Composition and Antimicrobial Activity of the Essential Oil of Achillea multifida

• Abstract
• Materials and Methods
• References

Abstract

The composition of the water-distilled essential oil of Achillea multifida (DC.) Boiss. (Compositae) was analysed by GC and GC/MS. Fifty-eight compounds were identified representing 93.9 % of the total oil. α-Thujone (60.9 %), β-thujone (9.1 %), sabinene (4.1 %) and camphor (3.7 %) were characterised as the main constituents. The essential oil was tested for its antimicrobial activity using a micro-dilution assay resulting in the inhibition (MIC: 62.5 - 250 μg/ml) of human pathogenic bacteria and yeast.

The genus Achillea of the family Compositae comprises 42 species and 47 taxa in Turkey, of which 23 taxa are endemic. Achillea multifida (DC.) Boiss. [Syn.: Achillea atrata L. subsp. multifida (D.C.) Heim.] grows on Uludag˘ Mountain in Bursa, Turkey. It is 10 - 30 cm tall, perennial, aromatic plant with green leaves and white-yellowish flowers, locally known as ”ebülmülk” or ”civanperçemi” [1], [2], [3], [4].

Achillea species have been used since ancient times for their medicinal, agricultural, cosmetic, and fragrance properties. In particular, the well-known A. millefolium L., which is known as yarrow or milfoil, has been used internally as herbal tea and externally in lotions and ointments as well as in herbal medications [5], [6].

Our research group has reported the composition of the essential oils of different Achillea species growing in Turkey [7], [8], [9], [10]. The previous essential oil studies on Achillea sp. including our studies were recently compiled by our group [10]. However, to the best of our knowledge, there is no previous study on the essential oil of A. multifida. The only previous study on this species reported the occurrence of flavones and sesquiterpene lactones and their antimicrobial activity. This paper [11] also indicates the traditional uses against pulmonary and upper respiratory system infections.

This study concerns the analysis of the essential oil by gas chromatography/mass spectrometry (GC/MS) (see Table [1]) and the antimicrobial evaluation against Gram (+) and Gram (-) human pathogenic bacteria and the yeast Candida albicans. A micro-broth dilution assay [12] was used to determine the minimum inhibitory concentrations (MIC) of the essential oil against nine different microorganisms (See Table [2]).

The GC/MS analysis resulted in the identification of fifty-eight compounds, representing 93 % of the total essential oil. Oxygenated monoterpenes were the major constituents (81 %). Thujones (compounds 13 and 14) comprised approximately 70 %. The structurally related monoterpenes sabinene (4), and camphor (17) were identified as the other major components in the essential oil.

Recent studies have shown that natural products and especially essential oils and components thereof display potential as antimicrobial agents for various uses in medical applications [13], [14]. Further literature search showed that Achillea sp., in particular exhibit antimicrobial properties against human and plant pathogenic bacteria, fungi and yeast [11], [15], [16]. In a more recent publication Achillea multifida was reported to contain flavones displaying inhibitory activity against E. coli and C. albicans [11].

In our present study, the essential oil of Achillea multifida was tested for its antimicrobial properties against human pathogenic bacteria and yeast. It showed good inhibitory activity against the Gram (-) human pathogens E. aerogenes (MIC 62.5 μg/ml), P. aeruginosa (MIC 125 μg/ml), and the yeast C. albicans (MIC 62.5 μg/ml). Some inhibition against the other tested bacteria was also observed when compared to the standard agent chloramphenicol. The detailed results of inhibitory concentrations against the used microorganisms can be seen at Table [2].

Materials and Methods

Plant material: The plant material was collected from Bursa: Uludag, ca. 1850 m, on 15 September 2000. Voucher specimens are kept at the Herbarium of the Faculty of Pharmacy (ESSE 13 284), Anadolu University, Eskişehir, Turkey.

Isolation of essential oil: Air dried aerial parts of the plant material (200 g) were subjected to hydro-distillation for 3 h using a Clevenger-type apparatus to yield the essential oil (1.98 ml). Essential oil yield was calculated based on a moisture-free basis as 1.06 % (w/v).

Physical properties of the oil were determined as follows. Specific rotation was determined using an Oriel Pol S-2 polarimeter [α]_D ²⁴: -1.4263°; refractive index was measured using an Abbe Refractometer (Shimadzu, Bausch & Lomb) as n _D ²⁴: 1.457; the density of the essential oil was determined using Drummond micro-capillaries (10 μl) as d²⁴ = 0.9248 g/g.

Chromatographic analysis: The essential oil was analysed by GC using a Hewlett Packard 6890 system. An HP-Innowax FSC column (60 m × 0.25 mm inner diameter, with 0.25 μm film thickness) was used with nitrogen as carrier gas (1 ml/min). The oven temperature was kept at 60 °C for 10 min and programmed to 220 °C at a rate of 4 °C/min, then kept constant at 220 °C for 10 min and then programmed to 240 °C at a rate of 1 °C/min. The injector temperature was at 250 °C. Relative percentages (i. e., percent peak area relative to total peak area) were obtained from electronic integration measurements using a flame ionization detector (FID, 250 °C). n-Alkanes were used as reference points in the calculation of Relative retention indices (RRI).

Indentification of compounds: The sample was analysed by GC/MS employing the same chromatographic conditions as described above, using a Hewlett Packard GCD system with helium as carrier gas (1 ml/min). Split ratio was adjusted at 50 : 1. MS were recorded at 70 eV. Mass range was from 35 to 425 m/z. Library search was carried out using the TBAM Library of Essential Oil Constituents. The individual compounds identified in the essential oil are given in Table [1].

Antimicrobial assay: The micro-dilution broth susceptibility assay [12] was used for the antimicrobial evaluation of the essential oil. A stock solution was prepared in dimethyl sulphoxide (DMSO) and the dilution series were prepared up to 0.97 μg/ml using sterile distilled water in 96-well microtiter plates. Overnight grown microbial suspensions in double strength Mueller-Hinton broth and yeast suspension of Candida albicans in yeast medium were standardized to approximately 10⁸ CFU/ml (using McFarland No: 0.5). 100 μl of each microbial suspension was then added to each well. The last row containing only the serial dilutions of antimicrobial agent without microorganism was used as negative control. Sterile distilled water and medium served as a positive growth control. After incubation at 37 °C for 24 h the first well without turbidity was determined as the minimal inhibition concentration (MIC). Human pathogens were obtained both from the culture collection of the Microbiology Department in Anadolu University, and from the culture collection of Osmangazi University, Medical Faculty, Microbiology Department. Chloramphenicol was used as standard antibacterial agent for the bacteria, whereas ketoconazol was used for C. albicans (see Table [2] for the results).

References

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• 2 Güner A, Özhatay N, Ekim T, Başer K HC. Flora of Turkey and The East Aegean Islands. Vol. 11 University Press Edinburgh; 2000
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• 4 Güleryüz G. Achillea multifida (DC.) Boiss. (Compositae).  The Karaca Arboretum Magazine. 1999;  5 43-4
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• 12 Koneman E W, Allen S D, Janda W M, Schreckenberger P C, Winn W C. Color Atlas and Textbook of Diagnostic Microbiology. Lippincott-Raven Pub Philadelphia, PA; 1997: p. 785-6
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• 13 Hammer K A, Carson J F, Riley T V. Antimicrobial activity of essential oils and other plant extracts.  J Appl Microbiol. 1999;  86 985-90
  CrossrefPubMedSearch in Google Scholar
• 14 Dorman H JD, Deans S G. Antimicrobial agents from plants: antibacterial activity of plant volatile oils.  J Appl Microbiol. 2000;  88 308-16
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• 15 Barel S, Segal R, Yashphe J. The antimicrobial activity of the essential oil from Achillea fragrantissima .  J Ethnopharmacol. 1991;  33 187-91
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• 16 Fiori A CG, Schwan-Estrada K RF, Stangarlin J R, Vida J B, Scapim C A, Cruz M ES. Antifungal activity of leaf extracts and essential oils of some medicinal plants against Didymella bryoniae .  J Phytopathol. 2000;  148 483-7
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. K. Hüsnü Can Başer

Medicinal and Aromatic Plant and Drug Research Centre (TBAM)

Anadolu University, 26470-Eskişehir, Turkey.

Email: khcbaser@anadolu.edu.tr or khcbaser@yahoo.co.uk

References

• 1 Davis P H. Flora of Turkey and the East Aegean Islands. Vol. 5 University Press Edinburgh; 1982: p. 229
  Search in Google Scholar
• 2 Güner A, Özhatay N, Ekim T, Başer K HC. Flora of Turkey and The East Aegean Islands. Vol. 11 University Press Edinburgh; 2000
  Search in Google Scholar
• 3 Baytop T. Türkiye’de Bitkiler ile Tedavi Geçmişten Bugüne (Therapy with Medicinal Plants in Turkey-Past and Present). 2nd Ed Nobel Tıp Basımevi Istanbul, Turkey; 1999: p. 177
  Search in Google Scholar
• 4 Güleryüz G. Achillea multifida (DC.) Boiss. (Compositae).  The Karaca Arboretum Magazine. 1999;  5 43-4
  PubMedSearch in Google Scholar
• 5 Chatzopoulou P, Katsiotis S T, Baerheim-Svendsen A. An ascaridole containing essential oil of the Achillea millefolium L. complex growing wild in northern Greece.  J Essent Oil Res. 1992;  4 457-9
  PubMedSearch in Google Scholar
• 6 Hanlidou E, Kokkalou E, Kokkini S. Volatile constituents of Achillea grandifolia .  Planta Med. 1992;  58 105-7
  PubMedSearch in Google Scholar
• 7 Küsmenoglu S, Başer K HC, Özek T, Harmandar M, Gökalp Z. Constituents of the Essential Oil of Achillea biebersteinii Afan.  J Essent Oil Res. 1995;  7 527-8
  PubMedSearch in Google Scholar
• 8 Başer K HC, Demirci B, Kaiser R, Duman H. Composition of the essential oil of Achillea phrygia Boiss.et Ball.  J Essent Oil Res. 2000;  12 327-9
  PubMedSearch in Google Scholar
• 9 Başer K HC, Demirci B, Duman H, Aytaç Z, Adıgüzel N. Composition of the essential oil of Achillea goniocephala Boiss. et Bal. from Turkey.  J Essent Oil Res. 2001;  13 219-20
  PubMedSearch in Google Scholar
• 10 Başer K HC, Demirci B, Duman H. Composition of the essential oils of two endemic species from Turkey: Achillea lycaonica and A. ketenoglui .  Chem Nat Comp. 2001;  37 245-52
  PubMedSearch in Google Scholar
• 11 Aljancic I, Vajs V, Menkovic N, Karadzic I, Juranic N, Milosavljevic S. et al . Flavones and sesquiterpene lactones from Achillea atrata subsp, multifida: antimicrobial activity.  J Nat Prod. 1999 ;  62 909-11
  CrossrefPubMedSearch in Google Scholar
• 12 Koneman E W, Allen S D, Janda W M, Schreckenberger P C, Winn W C. Color Atlas and Textbook of Diagnostic Microbiology. Lippincott-Raven Pub Philadelphia, PA; 1997: p. 785-6
  Search in Google Scholar
• 13 Hammer K A, Carson J F, Riley T V. Antimicrobial activity of essential oils and other plant extracts.  J Appl Microbiol. 1999;  86 985-90
  CrossrefPubMedSearch in Google Scholar
• 14 Dorman H JD, Deans S G. Antimicrobial agents from plants: antibacterial activity of plant volatile oils.  J Appl Microbiol. 2000;  88 308-16
  CrossrefPubMedSearch in Google Scholar
• 15 Barel S, Segal R, Yashphe J. The antimicrobial activity of the essential oil from Achillea fragrantissima .  J Ethnopharmacol. 1991;  33 187-91
  CrossrefPubMedSearch in Google Scholar
• 16 Fiori A CG, Schwan-Estrada K RF, Stangarlin J R, Vida J B, Scapim C A, Cruz M ES. Antifungal activity of leaf extracts and essential oils of some medicinal plants against Didymella bryoniae .  J Phytopathol. 2000;  148 483-7
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. K. Hüsnü Can Başer

Medicinal and Aromatic Plant and Drug Research Centre (TBAM)

Anadolu University, 26470-Eskişehir, Turkey.

Email: khcbaser@anadolu.edu.tr or khcbaser@yahoo.co.uk