Elsevier

Phytochemistry Letters

Volume 11, March 2015, Pages 418-423
Phytochemistry Letters

Antileishmanial activity of fucosterol recovered from Lessonia vadosa Searles (Lessoniaceae) by SFE, PSE and CPC

https://doi.org/10.1016/j.phytol.2014.12.019Get rights and content

Highlights

  • Fucosterol was recovered from Lessonia vadosa by SFE or PSE combined to CPC.

  • Fucosterol demonstrated promising antileishmanial activity.

  • No cytotoxicity against murine macrophages was noticed.

  • Scale-up is feasible with SFE and CPC for industrial application.

Abstract

Fucosterol, a triterpene derivative encountered in several alga species, provides a wide range of biological activities, such as protection against metabolic syndrome, or against UV-induced skin damage. We describe here the comparison of extraction by supercritical fluid (SFE) and pressurized solvent (PSE) of the brown alga Lessonia vadosa mainly abundant in the coastal water of Patagonia, followed by the isolation of fucosterol, using centrifugal partition chromatography (CPC), in association with HPLC–UV quantification. After collection, the seaweed was dried, ground and extracted either by PSE or by SFE under various conditions. The yield and the content in fucosterol of each extract were determined by HPLC–UV. Optimization of a biphasic solvent system and KD calculation led to the isolation of pure fucosterol with high recovery rate. Extraction by SFE using CO2 at 180 bar and 50 °C with 20 to 30 % of cellulose as modifier and CPC purification by cyclohexane/acetone/methanol/water 10/1/10/1 with lower layer as mobile phase led to the best results in terms of yield, purity, time and solvent consumption. Natural and semisynthetic steroid derivatives have been previously shown to be potential drug candidates against parasitic diseases including leishmaniasis. In this context fucosterol was evaluated and demonstrated noticeable antileishmanial activity (IC50 < 10 μM) against intracellular amastigotes with limited or no cytotoxicity in host cell macrophages. These results make this compound a valuable starting scaffold for pharmacomodulation. Adaptation of the procedure by slight modifications of the extraction and/or isolation conditions could permit the exploitation of other alga species as raw material. Since SFE and CPC are available for pilot and batch production, this work may serve as a model for further scale-up and industrial development.

Introduction

Seaweeds constitute a valuable source of bioactive compounds (Dhargalkar and Verlecar, 2009), polar such as fucoidans and alginates (Athukorala and Yuan, 2013, Chandia and Matsuhiro, 2008, Chandia et al., 2004), or apolar like fucoxanthine or sterols (Dambeck and Sandmann, 2014, Xiao et al., 2013). Indeed, as they generally present a less complex metabolic profile than plants, the extraction and recovery process may appear easier and more efficient.

Fucosterol is encountered in several algae species (Seung et al., 2012, Xiao et al., 2013). A wide range of biological activities has been reported for this natural product, especially on the fields of inflammation (Hwang et al., 2014a, Yoo et al., 2012) and protection against UV damages, metabolism (Hwang et al., 2014b, Kim et al., 2013), or cancer (Ji et al., 2014, Zhang et al., 2012).

Nevertheless, to our knowledge, this natural product has never been evaluated against trypanosomatid parasites, such as Leishmania, the aetiological agent of leishmaniasis. This pathology is transmitted by the bite of sandflies where the parasites in the promastigote form pass into the mammalian host and are phagocytosed by macrophages, where they turn into the non-motile proliferating amastigotes, in the phagolysosome (Chang and Fong, 1983). This disease is associated with high mortality and morbidity rates in developing countries, making the fight against this parasitic infection a priority in public health policy. Over 350 million people are at risk of infection and about two million new cases are considered to occur every year in the endemic zones of Latin America, Africa, the Indian subcontinent, the Middle East and the Mediterranean region. Currently there are no effective human vaccines for the prevention of this disease, whereas chemotherapy is often ineffective (Chatelain and Ioset, 2011, Croft et al., 2006). Thus, facing an urgent need to discover new drugs for the treatment of leishmaniasis, and as some natural and semisynthetic sterol derivatives (Pan et al., 2012, Ghosh et al., 2014, Guimaraes et al., 2009, Gros et al., 2006, Gigante et al., 2009) already demonstrated antileishmanial activity, fucosterol was targeted for screening against Leishmania strains.

The choice of the brown macroalga Lessonia vadosa Searles (Lessoniaceae, syn. L. flavicans Bory, L. fuscescens Bory; family Lessoniaceae) as starting material was supported by the presence in the genus Lessonia, distributed in the southern hemisphere (Chandia et al., 2004) of fucosterol as major component of the sterol fraction (Wächter et al., 2001). Moreover, this species is not endangered, abundant along Patagonia Argentina coast (Boraso, 2013) where it is currently collected for the production of alginic acid and other polysaccharides (Zambon et al., 2003).

All these considerations prompted us to develop a procedure of extraction and isolation following the green chemistry principles. Supercritical fluid extraction (SFE), pressurized solvent extraction (PSE) and centrifugal partition chromatography (CPC) are extraction and isolation techniques which require low amounts of solvents, and no polluting solid support such as silica. Thus, their environmental impact is considered as compatible with sustainable development criteria. Moreover, SFE and CPC are still available at industrial scale for batch production. Previous papers describe the obtainment of phytosterols from various natural sources using SFE, ultrasonic or microwave-assisted extraction and/or high-speed counter current chromatography (HSCCC) (Zhou et al., 2012, Nyam et al., 2010, Xiao et al., 2013, Seung et al., 2012, Schröder and Vetter, 2012). Our work depicts the first comparison of SFE versus PSE, in association with CPC.

Section snippets

Identification of fucosterol

Dried and ground aliquots (20 g) of L. vadosa were extracted by maceration in 200 mL of ethyl acetate (EtOAc) or cyclohexane (CHex) at room temperature under stirring for 1 h to furnish, respectively, 21 mg and 12 mg after concentration under reduced pressure. The extracts were analyzed by TLC (elution 97/3 CH2Cl2/MeOH v/v), GC–MS (Fig. 1) and HPLC–UV (Fig. 2). The presence of fucosterol as major compound was ascertained by comparison with references (TLC, HPLC–UV) or with database (GC–MS

Material and chemicals

L. vadosa Searles samples were collected on “La Tranquera” beach, Golfo San Jorge, Santa Cruz, Argentina (S 46° 01′ 54.86″ S, 67° 35′ 38.38″ W) in February 2007 in a 4–9 m depth. A voucher specimen is deposited at the Herbario Regional Patagónico, Facultad de Ciencias Naturales, UNPSJB, Argentina (HRP N° 7095). After drying, 1.6 kg of alga was ground using a table mill.

CHex, MeOH, EtOAc and acetone for extraction and CPC were purchased from Carlo Erba-SDS. CHex, EtOAc and acetone were distilled

Conclusion

Fucosterol was recovered from L. vadosa Searles, an abundant seaweed, using SFE or PSE coupled with CPC. The results of antileishmanial evaluation are quite promising, making this naturally occurring triterpene derivative a valuable starting scaffold for pharmacomodulation. Adaptation of the procedure by slight modifications of the extraction and/or isolation conditions could permit the exploitation of other alga species as raw material. Since SFE and CPC are available for pilot and batch

Conflict of interest statement

The authors have no conflict of interest to declare.

Acknowledgements

The authors thank the FP7-PEOPLE-2010-IRSES Marie Curie Actions, program “ChemBioFight” (exploring CHEMical BIOdiversity with innovative approaches for FIGHTing Chagas and leishmaniasis, grant number 269031) for financial support. Prof. A.-L. Skaltsounis and Dr. Maria Halabalaki are gratefully acknowledged for the management of the program. The authors thank the PICTO Golfo San Jorge PI 36871 for financial support and Dr. Alicia L. Boraso (UNPSJB) for the identification of the alga.

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    This paper is part of a special issue of selected presentations delivered at the 9th International Symposium on Chromatography of Natural Products, 26–29 May 2014, Lublin, Poland.

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