Ignite Creation Date:
2024-10-26 @ 3:37 PM
Last Modification Date:
2024-10-26 @ 3:37 PM
Study NCT ID:
NCT06548503
Status:
COMPLETED
Last Update Posted:
None
First Post:
2024-08-06
Brief Title:
Sylimarin Pyrroloquinoline Quinone Sodium Salt and Myricetin Assumption Effects
Sponsor:
None
Organization:
None
Study Overview
Official Title:
Assumption of Sylimarin Pyrroloquinoline Quinone Sodium Salt and Myricetin Effects on Alcohol Levels and Markers of Oxidative Stress
Status:
COMPLETED
Status Verified Date:
2024-08
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
No
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Alcohol abuse is one of the most common causes of mortality worldwide also associated with increased oxidative stress and end-organ damage in chronic assumption
This study intend to evaluate the effect of the intake of a product contain silymarin pyrroloquinoline quinone sodium salt and myricetin conventionally for this project SiPiMi on alcohol ethyl glucuronide EtG and markers of oxidative stress levels on regular wine assumption over a predefined time period
This study intend to evaluate the effect of the intake of a product contain silymarin pyrroloquinoline quinone sodium salt and myricetin conventionally for this project SiPiMi on alcohol ethyl glucuronide EtG and markers of oxidative stress levels on regular wine assumption over a predefined time period
Detailed Description:
The included subjectswill be randomized in a controlled single-blinded trial to evaluate the intake of a product containing silymarin pyrroloquinoline quinone sodium salt and myricetin conventionally for this project SiPiMi on alcohol levels and markers of oxidative stress
Inclusion criteria
age between 18 and 35 years
no history of alcohol abuse or other substances
Caucasian ethnicity
no smoker
good health condition no autoimmune endocrine infectious cardiac renal hepatic or metabolic diseases
no pregnancy or lactation condition Subjects will be asked to avoid the use of aspirin paracetamol or other anti-inflammatory drugs in the 7 days before and during the experiment and the assumption of caffeine in the 12 hours before the test Furthermore volunteers will be asked to avoid any alcoholic intake in the 7 days before and during the experiment except the one intended for the experiment
All subjects will sign a consent after receiving proper information about the risks and benefits of this study
Anthropometric data height weight age will be tracked at the beginning Venous blood samples will be drawn for standardized clinical hematological analyses such as mean cell volume MCV hepatic function including aspartate transferase AST alanine transferase ALT γ-glutamyltransferase γ GT total and fractionated bilirubin
Experimental protocol
After inclusion blind randomization using an electronic number generator will be performed into the following two groups
A placebo sham-control group B SiPiMi group Subject and experimenters will be blinded to the group assignation
221 Treatment and placebo SiPiMi group will be supplemented with a commercial complex AGfit3 Alchimia Innovazione srl Padova Italy AGfit3 contains active agents coming from two plants Silymarin and Myrica Cerifera traditionally used for treating the liver combined with the sodium salt of Bio-PQQ pyrroloquinoline quinone AGfit3 is added to an aqueous solution of orange granular freeze-dried orange citric acid sucrose Placebo adopted is instead an aqueous solution of ascorbic acid 1 g orange granular freeze-dried orange citric acid sucrose
In this experiment all subjects will be asked to drink a glass 150 ml of Cabernet red wine Cantine Ca Lustra Zanovello Cinto Euganeo PD Italia with an alcohol content of 125 05 corresponding to 1481 - 1540 g of ethanol according to a drink average dose as per current literature
Biological samples will be collected and measurements carried out as follows
Day 0 baseline before the experiment Blood saliva and urine samples collected basal measures
Day 1 to evaluate acute wine intake blood samples will be drawn 60 120 and 240 min after drinking 150 mL of red wine to establish ethanol metabolism curve Placebo or SiPiMi will be taken after the wine dose Saliva will be collected 120 and 240 min after drinking while urine after 240 min
Day 2-6 long term intake - volunteers will drink one wine glass at lunch and another one at dinner 300 mLdie corresponding to 30g ethanoldie approximately they will assume the wine dose at the beginning of the meal and then continue the diet with a balanced daily menu according to individual energy needs They will then take placebo or SiPiMi after the meal depending on the group Saliva will be collected in the morning
Day 7 biological samples will be collected in the same way as day 1 up to the end of the study
Blood saliva and urine samples After enrollment for each subject in the morning before breakfast venous blood samples about 5 mL will be drowned in EDTA and LH tubes Vacuette tube Greiner bio-one Kremsmünster Austria blood samples centrifugated Hettich MIKRO 200R centrifuge for 10 min to separate plasma and red blood cells RBC Multiple aliquots then will be immediately frozen and stored at -80 C Plasma samples will be collected to determine blood ethanol ROS TAC and Co Q10 levels Saliva samples will be collected to determine ROS and TAC while urine samples to determine Ethyl Glucuronoide ETG lipid peroxidation 8-iso-PGF2α and NO metabolites NOx creatinine neopterin and uric acid concentration Aminothiols redox status will be evaluated by an RBC analysis
Biomarker analysis
Blood Alcohol Level Blood ethanol level will be determined using an Agilent 7820 A series GC instrument and HP-Innowax 30 m 025 mm 025 μm capillary column with Helium flow 15 mLmin as carrier gas The injector the column and the detector will be maintained at 250 C 40 C and 250 C respectively The analysis will be carried out by isothermal elution n-Propanol will be used as internal standard Nitrogen hydrogen and air will be used as gas for FID detector at 25 40 and 400 mLmin respectively
Urine Ethyl Glucuronoide ETG ETG is a metabolic product of ethylic alchool coming from the reaction of ethanol and acid glucuronic ETG will be determined in urine samples using a commercial enzymatic immunoassay quantILAB n W1510011723 according to manufacturers instructions All measures will be assessed in duplicate The inter-assay coefficient of variation will be checked to fall in the range indicated by the manufacturer
Plasma ROS production An X-band electron paramagnetic resonance spectroscopy instrument EPR 93 GHz E-Scan Bruker Co Billerica MA USA will be used to detect ROS production in plasma and saliva samples Briefly spin probe CMH 1-hydroxy-3-methoxy-carbonyl-2255-tetramethylpyrrolidine will be used for ROS de-termination while a stable radical CP 3-carboxy2255-tetramethyl1-1-pyrrolidi-nyloxy will be adopted as an external reference to convert ROS recorded data into absolute quantitative levels µmolmin-1
Total Antioxidant Capacity TAC The 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid Trolox- equivalent antioxidant capacity assay a widely used kit-based commercial method Cayman Chemical Ann Arbor MI USA Item No 709001 will be used as previously described 22 24 Briefly 10 μL of plasma andor saliva will be added in duplicate to 10 μL of metmyoglobin and then 150 μL of the chromogen solution reactions will be started by the addition of 40 μL of hydrogen peroxide Reaction mixtures will be incubated for 3 min at room temperature then the absorbance signal at 750 nm will be determined by an Infinite M200 microplate reader spectrophotometer Tecan Austria TAC will be expressed as trolox equivalent antioxidant capacity concentration mM
8-Isoprostane 8-iso-PGF2α Lipid peroxidation will be assessed in urine samples using an immunoassay of 8-isoprostane concentration Cayman Chemical Ann Arbor MI USA Item No 516351 Samples and standard will be read in duplicate at a wavelength of 512 nm Results will be normalized by urine creatinine values
NO Metabolites NO derivatives nitrate and nitrite NO2 NO3 NOx will be measured in urine samples by a colorimetric method based on the Griess reaction using a commercial kit Cayman Chemical Ann Arbor MI USA Item No 780001 Samples will be read at 545 nm and the concentration assessed by a standard curve
Co Q10 coenzyme CoQ10 plasma levels will be quantified using Human Coenzyme Q10 Elisa Kit EKC33185 Biomatik Ontario Canada following the manufacturers instructions Briefly assay employs the competitive inhibition enzyme immunoassay technique the microtiter plate provided in this kit has been pre-coated with CoQ10 Standards or plasma samples are added to the appropriate microtiter plate wells with Horseradish Peroxidase HRP conjugated antibody preparation specific for CoQ10 The competitive inhibition reaction is launched between with pre-coated CoQ10 and CoQ10 in samples Samples will be read at 450 nm and the concentration was assessed by a standard curve
Thiols measurement Total tot and reduced red aminothiols Cyscysteine and GSH glutathione will be measured in erythrocytes RBC Thiol separation will be performed at room temperature by isocratic high-pressure liquid chromatography HPLC analysis on a Discovery C-18 column 250 46 mm ID Supelco Sigma-Aldrich St Louis MOS USA eluted with a solution of 01 M acetate buffer pH 40 methanol 8119 vv at a flow rate of 1 mLmin Fluorescence intensities will be measured with an excitation wavelength at 390 nm and an emission wavelength at 510 nm using a fluorescence spectrophotometer Jasco Japan A standard calibration curve will be used
Creatinine neopterin and uric acid Urinary creatinine neopterin and uric acid concentrations will be measured in urine by isocratic high-pressure liquid chromatography HPLC The calibration curves will be linear over the range of 0125-1 μmolL 0625-20 mmolL and 125-10 mmolL for neopterin uric acid and creatinine levels respectively Inter-assay and intra-assay coefficients of variation will be set at 5
Statistic analysis Kolmogorov-Smirnov test will be implemented to assess whether each variable followed a normal distribution and descriptive statistics will be calculated Two-way ANOVA with factors type product Active vs Placebo and time 1day vs 7day will be applied Then the one-way ANOVA will be applied to compare the trend values between the two groups Tukeys honest tests will be used for post-hoc analysis Probability levels of 005 will be considered significant Also the correlation between the investigated variables will be assessed using Spearman correlation coefficients ROS production was considered as the primary outcome no other parameters were taken into account and prospective calculations of power to determine the sample size were made using G power software GPower 31 At 80 power the sample size-calculated in preliminary studies - has been set at eleven thirteen subjects
Inclusion criteria
age between 18 and 35 years
no history of alcohol abuse or other substances
Caucasian ethnicity
no smoker
good health condition no autoimmune endocrine infectious cardiac renal hepatic or metabolic diseases
no pregnancy or lactation condition Subjects will be asked to avoid the use of aspirin paracetamol or other anti-inflammatory drugs in the 7 days before and during the experiment and the assumption of caffeine in the 12 hours before the test Furthermore volunteers will be asked to avoid any alcoholic intake in the 7 days before and during the experiment except the one intended for the experiment
All subjects will sign a consent after receiving proper information about the risks and benefits of this study
Anthropometric data height weight age will be tracked at the beginning Venous blood samples will be drawn for standardized clinical hematological analyses such as mean cell volume MCV hepatic function including aspartate transferase AST alanine transferase ALT γ-glutamyltransferase γ GT total and fractionated bilirubin
Experimental protocol
After inclusion blind randomization using an electronic number generator will be performed into the following two groups
A placebo sham-control group B SiPiMi group Subject and experimenters will be blinded to the group assignation
221 Treatment and placebo SiPiMi group will be supplemented with a commercial complex AGfit3 Alchimia Innovazione srl Padova Italy AGfit3 contains active agents coming from two plants Silymarin and Myrica Cerifera traditionally used for treating the liver combined with the sodium salt of Bio-PQQ pyrroloquinoline quinone AGfit3 is added to an aqueous solution of orange granular freeze-dried orange citric acid sucrose Placebo adopted is instead an aqueous solution of ascorbic acid 1 g orange granular freeze-dried orange citric acid sucrose
In this experiment all subjects will be asked to drink a glass 150 ml of Cabernet red wine Cantine Ca Lustra Zanovello Cinto Euganeo PD Italia with an alcohol content of 125 05 corresponding to 1481 - 1540 g of ethanol according to a drink average dose as per current literature
Biological samples will be collected and measurements carried out as follows
Day 0 baseline before the experiment Blood saliva and urine samples collected basal measures
Day 1 to evaluate acute wine intake blood samples will be drawn 60 120 and 240 min after drinking 150 mL of red wine to establish ethanol metabolism curve Placebo or SiPiMi will be taken after the wine dose Saliva will be collected 120 and 240 min after drinking while urine after 240 min
Day 2-6 long term intake - volunteers will drink one wine glass at lunch and another one at dinner 300 mLdie corresponding to 30g ethanoldie approximately they will assume the wine dose at the beginning of the meal and then continue the diet with a balanced daily menu according to individual energy needs They will then take placebo or SiPiMi after the meal depending on the group Saliva will be collected in the morning
Day 7 biological samples will be collected in the same way as day 1 up to the end of the study
Blood saliva and urine samples After enrollment for each subject in the morning before breakfast venous blood samples about 5 mL will be drowned in EDTA and LH tubes Vacuette tube Greiner bio-one Kremsmünster Austria blood samples centrifugated Hettich MIKRO 200R centrifuge for 10 min to separate plasma and red blood cells RBC Multiple aliquots then will be immediately frozen and stored at -80 C Plasma samples will be collected to determine blood ethanol ROS TAC and Co Q10 levels Saliva samples will be collected to determine ROS and TAC while urine samples to determine Ethyl Glucuronoide ETG lipid peroxidation 8-iso-PGF2α and NO metabolites NOx creatinine neopterin and uric acid concentration Aminothiols redox status will be evaluated by an RBC analysis
Biomarker analysis
Blood Alcohol Level Blood ethanol level will be determined using an Agilent 7820 A series GC instrument and HP-Innowax 30 m 025 mm 025 μm capillary column with Helium flow 15 mLmin as carrier gas The injector the column and the detector will be maintained at 250 C 40 C and 250 C respectively The analysis will be carried out by isothermal elution n-Propanol will be used as internal standard Nitrogen hydrogen and air will be used as gas for FID detector at 25 40 and 400 mLmin respectively
Urine Ethyl Glucuronoide ETG ETG is a metabolic product of ethylic alchool coming from the reaction of ethanol and acid glucuronic ETG will be determined in urine samples using a commercial enzymatic immunoassay quantILAB n W1510011723 according to manufacturers instructions All measures will be assessed in duplicate The inter-assay coefficient of variation will be checked to fall in the range indicated by the manufacturer
Plasma ROS production An X-band electron paramagnetic resonance spectroscopy instrument EPR 93 GHz E-Scan Bruker Co Billerica MA USA will be used to detect ROS production in plasma and saliva samples Briefly spin probe CMH 1-hydroxy-3-methoxy-carbonyl-2255-tetramethylpyrrolidine will be used for ROS de-termination while a stable radical CP 3-carboxy2255-tetramethyl1-1-pyrrolidi-nyloxy will be adopted as an external reference to convert ROS recorded data into absolute quantitative levels µmolmin-1
Total Antioxidant Capacity TAC The 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid Trolox- equivalent antioxidant capacity assay a widely used kit-based commercial method Cayman Chemical Ann Arbor MI USA Item No 709001 will be used as previously described 22 24 Briefly 10 μL of plasma andor saliva will be added in duplicate to 10 μL of metmyoglobin and then 150 μL of the chromogen solution reactions will be started by the addition of 40 μL of hydrogen peroxide Reaction mixtures will be incubated for 3 min at room temperature then the absorbance signal at 750 nm will be determined by an Infinite M200 microplate reader spectrophotometer Tecan Austria TAC will be expressed as trolox equivalent antioxidant capacity concentration mM
8-Isoprostane 8-iso-PGF2α Lipid peroxidation will be assessed in urine samples using an immunoassay of 8-isoprostane concentration Cayman Chemical Ann Arbor MI USA Item No 516351 Samples and standard will be read in duplicate at a wavelength of 512 nm Results will be normalized by urine creatinine values
NO Metabolites NO derivatives nitrate and nitrite NO2 NO3 NOx will be measured in urine samples by a colorimetric method based on the Griess reaction using a commercial kit Cayman Chemical Ann Arbor MI USA Item No 780001 Samples will be read at 545 nm and the concentration assessed by a standard curve
Co Q10 coenzyme CoQ10 plasma levels will be quantified using Human Coenzyme Q10 Elisa Kit EKC33185 Biomatik Ontario Canada following the manufacturers instructions Briefly assay employs the competitive inhibition enzyme immunoassay technique the microtiter plate provided in this kit has been pre-coated with CoQ10 Standards or plasma samples are added to the appropriate microtiter plate wells with Horseradish Peroxidase HRP conjugated antibody preparation specific for CoQ10 The competitive inhibition reaction is launched between with pre-coated CoQ10 and CoQ10 in samples Samples will be read at 450 nm and the concentration was assessed by a standard curve
Thiols measurement Total tot and reduced red aminothiols Cyscysteine and GSH glutathione will be measured in erythrocytes RBC Thiol separation will be performed at room temperature by isocratic high-pressure liquid chromatography HPLC analysis on a Discovery C-18 column 250 46 mm ID Supelco Sigma-Aldrich St Louis MOS USA eluted with a solution of 01 M acetate buffer pH 40 methanol 8119 vv at a flow rate of 1 mLmin Fluorescence intensities will be measured with an excitation wavelength at 390 nm and an emission wavelength at 510 nm using a fluorescence spectrophotometer Jasco Japan A standard calibration curve will be used
Creatinine neopterin and uric acid Urinary creatinine neopterin and uric acid concentrations will be measured in urine by isocratic high-pressure liquid chromatography HPLC The calibration curves will be linear over the range of 0125-1 μmolL 0625-20 mmolL and 125-10 mmolL for neopterin uric acid and creatinine levels respectively Inter-assay and intra-assay coefficients of variation will be set at 5
Statistic analysis Kolmogorov-Smirnov test will be implemented to assess whether each variable followed a normal distribution and descriptive statistics will be calculated Two-way ANOVA with factors type product Active vs Placebo and time 1day vs 7day will be applied Then the one-way ANOVA will be applied to compare the trend values between the two groups Tukeys honest tests will be used for post-hoc analysis Probability levels of 005 will be considered significant Also the correlation between the investigated variables will be assessed using Spearman correlation coefficients ROS production was considered as the primary outcome no other parameters were taken into account and prospective calculations of power to determine the sample size were made using G power software GPower 31 At 80 power the sample size-calculated in preliminary studies - has been set at eleven thirteen subjects
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None