Essential Oil Composition of Xanthium italicum From Serbia

Abstract

The essential oil isolated from stem, fresh and mature fruits of Xanthium italicum Moretti by hydrodistillation was analyzed by gas chromatography-mass spectrometry. In the oil of stem, fresh and mature fruits, 121 components were identified, representing 94.6%, 92.1%, and 91.3% of the total oil, respectively. The most abundant compounds in stem oil were limonene (23.0%), methyl eugenol (5.4%), β-cubebene (5.0%),and δ-cadinene (3.3%). The oil of fresh fruits contained germacrene B (28.7%), shyobunol (16.7%), and α-humulene (8.4%) as major components. There were 4 major constituents in X. italicum oil from mature fruits: germacrene B (31.3%),α-humulene (11.8%), δ-cadinene (3.2%),and γ-muurolene (2.9%). Percentages of sesquiterpenes in oils from fresh and mature fruits were very high, 85.8% and 73.8 %.

The genus Xanthium (family Asteraceae) is represented in the flora of Serbia by three species: X. strumarium L., X. italicum Moretti, and X. spinosum L. ¹ Xanthium italicum is an annual herb, 30 to 100 cm high, with a stout, hairy stem and spindle-like root. The species is an alien, very invasive, competitive weed, which has the ability to easily adapt to different environmental conditions once established. It is often found to form dense monocultures as long as light, moisture and nutrition are sufficient, which consequently results in adverse impacts on native plant communities. In oriental countries, Xanthium species have been used as traditional herbal medicines for a long time for treating numerous diseases because of their antiviral, anti-staphylococcal, anti-leishmanial, anti-trypanosomal, anthelmintic, and anti-inflammatory effects. ² All parts of Xanthium plants possess sedative, diaphoretic and diuretic properties.

In Western literature, Xanthium species are not described as medicinal herbs, but are well known as herbs toxic to grazing animals. A highly toxic glycoside, carboxyatractyloside, is present in the seeds and seedlings of Xanthium species. The toxin readily disappears after germination. This plant causes contact dermatitis in humans.

The most important chemical constituents of X. italicum include different xanthanolides and phytosterols. ^{2 -5} Some of these compounds exhibited phytotoxic activity. ⁶ Extracts of X. italicum showed strong biological activity ^4,6

There are reports on the chemical composition of X. italicum oil from Corsica ⁷ and China, ⁸ but no study has been conducted on X. italicum oil of mature and green fruits, and oils of Serbian origin. The objective of the present study was to perform a detailed compositional analysis of the essential oils of stem, fresh and mature fruits of this species from Serbia by gas chromatography (GC) and GC-mass spectrometry (MS).

In the stem oil, 85 components were identified, representing 94.6% of the total oil (Table 1). The most abundant compound was limonene (23%). Other important compounds were methyl eugenol (5.4%), β-cubebene (5.0%), and δ-cadinene (3.3%). The oil of fresh fruits (64 identified components) contained germacrene B (28.7%), shyobunol (16.7%), and α-humulene (8.4%) as major components. There were 4 major constituents of the oil (63 identified components) from mature fruits: germacrene B (31.3%), α-humulene (11.8%), δ-cadinene (3.2%), and γ-murolene (2.9%). However, the chemical composition of the stem oil was different from those of the fresh and mature fruits because it contained almost the same amount of monoterpenes (47.0%) and sesquiterpenes (42.9%), whereas the percentages of sesquiterpene compounds in the oils from fresh and mature fruits were very high (85.8% and 73.8 %). Also, the content of monoterpene compounds in oils obtained from fresh and mature fruits were very similar (2.5% and 2.7%).

Table 1

Chemical Composition of Essential Oils of Stem, Fresh Fruits and Mature Fruits of Xanthium italicum Achieved by gas chromatography (GC) and GC-mass spectrometry (MS) (%).

Peak	RI	RL	Compound	Stem (%)	Fresh fruits (%)	Mature fruits (%)
1	921	922	Tricyclene	tr	0.0	0.0
2	924	927	α-Thujene	tr	0.0	0.0
3	932	938	α-Pinene	1.9	tr	0.0
4	953	948	Camphene	0.6	tr	0.0
5	953	954	Thuja-2,4(10)-diene	tr	0.0	0.0
6	957	960	Benzaldehyde	0.0	tr	0.0
7	976	973	Sabinene	2.0	tr	0.0
8	980	976	β-Pinene	0.3	tr	0.0
9	991	991	β-Myrcene	0.4	tr	0.0
10	1003	1004	p-Mentha-1(7),8-diene	tr	0.0	0.0
11	993	1012	(E,E)-2,4-Heptadienal	tr	0.0	0.0
12	1014	1017	α-Terpinene	tr	0.0	0.0
13	1031	1029	Limonene	23.0	1.1	0.5
14	1048*	1044	Benzeneacetaldehyde	tr	tr	0.2
15	1049	1047	(E)-β-Ocimene	tr	0.0	0.0
16	1054	1060	γ-Terpinene	0.3	0.0	0.0
17	1065	1068	cis-Sabinene hydrate	0.3	0.0	0.0
18	1088	1090	α-Terpinolene	tr	0.0	0.0
19	1089	1090	p-Cymenene	tr	0.0	0.0
20	1098	1099	trans-Sabinene hydrate (IPP vs OH)	0.3	0.0	0.0
21	1100	1101	n-Nonanal	tr	0.5	0.7
22	1108*	1107	Pentyl 3-methylbutanoate	0.0	0.0	tr
23	1118*	1121	trans-p-Mentha-2,8-dienol	tr	tr	0.0
24	1122	1127	α-Campholenal	0.5	0.0	tr
25	1132	1134	cis-Limonene oxide (Me vs IPP)	tr	0.0	0.0
26	1135*	1140	trans-(-)-Pinocarveol	1.8	tr	0.0
27	1144	1146	trans-Verbenol	1.4	tr	0.0
28	1160	1164	Pinocarvone	1.2	0.0	0.2
29	1165	1167	endo-Borneol	1.5	0.4	0.0
30	1174	1179	Terpinen-4-ol	1.3	0.0	tr
31	1179	1186	p-Cymen-8-ol	tr	0.0	0.0
32	1184	1189	Ethyl-(E)- 4-octenoate	0.0	0.0	tr
33	1191	1198	Myrtenol	1.1	0.0	0.0
34	1202*	1204	α-Campholenol	0.3	0.0	0.0
35	1200	1205	Decanal	0.0	0.0	tr
36	1215*	1216	2-Methyl-2-nonen-4-one	tr	0.0	0.0
37	1223*	1221	β-Cyclocitral	1.0	tr	0.2
38	1242	1245	Carvone	tr	0.0	tr
39	1260*	1263	trans-2-Decenal	0.0	tr	0.5
40	1285	1287	(-)-Bornyl acetate	2.5	0.6	1.1
41	1294*	1292	1-Tridecene	tr	0.5	1.3
42	1298*	1301	Theaspirane A	0.0	tr	0.0
43	1310*	1308	Undecanal	0.0	tr	0.0
44	1314	1318	(E,E)-2,4-Decadienal	0.0	tr	0.3
45	1348*	1349	2-methylpropyl octanoate	0.0	tr	tr
46	1351	1353	α-Cubebene	tr	tr	0.3
47	1359	1360	Eugenol	tr	0.0	0.0
48	1357	1364	E-2-Undecenal	0.0	0.0	0.2
49	1370	1371	Cyclosativene	0.7	0.0	0.4
50	1376	1379	α-Copaene	0.7	0.3	0.4
51	1382	1384	Modheph-2-ene	tr	tr	tr
52	1384	1389	β-Bourbonene	tr	tr	0.2
53	1391	1393	β-Cubebene	5.0	1.1	1.6
54	1401	1405	Methyleugenol	5.4	0.5	0.8
55	1414*	1414	α-Gurjunene	0.0	0.6	0.6
56	1428*	1417	Aristolene	0.0	1.4	1.0
57	1418	1423	(E)-β-Caryophyllene	2.2	0.9	0.9
58	1430	1433	β-Copaene	0.0	tr	0.0
59	1434*	1437	γ-Elemene	0.0	0.9	0.7
60	1449*	1550	Dihydro-β-ionol	0.0	tr	0.0
61	1455	1460	α-Humulene	1.5	8.4	11.8
62	1461	1468	cis-Muurola-4(15),5-diene	0.0	tr	0.3
63	1471	1474	4,5-Di-epi-Aristolochene	tr	0.0	0.0
64	1475	1478	trans-Cadina-1(6),4-diene	tr	0.0	0.0
65	1477	1481	γ-Muurolene	tr	1.2	2.9
66	1480	1487	Germacrene D	12.8	3.9	1.6
67	1490	1492	Valencene	0.0	0.3	0.0
68	1485	1493	β-Selinene	1.0	0.0	0.4
69	1492*	1492	1-Pentadecene	0.0	0.0	0.5
70	1495	1496	2-Tridecanone	0.0	0.0	0.3
71	1493	1500	epi-Cubebol	2.3	0.0	0.0
72	1500	1501	Bicyclogermacrene	0.0	1.6	0.4
73	1504	1499	α-Muurolene	0.6	tr	1.9
74	1511	1511	Tridecanal	0.0	0.0	0.3
75		1513	7,8-Dehydro-α-acoradiene	0.4	0.0	0.0
76	1513	1518	γ-Cadinene	0.0	tr	0.9
77	1514	1520	Cubebol	0.9	1.0	0.0
78	1522	1528	δ-Cadinene	3.3	2.6	3.2
79	1533	1536	trans-Cadina-1,4-diene	0.0	0.0	0.2
80	1542	1543	Selina-3,7(11)-diene	0.0	1.3	1.2
81	1542	1547	α-Calacorene	tr	0.0	0.7
82	1549	1556	Elemol	0.0	0.5	0.0
83	1559	1567	Germacrene B	0.6	28.7	31.3
84	1564	1567	β-Calacorene	0.0	0.0	tr
85	1574	1582	Germacrene D-4-ol	1.1	3.5	0.0
86	1582	1589	(-)-Caryophyllene oxide	2.1	0.9	2.1
87	1595*	1599	4(14)-Salvialene-1-one	0.2	0.0	0.3
88	1593*	1603	Humulene epoxide I	0.0	0.0	0.2
89	1592*	1611	Copaborneol	1.3	0.3	0.0
90	1608	1615	Humulene epoxide II	1.0	0.7	1.7
91	1624*	1625	Selina-6-en-4-ol	0.4	0.0	0.0
92	1630	1634	Muurola-4,10(14)-dien-1-β-ol	1.0	0.0	0.6
93	1642*	1635	Agarospirol	0.0	1.0	0.0
94	1640	1647	T-Muurolol	0.3	2.2	2.0
95	1644	1651	α-Muurolol (Torreyol)	0.0	0.5	0.0
96	1646	1655	Desmethoxy encecalin	3.7	0.0	0.0
97	1653	1661	α-Cadinol	0.6	3.2	2.2
98	1666	1668	14-hydroxy-(Z)-Caryophyllene	0.0	0.0	0.9
99	1675	1668	trans-Calamenen-10-ol	0.7	0.4	0.0
100	1685	1693	Eudesma-4(15),7-dien-1β-ol	0.9	0.5	0.0
101	1700	1701	Eudesm-7(11)-en-4-ol	0.0	0.0	0.7
102	1692	1702	Shyobunol	0.3	16.7	0.0
103	1700*	1704	Juniper camphor	0.0	tr	0.0
104	1700*	1704	Heptadecane	tr	0.0	2.2
105	1762	1765	Aristolone	0.0	0.0	0.2
106	1766	1767	β-Costol	0.5	0.4	0.0
107	1767	1770	14-Oxy-a-muurolene	0.0	0.0	0.2
108	1775	1774	2-α-Hydroxy-amorpha-4,7(11)-diene	0.0	0.9	0.0
109	1774	1782	epi-Cyclocolorenone	0.2	0.0	0.0
110	1803	1808	14-Hydroxy-δ-cadinene	0.2	0.4	0.0
112	1845*	1846	Hexahydrofarnesyl acetone	0.4	0.0	0.8
113	1884*	1883	1-Hexadecanol	0.0	0.4	0.0
114	1922*	1919	(E,E)-7,11,15-Trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene	0.0	0.0	0.4
115	1969	1968	α-Springene	0.0	0.0	0.2
116	1987	1986	E,Z-Geranyl linalool	0.0	0.0	0.2
117	2042	2046	Kaurene	0.0	2.4	6.9
118	2116*	2116	Phytol	0.7	0.0	0.0
			Total	94.6	92.1	91.3
			Monoterpenoids	47.0	2.5	2.7
			Hydrocarbons	28.5	1.1	0.5
			Oxygenated	18.5	1.4	2.2
			Sesquiterpenoids	42.9	85.8	73.8
			Hydrocarbons	28.9	53.0	62.8
			Oxygenated	14.0	32.8	11.0
			Diterpene	0.7	2.4	7.0
			Hydrocarbons	0.0	2.4	7.0
			Oxygenated	0.7	0.0	0.0
			Others	4.0	1.4	7.8

Compounds are listed in order of elution on a HP-5MS column; RA: Adams retention indices; RI: Experimental retention indices relative to C8-C32 n-alkanes; (*): NIST Chemistry Web Book Retention indices [20-21]; tr: traces (<0.1%).

The concentration of germacrene B was significantly lower in the stem oil (0.6%) than in the oils obtained from fresh (28.7%) and mature fruits (31.3%). On the other hand, the relative amount of d-limonene was found to be 23.0% in the essential oil of stem, whereas in the fruit oils the concentration varied with the stage of maturity (1.1%, fresh; 0.5%, mature). Shyobunol was one of the most abundant compounds in the oil from fresh fruits, but was not detected in mature fruits.

In a report on the phytotoxic activity and chemical composition of the essential oil of X. italicum from China ⁸ (aerial parts of the plant), Shao et al identified 33 compounds, representing 94.9% of the total oil, which was found to be rich in monoterpene hydrocarbons (60.7%). Oxygenated monoterpenes represented only 7.0% of the total oil. In comparison with monoterpenes, sesquiterpene abundance was relatively low (23.9%). Among the 33 oil constituents, the most abundant compounds were limonene (51.6%), germacrene B (7.0%), δ-cadinol (5.9%), β-pinene (5.2%), α-Caryophyllene (5.1%) and bornyl acetate (3.1%). In the essential oil of X. italicum from Corsica, ⁷ the main compounds were limonene (35.3%), borneol (5.6%), sabinene (5.8%), germacrene D (2.5%), α-bisabolol (2.4%), and α-humulene (2.1%). Stem and leaf oils were characterized by the occurrence of limonene (47.9% and 22.9%, respectively) and germacrene D (4.6% and 15.8%, respectively). Flower oils displayed δ-selinene and germacrene D as the main components (22.4% and 17.0%, respectively). Xanthium italicum fruits yielded oil with high amount of oxygenated compounds (72.9%), particularly sesquiterpene compounds (61.5%) such as α-bisabolol (43.0%), α-cadinol (5.0%), and (E,E)-α-farnesol (4.3%).

Limonene is the most abundant component in the stem oil from Serbia, as well as in the oils from Corsica ⁷ and China. ⁸ Another similarity is noticeable with the oils from Corsica. ⁷ Like the Serbian fruit oils, those from Corsica had a high number of oxygenated compounds, particularly sesquiterpenes. The examined oil from China ⁸ was rich in monoterpene hydrocarbons (60.7%), as was the Serbian stem oil.

Experimental

Plant Material and Sample Preparation

Xanthium italicum Moretti plants were collected around the village Temska near Pirot city, Serbia, and identified according to Gajić. ¹ A voucher specimen (13277) was deposited in the “Herbarium Moesiacum Niš”, University of Niš (HMN). The air-dried aerial parts (500 g) were powdered and submitted for 2 hours to water-distillation using a Clevenger-type apparatus. The oil was dried over anhydrous sodium sulfate and, after filtration, stored at +4°C prior to analysis.

Gas Chromatographic and Gas Chromatographic-Mass Spectrometric Analysis

Gas chromatographic/mass spectrometric analyses were performed on an Agilent 7890 gas chromatograph with a 7000B GC/MS/MS triple quadrupole system, operating in MS1 scan mode, and equipped with a fused-silica capillary column (Agilent HP-5 MS [30 m × 0.25 mm i.d. ×0.25 µm film thickness]). The chromatographic analyses were carried out under the following conditions: He as carrier gas at a flow rate of 1.0 mL/min; GC oven temperature was kept at 45°C for 2.25 min and programmed to 290°C at a rate of 4°C/min; split ratio was adjusted at 40:1; injection volume 1 µL. Post run: back flash for 1.89 minutes, at 280°C, with helium pressure of 50 psi. The injector temperature was set at 230°C. Ionization mode was electronic impact at 70 eV. Mass range was set from 40 to 440 Da.

For gas chromatography with flame ionization detection analysis (GC/FID), the same column and chromatographic conditions were applied as described for GC/MS. FID temperature was 300°C. The percentage amounts of the separated compounds were calculated from the GC peak areas using the normalization method without correction factors.

The data are reported as mean value of 3 sample injections.

Identification of Components

Oil constituents were identified by comparison of their linear retention indices (relative to C8-C20 and C21-C44 alkanes ^9,10 on a HP-5MS column, with literature values), and their MS with those of authentic standards, as well as those from Wiley 6, NIST11, Agilent Mass Hunter Workstation B.06.00 software, ¹¹ and a homemade MS library with the spectra corresponding to pure substances and components of known essential oils by the application of AMDIS software. ¹²