Elsevier

Food Chemistry

Volume 221, 15 April 2017, Pages 959-968
Food Chemistry

A new exploration of licorice metabolome

https://doi.org/10.1016/j.foodchem.2016.11.068Get rights and content

Highlights

  • Profiles of flavonoids and saponins are different in Chinese and European licorice.

  • G. inflata revealed a chalconoid enriched metabolic profile.

  • Compounds found in Moraceae have been detected for the first time in G. glabra.

  • High number of significant compounds have been identified, proposing new markers.

Abstract

The roots and rhizomes of licorice plants (genus Glycyrrhiza L.) are commercially employed, after processing, in confectionery production or as sweetening and flavouring agents in the food, tobacco and beer industries. G. glabra, G. inflata and G. uralensis are the most significant licorice species, often indistinctly used for different productions. Licorice properties are directly related to its chemical composition, which determines the commercial values and the quality of the derived products. In order to better understand the characteristics and properties of each species, a chemical characterization of three species of licorice (G. glabra, G. inflata, G. uralensis) is proposed, through an untargeted metabolomic approach and using high-resolution mass spectrometry. The statistical analysis reveals new possible markers for the analyzed species, and provides a reliable identification of a high number of metabolites, contributing to the characterization of Glycyrrhiza metabolome.

Introduction

The dried roots and rhizomes of Glycyrrhiza species (Fabaceae family) are among the oldest and most frequently used herbal medicines in both Eastern and Western countries. The Glycyrrhiza genus contains about 30 species, widely distributed all over the world. Due to its sweet taste, licorice is also employed in the preparation of sweets and as a flavouring additive in the food and tobacco industries (Nassiri-asl & Hosseinzadeh, 2012). Various biological activities are associated with licorice extracts, such as anti-viral, anti-microbial, antioxidant, anti-inflammatory, anti-ulcer, anti-cancer and anti-HIV effects (Asl and Hosseinzadeh, 2008, Fiore et al., 2005). These activities are mainly related to two classes of compounds: triterpenic saponins and flavonoids. More than 50 triterpenic saponins have been identified in Glycyrrhiza (Zheng, Qi, Zhou, & Li, 2010), the most relevant of which is glycyrrhizin (1); 1 is an oleanane-type saponin which typically represents 5–10% of the roots and has been recognized as an efficient sweetening agent, that is 50 times sweeter than refined sugar (Isbrucker & Burdock, 2006). Among phenolics, the most abundant ones in licorice are typically liquiritigenin, isoliquiritigenin and their glycoside derivatives, which represent  1% of licorice aqueous extract. The content of bioactive principles in Glycyrrhiza varies considerably with the species, geographic origin, environmental conditions, harvesting and processing of the plant (Fiore et al., 2008). As changes in the chemical composition of a licorice plant are directly related to its quality and health effects, a comprehensive metabolite characterization of this plant is critical to ensuring the efficacy and safety of licorice roots in food and medicinal use. The three licorice species that are most widely used in commercial products are Glycyrrhiza glabra L. (typically cultivated in Europe, henceforth called European licorice), Glycyrrhiza uralensis Fisch. and Glycyrrhiza inflata Bat. (the two species are generally used in the traditional Chinese medicine and are known as Chinese licorice).

European licorice has a rich historical tradition, starting from the Scythians who discovered the pharmacological properties of its roots and taught them to the Greeks (Fiore, Calò, Ragazzi, Bielenberg, & Armanini, 2004). Nowadays, licorice (roots, dried extracts and derivatives) represents a typical and traditional Italian food product exported all over the world.

A number of studies have been published describing the different chemical composition of the Glycyrrhiza species. However, these studies generally focus only on a limited number of compounds (Farag et al., 2012, Liao et al., 2012). Indeed, the chemical composition of licorice has been studied by means of classical targeted analyses, especially in relation to traditional oriental medicine (Wang et al., 2011). Some recent studies have reported more extensive chemical characterizations, despite sometimes lacking in method standardization, identification criteria or biochemical evaluations. In this context, the untargeted metabolomic methodology allowing the simultaneous detection of hundreds of compounds without requiring any a priori knowledge of the identity of the detected metabolites could represent a powerful tool for the chemical investigation of licorice characteristics. The metabolomic approach has found applications in many fields, including natural product characterization, food quality assessment and traceability (Bernillon et al., 2013, Wahyuni et al., 2013), proving to be a fast and reliable method for the evaluation of metabolic differences between organisms and for the discovery of biological markers for species.

Nowadays, high-resolution mass spectrometry (HRMS) has become the mainstream technique in metabolomic analyses, due to its sensitivity, selectivity and accuracy, which permit the characterization of complex matrices. In this study, a high-throughput untargeted metabolomic analysis has been performed in order to assess the metabolic differences among the most widespread Glycyrrhiza species, namely G. glabra, G. uralensis and G. inflata. The HPLC (high performance liquid chromatography)-HRMS technique was employed to obtain a comprehensive characterization of the polar and semi-polar licorice metabolites, such as triterpenic saponins and flavonoids. Species-specific molecular markers were identified following a rigorous and standardized protocol, and significant differences among the species were highlighted by means of statistical methods. Our results offer a new insight into the biochemistry of licorice, contributing to the understanding of these widespread plants, and suggesting potential regulating mechanisms in the presence of species-specific compounds. The new molecular markers highlighted in this study could be useful for species identification, which is a priority problem for the traceability of licorice products considering the morphological similitude of the roots.

Section snippets

Sample preparation

The metabolite extraction procedure was based on the protocol described by De Vos et al., 2007. Ten different commercial samples of Glycyrrhiza dried roots were analyzed: 5 samples of G. glabra from Italy (Calabria region), 1 sample of G. glabra from Russia, 3 samples of G. uralensis and 1 sample of G. inflata from China. All of the commercial samples were made of more organisms. In particular, the G. inflata samples consisted of 1 kg of sliced dried roots. Thus, we can consider this sample as

Marker compounds and major metabolites

As an initial step for the investigation of the chemical composition of licorice roots, metabolites with the highest signal intensities (among the compounds that showed p < 0.01) for each species were selected and reported in Table 1.

The major metabolite of G. glabra and G. uralensis, is, as expected, glycyrrhizin (1), the most studied triterpenic saponin of licorice, characterized by a sweet taste and a range of biological activities. Glycyrrhizin showed a significantly higher signal intensity

Conclusions

In this study, an untargeted metabolomic analysis of three licorice species was performed, in order to identify the differences in the metabolic pattern of the plants.

Most of the identified compounds belong to the classes of flavonoids and saponins, which are known to have a large range of biological activity, as shown in previous studies. However, our metabolomic analysis elucidated the most important differences in the composition pattern of metabolites in the three analyzed species. By means

Conflict of interest statement

The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed

Acknowledgements

We thank Daniela Almansi for the English revision of the manuscript.

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