Variability in essential oil content and composition of Ocimum ciliatum accessions from Iran: evidence for three chemotypes

ABSTRACT

The chemical composition of essential oils isolated from aerial parts of 28 accessions of Ocimum ciliatum from Iran was investigated by gas chromatography and gas chromatography–mass spectrometry. The oil contents were found to be about 0.71–1.25% (w/w). In total, 48 compounds, accounting for 93.7–99.8% of the oils composition, were identified. The results from the composition suggested that the oils could be classified as aromatic/oxygenated monoterpene–type. Among the aromatic compounds, the main components were methyl chavicol (11.6–89.6%) and citral (0.5–50.7%), which were the main oxygenated monoterpenes in all essential oils. The chemotypes values of the essential oils composition were discussed according to the results of cluster analysis. The 28 accessions were divided into 3 groups, each comprising a chemotype. The different characterized chemotypes were chemotype I (methyl chavicol), chemotype II (citral), and chemotype III (methyl chavicol/citral).

KEYWORDS:

• Ocimum ciliatum
• Essential oil
• Chemical composition
• Cluster analysis
• Chemotype

Introduction

The genus Ocimum as one of the largest genera of the Lamiaceae family, comprises aromatic, annual and perennial herbs and shrubs from the tropical and subtropical regions of Asia, Africa, and South America.^[1] In many of Asian and African countries, some Ocimum species are used in traditional medicines for different applications.^[2,3] The Ocimum species are a source of valuable essential oils that comprise a complex mixture of monoterpenoids (carvone, cineole, fenchone, geraniol, linalool, myrcene, and thujone), sesquiterpenoids (caryophyllene and farnesol), a triterpenoid (ursolic acid), and phenolic compounds (flavonoids and phenolic acids).^[4–6] Basil essential oil contains the biological active constituents that possess antiallergic, antimicrobial, antifungal, and immuno-stimulatory properties, thus are extensively used in pharmacy, perfume, cosmetic, and food industries.^[7–9]

Basil essential oil compositions vary due to the leaf and flower colors, aroma, plant origin, and variation in chemotypes.^[4,10] Depending on basil species some of them have high camphor content, while others contain citral, geraniol, methyl chavicol, methyl cinnamate, methyl eugenol, linalool, eugenol, and thymol.^[2,4,10] Evaluation of genetic diversity among Iranian accessions of Ocimum spp. was done earlier.^[11] Iran is known as an origin location of Ocimum ciliatum. It is a known annual and aromatic herb which is grown in home and vegetable gardens by local people in every part of Iran, and the green leaves and herbaceous parts of stems are used as vegetable and culinary herb.

Because of interspecific hybridization and polyploidy of Ocimum genus, the taxonomy of this genus is complex, and also exhibit great chemical variability in its essential oils; so the Ocimum genus is characterized by a variability in its chemical composition that can exist as chemotypes.^[12] There have been many attempts to classify different species of Ocimum into chemotypes and use chemotaxonomy instead of morphological division.^[3] The following chemotypes have been described in O. basilicum: linalool, linalool/eugenol, methyl chavicol/linalool, and methyl chavicol^[13]; 1,8 cineole^[14]; and citral^[3]. Different chemotypes were reported in the other species of Ocimum such as methyl cinnamate-type in O. americanum, eugenol-type in O. gratissimum, and methyl eugenol-type in O. sanctum.^[15] Further work is required to describe the wide chemical variability range for different species of Ocimum.^[16] In this point, the various chemotypes, found in different herbal plants, play an important role in determining their utilization and value the chemical variability studies.^[15,17]

With consideration to the potential for economic interest in O. ciliatum plant and its continuing widespread traditional use, we believe further studies are warranted to characterize the chemical variations for finding favorable chemotypes, which may be found in this species of Ocimum genus. Therefore, this study was aimed: (a) to determine the essential oil quantity and quality of 28 O. ciliatum accessions from Iran, and (b) to identify the possible chemotypes in these accessions.

Materials and methods

Plant materials

This study was conducted in Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran, under semi-arid conditions. Seeds of 28 accessions belonging to O. ciliatum were collected from home and vegetable gardens across 28 sites from northern to southern Iran, which were located at altitudes between 5 and 1859 m (Fig. 1 and Table 1). To examine essential oil composition of the O. ciliatum accessions without environmental influences, the plants were grown under same environmental and soil conditions, so that the results are comparable and differences in chemical profiles should reflect genetic differences among the various accessions. Seeds were sown on a sandy clay soil in field in March 2014. For the essential oil isolation, the aerial parts of all samples were collected at the full flowering stage and dried in the shade at room temperature immediately after harvesting. Voucher specimens of all analyzed populations were deposited at the Herbarium of Tarbiat Moallem University.

Table 1. Ocimum ciliatum accessions included in this study, their province of origin, collection sites, climatic and geographical coordinates.

Number	Province	Collection site	Climate^a	Latitude (N)	Longitude (E)	Altitude (m)
1	Fars	Shiraz	Temperate–semi arid	29° 32′N	52º 36′ E	1484
2	Lorestan	Borujerd	Cool temperate–semi arid	33° 55′N	48° 45′ E	1629
3	Yazd	Yazd	Warm temperate-arid	31° 54′N	54° 17′ E	1237
4	Esfahan	Esfahan	Temperate–arid	32° 37′ N	51° 40′ E	1551
5	Bushehr	Bandar deylam	Warm–arid	30° 03′N	50° 09′ E	5
6	Fars	Nurabad	Cool temperate–semi arid	34° 03′N	48° 00′ E	1859
7	Kerman	Kerman	Temperate–arid	30° 17′N	54° 04′ E	1632
8	East Azarbayjan	Tabriz	Cool temperate–arid	38° 05′N	46° 17′ E	1361
9	Kohkilooie	Yasuj	Cool temperate–semi arid	30° 50′ N	51° 41′ E	1831
10	Golestan	Gorgan	Temperate–semi arid	36° 51′N	54° 16′ E	13
11	Semnan	Damghan	Temperate–arid	36° 10′N	54° 17′ E	1345
12	Razavi Khorasan	Khalilabad	Temperate–arid	35° 10′ N	58° 23′ E	1110
13	Ghom	Ghom	Temperate–arid	34° 42′ N	50° 51′ E	877
14	Esfahan	Khominishahr	Temperate–arid	32° 25′ N	51° 16′ E	1595
15	Kurdistan	Sanandaj	Cool temperate–semi arid	35° 20′ N	47° 00′ E	1373
16	Kerman	Zarand	Temperate–arid	30° 46′ N	56° 34′ E	1753
17	North Khorasan	Bojnord	Cool temperate–semi arid	37° 28′ N	57° 19′ E	1091
18	South Khorasan	Birjand	Temperate–arid	32° 52′ N	59° 12′ E	1491
19	Razavi Khorasan	Kashmar	Temperate–arid	35° 12′ N	58° 28′ E	1109
20	Razavi Khorasan	Kashmar	Temperate–arid	35° 12′ N	58° 28′ E	1109
21	Alborz	Karaj	Temperate–arid	35° 55′N	50° 54′ E	1313
22	Fars	Neyriz	Temperate–arid	29° 12′N	54° 20′ E	1632
23	Mazandaran	babol	Temperate- semi humid	36° 33′ N	52° 41′ E	22
24	Mazandaran	Sari	Temperate- semi humid	36° 34′N	53° 03′E	15
25	Qazvin	Ghazvin	Cool temperate–arid	36° 15′N	50° 03′ E	1279
26	Sistan	Zabol	Warm temperate-arid	31° 02′ N	61° 29′ E	489
27	Hormozgan	Bandar-Abbas	Warm–arid	27° 13′ N	56° 22′ E	32
28	Hormozgan	Bandar-Abbas	Warm–arid	27° 13′ N	56° 22′ E	32

^aYearly mean temperature in warm, temperate, and cool climates is 15–25, 10–15, and 0–10°C, respectively. Yearly mean rainfalls in humid, semi-humid, semi–arid, and arid climates are, respectively, >1400, 600–1400, 300–600, and 100–300 mm.

Figure 1. Collection sites of Ocimum ciliatum accessions.

Essential oil extraction

Gas chromatography

Gas chromatography (GC) analysis was performed using a Thermoquest gas chromatograph equipped with a flame ionization detector (FID) and a DB-5 fused-silica column (30 m × 0.25 mm i.d.; film thickness 0.25 µm). The oven temperature was programmed from 60 to 250°C at 4°C/min and held isothermal at 250°C for 10 min; injector temperature, 250°C; detector temperature, 300°C; carrier gas, N2 (1.1 ml/min); split ratio, 1:50. Quantity data were obtained from GC-FID area percentages without the use of correction factors.

Gas chromatography–mass spectrometry

The Gas chromatography–mass spectrometry (GC-MS) analysis was carried out with a Thermoquest-Finnigan Trace apparatus equipped with a DB-5 fused-silica column (60 m × 0.25 mm i.d., film thickness 0.25 µm). The oven temperature was programmed from 60 to 250° at 4°C/min and held isothermal at 250°C for 10 min; ion source and transfer-line temperature, 250°C; carrier gas, He (1.1 ml/min). The mass spectrum was acquired over the mass range of 35–465 a.m.u. with an ionization voltage of 70 eV.

Identification of compounds

The constituents of the essential oils were identified by the calculation of their RI under temperature programmed conditions for n-alkanes (C₆–C₂₄) on a DB-5 column. The identification of the individual compounds was made by comparing their mass spectra with those of the internal reference MS library (Wiley 7.0) or of authentic compounds and confirmed by comparing their RI values with those of authentic compounds or with those reported in the literature.^[18] Semi-quantitative data were obtained from the integration of FID area percentages without the use of correction factors.

Statistical analysis

Cluster analysis was used to classify and group the 28 accessions of O. ciliatum according to their essential oil components. Cluster analysis based on their essential oil components was calculated using the Euclidean distance to identify of the varieties. The computations were performed using SPSS package software (version 16.0).

Results and discussion

The accessions of medicinal plants growing in various geographical conditions might be different in their chemical composition.^[19,20] Therefore, the limitation of environmental effect aims to the better recognization of genetic impacts on the composition of essential oil and facilitate the plant selection for breeding programs, also in this condition; the results of different studies will be comparable with each other.^[16,21] In this study, 28 accessions of O. ciliatum were collected from northern to southern parts of Iran (Table 1 and Fig. 1) and were evaluated for chemical characterization. Possible chemo-diversity in these accessions was found without environmental effects. To the best of our knowledge, the chemical variability of essential oil from O. ciliatum has not been reported. All throughout the tables and figures of this discussion, the accessions are numbered from 1 to 28.

Essential oils of the dried aerial parts from 28 accessions of O. ciliatum were obtained by hydro-distillation, in a yield ranging from 0.71 to 1.25% (w/w). The chemical constituents of 28 accessions of O. ciliatum with their retention indices (RI) and percentage contents in the essential oils are listed in Table 2, according to their elution order on a DB-5 column. The characterization of these essential oils by GC-FID and GC/MS analyses allowed the identification of 48 volatile constituents, accounting for 93.7–99.8% of the total oil composition.

Table 2. Chemical composition of the essential oils of the 28 Ocimum ciliatum accessions.

			Accessions^b
Number	Compound	RI^a	1	2	3	4	5	6	7	8	9	10	11	12	13	14
1	Pineneα	936	–^c	–	–	0.8	–	–	–	–	–	–	–	–	–	–
2	Sabinene	975	0.4	0.4	0.4	0.4	0.4	–	0.3	0.4	0.3	0.4	0.4	0.3	0.4	0.5
3	β-Pinene	982	0.2	0.2	0.1	–	0.1	–	–	–	–	–	0.2	–	0.4	–
4	6-Methyl-5-hepten-2-one	989	0.2	0.2	0.3	0.4	0.1	–	0.3	0.4	0.3	0.2	0.2	0.3	0.5	0.4
5	β-Myrcene	993	–	0.1	0.1	0.2	0.2	–	0.1	–	–	0.1	0.2	0.1	–	–
6	Limonene	1033	–	–	–	–	–	–	–	–	–	–	–	–	–	–
7	1,8-Cineole	1037	–	–	–	–	–	–	–	–	–	–	0.2	–	–	–
8	Cis-β-ocimene	1048	–	0.1	–	–	–	–	0.2	–	0.2	–	0.2	0.2	–	–
9	Trans-linalool oxide	1075	–	0.1	–	0.2	0.2	–	–	–	–	–	–	–	–	–
10	Cis -linalool oxide	1092	–	–	–	–	–	–	–	–	–	–	–	0.1	–	–
11	Fenchone	1094	0.6	0.4	0.6	0.2	0.2	0.9	0.5	0.9	0.5	0.3	0.5	0.5	0.8	0.7
12	Linalool	1107	0.4	0.6	1	0.3	0.4	1	0.7	1.3	0.9	0.4	0.8	0.9	0.3	1
13	Trans-chrysanthemal	1155	0.6	0.2	0.2	–	–	0.5	0.2	0.6	0.2	–	0.2	0.3	0.3	0.6
14	Camphor	1157	–	0.6	0.9	0.2	0.4	0.9	0.7	1.2	0.7	0.4	0.8	0.8	0.6	1
15	Methyl chavicol	1206	51.6	47.5	30.3	56.3	67.4	14.2	46.1	13.8	38.4	67.3	40.4	35	36.3	18.9
16	Nerol	1230	1.7	1.8	2.6	1.5	0.9	4	2.9	2.4	3	1.2	2.4	3.5	2.6	3.6
17	Neral (Z-citral)	1248	11	11.3	16.8	9	6.7	20.4	12.8	20.6	14.2	6.8	13	15.5	13.1	18.7
18	Geraniol	1253	25.9	26.7	39.8	21.1	17	–	30.2	–	34.3	17.4	31	36.6	31.2	–
19	Trans-myrtanol	1263	–	–	–	0.3	–	1.4	–	2.1	–	–	–	–	2.7	2
20	Geranial (E-citral)	1274	14.9	15.4	23	12.1	9.3	28.4	17.4	28.9	20.1	9.6	18	21.1	18.1	25.8
21	Bornyl acetate	1286	0.2	–	—	–	–	–	–	–	–	–	0.1	–	0.3	0.3
22	Methyl (Z)-cinnamate	1313	–	–	–	0.5	–	–	–	0.5	–	–	–	–	–	–
23	Eugenol	1357	0.7	0.8	1.2	0.9	0.3	1.6	0.5	1.8	0.8	0.4	0.8	1.1	1.2	1.2
24	Neryl acetate	1362	0.2	0.2	0.4	0.3	0.2	0.9	–	1	0.2	–	0.3	0.3	0.6	0.4
25	Geranyl acetate	1382	0.4	0.5	0.6	0.2	0.3	–	0.4	0.8	0.6	0.3	0.6	0.5	0.6	0.8
26	β-Elemene	1395	–	–	–	0.4	–	–	–	–	–	–	–	–	–	–
27	Methyl (E)-cinnamate	1396	–	–	–	0.2	–	–	–	–	4.4	–	–	–	0.2	–
			Accessions
Number	Compound	RI	1	2	3	4	5	6	7	8	9	10	11	12	13	14
28	Methyl eugenol	1406	3.5	4.7	5	0.4	2.7	7.7	4.1	6.8	–	2.9	5	4.5	5.5	6.4
29	Trans-caryophyllene	1425	1.1	1.3	1.3	1.2	0.8	2.1	1	1.9	1.3	0.8	1.4	1.1	1.6	1.7
30	Trans-α-bergamotene	1439	–	0.1	0.1	–	–	0.8	–	–	–	–	0.6	–	–	0.7
31	α-Guaiene	1441	0.4	0.6	0.6	0.2	0.3	–	0.4	0.8	0.5	0.3	2.4	0.5	0.7	–
32	Tran-β-farnesene	1456	1.6	2.4	2.5	0.3	1.3	3.5	2	3.1	2.2	1.4	0.2	2.2	2.5	3.1
33	α-Humulene	1461	0.2	0.2	0.1	0.2	0.1	–	0.2	–	0.1	0.2	2.1	–	–	–
34	Germacrene-D	1489	1.5	2	2.2	0.2	1.2	3.1	1.8	2.5	2.1	1.2	0.3	2.1	2.6	2.7
35	Bicyclogermacrene	1503	0.3	0.1	0.3	0.2	–	–	0.3	–	0.3	0.2	–	0.3	0.4	–
36	δ-Guaiene	1510	–	0.4	–	0.2	–	–	–	–	–	–	–	–	–	0.5
37	γ-Cadinene	1521	3.2	4.4	4.2	1	2.6	5.6	3.5	4.8	4.1	2.7	4.3	4	5.1	5.2
38	Germacrene- B	1549	0.1	–	–	1.2	–	–	–	–	–	–	–	–	–	–
39	Trans-α-bisabolene	1553	0.7	0.7	1.1	4.7	0.4	1.4	0.6	1.6	0.9	0.4	0.7	1	1.2	1.6
40	Trans-nerolidol	1572	–	–	–	0.2	–	0.5	–	–	–	–	–	–	–	–
41	3-Methoxycinnamaldehyde	1591	0.3	–	0.4	2	0.1	–	0.3	0.6	0.4	0.2	0.3	0.3	0.4	0.6
42	caryophyllene oxide	1599	0.3	–	0.3	0.4	0.2	0.8	0.3	0.5	0.3	0.3	0.3	0.2	0.5	0.8
43	Humulene oxide	1627	–	–	–	–	–	–	–	0.4	–	–	–	–	–	–
44	T-muurolene	1632	–	–	–	–	–	–	–	–	–	–	–	–	–	–
45	α-Cadinol	1661	–	–	–	–	–	–	–	–	–	0.1	–	–	–	–
46	α-Eudesmol	1668	0.3	–	0.1	0.2	0.1	–	0.2	–	0.1	0.3	0.1	0.1	0.3	0.5
47	(Z,E)-farnesol	1693	–	–	–	–	–	–	–	–	–	–	–	–	–	–
48	β-Sinensal	1699	0.2	0.2	–	–	–	–	–	–	–	0.2	0.2	–	–	–
	Monoterpene hydrocarbons		0.6	0.8	0.6	1.4	0.7	0	0.6	0.4	0.5	0.5	1	0.6	0.8	0.5
	Oxygenated monoterpenes		30	31.1	46.1	24.3	18.6	58.4	35.6	59.8	40.4	19	36.9	43.5	40	54.9
	Sesquiterpene hydrocarbons		9.1	12.2	12.4	9.8	6.7	16.5	9.8	14.7	11.5	7.2	12	11.2	14.1	15.5
	Oxygenated sesquiterpenes		0.8	0.2	0.4	0.8	0.3	1.3	0.5	0.9	0.4	0.9	0.6	0.3	0.8	1.3
	Aromatic compound		56.1	53	36.9	60.3	70.5	23.5	51	23.5	44	70.8	46.5	40.9	43.6	27.1
	Total		96.8	97.4	96.9	97	97	99.8	97.7	99.6	97.1	98.7	97	96.8	99.8	99.6
	Oil content (w/w%)		1.03	0.87	0.92	0.71	0.87	0.71	0.88	0.83	0.85	0.87	0.88	0.87	0.85	0.92
			Accessions^b
Number	Compound	RI^a	15	16	17	18	19	20	21	22	23	24	25	26	27	28
1	Pineneα	936	–^c	–	0.4	–	–	–	–	–	t^d	0.3	0.4	–	1.7	1.7
2	Sabinene	975	0.4	0.4	0.3	0.4	0.4	0.4	0.4	0.3	–	–	–	–	–	–
3	β-Pinene	982	0.2	0.1	0.1	0.1	0.2	0.3	0.2	0.2	–	–	–	–	–	–
4	6-Methyl-5-hepten-2-one	989	0.3	0.3	0.3	0.4	0.3	0.3	0.1	0.1	t	0.2	0.4	–	1	1.1
5	β-Myrcene	993	0.1	0.1	–	0.1	0.1	0.3	0.2	0.1	–	0.4	0.1	–	–	–
6	Limonene	1033	0.6	–	–	–	–	–	–	1.8	–	–	–	–	–	–
7	1,8-Cineole	1037	–	–	–	–	0.2	–	0.8	–	–	–	–	–	0.5	0.5
8	Cis-β-ocimene	1048	–	0.2	–	0.1	0.3	–	–	–	–	–	0.2	0.1	–	0.5
9	Trans -linalool oxide	1075	–	–	0.3	–	0.1	–	–	0.2	–	–	0.3	0.2	2.6	2.7
10	Cis-linalool oxide	1092	–	–	–	–	–	–	–	–	–	–	–	–	–	–
11	Fenchone	1094	0.5	0.4	0.1	0.6	0.6	0.6	0.3	–	–	0.6	0.3	0.3	–	–
12	Linalool	1107	0.7	0.6	0.1	0.8	0.9	1	0.4	–	–	0.4	0.5	0.5	–	–
13	Trans-chrysanthemal	1155	0.2	0.3	–	0.4	0.3	0.4	0.1	–	–	–	0.1	0.3	0.6	0.6
14	Camphor	1157	0.6	0.6	0.1	0.7	0.7	0.7	0.3	–	–	0.7	0.2	0.1	0.6	0.6
15	Methyl chavicol	1206	46	42.9	76.3	27.5	28.8	29.2	63.7	89.6	70.4	45.6	40	53	11.6	11.8
16	Nerol	1230	2	2.1	0.3	2.9	3.1	3.1	1.2	–	–	1.7	0.2	2.1	–	–
17	Neral (Z-citral)	1248	12.3	12.5	4.4	16.9	16	16.2	7.2	–	8.7	12.8	13.2	9.2	21.2	21.6
18	Geraniol	1253	29.3	29.3	10.3	39.8	38	38.5	16.8	0.5	18.9	30.3	31.9	22	1.8	1.7
19	Trans-myrtanol	1263	–	–	0.7	–	–	1.9	–	–	–	–	–	–	1.1	1.1
20	Geranial (E-citral)	1274	17	16.8	5.9	22.9	22	22.3	9.6	0.5	10.2	17.5	18.7	12.8	28.6	29.1
21	Bornyl acetate	1286	–	–	0.1	–	–	–	–	–	–	–	–	–	3.3	3.3
22	Methyl (Z)-cinnamate	1313	–	–	0.4	0.1	–	–	–	–	–	–	–	–	–	–
23	Eugenol	1357	1.1	0.9	1.3	1.1	1	1	0.5	–	–	–	–	–	–	–
24	Neryl acetate	1362	0.3	0.5	0.5	0.3	0.3	0.3	0.1	–	0.9	–	–	–	–	0.7
25	Geranyl acetate	1382	0.5	0.6	–	0.7	0.8	0.8	0.4	–	0.3	1.2	–	–	–	–
26	β-Elemene	1395	–	–	0.6	–	–	–	–	–	–	–	–	–	11.2	11.3
27	Methyl (E)-cinnamate	1396	–	–	0.1	–	–	–	–	–	–	–	0.6	0.6	0.6	0.6
			Accessions
No.	Compound	RI	15	16	17	18	19	20	21	22	23	24	25	26	27	28
28	Methyl eugenol	1406	4.3	4.8	–	5.4	5.5	5.6	3.4	1.7	–	0.4	0.3	0.2	0.5	0.5
29	Trans-caryophyllene	1425	1.2	1.3	0.7	1.6	1.6	1.6	1.1	0.4	1.6	–	–	–	–	–
30	Trans-α-bergamotene	1439	–	0.1	–	0.2	0.2	–	0.1	–	0.7	–	0.5	0.3	–	–
31	α-Guaiene	1441	0.5	0.5	0.1	0.6	0.6	0.6	0.4	0.2	–	–	0.1	–	1.1	1.1
32	Tran-β-farnesene	1456	2	2.4	0.1	2.6	2.6	2.6	1.6	0.7	t	4.5	6.9	3.8	1.7	1.7
33	α-Humulene	1461	–	–	–	–	–	–	–	–	0.8	0.9	–	0.6	–	–
34	Germacrene-D	1489	1.8	2.4	0.2	2.1	2.2	2.2	1.5	0.9	t	0.5	–	0.4	1	1
35	Bicyclogermacrene	1503	0.2	0.3	–	0.4	0.3	0.3	0.2	0.1	–	2.6	0.5	–	–	–
36	δ-Guaiene	1510	–	–	–	–	0.1	–	–	–	–	–	–	2.5	–	–
37	γ-Cadinene	1521	3.5	3.6	1.3	4.8	4.5	4.6	3.1	1.6	–	–	–	–	–	–
38	Germacrene- B	1549	–	–	–	–	–	–	–	–	–	–	1.8	2.1	–	–
39	Trans-α-bisabolene	1553	0.6	0.9	2.6	1.6	1.4	1.4	0.6	–	1.8	–	–	–	–	–
40	Trans-nerolidol	1572	–	–	–	–	–	–	–	–	–	–	0.3	0.3	–	–
41	3-Methoxycinnamaldehyde	1591	0.2	0.4	1	0.6	–	0.6	0.2	–	0.1	–	–	–	–	–
42	caryophyllene oxide	1599	0.2	0.3	0.1	0.3	0.4	0.7	0.2	–	2.5	–	4.8	4.4	–	–
43	Humulene oxide	1627	–	–	0.2	0.1	–	–	–	–	0.5	2.1	–	–	–	–
44	T-muurolene	1632	–	–	0.2	–	–	–	–	–	–	0.3	0.2	–	–	1.1
45	α-Cadinol	1661	–	–	–	–	–	–	–	–	–	–	1.9	1	4.4	4.4
46	α-Eudesmol	1668	0.1	–	–	0.1	–	–	–	–	–	4.6	–	–	–	1.2
47	(Z,E)-farnesol	1693	–	–	–	–	–	–	–	–	0.1	1.1	0.4	0.3	–	–
48	β-Sinensal	1699	–	–	–	–	–	–	–	–	–	0.4	0.1	0.1	–	–
	Monoterpene hydrocarbons		1.3	0.8	0.8	0.7	1	1	0.8	2.4	t	0.7	0.7	0.1	1.7	2.2
	Oxygenated monoterpenes		34.1	34.4	12.5	46.2	45	47.3	20.4	1	20.1	35.3	34.2	26.7	60.3	61.9
	Sesquiterpene hydrocarbons		9.8	11.5	5.8	13.9	13.5	13.3	8.6	3.9	4.9	8.8	10	9.7	15	16.2
	Oxygenated sesquiterpenes		0.3	0.3	0.3	0.5	0.4	0.7	0.2	0	3.1	8.2	7.5	6.1	4.4	5.6
	Aromatic compound		51.6	49	79.1	34.7	35.3	36.4	67.8	91.3	70.5	46	40.9	53.8	12.7	12.9
	Total		97.6	96.3	99.1	96.6	95.5	98.9	98.2	98.7	98.8	99.4	93.7	96.5	94.9	99.6
	Oil content (w/w%)		0.81	0.81	0.92	0.81	0.71	0.74	1.16	1.25	0.81	1.03	0.92	1.03	0.92	0.90

^aRI, retention index determined relative to n-alkanes (C₆–C₂₄) on a DB-5 column.

^bDetails concerning the accession numbers and codes are given in Table 1.

^cNot detected.

^dttrace (less than 0.1%).

Across the accessions analyzed, we identified four main essential oil components (compounds found in concentrations higher than 10%, calculated as % of peak area). The main constituents were methyl chavicol, citral (neral+geranial), and β-elemene. As listed in Table 2, all essential oils of the 28 accessions chiefly consisted of methyl chavicol (11.6–89.6%) and citral (0.5–50.7%), indicating that they were the common compounds in all the O. ciliatum accessions.

The chemical compositions of basil oil have been the subject of considerable studies.^[3,14,22] The comparison of our results with literature indicated that the components approximately varied from the other species in this genus. There are several chemotypes that have been established from various phytochemical investigations. The recurring polymorphism determines a large number of subspecies, different varieties, and forms producing essential oils with varying chemical composition.^[23] Safari Dolatabad et al.^[24] in their study showed that citral and methyl chavicol were the main compounds of O. ciliatum essential oil. In addition, 28 compounds were found in concentrations higher than 1%, and 16 compounds were found in concentrations less than 1%.

The main components of basil reported were different based on the region of growing plant. For example, reunion basil chemotypes are distinguished by a high concentration of methyl chavicol, while most tropical chemotypes of basil have methyl cinnamate as a main constituent of the essential oil. Another frequent chemotype high in eugenol is grown in North Africa, Russia, Eastern Europe, and parts of Asia.^[25]

Aromatic compounds (12.7–91.3%) represented by methyl chavicol and methyl eugenol were the main compounds in all accessions; also in the overall terpene profile, contents of oxygenated monoterpenes were found significantly high (1.0–61.9%) that are represented by citral and nerol. Besides, sesquiterpene hydrocarbons (3.9–16.5%) represented by β-elemene, trans-β-farnesene, and γ-cadinene; oxygenated sesquiterpenes (0.0–8.2%) represented by α-cadinol and α-eudesmol; monoterpene hydrocarbons (0.0–2.4%) represented by α-pinene were other components in the essential oil of accessions. These volatiles together were the compounds for discriminating the accessions.

There were variations among the accessions for the quantity and quality of majority of constituents. Variations in volatile oil composition have also been reported in different medicinal plants such as O. basilicum^[13], Artemisia Herba-alba^[26], A. dracunculus^[27], Mentha haplocalyx, and M. spicata^[28]. In this study, the number of compounds per accession ranged from 16 (in accession number 22) to 35 (in accessions number 4). The essential oil components except two main constituents (methyl chavicol and citral) were especially variable in occurrence and concentration among the different accessions, ranging from absent in some varieties to more than 10% of the total essential oil composition in others. β-Elemene is one of the essential oils constituent that is poorly represented in the most accessions and its occurrence is limited in two accessions (accession number 27 and 28) in which its content is higher than 10%. In agreement with these results, Grayer et al.^[21] reported that the essential oil compositions appeared to be very different in the various accessions of O. basilicum, and some compounds were specific to a single variety; similar results were also obtained by Bhatia et al.^[29]. Variation in the metabolites concentration among different chemotypes of Withania somnifera fruits suggested that specific chemotypes can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.^[29] So, the concentration of active components is one of the major factors that affected the medicinal plants’ quality, as well as the minor compounds are important in respect of biological activity and they are critical for the activity of the oil and may have synergistic effects.^[10,30]

To characterize and verify the variations of the essential oils and identify the different possible chemotypes in the accessions of O. ciliatum, their compositions were analyzed by cluster analysis. Grayer et al.^[21] reported that the use of one single volatile oil to describe a chemotype is problematic, since there are often two or more major compounds which might be present in nearly equal amounts. They proposed a system of chemotype study based on the combination of compounds rather than the sole dominant compounds. Therefore, in this study we used the main and minor constituents for chemodiversity analysis.

As shown in Fig. 2, cluster analysis allowed to separate the 28 O. ciliatum accessions into 3 groups, each representing a chemotype. The dendrogram exhibited a distinct separation of three chemotypes included: methyl chavicol chemotype, citral chemotype, and methyl chavicol/citral chemotype. The detailed characteristics of each chemotype are described below.

Figure 2. Dendrogram obtained by cluster analysis of the essential oil components of the 28 accessions of O. ciliatum under study based on Euclidean distances.

Characterization of chemotype I (methyl chavicol)

The composition results suggested that the 21% of studied oils could be classified as aromatic compound/methyl chavicol-type. Methyl chavicol represented 63.7–89.6% of the total oil. The methyl chavicol of essential oil is responsible for aniseed-like aroma and sweet taste of basil plants.^[31] Although methyl chavicol showed a probable carcinogenic effect^[32], it has a relaxant and anticonvulsive effect as well as fungistatic and antifungal activity.^[33] The accessions of this chemotype, based on the other main components, were separated into two different sub-chemotypes. The high content of citral (10.3–18.9%) in five accessions (5, 10, 21, 23, 17) differentiated them as a sub-chemotype (A) in comparison with accession number 22 (sub-chemotypes B) that its citral content was significantly low (0.5%). In fact, the abundance of methyl chavicol in Ocimum genus has been reported previously. Pirmoradi et al.^[22] reported that methyl chavicol (33.6–49.1%) was the main component in all essential oils of 21 accessions of Iranian sweet basil (O. basilicum). Vieira and Simon^[3] reported the methyl chavicol chemotypes in O. basilicum accessions from Turkey.

The literature survey revealed that the main source of methyl chavicol is Agastache (Agastache foeniculum). In this medicinal plant, the content of methyl chavicol was reported to be 74.6–91.7%.^[34,35] Therefore, according to our results, the content of methyl chavicol in methyl chavicol chemotype of O. ciliatum was comparable with Agastache plant. So, in this respect, the distinct composition of essential oils produced in each chemotype indicates each chemotype’s potential value in future growing and breeding programs.

Characterization of chemotype II (citral)

Five accessions (6, 8, 14, 27, 28) belonged to this chemotype and consisted 18% of studied accessions. This chemotype was characterized by a high content of citral, which ranged from 44.5 to 50.7%; also, β-elemene, methyl eugenol, trans-β-farnesene, and γ-cadinene were other major components in this chemotype. According to the content of mentioned constituents in this chemotype, we could observe two sub-chemotypes. Accessions 27 and 28 were distinguished from the others as sub-chemotype C, because β-elemene was found in the highest content (11.2–11.3%) in these accessions, but it was in low amounts or non-existent in the other accession oils. Accessions 6, 8, and 14 were clustered into sub-chemotype D, due to the fact that the contents of methyl eugenol (6.4–7.7%), trans-β-farnesene (3.1–3.5%), and γ-cadinene (4.8–5.6%) were higher than the other studied accessions. According to the results of previous researches, 80% of the citral chemotype was investigated in Ocimum genus.^[16] Moreover, 33.6% citral concentration in O. basilicum accessions^[3], the 68% and 78% citral concentrations in Ocimum accessions^[36] and in O. citriodorum populations^[21] were reported, respectively.

Characterization of chemotype III (methyl chavicol/citral)

We have also identified a mixed aromatic + oxygenated monoterpene chemotype with elevated levels of methyl chavicol and citral. By comparing the main components in the different studied accessions of O. ciliatum, the high percentage of methyl chavicol (27.5–56.3%) followed by citral (21.1–39.8%) differentiated most accessions as a distinct chemotype. Among 17 accessions in this chemotype, 8 were grouped in sub-chemotype E, for high content of citral than methyl chavicol. Also in this group the contents of methyl eugenol and γ-cadinene were high. The other nine accessions for high content of methyl chavicol in comparison with citral content were clustered in sub-chemotype F.

According to our results, aromatic and oxygenated monoterpenes compounds have been shown to play a significant role in the establishment of chemotypes as well as reported in different species such as Ocimum spp.^[3,13], Leptospermum scoparium^[37], and Artemisia spp.^[38] Altogether, our results show the chemical variability of O. ciliatum accessions. Ocimum essential oil is known for its high variability and many chemotypes from different accessions which have been investigated.^[15,16] Therefore, this genus has a characteristic flavor because of variety of essential oil components that affect their nutritional and medical functioning.^[15] From practical standpoint, the present studied accessions might be considered as consolidated sources of some bioactive compounds (methyl chavicol and citral) for being widely used in agro foods, pharmaceutical, cosmetic, and perfume industries.

The chemical composition of essential oil from most herbal plants such as basil is dependent on genetic, ontogenetic, and environmental factors, similarly as in other oil plants.^[20] Liber et al.^[13] reported that the components of essential oils were predominantly determined by the plants’ genotype, so the limitation of environmental effects make it possible to be investigated in future growing and breeding programs. Also it would be extremely valuable to be able to grow accessions with a known composition and ratio of individual essential oil constituents for which a constant yield could be secured.^[13]

Conclusion

The essential oils obtained from aerial parts of 28 accessions of O. ciliatum from Iran were investigated by GC and GC-MS. The oxygenated monoterpenes represented by methyl chavicol and citral were the main compounds in all essential oils. According to the results of cluster analysis based on essential oils constituents, we identified three chemotypes (methyl chavicol, citral, and methyl chavicol/citral), and due to the uniformity of cultivation and climatic conditions, variation of studied traits was related to genetic factors; therefore, selection would be useful for their genetic improvement.