Phytochemical investigations and antiproliferative secondary metabolites from Thymus alternans growing in Slovakia

Figures & data

Figure 1. Distribution of the collection sites of T. alternans in Slovakia.

Table 1. Non-volatile compounds extracted from T. alternans, tentatively identified on the basis of mass fragmentation spectra.

Identified compound	[M-H]^− and fragments
Luteolin-3-O-glucopyranoside^a	447, 285,175
Luteolin-7-O-glucopyranoside^a	447, 285,175
Luteolin-7-O-rutinoside^a	593, 285, 175
Chrysoeriol-hexoside	461, 446, 299, 284, 256
Methoxy luteolin-hexoside	461, 447, 285, 256, 175
Chrysoeriol-7-O-hexosyl-deoxyhexoside	923, 461, 285, 175
Rosmarinic acid hexoside	521, 367, 359, 191
Esculin^a	339, 177
Rosmarinic acid^a	359, 161
Caffeic acid-hexoside	341, 179, 89
3′-O-(8″-Z-Caffeoyl) rosmarinic acid^b	537, 493, 359
Caffeic acid derivative	377, 341, 179
	647, 601, 341, 179
3’-O-methyl- rosmarinic acid^b	377, 161, 133, 105
Apigenin-7-O-glucopyranoside^a	431, 269, 175
Rosmarinic acid derivative	493, 359, 161, 133

a Structure identity confirmed by injection of reference compound.

b The compound was assigned on the basis of literature data.

Figure 2. Structures of the isolated phenolic derivatives.

Figure 3. Structures of the isolated triterpenes.

Table 2. Composition of EOs obtained from three populations of Thymus alternans growing in Slovakia (NS – village of Nová Sedlica, RUN – village of Runina, POL – village of Topoľa. For details of localities see part Plant material).

N.	Component^a	RI^b	RI LIT.^c	Area%^d			ID^e
				NS	RUN	POL
				1	α-Thujene	916		924	0.1	tr^f		RI, MS
2	α-Pinene	921	932	0.3	0.1	tr	Std
3	Camphene	934	946	0.5	0.1	0.1	Std
4	Sabinene	959	969	tr	tr	tr	RI, MS
5	β-Pinene	962	974	0.3	0.2	0.1	Std
6	1-Octen-3-one	968	972	0.2	0.1	tr	RI, MS
7	1-Octen-3-ol	974	974	1.6	1.1	1.4	Std
8	3-Octanone	979	979	0.2	0.1	0.1	RI, MS
9	Myrcene	981	988	4.0	1.9	1.3	Std
10	3-Octanol	990	988	0.2	0.2	0.1	RI, MS
11	Α-terpinene	1009	1014	0.4	0.3	0.1	RI, MS
12	p-Cymene	1017	1020	0.3	0.3	0.1	Std
13	Limonene	1020	1024	0.4	0.3	0.2	Std
14	1,8-Cineole	1021	1026	0.7	0.3	0.4	Std
15	(Z)-β-Ocimene	1032	1032	1.0	0.5	0.4	RI, MS
16	(E)-β-Ocimene	1041	1044	7.0	4.3	2.6	RI, MS
17	γ-Terpinene	1050	1054	1.0	1.0	0.2	Std
18	cis-Sabinene hydrate	1057	1065	2.4	2.3	0.7	RI, MS
19	Terpinolene	1079	1086	0.2	0.2	0.1	Std
20	trans-Sabinene hydrate	1089	1098	0.3	0.3	0.1	RI, MS
21	Linalool	1095	1095	2.7	1.7	6.4	Std
22	n-Undecane	1100	1100	0.2	0.1		Std
23	1-Octen-3-yl acetate	1110	1110	0.1	0.6	0.1	RI, MS
24	cis-p-Menth-2-en-1-ol	1113	1118	tr	0.2	0.1	RI,MS
25	allo-Ocimene	1124	1128	0.1	tr		Std
26	Camphor	1132	1141	1.1	0.4	1.1	Std
27	trans-Chrysanthemal	1141	1153	0.2	0.3	0.2	RI, MS
28	Nerol oxide	1149	1154	tr	0.1	0.1	RI, MS
29	Isoborneol	1154	1155	1.1	0.5	1.0	Std
30	Terpinen-4-ol	1166	1174	1.8	1.8	1.0	Std
31	Isogeranial	1178	1174	0.4	0.4	0.4	RI, MS
32	α-Terpineol	1181	1186	0.5	0.3	0.5	Std
33	Nerol	1222	1227	1.9	4.9	3.1	Std
34	Citronellol	1225	1223	0.1	0.1	0.1	Std
35	Thymol, methyl ether	1229	1232	tr	tr	tr	RI, MS
36	Neral	1234	1235	5.1	5.4	4.9	Std
37	Carvacrol, methyl ether	1236	1241		0.1		RI, MS
38	Geraniol	1251	1249	3.3	6.0	6.2	Std
39	Linalool acetate	1254	1254	2.9	tr	2.4	RI, MS
40	Geranial	1265	1264	6.8	7.7	7.2	Std
41	Isobornyl acetate	1276	1283	tr	tr	tr	Std
42	Carvacrol	1299	1298		0.5		Std
43	δ-Elemene	1325	1335	0.1	0.1		RI, MS
44	Neryl acetate	1360	1359	3.6	1.5	2.3	Std
45	β-Bourbonene	1369	1387	1.8	1.0	1.3	RI, MS
46	Geranyl acetate	1379	1379	0.3	0.9	1.1	RI, MS
47	(E)-caryophyllene	1402	1417	1.5	1.0	1.6	Std
48	β-Copaene	1413	1430	0.2	0.2	0.2	RI, MS
49	α-Humulene	1436	1454	0.2	0.4	0.2	Std
50	allo-Aromadendrene	1443	1458	0.7	0.1	0.1	RI, MS
51	cis-Cadina-1(6),4-diene	1449	1461			0.1	RI, MS
52	(E)-β-Farnesene	1450	1454		0.1		RI, MS
53	Germacrene D	1465	1484	6.7	7.4	7.4	RI, MS
54	Bicyclogermacrene	1480	1500	0.5	0.5	0.2	RI, MS
55	α-Muurolene	1487	1500	0.2	tr	0.1	RI, MS
56	β-Bisabolene	1498	1505	1.5	0.7	1.3	RI, MS
57	(E,E)-α-Farnesene	1501	1505	0.3	0.1	0.1	RI, MS
58	δ-Cadinene	1510	1522	1.1	0.2	0.4	RI, MS
59	β-Sesquiphellandrene	1512	1521	0.2	0.4	tr	RI, MS
60	α-Cadinene	1533	1537	0.1	tr		RI, MS
61	Hedycaryol	1536	1546	2.9	3.6	1.4	RI, MS
62	(E)-Nerolidol	1559	1561	15.8	26.4	31.4	Std
63	Germacrene D-4-ol	1559	1574	tr	tr	tr	RI, MS
64	Viridiflorol	1584	1590	0.2			Std
65	γ-Eudesmol	1615	1630	0.4	0.1	0.2	RI, MS
66	epi-α-Cadinol	1626	1638	0.4	0.1	0.1	RI, MS
67	epi-α-Muurolol	1626	1640	0.4	0.3	0.2	RI, MS
68	β-Eudesmol	1631	1649	0.2	0.2	0.1	RI, MS
69	α-Eudesmol	1635	1652	0.3	0.3	0.2	RI, MS
70	α-Cadinol	1648	1652	1.8	0.8	0.6	RI, MS
71	n-Nonacosane	2900	2900	0.1	0.2	0.3	Std
	Total identified (%)			91.0	91.4	93.2
	Oil yield (%)			0.2	0.2	0.1
	Grouped compounds (%)
	Monoterpene hydrocarbons			15.6	9.2	5.1
	Oxygenated monoterpenes			35.3	34.6	39.1
	Sesquiterpene hydrocarbons			15.1	12.2	12.9
	Oxygenated sesquiterpenes			22.5	32.1	34.1
	Others			2.6	2.4	2.0

a Compounds are in order of elution from a HP-5MS column.

b Retention index on HP-5MS column.

c Retention index taken from literature (Adams, 2007; NIST 08, 2008; FFNSC 2, 2012).

d Percentage values are means of three determinations, with a RSD% for the main components below 10% in all cases.

e Identification methods: MS, by comparison with the mass libraries Wiley, Adams and NIST 08; RI, by comparison with RIs-containing libraries (Adams, 2007; NIST 08, 2008; FFNSC 2, 2012); Std, by comparison with authentic standard.

f tr, traces (mean value below 0.1%).

Table 3. In vitro cytotoxic activity of triterpenes isolated from aerial parts of T. alternans collected in Nová Sedlica, Slovakia. Cells (5–8 × 104·mL ^− 1) were treated for 72 h with increasing concentrations of tested compounds.

IC50(μM) ± S.D.
Compound	A375	HCT-15	A431	BxPC3	U1810
T1	1.57 ± 0.72	0.88 ± 0.22	4.54 ± 1.06	0.52 ± 0.24	2.86 ± 0.83
T2	1.26 ± 0.12	1.46 ± 0.13	4.98 ± 1.14	1.84 ± 0.31	2.25 ± 1.05
T3	0.48 ± 0.67	0.67 ± 0.09	2.03 ± 0.98	0.42 ± 0.13	0.98 ± 0.21
T4	1.45 ± 0.56	1.69 ± 0.72	4.85 ± 1.32	1.42 ± 0.73	3.59 ± 1.31
T5	0.87 ± 0.32	2.42 ± 1.05	3.28 ± 1.09	1.84 ± 0.91	2.93 ± 1.13
T6	0.45 ± 0.10	0.54 ± 0.08	2.16 ± 0.83	0.19 ± 0.09	1.62 ± 0.85
Betulinic acid	ND	6.85 ± 2.18	8.96 ± 2.83	4.28 ± 1.35	ND
Cisplatin	2.06 ± 1.01	15.53 ± 2.48	1.96 ± 0.84	10.17 ± 1.65	0.76 ± 0.85

S.D. = standard deviation. ND = not detected. Cell viability was evaluated by means of MTT test. IC₅₀ values were calculated by the dose–response curves by means of four parameter logistic model (p < 0.05).

Table 4. In vitro cytotoxic activity of T. alternans EO^a.

IC50 (μg/ml) ± S.D.^b
Compound	A375^c	MDA-MB 231^d	HCT116^e
Essential oil	5.51 ± 0.34	5.9 6 ± 0.46	8.45 ± 0.61
(E)-Nerolidol	2.92 ± 0.09	5.76 ± 0.29	4.13 ± 0.18
	(13.13 ± 0.43)	(25.90 ± 1.31)	(18.57 ± 0.81)
Neryl acetate	28.05 ± 1.27	27.37 ± 1.87	45.61 ± 2.83
	(142.9 ± 6.47)	(139.5 ± 9.53)	(232.4 ± 14.4)
Nerol	75.83 ± 3.97	24.44 ± 1.66	33.49 ± 4.53
	(491.6 ± 25.7)	(158.4 ± 10.8)	(217.1 ± 29.4)
Positive control
Cisplatin	0.43 ± 0.045	2.94 ± 0.23	2.42 ± 0.21
	(1.43 ± 0.15)	(9.80 ± 0.76)	(8.06 ± 0.70)

a Sample was obtained from population growing in Nová Sedlica.

b IC₅₀= The concentration of compound that affords a 50% reduction in cell growth (after 72 h of incubation).

c Human malignant melanoma cell line.

d Human breast adenocarcinoma cell line.

e Human colon carcinoma cell line. Bracketed IC₅₀ values are expressed in μM ± S.D.

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