Phenylpropanoids and their metabolites are the major compounds responsible for blood-cell protection against oxidative stress after administration of Lippia citriodora in rats

Abstract

Lippia citriodora (lemon verbena) has been widely used in folk medicine for its pharmacological properties. Verbascoside, the most abundant compound in this plant, has protective effects associated mostly with its strong antioxidant activity. The purpose of this study was to test the effect of L. citriodora extract intake on the antioxidant response of blood cells and to correlate this response with the phenolic metabolites found in plasma. For this purpose, firstly the L. citriodora extract was characterized and its radical scavenging activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Then, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GRed) activities were determined in lymphocytes, erythrocytes, and neutrophils isolated from rats after acute intake of L. citriodora. Phenolic metabolites were analyzed in the same plasma samples by HPLC–ESI-TOF-MS. Myeloperoxidase (MPO) activity in neutrophils, which has been proposed as a marker for inflammatory vascular damage, was also determined. After L. citriodora administration, the antioxidant enzymes activities significantly accelerated (p < 0.05) while MPO activity subsided, indicating that the extract protects blood cells against oxidative damage and shows potential anti-inflammatory and antiatherogenic activities. The main compounds found in plasma were verbascoside and isoverbascoside at a concentration of 80 ± 10 and 57 ± 4 ng/ml, respectively. Five other metabolites derived from verbascoside and isoverbascoside were also found in plasma, namely hydroxytyrosol, caffeic acid, ferulic acid, ferulic acid glucuronide, and homoprotocatechuic acid, together with another eight phenolic compounds. Therefore, the phenylpropanoids verbascoside and isoverbascoside, as well as their metabolites, seem to be the responsible for the above-mentioned effects, although the post-transcriptional activation mechanism of blood-cell antioxidant enzymes by these compounds needs further investigation.

Introduction

Lippia citriodora (lemon verbena), a shrub indigenous to South America, was introduced into Europe at the end of the 17th century and has been widely used in infusions for its antispasmodic, antipyretic, sedative, and digestive properties (Carnat et al., 1999, Pascual et al., 2001, Valentao et al., 1999). L. citriodora leaves contain a large number of polar compounds such as phenylpropanoids, flavonoids, phenolic acids, and iridoid glycosides, verbascoside being the most abundant (Quirantes-Pine et al. 2009). Several properties have been described for this compound, such as anti-inflammatory (Deepak and Handa, 2000, Diaz et al., 2004), antimicrobial (Avila et al. 1999), and antitumor (Ohno et al. 2002) activity. These protective effects have been attributed, among other factors, to its antioxidant activity (Valentao et al., 2002, Wong et al., 2001).

Reactive oxygen species (ROS) have been associated with the mediation of several pathological processes, including inflammatory diseases, cancer, and atherosclerosis. Phenolic compounds can help to limit the oxidative damage caused by ROS either by acting directly on ROS or by stimulating endogenous defence systems. These defence systems include antioxidant enzymes, namely catalase (CAT), glutathione reductase (GRed), and glutathione peroxidase (GPx), which act as scavengers of the ROS. CAT catalyses the conversion of H₂O₂ to water, preventing the generation of hydroxyl radicals, GRed reduces glutathione disulfide to the sulfhydryl form, and GPx reduces lipid hydroperoxides to their corresponding alcohols and free hydrogen peroxide to water (Aymoto Hassimotto et al. 2008).

Other studies in humans have reported that the intake of L. citriodora extract promotes the protection of blood cells by activating GRed and CAT in erythrocytes and lymphocytes, and by decreasing sport-induced oxidative damage in neutrophils (Carrera-Quintanar et al., 2012, Funes et al., 2011). Nevertheless, these studies have not reported any metabolite derived from L. citriodora in the plasma of human volunteers. Therefore, there is a lack of knowledge about the effects of L. citriodora metabolites on the antioxidant defences of white and red blood cells.

The in vivo antioxidant activity of L. citriodora depends on its absorption and metabolism in the gut. Although the pharmacokinetics of verbascoside has been investigated (Funes et al., 2009, Wu et al., 2006), little is known about its metabolism as well as the bioavailability of the other compounds present in this plant.

Therefore, the aim of this study was to test the effect of L. citriodora extract intake on the antioxidant response of lymphocytes, erythrocytes, and neutrophils, and to correlate it with the phenolic metabolites found in plasma. In this way, the metabolites present in plasma samples and probably related to blood-cell protection activity against oxidative stress by L. citriodora were determined.