Fatty acid composition and tocopherol content in four Tunisian Hypericum species: Hypericum perforatum, Hypericum tomentosum, Hypericum perfoliatum and Hypericum ericoides Ssp. Roberti

2.1 Plant material

The aerial parts of H. perforatum, H. perfoliatum, H. tomentosum and H. ericoides Ssp Roberti (top of 2/3 plants) were collected at the full flowering stage, during June 2006 from wild populations located in El Feidja (North-western Tunisia; latitude 36°29′ (N); longitude 8°15′ (E); altitude 779 m). The site was characterized by annual precipitation of 1600 mm and mean annual temperature of 16.8 °C. The sampling site was not grazed or mown during the period when the plants were gathered. The sampling was done by a randomized collection of 15–20 plants. To avoid the sampling on the same plants, minimum distance of 10 m was adopted. The plant material was botanically characterized by Prof. Mohammed El Hedi El Ouni (Department of Biology, Faculty of Sciences, Bizerte, Tunisia) and according to the morphological description presented in Tunisian flora (Pottier-Alapetite, 1981). The harvested material was air-dried at room temperature (20 ± 2 °C) in the dark for 1 week and subsequently essayed for their fatty acid composition and tocopherol content.

2.2 Extraction and analysis of fatty acids

Samples of ground powder (1 g) in triplicate were weighed and extracted with chloroform: methanol (2:1, v/v) (LabScan, Dublin, Ireland) following the modified procedure of Bligh and Dyer (1959). The mixture was shaken and centrifuged (Eppendorf 5810R, LePecq, France) at 3000g for 10 min to allow phase development. The bottom (organic) layer was collected and filtered. The total extracted lipid material was recovered after the solvent was removed in a stream of nitrogen, weighed and stored at 0 °C for further analysis.

Fatty acid methyl esters (FAMEs) were prepared by using sodium methoxide (Sigma–Aldrich, Buchs, Switzerland) according to the method of Cecchi et al. (1985). Methyl nonadecanoate (C19:0) was used as internal standard.

The FAMEs were analyzed on a HP 6890 gas chromatograph (Agilent Palo Alto, CA, USA) equipped with a flame ionization detector (FID). The esters were separated on a RT-2560 capillary column (100 m length, 0.25 mm i.d, 0.20 mm film thickness). The oven temperature was kept at 170 °C for 2 min, followed by a 3 °C/min ramp to 240 °C and finally held there for an additional 15 min. Nitrogen was used as carrier gas at a flow rate of 1.2 mL/min. The injector and detector temperature was maintained at 225 °C. Identification of FAMEs was made by comparison of their retention time with those of reference standards purchased from Fluka (Steinheim, Germany).

2.3 Determination of tocopherols

Tocopherols were extracted from total lipids with hexane containing 0.01% of butylated hydroxytoluene (BHT ⩾ 99%, Sigma–Aldrich, which was added to inhibit the oxidative degradation of tocopherols during extraction) (Sivakumar et al., 2005). This solution (20 μL) was injected into the high-performance liquid chromatography system. The HPLC system consisted of a Shimadzu liquid chromatograph (Shimadzu Corp, Kyoto, Japan) equipped with a LC-20AT quaternary pump, a DGU-20A3 degasser, an SPD-M20A diode array detector (DAD) and a manual rheodyne injector with a 20 mL loop. The analytical column was a C18 reverse phase Hypersil ODS, 250 mm × 4.6 mm with a packing material of 5 mm of particle size. Separation of tocopherols was based on isocratic elution with methanol: acetonitrile (9/1) at a flow rate of 0.8 mL/min and the wavelength was set at 292 nm. Tocopherols were identified by comparing their retention times with those of authentic standards obtained from Sigma–Aldrich (St Louis, MO, USA) and they were quantified using the external standard method.

2.4 Statistical analysis

Data on total lipids, fatty acid composition and tocopherols were reported as mean ± standard deviation (SD) from triplicate determinations for each sample. Mean comparison was performed by analysis of variance (ANOVA) followed by Tukey post hoc test at the significance level of p ⩽ 0.05. The multivariate statistical analyses, i.e., the principal component analysis (PCA) and the hierarchical cluster analysis (HCA) were applied to examine the inter-relationships between the investigated species, utilizing the content of all identified fatty acids. All statistical analyses were carried out using the Statistica v 5.5 software (Statsoft, 1998).

3.1 Total lipids and fatty acid profile

As illustrated in Fig. 1, a statistically significant (p < 0.05) variation was determined in the total lipid content between the Hypericum species. H. perfoliatum and H. tomentosum were outstanding with their markedly higher total lipid content with their values 3.5 and 3.2 mg/g dw, respectively. For H. ericoides Ssp. Roberti and H. perforatum, the total lipid content ranged from 0.6 to 1.2 mg/g dw. The total lipid content in the present samples was apparently lower than those reported for Hypericum triquetrifolium Turra. (Hosni et al., 2007). Taking into account that the studied species grow under the same pedoclimatic circumstances and processed under the same conditions, we can argue that the observed discrepancy among Hypericum species might be due their genetic makeup (Richards et al., 2008; Hosni et al., 2010).

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Figure 1 Total lipid content (mg/g dw) in four Hypericum species. Data are expressed as mean ± SD (n = 3). Data marked with different superscript are significantly different (p < 0.05).

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The fatty acid composition of the four investigated Hypericum sp. is given in Table 1. The proportional composition of the analyzed fatty acids displayed a significant (p < 0.05) variation between species. In fact, oleic (23.27%), palmitic (17.43%) and linoleic acids (11.21%) were the most abundant fatty acids in H. perforatum. The same fatty acids were also found as the major fatty acid compounds in H. perfoliatum but with different proportions (11.2%, 37.62% and 16.8% for oleic, palmitic and linoleic acids, respectively). The Fatty acid profile of H. tomentosum was characterized by the abundance of palmitic (20.67%) followed by oleic (17.33%), stearic (14.8%) and α-linolenic (13.3%) acids. Linoleic acid (36.47%) followed by capric (20.74%), oleic (15.1%) and myristic (10.82%) acids were the most abundant compounds in H. ercoides Ssp. Roberti. The striking differences in the proportion of individual fatty acids were also reflected in the ratio saturated to unsaturated fatty acids (1.54, 0.91, 1.27 and 0.84 for H. perforatum, H. perfoliatum, H. tomentosum and H. ericoides Ssp. Roberti, respectively).

To understand the relationships between the studied Hypericum species with respect to their fatty acid composition, a principal component analysis (PCA) was carried out. As shown in Fig. 2, along the principal component 1 (PC1) which accounts for 62% of total variance, all species were positively related. Along PC2 axis, accounting for a further 25.65% of the total variance, H. perforatum and H. tomentosum were negatively related to H. perfoliatum and H. ericoides. Accordingly, the PCA distinguished two main groups; the first group includes H. perfoliatum and H. ericoides Ssp. Roberti (characterized by their relatively higher linoleic and capric acids) while the second group includes H. perforatum and H. tomentosum (characterized by their relatively higher palmitic and stearic acids). Grouping the samples observable from the PCA plot was in general agreement with the results of hierarchical cluster analysis HCA (Fig. 3).

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Figure 2 Principal component analysis of four Hypericum species on the basis of their fatty acid composition.

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Figure 3 Dendrogram of four Hypericum species obtained by cluster analysis using square Euclidean distance.

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To the best of our knowledge, the fatty acid composition of H. perfoliatum, H. tomentosum and H. ericoides Ssp. Roberti is reported herein for the first time. Nevertheless, the fatty acid composition of some Hypericum species has been previously reported. For example, palmitic, α-linolenic and oleic acids were reported as the prominent fatty acids in H. perforatum (Omarovam and Artamonovam, 1999). Alpha-linolenic and palmitic acids were reported as the major compound of Turkish Hypericum lysimachioides var. lysimachioides (Özen et al., 2004a). The latter authors have also analyzed the fatty acid profiles of H. perforatum and H. Hypericum retusum Aucher and found that palmitic acid and linolenic acid were the major components (Özen et al., 2004b). They also found large amounts of 3-hydroxy fatty acid mainly 3-hydroxytetradecanoic acid (3-OH-C14:0) and 3-hydroxyoctadecanoic acid (3-OH-C18:0) in their oil samples. Report from the same country had indicated that linolenic and linoleic acids were the most abundant fatty acids in Hypericum scabrum, while linolenic and palmitic acids were found as the most plentiful ones in Hypericum scabroides and Hypericum amblysepalum (Özen and Başhan, 2003). In the same year, Stojanovic et al. (2003) compared the fatty acid profile of H. perforatum, Hypericum maculatum and Hypericum olympicum and found that linoleic followed by palmitic and oleic acids were the most abundant fatty acids in H. maculatum and H. olympicum. In contrast, palmitic, oleic and lignoceric acids were found as the major fatty acids in H. perforatum. Linoleic, linolenic and palmitic acids were also reported as the main fatty acid in the lipidic fraction of Hypericum androseumum seeds and H. elatum root bark (Hargreaves et al., 1967). In our previous work on fatty acid composition of H. triquetrifolium, we have reported the abundance of α-linolenic, linoleic, aleic and palmitic acids. Moreover, we have successfully identified the stearidonc acid (C18:4) which is an unusual fatty acid in the oil of this species (Hosni et al., 2007). More recently, Shafagat (2011), has reported that omega-3 fatty acid followed by linoleic and palmitic acids were the main components of the hexane extracts of H. scabrum from Iran. Based on these earlier reports and the present study, it can be inferred that the fatty acid composition of Hypericum sp. varies depending on the species and the origin of plant.

3.2 Tocopherol content

The tocopherol contents of the investigated Hypericum species are presented in Fig. 4. Three isoforms, α-, γ- and δ-tocopherols were detected and identified in all samples in significantly (p < 0.05) varying degrees. Individual tocopherol contents exhibited great variations among the studied species. The isoform δ-tocopherol had the highest content followed by γ- and α-tocopherol. In terms of variations in different isoforms between species, H. ericoides had the highest content of tocopherols (22.32, 4.28 and 4.89 μg/g dw for δ-, γ- and α-tocopherol, respectively), while the lowest content of these isoforms was observed for H. tomentosum (2.69, 0.61 and 0.54 μg/g dw for δ-, γ- and α-tocopherol, respectively). H. perforatum and H. perfoliatum have showed intermediate values. The tocopherols are suggested to be synthesized by the action of γ-tocopherol methyl-transferase to either γ- or δ-tocopherol which can be further synthesized to α- and β-tocopherol, respectively (Collakova and DellaPenna, 2003). Our findings indicate that the biosynthesis of δ-tocopherol is a major activity in the aerial part of the studied Hypericum species. Published data related to the tocopherol content in Hypericum species are not available and comparison is not possible. However, the tocopherol constituents of some species of the closely related family Clusiaceae have previously been reported. In this context, Crane et al. (2005) have compared the tocopherol content of two Calophyllum species from Guadeloupe and found that α-tocopherol was the main isoform in the Calophyllum calaba, whereas, the isoform γ-tocopherol was the main one in Calophyllum inophyllum. Another report from Vietnam had revealed that α-tocopherol was the main isoform in the oil of C. inophyllum (Matthaus et al., 2003). In another Clusiaceae species, Caraipa densifolia, only α- and γ-tocopherols were detected in its hexane extract (da Silveira et al., 2010).

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Figure 4 Tocopherol content (μg/g dw) in four Hypericum species. Data are expressed as mean ± SD (n = 3).

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In general, data regarding the tocopherol content in different species were conflicting and variable depending on species, origin, plant part, plant physiology and environmental factors (Bruni et al., 2004; Symańska and Kruk, 2008). At this point, the latter authors have showed that α-tocopherol is the most abundant isoform in leaves with only some exceptions reported for lettuce, spinach, dodder shoots, or some seedling where γ-, δ-tocopherols were found at the highest amounts. They also reported that δ-tocopherol was the dominant isoform in Cuscuta epithymum and Cuscuta japonica (Symańska and Kruk, 2008). In Brassica napus and Brassica juncea, the tocopherol content was found to be deeply influenced by environmental factors namely the daily temperature and rainfall (Richards et al., 2008).

From a chemical perspective, it is well recognized that tocopherols interact with polyunsaturated acyl groups, protecting the lipids (especially polyunsaturated fatty acids) from oxidative damage by scavenging lipid peroxy radicals and quenching or chemically reacting with reactive oxygen species (DellaPenna and Pogson, 2006). Therefore, it seems logical to speculate that the higher content of tocopherols in H. ericoides might have such physiological role since it presents the higher content of polyunsaturated fatty acids. This species might be considered as a potential source of natural antioxidant due to its high δ-tocopherol content (Fazio et al., 2013). It is worthy to note that the present tocopherol composition might be useful for the taxonomical purposes for the infrageneric classification of the genus Hypericum.

In summary, the present contribution provided a new insight into the chemistry of H. perforatum, H. tomentosum, H. perfoliatum and H. ericoides Ssp. Roberti. It provides base line information on the fatty acid composition and tocopherol content in the aforementioned species.