The aim of the present study was to investigate the effect of a micronized formulation of trans-resveratrol in humans with non-alcoholic fatty liver disease (NAFLD). Trans-Resveratrol has been used in the form of micronized formulation, which is better absorbed, has strong antioxidants effects, is more effective than plain resveratrol formulations and is circulated on the market as a food supplement. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a stilbenoid and a phytoalexin produced by several plants. NAFLD is an increasing clinical problem involving the liver for which effective treatments are required. The present study was based on two patient groups. The study, which commenced on April 2013 and finished on April 2015, included 44 patients, aged 29–70 years, with an average weight of 84.6 kg (n=22 per group; 28 men and 16 women) who were randomly assigned to groups and given 50 mg Evelor capsule (n=22) and 200 mg Evelor H tablet (n=22) correspondingly on a daily basis. The patients were followed up for 6 months. Quantity fat measurements, with ultrasound on the liver and kidney, were carried out. There was an initial measurement (time 1) and one after six months (time 2). The study results showed the effects of Trans-resveratrol micronized formulation in reducing the liver fat, as well as decreasing hepatic enzymes, serum glutamate pyruvic transaminase (SGPT) and gamma-glutamyl transpeptidase (g-GT) and insulin resistance. At the end of the study, the statistical analysis showed a statistically significant reduction on the liver fat. These data demonstrate that use of Trans-resveratrol micronized formulation improves features of NAFLD, and prevents liver damage. Thus, Trans-resveratrol micronized formulation can be a new treatment method for NAFLD.
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. It affects up to 30% of adults in Western countries and 15% in Asian countries and also an increasing number of children (
NAFLD is becoming a major health issue worldwide, not only for its prevalence, but also for its metabolic complications. The underlying insulin resistance is associated with hypertension, hyperlipidemia, cardiovascular disease, type 2 diabetes mellitus (T2DM), chronic kidney disease and recently with carotid atherosclerosis (
The polyphenol resveratrol (RSV) is a potential therapeutic candidate. RSV is a stilbenoid and a phytoalexin produced by several plants in response to injury or when the plant is under attack by pathogens such as bacteria or fungi (
Through clinical practice, it has been found that, in patients who suffer from arterial hypertension and elevated hepatic enzymes, treated with anti-hypertensive drugs and a strong antioxidant, such as micronized trans-resveratrol, hepatic enzymes were significantly improved. Thus, it was decided to start a clinical trial regarding the effect of micronized trans-resveratrol in patients with Non-alcoholic fatty liver disease (NAFLD).
In the present study, 50 or 200 mg RSV (Evelor and Evelor H, a food supplement manufactured by Agetis Supplements Ltd., Cyprus) was added to the standard treatment of patients with NAFLD daily for a period of 6 months and the therapeutic efficacy of RSV was investigated.
The study was approved by the Ethics Committee of Cyprus (file no. ΕΕΒΚ/ΕΠ/2010/12, date 06/06/2013). All the patients who participated were volunteers and they were asked to fill out a written consent form.
The patients were selected after they had initially been diagnosed with non-alcoholic fatty liver disease. The primary inclusion criterion was evidence of fatty liver on ultrasonography (US), which is the most commonly used imaging technique with remarkable sensitivity.
The US findings in non-alcoholic fatty liver disease are: i) Diffuse enhancement of near field echo in the hepatic region (stronger than in the kidney and in the spleen region) and gradual attenuation of the far field echo, ii) unclear display of intra-hepatic lacuna structure, iii) mild to moderate hepatomegaly with a round and blunt border, and iv) unclear display of right liver lobe and diaphragm (
The study excluded patients with one or more of the following features: i) Any known causes of steatosis, ii) cirrhosis, ii) malignant tumor or any other diseases which significantly decrease the patient's lifespan, iii) symptoms of heart failure or acute coronary syndrome, iv) chronic kidney disease, v) psychiatric disorders, vi) participating in other similar studies or participating in other studies that were completed in the last 6 months, and vii) using food supplements, they should stop the supplement and wait for two weeks before participating in the study.
A GE LogIQ5 expert Ultrasound Machine (General Electric, CA, USA), equipped with Ultrasound Transducer Probe (GE 3.5C model 2050357) was used. The equipment can measure the Echo Level (EL) at specific areas and depths. EL measures the mean intensity of pixels within a user-defined area (region of interest). Raw data provide the average sum (intensity per pixel)/pixels. The ultrasound depicts on screen the area (in cm2), mean (intensity dB) and standard deviation (dispersion). Phantom Model 040GSE (CIRS-Multi Purpose, Multi Tissue u/s Phantom) was used to calibrate the LogIQ5 machine. The specific phantom simulates the human liver and kidney organs and serves for calibration.
Regarding the parameters ‘liver/kidney value’ and ‘liver/kidney depth’, we used the same methodology used by Xia
Standardization of ultrasound quantitative parameters was performed using an abdominal phantom.
Attenuation measurements were taken at 2 depths, at the ROI xxcm (liver) and yycm (kidney). Attenuation was calculated by subtracting the EL (liver)-EL (kidney)=Hepato-Renal Index Difference. EL was measured in dB and was linear to the intensity; thus, linear regression was employed to compute normalized values (
In sagittal liver/right kidney view, an ROI of 1.5×1.5 cm (1,296 pixels) in the liver parenchyma was selected. The ROI had to be as uniform as possible, excluding blood vessels, bile ducts, and other focal hypo/hyper echogenicity. Another ROI of 0.5×0.5 cm (144 pixels) was identified in the right renal cortex with no large vessels, renal sinus or medulla. To avoid the interference of depth-dependent echo-intensity attenuation and the borderline echo distorting effects, the boundary between liver and right kidney area was placed near the center of the image, and the liver and right kidney ROIs were selected at the same depth of the ultrasound images. The gray scale mean value of the pixels within the two ROIs was used as measurement of echo intensity. Then we subtracted the average hepatic gray scale from the average renal cortex gray scale to calculate the US hepatic/renal value.
In the right intercostal view at the anterior axilla line, a tangent line of the sector ultrasound image was drawn and the ultrasound wave transmission line was determined, starting from the point of tangency and perpendicular to the tangent line. Two ROIs of 1.5×1.5 cm (1,296 pixels) were selected in liver homogeneous regions along the ultrasound transmission line near the liver anterior margin (depth, 4–6 cm) and the liver posterior margin, respectively. The linear distance between the two ROIs was also measured. The echo intensity of the ultrasound wave was attenuated exponentially, as shown in the equation:
where
The ratio of the average echo intensity in the liver near-field ROI to liver far-field ROI was then calculated based on the equation 1:
where
Then the formula for ultrasound hepatic echo-intensity attenuation rate was deduced from the equation 2:
where Δ
To standardize the measured values of US H/R value and hepatic echo-intensity attenuation rate among different ultrasound machines, a 3D abdominal phantom, containing mimic abdominal organs, was used for standardization in this research.
Participants underwent clinical examination, electrocardiogram and abdominal ultrasound. Blood tests were carried out and the following parameters were measured: Serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (g-GT), glucose, total cholesterol levels, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), insulin, insulin resistance, liver value, liver depth, kidney value, kidney depth and difference liver-kidney values.
Then, the patients were divided into two groups (randomly assigned) according to the treatment administered: 22 patients were given treatment A (Evelor, 50 mg RSV) and 22 patients were given treatment B (Evelor H, 200 mg RSV). The observation period lasted for 6 months. There was a clinical examination, blood tests and an abdominal ultrasound at the beginning of the study (time 1) and in 6 months (time 2). All the patients were on low fat diet and were followed up by a nutritionist.
The results obtained by the study were used to examine the following parameters: i) The count of hepatic enzymes, ii) insulin resistance, and iii) liver fat.
Two-way ANOVA and Bonferroni post-hoc test, with interaction (time and group) was used to determine whether there were differences among dose levels, time levels and a possible interaction among them. However, we did not discover any significant interactions and we used standard two-way ANOVA without interactions. Moreover, an independent t-test was conducted to examine whether there were any differences between the values of physiological parameters at the beginning and the end of the study. P<0.001 was considered to indicate a strongly statistically significant difference. The statistical program used for statistical analysis was R3.2.1.
A total of 44 patients participated in the study; 28 men and 16 women from the ages of 29 to 70 with a mean weight of 84.6 kg. The patients were divided into two groups of 22 individuals each, according to the treatment administered: Group A was treated with micronized trans-Resveratrol 50 mg (Evelor), and Group B was treated with micronized trans-Resveratrol 200 mg (Evelor H). Measurements were made at the beginning (time 1) and after 6 months (time 2) to compare results between the two different dose groups as well as within the group itself.
Based on the fact that there was no interaction between dose and time for all variable considered, we also implemented a two-way ANOVA model but without interaction.
The results show the same conclusions as in the case of a model that includes interactions: i) There were strongly statistically significant differences between Liver value and Kidney value across time. Both of these measurements, liver value and kidney value decreased (time 2). ii) The difference liver-kidney decreased as time progressed. iii) There were statistically significant differences between the two dose levels for ALP and TG. iv) There are significant differences between the dose levels for SGOT, glucose and HDL.
Liver is an important organ because of its unique metabolism. Its main function is to take up nutrients, to store and/or provide them to the other organs. At the same time, it is a clearance and recycling organ. That means it is also a frequent target for a number of toxicants. The resulting hepatic injury is characterized by leakage of cellular enzymes into the blood stream. Elevation of SGPT, SGOT, g-GT in the blood often reflects hepatocellular damage (
Normal ranges of SGOT/SGPT in blood are 5–40 or 7–56 U/l and these are sensitive indicators of liver damage from different types of diseases. SGPT is primarily located in the liver and represents more specifically an injury to the organ as compared to SGOT, which is found in decreasing order of concentration in liver, cardiac muscle, skeletal muscle, kidneys, lungs, and brain.
Gamma-GT normal range in blood is 5–55 U/l. It is primarily present in kidney, liver, and pancreatic cells. Small amounts are present in other tissues. Even though renal tissue has the highest level of g-GT, the enzyme present in the serum appears to originate primarily from the hepatobiliary system, and g-GT activity is elevated in any and all forms of liver disease. It is currently the most sensitive enzymatic indicator of liver disease (
The common causes of elevated SGPT, SGOT, g-GT are alcohol abuse, drugs, chronic hepatitis B and C, autoimmune hepatitis, congenital metabolic disorders and fatty liver disease. In the United States, the majority of unexplained cases of elevated transaminases are strongly associated with non-alcoholic fatty liver disease (NAFLD) (
NAFLD is the most common form of chronic liver disease and a major health burden in developed countries with a prevalence of up to 30% (
Recent studies have shown an association between NAFLD and metabolic syndrome, as it seems to have a common pathogenic factor, insulin resistance (IR) (
Although clinical studies have tried several pharmacological treatments, there is currently no satisfactory therapy for NAFLD. Therefore, investigators focus on the management of metabolic syndrome (
Natural polyphenols are a potential therapeutic option for NAFLD. They have been proposed for the treatment of different metabolic disorders, because of their anti-inflammatory and anti-oxidative properties. Additionally, they have an effect on glucose and lipid metabolism (
The polyphenol resveratrol (RSV3,5,4′-trihydroxystilbene) is a stilbenoid produced by several plants in response to injury or when the plant is under attack by pathogens. RSV has multiple biochemical and physiological actions. One of the most important is that RSV mimics a condition of caloric restriction (CR) (
AMPK is a protein consisting of three subunits, one catalytic and two non-catalytic. AMPK is activated, by phosphorylation, as a response to changes in the cellular AMP/ATP ratio (
RSV is also an activator of SIRT1, which is a NAD+ (oxidized nicotinamide adenine dinucleotide)-dependent protein deacetylase. SIRT1 plays a key role in lipid and glucose homeostasis and in insulin secretion sensitivity via CR. Furthermore, SIRT1 is an inhibitor of inflammation, reduces oxidative stress and improves endothelial function (
In this study, the results indicated that trans-resveratrol in micronized formulation supplementation prevents and improves liver damage. The mechanism that RSV mimics CR is not fully understood yet, but the activation of AMPK and SIRT1 has a key role. The reduction of TG accumulation and the improvement of IR serve to protect the liver from NAFLD. Therefore, our data suggest that trans-resveratrol in micronized formulation is a hepatoprotective agent in humans, and a new therapeutic option for NAFLD.
Not applicable
This study was financially supported by the ‘Program Sustainable Development and Competitiveness’.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
MT designed and performed the study. AC, AA and AM were responsible for the collection and analysis of the data. KF, DM and RK performed data analysis and interpretation. JD and ES participated in analysing the data and revised the manuscript. All authors read and approved the final manuscript.
All patients provided written informed consent for the use of their data. The study protocol was approved by the Cyprus National Bioethics Committee.
Not applicable.
The authors declare that they have no competing interests.
Main demographic variables.
Variables | Mean | SD | Median | Minimum | Maximum |
---|---|---|---|---|---|
Age (years) | 54.16 | 9.92 | 55 | 29 | 70 |
Weight (kg) at time 1 | 84.55 | 11.42 | 83.30 | 58 | 105 |
Weight (kg) at time 2 | 82.33 | 11.73 | 80 | 57 | 103 |
Height (cm) | 170 | 7 | 170 | 155 | 183 |
BMI (kg/cm2) at time 1 | 27.01 | 3.02 | 28 | 20 | 31 |
BMI (kg/cm2) at time 2 | 27.93 | 3.35 | 28.50 | 21 | 35 |
BMI, body mass index.
Classification of the participants by sex and treatment.
Sex/Dose | 50 mg RSV | 200 mg RSV |
---|---|---|
Male | 12 | 16 |
Female | 10 | 6 |
RSV, resveratrol.
Statistical comparisons based only on 50 mg (Evelor) dose across time.
Variables | Mean at Time 1 | Mean at Time 2 | t-test (P-value) |
---|---|---|---|
SGPT | 36.09 | 37.24 | 0.871 |
SGOT | 24.95 | 26.33 | 0.603 |
ALP | 63.63 | 59.23 | 0.593 |
g-GT | 30.95 | 27.00 | 0.770 |
GLU | 105.95 | 96.57 | 0.122 |
CH | 194.50 | 185.67 | 0.656 |
HDL | 48.60 | 47.67 | 0.905 |
LDL | 119.45 | 116.19 | 0.932 |
TG | 132.50 | 109.76 | 0.301 |
Insulin | 13.58 | 12.69 | 0.206 |
Insulin resistance | 1405.81 | 1226.04 | 0.135 |
Liver value | 55.20 | 45.42 | <0.001 |
Liver depth | 5.60 | 5.79 | 0.034 |
Kidney value | 32.49 | 28.42 | <0.001 |
Kidney depth | 7.06 | 7.30 | 0.008 |
Difference L-K value | 22.71 | 17.00 | 0.170 |
SGPT, serum glutamate pyruvic transaminase; SGOT, serum glutamic oxaloacetic transaminase; ALP, alkaline phosphatase; g-GT, gamma-glutamyl transpeptidase; GLU, glucose; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TG, triglycerides.
Statistical comparisons based only on 200 mg (Evelor H) dose across time.
Variables | Mean at Time 1 | Mean at Time 2 | t-test (P-value) |
---|---|---|---|
SGPT | 47.05 | 41.70 | 0.631 |
SGOT | 32.81 | 30.90 | 0.887 |
ALP | 74.50 | 71.20 | 0.804 |
g-GT | 29.20 | 25.35 | 0.434 |
GLU | 121.18 | 112.50 | 0.786 |
CH | 194.27 | 181.15 | 0.258 |
HDL | 41.14 | 41.70 | 0.832 |
LDL | 118.75 | 105.95 | 0.408 |
TG | 205.13 | 167.80 | 0.465 |
Insulin | 12.62 | 13.01 | 0.091 |
Insulin resistance | 1541.04 | 1489.52 | 0.151 |
Liver value | 58.77 | 43.33 | <0.001 |
Liver depth | 5.20 | 5.63 | 0.792 |
Kidney value | 32.30 | 25.21 | <0.001 |
Kidney depth | 7.04 | 7.26 | 0.795 |
Difference L-K value | 26.46 | 18.115 | 0.795 |
SGPT, serum glutamate pyruvic transaminase; SGOT, serum glutamic oxaloacetic transaminase; ALP, alkaline phosphatase; g-GT, gamma-glutamyl transpeptidase; GLU, glucose; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TG, triglycerides.
Statistical comparisons based on different doses and across time.
Variables | Time effect (P-value) | Dose effect (P-value) | Interaction between time and dose (P-value) |
---|---|---|---|
SGPT | 0.842 | 0.052 | 0.576 |
SGOT | 0.991 | 0.022 | 0.686 |
ALP | 0.563 | <0.001 | 0.883 |
g-GT | 0.567 | 0.377 | 0.798 |
GLU | 0.336 | 0.005 | 0.963 |
CH | 0.215 | 0.563 | 0.863 |
HDL | 0.983 | 0.004 | 0.764 |
LDL | 0.479 | 0.190 | 0.697 |
TG | 0.183 | <0.001 | 0.898 |
Insulin | 0.027 | 0.782 | 0.887 |
Insulin resistance | 0.021 | 0.179 | 0.914 |
Liver value | <0.001 | 0.621 | 0.383 |
Liver depth | 0.136 | 0.821 | 0.376 |
Kidney value | <0.001 | 0.287 | 0.778 |
Kidney depth | 0.051 | 0.330 | 0.350 |
Difference L-K value | 0.001 | 0.681 | 0.424 |
SGPT, serum glutamate pyruvic transaminase; SGOT, serum glutamic oxaloacetic transaminase; ALP, alkaline phosphatase; g-GT, gamma-glutamyl transpeptidase; GLU, glucose; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TG, triglycerides.
Statistical comparisons based on different doses and across time.
Variables | Time effect (P-value) | Dose effect (P-value) |
---|---|---|
SGPT | 0.841 | 0.051 |
SGOT | 0.991 | 0.022 |
ALP | 0.556 | <0.001 |
g-GT | 0.563 | 0.374 |
GLU | 0.330 | 0.004 |
CH | 0.211 | 0.460 |
HDL | 0.983 | 0.004 |
LDL | 0.475 | 0.187 |
TG | 0.179 | <0.001 |
Insulin | 0.025 | 0.781 |
Insulin resistance | 0.020 | 0.176 |
Liver value | <0.001 | 0.621 |
Liver depth | 0.136 | 0.821 |
Kidney value | <0.001 | 0.284 |
Kidney depth | 0.051 | 0.331 |
Difference L-K value | 0.001 | 0.681 |
SGPT, serum glutamate pyruvic transaminase; SGOT, serum glutamic oxaloacetic transaminase; ALP, alkaline phosphatase; g-GT, gamma-glutamyl transpeptidase; GLU, glucose; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TG, triglycerides.