We examined
The French oak wood extract Robuvit (Horpag Research Ltd.) is a registered proprietary water extract obtained from the wood of
The oak wood used for Robuvit originates exclusively from oak trees grown in France. Oak wood contains a specific profile of tannins named roburins that are part of the ellagitannins (ETs). Robuvit is standardized and specified to contain at least 20% of roburins (A, B, C, D, and E) including grandinin. The two most abundant ETs in the Robuvit are stereoisomers vescalagin and castalagin, which were originally isolated and described by Mayer et al. [
Owing to their unique molecular structure roburins are very potent antioxidants. Humans have been exposed to these compounds for centuries from wine and spirits that matured in oak wood barrels. Oak wood is currently the only known food source of roburins, and, according to this specificity, the major source of roburins in human diets results from the consumption of wine and spirits (cognac and whiskey) traditionally matured, aged, and stored in oak barrels [
Little is known about roburins bioavailability and biological effects. Natella et al. [
The aim of our study was to examine
Twenty healthy volunteers (12 women and 8 men, mean age
The 8-week experiment was divided into three periods. In the first period (run-in period) volunteers were instructed to control their diet for 2 weeks. No additional antioxidants like vitamins C, E or coenzyme Q or excess of chocolate, red wine, or beer should be consumed. Drinking a cup of green tea, 2 dL red wine, or 1 beer daily was allowed. After run-in period, Robuvit administration began (week 0). Volunteers took 1 capsule (100 mg) of extract Robuvit three times a day (300 mg daily) for 4 weeks, followed by two-week wash-out period in which capsules were not administered. The extract was supplied by Horphag Research Ltd., Switzerland.
The venous blood was collected after 12-hour overnight fast into commercial serum tubes and EDTA coated tubes. Within 1 hour of collection, blood was centrifuged (700 ×g, 5 min); serum and plasma were aliquoted and frozen at −80°C until analysis. For isolation of erythrocytes, blood was washed three times with 0.15 mol/L NaCl solution. After final centrifugation (700 ×g, 7 min), erythrocytes were haemolysed by addition of triple volume of distilled water and haemolysate was frozen at −20°C until analysis.
The samples of blood were taken after run-in period (week 0), at the end of intervention period (week 4) and at the end of wash-out period (week 6).
In the serum of the basic biochemical parameters, the concentrations of advanced oxidation protein products (AOPP) and lipid peroxides (LP) were determined; in the plasma the levels of advanced glycation end-products (AGEs), 8-isoprostanes (8-isoP), protein carbonyls, and total antioxidant capacity were examined. In isolated lymphocytes marker of oxidative damage to DNA and 8-oxoguanin (8-oxoG) was assessed. The concentration of haemoglobin by Drabkin method and SOD, CAT, and GPx activities were measured in haemolysate of erythrocytes.
ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt], Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), dextran sulphate, chloramine T, igepal, o-phenylenediamine, guanidine hydrochloride, Tween 20, bovine serum albumin (BSA), histopaque 1083, low melting point (LMP) agarose, normal melting point (NMP) agarose, Triton X-100, hydrogen peroxide, AAPH [2,2′-azobis (2-amidinopropane) dihydrochloride], and formamidopyrimidine-DNA glycosylase (Fpg) were purchased from Sigma-Aldrich (USA); sodium azide and DAPI (4,6-diamidino-2-phenylindole dihydrochloride) were purchased from Merck (Germany); TPTZ (2,4,6-tripyridyl-s-triazine), disodium fluorescein, 2,4-dinitrophenylhydrazine (DNPH), and glycerol were purchased from Fluka (Germany); all other chemicals were purchased from Lachema (Czech republic); Robuvit was obtained from Horphag Research Ltd. (Geneva, Switzerland).
Basic biochemical parameters (bilirubin, glucose, gamma-glutamyl transferase (GMT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid (UA), total proteins (TP), total cholesterol (TCh), triacylglycerols (TAG), HDL-cholesterol (HDL-chol), LDL-cholesterol (LDL-chol), and VLDL-cholesterol (VLDL-chol)) were determined at an accredited clinical biochemistry and haematology laboratory using a Hitachi 911 analyzer by Roche diagnostics kits (Switzerland).
57.8 mg of Robuvit was dissolved in 10 mL of distilled water, vortexed for 5 min, centrifuged (3000
Serum concentration of AOPP was measured by modified method according to Witko-Sarsat et al. [
The concentration of protein carbonyls in plasma was measured by ELISA method according to Buss et al. [
The concentration of plasma AGEs was determined by modified method according to Kalousová et al. [
Oxidative DNA damage in lymphocytes was evaluated by the enzymatically modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) recognizing oxidized purines [
8-isoP were determined by the commercial EIA kit (Cayman Chemical Company, USA). This assay is based on the competition between 8-isoP and an 8-isoprostane-acetylcholinesterase (AChE) conjugate (8-isoprostane Tracer) for a limited number of 8-isoprostane-specific rabbit antiserum binding sites. Because the concentration of the 8-isoprostane Tracer is held constant while the concentration of 8-isoP varies, the amount of 8-isoprostanes Tracer that is able to bind to the rabbit antiserum will be inversely proportional to the concentration of 8-isoP in the well. The plate is washed to remove any unbound reagents and then Ellman’s reagent (which contains the substrate to AchE) is added to the well. The product of enzymatic reaction has a distinct yellow colour and absorbs the light at 405 nm. The concentration of 8-isoP is expressed in pg/mL.
Concentration of LP in serum was measured by method according to El-Saadani et al. [
Activity of SOD was determined by the commercial kit number 19160 (Fluka, Germany), using bovine Cu/Zn-SOD as a standard (Sigma, Germany). Superoxide radical is generated from xanthine and oxygen by xanthine oxidase. Superoxide radical reduces Dojindo’s highly water-soluble tetrazolium salt, WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) to form a yellow water-soluble formazan dye with maximum absorption at 450 nm. At the presence of SOD the reduction of WST-1 is decreased. 1 U of SOD activity is defined as the enzyme activity causing 50% inhibition of reduction of WST-1. SOD activity is expressed in U SOD/mg Hb.
Catalase activity was determined by modified method of Bergmeyer [
Activity of GPx was determined by the commercial kit (Sigma-Aldrich, USA). This kit uses an indirect determination method. It is based on the oxidation of glutathione (GSH) to oxidized glutathione (GSSG) catalyzed by GPx, which is then coupled to the recycling of GSSG back to GSH utilizing glutathione reductase and NADPH. The decrease in NADPH absorbance measured at 340 nm during the oxidation of NADPH to NADP+ is indicative of GPx activity. Activity of GPx is expressed in U/g Hb.
The ORAC assay was measured by modified method according to Huang et al. [
The FRAP was determined by the method according to Benzie and Strain [
Results are presented as a mean ± standard deviation (SD) or median and interquartile range (IQ, 1st quartile, 3rd quartile). Parametric Student’s paired
Administration of Robuvit for 4 weeks decreased weight of volunteers, but because of high interindividual variability of body weight this change (−0.7 kg) has no relevance. Values of BMI were not significantly changed after week 4 and week 6.
Basic biochemical parameters were investigated in fasting venous blood in examination periods 0, 4, and 6: bilirubin, glucose, GMT, ALP, AST, ALT, UA, TP, TCh, TAG, HDL-chol, LDL-chol, and VLDL-chol. All values of mentioned biochemical parameters were in the physiological ranges.
We did not observe serious unwanted effects of Robuvit administration.
Concentrations of markers of oxidative damage to proteins (AOPP, protein carbonyls, and AGEs), DNA (8-oxoG), and lipids (8-isoP and LP), activities of antioxidant enzymes (SOD, CAT, and GPx), and total antioxidant capacity (ORAC and FRAP) are summarized in Table
Markers of oxidative stress, activity of antioxidant enzymes, and total antioxidant capacity after weeks 0, 4, and 6.
Parameter | Week 0 | Week 4 |
|
Week 6 |
|
---|---|---|---|---|---|
AOPP ( |
58.06 |
33.62 |
<0.0001 | 33.18 |
<0.0001 |
PC (ng/mL) | 0.296 ± 0.059 | 0.288 ± 0.052 | n.s. | 0.273 ± 0.054 | 0.0037 |
AGEs (AU/g prot.) | 273.17 |
296.17 |
n.s. | 286.07 |
n.s. |
8-oxoG/106 G | 0.61 |
0.79 |
n.s. | 0.78 |
n.s. |
8-isoP (pg/mL) | 24.59 |
34.02 |
n.s. | 31.63 |
n.s. |
LP (nmol/mL) | 42.55 |
33.39 |
0.0174 | 34.13 |
0.0038 |
SOD (U/mg Hb) | 42.42 ± 6.83 | 47.41 ± 9.93 | 0.0198 | 48.03 ± 5.27 | 0.0240 |
CAT ( |
5.60 |
6.47 |
0.0012 | 4.59 |
n.s. |
GPx (U/g Hb) | 39.55 ± 9.16 | 40.74 ± 8.23 | n.s. | 36.21 ± 8.96 | n.s. |
ORAC (mmol/L) | 8.97 |
8.72 |
n.s. | 8.69 |
n.s. |
FRAP (mmol/mL) | 0.586 ± 0.098 | 0.623 ± 0.115 | 0.0091 | 0.603 ± 0.124 | n.s. |
Data are presented as mean ± SD or median and IQ range (1st quartile; 3rd quartile); AOPP: advanced oxidation protein products; PC: protein carbonyls; AGEs: advanced glycation end-products; AU: arbitrary units; 8-isoP: 8-isoprostanes; LP: lipid peroxides; SOD: Cu/Zn-superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; ORAC: oxygen radical absorbance capacity; FRAP: ferric reducing ability of plasma;
The concentrations of protein carbonyls, AGEs, 8-oxoG, and 8-isoP were not significantly changed after week 4. Only after week 6 we found out significantly decreased level of protein carbonyls by 7.77% in comparison to week 0.
We measured activities of SOD, CAT, and GPx in haemolysate of erythrocytes. Administration of Robuvit significantly increased activity of SOD by 11.76% and this effect persisted after week 6 where the activity of SOD was even more increased by 13.22%. We also found a significantly higher activity of CAT after week 4 by 15.54%. Activity of GPx was not influenced by intake of Robuvit.
Total antioxidant capacity of plasma was determined by two methods with different mechanism of action: ORAC and FRAP. We did not find significant changes in ORAC values in weeks 4 and 6 in comparison to week 0. However, we found out significantly increased level of FRAP values by 6.31% after week 4.
We have studied
We have determined
In our human trial we have found that Robuvit was able to protect significantly proteins and lipids against oxidation as we found significantly decreased levels of AOPP and LP in serum.
In addition, 4-week intake of Robuvit significantly increased the intracellular defence against oxidative stress by enhancing the activities of SOD and CAT in erythrocytes. We also found significantly increased FRAP values of total antioxidant capacity in the plasma (by 6.3%) after week 4 in healthy volunteers, but this small change may not have a biological relevance. Also Natella et al. [
The mechanism for the antioxidant effect of polyphenols in Robuvit
Following consumption of the oak wood extract Robuvit, besides EA and GA, three different urolithins were identified in plasma of volunteers [
In conclusion, we have confirmed for the first time that the intake of the French oak wood extract Robuvit is associated with a decreased damage to proteins and lipids, a stimulation of antioxidant enzymes (SOD and catalase), and a moderate increase of total antioxidant capacity of plasma in humans.
The authors declare that there is no conflict of interests.
The study was partially supported by Horphag Res. Ltd. and by Mind and Health, Civil Association. The authors wish to thank Assoc. Professor P. Blazicek, PhD., for the assistance at basic biochemical parameters determination, a.o. Univ. Professor Karl-Heinz Wagner, PhD., for the assistance at ORAC measurement, Zuzana Kralovicova for FRAP determination and for technical and clinical assistance, D. Opalena, L. Chandogova, and E. Palaghyova.