Sterols from the brown alga Cystoseira foeniculacea: Degradation of fucosterol into saringosterol epimers

1 Introduction

Brown seaweeds constitute an important part of the marine biomass. However, if the exploitation of hydrocolloids for the food industry is flourishing, the valorization of algal metabolites remains under-exploited. Indeed, more than 1300 chemical compounds have been isolated from brown algae and this chemical group represents more than 5% of all natural products described to date from marine sources (MarinLit, 2013). In this phylum, the phytochemical composition of species belonging to the Sargassaceae and Dictyotaceae families has been widely studied: more than 1100 secondary metabolites have been described from such brown algae. More particularly, among the species of Sargassaceae, a number of them are thus recognized for their ability to produce high amounts of terpenoids, especially acyclic diterpenoids and meroditerpenoids.

Cystoseira foeniculacea (Linnaeus) Greville is a marine brown alga (Fucales, Sargassaceae) which is distributed extensively, as most of the Cystoseira species, in the Mediterranean Sea and to a lesser extent, along the northwestern coasts of the Atlantic Ocean (Guiry and Guiry, 2013). According to the literature review, there had been no specific study conducted on the chemical composition of this species.

As part of our ongoing research on phytochemical investigation of brown algae (Bouzidi et al., 2008; El Hattab et al., 2008, 2009; Mokrini et al., 2008; Viano et al., 2011; Othmani et al., 2014), the present paper describes the chemical analysis of organic extracts obtained from Mediterranean samples of C. foeniculacea collected off the Algerian coasts. This work highlights the absence of terpenoids and allows the isolation and characterization of three known sterols, fucosterol (1) and saringosterols (2 and 3) (Fig. 1), for the first time from this species. In this study, a simple chromatographic method for the analysis of algal sterols by RP-C₈ HPLC is described. The oxidative degradation of fucosterol (1) into a C-24 epimeric mixture of saringosterols (2 and 3) is as well revealed.

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Figure 1

Chemical structures of compounds 1–3.

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2 Material and methods

3 Results

The extraction step allowed the obtaining of a lipidic extract from C. foeniculacea air-dried material in a yield of 1.7% (weight of extract/dry algal weight) which ranged in typical values found for algae of the genus Cystoseira (Valls et al., 1993). After partition of the crude extract, its organic part was fractionated by column chromatography. Then, each fraction was analyzed by ¹H NMR spectroscopy in order to carry out a large scale chemical screening and to determine the overall chemical composition of C. foeniculacea. Most of the fractions were found to be constituted by simple lipids, e.g., fatty acids and their derivatives, but one of them showed typical ¹H NMR signals of steroids. This fraction was further investigated by reversed-phase HPLC on a C₈ column (RP-C₈) in order to characterize its main chemical components. The chromatographic profile of this sterol fraction showed two main peaks which were separated and obtained in sufficient amounts, by repetitive chromatographic runs, for further NMR study.

The complete analysis of the NMR data of the two sub-fractions along with a comparison with literature data allowed their structure to be elucidated as fucosterol (1) (Hwang et al., 2012) and an epimeric mixture of saringosterols (2 and 3) (Ayyad et al., 2003; Catalan et al., 1983; Huh et al., 2012). The duplicate signals (C-17, C-20, C-22 to C-24, C-27 to C-29) in the ¹³C NMR spectrum of 2/3 were in accordance with the occurrence of the two C-24 epimers. Further analysis of 2D NMR data, in particular HMBC and ¹H–¹H COSY spectra allowed corrections of some attributions for the ¹³C NMR data of these compounds (Table 1) in comparison with those reported in the literature (Ayyad et al., 2003; Huh et al., 2012). Moreover, after its storage during 24 h at room temperature in an unsealed vial, a further NMR analysis of fucosterol (1) was performed and additional signals were then observed on its ¹H and ¹³C NMR spectra. A thorough examination revealed that these new signals were the same as those reported for saringosterols (Fig. 2). This sample was also analyzed by RP-C₈ HPLC under the same conditions as above; the chromatogram revealed the existence of two peaks eluted at retention times which were exactly the same as those of 1 and 2/3. These data therefore confirmed the spontaneous oxidative degradation of fucosterol (1) into saringosterols (2 and 3). Thus, saringosterols (2/3) isolated in this study from the crude extracts of C. foeniculacea could be considered as artifacts formed throughout the various stages of the process that led to the obtaining of pure compounds from these algal samples.

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Figure 2

Details of ¹H NMR spectra of: (a) fucosterol (1), (b) mixture of saringosterol C-24 epimers (2–3), and (c) naturally oxidized fucosterol.

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4 Discussion

From a chemotaxonomic aspect, this work states that, as several other Cystoseira species (e.g., Cystoseira humilis and Cystoseira compressa), C. foeniculacea could be classified into a “chemical group” holding none lipophilic secondary metabolites (e.g., diterpenoids or meroditerpenoids) (Amico, 1995; Jégou et al., 2010; Valls and Piovetti, 1995). These results were in accordance with a recent study which described Cystoseira as a polyphyletic genus where all these terpenoid non-producing species were gathered into the same clade (Draisma et al., 2010).

Concerning the chromatographic method developed here for the analysis of the sterol fraction of C. foeniculacea, it may be pointed out that algal sterols, and more generally plant sterols, could be detected and quantified through a variety of analytical techniques (Abidi, 2001). The final isolation of sterols from algal lipophilic extracts was mainly conducted by RP-C₁₈ HPLC (Hwang et al., 2012; Terasaki et al., 2009). In this work, a chromatographic method based on the use of a stationary phase with a smaller hydrophobic selectivity (RP-C₈ instead of RP-C₁₈) was developed: it allowed a quick and easy separation of the main steroidal components from algal extracts. In the case of sterols with few polar groups, which were highly retained on a C₁₈ stationary phase, this method led to a similar separation with a significant economy of time and mobile phase.

Finally, with such a methodology it was then possible to characterize easily the main components of the sterol fraction of C. foeniculacea by NMR as fucosterol (1) and saringosterols (2 and 3). Fucosterol occurs as the predominant sterol in brown algae (Terasaki et al., 2009) and this characteristic component displays a wide variety of biological activities (Balboa et al., 2013). In Sargassaceae, fucosterol has been described in several species, particularly, in some representatives of the genus Cystoseira, such as C. usneoides, C. nodicaulis, C. tamariscifolia (Lopes et al., 2011), C. hakodatensis (Abidi, 2001), C. amentacea var. stricta (Piovetti et al., 1991), C. barbata and C. crinita (Milkova et al., 1997).

Saringosterols were noted in a number of Phaeophyta (Ikekawa et al., 1966; Knights, 1970; Liu et al., 2009; Okano et al., 1985; Wachter et al., 2001) and in rare cases, they were described in both red and green algae (Huang et al., 2007; Shi et al., 2011). Saringosterols have been previously shown to exhibit wide-ranging biological activities (Chen et al., 2014; Hoet et al., 2007; Huh et al., 2012; Kim et al., 2014; Wachter et al., 2001).

Fucosterol is highly reactive and its degradation was already mentioned. Thereby, it has been shown that the presence of saringosterols in the brown algae Ascophyllum nodosum (Knights, 1970) or Cystoseira crinita (Milkova et al., 1997) was in fact an artefact arising from the auto-oxidation during the air-drying of the alga, while, the fresh material would contain only fucosterol. Thus, chemical analysis of fucosterol, and more generally of sterol fractions and/or crude extracts containing fucosterol, might absolutely take into account the chemical conversions of this compound, in particular through degradation process, they necessarily lead to biased results otherwise.

5 Conclusion

The present study investigated the separation and the characterization of fucosterol (1) and a mixture of 24R-(2) and 24S-(3) saringosterols from the brown alga C. foeniculacea by using a simple and easy method based on RP-C₈ chromatography. The analysis by 1D and 2D NMR experiments allowed the reassignment of some attributions of chemical shift of the isolated sterols 2 and 3 by comparison with those reported in the literature. We have also confirmed the oxidative degradation by HPLC analysis of a degraded sample of fucosterol. Indeed, the NMR analysis of pure 1 stored for few times revealed the presence of new signals corresponding to those of the 2/3 mixture. Importantly, we highlighted in this work the rapid oxidation of fucosterol (1) into saringosterols (2 and 3).