not detected.

Table 2. Chemical composition of the essential oil of A. absinthium.

Compound	RI^a	RI^b	%^a	%^b
Tricyclene	932	1014	0.7	0.5
α-Thujene	937	1084	0.3	0.2
α-Pinene	943	1021	0.4	0.3
Camphene	957	1052	1.8	2.1
Thuja-2,4(10)-diene	960	1145	0.5	0.3
β-Pinene	985	1124	1.1	1.0
α-Terpinene	1022	1131	0.5	0.7
p-Cymene	1030	1242	0.4	0.6
Limonene	1036	1165	1.6	1.3
1,8-Cineole	1139	1175	1.3	1.9
(E)-β-Ocimene	1058	1195	3.5	2.9
γ-Terpinene	1065	1195	0.8	0.5
cis-Sabinene hydrate	1073	1461	0.3	0.9
Terpinolene	1092	1258	0.7	0.5
Linalool	1105	1563	0.4	0.7
cis-p-Menth-2-en-1-ol	1129	1581	0.3	0.2
Camphor	1150	1541	2.9	2.3
β-Pinene oxide	1163	1942	0.9	1.3
Pinocarvone	1170	2156	1.1	1.2
Borneol	1073	1744	18.7	16.7
Terpinen-4-ol	1181	1656	2.8	2.5
cis-Pinocarveol	1189	1693	1.4	1.4
α-Terpineol	1193	1736	0.6	0.5
Myrtenol	1199	1718	1.0	0.8
trans-Piperitol	1213	1788	1.0	1.2
(Z)-Ocimenone	1240	1901	1.2	1.5
Cumin aldehyde	1249	1832	1.6	1.3
Piperitone	1258	1782	0.7	0.5
Perilla aldehyde	1279	1845	2.1	2.7
Isobornyl acetate	1293	1605	4.0	4.7
Thymol	1295	2225	1.7	1.4
Carvacrol	1304	2252	0.6	0.8
6-Hydroxycarvotanacetone	1311	1796	t	0.1
Eugenol	1361	2217	t	t
Cyclosativene	1379	1454	0.3	0.5
β-Cubebene	1393	1499	t	t
Cyperene	1407	1545	0.3	0.1
α-Gurjunene	1416	1552	0.2	0.1
β-Caryophyllene	1426	1627	0.1	0.3
β-Gurjunene	1441	1532	3.8	4.4
α-Humulene	1457	1710	0.3	0.5
Seychellene	1468	1661	0.5	0.2
γ-Gurjunene	1479	1618	0.7	0.6
γ-Curcumene	1488	1725	0.4	0.1
Germacrene D	1493	1760	0.1	0.2
ar-Curcumene	1491	1817	0.4	0.6
β-Selinene	1493	1766	0.2	0.1
cis-β-Guaiene	1499	1575	0.7	0.9
epi-Cubenol	1506	1939	1.1	0.9
Bicyclogermacrene	1507	1672	0.9	0.7
α-Muurolene	1504	1753	0.5	0.3
δ-Cadinene	1533	1802	0.4	0.5
β-Vetivenene	1533	–	0.9	–
6,11-oxido-Acor-4-ene	1539	1918	0.1	t
α-Calacorene	1545	1961	0.4	0.3
β-Calacorene	1571	1975	0.2	0.6
Germacrene-D-4-ol	1583	2103	0.6	0.5
Caryophyllene oxide	1589	2049	3.7	4.3
Guaiol	1612	2137	0.6	0.2
Cubenol	1652	1993	1.9	2.8
α-Muurolol	1658	2230	0.6	0.1
β-Eudesmol	1661	2274	0.4	0.1
α-Cadinol	1666	2240	2.6	2.8
Methyl hinokiate	1714	2354	11.9	12.9
Total identified			91.7%	90.1%
Monoterpene hydrocarbons			12.3%	10.2%
Oxygenated monoterpenes			39.7%	41.1%
Sesquiterpene hydrocarbons			11.3%	10.7%
Oxygenated sesquiterpenes			23.5%	24.6%
Phenyl derivatives			3.9%	3.5%

t: trace (<0.1%).

^aCP Sil-8-CB (30 m × 0.25 mm i.d., 0.25 µm film thickness) column.

^bBP 21 (60 m × 0.25 mm i.d., 0.25 µm film thickness) column.

In vitro antimicrobial activity of the essential oil of A. absinthium is shown in Table 3. The organism Micrococcus luteus was found to be more susceptible to the oil with an MIC value of 25 ± 4 µg/mL, followed by Micrococcus flavus, Bacillus subtilis, Penicillium chrysogenum and Aspergillus fumigatus with MIC values of 58 ± 8, 65 ± 8, 84 ± 15 and 91 ± 13 µg/mL. The organisms like Aspergillus niger and Staphylococcus epidermidis were found less susceptible to the oil with MIC values of 183 ± 27 and 238 ± 36 µg/mL, respectively. No activity was observed against the microorganisms Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhimurium and Enterobacter aerogenes at a range of 250–259 µg/mL.

Table 3. Antimicrobial activity (MIC values µg/mL) of the essential oil of A. absinthium.

	MIC mean ± SEM (µg/mL)
Microbial strains	A. absinthium	Erythromycin	Amikacin	Amphotericin B
Gram-positive
Staphylococcus aureus	NA	2 ± 1	NT	NT
Staphylococcus epidermidis	238 ± 36	2 ± 1	NT	NT
Micrococcus flavus	58 ± 8	2 ± 1	NT	NT
Micrococcus luteus	25 ± 4	1 ± 1	NT	NT
Bacillus subtilis	65 ± 8	1 ± 1	NT	NT
Gram-negative
Escherichia coli	NA	NT	9 ± 4	NT
Klebsiella pneumoniae	NA	NT	5 ± 2	NT
Serratia marcescens	NA	NT	5 ± 3	NT
Proteus vulgaris	NA	NT	5 ± 3	NT
Proteus mirabilis	NA	NT	2 ± 1	NT
Pseudomonas aeruginosa	NA	NT	4 ± 3	NT
Salmonella typhimurium	NA	NT	2 ± 2	NT
Enterobacter aerogenes	NA	NT	9 ± 4	NT
Fungi
Aspergillus niger	183 ± 27	NT	NT	1 ± 1
Aspergillus fumigatus	91 ± 13	NT	NT	1 ± 1
Penicillium chrysogenum	84 ± 15	NT	NT	1 ± 1

Values are mean ± standard deviation of six experiments in replicate. NT: Not tested. NA: Not active.

According to Wan et al. (1998), the majority of the essential oils assayed for their antibacterial properties showed a more pronounced effect against the Gram positive bacteria. The resistance of Gram negative bacteria to essential oil has been ascribed to their hydrophilic outer membrane, which can block the penetration of hydrophobic compounds into target cell membrane (Inouye et al., 2001; Joshi, 2010).

Conclusions

The findings demonstrate that the essential oil of A. absinthium, methyl hinokiate has been reported for the first time. The oil showing antimicrobial activity against bacteria and fungi could be attributed the traditional use of the plant as an antiseptic.

Declaration of interest

The author reports no conflicts of interest. The author alone is responsible for the content and writing of this article.

Acknowledgements

The author is grateful to the Dr S. D. Kholkute, Director-in-Charge, Regional Medical Research Centre (ICMR), Belgaum, Karnataka, India, for providing necessary facilities and thankful to Miss Vijaylaxmi Badakar, Lab Assistant, for carrying out antimicrobial activity.