In vitro and in vivo transformations of Centaurium erythraea secoiridoid glucosides alternate their antioxidant and antimicrobial capacity

Highlights

• Enzymatic hydrolysis alternates phytochemical profile of Centaurium erythraea extract.

• Aglycones erythrocentaurin and gentiopicral predominated in the hydrolyzed extract.

• Extracts and pure secoiridoids displayed strong antimicrobial and antioxidant activity.

• Hydrolysis boosted swertiamarin and sweroside antioxidant potential.

• Secoiridoid glucosides were efficiently metabolized by Penicillium funiculosum.

Abstract

The present study was principally aimed at ascertaining the differences in metabolomics profiles and biological activities between non-hydrolyzed (ME) and hydrolyzed methanol extract (HME) of Centaurium erythraea Rafn. UHPLC–MS/MS Orbitrap analysis showed that the enzymatic hydrolysis of the extract caused changes in β-d-glycoside/aglycone ratio of both flavonoid and secoiridoid compounds. UHPLC/DAD/+HESI−qqqMS characterization and/or quantification of secoiridoid glucosides (SGs) and their aglycones in both ME and HME revealed gentiopicral and erythrocentaurin as the major aglycones, the same metabolic products which appear after the hydrolysis of pure swertiamarin, the dominant secoiridoid glucoside of C. erythraea. SGs played an antioxidant role only in ABTS assay, whilst the remarkable antioxidant potential of C. erythraea methanol extract is ascribed chiefly to phenolics detected in it. Interestingly, antioxidant activities of swertiamarin and sweroside recorded in ABTS assay increased after the compounds have been hydrolyzed, which highlighted their possible antioxidant role during ingestion. Strong antimicrobial activities of ME and HME against a vast array of pathogens, which exceed the effects of the reference antibiotics and antimycotics, largely depended on the amount of secoiridoids in either of the glycosylation forms. Extracts and pure secoiridoids were especially effective against most of the tested Penicillium species. On the other hand, P. funiculosum has evolved an efficient mechanism of detoxification of sub-lethal concentrations of secoiridoid glucosides, involving their biotransformation and complete digestion. The presented findings will contribute to clarify the fate and role of the SGs after C. erythraea ingestion within in vivo systems, and to further promote this remarkable plant as a food preservation additive with significant health benefits.

Introduction

Centaurium erythraea Rafn (Gentianaceae), commonly known as “centaury”, is a biennial herbaceous plant species widely distributed in Europe from Sweden to Mediterranean basin, and east to Asia, while naturalized in America and Australia (Marhold, 2011, U.S. National Plant Germplasm System, 2017). It is one of the most widely used bitter herbs, medicinally important due to the production of bioactive secondary metabolites. Pharmacological effects of centaury include anti-inflammatory and anti-pyretic (Berkan et al., 1991), hypoglycemic (Stefkov et al., 2014), antioxidant (Đorđević et al., 2017, Sefi et al., 2011, Šiler et al., 2014), antimicrobial (Šiler et al., 2014), hepatoprotective (Mroueh et al., 2004), gastroprotective (Tuluce et al., 2011), and many others, recently reviewed in Šiler and Mišić (2016). Many previous attempts have been conducted with the aim to introduce this plant into cultivation (Pataczek et al., 2017, Radušienė, 1995, Uzundzhalieva et al., 2013), and thus overcome the problem of its decreasing natural populations as the consequence of overexploitation and degradation of biological resources. This species is considered endangered in many European countries (e.g. Colling, 2005, Niklfeld and Schratt-Ehrendorfer, 1999, Węglarz et al., 2009). New concepts to alternatively propagate this plant and produce its bioactive compounds have been implemented, including cell and hairy root culture systems of C. erythraea, as well as of related species (e.g. Mišić et al., 2013, Piatczak et al., 2006, Subotić et al., 2009). Although some attempts to scale-up production of secoiridoids in bioreactors have been made (Mišić et al., 2013, Piatczak et al., 2006, Radović et al., 2013), a better understanding of elicitation mechanisms is still needed prior commercial production of these bioactive compounds on industrial scale.

Secoiridoid glucosides and phenolics (xanthones, flavonoids and phenolic acids) are reported to be the most abundant secondary metabolites in aerial parts of C. erythraea (Aberham et al., 2011, Banjanac et al., 2017). Among the secoiridoid glucosides (SGs) swertiamarin (SWM), gentiopicrin (GP) and sweroside (SW) are most commonly found. SGs are monoterpene compounds, with the basic hydrocarbon skeleton composed of two isoprene units (C10) conjugated with glucose. Their biosynthesis starts with geraniol, which is further through the series of oxidation, reduction, glycosylation, cyclization, methylation steps and a sequence of intermediates (8-hydroxygeraniol, 8-oxogeraniol, nepetalactol, iridotrial, 7-deoxyloganetic acid, 7-deoxyloganic acid, loganic acid, and loganin) converted to secologanin, the first secoiridoid in the pathway. Genes involved in this part of the biosynthetic pathway has been studied in Catharanthus roseus (Miettinen et al., 2014, O’Connor, 2012, Salim et al., 2014), Gentiana macrophylla (Hua et al., 2014), Rauwolfia serpentine (Ikeda et al., 1991), Olea europea (Alagna et al., 2016), and Lonicera japonica (Yamamoto et al., 2000). Biosynthetic branch leading from secologanin to SW, SWM, and GP is still unresolved, as well as are the steps involved in the catabolism of these compounds.

Iridoid glycosides, including secoiridoid glucosides, act as prodrugs, and their activities are induced when the compounds are activated by enzymes or by acid hydrolysis (Zeng et al., 2014). The β-glucosidases catalyze hydrolysis of the bond between iridoid and sugar moiety, thus converting less reactive glucosides into highly reactive aglycones (Pankoke et al., 2013). Some pharmacokinetic studies demonstrated that aglycone metabolites of some glucosides were more readily absorbed and more bioavailable in plasma; these glucosides are, after ingestion, metabolized in the gastrointestinal tract by the intestinal flora and/or endogenous β-glucosidases (Otieno and Shah, 2007, Setchell et al., 2001). Secoiridoid aglycones are relatively rarely identified within the Gentianaceae family (Parra et al., 1985, Zeng et al., 2014), probably due to high instability and limitations of the analytical methods applied. Although aglycones have not been previously prepared using either enzymatic or acid hydrolysis, biotransformation of SWM, GP, and SW by microorganisms (El-Sedawy et al., 1990, Jun et al., 2008, Zeng et al., 2014) has resulted in the production of several metabolites, including erythrocentaurin and gentiopicral, the common products of SWM and GP hydrolysis.

The study was basically aimed at ascertaining the differences in metabolomics profiles between non-hydrolyzed and hydrolyzed extract of Centaurium erythraea. Hydrolytic degradation of glycosylated compounds is a basic chemical reaction which is taking place after plant ingestion, and aglycones may differ from the corresponding glycosides in biological activity. Therefore, our next aim was to find if the non-hydrolyzed and hydrolyzed extracts demonstrate differences in antioxidant and antimicrobial activities and to figure out which of the profiled compounds may be mostly responsible for such extract’s behavior. We were also interested to discover the fate of the secoiridoid compounds that some microorganisms readily digest.