Essential oil content and composition of Achillea biebersteinii Afan. in different plant parts and phenological stages

Abstract

In this study, the essential oil content and composition of Achillea biebersteinii Afan. at different phenological stages including vegetative, floral budding, full flowering (leaves, stems and flowers) and fruit set were studied by means of gas chromatography (GC) and gas chromatography–mass spectometry (GC–MS). All oil samples from different plant parts and phenological stages were mostly made up of monoterpenoid compounds (88.6 – 99.6%), especially oxygenated ones (52.4 – 82.4%). The oil of the vegetative stage contained high amounts of limonene, 4aα-7β-7aα-nepetalactone, p-cymene and 1,8-cineole. The major constituents in the flower budding stage oil were found to be limonene, 1,8-cineole and 4aα-7β-7aα-nepetalactone. In the oil of the fruit set stage, γ-terpinene, p-cymene and cis-chrysanthenyl acetate were the predominant constituents. On the other hand, the most important compounds from the stem oil were 4aα-7α-7aα-nepetalactone, 1,8-cineole, 4aα-7β-7aα-nepetalactone and camphor. 4aα-7α-7aα-nepetalactone, limonene, 1,8-cineole and cis-p-menth-2-en-1-ol were found in high concentration in the oil of leaves, whereas 4aα-7α-7aα-nepetalactone, 4aα-7β-7aα-nepetalactone, limonene and p-cymene were present in large amounts in the oil of flowers.

Keywords:

• Achillea biebersteinii Afan.
• essential oil
• phenological stages
• limonene
• 4aα-7α-7aα-nepetalactone
• 4aα-7β-7aα-nepetalactone

Introduction

The genus Achillea L. (commonly known as yarrow) belongs to the family Asteraceae and comprises more than 100 species worldwide (1). These often medicinal and rhizomatous perennial plants are native to Europe, Western Asia and North Africa, although they are also found in Australia, New Zealand and North America (2, 3). In traditional systems of medicine, Achillea species have a long history of use as medicinal plants mainly due to their anti-inflammatory, anti-spasmodic, diaphoretic, diuretic, carminative, tunic, vermifugal and emmenagogic properties and are used as a cure for hemorrhage, pneumonia, rheumatic and abdominal pains, stomach-ache and wounds (4, 5, 6). Nowadays, different medicinal properties of these plants such as spasmolytic, choleretic, anti-inflammatory and wound healing are documented (7). In recent years, anticancer activity of essential oils isolated from some Achillea species has been reported and it has been shown that they can modulate macrophage activities (8). Due to their hair growth promotion properties, yarrow essential oils are used in cosmetic industries for production of hair shampoos as well creams (9, 10).

In the flora of Iran, the genus Achillea is represented by 19 species of which seven are endemic (3). One of these species is A. bieberstenii Afan. which occurs naturally in many parts of the country in the central, north, northwest, west and northeast with the local name of ‘Bumadarane Zard’ (3, 11). This plant is a perennial villous herb with 10 – 100 cm height and radiate heads which are borne in large dense compound corymbs on the erect stems (3). To date, many investigations considered the volatile oil of A. biebersteinii from the chemical constituents to biological activities points of view (1, 5, 6, 12–15). Based on the results of these studies, there is a considerable chemical polymorphism in the essential oil of this plant. These oils show different biological activities including antibacterial, antifungal, antioxidant, insecticidal, herbicidal and wound healing (6, 13, 16, 17).

It is clear that the essential oil content and composition of medicinal and aromatic plants and so their biological activities are influenced by both intrinsic and external factors such as genetic background, climatic conditions, phenological stages, type of plant part, processing of plant materials and method of oil extraction (18–22). Although in a previous study on this plant, essential oil compositions of different aerial parts (leaves, flowers and stems) from it were reported (5) as far as we know there is no other previous report on the chemical analysis of essential oils in different phenological stages.

This paper deals with the results of chemical analyses of the oils obtained from different phenological stages of A. biebersteinii including flowering (leaves, stems and flowers), floral budding, vegetative and fruit set. The results can be used for determining optimal harvesting times of this plant for relevant industries.

Experimental

Plant materials and extraction of essential oils

Fresh aerial parts of A. biebersteinii were collected in May and June 2009 at different developmental stages (vegetative, floral budding, flowering and fruit set) from its natural habitat in the Dizin zone, northwest of Tehran, Iran (Latitude: 36° 4´ 52´´ N, Longitude: 51° 22´ 46´´E, Altitude: 2325–2425 m). Voucher specimens were deposited at the Herbarium of Ferdowsi University of Mashhad (FUMH), Mashhad, Iran.

For extraction of the essential oil, the air dried sample of each plant part and phenological stage (50 g) was separately hydrodistilled using a Clevenger-type apparatus for 3 hours, according to the method recommended in the British Pharmacopoeia (23). To determine the oil content of plant materials, the experiment was repeated three times. After isolation, all essential oils were dried over anhydrous sodium sulfate and stored in tightly closed dark vials at 4°C until analysis.

GC and GC–MS conditions

GC analyses were performed using a Shimadzu GC-9 gas chromatograph equipped with a DB-5 (dimethylsiloxane, 5% phenyl) fused silica column (J & W Scientific Corporation) (30 m × 0.25 mm i.d., film thickness 0.25 μm). Oven temperature was held at 50°C for 5 min and then programmed to rise to 240°C at a rate of 3°C/min. The flame ionization detector (FID) temperature was 265°C and injector temperature was 250°C. Helium was used as carrier gas with a linear velocity of 32 cm/s. The percentages of compounds were calculated by the area normalization method, without considering response factors.

GC–MS analyses were carried out in a Varian 3400 GC-MS system equipped with a DB-5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 μm); oven temperature was 50–240°C at a rate of 4°C/min, transfer line temperature 260°C, carrier gas, helium, with a linear velocity of 31.5 cm/s, split ratio 1:60, ionization energy 70 eV, scan time 1 s, and mass range 40–300 amu.

Identification of volatile components

The components of the oils were identified by comparison of their mass spectra with those of a computer library or with authentic compounds and confirmed by comparison of their retention indices, either with those of authentic compounds or with data published in the literature (24, 25). Mass spectra from the literature were also compared (24, 25). The retention indices were calculated for all volatile constituents using a homologous series of n-alkanes.

Results and discussion

The percentage compositions of all essential oils are listed in Table 1. There were significant differences among the essential oil contents of A. biebersteinii plants in different phenological stages and parts. Based on the dry weight of samples, the yield of essential oil ranged from 0.41% (whole plant in fruit set stage) to 1.12% (flowers in full flowering stage). In previous studies, the essential oil content of A. biebersteinii collected from different parts of the world at the flowering stage has been reported to be 0.20% (Jordan) (12), 0.63–0.70% (Turkey) (6) and 0.42–2.70% (Iran, transferred to the field conditions) (1).

Table 1. Percentage composition of essential oil samples isolated from different parts and morphological stages of Achillea biebersteinii Afan.

NO.	Compounds	RIs	RI	Area (%)						Method of identification
				At Flowering			Phenological stage
				Flowers	Leaves	Stems	V	F.B	F.S
1	α-Pinene	939	939	0.6	2.5	0.6	1.5	3.1	0.3	RI, MS, CoI
2	Camphene	954	952	0.4	1.9	0.6	1.9	0.9	0.1	RI, MS, CoI
3	Yomogi alcohol	999	998	0.6	0.6	1.2	2.6	4.1	2.3	RI, MS
4	α-Phellandrene	1003	1002	0.3	1.1	0.5	0.5	1.1	-	RI, MS
5	α-Terpinene	1017	1018	0.3	-	0.4	-	-	0.2	RI, MS
6	p-Cymene	1025	1025	8.3	-	0.6	14.4	-	13.8	RI, MS, CoI
7	Limonene	1029	1031	11.0	15.4	3.6	28.9	35.5	-	RI, MS, CoI
8	1,8- Cineole	1031	1033	4.2	15.3	18.8	12.3	25.2	7.1	RI, MS, CoI
9	γ-Terpinene	1060	1061	-	-	-	-	-	63.7	RI, MS
10	Artemisia ketone	1062	1063	0.2	-	-	0.5	0.5	-	RI, MS
11	Artemisia alcohol	1084	1083	-	-	-	-	-	1.2	RI, MS
12	trans-Sabinene hydrate	1098	1096	0.1	-	-	-	-	-	RI, MS
13	Linalool	1097	1098	0.2	0.4	0.4	0.3	-	-	RI, MS, CoI
14	cis-p-Menth-2-en-1-ol	1122	1120	5.5	7.7	5.9	3.0	1.6	0.6	RI, MS
15	Chrysanthenone	1128	1125	1.7	3.6	2.6	0.6	0.4		RI, MS
16	Camphor	1146	1143	1.8	5.3	7.5	4.2	1.6	1.2	RI, MS, CoI
17	Terpinen-4-ol	1177	1177	0.6	1.3	2.8	1.6	1.9	-	RI, MS, CoI
18	α-Terpineol	1189	1189	-	-	1.0	-	1.1	-	RI, MS
19	cis-Chrysanthenyl acetate	1265	1262	-	-	-	-	-	4.4	RI, MS
20	4aα-7α-7aα-Nepetalactone	1360	1358	33.6	34.1	32.7	5.7	3.5	-	RI, MS
21	4aα-7α-7aβ-Nepetalactone	1387	1385	0.5	1.2	1.2	1.7	1.9	4.3	RI, MS
22	4aα-7β-7aα-Nepetalactone	1392	1390	29.1	5.9	8.4	20	11.1	-	RI, MS
23	Spathulenol	1578	1576	-	-	1.9	-	-	-	RI, MS
24	Caryophyllene oxide	1579	1579	-	-	1.6	-	-	-	RI, MS
25	Globulol	1585	1583	-	-	3.3	-	-	-	RI, MS
Monoterpene hydrocarbons				20.8	20.9	6.3	47.2	40.6	78.1
Oxygenated monoterpenes				78.2	75.5	82.4	52.4	52.9	21.1
Oxygenated Sesquiterpenes				-		6.5	-	-
Total identified				99.0	96.4	95.2	99.6	93.5	99.2
Essential oil content (v/w %)				1.12	1.05x	1.05	0.54	0.61	0.41
Notes: RI, retention indices in elution order from DB-5 column; RIs, Standard RI from Adams, 2007; MS, mass spectrometry; CoI, co-injection. V, F.B, F.S are samples from stages of vegetative, floral budding and fruit set, respectively.

GC and GC-MS allowed the identification of 25 constituents in the essential oil of all samples representing about 93.5% to 99.6% of the total composition. Only seven components were common to all oils. The highest and lowest number of components were identified in the oils of stems (20 constituents) and fruit set stage (12 compounds), respectively. The identified constituents and their relative percentages are presented in Table 1, where the components are listed in order of their elution from the DB-5 column. There were similarities and significant differences among these oils. Oxygen-containing monoterpenes constituted the principal fraction of all samples with percentages ranging from 52.4% (vegetative stage) to 82.4% (stem part), except oil of the fruit set stage in which monoterpene hydrocarbons (78.1%) were dominant. Monoterpene hydrocarbons were the second main group of constituents (6.3–78.1%), except oil of stems in which oxygenated sesquiterpenes (6.7%) were dominant. These components were exclusively produced in stems at full flowering stage.

The major constituents identified in the oil of the vegetative stage were limonene (28.9%), 4aα-7β-7aα-nepetalactone (20%), p-cymene (14.4%) and 1,8- cineole (12.3%). The oil of the floral budding stage was dominated by limonene (35.5%), 1,8- cineole (25.2%) and 4aα,7-β,7a-α-nepetalactone (11.1%). The main components found in the oil of the flowering stage were 4aα-7α-7aα-nepetalactone (54.3%), cis-p-menth-2-en-1-ol (7.9%), limonene (7.5%), 1,8-cineole (7%) and 4aα,7-β,7a-α-nepetalactone (6.5%). The oil of the fruit set stage was dominated by γ-terpinene (63.7%), p-cymene (13.8%) and cis-chrysanthenyl acetate (4.4%), while two components of γ-terpinene and cis-chrysanthenyl acetate were detected only in this stage. The stem oils were rich in 4aα-7α-7aα-nepetalactone (32.7%), 1,8- cineole (18.8%), 4aα-7β-7aα-nepetalactone (8.4%) and camphor (7.5%), whereas the leaf oils were rich in 4aα-7α-7aα-nepetalactone (34.1%), limonene (15.4%), 1,8-cineole (15.3%) and cis-p-menth-2-en-1-ol (7.7%). In the oil of inflorescence, 4aα-7α-7aα-nepetalactone (33.6%), 4aα-7β-7aα-nepetalactone (29.1%), limonene (11%) and p-cymene (8.3%) were dominant.

The biosynthesis of secondary metabolites is a highly coordinated and regulated process and involves both metabolon formation and metabolic channeling (26). From the secondary metabolism point of view, the chemical composition of medicinal and aromatic plants can be altered in response to different environmental conditions and developmental stages or in different plant parts (18, 20, 21, 27).

Comparing results of the present study on the essential oils of A. biebersteinii plants with those reported earlier, Kordali et al. (14) reported similarly high levels of oxygenated monoterpenes (84.0%), with 1,8-cineol (38.1%) and camphor (23.6%) as the major components of a sample from Erzurum, Turkey, along with a small amount of sesquiterpenes (6.4%). Accordingly, 1,8-cineol (9.6–22.3%), camphor (4.7–38.1%), chrysanthenone (8.2%) and p-cymene (31.6%) have been reported by several other authors as the principal constituents of A. biebersteinii essential oils. In some cases, however, the analyzed essential oils were dominated by piperitone, ascaridole, borneole and carvenone oxide which were not found in our samples at all (5, 6, 12–15).

Considering the limited distributions of nepetalactons in the plant kingdom (28, 29), the presence of this component in the essential oil of A. biebersteinii including our studied samples is interesting (6). To the best of our knowledge, this plant is the only species of the genus Achillea which produces nepetalactones. Except essential oil of the fruit set stage, all essential oils contained them as one of the major components.

Results of this study show that the pathway(s) for synthesis and pattern of accumulation of terpenes differs considerably between the different parts and different phenological stages of A. biebersteinii. It is seems that there is a correlation between synthesis of certain compounds and a given type of organ and/or phenological stage. Example in this study, for γ-terpinene was synthesized and accumulated in large amounts in the fruit set stage, whereas it was not contained in any part or any stage of this plant. Similarly, spathulenol, caryophyllene oxide and globulol existed only in stems. Therefore, the results of the present study offer a system to study genetic control mechanisms regulating terpene biosynthesis in an organ-specific or stage-specific manner.

Since aroma, taste and biological activities of essential oils are determined by their chemical compositions (27, 30), further studies are therefore needed to evaluate the potential applications of A. biebersteinii essential oils at different phenological stages in relevant industries.

Acknowledgements

Special thanks to Mr Morteza Akramian (Department of Horticultural Sciences, Faculty of Agriculture, University of Tehran) for helpful assistance in collecting plant material and for his valuable guidance in this work.