Chemical composition of the essential oil of Stachys menthifolia Vis.

Abstract

Stachys menthifolia Vis. (Lamiaceae) is an endemic species from the Balkan Peninsula spread throughout Albania, Greece, Montenegro, and Croatia. This article presents the first investigation of the essential oil composition of this species from Croatia. Aerial parts of the plant were collected from three different natural habitats in the region of Biokovo Mountain. The studied populations showed similarity in qualitative, but not in quantitative, composition of their essential oils. Hydrodistilled volatile oil obtained from the plant material of S. menthifolia was subjected to gas chromatographic analysis coupled to mass spectrometry. More than 100 compounds were identified in the three samples, representing 86.8–90.8% of the total oil. The terpene profile of S. menthifolia is characterized by a high content of oxygenated sesquiterpenes (48.4–58.9%) and diterpene hydrocarbons (3.5–25.2%), with 8-α-acetoxyelemol (6.9–21.3%), abietatriene (3.5–21.1%), and 4′-methoxyacetophenone (4.5–17.0%) as the main constituents.

Keywords::

• Stachys menthifolia Vis.
• 8-α-acetoxyelemol
• abietatriene
• essential oil composition
• GC-MS

Introduction

Stachys L. (Lamiaceae) is a large genus of herbs and shrubs comprising about 300 species, distributed in temperate and tropical regions of the world (Mabberley, 1997). Plants of this genus have been used in folk medicine for centuries to treat genital tumors, sclerosis of the spleen, inflammatory diseases, cough, ulcers, and infected wounds (Hartwell, 1982). Like most plants used for medicinal purposes, Stachys taxa have been submitted to several investigations in order to determine the content of biologically active compounds. As many other representatives of the family Lamiaceae, Stachys species also produce essential oils, but in spite of the large size of this genus, the composition of volatile compounds is known only in a small number of species (Maly, 1985; Cakir et al., 1997; Mariotti et al., 1997; Pélissier et al., 1999; Chalchat et al., 2000, 2001; Skaltsa at al., 2003; Tirillini et al., 2004; Bilusic Vundac et al., 2006; Radulovic et al., 2007).

More than 175 years ago, Roberto Visiani described the endemic species Stachys menthifolia Vis. of the West Balkan region at the locality of Ascrivium – the classical name for Kotor (Montenegro). This species was also found in Croatia, in the Biokovo area (Šilić & Šolić, 2002).

This article presents the first investigation of the essential oil composition of S. menthifolia collected from three localities in the Biokovo area, which represents the only certain and verified place where this plant is found in Croatia.

Material and methods

Plant material

Three specimens of S. menthifolia were gathered from three natural habitats on the eastern slopes of the Biokovo area. Details of the collection localities are presented in Table 1. All samples were collected in the course of flowering on the very same day in June 2005. Voucher specimens of each population were identified by Dr. Marija Edita Šolić, and deposited in the scientific collection of the Institute “Mountain and Sea,” in the Herbarium of the Biokovo region (MAKAR) in Makarska, Croatia, and at the Department of Chemistry, Faculty of Science, University of Sarajevo, Bosnia and Herzegovina.

Table 1.  Origin of plant material of Stachys menthifolia Vis.

Sample	Voucher specimen (MAKAR)	Locality	Altitude^a (m s. m.)	Slope (°)	Exposure
1	KU-2309	Vrgorac	180	60–90	E-SE
2	KU-2310	Vrgorac	30–130	0–30	S
3	KU-2311	Ploče	60	30	NW

^am s. m. - meter ‘supra mare’ (lat.)

Reagents

All applied reagents were of the highest purity available and purchased from the Sigma-Aldrich Chemical Co.

Isolation of volatile oil

Essential oils from the air-dried parts of S. menthifolia were isolated by hydrodistillation for 2 h. The oils were extracted with dichloromethane and dried over anhydrous sodium sulfate and stored at 4°C in the dark.

Gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) was carried out on a Hewlett-Packard (HP) 6890 Series II gas chromatograph fitted with a fused silica HP-5 (5% phenyl methyl siloxane) capillary column (30 m × 0.252 mm, 0.25 μm film thickness), coupled to a HP 6890 Series II mass selective detector (MSD). The column temperature was programmed from 60 to 240°C at 3°C min⁻¹, and helium was used as the carrier gas. Other operating conditions were as follows: inlet pressure 9.43 psi, injector temperature 250°C, detector temperature 280°C, split ratio 1:25, injection volume 1 μL. Ionization of the sample components was performed in electron ionization (EI) mode (70 eV), with scan range 20–555 amu, and scan time 1.60 s.

Qualitative and quantitative analysis

The linear retention indices (RIs) for all compounds were determined by co-injection of hexane solution containing the homologous series of C₈–C₂₆ n-alkanes (Van Del Dool & Kratz, 1963). Identification of the essential oil constituents was accomplished by visual interpretation, comparing their RIs and mass spectra with literature data (Adams, 2007) from computer library search (HP Chemstation computer library NBS75K.L, NIST/EPA/NIH Mass Spectral Library 2.0, and Mass Finder 3 Computer Software and Terpenoids Library), and the laboratory’s own database. Semiquantitative analysis was carried out directly from peak areas in the GC profile.

Results and discussion

The essential oils were extracted by hydrodistillation from the aerial parts of S. menthifolia. The content of essential oil based on the dry weight of the plant material was 0.07, 0.14, and 0.13% for sample 1, 2, and 3, respectively.

The total ion chromatograms of essential oils are displayed in Figure 1. The figure shows the heterogeneity of the mixtures, and also similarity in qualitative, but not in quantitative, composition of the samples. The components identified in S. menthifolia, their retention indices, and percentage compositions are summarized in Table 2, where all compounds are arranged in order of their elution on the HP-5 column.

Figure 1.  Total ion chromatogram (TIC) of the essential oils of Stachys menthifolia Vis. with mass spectra of the major constituents.

Table 2.  Essential oil composition of Stachys menthifolia Vis.

No.	RI	Compound	Sample 1 RA (%)	Sample 2 RA (%)	Sample 3 RA (%)
1	801	Hexanal	0.2	0.2	t
2	848	(2E)-Hexenal	0.1	0.2	t
3	901	Heptanal	t	t	t
4	921	α-Thujene	—	0.2	t
5	927	α-Pinene	—	1.0	t
6	943	(3Z)-Heptenal	t	t	—
7	945	(2E)-Heptenal	t	t	—
8	952	Thuja-2,4(10)-diene	t	—	t
9	953	Benzaldehyde	—	t	t
10	975	1-Octen-3-ol	0.5	0.2	t
11	982	6-Methyl-5-hepten-2-one	t	t	t
12	991	β-Myrcene	—	t	t
13	994	3-Octanol	0.1	t	t
14	997	(2E, 4E)-Heptadienal	t	t	t
15	1020	p-Cymene	t	0.2	t
16	1024	Limonene	0.1	3.4	0.3
17	1029	1,8-Cineole	t	t	—
18	1031	(3E)-Octen-2-one	t	t	—
19	1039	Benzene acetaldehyde	t	0.1	t
20	1053	γ-Terpinene	—	t	t
21	1068	cis-Linalool oxide (furanoid)	t	t	—
22	1085	trans-Linalool oxide (furanoid)	t	t	—
23	1096	Linalool	0.9	1.7	0.7
24	1102	n-Nonanal	t	t	—
25	1111	Phenyl ethyl alcohol	t	t	—
26	1113	6-Camphenol	t	t	—
27	1122	α-Campholenal	t	t	t
28	1133	cis-p-Mentha-2,8-dien-1-ol	t	t	—
29	1136	trans-Pinocarveol	t	t	—
30	1140	trans-Verbenol	0.3	t	0.2
31	1173	Terpinen-4-ol	0.9	0.5	0.4
32	1184	p-Cymen-8-ol	t	t	t
33	1186	cis-Pinocarveol	t	t	—
34	1187	α-Terpineol	0.3	0.2	t
35	1189	Methyl salycilate	t	t	—
36	1193	Myrtenol	t	t	—
37	1194	Myrtenal	t	t	t
38	1197	cis-Piperitol	t	t	—
39	1208	Verbenone	t	—	t
40	1216	trans-Carveol	0.3	t	0.2
41	1240	Carvone	0.2	t	0.2
42	1270	Pregeijerene B	—	t	0.3
43	1350	4’-Methoxyacetophenone	17.0	7.3	4.5
44	1352	Thymol acetate	t	t	t
45	1372	α-Copaene	t	0.3	0.2
46	1381	β-Bourbonene	t	t	—
47	1405	Longifolene	—	t	t
48	1415	β-Caryophyllene	0.5	1.4	2.8
49	1436	β-Guaiene	—	t	t
50	1449	α-Humulene	t	t	0.2
51	1451	Geranyl acetone	—	t	t
52	1462	cis-Cadina-1,6(4)-diene	0.3	t	0.2
53	1481	ar-Curcumene	t	0.2	t
54	1485	(E)-β-Ionone	t	—	t
55	1492	10,11-Epoxy-calamenene	t	t	t
56	1497	α-Muurolene	t	0.2	0.2
57	1507	α-Bisabolene	—	t	0.1
58	1511	γ-Cadinene	t	0.4	0.4
59	1514	endo-1-Bourbonanol	t	0.2	0.3
60	1521	δ-Cadinene	0.4	1.5	1.6
61	1524	Dihydroactinidiolide	0.4	t	t
62	1534	α-Cadinene	t	t	0.2
63	1540	α-Calacorene	0.2	0.2	0.2
64	1547	Elemol	2.2	3.6	2.8
65	1563	Palustrol	t	0.2	t
66	1566	β-Calacorene	t	t	t
67	1568	(3Z)-Hexenyl benzoate	t	t	t
68	1574	Spathulenol	1.5	1.7	0.7
69	1579	Caryophyllene oxide	2.8	2.4	5.2
70	1588	Globulol	1.5	2.4	1.9
71	1594	Viridiflorol	t	t	t
72	1600	Guaiol	0.3	—	t
73	1605	5-epi-7-epi-α-Eudesmol	t	0.3	0.4
74	1608	β-Oplopenone	t	t	t
75	1615	10-epi-γ-Eudesmol	t	t	t
76	1617	1,10-di-epi-Cubenol	—	t	t
77	1626	trans-Isolongifolanone	1.2	1.6	1.2
78	1628	γ-Eudesmol	1.1	1.9	1.4
79	1633	Caryophylla-4(12),8(13)-dien-5-α-ol	0.4	t	0.3
80	1640	epi-α-Muurolol	1.8	2.6	2.0
81	1643	Hinesol	t	t	t
82	1647	β-Eudesmol	2.4	4.8	5.0
83	1650	α-Eudesmol	1.4	3.2	3.3
84	1652	α-Cadinol	2.3	3.1	2.0
85	1664	14-Hydroxy-β-caryophyllene	t	t	t
86	1668	Valeranone	3.4	5.7	4.6
87	1762	(Z)-Lanceol	t	t	t
88	1674	n-Tetradecanol	—	0.6	0.5
89	1675	Khusinol	0.4	—	—
90	1682	Germacra-4(15),5,10(14)-tien-1-α-ol	0.4	0.3	t
91	1688	Eudesma-4(15),7-dien-1-β-ol	0.3	t	0.2
92	1699	Amorpha-4,9-dien-2-ol	t	0.2	0.5
93	1734	Oplopanone	2.3	0.8	0.5
94	1742	8-α-11-Elemodiol	1.9	0.3	0.5
95	1760	Cadalene	0.8	0.2	0.4
96	1772	α-Costol	0.8	0.2	—
97	1792	8-α-Acetoxyelemol	21.3	6.9	12.8
98	1806	Eudesm-11-en-4-α,6-α-diol	0.6	—	—
99	1811	Cryptomeridiol	6.7	4.3	0.5
100	1824	Khusinol acetate	—	t	t
101	1902	Isopimara-9(11),15-diene	0.5	0.9	1.1
102	1953	Pimaradiene	0.2	0.6	0.8
103	1983	Manool oxide	0.7	2.3	—
104	2002	(E)-Labda-7,12,14-triene	—	0.5	2.7
105	2005	13-epi-Dolabradien	—	0.3	0.6
106	2050	Abietatriene	3.5	11.8	21.1
107	2072	Abietadiene	—	t	0.8
108	2141	Abienol	—	0.9	0.7
109	2206	2-Keto-manool oxide	0.7	1.2	0.7
110	2225	7-α-Hydroxy-manool	—	0.3	0.2
111	2237	n.i. 1	1.5	0.9	0.9
112	2258	Larixol	0.4	1.1	0.7
113	2284	n.i. 2	4.3	6.2	2.6
114	2287	Dehydroabietal	—	—	1.5
115	2298	n-Tricosane	—	t	t
116	2398	n-Tetracosane	0.1	t	t
117	2499	n-Pentacosane	0.2	t	t
118	2598	n-Hexacosane	0.2	t	t
		Total identified	87.0	86.8	90.8

RI, retention indices calculated on HP-5 column; RA, relative area; t, traces (<0.1%); n.i., not identified; n.i. 1: m/z (%) = 27 (12), 43 (100), 55 (64), 67 (46), 81 (55), 95 (45), 107 (37), 121 (36), 135 (53), 147 (25), 161 (13), 175 (37), 190 (33), 207 (21), 218 (3), 227 (1), 236 (2), 243 (4), 255 (50), 273 (7), 291 (67); n.i. 2: m/z (%) = 27 (19), 43 (100), 55 (75), 69 (63), 81 (46), 95 (47), 109 (38), 123 (70), 137 (29), 149 (28), 159 (21), 177 (21), 187 (16), 205 (39), 215 (16), 225 (2), 233 (7), 243 (17), 255 (5), 261 (19), 271 (7), 291 (5), 304 (2).

Ninety-eight compounds were identified in sample 1, representing 87.0% of the total oil. The most abundant compounds were 8-α-acetoxyelemol (21.3%), 4’-methoxyacetophenone (17.0%), and cryptomeridiol (6.7%). One hundred-eleven chemical constituents were identified in sample 2, representing 86.8% of the total volatiles. Major compounds in this sample were abietatriene, 4’-methoxyacetophenone, and 8-α-acetoxyelemol, with percentages of 11.8, 7.3, and 6.9%, respectively. Ninety-eight identified components in sample 3 represented 90.8% of the total essential oil. The study of chemical composition of this sample revealed richness in abietatriene (21.1%), 8-α-acetoxyelemol (12.8%), and caryophyllene oxide (5.2%).

The distribution of the terpenoid compounds in the studied S. menthifolia essential oils is given in Table 3. Oxygenated sesquiterpenes were predominant in all samples, with a content of 58.9% in sample 1 and the same relative amount of 48.4% in samples 2 and 3. The next most abundant class of compounds in sample 1 were aromatic compounds, representing 17.0% of the total oil, while samples 2 and 3 showed a high abundance of diterpene hydrocarbons, 12.6 and 25.2%, respectively.

Table 3.  Classes of compounds identified in essential oil of Stachys menthifolia Vis.

Class of compounds	Sample 1 (%)	Sample 2 (%)	Sample 3 (%)
Aliphatic compounds	1.4	1.2	0.5
Aromatic compounds	17.0	7.6	4.5
Monoterpene hydrocarbons	0.1	4.6	0.3
Oxygenated monoterpenes	2.9	2.4	1.7
Sesquiterpene hydrocarbons	1.4	4.2	6.4
Oxygenated sesquiterpenes	58.9	48.4	48.4
Diterpene hydrocarbons	3.5	12.6	25.2
Oxygenated diterpenes	1.8	5.8	3.8
Total	87.0	86.8	90.8

It is highly possible that the observed differences in content and composition of the essential oils of three S. menthifolia populations, collected from the fissures of limestone or open, limy, rocky ground in a relatively small area of Biokovo Mountain, are the result of different geographical parameters, such as altitude, slope, and exposure (Table 1). To be more precise, the highest altitude and solar exposure might have had some influence on the total amount of oxygen-containing compounds in sample 1 being extremely high (80.6%). In contrast, the content of terpenoid hydrocarbons in this sample (5.0%) was significantly lower than in essential oil obtained from sample 2, collected from a lower altitude and more southern exposure (21.4%). In addition, essential oil from sample 3, originating from a natural habitat found near the sea-coast, at low altitude, had the highest percentage of terpenoid hydrocarbons (31.9%), probably due to the north-western, less solar exposure.

The literature survey on studies related to essential oils obtained from plants belonging to the Stachys genus from the Balkan Peninsula showed significant variability in their chemical compositions depending on the location and stage of plant development. Essential oil obtained from Stachys milanii Petrovic (Palic et al., 2006) had borneol and terpinen-4-ol as the most abundant compounds, while the major constituents of essential oil from Bosnian Stachys alpina L. spp. dinarica Murb. were β-caryophyllene and germacrene D (Kukic et al., 2006). Essential oil obtained from Stachys officinalis L. (Trevis) possessed a similar chemical constitution, with a mixture of isocaryophyllene and β-caryophyllene as the principal components (Chalchat et al., 2001). Dehydroabietatriene was the predominant compound in the essential oil of Stachys plumose Griseb. (Petrovic et al., 2006). Sesquiterpene hydrocarbons were the main group of constituents in Croatian Stachys species (S. officinalis, S. palustris, S. recta, and S. salviifolia) except in S. alpina, which had a higher content of oxygenated sesquiterpenes (Bilusic Vundac et al., 2006).

According to the presence and quantity of dominant compounds, the essential oil of investigated populations of S. menthifolia significantly differs from the only previously published data concerning this species (Skaltsa et al., 2003). Essential oil obtained from S. menthifolia of Greek origin had abietatriene (13.7%), kaurene (9.0%), and 13-epi-manoyl oxide (7.5%) as the most abundant compounds. In general, this oil was characterized by a high content of terpenoid hydrocarbons (42.3%), while Croatian samples of this species had a smaller amount of this class of compounds (5.0–31.9%).

Although previously examined, S. menthifolia from Greece had 13-epi-manoyl oxide as one of the principal constituents of its oil, the total amount of oxygen-containing compounds was significantly low (28.2%) in comparison to Croatian specimens (58.4–80.6%). Moreover, the main components of essential oil from Greek S. menthifolia were not found in Croatian specimens, with the exception of abietatriene, found as one of the major constituents of the essential oil investigated in the present work. In conclusion, the observed differences in qualitative and quantitative composition of the essential oils obtained from these two geographically isolated populations of S. menthifolia confirm the influence of environmental conditions on the nature of the plant chemical composition.

Based on the profile of the principal terpene constituents, the studied populations of Croatian S. menthifolia showed a unique essential oil composition among species of Stachys genus. In general, all samples were characterized by a high content of oxygenated sesquiterpenes. This study presents the first report of an essential oil with high percentage of 8-α-acetoxyelemol, obtained from a plant that belongs to the Lamiaceae family. Up to now, this oxygenated sesquiterpene has been known as one of the main characteristics of essential oils of the Juniperus genus (Cupressaceae), (Adams, 2004; Adams et al., 2006).

To summarize, GC-MS analysis of the volatile constituents of three S. menthifolia populations growing wild in the Biokovo area of Croatia indicated similarities in qualitative, but differences in quantitative, composition of their essential oils. From the presented results it can be concluded that two of the three populations clearly belong to the abietatriene chemotype, while the third population appears to belong to a new 8-α-acetoxyelemol chemotype.

Acknowledgement

Part of this article was presented as a poster at the 14th International Symposium on Separation Sciences “New Achievements in Chromatography,” Primošten, Croatia, 30th September–3rd October 2008.

Declaration of interest: The authors report no conflicts of interest.