Correlation between plasma antioxidant capacity and verbascoside levels in rats after oral administration of lemon verbena extract

Food Chemistry 117 (2009) 589–598

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Correlation between plasma antioxidant capacity and verbascoside levels in rats after oral administration of lemon verbena extract L. Funes a,*, S. Fernández-Arroyo a, O. Laporta a, A. Pons b, E. Roche c, A. Segura-Carretero d, A. Fernández-Gutiérrez d, V. Micol a,* a

Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202-Elche, Alicante, Spain Biologia Fonamental i Ciéncies de la Salut, University of the Balearic Islands, Palma de Mallorca, Spain c Instituto de Bioingeniería, Departamento de Biología Aplicada-Nutrición, Universidad Miguel Hernández, 03202-Elche, Alicante, Spain d Departamento de Química Analítica, Facultad de Ciencias, Universidad de Granada, Granada, Spain b

a r t i c l e

i n f o

Article history: Received 28 January 2009 Received in revised form 1 April 2009 Accepted 14 April 2009

Keywords: Verbascoside Antioxidant capacity TEAC ORAC MDA HPLC-MS/MS SOD FRAP Pharmacokinetics Malondialdehyde Biological membranes

a b s t r a c t Phenylpropanoids are the main class of compounds from lemon verbena which have shown a wide biological activity, verbascoside being the most abundant one. In this work, the composition of a lemon verbena extract was elucidated by HPLC-ESI-MS/MS and one flavone and one methoxylated phenylpropanoid were found in this source for the first time. The antioxidant activity of the lemon verbena extract was fully characterised by several methodologies. Unexpectedly, the extract was especially active in lipophilic environments and lipid peroxidation inhibition assay, as it was found for pure verbascoside. The lemon verbena extract, containing verbascoside as its major bioactive compound, was acutely administered to rats and verbascoside was the only metabolite detected in plasma samples as measured by HPLC mass spectrometry. The correlation between the highest verbascoside concentration in plasma and maximum plasma antioxidant capacity was observed at 20 min as measured by different techniques, i.e. minimum malondialdehyde (MDA) generation, highest ferric-reducing ability of plasma (FRAP value) and maximum superoxide dismutase activity (SOD). Therefore, the in vitro measurements of the antioxidant activity of lemon verbena extract may significantly support the antioxidant activity observed in vivo in this work. Moreover, neither evidence of acute oral toxicity nor adverse effects were observed in mice when the lemon verbena extract containing 25% verbascoside was used at a dosage of 2000 mg/kg. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Lemon verbena (Aloysia triphylla or Lippia citriodora) is an herb mainly used as a spice and a medicinal plant. It grows spontaneously in South America and is cultivated in North Africa and South-

Abbreviations: AAPH, 2,20 -azobis (2-methyl-propionamine) dihydrochloride; ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt; ANOVA, analysis of variance; AUC, area under curve; BHT, butylated hydroxytoluene; BPC, base peak chromatogram; DAD, diode-array detector; EIC, extracted-ion chromatogram; ESI, electrospray ionisation; EtOH, ethanol; EYPC, egg yolk phosphatidylcholine; FL, fluorescein; FRAP, ferric-reducing ability of plasma; HPLC–MS, high-performance liquid chromatography–mass spectrometry; LD50, lethal dose 50%; LOD, limit of detection; LOQ, limit of quantification; MDA, malondialdehyde; ORAC, oxygen radical absorbance capacity; SOD, superoxide dismutase; TBA, thiobarbituric acid; TCA, trichloroacetic acid; TE, Trolox equivalents; TEAC, Trolox equivalent antioxidant capacity; TEP, 1,1,3,3-tetraethoxypropane; TPTZ, 2,4,6,tripyridyl-S-triazine; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; UV, ultraviolet–visible wavelength. * Corresponding authors. Address: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avda. de la Universidad s/n. 03202-Elche, Alicante, Spain. Tel.: +34 96 6658430; fax: +34 96 6658758. E-mail address: [email protected] (V. Micol). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.04.059

ern Europe. Lemon verbena leaves are used to add a lemony flavour in many culinary purposes such as fish and poultry dishes, vegetable marinades, salad dressings, jams, puddings, and beverages. It is also used to make herbal teas and refreshing sorbets. Therefore, lemon verbena products and their compounds can be considered into the food category. The leaves of these species are reported to possess digestive, antispasmodic, antipyretic, sedative and stomachic properties. It has traditionally been used in infusions for the treatment of asthma, cold, fever, flatulence, colic, diarrhoea and indigestion (Newall, Anderson, & Phillipson, 1996). Phenylpropanoids represent the main class of compounds of this plant as verbascoside being the most abundant one (Fig. 1) (Bilia, Giomi, Innocenti, Gallori, & Vincieri, 2008; Laporta et al., 2004). Several properties have been described for this compound like anti-inflammatory (Deepak & Handa, 2000; Diaz et al., 2004; Hausmann et al., 2007), antimicrobial (Avila et al., 1999) and antitumor (Ohno, Inoue, Ogihara, & Saracoglu, 2002). Among several factors, verbascoside antioxidant activity may suppose a significant contribution to its protective effects (Liu et al., 2003; Siciliano et al., 2005; Valentao et al., 2002; Wong, He, Huang, & Chen, 2001).

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L. Funes et al. / Food Chemistry 117 (2009) 589–598 OH

OH

HO OH

OH

HO

(1) Luteolin-7-diglucuronide

HO

HO

O

COOH

O

OH

O

O

O

OH O

COOH

OH HO OCH3

COOH

O

OH OH

O OH

O

O

O

O

OH

(2) Apigenin-7-diglucuronide (Clerodendrin)

_ R2O CH2

OH

O

OH

COOH

O

OH

HO

OH

(3) Chrysoeriol-7-diglucuronide

HO O

R1O

COOH

O

H3C

OH

O

O

O

O

(4) Verbascoside: R1= caffeic acid; R2= H (6) Isoverbascoside: R1= H; R2= caffeic acid

O OH O

OH

HO OH

COOH

OH

(7) Eukovoside: R1= ferulic acid; R2= H

_ HO CH2

O

O

OH

O HO O H3C

O

HO

OCH3

O

H3CO

(9) Martynoside HO

OH OH

Fig. 1. Chemical structures of the major compounds found in lemon verbena leaves extract.

Due to the presence of lemon verbena in food products, it may be necessary to know its pharmacokinetics and its possible antioxidant effects in vivo. So far, most of the previous results on lemon verbena derive from in vitro assays. Till now, detailed information about the pharmacokinetics of verbascoside is very limited and derives from plants which are different from lemon verbena (Wu, Lin, Sung, & Tsai, 2006; Wu, Tsai, Lin, & Tsai, 2007). Only one report has shown pharmacokinetics of this compound after oral administration and its bioavailability was found to be very low (Wu et al., 2006). In this work, the composition and in vitro antioxidant activity of an extract from lemon verbena standardised in 25% verbascoside (w/w) was determined. Moreover the pharmacokinetics of this compound in rats after oral administration of the above mentioned extract was studied. Significant amounts of verbascoside were found in plasma of rats which was readily available 20 min after oral administration. In addition, a correlation of the plasma antioxidant status in rats, as measured by different antioxidant methods, and the concentration of verbascoside is shown for the first time. Finally the toxicity of the extract in mice was also studied.

(TCA); thiobarbituric acid (TBA); 1,1,3,3-tetraethoxypropane (TEP); 2,4,6, tripyridyl-S-triazine (TPTZ); ferric chloride (FeCl3); Trolox (6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid); 2,20 azino-bis(3-ethylbenzothiazoline-6 sulphonic acid) (ABTS); 2, 20 -azobis (2-methyl-propionamine) dihydrochloride (AAPH); fluorescein (FL), xanthine; xanthine oxidase and cytochrome c were purchased from Sigma–Aldrich Corp. (St. Louis, MO, USA). Natural lipid egg yolk phosphatidylcholine (EYPC) was obtained from Avanti Polar Lipids (Birmingham, AL, USA), dissolved in chloroform/methanol (1:1, v/v) and stored at 20 °C. Lemon verbena extract (25% verbascoside, w/w), Citrus aurantium peel extract (25% naringin, 25% neohesperidin, w/w) and olive leaf extract (25% oleuropein, w/w) were kindly provided by Monteloeder, S.L. (Elche, Spain). All other compounds were of analytical, spectroscopic or chromatographic reagent grade and were obtained from Merck KGaA (Darmstadt, Germany). Double-distilled and deionized water was used throughout this work.

2.2. Animals 2. Materials and methods 2.1. Reagent and chemicals Sulphatase and glucuronidase (EC 232-772-1) from Helix pomatia type H-1, butylated hydroxytoluene (BHT); trichloroacetic acid

Twenty-eight male Wistar rats (280–380 g) were housed in standard cages at room temperature with free access to food and water for two weeks. Throughout the experiments, animals were processed according to the suggested ethical guidelines for the care of laboratory animals (Morton et al., 2001).

L. Funes et al. / Food Chemistry 117 (2009) 589–598

2.3. Treatment of animals Rats were orally treated with lemon verbena extract (2180 mg/ kg, corresponding to 545 mg/kg of verbascoside) via gastric gavage. For the administration, the extract was suspended in saline serum (3 mL). The control group received only saline serum. Rats were subjected to ketamine/xylazine anesthesia and the blood samples were withdrawn via cardiac puncture into heparinized tubes at 20, 40, 55, 75, and 100 min post dosing. All blood samples were centrifuged at 1000g for 15 min at 4 °C, and then plasma was stored at 80 °C. 2.4. Measurement of the Trolox equivalent antioxidant capacity (TEAC) The Trolox equivalent antioxidant capacity (TEAC) assay, which measures the reduction of the radical cation of ABTS by antioxidants, was performed as previously described (Laporta, Pérez-Fons, Mallavia, Caturla, & Micol, 2007). Compounds were assayed at five different concentrations determined within the linear range of the dose–response curve. A calibration curve was prepared with different concentrations of Trolox (0–20 lM). Results were expressed in mM of Trolox per mM pure compound or g of extract. 2.5. Radical-scavenging capacity by measurement of inhibition of malondialdehyde (MDA) generation The quantitative evaluation of the antioxidant capacity of the compounds/extracts against lipid peroxidation was determined through the measurement of the inhibition of MDA generation by using small unilamellar vesicles formed by sonication of multilamellar vesicles of egg yolk phosphatidylcholine (EYPC). Oxidation conditions, extraction and quantitation of TBA–MDA chromogen were performed as previously described (Laporta et al., 2007). Briefly, the chromogen was determined using HPLC coupled to fluorescence detection. The analysis were conducted by injecting 20 lL of sample into a reverse phase column LiChrospherÒ 100 RP-18 (5 lm, 250 x 4 mm i.d.) from Merck KGaA (Darmstadt, Germany) using isocratic mode with methanol-50 mM potassium phosphate buffer, pH 6.8 (40:60, v/v), and a flow rate of 1 mL/ min. The TBA–MDA product was monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Results were expressed in mg/L for extracts or lM of pure compound which were able to inhibit 50% of the generation of MDA (nmol MDA/mg EYPC). 2.6. Assay of the oxygen radical absorbance capacity (ORAC) To assay the capacity of the compounds/extracts to scavenge peroxyl radicals a validated ORAC method, which uses fluorescein (FL) as the fluorescent probe (ORACFL), was utilised (Ou, HampschWoodill, & Prior, 2001) with minor modifications (Laporta et al., 2007). ORAC values were expressed as micromole Trolox equivalents per gram of antioxidant substance. 2.7. Enzymatic hydrolysis and determination of verbascoside in plasma Plasma (50 lL) was mixed with 50 lL of enzyme mixture in 100 mM acetate buffer (pH 5.0) containing 12.5 units of sulphatase and 270 units of b-glucuronidase activity. The mixture was incubated at 37 °C for 45 min. Soon after 200 lL of 0.83 M acetic acid in methanol were added. The mixture was vortexed, sonicated, and centrifuged (7400g for 5 min at 4 °C), and the supernatant was analysed by HPLC-DAD-MS/MS system. The LC/MS system consisted of an Agilent LC 1100 series (Agilent Technologies Inc., Palo Alto, CA, USA) controlled by the Chemstation software. The HPLC instrument was coupled to an Esquire

591

3000 + (Bruker Daltonics, GmbH, Germany) mass spectrometer equipped with an ESI source and ion trap mass analyser, and controlled by Esquire control and data analysis software. A Merck LiChrospher 100 RP-18, 5 lm, 250  4 mm (i.d.) column was used for analytical purposes. For the analysis of verbascoside, a 20 min linear gradient from 5% to 30% acetonitrile in 1% formic acid in water was used. For the accurate performance of the LC–MS pump, 10% of organic solvent was premixed in the water phase. The flow rate was 0.5 mL/min (Maatta, Kamal-Eldin, & Torronen, 2003). Diode-array detection was set at 280 and 330 nm. Mass spectrometry operating conditions were optimised in order to achieve maximum sensitivity values. The ESI source was operated in negative mode to generate [MH] ions using the following conditions: desolvation temperature at 350 °C and vapourizer temperature at 400 °C; dry gas (nitrogen) and nebulizer were set at 5 L min1 and 65 psi, respectively. The MS data were acquired as full scan mass spectra at 150–1000 m/z by using 50 ms for collection of the ions in the trap. 2.8. Peaks identification and validation of the verbascoside assay method Identification of all constituents was performed by HPLC-DADand –MS/MS analysis, comparing the retention time, UV and MS spectra of the peaks in the samples with those authentic reference samples or data reported in the literature. The purity of peaks was checked comparing the UV spectra of each peak with those of authentic reference samples and by examination of the MS and MS/MS spectra. The accuracy of the method for the determination of verbascoside in plasma was further assessed with recovery studies by spiking verbascoside into blank plasma and water in triplicates. The linearity range of the responses was determined on eight concentration levels with three injections for each level. Calibration graphs for HPLC were recorded with sample amount ranging from 0.25 lg/mL to 0.25 mg/mL (r2 > 0.9999). Quantitative evaluation of verbascoside was performed by means of a six-point regression curve (r2 > 0.996) in a concentration range between 0.25 lg/mL and 0.1 mg/mL, using verbascoside as reference external standard and evaluated at 330 nm. LOD (Limit of Detection) was 0.10 lg/ mL and Limit of Quantification (LOQ) was 0.25 lg/mL. 2.9. HPLC analysis of plasma MDA Briefly, 50 lL of plasma were mixed with 50 lL of 0.05% BHT in ethanol and 50 lL of TCA 20% in HCl 0.6 M. The samples were incubated 15 min on ice and then were centrifuged at 5000g during 15 min at 4 °C. Then 100 lL of TBA 0.6% in water were added to 100 lL of supernatant. At follow, the mixture was incubated at 97 °C for 1 h, let to cool down and extracted with 300 lL of n-butanol through vigorous shaking, and then samples were centrifuged at 10000g for 3 min. The TBA–MDA chromogen was determined using an HPLC and fluorescence detection system as previously described in paragraph 2.5 of this section. 2.10. Ferric-reducing ability of plasma To assess plasma antioxidant capacity, the ferric-reducing ability of plasma (FRAP) was measured as previously described (Benzie & Strain, 1996). Briefly, 40 lL of diluted plasma samples (1:2) were mixed on a 96-well plate with 250 lL of freshly prepared FRAP reagent. Samples were incubated for 10 min at 37 °C and then absorbance at 593 nm was recorded during 4 min on a microplate reader (SPECTROstar Omega, BMG LabTech GmbH, Offenburg, Germany). FRAP values were calculated using FeSO47H2O as standard.

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2.11. Superoxide dismutase (SOD) activity in plasma SOD activity was measured through an adaptation of the method of McCord and Fridovich (1969). The xanthine/xanthine oxidase system was used to generate the superoxide anion. This anion produced the reduction of cytochrome c, which was monitored at 550 nm. The SOD activity in the sample removed the superoxide anion and produced an inhibition of the cytochrome c reduction. The activity was determined with a SPECTROstar Omega microplate reader at 37 °C. 2.12. Acute oral toxicity assessment Ten-week-old male and female ICR strain mice were used for the study. The animals were housed in cages in a temperature-controlled animal room (23 ± 1 °C) with a relative humidity of 55 ± 5% and were fed a standard diet. The animal care and handling was done according to the regulations of Council Directive 86/609/ EEC about Good Laboratory Practice (GLP) on animal experimentation. The animals were allowed to fast by withdrawing the food and water for 18 h and divided into four groups of eight individuals each (two groups for the control and two groups for the test substance) before the administration. Briefly, the powder (lemon verbena extract, 25% verbascoside) was dissolved in 0.9% NaCl sterile solution, pH 7.4 at a concentration of 80 mg/ml and centrifuged to eliminate insoluble particles. The test substance was administered by oral gavage in a total volume of 0.5 mL at a single dose of 2000 mg/kg. Weight control and observations were continued for 14 days. On day 14, the mice were anesthetized, killed by exsanguinations, and examined by necropsy. The assay was performed following the recommend regulations (OECD Test Guideline 420; Fixed Dose Procedure). 2.13. Statistical analysis Statistical analysis was performed using the statistical package SPSS 13.0 for Windows. The effects of lemon verbena extract consumption on the investigated parameters were evaluated by comparing the values obtained at different time intervals with the control (0 time) using one way ANOVA test. Depending on the results, significance values of p < 0.01, p < 0.05 and p < 0.10 were considered statistically significant.

3. Results and discussion 3.1. Composition of lemon verbena extract by HPLC–MS/MS The composition of the lemon verbena extract utilised in this work was determined by means of HPLC-DAD-ESI-MS/MS. The identification of the peaks was based on the analysis of their retention time, UV spectra and MS/MS data as mentioned in the Materials section. Fig. 2 shows the high-performance liquid chroma- tography profile at 330 nm of lemon verbena extract (Fig. 2A) and its corresponding base peak chromatogram (BPC) (Fig. 2B), which is indicative of peak purity detection in data generated with LC–MS. We were able to identify seven major phenolic compounds in the extract (see Fig. 1 and Table 1), namely four phenylpropanoids, and three glycosylated flavones. Nevertheless, no iridoid derivatives were identified in the extract although other authors have found them in lab-scale ethanolic extractions from lemon verbena leaves (Bilia et al., 2008). Among the phenylpropanoids, verbascoside was clearly the most abundant compound (around 25%, w/w). In contrast to previously reported (Bilia et al., 2008), its positional isomer, isoverbascoside, was found in very low amounts (Fig. 2). It has been reported that isoverbascoside can be obtained from the

deacetylation and caffeoyl migration of verbascoside under hydrolytic environment, so it might derive from drastic extraction conditions (Kawada, Asano, Makino, & Sakuno, 2002). The methoxylated phenylpropanoid eukovoside was also present in the lemon verbena extract, as previously reported (Bilia et al., 2008; Shuya, Shengda, Xingguo, & Zhide, 2004; Sticher, Salama, Chaudhuri, & Winkler, 1982). Martynoside, another methoxylated phenylpropanoid (Kirmizibekmez et al., 2005) was indentified for the first time in the lemon verbena extract. Three flavones were also identified in minor quantities in the lemon verbena extract (Fig. 1), all of them in their diglucuronide form, luteolin-7-diglucuronide (Carnat et al., 1995) and apigenin7-diglucuronide (Bilia et al., 2008; Ragone, Sella, Conforti, Volonte, & Consolini, 2007), both previously reported in ethanolic extracts of lemon verbena. Moreover, one methoxylated flavone, chrysoeriol-7-diglucuronide is reported for the first time in lemon verbena. Total mass data and fragmentation patterns of these flavones were identical to those ones reported in other plant sources (Wang et al., 2008). 3.2. Antioxidant activity of lemon verbena extract The antioxidant activity of lemon verbena extract was fully characterised in vitro by using several antioxidant measurements. First, the antioxidant activity by means of the Trolox equivalent antioxidant capacity (TEAC) assay was studied and compared to other antioxidant extracts and pure compounds. Quercetin, one of the most potent compounds under this type of assay, was used as a reference, which showed TEAC values that agreed to those found by other authors (Miller & Begoña Ruiz-Larrea, 2002; RiceEvans, Miller, & Paganga, 1996; Soobrattee, Neergheen, LuximonRamma, Aruoma, & Bahorun, 2005) (Table 2). Lipophilic TEAC value obtained for lemon verbena extract was considerably high, i.e., 1.15 ± 0.07 (mM Trolox/g). Pure verbascoside showed a lipophilic TEAC value of 1.28 ± 0.11 mM Trolox/mM (i.e., 1.28 mmol Trolox/ mmol or 2.05 mmol Trolox/g), which was consistent with the value previously found by other authors (Siciliano et al., 2005). In spite of the extract only contained 25% verbascoside and minor quantities of other phenylpropanoids and flavones (Fig. 2), TEAC value found for lemon verbena extract accounted for more than the half of the value determined for pure verbascoside per gram. The theoretical TEAC value of the extract due to verbascoside contribution would have been 0.51 mM Trolox/g but unexpectedly more than double of this value was obtained. From this result it could be postulated that other minor components of the extract such as the flavones could contribute to enhance the antioxidant capacity of the extract probably in a synergistic manner. Furthermore, lemon verbena extract showed stronger TEAC values than other strong antioxidant extracts such as olive leaf extract (25% oleuropein) or orange flavonoid extract (25% naringin, 25% neohesperidin). Surprisingly, lemon verbena extract showed a higher TEAC value in a lipophilic environment than in water, which has been shown for other hydrophilic and glycosylated phenolic antioxidants such as hypoxoside (Laporta et al., 2007). As it has been previously reported, this fact may be related to its stronger capacity to scavenge free radicals in a lipophilic environment. This behaviour was also observed for the orange flavonoid extract, which also contains minor quantities of flavones that could contribute to this antioxidant behaviour. In contrast, olive leaf extract showed similar TEAC value in a lipophilic environment (ethanol) than in a hydrophilic medium (water). In order to determine the contribution of the radical scavenging activity of the single compounds which are part of the verbascoside molecule, i.e., caffeic acid and hydroxytyrosol, TEAC and MDA inhibition values of these compounds were also evaluated in comparison to verbascoside. Lipophilic and hydrophilic TEAC and MDA

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Fig. 2. High performance liquid chromatography profile of lemon verbena extract (A) at 330 nm, and (B) base peak chromatogram of 50–800 m/z. Intensity scale was magnified to show the less abundant compounds.

Table 1 List of the main phenolic compounds in the lemon verbena extract identified by HPLC-DAD-ESI-MS/MS. Retention time (min)

Peak no. Lemon verbena extract

Compound

UVMax (nm)

Mol. ion [MH]

Primary fragment (MS/MS)

12.1 14.0 17.3 18.0 19.0 20.2 22.3 26.7 27.7

1 2 3 4 5 6 7 8 9

Luteolin-7-diglucuronide Apigenin-7-diglucuronide (Clerodendrin) Chrysoeriol-7-diglucuronide Verbascoside Not identified Iso-verbascoside Eukovoside Not identified Martynoside

348 340 340 330 350 330 330 340 330

637 621 651 623 566 623 637 711 651

351, 285 351 351 461 – 461 351 – 475

Table 2 Lipophilic and hydrophilic TEAC values and IC50 values for MDA generation inhibition of several extracts and pure compounds. Quercetin is shown as reference value. Extract or compound

Lemon verbena Olive leaf Orange flavonoid Verbascoside Caffeic acid Hydroxytyrosol Quercetin a b c d e f g

Antioxidant capacity TEAC mM (H2O)a

TEAC mM (EtOH)b

MDA inhibition (IC50c)

0.81 ± 0.04d 0.92 ± 0.12 0.51 ± 0.01 1.03 ± 0.15e 0.59 ± 0.03 0.66 ± 0.12 3.00 ± 0.30

1.15 ± 0.07 0.94 ± 0.11 0.71 ± 0.08 1.28 ± 0.11 0.73 ± 0.14 0.80 ± 0.10 2.26 ± 0.07

6.91 ± 0.21f 7.00 ± 1.20 15.6 ± 1.64 3.82 ± 0.5g 7.41 ± 1.10 15.42 ± 2.70 3.66 ± 0.20

mM Trolox equivalent antioxidant capacity determined in H2O (n = 3). mM Trolox equivalent antioxidant capacity determined in EtOH (n = 3). Concentration corresponding to the 50% inhibition of lipid peroxidation determined by HPLC–MDA measurement (n = 3). mM Trolox/g of extract. mM Trolox/mM of pure compound. mg/L of extract. lM of pure compound which were able to inhibit the 50% of the production of MDA (nmol MDA/mg lipid).

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inhibition values of hydroxytyrosol were in agreement to previously reported (Laporta et al., 2007). Among the tested pure compounds, verbascoside showed higher TEAC values than caffeic acid or hydroxytyrosol, but it was less potent than quercetin. Verbascoside exhibited the same behaviour shown before, i.e., a higher TEAC value in EtOH than in water, what was consistent with the behaviour observed for the lemon verbena extract. Caffeic acid and hydroxytyrosol also showed a similar behaviour, but it was less pronounced. This fact might reveal the efficacy of verbascoside to scavenge free radicals within a hydrophobic environment. In contrast, quercetin evidenced a lower TEAC value in a lipophilic environment (ethanol) than in a hydrophilic medium (water). The capacity of the lemon verbena extract to inhibit lipid peroxidation was also determined by HPLC determination of MDA generation (Table 2). Lemon verbena extract showed an IC50 value of 6.91 ± 0.21 mg/L, which revealed stronger antioxidant activity than other antioxidant extracts, such as olive leaf and tea catechins, determined in the same conditions (Laporta et al., 2007), and similar potency to the previously reported African potato extract, i.e., 7.5 mg/L (Hypoxis hemerocallidea). This fact may also be in relation to the stronger capacity showed by this extract in the lipophilic TEAC assay, what may also reveal the capability of lemon verbena extract (and therefore verbascoside) to scavenge free radicals under a specific environment such as the biological membranes. Some apparently hydrophilic antioxidants such as hypoxoside (Laporta et al., 2007), have shown to be effective antioxidants in lipophilic environments and exerted strong effects in biological membranes (Laporta, Funes, Garzón, Villalaín, & Micol, 2007). When the capacity to inhibit lipid peroxidation was measured by MDA inhibition assay, verbascoside was much stronger than hydroxytyrosol or caffeic acid, and as potent as quercetin (Table 2). This result reveals that even though verbascoside is a water-soluble compound, it seems to be quite efficient to avoid lipid peroxidation, probably establishing some kind of molecular interactions with the lipid membrane surface, as it has been proposed for other hydrophilic antioxidants (Laporta et al., 2007). The antioxidant capacity of the lemon verbena extract was also determined by using the well-known ORAC-FL assay (Ou et al., 2001), which accounts for the scavenging of peroxyl radicals. The antioxidant dose–response behaviour of this extract was determined through the measurement of the area under the fluorescence decay curve (AUC) of the sample as compared to that in the absence of antioxidant. Lemon verbena extract showed an ORACFL value of 4075 ± 234 TE/g dw, which is a significant antioxidant value compared to other antioxidant powdered extracts measured in the same conditions (Ou et al., 2001) and similar to the values previously reported for green tea extract (70% catechins) and olive leaf extract (25% oleuropein), and performed in identical conditions (Laporta et al., 2007). Since verbascoside was the major compound present in lemon verbena extract, we might assume that the in vitro antioxidant capacity of the extract shown in the previous assays was mainly due to this compound, although synergistic effects of verbascoside with the rest of minor compounds (other phenylpropanoids and flavones) cannot be discarded.

was 17.3 min. The mass spectrum revealed a base peak at m/z 623 corresponding to [MH], which was corroborated with the use of a commercially available verbascoside standard (Fig. 3B). Moreover, a fragment ion at m/z 461 ([MH163]) was consistent with the loss of the rhamnosyl moiety. The pharmacokinetic study was performed using five individuals for each of five times data collection plus controls (Table 3). The pharmacokinetic parameters revealed an AUC of 3085 ± 1400 ng min/mL for an oral administration of total extract corresponding to a verbascoside dose of 545 mg/kg. The maximum verbascoside concentration found in plasma after oral administration was reached within the first 20 min, meaning that this compound was quickly absorbed from the gastrointestinal tract. In this study, the maximum verbascoside concentration found in plasma samples was 1431 ± 300 ng/mL (around 2.3 lM), which means 15 times more than that one previously reported by another study which used a different plant source containing verbascoside (Wu et al., 2006). Nevertheless, it can be concluded that oral bioavailability of verbascoside in rats is still rather low. However, the presence of other metabolites deriving from verbascoside or flavones and still non-determined in plasma could contribute to the in vivo biological activity of the lemon verbena extract. Absorption and pharmacokinetics of verbascoside have been proposed to be similar to other phenolic compounds such as rosmarinic acid or some tea catechins (Wu et al., 2006). The plasma concentration of these and other polyphenolic compounds such as phenolic acids, flavonoids, stilbenes and lignans in plasma, as determined in different mice and human studies have been estimated to be in the low micromolar range (Manach, Scalbert, Morand, Remesy, & Jimenez, 2004; Manach, Williamson, Morand, Scalbert, & Remesy, 2005; Rice-Evans, Miller, & Paganga, 1997; Yang, Sang, Lambert, & Lee, 2008). Nevertheless, a large body of evidences has demonstrated the biological activity of several phenylpropanoids at the low micromolar range (Korkina, 2007). The antiinflammatory activity of several phenylpropanoids has been proven in several cellular models at low micromolar values (Diaz et al., 2004; Matsuda, Morikawa, Managi, & Yoshikawa, 2003; Matsuda, Pongpiriyadacha, Morikawa, Ochi, & Yoshikawa, 2003). In addition, several phenylpropanoids, verbascoside among them, have shown strong antioxidant capacity at low micromolar concentrations in microsomal and LDL lipid peroxidation assays (Cos et al., 2002; Wong et al., 2001). Moreover the antitumour activity of phenylpropanoids has been shown in the micromolar range in cultured cells (Chen et al., 2005; Inoue, Sakuma, Ogihara, & Saracoglu, 1998; Zhang et al., 2002) and some phenylpropanoids have been able to suppress the growth and metastasis of tumour xenografts in nude mice in vivo (Chung et al., 2004). Moreover, the in vivo hepatoprotective effect of verbascoside against CCl4 in mice and the antioxidant effects in rats were achieved at relatively low dosages, i.e., 30 and 3 mg/kg, respectively (Lee et al., 2004). Therefore it seems reasonable to postulate that low verbascoside concentrations, i.e., within the low micromolar range, maintained for a long term in plasma might be able to exert some effects at their cellular targets.

3.3. Pharmacokinetic study of verbascoside from lemon verbena in rats

3.4. Plasma antioxidant capacity in rats after lemon verbena oral administration

A pharmacokinetic study after oral administration of lemon verbena extract was performed in order to determine the presence of the compounds from the extract in the plasma of rats. Verbascoside was detected in rat plasma using LC–MS/MS with electrospray ionisation. Fig. 3 shows the HPLC-DAD-MS profile of a rat plasma sample 20 min after the oral administration of lemon verbena extract and the product ion scan spectrum using electrospray negative-ion mode. Retention time of verbascoside in plasma samples

The antioxidant activity of verbascoside has been previously documented in several in vitro and ex vivo systems (Liu et al., 2003; Seidel et al., 2000; Siciliano et al., 2005; Valentao et al., 2002; Wang et al., 1996; Wong et al., 2001). Therefore, in this work, the ex vivo antioxidant activity of plasma samples of rats previously fed with lemon verbena extract was determined. Several measurements were performed in the same samples in which the pharmacokinetic study was done: determination of the level

L. Funes et al. / Food Chemistry 117 (2009) 589–598

595

Fig. 3. HPLC-DAD-ESI-MS profile of rat plasma sample containing verbascoside collected 20 min after oral administration of lemon verbena (A) at 330 nm and (B) extractedion chromatogram (EIC) at 623 m/z and MS spectra of the peak corresponding to the same sample.

of plasma malondialdehyde (MDA) by HPLC, the ferric-reducing ability of plasma (FRAP value) and the superoxide dismutase activity (SOD). Fig. 4A shows the level of MDA determined by HPLC with fluorescence detection in rat plasma samples corresponding to the different time data points of the pharmacokinetic study (bars). The figure also shows the concentration of verbascoside determined in the same samples as measured by HPLC-DAD-ESI-MS/MS. The figure shows a clear correlation between the maximum peak of verbascoside concentration at 20 min (1431 ± 300 ng/mL) and the

Table 3 Pharmacokinetic data of verbascoside after oral administration of lemon verbena extract in rats. Parameter

545 mg/kg, p.o.

Tmax (min) Cmax (ng/mL) AUC0–100 (ng min/mL) Kel (ng min/mL)

20 1431 ± 300 3085 ± 1400 0.016 ± 0.005

Tmax and Cmax were obtained from experimental observation. The AUC0t was calculated using trapezoidal rule to the last point, and elimination rate constant (Kel) was calculated by the residual method.

minimum MDA value. The differences of MDA values determined at 20 and 40 min were statistically significant compared to the control. To further characterise the antioxidant activity of the plasma samples of rats subjected to a single oral dose of lemon verbena extract, the ferric-reducing ability of plasma, FRAP, was determined in vitro (Fig. 4B). In this case, maximum antioxidant activity was observed for samples corresponding to 20 and 40 min extractions, with a significance of p < 0.01 compared to control. In addition, the capability of rat plasma samples to scavenge superoxide radicals was determined by using the xanthine/xanthine oxidase system coupled to superoxide dismutase (Fig. 4C). The results showed that the maximum capacity to inhibit the formation of the superoxide anion radical, i.e., SOD activity, also correlated to the rat plasma sample obtained at 20 min after the oral ingestion of lemon verbena extract, at which it was also observed the highest verbascoside concentration by LC–MS/MS. Therefore, all these results obtained using three different complementary antioxidant measurements lead to the assumption that the antioxidant activity shown in the plasma samples of rats subjected to oral ingestion of lemon verbena extract must be mainly due to the presence of verbascoside or its derivatives, which may also exert other biological effects after reaching the appropriate

L. Funes et al. / Food Chemistry 117 (2009) 589–598

MDA

Verbascosid e

0.4 1500

*

0.3

1000

** 500

0.2

0

0.1 Control

20

40

55

75

B

2000

Verbascoside (ng/mL plasma)

MDA (nmol/mL plasma)

A

1000

*

*

20

40

800

FRAP (µM/L)

596

600 400 200 0 Control

100

75

100

Time (min)

C

4

nKat/mL plasma

Time (min)

55

3

*

2

1

0

Control

20

40

55

75

100

Time (min) Fig. 4. (A) Inverse correlation between MDA values determined by HPLC-fluorescence (bars) and plasmatic levels of verbascoside determined by HPLC-DAD-ESI-MS/MS (filled circles) in the same rat plasma samples. Each bar represents the mean ± SD (n = 8); ** (p < 0.05) and * (p < 0.1) indicate statistically significant differences compared to control. (B) Ferric-reducing capacity of plasma (FRAP) in rats at 20, 40, 55, 75 and 100 min after being orally treated with lemon verbena extract. Each bar represents the mean ± SD (n = 12); * (p < 0.01) indicates statistically significant differences compared to control. (C) Superoxide dismutase activity in plasma of rats at the same time intervals than those in B after the oral ingestion of lemon verbena extract. Each bar represents the mean ± SD (n = 8); * (p < 0.05) indicates statistically significant differences compared to control.

cellular targets. As deduced from the in vitro antioxidant measurements, verbascoside might be also able to reach targets on lipophilic environments. It could also be postulated that the presence of verbascoside, or other related but non-detected metabolites, could increase the activity of antioxidant enzymes such as catalase, superoxide dismutase or glutathione peroxidase at post-translational level. Moreover, any change at the level of gene expression regulation must be discarded due to the short time of the observed effect. However, further research is needed in order to find any possible cellular metabolites from phenylpropanoids and to determine the presence of the metabolites deriving from lemon verbena in humans. 3.5. Oral Acute Toxicity of lemon verbena extract Although lemon verbena is an edible plant and is commonly used to prepare infusions and spices, the oral acute toxicity of an extract containing a high content of verbascoside was performed in mice in order to establish the approximate oral LD50. A single dose of 2000 mg/kg b.wt. of lemon verbena extract (25% verbascoside) was utilised and mice were observed through a 2-week period. No deaths occurred in either the control or the lemon verbena groups during treatment. No significant alterations in the body weight of the lemon verbena-treated groups (male and female) compared with their respective controls were observed (not shown). Once the assay was completed, two individuals (one from each sex) treated with the extract were sacrificed and compared with their respective controls through postmortem and pathological examinations. Observation comprised examination of the external

surface of the body (skin), all orifices, mucous membranes and the cranial, thoracic and abdominal cavities and their contents. The postmortem analysis of either male or female individuals subjected to treatment did not show abnormalities on vital organs such as brain, heart, lungs, liver, spleen, kidneys or intestines. Digestive system (stomach, duodenum, ileum, etc.) was also completely normal compared to controls. In addition, no particular gender-related effects were observed since no toxicity was noticed in male or female groups. A complete absence of toxicity at a concentration as high as 2000 mg/kg b.wt. was observed meaning that the substance may be classified either as a very low toxicity substance, i.e., GHS Category 5 (Globally Harmonized System) or unclassified. Considering that the lemon verbena extract used contained 25% verbascoside, an LD50 value for the pure compound, verbascoside, of P500 mg/kg b.wt. might be extrapolated.

4. Conclusions To conclude, lemon verbena extract containing 25% verbascoside showed strong antioxidant capacity, especially in a lipophilic environment, which was higher than expected as concluded from the antioxidant capacity of pure verbascoside, probably due to synergistic effects. The capacity of verbascoside to act as an effective radical scavenger in lipophilic environments was also shown. Moreover, verbascoside was found as the only metabolite identified by HPLC-ESI-MS/MS in the plasma samples deriving from rats fed with the lemon verbena extract, obtaining a Cmax at 20 min of 2.3 lM. Verbascoside peak correlated to maximum plasma antioxidant activity as measured by malondialdehyde (MDA)

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determination, ferric-reducing ability of plasma (FRAP value) and superoxide dismutase (SOD) measurements. In addition, no signs of toxicity were observed in mice at a verbascoside dosage of 500 mg/kg. Therefore, lemon verbena extract may suppose a nontoxic source of verbascoside. Verbascoside or acteoside is present in many vegetable materials and by-products. For instance, recent work has found verbascoside to be the most potent antioxidant compound in olive mill waste extract (Obied, Prenzler, & Robards, 2008), then verbascoside-enriched extracts, such as lemon verbena extract, might have interesting applications in cosmetic, nutraceuticals or functional food. Acknowledgements This investigation has been supported by Grants AGL200760778, AGL2007-62806 and AGL2008-05108-C03-03, and FPI fellowship to L. Funes from MEC. We also thank MONTELOEDER, S.L. for providing us with the lemon verbena extract. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2009.04.059. References Avila, J. G., de Liverant, J. G., Martinez, A., Martinez, G., Munoz, J. L., Arciniegas, A., et al. (1999). Mode of action of Buddleja cordata verbascoside against Staphylococcus aureus. Journal of Ethnopharmacology, 66(1), 75–78. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: The FRAP assay. Analytical Biochemistry, 239(1), 70–76. Bilia, A. R., Giomi, M., Innocenti, M., Gallori, S., & Vincieri, F. F. (2008). HPLC-DADESI-MS analysis of the constituents of aqueous preparations of verbena and lemon verbena and evaluation of the antioxidant activity. Journal of Pharmaceutical and Biomedical Analysis, 46(3), 463–470. Carnat, A., Carnat, A. P., Chavignon, O., Heitz, A., Wylde, R., & Lamaison, J. L. (1995). Luteolin 7-diglucuronide, the major flavonoid compound from Aloysia triphylla and Verbena officinalis. Planta Medica, 61(5), 490. Chen, M., Xiao, S. P., Cui, G. H., Zhang, S. J., Wu, Z. G., Huang, L. Q., et al. (2005). The determination of echinacoside and acteoside in herbs of Cistanche tubulosa. Zhongguo Zhongyao Zazhi, 30(11), 839–841. Chung, T. W., Moon, S. K., Chang, Y. C., Ko, J. H., Lee, Y. C., Cho, G., et al. (2004). Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: Complete regression of hepatoma growth and metastasis by dual mechanism. FASEB Journal, 18(14), 1670–1681. Cos, P., Rajan, P., Vedernikova, I., Calomme, M., Pieters, L., Vlietinck, A. J., et al. (2002). In vitro antioxidant profile of phenolic acid derivatives. Free Radical Research, 36(6), 711–716. Deepak, M., & Handa, S. S. (2000). Antiinflammatory activity and chemical composition of extracts of Verbena officinalis. Phytotherapy Research, 14(6), 463–465. Diaz, A. M., Abad, M. J., Fernandez, L., Silvan, A. M., De Santos, J., & Bermejo, P. (2004). Phenylpropanoid glycosides from Scrophularia scorodonia: In vitro antiinflammatory activity. Life Sciences, 74(20), 2515–2526. Hausmann, M., Obermeier, F., Paper, D. H., Balan, K., Dunger, N., Menzel, K., et al. (2007). In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulphate sodium-induced colitis. Clinical and Experimental Immunology, 148(2), 373–381. Inoue, M., Sakuma, Z., Ogihara, Y., & Saracoglu, I. (1998). Induction of apoptotic cell death in HL-60 cells by acteoside, a phenylpropanoid glycoside. Biological and Pharmaceutical Bulletin, 21(1), 81–83. Kawada, T., Asano, R., Makino, K., & Sakuno, T. (2002). Synthesis of isoacteoside, a dihydroxyphenylethyl glycoside. Journal of Wood Science, 48, 512–515. Kirmizibekmez, H., Montoro, P., Piacente, S., Pizza, C., Donmez, A., & Calis, I. (2005). Identification by HPLC-PAD-MS and quantification by HPLC-PAD of phenylethanoid glycosides of five Phlomis species. Phytochemical Analysis, 16(1), 1–6. Korkina, L. G. (2007). Phenylpropanoids as naturally occurring antioxidants: From plant defense to human health. Cellular and Molecular Biology, 53(1), 15–25. Laporta, O., Funes, L., Garzón, M. T., Villalaín, J., & Micol, V. (2007). Role of membranes on the antibacterial and anti-inflammatory activities of the bioactive compounds from Hypoxis rooperi corm extract. Archives of Biochemistry and Biophysics, 467(1), 119–131. Laporta, O., Pérez-Fons, L., Balan, K., Paper, D., Cartagena, V., & Micol, V. (2004). Bifunctional antioxidative oligosaccharides with antiinflammatory activity for joint health. AgroFOOD Industry Hi-tech, 15, 30–33.

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