Microwave-Assisted Extraction and HPLC-UV-CD Determination of (S)-usnic Acid in Cladonia foliacea.

Valeria Cavalloro,Rita Stabile,Daniela Rossi,Pasquale Linciano,Silvia Assini,Giorgio Marrubini,Emanuela Martino,Simona Collina,Gabriele Gheza

doi:10.3390/molecules26020455

Valeria Cavalloro, Rita Stabile + Show 7 more

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https://doi.org/10.3390/molecules26020455

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Abstract

During the years, many usnic acid (UA) conjugates have been synthesized to obtain potent endowed with biological properties. Since (S)-UA is less abundant in nature than (R)-enantiomer, it is difficult to source, thus precluding a deeper investigation. Among the lichens producing UA, Cladonia foliacea is a valuable (S)-UA source. In the present work, we report on a rapid HPLC-UV/PAD-CD protocol suitable for the analysis and the identification of the main secondary metabolites present in C. foliacea extract. Best results were achieved using XBridge Phenyl column and acetonitrile and water, which were both added with formic acid as mobile phase in gradient elution. By combining analytical, spectroscopical, and chiroptical analysis, the most abundant analyte was unambiguously identified as (S)-UA. Accordingly, a versatile microwave-assisted extractive (MAE) protocol, assisted by a design of experiment (DoE), to quantitatively recover (S)-UA was set up. The best result in terms of UA extraction yield was obtained using ethanol and heating at 80 °C under microwave irradiation for 5 min. Starting from 100 g of dried C. foliacea, 420 mg of (S)-UA were achieved. Thus, our extraction method resulted in a suitable protocol to produce (S)-UA from C. foliacea for biological and pharmaceutical investigation or commercial purposes.

Highlights

Metabolites produced by lichens are still poorly investigated, these organisms are commonly used in folk medicine to treat pathological conditions [1,2,3,4,5,6,7,8,9]
Cladonia species ically host this andIn other the present work, we report on a rapid High-Performance Liquid ChromatogChromatography (HPLC)-UV/PAD-CD analytical methodology suitable for the analysis and the identification of the main secondary metabolites present in C. foliacea crude extract
By combining analytical (HPLC), spectroscopical, and chiroptical analysis (ECD and optical rotation value) and by comparison with the corresponding commercially available (R)-enantiomer used as standard, the usnic acid (UA) produced by the C. foliacea was unambiguously identified as (S)-UA with an optical purity > 99%

Summary

Introduction

Metabolites produced by lichens are still poorly investigated, these organisms are commonly used in folk medicine to treat pathological conditions [1,2,3,4,5,6,7,8,9]. UA is a dibenzofuran derivative, and it is characterized by the presence of a stereogenic center (Figure 1). Both enantiomers occur in nature, depending on the producing organism. (S)-UA was isolated as an exclusive enantiomer in few species of Cladonia (C. uncialis and C. foliacea) and Alectoria (A. lata and A. ochroleuca) [10]. In another few species of lichens, UA was detected as a mixture of (R)- and (S)-isomers in different ratios [10]

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