Chemical Composition and Antimicrobial Activities of the Essential Oils of Teucrium chamaedrys. subsp. chamaedrys., T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium.

Abstract

The composition of the essential oils obtained from the air-dried Teucrium chamaedrys. L. subsp. chamaedrys., Teucrium orientale. L. var. puberulens., and Teucrium chamaedrys. L. subsp. lydium. (Lamiaceae) were analyzed by GC-MS. Thirty-six, 35, and 33 components were identified in the essential oils, and germacrene D (16.7%) was the most abundant constituent in T. chamaedrys. subsp. chamaedrys., and β-caryophyllene was the most abundant component in both T. orientale. var. puberulens. and T. chamaedrys. subsp. lydium. in the ratios 21.7% and 19.7%, respectively. The isolated essential oils of the plants were tested for antimicrobial activity and showed moderate antibacterial activity against Gram-positive and Gram-negative bacteria, but no antifungal activity was observed against two yeast-like fungi.

Keywords:

• Antimicrobial activity
• essential oil
• GC-MS
• Lamiaceae
• Teucrium chamaedrys. subsp. chamaedrys
• T. chamaedrys. subsp. lydium
• T. orientale. var. puberulens

Introduction

The genus Teucrium. L. (Lamiaceae) is represented by 32 species in Turkey, which are annual or biennial herbs or shrubs (Ekim, 1982; Davis, 1988; Güner et al., 2000). Representatives of Teucrium. genus have been used for more than 2000 years as medicinal herbs (Grieve, 1996; Ulubelen et al., 2000; Bedir et al., 2003). Teucrium flavum. L., T. montanum. L., and T. chamaedrys. L. have been used in folk medicine to treat ulcers and diabetes and to combat obesity in Turkey (Zeybek & Zeybek, 1994; Baytop, 1999; Ulubelen et al., 2000). T. chamaedrys. is distributed throughout Turkey with six subspecies, among which T. chamaedrys. L. subsp. chamaedrys., a rhizomatous perennial herb, is mainly distributed in northern and central Anatolia (Ekim, 1982; Davis, 1988; Güner et al., 2000). T. orientale. L. var. puberulens. is a nice-smelling nonendemic member of this genus and distributed mainly in central Anatolia (Davis, 1988; Güner et al., 2000). T. chamaedrys. L. subsp. lydium. O. Schwarz is distributed mainly in the west and southwest of Anatolia (Davis, 1988; Güner et al., 2000).

Previous phytochemical studies on T. chamaedrys. subsp. chamaedrys. have shown the presence of different natural compounds including 24α-ethylcholesta-5,25-dien-3β-ol, sitosterol, α-amyrin, ursolic acid, apigenin, naringenin, pectolinarigenin, circiliol, 3β-hydroxystigmast-24,25-dien-24-al, and 3β-hydroxy-24α-ethylcholesta-5,25-dien-7-one (Ulubelen et al., 1994). T. chamaedrys. is known to contain neo-clerodane diterpenoids and phenylethanoid glycosides (Bedir et al., 2003).

Volatile constituents and biological activities studies are available in the literature on Teucrium. species: T. haenseleri. Boiss. (Gaspar et al., 1997), T. chamaedrys. (Corovic et al., 1965; Martonfi & Cernaj, 1989; Kovacevic et al., 2001), T. fruticans. L. (Flamini et al., 2001), T. salviastrum. Schreb. (Cavaleiro et al., 2002), T. pumilum. L. (Perez et al., 2000), T. marum. L. (Ricci et al., 2005), T. leucocladum. Boiss. (El-Shazly & Hussein, 2004), T. polium. L. (Wassel & Ahmed, 1974 Vokou & Bessiere, 1984; Abdollahi et al., 2003; Proestos et al., 2006;), T. lusitanicum. Schreb. and T. algarbiensis. Cout. (Cavaleiro et al., 2004), T. orientale. L. var. orientale. (Javidnia & Miri, 2003; Yιldιrιm et al., 2004), T. chamaedrys. (Morteza-Semnani et al., 2005), T. capitatum. L. (Antunes et al., 2004), T. libanitis. Schreb. and T. turredanum. Losa and Rivas Goday (Blazquez et al., 2003), T. divaricatum. Heldr. subsp. divaricatum. (Tzakou et al., 1997), T. flavum. (Baher & Mirza, 2003), T. melissoides. Boiss. (Ahmadi et al., 2002), T. carolipaui. Pau (Pala-Paul et al., 2001), T. stocksianum. Boiss. (Al Yousuf et al., 2002), T. puechiae. Greuter and Burdet (Allain et al., 1994), T. flavum. L. subsp. flavum., T. polium., T. chamaedrys. L. var. Illyricum, and T. marum. (Pelissier et al., 1996), T. Be lion. Schreb. (Willar et al., 1980), T. arduini. L., T. botrys. L., T. chamaedrys., T. flavum., T. montanum., T. polium., T. scordium. L. (Kovacevic et al., 2001), H. perforatum. L. and T. chamaedrys. (Chialva et al., 1981), T. scorodonia. L. and T. oxylepis. Font Quer (Velasco-Negueruela & Perez-Alonso et al., 1990), T. heterophyllum. L'Her. (Barroso et al., 1996), T. cyprium. subsp. cyprium. Boiss., T. micropodioides. Rouy, T. divaricatum. subsp. canescens. Sieber, and T. kotschyanum. Poech (Arnold et al., 1991), T. polium., T. polium. L. var. album., and T. polium. L. var. pitpilosum. (Kamel & Sandra, 1994), T. puechiae. Greuter and Burdet (Allain et al., 1994), and T. marum. (Sanz et al., 2000).

To our knowledge, there is no published report on the essential oil analysis and antimicrobial activity of T. chamaedrys. subsp. chamaedrys, T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium.. As part of this systematic research, the essential oil constituents of the plants were obtained by the widely used hydrodistillation method in a Clevenger-type apparatus (Tunalier et al., 2002; Sefidkon et al., 2004; Yaylι et al., 2005). The obtained crude essential oils were then investigated by GC-MS technique. Identification of the compounds was made by a typical library search (NIST, Wiley) and literature comparison (Blank, 1989; Adams, 1995; Tzakou et al., 1997; Rychlik, 1998; Skaltsa et al., 2000, 2003; Tkachev & Dobrotvorsky, 2000; Flamini et al., 2001, 2002; Blazquez et al., 2003; Ertugrul et al., 2003; Jovanovic et al., 2004.; Sefidkon et al., 2004; Figueredo et al., 2005; Javidnia et al., 2005).

The purpose of this work is to investigate the chemical composition and antimicrobial activity of the essential oils from the three Teucrium. species: T. chamaedrys. subsp. chamaedrys., T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium..

Materials and Methods

Plant material

T. chamaedrys. subsp. chamaedrys. was collected in Kelkit, Pekün, Haneke plateau-Gümüşhane (Alpine meadows at a height of ∼2000 m) in the northeastern part of Turkey in July 2004. T.. orientale. var. puberulens. was collected in Tersun mountain, Şiran-Gümüşhane (at a height of ∼2000 m) in the northeastern part of Turkey in August 2004. T. chamaedrys. subsp. lydium. was collected in Çamlι plateau-Mersin (alpine meadows at a height of ∼3000 m) in the southeastern part of Turkey in July 2004. The plants were authenticated immediately after collection (Ekim, 1982; Davis, 1988; Güner et al., 2000) and air-dried at room temperature for later analysis. Voucher specimens (No. Coşkunçelebi 490-2004, 518-2004, and 535-2004 KTUB) were deposited in the herbarium of the Department of Biology, Karadeniz Technical University, Turkey.

Isolation of the essential oils

The air-dried plants of T. chamaedrys. subsp. chamaedrys. (42 g), T. orientale. var. puberulens. (45 g), and T. chamaedrys. subsp. lydium.(32 g) were hydrodistilled in a Clevenger-type apparatus using an ice bath for cooling system (3 h). The oils were taken by dissolving in HPLC grade n.-hexane (0.5 mL) and kept at 4°C in a sealed brown vial. One microliter of the extract was directly injected into the GC-MS instrument. The percentage yields of the oils from T. chamaedrys. subsp. chamaedrys., T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium., calculated on a moisture-free basis, were 0.15%, 0.25%, and 0.18% (v/w), respectively.

Gas chromatography

GC-MS analyses were performed using an Agilent-6890 GC System equipped with an Agilent-5973 MS system (Agilent Technologies, Palo Alto, CA, USA). The mass spectrometer with an ion trap detector was used in full scan mode under electron impact ionization (70 eV). The chromatographic column used for the analysis was an HP-5 capillary column (30 m × 0.32 mm i.d., film thickness 0.25 µm). Helium was used as carrier gas at a flow rate of 1 mL/min. The injections were performed in splitless mode at 230°C. One microliter essential oil solution in hexane (HPLC grade) was injected and analyzed with the column held initially at 60°C for 2 min and then increased to 260°C with a 5°C/min heating ramp and subsequently kept at 260°C for 13 min. The relative percentage amounts of the separated compounds were calculated from total ion chromatograms by a computerized integrator.

Identification of components

The components of the oil were identified by comparison of their mass spectra with those of mass spectral libraries (NIST and Wiley) and confirmed by comparison of their retention indices with data published in the literature (Adams, 1995; Skaltsa et al., 20002003; Tkachev & Dobrotvorsky, 2000; Flamini et al., 2002; Blazquez et al., 2003; Ertuğrul et al., 2003; Javidnia & Miri, 2003; Jovanovic et al., 2004; Yu, 2004; Figueredo et al., 2005; Javidnia et al., 2005; Morteza-Semnani et al., 2005; Yaylι et al., 2005).

Antimicrobial activity assessment

All test microorganisms were obtained from Refik Saydam Hifzissihha Institute (Ankara, Turkey) and were as follows: Escherichia coli. ATCC 35218, Klebsiella pneumoniae. ATCC 13883, Yersinia pseudotuberculosis. ATCC 911, Serratia marcescens. ATCC 13880, Enterococcus faecalis. ATCC 29212, Staphylococcus aureus. ATCC 25923, Bacillus subtilis. ATCC 6633, Candida albicans. ATCC 60193, Candida tropicalis. ATCC 13803. Essential oil was dissolved in acetone to prepare the stock solutions of 1000 and 500 µg/mL.

Agar-well diffusion method

A simple susceptibility screening test using the agar-well diffusion method as adapted earlier was used (Perez et al., 1990, 1999; Erdemoğlu et al., 2003; Bagamboula et al., 2004). Each microorganism was suspended in brain heart infusion (BHI) (Difco, Detroit, MI, USA) broth and diluted approximately 10⁶ colony forming unit (CFU) per milliliter. They were “flood-inoculated” onto the surface of BHI agar and Sabouraud dextrose agar (SDA) (Difco) and then dried. For C. albicans. and C. tropicalis., SDA was used. Five-millimeter-diameter wells were cut from the agar on plates using a sterile cork-borer, and 100 µL of the stock solution was delivered into the wells. The plates were incubated for 18 h at 35°C. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test microorganism. Ceftazidime (Fortum) (10 µg) and Triflucan (5 µg) were used as the standard drugs for antibacterial and antifungal activities, respectively. Acetone was used as solvent control. The tests were carried out in duplicate. Results were interpreted in terms of diameter of inhibition zone: (−): < 5.5 mm; (+): 5.5–10 mm; (+ +): 11–15 mm; (+ + +): ≥ 16 mm.

Results and Discussion

The composition of essential oils of T. chamaedrys. subsp. chamaedrys, T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium. were analyzed by GC-MS with an HP-5 column. The general chemical profile of the essential oils, the percentage content, and the retention indices of the constituents are summarized in Table 1. A total of 36, 35, and 33 components were characterized on the basis of a typical library search and literature data (Adams, 1995; Blank, 1989; Tzakou et al., 1997; Rychlik, 1998; Skaltsa et al., 20002003; Tkachev et al., 2000; Flamini et al., 20012002; Blazquez et al., 2003; Ertugrul et al., 2003; Javidnia & Miri, 2003; Jovanovic et al., 2004; Sefidkon et al., 2004; Figueredo et al., 2005; Javidnia et al., 2005; Morteza-Semnani et al., 2005; Yaylι et al., 2005) with selecting only the components showing matches exceeding 85%, which represented about 79.2%, 85.2%, and 76.8% of the essential oils from T. chamaedrys. subsp. chamaedrys, T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium., respectively.

Table 1. Identified components in the essential oil of T. chamaedrys. subsp. chamaedrys, T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium.^a..

		T. chamaedrys. subsp. chamaedrys.		T. orientale. var. puberulens.		T. chamaedrys. subsp. lydium.
No.	Compounds	% Area	Q. (%)	% Area	Q. (%)	% Area	Q. (%)	Exp. RI	Ident.(LRI/MS)
1	α-Thujene	0.2	98	—	—	0.1	91	928	929
2	α-Pinene	15.8	96	—	—	12.5	96	939	939
3	Camphene	0.1	98	—	—	0.1	98	954	952
4	(E.)-2-Heptenal	—	—	tr	92	—	—	958	961
5	β-Pinene	8.9	97	—	—	6.6	97	983	982
6	β-Myrcene	4.1	96	—	—	0.5	96	985	988
7	(E.,E.)-2,4-Heptadienal	0.1	94	—	—	—	—	997	1008
8	Phenyl oxirane	—	—	tr	87	—	—	1119	MS
9	(E.)-2-Octenal	0.1	85	—	—	—	—	1127	MS
10	2-Methyl cumarone	—	—	20.0	86	—	—	1188	MS
11	α-Terpineol	—	—	0.9	91	—	—	1196	1195
12	Safranal	—	—	0.1	95	—	—	1198	1201
13	Decanal	0.1	87	—	—	0.1	85	1205	1205
14	p.-Menth-1-en-9-al	—	—	1.0	90	—		1217	MS
15	β-Cyclocitral	0.2	92	—	—	—		1222	1218
16	Nerol	—	—	0.2	97	—		1231	1228
17	Carvone	—	—	—	—	0.2	97	1243	1241
18	Citrol	—	—	—	—	0.1	86	1255	MS
19	Geraniol	—	—	0.6	97	—	—	1257	1255
20	(E.)-2-Decenal	0.6	90	—	—	—	—	1263	1260
21	Nonanoic acid	—	—	0.1	90	—	—	1274	1275
22	Dihydroedulan I	—	—	0.2	94	—	—	1294	1289
23	Carvacrol	—	—	—	—	0.5		1299	1299
24	Tridecane	0.1	87	—	—	—		1300	1300
25	p.-Vinylguaiacol	—	—	0.3	97	—		1314	1323
26	(E.,E.)-2,4-Decadienal	0.4	94	—	—	0.1	86	1317	1316
27	Bicycloelemene	0.2	87	0.3	98	—		1340	MS
28	α-Cubebene	—	—	0.7	99	—		1353	1353
29	Eugenol	—	—	0.1	96	—		1359	1359
30	Decanoic acid	—	—	0.5	95	—		1373	1373
31	α-Copaene	0.5	99	1.3	99	0.6	99	1378	1378
32	β-Bourbonene	3.1	99	2.4	96	3.6	99	1387	1388
33	β-Cubebene	—	—	1.5	99	—	—	1392	1392
34	γ-Caryophyllene	0.2	99	0.5	99	—		1408	MS
35	β-Caryophyllene	11.8	99	21.7	99	19.7	99	1424	1424
36	epi.-Bicyclosesquiphellandrene	—	—	—	—	0.7	93	1430	MS
37	Aromadendrene	—	—	0.2	99	—	—	1442	1438
38	α-Humulene	1.0	99	4.8	99	1.7	99	1457	1457
39	(E.)-β-Farnesene	1.8	98	—	—	2.1	96	1460	1460
40	allo.-Aromadendrene	—	—	1.1	90	—	—	1465	1461
41	Germacrene D	16.7	99	10.6	99	9.3	99	1481	1481
42	Bicyclogermacrene	2.4	99	3.4	99	1.5	99	1497	1496
43	(E.,E.)-α-Farnesene	0.3	94	—	—	0.3	97	1509	1508
44	β-Bisabolone	—	—	0.4	98	—	—	1510	1509
45	γ-Cadinene	0.4	95	0.5	99	0.7	97	1516	1514
46	δ-Cadinene	2.1	99	4.1	99	1.7	99	1526	1526
47	Cadina-1,4-diene	0.2	89	—	—	—	—	1534	1532
48	α-Calacorene	0.2	95	—	—	0.4	98	1546	1546
49	Spathulenol	0.7	98	1.0	98	—	—	1580	1578
50	Caryophyllene oxide	2.4	94	2.7	94	6.1	94	1585	1584
51	Viridiflorol	—	—	0.5	99	—	—	1594	1594
52	Fonenol	—	—	0.4	90	—	—	1621	MS
53	Caryophylla-4(12),8(13)- dien-5-β-ol	—	—	—	—	0.7	99	1637	MS
54	tau-Muurolol	0.8	93	1.6	96	1.0	95	1645	1643
55	α-Cadinol	0.8	98	1.1	95	1.0	91	1657	1656
56	Caryophyllenol-II	—	—	—	—	1.1	86	1671	MS
57	Vulgarol B	—	—	0.4	87	—	—	1674	MS
58	di-epi-α-Cedrene	0.9	83	—	—	—	—	1677	MS
59	Heneicosane	0.2	83	—	—	—	—	2100	2100
60	cis.-Phytol	1.0	90	—	—	1.0	90	2113	2114
61	Tricosane	0.2	98	—	—	0.4	98	2300	2300
62	Tetracosane	—	—	—	—	0.3	96	2400	MS
63	Pentacosane	0.4	90	—	—	1.0	98	2500	2500
64	Heptacosane	0.2	95	—	—	0.6	98	2700	2700
65	Nonacosane	—	—	—	—	0.5	95	2900	MS

Unknown	RI	m./z. (%)	A	B	C
Un-1	1037	136(24), 121(26), 107(24), 93(78), 79(38), 68(100), 53(24)	5.4	—	5.1
Un-2	1044	136(2), 121(12), 105(18), 93(100), 79(38), 67(12), 53(12)	0.6	—	—
Un-3	1087	136(6), 121(24), 93(68), 80(30), 71(100), 67(20), 55(56)	0.6	—	—
Un-4	1089	154(5), 136(12), 121(24), 93(74), 80(32),71(100), 55(54)	—	4.2	—
Un-5	1090	207(6), 114(8), 98(32), 82(32), 70(36), 57(100), 53(8)	0.6	—	—
Un-6	1179	135(7), 131(2), 128(100), 102(6), 75(4), 64(8), 51(8)	—	0.6	—
Un-7	1189	149(4), 136(45), 126(56), 90(66), 81(42), 67(20), 59(100)	—	0.8	—
Un-8	1190	152(5), 107(57), 91(55), 79(100), 67(20), 55(23)	—	—	2.1
Un-9	1195	152(3), 135(8), 107(44), 91(54), 79(100), 67(14), 53(14)	0.6	—	—
Un-10	1432	204(8), 161(100), 133(12), 119(20), 105(37), 91(36), 79(19)	0.8	0.7	—
Un-11	1516	204(20), 189(24), 161(40), 105(50), 98(100), 69(75), 55(48)	—	—	2.3
Un-12	1552	205(12), 177(25), 121(40), 91(80), 79(100), 69(60), 55(62)	—	—	2.6
Un-13	1599	220(4), 134(44), 119(99), 107(76), 93(100), 79(84), 71(12)	—	0.5	—
Un-14	1641	246(6), 205(20), 159(36), 119(100), 105(64), 91(88), 79(40)	—	0.9	—
Un-15	1685	220(43), 177(37), 159(92), 109(100), 91(95), 79(82), 55(66)	—		2.7
Un-16	1688	220(30), 159(80), 109(80), 91(100), 79(64), 67(46), 55(58)	0.7	—	—
Un-17	1847	268(4), 250(8), 109(30), 95(28), 85(32), 71(56), 58(100)	1.3	1.0	—
Un-18	1960	256(14), 223(20), 149(100), 129(40), 73(22), 57(18)	—		2.5
Un-19	2113	278(5), 226(5), 123(27), 95(16), 81(27), 71(100), 57(32)	1.0	—	—
Total			11.6	8.7	17.3

Total isolate	79.2	85.2	76.8
Total unknown	11.6	8.7	17.3
TOTAL	90.8	93.9	94.1

RI, retention index; LRI, literature retention index; tr, trace, less than 0.05%; Q, quality; A, T. chamaedrys. subsp. chamaedrys.; B, T. orientale. var. puberulens;. C, T. chamaedrys. subsp. lydium..

^a.Compounds are listed in order of elution. RI values are calculated from retention times relative to that of n.-alkanes (C₆–C₃₂) on the nonpolar HP-5 column.

Germacrene D (16.7%), α-pinene (15.8%), β-caryophyllene (11.8%), β-pinene (8.9%), and β-myrcene (4.1%) were the main constituents of the essential oil of T. chamaedrys. subsp. chamaedrys.. The main components of the essential oil of T. orientale. var. puberulens. were β-caryophyllene (21.7%), 2-methyl cumarone (20.0%), germacrene D (10.6%), α-humulene (4.8%), and δ- cadinene (4.1%). The major compounds in the essential oils of T. chamaedrys. subsp. lydium. were β-caryophyllene (19.7%), α-pinene (12.5%), germacrene D (9.3%), β-pinene (6.6%), and caryophyllene oxide (6.1%).

The chemical class distribution of the essential oil components of the plants are reported in Table 2. The compounds were separated into six classes, which were monoterpenes, monoterpenoids, sesquiterpenes, sesquiterpenoids, diterpenoids, and others (Table 2). Eleven compounds were common to all three species with the total ratio of 42.0%, 54.2%, and 46.9% in T. chamaedrys. subsp. chamaedrys, T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium., respectively.

Table 2. The chemical class distribution of the essential oil components of T. chamaedrys. subsp. chamaedrys., T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium..

	T. chamaedrys. subsp. Chamaedrys.		T. orientale. var. puberulens.		T. chamaedrys. subsp. lydium.
Compound class	% Area	Number of compounds	% Area	Number of compounds	% Area	Number of compounds
Monoterpenes	29.1	5	—	—	19.8	5
Monoterpenoids	0.2	1	2.9	6	0.8	3
Sesquiterpenes	41.8	15	53.5	15	42.3	12
Sesquiterpenoids	4.7	4	7.7	7	9.9	5
Diterpenoids	1.0	1	—	—	1.0	1
Others	2.4	10	21.1	7	3.0	7
The common compounds	42.0	11	54.2	11	46.9	11
Total isolate	79.2	36	85.2	35	76.8	33

The major compounds for the chemical class distributions in the essential oils of T. chamaedrys. subsp. chamaedrys., T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium. are reported in Table 3; germacrene D (16.7%) was the most abundant constituent in T. chamaedrys. subsp. chamaedrys., and β-caryophyllene was the most abundant component in both T. orientale. var. puberulens. and T. chamaedrys. subsp. lydium. in ratios 21.7% and 19.7%, respectively. Comparing the two oil compositions, a similar biosynthetic trend is evident in both varieties of T. chamaedrys., at least for the main constituents in the ratio of 72.7% out of 76.8%. However, the qualitative and quantitative difference between the two essential oils allows the differentiation between the two varieties in agreement with the morphological observations described above.

Table 3. The major components in the chemical class distribution of the essential oil constituents of T. chamaedrys. subsp. chamaedrys., T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium..

	T. chamaedrys. subsp. chamaedrys.			T. orientale. var. puberulens.			T. chamaedrys. subsp. lydium.
Compound class	Major component	% Area	RI	Major component	% Area	RI	Major component	% Area	RI
Monoterpenes	α-Pinene	15.8	939	—	—	—	α-Pinene	12.5	939
Monoterpenoids	β-Cyclocitral	0.2	1222	α-Terpineol	0.9	1196	Carvacrol	0.5	1299
Sesquiterpenes	Germacrene D	16.7	1481	β-Caryophyllene	21.7	1424	β-Caryophyllene	19.7	1424
Sesquiterpenoids	Caryophyllene oxide	2.4	1585	Caryophyllene oxide	2.7	1585	Caryophyllene oxide	6.1	1585
Diterpenoids	cis.-Phytol	1.0	2113	—	—	—	cis.-Phytol	1.0	2113
Others	(E.)-2-Decenal	0.6	1263	2-Methyl cumarone	20.0	1188	Heptacosane	0.6	2700

RI, retention index.

The antimicrobial activities of the essential oils of T. chamaedrys. subsp. chamaedrys, T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium. were tested in. vitro. by using the agar-well diffusion method with the microorganisms as seen in Table 4. The essential oils showed antibacterial activity against Gram-positive and Gram-negative bacteria, but no antifungal activity was observed against the two yeast-like fungi.

Table 4. Screening results for antimicrobial activity of the essential oils from T. chamaedrys. subsp. chamaedrys., T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium..

		Microorganisms and inhibition zone (mm)
Sample	Stock (µg/mL)	Ec	Yp	Kp	Sm	Ef	Sa	Bs	Ca	Ct
T. chamaedrys. subsp. chamaedrys.	1000	+	+ +	—	+	+	+	—	—	—
T. orientale. var. puberulens.	500	—	—	—	—	+ +	+	+	—	—
T. chamaedrys. subsp. lydium.	500	—	—	—	—	+	+ +	+ +	—	—
Ceftazidime	10	+ + +	+ + +	+ + +	+ + +	+ + +	+ + +	+ + +
Triflucan	5								+ + +	+ + +

Results were interpreted in terms of the diameter of the inhibition zone. (−): < 5.5 mm; (+): 5.5–10 mm; (+ +): 11–15 mm; (+ + +): ≥ 16 mm.

Ec, Escherichia coli. ATCC 35218; Yp, Yersinia pseudotuberculosis. ATCC 911; Kp, Klebsiella pneumoniae. ATCC 13883; Sm, Serratia marcescens. ATCC 13880; Ef, Enterococcus faecalis. ATCC 29212; Sa, Staphylococcus aureus. ATCC 25923; Bs, Bacillus subtilis. ATCC 6633; Ca, Candida albicans. ATCC 60193; Ct, Candida tropicalis. ATCC 13803. Ceftazidime and triflucan are used as antibacterial and antifungal standards, respectively.

The test samples showed better antimicrobial activity against the Gram-positive bacteria when compared with the Gram-negative bacteria. The essential oils of T. chamaedrys. subsp. chamaedrys. showed antibacterial activity against Escherichia coli. ATCC 35218, Yersinia pseudotuberculosis. ATCC 911, Serratia marcescens. ATCC 13880, Enterococcus faecalis. ATCC 29212, and Staphylococcus aureus. ATCC 25923. However, no antimicrobial activity was observed against the other four microorganisms tested, two bacteria and two fungi (Table 4). The essential oils of T. orientale. var. puberulens. and T. chamaedrys. subsp. lydium. showed antibacterial activity against Enterococcus faecalis. ATCC 29212, Staphylococcus aureus. ATCC 25923, and Bacillus subtilis. ATCC 6633. However, no antimicrobial activity was observed against the other six microorganisms tested, four bacteria and two fungi (Table 4).

Some chemical differences on the composition of the essential oils of T. chamaedrys. subsp. chamaedrys., T. orientale. var. puberulens., and T. chamaedrys. subsp. lydium. were found and probably related to the different subspecies and/or to the geographical origin of the plants.

Acknowledgments

This study was supported by grants from Karadeniz Technical University Research Fund and State Planning Agency (DPT) of Turkey.