The genus Hypericum (Hypericaceae) is represented by nearly 100 taxa grouped under 19 sections in Turkey. Among them, 45 taxa are endemic. In the traditional medicine of Turkey, the genus is known as “sari kantaron, kantaron, binbirdelik otu, mayasil otu” and most of them, especially H. perforatum, have been used for the treatment of burns, wounds, hemorroids, diarrhorea and ulcers [1-5].

Moreover, aqueous extracts prepared from the flowering aerial parts of the Hypericum species are being used in the treatment of psychological diseases such as neuralgia, anxiety, neurosis and depression [6]. The preparative forms of the Hypericum perforatum (St. John’s Wort) are sold for the treatment of mild to moderate depression in the USA and Europe. The chemical composition of some Hypericum species includes naphthodianthrones (especially hypericin and pseudohypericin), acylphloroglucinol derivatives (especially hyperforin and adhyperforin), flavonoids (especially quercetin, quercitrin, hyperoside and biapigenin), tannins, n-alkanes, xanthones and essential oil [7-9].

The essential oil compositions of about 50 different Hypericum species have so far been identified [10-13].

In this study, the oils of 5 endemic Hypericum species were obtained by hydro-distillation and analyzed by GC and GC/MS.

With the exception of H. thymopsis and H. scabroides the chemical composition of the essential oils of H. uniglandulosum, H. kotschyanum and H. salsugineum have not been reported to date [13,14].

The aim of this study was to investigate volatile constituents of five endemic Turkish Hypericum species, thus adding for the database of phytochemical knowledge for the genus. In addition to this, a comparison between the volatile oil composition of H. thymopsis and H. scabroides from different locations was performed.

Compounds were calculated automatically from peak areas of the total ion chromatogram (TIC). n-alkanes were used as reference points in the calculation of relative retention indexes (RRI). A library search was carried out using Wiley GC/MS Library, MassFinder, Adam’s Library [15,16] and in-house “Baser Library of Essential Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature data [17,18], was also used for the identification.

Plant material

Flowering aerial parts of H. uniglandulosum were collected from east Anatolia, namely, Erzincan: Erzincan-Eski Çayirli road, 10 km to Eski Çayirli, 1450 m, 15.07.2006, that of H. scabroides were collected from east Anatolia, namely, Erzincan: Erzincan-Kelkit, 15 km to Kelkit, 1550 m, 14.07.2006, that of H. kotschyanum were collected from south Anatolia, namely, Içel: North-west of Arslanköy, 1840 m, 15.06.2006, that of H. salsugineum were collected from central Anatolia, namely, Konya: around The Salt Lake, on 01.07.2005, H. thymopsis were collected from central Anatolia, namely, Sivas: Sivas-Malatya road, Ziyaret Tepe, 1350 m, 10.07.2005. Specimens were identified and vouchers were deposited in the Herbarium of Istanbul University, Faculty of Pharmacy (Istanbul Üniversitesi Eczacilik Fakültesi Herbaryumu, Istanbul, Turkey) under code numbers of ISTE 85344, ISTE 85343, ISTE 83979, ISTE 85341 and ISTE 85342, respectively.

Isolation of the essential oil

Air dried and powdered plant materials were subjected to hydrodistillation in a Clevenger-type apparatus according to the method recommended in the European Pharmacopoeia [19]. The oils obtained were stored at +4°C until analyzed.

Gas chromatography (GC)

The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300°C. To obtain the same elution order with GC-MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. The analysis results are given in table 1.

Table 1: Percentage of volatiles of 5 endemic Hypericum species.

Gas chromatography/mass spectrometry (GC/MS)

The GC/MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60 m×0.25 mm, 0.25 μm film thickness) was used with helium as carrier gas (0.8 mL/min). GC oven temperature was kept at 60°C for 10 min and programmed to 220°C at a rate of 4°C/min and kept constant at 220°C for 10 min and then programmed to 240°C at a rate of 1°C/min. Injection volume was 1 μL (10%) in hexane. Split ratio was adjusted at 40:1. The injector temperature was set at 250°C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450. Relative percentage amounts of the separated compounds were calculated automatically from peak areas of the total ion chromatogram (TIC). n-Alkanes were used as reference points in the calculation of relative retention indexes (RRI). A library search was carried out using Wiley GC/MS Library, MassFinder, Adam’s Library [15,16] and in-house “Baser Library of Essential Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature data [17,18], was also used for the identification.

Volatile oils were obtained from the aerial parts of the H. uniglandulosum, H. scabroides, H. kotschyanum, H. salsugineum and H. thymopsis with yields of 0.67% (v/w), trace (in hexane), 0.67% (v/w), trace (in hexane), 0.67% (v/w), respectively. The analyses were performed and are given in table 1.

Fifty-eight constituents corresponding to the 72.7% of the oil from the H. uniglandulosum, thirty-two constituents corresponding to the 75.3% of the oil from H. scabroides, forty-five constituents corresponding to the 92.4% of the oil from the H. kotschyanum, fiftyfour constituents corresponding to the 96.9% of the oil from the H. salsugineum, seventeen constituents corresponding to the 99.9% of the oil from the H. thymopsis were identified.

2,6-Dimethyl-3,5-heptadien-2-one (40.7%), nonacosane (3.2%), hexadecanoic acid (2.7%) and α-pinene (2.7%) were characterized as the main components of the H. uniglandulosum. Hexadecanoic acid (17.7%), spathulenol (5.3%), nonacosane (4.4%), dodecanoic acid (4.1%), baeckeol (4.1%) and γ-muurolene (3.9%) were characterized as the main components of the H. scabroides (SB). α-pinene (14.4%), nonacosane (11.1%), hexadecanoic acid (9.2%), β-pinene (8.7%), spathulenol (6.3%) and limonene (5.1%) were characterized as the main components of the H. kotschyanum. Nonacosane (42.7%), hexadecanoic acid (23.2%) and baeckeol (6.1%) were characterized as the main components of the H. salsugineum. α-pinene (44.0%), baeckeol (32.9%), spathulenol (8.0%), limonene (7.6%) and camphene (5.2%) were characterized as the main components of the H. thymopsis. The chemical class distribution of the volatile oils of 5 different species is given in table 2.

Table 2: The chemical class distribution of the oil components of 5 endemic Hypericum species.

It has been observed that, the oil of H. uniglandulosum was rich in terms of carbonylic compounds and fatty acids. The oil of H. scabroides was rich in terms of sesquiterpene hydrocarbons and fatty acids. The oil of H. kotschyanum, on the other hand, was dominated by monoterpene hydrocarbons, alkanes, oxygenated sesquiterpene hydrocarbons and fatty acids. The oil of H. salsugineum contained alkanes and fatty acids. The oil of H. thymopsis was found to be rich in monoterpene hydrocarbons and a phenolic-compound.

As a result of this research α-pinene-2,6-dimethyl-3,5-heptadien- 2-one, baeckeol, nonacosane and hexadecanoic acid were identified as major volatile constituents ( >10%) in Hypericum species.

Comparing the main constituent of the H. uniglandulosum oil to the other studies, the following are observed: 2,6-Dimethyl-3,5-heptadien- 2-one was found to be the major component only in H. tetrapterum from Serbia [20].

Hexadecanoic acid and spathulenol were detected in high amount in H. scabroides in this study, however, the δ-3-carene and sabinene were reported as the main constituents of the H. scabroides collected from different location [14].

Nonacosane was found to be the major component only in two species, namely H. salsugineum and H. davisii [21].

It was observed that α-pinene was found to be the main component in H. kotschyanum and H. thymopsis. Among the previous studies about Hypericum essential oils from Turkey, many taxa were characterized by the high amount of α-pinene [11,20,22-30], namely H. calycinum, H. cerastoides, H. montbretii, H. scabrum, H. perforatum [10,31], H. hyssopifolium subsp. elongatum var. elongatum, H. capitatum var. capitatum, H. aviculariifolium subsp. depilatum var. depilatum and H. apricum [21].

α-pinene, baeckeol, limonene and spathulenol were identified as major components in H. thymopsis, although baeckeol and limonene were not determined in a previous study. α-pinene, germacrene D, δ-cadinene, γ-cadinene, spathulenol, α-cadinol, eudesma-4(15),7- dien-1β-ol, nonacosane and hexadecanoic acid were identified both in this study and the previous one [13].

Essential oil compositions of species show difference in the sense of collecting regions and dates. Chemical profiling using volatiles may be useful in taxonomical classifications.

The authors would like to thank Tuba Kiyan for her assistance.