A randomized, double-blind, placebo-controlled trial



Phytonutrients and vitamin and mineral supplements have been reported to provide increased antioxidant capacity in humans; However, there is still controversy. In the current clinical trial, we investigated the antioxidant and DNA protection capabilities of a plant-based, multi-vitamin / mineral and phytonutrient (PMP) supplement in healthy adults usually low in fruit and vegetable consumption. This study was an eight-week, double-blind, randomized, parallel-arm, and placebo-controlled study. Eight weeks of PMP supplementation reduced reactive oxygen species (ROS) and prevented DNA damage without altering the endogenous antioxidant system. Plasma vitamins and phytonutrients were significantly correlated with ROS capture and DNA damage. In addition, gene expression analysis in PBMC showed subtle changes in superoxide metabolic processes. In this study, we demonstrated that supplementation with a PMP significantly enhanced ROS purification activity and prevented DNA damage. However, further research is still needed to further identify mechanisms of action and the role of circulating phytonutrient metabolites.

Keyword: phytonutrients, ROS removal, DNA damage, antioxidant capacity, human clinical study

1. Introduction

Clinical and epidemiological studies have shown that oxidative stress is related to cardiovascular disease, cancer and other chronic diseases that account for the majority of mortality[[[[1.2.3]. Several studies have shown that multivitamin and mineral supplements can help provide essential nutrients, maintain health, reduce the risk of various diseases and support normal functions[[[[]. However, there are several publications of clinical trials that report no health benefits of multivitamin and mineral supplements[[[[].

In addition to vitamins and minerals, plants contain a wide range of phytonutrients that have been reported to be involved in chronic disease prevention.[[[[14]. Unlike vitamins and minerals, it is known that some phytonutrients (ie polyphenols) are poorly absorbed in the small intestine[[[[15]. Polyphenols are typically found in low concentrations (nmol / l to mmol / l range) in both plasma and urine[[[[16], and detection requires sensitive analytical tools to have reliable and reproducible quantification. We have previously shown that the contribution of phytonutrients to the total antioxidant capacity was relatively higher than the vitamins alone.[[[[17]. In another study, we reported that supplements with a phytonutrient-containing, multivitamin / mineral increased plasma folate levels and folate metabolism in people with usually low intakes of fruits and vegetables.[[[[18]. In addition, the subjects in this study demonstrated resistance to DNA damage while maintaining endogenous oxidative defense ability.[[[[19]. However, the direct link between plant phytonutrients and ROS removal and mechanisms of action has not been fully elucidated.

In the current clinical trial, we investigated the antioxidant and DNA protection capabilities of a plant-based multivitamin / mineral and phytonutrient supplement in healthy adults usually low in fruit and vegetable consumption. To recruit people who had low consumption of fruits and vegetables, Recommended Food Point (RFS) was used[[[[20]. RFS was validated to be related to antioxidant capacity, and in our previous study, subjects with low RFS DNA repair with antioxidant nutrients improved[[[[19]. In addition, associations between antioxidant capacity and plasma vitamins and phytonutrients and potential antioxidant mechanisms based on gene array and network analysis are reported.

2. Materials and Methods

2.1. Investigate product

A plant-based multivitamin / mineral and phytonutrient supplement (PMP) and a color-matched placebo were provided by Access Business Group International, LLC (Buena Park, CA, USA). The PMP supplement (12 tablets) contained the following micronutrients: 14 vitamins (700 μg retinol equivalents A, 2.4 mg B1, 2.8 mg B2, 3 mg B6, 4.8 μg B12, 200 mg C, 10 μg D, 22 mg of α-tocopherol equivalents E, 55 μg K, 3 mg β-carotene, 60 μg biotin, 500 μg folate, 30 mg niacin and 10 mg pantothenic acid) and 10 minerals (700 mg calcium, 50 μg chromium, 0.4 mg copper , 75 μg iodine, 6 mg iron, 3 mg manganese, 220 mg magnesium, 25 μg molybdenum, 55 μg selenium and 12 mg zinc). The PMP supplement also contained phytonutrients from extracts or powders of acerola, alfalfa, blackcurrant, blueberry, strawberry, grape, grapefruit, kelp, lemon, mandarin orange, marigold, onion, orange, parsley, peppermint, rosemary, spinach, tomato, turmeric, and wells and granules with quercetin. Most phytonutrients were from botanical extracts, ie. botanical raw materials that were further concentrated by removing other parts of the trouser structure while preserving phytonutrients. A few ingredients were from dehydrates, ie. dried botanical raw material powders where all water was removed from the plant while all other parts of the plant (e.g. fiber, cell walls, sugars and phytonutrients) remain. The placebo sample for this study was formulated to match the shape and color of the PMP tablets. The placebo was composed of microcrystalline cellulose, silica, magnesium stearate and dyes.

2.2. topics

Healthy adults (25-69 years old) with usually low intake of fruits and vegetables as determined by RFS ≤ 36 (scale, 0–47)[[[[20]and body fat ≥ 20% (InBody; Biospace, Seoul, Korea) were eligible. All inclusion and exclusion criteria are presented in Table S1. One hundred and thirty subjects were recruited from Ewha Womans University (Seoul, Korea). Twelve subjects were excluded for not meeting the eligibility criteria, 25 subjects withdrew their consent, and the remaining 96 subjects were enrolled in the trial and received basic assessment. A Consolidated Standards for Reporting Experiments (CONSORT) Flowchart is presented in[[[[21]. All subjects provided written informed consent prior to enrollment. The study protocol was approved by Institutional Review Boards of Ewha Womans University (IRB No.119-16) and registered in the WHO International Clinical Trials Registry Platform (KCT0002055).

CONSORT flow chart summarizing the subject’s offerings for the study.

2.3. study Design

The clinical trial was an eight-week, double-blind, randomized, parallel-arm and placebo-controlled trial. During the two-week start and eight-week trial, subjects were instructed by dietitians to maintain dietary and lifestyle habits, including alcohol intake, physical activity and sleep time, but to avoid certain foods that were rich in antioxidant nutrients. After the two-week run-in period, subjects were randomly assigned to either placebo (n = 48) or PMP (n = 48) group and balanced by age and gender (Figure S1). The subjects were assigned to take six tablets, twice daily, for a total of 12 tablets per day. Day of the eight week period, preferably with water in the middle of a meal. Remaining tablets were counted to assess compliance at weeks 4 and 8 during site visits. To assess nutritional intake, lifestyle, and monitor diet compliance during the trial, subjects were given dietary instructions and provided a three-day diet recording (two weekdays and one weekend) at baseline and weeks 4 and 8 using a smart phone application. In the evening before each blood draw (baseline and week 8), subjects consumed a standardized meal to reduce the potential confounding effect of the previous meal. The standardized meal (about 640 kcal; carbohydrate: protein: lipid% ratio = 65:23:12) consisted of steamed rice (340 kcal in 170 g) and Bulgogi (roasted beef marinated with soy bean sauce; 300 kcal in 170 g) . After a quick 12 hours overnight, venous blood was collected in tubes with ethylenediaminetetraacetic acid (EDTA). The plasma and erythrocytes were separated by centrifuge at 4 ° C (1500 × g for 10 minutes). Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density centrifugation using Histopaque®-1077 reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. All samples were stored at -80 ° C until analyzed. The site visit schedule is illustrated in Figure S1.

2.4. Oxidative stress-related biochemical analysis

Plasma reactive oxygen species (ROS) level was measured by luminol-amplified chemiluminescence (5-amino-2,3-dihydro-1,4-phthazinedione, Sigma-Aldrich, St. Louis, MO, USA) under use of a chemiluminescence Fluoroscan Ascent FL (Thermo Fisher, Vantaa, Finland) at 37 ° C[[[[22]. Briefly, 50 µl of plasma and 200 µl of 2 mM luminol (dissolved in 0.05 M NaOH solution) were pipetted to a 96-well microplate (SPL Life Sciences Co., Ltd., Pocheon-si, Gyeonggi-do, Korea) , and then read for 1 minute to measure background. Then 100 µl of 10 mM H2ISLAND2 was added and light emission was analyzed for 15 minutes at 1 minute intervals. After adjusting for background levels, the area under the curve (AUC) for ROS was calculated using the trapezoidal rule.

Comet assay was performed using a single cell gel electrophoresis assay as previously described[[[[23]. Briefly, PBMC was resuspended in 2 ml of cold phosphate buffered saline to a concentration of 1 × 105 cells / ml using an automated cell counter (TC 10TM; BIO-RAD Laboratories, Inc., Hercules, CA, USA). 50 microliters was mixed with 150 µl of 1% low-gelling temperature agarose (Sigma-Aldrich, St. Louis, MO, USA) and immediately dispersed on Comet slides (Trevigen Inc., Gaithersburg, MD, USA) and incubated at 4 ° C for 30 minutes. Slides were then immersed in a light solution (2.5 M NaCl, 100 mM Na 2 EDTA, 10 M Trizma base, 1% (v/vTriton X-100 and 10% (v/vDMSO, pH 10) for 1 hour at 4 ° C. The slides were transferred to cold alkaline electrophoresis solution (10 N NaOH, 200 mM EDTA, pH> 13) and then electrophoresed at 31 V for 30 minutes at 4 ° C. Electrophoresis, the slides were immersed in distilled water twice for 10 min, then 5 min in 70% ethanol and dried overnight at room temperature. DNA was stained with 50 μL SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA) diluted 1: 10,000 in Tris – EDTA buffer (pH 7.5) for 5 minutes at 4 ° C and visualized with a fluorescence microscope (Nikon, Tokyo, Japan) at 4 × magnification. Images from 50 comets on each slide were analyzed with COMET VI image analysis software (Perceptive Instruments, Suffolk, UK).

Total plasma malondialdehyde (MDA) levels were quantified by high performance liquid chromatography fluorescence detection system (HPLC-FLD; Shiseido Co., Ltd., Tokyo, Japan). Fifty microliters of plasma was mixed with 300 μL of 0.44 M phosphoric acid (Duksan Pure Chemicals Co., Ltd., Ansan-si, Gyeonggi-do, Korea) and 150 μL of 42 mM 2-thiobarbituric acid (TBA; Sigma-Aldrich, St. Louis, MO, USA). After incubating plasma samples at 95 ° C for 1 hour, the samples were cooled to 4 ° C followed by centrifugation at 2500 × g for 3 min. The supernatants were filtered with a 0.45 µm PTFE syringe filter (Woongki Science, Seoul, Korea). Ten microliters of the TBA-MDA adduct was injected into a Capcell Pak C18 column (UG120 type, 4.6 mm i.d. × 250 mm, 5 µm particle size; Shiseido Co., Ltd., Tokyo, Japan). The mobile phase was 50 mM phosphate: methanol buffer (7: 3) v/vpH 6.8) at an isocratic flow rate of 1 ml / min. MDA levels were determined from a standard curve using 1,1,3,3-Tetraethoxypropane (Sigma-Aldrich, St. Louis, MO, USA).

Plasma oxidized low density lipoprotein (Ox-LDL) was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instruction (Mercodia, Uppsala, Sweden). The levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and antioxidant enzyme activities (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) in erythrocytes were measured spectrophotometrically using commercially available kits (Cayman, Ann Arbor, MI, USA) follow the manufacturer’s instructions.

2.5. Western Blot

Plasma lysates were prepared and Western blot was performed as previously described[[[[24]. Briefly, plasma from ten individuals from each group was prepared by lysing in radioimmunoprecipitation assay buffer (50 mmol / L Tris, pH 7.3, 150 mmol / L NaCl, 1 mmol / L EDTA, 1% Triton X-100, 0.5% Na-deoxycholate and 0.1% SDS) with protease inhibitors, NaVO4 and NaF. Hundreds of micrograms of plasma lysates were dissolved in 10% SDS-PAGE and then transferred to PVDF membranes. Equal loading and transfer of proteins was verified by Ponceau red staining of the membranes. Blots were incubated using the following antibodies: anti-pCHK1 (Ser345, 1: 500 dilution, Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (1: 5000 dilution, Cell Signaling Technology, Danvers, MA, USA)). Proteins were detected by chemiluminescence detection (Pierce ECL Western blot substrate, Thermo Fisher Scientific Inc., Rockford, IL, USA) and analyzed by FUSION Solo (Vilber Lourmat, Collégien, France).

2.6. Vitamin and phytonutrient analysis

Prior to analysis of plasma phytonutrients, a chemical fingerprint of the PMP study product with ultra-performance liquid chromatography-quadrupole / mass spectrometry for flight time (UPLC-Q-TOF-MS) was formed.[[[[25]and ultra high performance liquid chromatography linear trap quadrupole ion trap tandem mass spectrometry (UHPLC-LTQ-IT-MS / MS)[[[[26]. Briefly, six tablets were pulverized in a mortar and pestle, extracted with 1 ml of methanol and mixed for 1 hour and then centrifuged at 2370 × g for 5 minutes at 4 ° C. The supernatants were then filtered through a 0.2 µm PTFE filter and evaporated at a rate (Modulspin 31; Biotron, Bucheon-si, Gyeonggi-do, Korea). Has microliters (50 mg / ml weight-/v) of each sample was injected into LC-MS.

For analysis of plasma vitamins and phytonutrients, 200 μL of plasma was taken from each subject into four samples containing plasma from 12 individuals (due to limited plasma volume). Eight hundred microliters of plasma was extracted with 3.2 ml of methanol with an MM400 mixing mill (Retsch®, Haan, Germany) with a frequency of 30 s-1 for 10 minutes. After centrifugation at 4 ° C (12,578 × g for 10 minutes), supernatants were filtered through 0.2 µm PTFE filters and then evaporated at a rate vac. The final concentration of each sample was 50 mg / ml (weight-/v).

LC triple Q-MS analysis was performed on Nexera2 LC system (Shimadzu Corp., Kyoto, Japan) combined with a triple quadrupole MS equipped with an electrospray source (LC-MS 8040, Shimadzu). Five microliters were injected into a Kinetex C18 column (100 × 2.1 mm, 2.6 µm, Phenomenex, Torrance, CA, USA) with a mobile phase containing 0.1% formic acid (solvent A) and acetonitrile containing 0 1% formic acid (solvent B) at a flow rate of 300 μL / min. The gradient was 5% solvent B for 1 minute and increased linearly from 5% to 100% over 9 minutes and then decreased to 5% for 1 minute. MS was operated under the following conditions: capillary voltage -3000 V, capillary temperature 350 ° C, evaporator temperature 300 ° C, shear gas 3 L / min, ion sweep gas 2.0 Arb, Aux gas 10 Arb and drying gas 8 L / min. The following multiple reaction monitoring transitions were used: 220> 95 for pantothenic acid, 175> 115 for ascorbic acid, 442> 295 for folic acid, 359> 161 for rosmarinic acid, 609> 301 for hesperidin, 387> 206 for tuberonic acid glucoside 595> 287 for cyanidine 3-ISLAND-glucoside, 609> 301 for routine, 593> 285 for kaempferol rutinoside, 463> 301 for quercetin 3-ISLAND-glucoside, 625> 463 for quercetin diglucoside, 579> 271 for naringin, 301> 151 for quercetin, 463> 301 for peonidine 3-glucoside, 337> 119 for demethoxycurcumin, 367> 217 for curcumin, 283> 268 for wogonin, 418> 356 for gamma-tocopherol, 331> 287 for carnosic acid and 273> 149 for phloretin.

2.7. Quantitative PCR array on peripheral blood mononuclear cells (PBMC)

Quantitative polymerase chain reaction (qPCR) alignment was performed using AccuPower® Customized qPCR panel kit (Bioneer, Daejeon, Korea) as previously described[[[[27]. Briefly, total RNA was extracted using a TRIZOL reagent (Invitrogen Co., Carlsbad, CA, USA). Total RNA concentrations and the 260/280 nm ratio were evaluated using a spectrophotometer (Biospecnano; Shimadzu Corp, Kyoto, Kyoto Prefecture, Japan). Only samples with a 260/280 nm ratio between 1.7 and 2.1 were further processed. cDNA was generated using one AccuPower®Rocket ScriptTMCycle RT PreMix (Bioneer, Daejeon, Korea). qPCR was performed with a Step-One-Plus RT-PCR system (Applied Biosystems, Foster City, CA, USA) in a 96-well microplate using a final volume of 20 μL of the following components: 1 μL ROX dye , 1 µL template, 10 µL 2 × GreenStar qPCR master mix and 8 µL nanofiltered water (Bioneer, Daejeon, Korea) per well. Amplifications were performed using a 10-minute template pre-denaturation step at 95 ° C, followed by 40 cycles of 95 ° C for 5 seconds, 58 ° C for 25 seconds, and 72 ° C for 30 seconds. A total of 88 genes were classified into the following categories and are shown in Table S2: inflammatory mediators and signaling molecules (47 genes), plaque formation and coagulation (3 genes), antioxidant (14 genes), blood cell differentiation (2 genes), and lipid / lipoprotein metabolism (22 genes). The relative amounts of mRNA were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the relative amount of RNA was calculated using the comparative CT Course of action.

2.8. Core interaction network of qPCR analysis

The network-based enrichment analysis of selected genes up-regulated or down-regulated after PMP supplementation was performed using the EnrichNet database (http://enrichnet.org) by default[[[[28]. Gene interaction subnetworks for selected gene set predicted to be functionally associated with genontology terms. Each node represents a physical entity. Each edge represents a gene regulatory interaction.

2.9. Statistical analysis

The sample size was estimated to be 48 individuals per day. Group to provide an 80% strength to demonstrate a significant difference in tail intensity, based on our previous study[[[[19]and a waiver of 20%. All data were analyzed based on a PR protocol principle in accordance with the predefined inclusion criteria. All variables at each time point were tested for normal distribution with the Shapiro – Wilks test, and skewed data were normalized by square root transformation. Differences in baseline characteristics between the placebo and PMP groups were assessed with the help of students t-continuous variables test and chi-square tests for categorical variables. Differences in means of results were analyzed using a linear mixed-effects (LME) model considering a random effect (participant), a random error (within participant), fixed effects (group, week, and the interaction between group and week)) and covariates. Age, gender, body mass index, RFS, total energy intake, smoking, alcohol and dietary β-cryptoxanthin and flavanon intake were used as covariates. In addition, pooled pvalue was derived from multivariate linear mixed-effects model in the combined comet assay data for the effect of PMP on overall DNA damage. Prior to analysis, each parameter was standardized using a z-score transformation by subtracting the mean and then dividing by the standard deviation. The Pearson correlation analysis was used to analyze the association between plasma vitamins and phytonutrients and biomarkers. All statistical analyzes were performed using SAS 9.4 (SAS Institute, Cary, NC, USA) and pvalue <0.05 was considered significant.

3. Results

3.1. Subject characteristics throughout the study

Of 96 eligible subjects, 84 (42 in the placebo group and 42 in the PMP group) completed the eight-week supplement to be included in the analysis (). Nine subjects with Andrew gave their consent for personal reasons, two and one subject were excluded due to the opinion of medicine and investigators respectively. There was no significant difference in baseline characteristics between the two groups (). Participants in this study had RFS of 19.1 ± 1.3 for placebo and 19.1 ± 1.5 for the PMP group, which were categorized as low consumers of fruits and vegetables. Lifestyles including amount of alcohol consumption, physical activity level, and overall sleep time were unchanged during the study (Table S3). The mean compliance for all subjects was 92.8%. No serious or serious side effects were observed.

Table 1

The baseline characteristics of Persia 1.

variable placebo PMP p-value 2
(n = 42) (n = 42)
Age (years) 41.6 ± 1.7 38.2 ± 1.7 0169
Gender Male Female, n) 13/29 13/29 1000
Recommended food score 19.5 ± 1.3 19.1 ± 1.5 0830
Body weight (kg) 67.4 ± 2.1 65.1 ± 2.2 0462
Body mass index (kg / m2) 24.8 ± 0.6 23.7 ± 0.6 0202
Percentage of body fat (%) 31.7 ± 0.9 30.1 ± 1.0 0258
smoking, n (%) 3 (7.1) 4 (9.5) 0693
Alcohol drinks, n (%) 22 (52.4) 24 (57.1) 0661
Blood pressure (mmHg)
Systolic blood pressure 119.1 ± 2.1 116.7 ± 2.0 0414
Diastolic blood pressure 79.5 ± 1.6 79.0 ± 1.5 0786
Blood lipid profiles (mg / dL)
Total triglyceride 142.2 ± 18.1 121.4 ± 9.2 0311
Total cholesterol 189.5 ± 5.7 187.0 ± 4.3 0729
LDL cholesterol 119.4 ± 5.7 120.6 ± 4.2 0856
HDL cholesterol 53.2 ± 2.2 54.1 ± 1.8 0752

3.2. DNA oxidative damage

DNA damage in PBMC was significantly reduced for tail length after PMP supplementation compared with placebo group (p = 0.042) (A). Although there were no significant differences in tail intensity and tail torque changes between placebo and PMP treated group, when all comet parameters were combined and analyzed using the multivariate linear mixed-effects model, overall pvalue was 0.032 in PMP compared to placebo. In addition, we assessed DNA damage and repair response by measuring plasma phosphorylated checkpoint kinase 1 (pCHK1-Ser345) protein level after supplementation. Among 10 plasma samples for each group, eight placebo and six PMP group samples were used for Western blot analysis. After eight weeks of supplementation, pCHK1 protein levels had an increase in PMP group compared to baseline (B; p = 0.091). Taken together, PMP supplementation induced DNA repair cell signaling and increased plasma DNA damage and repair response.

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Effect of Eight Week Grants on DNA Damage and Repair: (One) Comet Assay at PBMC; and (B) Representative Western blot of plasma phosphorylated checkpoint kinase 1 (pCHK1-Ser345). Change in DNA damage and repair mechanisms is presented as a box and scatter plot. The ends of the box are the upper and lower quartiles. The medians are marked with a vertical line inside the boxes. The erasers are the two lines outside the box that extend to the highest and lowest value. Dots are individual values ​​for each individual. For Western blot, representative samples (n = 8 for placebo and n = 6 for PMP) was analyzed. CT (a sample from the PMP group) indicates a load check to analyze quantitation data, 0 indicates before supplementation and 8 indicates after supplementation. Statistical significance of comet assay was determined by a linear model with mixed effects. In the case of Western blot, mixed-model linear model was used to compare the difference within each group (* p <0.05). LS means ().

3.3. ROS Scavenging

Luminol-dependent chemiluminescence was used to determine plasma ROS levels after placebo and PMP supplementation for eight weeks. PMP group had significantly lower ROS AUC compared to placebo group (p = 0.018) (). However, other plasma biomarkers measured in this study, such as MDA and Ox-LDL levels and erythrocyte SOD and GPx activity, were not significantly different between the two groups ().

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Box and scatter plot for the change in plasma ROS AUC after eight weeks of supplementation. ROS AUC was calculated using the trapezoidal rule. The ends of the crates are the upper and lower quartiles. The median is marked by a vertical line inside the box. The erasers are the two lines outside the box that extend to the highest and lowest value. Dots are individual values ​​for each individual. Statistical significance was determined by a linear model with mixed effects (* p <0.05). Reactive Oxygen Species (ROS); area under the curve (AUC); LS means ().

Table 2

The effect of placebo and PMP supplementation on lipid oxidation and endogenous antioxidant defense 1.

variable Placebo (n = 42) PMP (n = 42) discretion 2 p-value
Week 0 Week 8 Week 0 Week 8
SOD activity (U / ml) 200.25 ± 4.32 192.76 ± 3.63 205.87 ± 4.32 206.02 ± 3.63 7637 0250
GPx activity (µmol / min / ml) 1.12 ± 0.04 1.10 ± 0.04 1.11 ± 0.04 1.11 ± 0.04 0017 0559
MDA (µmol / L) 2.97 ± 0.14 2.99 ± 0.14 2.96 ± 0.14 3.02 ± 0.14 0040 0774
Oxidized LDL (U / L) 41.18 ± 1.84 39.89 ± 1.73 43.25 ± 1.84 41.78 ± 1.73 -0,176 0914

3.4. qPCR RNA Array Analysis

Of the 88 genes analyzed in the PCR matrix, 87 genes amplified, all except NOS2, but there were no significant differences between the two groups in either gene (Table S4). Although no statistical significance was found, network enrichment analysis was performed to analyze interactions between genes and to investigate biological pathways potentially modulated by PMP supplementation. Genes that were not altered in placebo but altered in PMP supplementation were selected for network analysis. Among 87 genes, 52 changed over 20% in the placebo group, which was then removed from network analysis. Of the remaining 35 genes that showed no changes in the placebo group, the PMP supplemented group showed up-regulation of 32 genes and down-regulation of 3 genes. In addition to the genes measured in this study (blue and green circles), associated genes (red circle) are shown in. Network enrichment analysis with GO annotation system indicated that among up-regulated genes, SOD2, CYBA and CYBB are involved in superoxide metabolic processes (). In addition, CCS, CBS, CYB5R4, NCF1, NCF2, NOS2, NOX, NOXA1, NOXO1, PREX1, SH3PXD2A, SH3PXD2B, SOD1, and SOD3 were associated with up-regulated genes, which are also genes involved in superoxide metabolic processes. In the GO pathway analysis, several pathways related to ROS, such as superoxide metabolic pathway and superoxide anion generation, were ranked in the Top 10 altered pathways with statistical significance (q <0.05; Table S5).

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Network of up-regulated genes altered by PMP supplementation for eight weeks. Genes were selected based on differential expression pattern compared with placebo. Each node represents a physical entity. Each edge represents a gene regulatory interaction. Blue circles are genes that are targeted and analyzed by PCR analysis. Red circles are the general process provided by the EnrichNet database (http://enrichnet.org), but not analyzed. Green circles denote overlapped gene between blue and red genes.

3.5. Measurement of vitamin and phytonutrient

One vitamin and 19 phytonutrients were identified in PMP product by UPLC-Q-TOF-MS and UHPLC-LTQ-IT-MS / MS analysis (A and Table S6). Three vitamins and three phytonutrients were detected in the plasma by LC triple-Q-MS analysis (B). Pantothenic acid increased significantly (p = 0.007) in PMP compared to placebo group. All other phytonutrients and vitamins were not significantly different. The correlation between biomarkers and phytonutrients and vitamins analyzed in plasma is illustrated by a heat map () and r values ​​with p-values ​​(). Plasma folic acid and hesperidin levels were negatively correlated with ROS AUC (p <0.05). Ascorbic acid and rosemary acid were negatively correlated with DNA tail intensity (p <0.05). Plasma ascorbic acid, rosmarinic acid and hesperidin levels were negatively correlated with tail length (p <0.05). Plasma pantothenic and ascorbic acid levels were negatively correlated with tail torque (p <0.05). Rosemary acid in plasma was negatively correlated with SOD levels (p <0.05).

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(One) UPLC-Q-TOF-MS chromatographic fingerprint of the PMP study product; and (B) change in plasma after eight weeks of supplementation. The y-axis of box diagrams indicates the change in the relative peak area of ​​each vitamin and phytonutrient. Statistical significance was determined by a linear model with mixed effects (* p <0.05). LS funds ().

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Heat map illustrating Pearson correlation coefficients between plasma and erythrocyte biomarkers and plasma vitamin and phytonutrients. Reactive Oxygen Species (ROS); area under the curve (AUC); superoxide dismutase (SOD); glutathione peroxidase (GPx); malondialdehyde (MDA); oxidized low density lipoprotein (Ox-LDL). * p <0,05.

Tabel 3

Korrelation mellem biomarkører og plasma-vitaminer og phytonutrients 1.

Pantothensyre Ascorbinsyre Folinsyre Rosmarinsyre hesperidin Tuberonsyre glukosid
r p
r p r p r p r p r p
ROS AUC -0227 0,0549 -0194 0,1022 -0233 0,0485 -0,029 0,8070 -0365 0,0016 -0113 0,3431
Haleintensitet -0148 0,1969 -0471 <0,0001 0,006 0,9618 -0338 0,0025 -0127 0,2680 -0038 0,7397
Hale længde -0187 0,0921 -0390 0,0003 -0032 0,7777 -0221 0,0464 -0217 0,0497 -0178 0,1100
Hale øjeblik -0238 0,0367 -0608 <0,0001 -0087 0,4529 -0,158 0,1713 -0211 0,0659 -0186 0,1061
SOD-aktivitet 0,121 0,2730 -0222 0,0420 -0019 0,8667 -0268 0,0139 0,073 0,5088 0,026 0,8170
GPx-aktivitet 0,008 0,9417 -0138 0,2109 0,074 0,5013 0,058 0,6020 0,073 0,5081 -0,021 0,8484
Plasma MDA 0,075 0.4964 0.165 0.1329 −0.073 0.5102 0.138 0.2118 −0.125 0.2578 −0.001 0.9919
Ox-LDL −0.078 0.4786 0.036 0.7422 −0.039 0.7261 0.069 0.5358 0.111 0.3150 0.046 0.6794

4. Discussion

In our previous clinical trial, nutritional supplementation with a multivitamin and mineral containing phytonutrients was effective in reducing oxidative damage while maintaining endogenous ROS homeostasis[[[[19]. In this trial, PMP supplementation reduced DNA damage without altering endogenous antioxidant enzyme activities, and increased ROS scavenging, which is consistent with our previous study. A certain amount of oxidative stress is useful to the body for growth and cell signaling, and our body has a defense system for controlling low level of oxidative stress such as glutathione, vitamin C, vitamin E, and antioxidant enzymes[[[[29]. It has been reported that low grade level of oxidative stress is crucial to maintaining and priming our endogenous antioxidant system against high levels of oxidative stress and damage[[[[30]. Based on our two human intervention studies, PMP supplementation was effective on scavenging ROS and preventing DNA damage without stimulating antioxidant enzymes.

Single-cell electrophoresis (also known as the comet assay) is widely used for measuring ROS-induced DNA damage and fragmentation[[[[31]. There are several reports that DNA repair is enhanced by fruit and vegetables[[[[32.33.34]. According to our previous study, comet assay on PBMC from subjects who had low RFS showed improved DNA repair after supplementation with antioxidant and phytonutrients[[[[19]. In this current study, we showed that PMP supplementation reduced DNA tails length in PBMC. Although comet assay has been widely used for testing DNA repair, there is no standard reference value. Therefore, in this study, we tested comet assay before and after PMP and placebo supplementation and compared the changed values between placebo and PMP groups. In addition, compared to our previous study, we examined the protein expression of plasma pCHK1-Ser345 levels and detected modest increases in protein levels after PMP supplementation. Cellular responses to DNA repair are initiated by the ATR-CHK1 pathway, which is activated by single-stranded DNA breaks according to oxidative stress in a baseline of physiology status[[[[35]. These data suggest that the PMP supplementation might provide protection against ROS-induced DNA damage by initiating single-strand break repair signaling responses via ATR-CHK1 pathway[[[[36.37]. The effect of PMP supplements such as components of folic acid and ascorbic acid may be attributed in part to the induced pCHK1 protein expression of DNA damage and repair pathway[[[[38].

To examine whether circulating oxidative stress genes were modulated by PMP supplementation, a qPCR array containing oxidative and inflammatory stress genes was performed on PBMC. PMP supplementation did not significantly stimulate or suppress genes involved in oxidative defenses. In addition, PMP supplementation did not alter the expressions of endogenous antioxidant genes. However, according to enriched network analysis, subtle changes of each gene might have induced relationships between genes and proteins with similar functions. In the PMP group compared to placebo, genes such as SOD2, CYBA, and CYBB were modestly upregulated and these genes are involved in superoxide metabolic processes. It has been reported that CYBB deficiency enhances multiple inflammatory cascades and deficiency of NCF1 ameliorates the disease[[[[39].

Phytonutrients are known to be poorly absorbed in small intestine and many are metabolized in the gut mucosa and/or liver followed by conjugation to glucuronide, sulfate and/or methyl groups[[[[40]. In addition, phytonutrients reaching the colon are extensively transformed by the microbiota and then excreted in bile and urine, usually within 24–48 h[[[[16]. In this study, the levels of plasma phytonutrient were low and there is limited information on their metabolized forms. Rosmarinic acid and hesperidin have been reported to be metabolized by colonic bacteria[[[[41.42]. When orally ingested, hesperidin cannot be metabolized by β-glucosidase in the small intestine but hydrolyzed to hesperetin aglycon by colonic microbiota[[[[42]. Rosmarinic acid is known to be degraded into caffeic acid and 3-(3,4-dihydroxyphenyl)lactic acid[[[[43.44]. Therefore, it was difficult to quantify phytonutrients in plasma as intact form. However, with limitation for obtaining standard compounds for each metabolite, in this study, several identified phytonutrients containing in PMP were analyzed in pooled plasma samples. Among phytonutrients, rosmarinic acid, hesperidin, and tuberonic acid glucoside, and among vitamins, pantothenic acid, ascorbic acid, and folic acid were negatively correlated with ROS and DNA damage. Rosmarinic acid and hesperidin showed significant negative correlation to tail intensity and length. In addition, hesperidin also showed significant negative correlation to ROS scavenging. Rosmarinic acid and hesperidin have been reported to prevent DNA damage and scavenge ROS; however, these studies are in vitro and animal studies[[[[]. Although mechanisms of action of these flavonoids and their metabolites could not be revealed in the present trial, this is the first clinical study reporting a relationship between the level of rosmarinic acid and hesperidin to DNA damage and ROS scavenging.


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