Hesperidin and hesperetin membrane interaction: Understanding the role of 7-O-glycoside moiety in flavonoids

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Abstract

Citrus species contain various typical flavonoids. However, absorption and metabolism of flavonoids are complex processes that determine its bioavailability which remain not clear until now.

The aim of this study was to investigate the interactions among dimyristoyl-phosphatidyl choline (DMPC) liposomes and the flavanones hesperidin (glycoside) and hesperetin (aglycone). The results describe the molecular details of these interactions and the consequences for the membranes properties, by using differential scanning calorimetry (DSC), atomic force microscopy (AFM), fluorescence (using MC540 as probe), X-ray diffraction and theoretical study.

The results show that hesperetin interacts with membranes stronger than hesperidin. It is possible to hypostatize that hesperidin, due to its rutinoside moiety, is located at the level of polar head whereas hesperetin interacts better with acyl chains and adopts a more planar conformation. The findings of this work may contribute to explain the high bioavailability of aglycones due to better membrane interaction.

Introduction

Citrus fruits, such as orange (Citrus sinensis), grapefruit (Citrus paradisi) and tangerine (Citrus reticulata) are widely consumed around the world. Citrus products have recently received attention because of their potential therapeutic benefits associated with high content of flavonoids, reporting to have antiallergic [1], antioxidant [2], anticancer and antinflammatory effects [3], as well as prevention of bone loss [4].

Citrus species contain various typical flavonoids such as flavanones, flavanone glycosides, and polymethoxyflavones, which occur rarely in other plants [5], [6]. The predominant flavanone glycoside in grapefruit is naringin and in oranges is hesperidin. Citrus fruits contain only a very low amount of hesperetin (hesperidin aglycone), and most of the flavanones exist as glycosides [6].

Nowadays, there is much controversy as to whether natural flavonoid glycosides can be absorbed by the gastrointestinal tract, or whether they are hydrolyzed in the small intestine prior to absorption. Thus, absorption and metabolism of flavonoids are complex processes that determine its bioavailability which remain not clear until now [7]. A number of early studies hypothesized that flavonoids would not enter the circulation, either as the natural glycosides or as the aglycone hydrolysis products, but as phenolic acid fission products after enteric bacteria action [8]. However, further studies have detected quercetin-3-rhamnoglucoside (rutin) in the circulation after the consumption of apple or onion [9]. It also has been reported that, in human, isoflavone aglycones are transported into enterocytes more efficiently than their glucoside counterparts because of their moderate lipophilicity [10]. The same properties appear to govern the transport of quercetin and its glucosides [11]. It is recognized that flavonoid diglycosides like rutinosides and neohesperidosides, pass almost intact through the small intestine [12]. In contrast to glucosides, which can be hydrolyzed by glucosidases available throughout the intestinal tract, rutinosides diglycosides are hydrolyzed only in colon by rahmnosidases produced by enterobacteria [13].

Recently, tree works, almost simultaneously published, studied the absorption process of hesperetin and its rutinoside hesperidin in Caco-2 cell model. Serra et al. reported that permeation was not detected for the glycosides in either the apical to basolateral or basolateral to apical directions confirming the need for metabolism before absorption through the intestinal membrane [14]. The glucose transporter, suggested for other flavonoids glycosides, was not confirmed for the compounds in this study. The aglycones permeated in both directions and glucuronide conjugates of them were also detected. On the other hand, Kobayashi et al. reported that hesperetin is efficiently absorbed from the intestine through proton-coupled and energy-dependent polarized transport Na+ independent, whereas hesperidin is poorly transported via the paracellular pathway and its transport would be highly dependent on conversion to hesperetin via the hydrolytic action of microflora [7], [15].

Indeed, a biophysical study in order to understand hesperidin/hesperetin membrane interaction has not been performed and it would give novel insight in the discussion. Serra et al. reported the use of phospholipids mimetic membrane (parallel artificial membrane permeability assay-PAMPA) to study absorption of flavonoids. The model shows results correlated with Caco-2 cell model. Artificial membranes are commonly used as a model for natural membranes to study drug–membrane interactions. In eucaryotic the major constituents of cell membranes are phosphatidylcholines. They are responsible for several features of the bilayer like stability and semi-permeable properties. The interactions of drugs with phospholipids can change the physicochemical properties of the membranes, which may be essential to understand the dynamics of passing through them.

The aim of this study was to investigate the interactions between dimyristoyl-phosphatidyl choline (DMPC1) liposomes and flavonoids hesperidin and hesperetin. The results describe the molecular details of these interactions and the consequences for the membranes properties, by using z potential measurement, atomic force microscopy (AFM), differential scanning calorimetry (DSC), fluorescence (using MC540 as a probe), X-ray and theoretical study.

Section snippets

Chemicals

Hesperidin and hesperetin were isolated from citrus peel as we reported recently [16]. Briefly, an ultrasound assisted extraction method allowed isolation of hesperidin from citrus peels (purity >99% by HPLC/MS). Hesperidin was submitted to acid hydrolysis to obtain hesperetin (purity >98% by HPLC/MS). Reference standards of flavonoids were purchased from CrhomaDex Inc. (Irvine, USA). DMPC (1,2-dimyristoyl-phosphatidylcholine) was purchased from Avanti Polar Lipids (Alabaster, AL, USA).

Flavonoid incorporation in liposomes and effect on z potential and size

It is well known and documented that incorporation of drug molecules in liposomes might significantly modify basic vesicle properties as surface charge [22], [23], [24], size [25], or membrane integrity [26].

In this study, the flavonoids HD and HT were incorporated in DMPC liposomes at different initial amounts of flavonoids. The percentage incorporation values are presented in Table 1. As expected from the relevant lipophilicity value of the HT (Fig. 1), incorporation in liposomes follows the

Discussion

Generally, flavonoids are absorbed after transport by passive diffusion and attachment to the lipid bilayer of the intestinal epithelial cell surface [10], [11]. In in vivo studies, orally administered flavonoids such as rutin, hesperidin, naringin and narirutin, which contain rutinoses or neohesperidoses, are absorbed only in the distal part of intestine, after hydrolysis by intestinal enzymes of colonic microflora [12], [33]. Izumi et al. [34] have reported that, in human, soy isoflavone

Acknowledgments

This work was supported by Grants from Science, Technology and Innovation Department – COLCIENCIAS, Colombia (project 111534319212) and Ministry of Agriculture and Rural Development, Colombia (project 2008L3788). J.L. has fellowship from COLCIENCIAS and CNPq (Brasil). Authors are grateful to André A. Pasa (Laboratório de Filmes Finos e Superfícies) and João C. De Lima (Laboratório de Preparação de Materiais Fora do Equilibrio por Mecanosíntese), from Physic Department at Universidade de Santa

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