INTRODUCTION:

Artemisia dracunculus L. (Asteraceae) is a perennial, green erect herb, grooved and ribbed leaves 2.5-3.8 Cm long. It is found at an altitude of 4200-4800 mtrs in Uttarakhand¹. The plant is commonly used in antiallergic anti-inflammatory agent and as a mental tonic and also used in retinopathy and cardiovascular disease^2,3. In phytochemical study of aerial part of Artemisia dracunculus, 4,5-di -o- Caffeoylquinic acid, Davidigenin, 6- demethoxy Capillarisin and 2, 4, dihydroxy-4-methoxy dihydroxy chalcone were isolated from the ethanol extract⁴ and some coumarins and their bioactivity from A.dracunculus⁵. Flavonoids, flavones, which were extracted from different places by different workers, were analysed earlier^6,
7 but no proper essential oil investigation has been carried out from A. dracunculus in Uttarakhand. This prompted us to find out the detailed phytochemical investigation of A. dracunculus growing in wildly in Dhanolti region, Tehri Garhwal at an altitude of 1650 mtrs.

MATERIAL AND METHODS:

Plant Material:

The fresh aerial parts of Artemisia dracunculus were collected from Dhanolti region of Tehri Garhwal in the month January-February 2010.

A voucher specimen was deposited at Department of Botany, HNB Garhwal University Campus Badshahi thaul, Tehri.

Oil Extraction:

The fresh aerial parts (400 g) were dried at 25 °C in the shade and subjected to hydro distillation, using a Clevenger-type apparatus for 5 h. The oil was dried with anhydrous sodium sulphate, weighed and stored at 4 – 6 °C until use.

GC/MS Analysis:

The GC-MS analysis of essential oils (0.2 µl) were performed using gas chromatograph HP 6890 with mass selective detector MS 5973 (Agilent Technologies, USA) fitted with a HP-5ms fused silica column (30 m × 0.25 mm; 0.25 µm film thickness), with electronic pressure control and split-splitless injector. Helium flow rate through the column was 1 ml/min in constant flow mode. The initial column temperature was 50⁰C rising 250⁰C at a rate 5⁰C/min. The MS detector acquisition parameters: transfer line held at 260⁰C and detector was held at 280⁰C. Detection was performed in the full scan mode from m/z 41 to 450. A hexane solution of C₈-C₂₈ n-alkanes was previously separated under the above conditions, and their retention times were determined. Linear temperature programmed retention indices (LTPRI) were calculated from the results of the separation of the essential oils and n-alkanes according to eq.:

Table-1: Chemical composition of the essential oil of Artemisia dracunculus L.

Compounds	LTPRI^Calc	LTPRI^Lit	Relative composition, %	Identification
Linalool	1098	1098	0.9	MS, GC
Camphor	1135	1143	0.1	MS, GC
Citronellol	1229	1228	1.4	MS, GC
NN^a (monoterpenoid? m/z 41,69,81,55,95,138)	1233	-	0.1	MS
Geraniol	1256	1252	4.2	MS, GC
Citronellol acetate	1357	1354	26.0	MS, GC
Eugenol	1359	1356	0.5	MS, GC
Sesquiterpene C₁₅H₂₄ (m/z 93,69,41,91,121)	1364	-	1.2	MS
Cyclosativene	1367	1368	0.2	MS, GC
γ –Elemene	1378	1380	0.3	MS, GC
Geranyl acetate	1390	1386	50.4	MS, GC
(Z)-β-Caryophyllene	1410	1408	0.2	MS, GC
β –Guaiene	1436	1432	0.1	MS, GC
Aromadendrene	1444	1441	0.7	MS, GC
Alloaromadendrene	1469	1462	0.5	MS, GC
α-Amorphene	1472	1475	0.3	MS, GC
γ-Himachalene	1476	1476	trace^b	MS, GC
Sesquiterpene C₁₅H₂₄ (m/z 161,91,105,41,204)	1487	-	0.9	MS
α –Muurolene	1492	1499	1.7	MS, GC
d-Cadinene	1505	1515	1.6	MS, GC
γ-Cadinene	1517	1522	6.1	MS, GC
Cadina-1,4-diene?	1529	1532	0.4	MS, GC
α –Calacorene	1534	1542	0.2	MS, GC
(E)- γ-Bisabolene	1542	1533	2.1	MS, GC
β-Calacorene	1554	1563	trace	MS, GC
TOTAL			100.0

^a Not identified; ^b below of 0.1 % of TIC

LTPRI = 100(t_x – t_n)/ (t_n+1– t_n) + 100n,

Where, t_x, t_n and t_n+1are the retention times of component x, and n-alkanes with the number of carbon atoms in the molecule n and n+1, respectively. After integration the fraction of each component in the total ion current (TIC) was calculated. Components of essential oils were identified with the aid of an automatic system of processing data of GC-MS supplied by NIST mass spectra library. Identification was considered reliable if the calculated values of LTPRI confirmed the results of computer search at mass spectra library (LTPRI^Calc – LTPRI^Lit ≤ 5 index units). (LTPRI^Lit)^{8, 9}

Quality evaluation by Physico-Chemical Analysis:

To evaluate the quality of essential a number of physico-chemical methods are used by which reproducible qualitative and quantitative result can be obtained. Many organizations are engaged can be setting standard for natural essential oils. In India, Bureau of Indian Standard (BIS) and similar by, at international levels, international organization of standardization (ISO) are actively working in this direction. The physical characteristic introduced by Otto Wallach^{10, 11} and his contemporaries for the analysis of essential oil include boiling point, refractive index, density, optical rotation and solubility. The chemical analysis is usually expressed as indices and values such as acid value, saponification value, hydroxyl value, carbonyl value etc. These chemical methods permit abroad determination of a functional grouping only. These methods have proved of great value in the essential oil industry and continue to do so due to their simplicity and rapidity. These method, were extensively used by Wallach, Ruzicka, Semmler and

Barbier^{12, 13}in their work on analysis of the essential oils. The employed physico-chemical characteristics are Boiling point, Specific Gravity, Refractive Index, Optical rotation, Freezing point, Solubility, Acid value, Alcohol value, Ester value, Carboxyl value

Antimicrobial activity:

The antimicrobial and antifungal activities of the essential oil were determined against Staphylococcus aureus (ATCC 29737), Echerichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Saccharomyces cerevisiae (ATCC 16404) and Candida albicans (ATCC 14053). Bacterial and fungal strains were tested on soybean casein digest agar and Sabouraud dextrose agar, respectively. Sterilized paper disks were loaded with different amount of A. dracuculus L. essential oil (0.25, 0.5, 1, 2, 4, 8, 16, 32 and 64 mg/ml) and applied on the surface of agar plates. All plates were incubated at 37°C for 24 h for bacteria; at 25 °C for 24 h for C. albicans. The MIC was defined as the lowest drug concentration, resulting in a clear zone of growth inhibition around the disk after conventional incubation period. 23 Paper disks containing different concentrations of fluconazole and gentamycin (Sigma Chemical Co.) were applied over the test plates as a comparative positive control.

RESULTS AND DISCUSSION:

Volatile oil from the aerial parts of Artemisia dracunculus was analyzed by GC-MS. Twenty five compounds were identified by retention index, relative composition (Table-1) of which the major were Geranyl acetate (50.4%), Cetronellol acetate (26.0%), g-Cadene (6.1%), Geraniol (4.2%), (E)- g-Bisabolene (2.1%), a-Muurolene (1.7%) and d-Cadinene (1.6%).The present oil was found more or less similar with other in respect of the presence of Geranyl acetate, Geraniol but differs in respect of a-Muurolene and (E)-g-Himachalene, not reported. These variations in the chemical composition may due to chemical races, geographical variations and differences in maturity. In Order to develop suitable parameters for quality evaluations of this essential oil following Physico-chemical properties were determines by literature.¹⁴(Table-2).

Table-2: Physico-Chemical Properties of Essential Oils of Artemisia dracunculus L.

S. No. Physico-Chemical properties Values

1. Color Pale yellow

2. Odor Pungent – smell

3. Refractive index 1.4775 at 25⁰c

4. Optical rotation 6.1⁰

5. Specific gravity 0.9286 c/c at 18.27⁰c

6. Solubility 1:15 (Clear), oil 80% EtOH

7. Alcohol percentage 1.0165

The results obtained in the antimicrobial assay are shown in (Table-3). The oil showed antimicrobial activity against all the tested microorganisms, excepted Pseudomonas aeruginosa. Maximum activity was observed against fungal microorganisms Saccharomyces cerevisiae (MIC = 2 mg/ml) and Candida albicans (MIC = 2 mg/ml). Moderate inhibitory activity of the oil against Staphylococcus aureus and Echerichia coli were also determined with MIC value of 32 mg/ml and 64 mg/ml respectively. No activity was observed against Pseudomonas aeruginosa. In the present study Gram-positive bacteria Staphylococcus aureus was more susceptible than Gram-negative bacteria strains. It has frequently been reported that Gram-negative bacteria were resistant to the inhibitory effects of essential oils and their components¹⁵. This resistance has been attributed to the presence of cell wall lipopolysaccharides, which can screen out the essential oils; the lipids are thus prevented from accumulating on the transporting cell membrane, and from entering the cells.

Table 3. Antimicrobial activity (MIC) of essential oil of Artemisisa dracunculus L. (mg/ml).

Strains	Essential oil	MIC (mg/ml) Gentamycin	Fluconazole
Staphylococcus aureus (ATCC 29737)	32	4×10^-3	ND
Echerichia coli (ATCC 8739)	64	1×10^-3	ND
Pseudomonas aeruginosa (ATCC 9027)	-	8×10^-3	ND
Saccharomyces cerevisiae (ATCC 16404)	2	ND	10×10^-3
Candida albicans (ATCC 14053)	2	ND	10×10^-3

^aND, not determined.

CONCLUSION:

Based on the above study, it may be summarized that the aerial parts of A. dracunculus may be utilized for separation of the essential oil and a source of Cetronellol acetate, a-Muurolene, and (E)- g-Bisabolene. The data presented confirm the antimicrobial potential of A. dracunculus essential oil. The essential oils tested represent an inex-pensive source of natural antibacterial substances for use in pathogenic systems to prevent the growth of bacteria and fungus.

ACKNOWLEDGMENTS:

The Authors thanks to Prof. Valery A. Isidorov, Institute of Chemistry, Bialystok University, Bialystok, Poland for GC-MS analysis and Dr. J. Kumar, Biochemistry division, G.B. Pant University, Hill Campus Ranichauri, Pantnagar, Tehri Garhwal, India for antimicrobial activity.

REFERENCES:

1. Welth of India (CSIR), New Delhi, Revised Edition, 1A, 2006, 435.

2. Yabe Nishimura C. Pharmacol. Rev. 1998; 50 (1): 21-23.

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7. Gerger H. et.al, Phytochem. 1977; 16: 795.

8. R.P. Adams, Identification of Essential Oil Components by GC – MS. Allurd, Carol Stream, IL, 1995.

9. NIST Chemistry WebBook, National Institute of Standards and Technology, Gaitherburg, MD,

10. Wallach O. Method of analysis of constant of essential oil syndicate National 7, Rue Gazan, Grasses, France, 1959.

11. Teisserie P. Recherches, 1964; 13.

12. Wallach O. Terpene and camphor, 1914, 2^nd Edi; (Verlay Von Vein and Co., Leipzig).

13. Semmler, The essential oil and their constituents, (Verlay Von Vein and Co., Leipzig), 1906-1907.

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15. Smith-Palmer A, Stewart J, Fyfe L. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett. Appl. Microbiol. 1998; 26: 118-122.

Received on 28.04.2010 Modified on 10.05.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 755-757