GC-MS Analysis of Chloroform Extracts in Flowers, Stems, and Roots of Tripleurospermum callosum.

ABSTRACT

The chemical composition of the chloroform extracts of flowers, stems, and roots of Tripleurospermum callosum. (Boiss. & Heldr) E. Hossain growing along the northeastern coast of Turkey was studied by gas chromatography-mass spectrometry (GC-MS). The GC-MS analyses of the chloroform extracts from air-dried parts of T. callosum. yielded the identification of 93 compounds. These compounds were separated into eight classes that were terpenes-terpenoids, hydrocarbons, alcohols, acids, oxygen-containing compounds, steroids, aromatics, and others. Major compounds were moretenol (11.71%) in flower oil: linoleic acid (16.18%), n.-hexadecanoic acid (17.88%), and 1-tricosene (13.41%) in stem oil: and n.-hexadecanoic acid (6.18%) in root oil. Similarity, of chloroform extracts in flowers, stems, and roots of T. callosum. were in the ratio of 13.77%, 45.99%, and 30.16%, respectively.

Keywords:

• Asteraceae
• flowers
• GC-MS
• roots
• stems
• Tripleurospermum callosum

Introduction

The genus Tripleurospermum. Sch. Bip. belongs to the Asteraceae (Compositae) family, which has 38 species in Europe and the regions of Asia and a few species in North America and North Africa (Bremer & Humphries, 1993). Most of them are endemic. One of the endemic species of the family is Tripleurospermum callosum. (Boiss. & Heldr) E. Hossain, which is distributed naturally in northern Turkey (Grierson, 1975).

Prior to this study, it was found that there were no chemical studies of T. callosum. in the literature. As part of this systematic research on the chemical constituents of T. callosum., the flowers, stems, and roots of T. callosum. were extracted with chloroform. The obtained crude extracts were then investigated by a gas chromatography-mass spectrometry (GC-MS) technique. Various types of 93 compounds were identified from T. callosum.. Therefore, the objective of this study was to identify and quantify the major components to see the differences in flowers, stems, and roots of T. callosum. (Guillen & Manzanos, 1998; Yayli et al., 2001).

Materials and Methods

Samples and extraction

Tripleurospermum callosum. (Boiss. & Heldr) E. Hossain were collected from Ikizdere and Ayder (∼1800 m) in the northern region of Turkey in July 2002 (voucher no. Inceer 137, KTÜB). The plants were air-dried in a shaded place at room temperature for 20 days and separated into three parts; flowers, stems, and roots. The smaller amount of flowers (2.35 g), stems (2.46 g), and roots (2.24 g) of T. callosum. were each extracted with chloroform for 4 days. Chloroform extracts were filtered, and the filtrates were concentrated on a rotary evaporator at 30°C to obtain the crude mixtures.

GC-MS analysis

GC-MS analyses were performed using an Agilent 5973–6890 N instrument equipped with a HP-5 (crosslinked 5% PH ME siloxane) capillary column (30 m, 0.25 mm i.d., 0.25-µm film thickness). For GC-MS detection, an EI mode was used with ionization energy of 70 eV. Helium was the carrier gas at a flow rate of 1 ml/min. Injector temperature was 230°C. Column temperature was kept at 60°C initially for 5 min. It was then gradually increased to 120°C with a heating rate of 3°C/min, held for 2 min at this temperature, and increased to 200°C with a heating rate of 2°C/min, held for 2 min at this temperature, and finally increased to 320°C with a heating rate of 3°C/min and held at that temperature for 2 min. Samples of 1 µl were injected manually in split mode. Ratio of sample in split mode was 49:1.

Results and Discussion

In this study, the flowers, stems, and roots of T. callosum. were extracted with CHCl₃ separately. Lipid components in flowers, stems, and roots of T. callosum. were compared (Guillen & Manzanos, 1998; Yayli et al., 2001). Crude extracts of the flowers, stems, and roots of the plant showed different colors that were yellow, green, and yellow-brown, respectively. Therefore, a capillary gas chromatographic-mass spectrometric method was employed to identify the lipid components of the flowers, stems, and roots of T. callosum. (Guillen & Manzanos, 1998). The compounds were identified by comparing the retention indices (RI) and experimental mass spectra with literature data and mass spectral libraries of Wiley and NIST (Adams, 1995; Massada, 1976; Egerton-Warburton & Ghisalberti, 1995). The GC-MS analyses of the chloroform extracts from air-dried parts of the flowers, stems, and roots of T. callosum. resulted in the identification of a total of 93 compounds, which constituted 82.64%, 91.74%, and 69.05% of the total oils, respectively (Table 1). The GC-MS results for oils from the flowers, stems, and roots of T. callosum. are shown in Table 1.

Table 1.. Lipid components in the flowers, stems, and roots of T. callosum. by GC-MS.

No.	Compounds	RI	Flower (%)	Stem (%)	Root (%)
1.	2-Methyl-naphthalene	1208	0.26	0.23	0.73
2.	(4α-trans.)-Decahydro-4α- methyl-1-methylene-7-(1-methylethylidene)-naphthalene	1281	—	—	0.45
3.	Isocomene	1347	0.14	0.52	3.07
4.	trans.-β-Farnesene	1469	0.45	—	—
5.	1-Pentadecene	1513	—	—	0.69
6.	p.-Methoxy-β-cyclopropylstyrene	1543	0.70	0.28	4.08
7.	(+)-Spathulenol	1578	0.28	0.14	2.42
8.	(−)-Caryophyllene oxide	1581	—	—	0.94
9.	Geranyl isovalerate	1588	—	—	1.04
10.	n.-Hexadecane	1600	0.40	0.07	0.40
11.	Dec-4,6,8-triyn-2-enoic acid, methyl ester	1622	—	—	0.26
12.	4-Methyl-2-(2-methyltetrahydrofuran-2-yl)phenol	1629	—	—	0.54
13.	α-Elemene	1650	—	—	1.15
14.	Cyclododecyne	1662	—	—	0.47
15.	Heptadecane	1700	0.18	0.42	—
16.	6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalene-2-ol	1714	—	—	0.66
17.	1-(1,1-Dimethylethyl)-4-ethoxy-benzene	1728	—	—	0.75
18.	2-Pentadecanone,6,10,14-trimethyl	1731	—	0.15	—
19.	2-(2-oxoethyl)-cis.-Bicyclo[3.3.0]octane-3,7-dione	1734	—	—	1.35
20.	Tetradecanioc acid	1768	0.33	0.19	0.27
21.	n.-Octadecane	1800	0.13	—	0.24
22.	Isobutylphthalate	1866	0.37	—	—
23.	Pentadecanoic acid	1867	—	—	0.49
24.	2,8-Dimethyldibenzothiophene	1868	0.30	0.43	—
25.	4,9-Dimethyl-naptho[2,3β]thiophene	1896	—	0.21	—
26.	Nonadecane	1900	—	0.07	—
27.	1-Phenyl-3-ethyl-4-hexen-1-yn-3-ol	1906	—	—	2.85
28.	2,5-Dimethyl-phenanthrene	1952	—	0.91	—
29.	Unknown	1992	—	—	7.28
30.	n.-Eicosane	2000	—	0.93	—
31.	n.-Hexadecanoic acid	2024	5.35	17.88	6.18
32.	[1,1′-Diphenyl]-3-amine	2031	—	—	4.71
33.	Tricyclo[7.3.0.0(3,8)]dodec-1(9)-en-12-one,2,2-dicyano, (Z.)	2049	1.35	1.38	—
34.	7-Isopropyl-1,3,4-trimethylazulene	2081	—	—	0.27
35.	Isofraxidin	2085	—	—	1.32
36.	Phytol	2089	—	3.09	—
37.	n.-Heneicosane	2100	0.38	—	0.57
38.	8-epi-1,2-Dihydroartemisin	2107	—	—	4.82
39.	Linoleic acid	2118	3.30	16.18	0.72
40.	Artemisia acetate	2119	—	—	0.44
41.	9,12-Octadecadien-1-ol	2185	0.77	0.62	0.17
42.	Z.,Z.-10,12-Hexadecadienal	2191	—	—	0.29
43.	1,8-Diaza-2,9-diketocyclotetradecane	2196	1.27	—	2.04
44.	8-Oxo-2-nonenal	2197	—	0.64	—
45.	n.-Docosane	2200	0.26	—	0.29
46.	(Z.)-9-Tricosene	2299	0.17	0.49	—
47.	n.-Tricosane	2300	1.46	0.28	—
48.	6-Nitro-cyclohexadecane-1,3-dione	2386	—	0.28	—
49.	(Z.)-9-Octadecenamide	2397	0.28	—	0.78
50.	n.-Pentacosane	2500	4.77	—	0.48
51.	di-(2-Ethylhexyl)phthalate	2519	0.17	—	—
52.	di-n.-Octyl phthalate	2578	—	—	0.43
53.	Cyclotetracosane	2589	0.82	4.05	5.88
54.	n.-Heptacosane	2700	4.52	—	—
55.	1,19-Eicosadiene	2719	0.14	0.27	—
56.	1-Eicosanol	2757	—	4.50	—
57.	1-Nonadecene	2762	0.23	0.28	0.71
58.	11-Butyl-docosane	2779	0.72	—	—
59.	n.-Octacosane	2800	0.32	0.19	—
60.	Squalene	2829	0.64	—	—
61.	n.-Nonacosane	2900	11.04	—	—
62.	(E.)-1-Methoxy-3,7-dimethyl-2,6-octadiene	2910	0.15	—	—
63.	Hexadecyl-oxirane	2912	—	—	0.44
64.	1-Tricosene	2936	—	13.41	—
65.	Cholest-5-en-3-ol(3β)	2946	0.63	—	—
66.	4,4-Dimethyl-2-oxazolidinethione	2970	—	0.21	—
67.	Stigmastan-3,5,22-trien	2981	—	0.61	—
68.	Cholesterol	2995	—	0.18	1.75
69.	Stigmasta-5,22-dien-3-ol	2995	0.99	3.51	4.04
70.	(23S.)-Ethylcholest-5-en-3-β-ol	2999	4.21	—	—
71.	n.-Triacontane	3000	2.35	—	—
72.	1-Hentetracontanol	3000	—	0.67	—
73.	2-(2′-Furyl)-3-phenyl-1H-indol	3019	—	—	0.21
74.	β-Amyrin	3019	5.53	—	—
75.	Campesterol	3033	0.97	2.04	1.49
76.	9,10-Dihyro-9,10,11-trimethyl-9,10-methanoanthracen-11-ol	3036	—	—	0.18
77.	Ergostanol	3036	—	0.09	—
78.	Stigmasterol	3048	—	0.22	3.09
79.	Lupenol	3057	1.11	—	—
80.	22,23-Dihydro-stigmasterol	3064	—	—	2.80
81.	α-Amyrin	3066	2.48	0.71	—
82.	γ-Sitosterol	3066	—	7.17	—
83.	23(Z.)-Ethylcholestanol	3068	—	0.85	—
84.	Δ-8(14)-Stigmasterol	3082	—	0.43	—
85.	(3β,5α)-Stigmast-7-en-3-ol	3082	0.27	—	—
86.	Stigmast-4-en-3-one	3108	—	—	0.93
87.	24-Methylenecycloartanol	3108	0.52	1.31	—
88.	Vitamin E	3111	0.44	0.55	—
89.	d,α.-Tocopherol	3111	0.44	0.34	—
90.	Moretenol	3120	11.71	—	—
91.	Friedelinol	3123	—	—	1.17
92.	6-Aza-β-homo-5-α-cholestano[6,7-d]tetrazole	3124	8.33	—	—
93.	Neophytadiene	3158	—	5.12	—
Total Identification %		82.64%	91.74%	69.05%

RI, retention indices on HP-5 capillary column.

The GC-MS analysis of the oils from flowers, stems, and roots of T. callosum. allowed the identification of 47 components in the flower oil, 44 components in the stem oil, and 47 components in the root oil. These compounds were separated into eight classes: terpenes-terpenoids, hydrocarbons, alcohols, acids, oxygen-containing compounds, steroids, aromatics, and others (Table 2).

Table 2.. Classification of lipid components of T.callosum..

	Chemical classes	Flower %	Stem %	Root %
1.	Terpenes and terpenoids	22.78	9.92	9.77
2.	Hydrocarbons	27.89	20.46	10.34
3.	Alcohols	1.28	5.79	0.35
4.	Acids	8.98	34.25	7.66
5.	Oxygen-containing compounds	0.15	0.94	9.52
6.	Steroids	16.86	16.74	11.01
7.	Aromatics	1.80	1.87	14.66
8.	Others	2.90	2.06	3.07
Total identification		82.64	91.74	69.05

Terpenes-terpenoids constituted 22.78% in flower oil, 9.92% in stem oil, and 9.77% in root oil of T. callosum.. The major compound of terpene-terpenoid was moretenol (11.71%) in the flower oil, neophytadiene (5.12%) in the stem oil, and isocomene (3.07%) in the root oil of T. callosum.. The ratio of hydrocarbons was 27.89% in flower oil, 20.44% in the stem oil, and 10.34% in the root oil of T. callosum.. The main component of hydrocarbon was n.-nonacosane (11.04%) in the flower oil, 1-tricosene (13.41%) in the stem oil, and cyclotetracosane (5.88%) in the root oil of T. callosum.. Alcohols constituted 1.28% in the flower oil, 5.79% in the stem oil, and 0.35% in root the oil of T. callosum.. The major representative of alcohol was 9,12-octadecadien-1-ol (0.77%) in the flower oil, 1-eicosanol (4.50%) in the stem oil, and 9,10-dihyro-9,10,11-trimethyl-9,10-methanoanthracen-11-ol (0.18%) in the root oil of T. callosum.. The ratio of acids was 8.98% in the flower oil, 34.25% in the stem oil, and 7.66% in the root oil of T. callosum.. The major representative of acid was n.-hexadecanoic acid (5.35%) in the flower oil, linoleic acid (16.18%) and n.-haxadecanoic acid (17.88%) in the stem oil, and n.-hexadecanoic acid (6.18%) in the root oil of T. callosum.. Oxygen-containing compounds constituted 0.15% in the flower oil, 0.94% in the stem oil, and 9.52% in the root oil of T. callosum.. The main component of oxygen-containing compound was (E.)-1-methoxy-3,7-dimethyl-2,6-octadiene (0.15%) in the flower oil, 8-oxo-2-nonenal (0.64%) in the stem oil, and 8-epi-1,2-dihydroartemisin (4.82%) in the root oil of T. callosum.. The ratio of steroids was 16.86% in flower oil, 16.74% in the stem oil, and 11.01% in the root oil of T. callosum.. The major representative of steroid was 6-aza-β-homo-5-α-cholestano[6,7-d] tetrazole (8.33%) in the flower oil, γ-sitosterol (7.17%) in the stem oil, and stigmasterol (3.09%) in the root oil of T. callosum.. Aromatics constituted 1.80% in the flower oil, 1.87% in the stem oil, and 14.66% in the root oil. The main component of aromatic was p.-methoxy-β-cyclopropylstyrene (0.70%) in the flower oil, 2,8-dimethyldibenzothiophene (0.43%) in the stem oil, and [1,1′-biphenyl]-3-amine (4.71%) in the root oil of T. callosum. (Table 1). The ratio of the other compounds was 2.90% in the flower oil, 2.06% in the stem oil, and 3.07% in the root oil of T. callosum. (Table 2).

Thirteen of the components exist in all three parts of the plant (Table 1). These compounds constituted 13.77% in the flowers, 45.99% in the stems, and 30.16% in the roots of T. callosum.. Generally, GC-MS analysis of chloroform extracts of the plant showed differences in chemical composition. The identified components and their percentages are given in Table 2, where the components are grouped by chemical classes. An immediate observation was the presence of high level of hydrocarbons (27.89%) in the flower, acids (34.25%) in the stem, and aromatics (14.66%) in the root oil of T. callosum. (Table 2).

The results of the analyses showed that the roots included more unknown compounds (69.05%) when compared to the other two in total identification (Table 1). This study also showed that each part of the plant had abundant terpenes-terpenoids, hydrocarbons, and steroid compounds. This is the first lipid contents report for the flowers, stems, and roots of T. callosum. studied by GC-MS.

Acknowledgments

This study was supported by a grant from Karadeniz Technical University and the State Planning Agency (DPT) of Turkey.