Sixty-nine healthy subjects (aged 20–65 years) were recruited during routine check-ups at a health promotion center at Yonsei University Hospital from May to November in 2009. Participants were asked to visit four times (1 week before and at 0, 4, and 8 weeks). Blood samples were taken at weeks 0 and 8, and diet and capsule intakes were assessed at weeks 0, 4, and 8. Healthy subjects who either used cigarettes or consumed alcohol (or both) were enrolled in this study at the Laboratory of Clinical Nutrigenetics/Nutrigenomics at Yonsei University. The exclusion criteria were the consumption of more than three servings of vegetables or fruit per day, antioxidant or vitamin/mineral supplementation, history of chronic disease (e.g., diabetes, heart disease, renal disease), or the use of medication (e.g., lipid-lowering or antihypertensive medications, anti-platelets, or disease-related medications). The participants were interviewed to determine their smoking and drinking habits. Smoking status was categorized as “current smoker” or “nonsmoker,” and alcohol consumption was categorized as “current drinker” or “nondrinker.” Nonsmokers or nondrinkers included both ex-consumers and those who had never consumed. Ex-smokers and ex-drinkers were defined as subjects who stopped smoking or drinking at least 1 year prior to participating in the study. Current smokers were asked the number of cigarettes smoked per day. Current drinkers were asked about the type and amount of alcohol consumed per day, and the amount of alcohol was then calculated as grams per day.
The study subjects number calculation assumed a two-tailed alpha = 0.05 and 1-β = 90% to detect a 10% difference with a standard error of 0.28 in the comet assay which is primary outcome and an attrition rate (20%). Biomarkers for testing antioxidant/oxidative effects and the number of subjects were determined by following the criteria recommended by the Korea Food and Drug Administration. Of the 69 study subjects, 12 subjects (placebo group, n = 4; low-dose group [3 g KRG/day], n = 4; high-dose group, [6 g KRG/day], n = 4) discontinued the study for personal reasons. No adverse reactions (e.g., fever, hot flush, nausea, vomiting, diarrhea) due to KRG supplementation were observed among the 57 subjects who completed the study.
Test capsule and study design
Identical-looking capsules contained red ginseng (low dose, 300 mg; high dose, 600 mg) or placebo (300 mg of KRG-flavored capsule containing corn starch). Red ginseng and placebo capsules were provided by the Korea Ginseng Cooperation (KGC, Daejeon, Korea). The red ginseng contained 16.58 mg/g total ginsenosides, and the ratio of protopanaxadiol ginsenosides (Rb1, Rb2, Rc, Rd, and Rg3) to protopanaxatriol ginsenosides (Rg1, Re, and Rf) was 1.65. Analyses of common ginsenosides were performed in quadruplicate using standard HPLC-UV techniques  at the Korean Ginseng Research Institute in Daejeon, Korea.
This study design was the randomized double-blind, placebo-controlled intervention trial for 8 week and was approved by the Institutional Review Board of Yonsei University (#RA-2009-650). After written informed consent was obtained, subjects were randomly assigned to placebo (n = 23) or KRG (low-dose [n = 24] or high-dose [n = 22]) groups. All subjects were asked to take 10 capsules in total per day immediately after any main meals. For example, three capsules after breakfast, three capsules after lunch, and four capsules after dinner. Compliance of KRG consumption was assessed at the end of the study by counting capsules remaining and self-recording. If compliance was beneath 75%, the subject dropped out. All participants were encouraged to maintain their usual lifestyle and dietary habits.
Assessment of dietary intake and level of physical activity
The subjects’ usual diet information was obtained using both a 24-hour recall method and a semi-quantitative food frequency questionnaire (SQFFQ), which has been tested previously for validity . We used the former to carry out analyses and the latter to check if the data collected by the 24-hour recall method was representative of the usual dietary pattern.
All the subjects were given written and verbal instructions by a registered dietitian for the completion of a 3-day (two weekdays and one weekend) dietary record every 4 weeks. On the sheet, the subjects were instructed to record the amount of food before ingestion and any remaining after ingestion by weighing the foods. One week before the start of the study, all the participants were advised to continue their usual diet for a week and were instructed to complete a 3-day dietary record as the baseline measurement, which was compared with the record obtained by 24-hour recall methods at a previous visit to check their records. The participants were also instructed to record their physical activity in a diary for 24 hours every 4 weeks. To check participants’ compliance during the whole study period, the dietitian interviewed them biweekly by telephone. During the study period, all participants were also encouraged to maintain their usual lifestyle.
Dietary energy values and nutrient content from the 3-day food records were calculated using the Computer Aided Nutritional analysis program (CAN-pro 2.0, Korean Nutrition Society, Seoul, Korea). Total energy expenditure (kcal/day) was calculated from activity patterns, including basal metabolic rate, physical activity for 24 hours , and specific dynamic action of food. Basal metabolic rates for each subject were calculated with the Harris-Benedict equation .
Anthropometric parameters, blood pressure, and blood and urine collection
Body weight and height were measured without clothes or shoes in the morning to determine the body mass index (BMI; kg/m2). Blood pressure was measured with an automatic blood pressure monitor (TM-2654, A&D, Tokyo, Japan) in triplicate using the left arm of a seated subject after a 20-min rest. After a 12 h fasting period, venous blood specimens were collected in EDTA-treated and plain tubes, centrifuged to produce plasma or serum, and stored at −70°C until analysis. Urine was collected in polyethylene bottles containing 1% butylated hydroxytoluene after 12 h of fasting. The tubes were immediately covered with aluminum foil and stored at −70°C until analysis.
Serum lipid profile and fasting glucose
Fasting total cholesterol and triglycerides were measured using commercially available kits on a Hitachi 7150 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). After precipitation of chylomicrons with dextran sulfate magnesium, concentrations of LDL and high-density lipoprotein (HDL) cholesterol in the supernatants were determined enzymatically. In participants with serum triglyceride concentrations below 400 mg/mL, LDL cholesterol was indirectly measured using the Friedewald formula. Fasting glucose levels were determined by the glucose oxidase method using a Beckman glucose analyzer (Beckman Instruments, Irvine, CA, USA).
Serum Blood Urea Nitrogen (BUN) and creatinine concentrations were measured by a colorimetric method using commercially available kits on a Hitachi 7180 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by a kinetic UV method based on recommendations by the International Federation of Clinical Chemistry (IFCC) using commercially available kits on a Hitachi 7180 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). A complete blood count (CBC) test was determined using the HOROBA ABX diagnostic (HORIBA ABX SAS, Parc Euromedicine, France).
Alkaline comet assay for DNA damage
As a primary outcome measure, DNA damage was assessed using the comet method described by Green et al. . Whole blood was mixed with phosphate buffered saline and poured gently over a lymphocyte separation solution (Histopaque-1077, Sigma Chemical Co., Korea). After centrifugation at 1,450 rpm for 4 min, lymphocytes were transferred to another tube, mixed with 0.7% low-melting agarose, and then added to slides pre-coated with 0.5% agarose. The slides were immersed in freshly prepared cold lysing solution and subjected to electrophoresis. The slides were then washed with neutralizing buffer and treated with ethanol. Tail length (μm) and tail moment (% of DNA in the tail × tail length) were evaluated using image analysis software (Komet 5.0, Kinetic Imaging, UK) and fluorescence microscopy (Leica, Germany) equipment with filters. Images from 50 cells from each slide were analyzed. All steps were performed in dim light, and the electrophoresis tank was covered with black paper to avoid additional light-induced DNA damage.
Antioxidative enzyme activities
Plasma and erythrocyte antioxidative enzyme activities were measured as secondary outcomes. Plasma and erythrocyte SOD activity was determined based on the generation of superoxide radicals produced by xanthine and hypoxanthine. Superoxide radicals react with tetrazolium chloride salts to produce a red formazan dye. SOD activity was estimated by the inhibition of this colorimetric reaction using an assay kit (Cayman Chemical, Ann Arbor, MI, USA), and the absorbance at 450 nm was measured with a Wallac Victor2 multilabel counter (Perkin Elmer Life Sciences, Turka, Finland). The intra-assay and inter-assay coefficients of variance were 4.43% and 8.26%, respectively. Plasma GPx activity was indirectly measured using an assay kit that involved a coupled reaction with glutathione reductase (Cayman Chemical). Oxidized glutathione, generated by the reduction of hydroperoxide by GPx, was restored to its reduced state by glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH). NADPH oxidation was monitored at a lower 340 nm absorbance, which was measured with the Wallac Victor2 multilabel counter. The intra-assay and inter-assay coefficients of variance were 6.01% and 7.68%, respectively. Plasma and erythrocyte catalase activity was measured based on the generation of formaldehyde from methanol in the presence of H2O2 using an assay kit (Cayman Chemical). Formaldehyde forms a bicyclic heterocycle with the chromogen Purpald, which changes from colorless to a purple color when oxidized. The absorbance was measured at 540 nm with the Wallac Victor2 multilabel counter. The intra-assay and inter-assay coefficients of variance were 5.52% and 6.78%, respectively.
Plasma oxidized LDL concentration and Urinary 8-epi-PGF2α
As one of the secondary outcome measures, plasma oxidized LDL concentration and urinary 8-epi-PGF2α excretion. Plasma oxidized LDL was determined with an enzyme immunoassay (Mercodia, Uppsala, Sweden), and the color reaction was measured at 450 nm with the Wallac Victor2 multilabel counter. The intra-assay and inter-assay coefficients of variance were 6.52% and 8.15%, respectively. Urinary 8-epi-PGF2α was measured using a colorimetric enzyme immunoassay (Bioxytech urinary 8-epi-PGF2α assay kit; OXIS International Inc., OR, USA). The resulting color was measured at 650 nm using the Wallac Victor2 multilabel counter. Urinary creatinine was determined using a Hitachi 7180 Autoanalyzer (Hitachi Ltd., Tokyo, Japan) by the alkaline picrated (Jeffe) reaction. Urinary 8-epi-PGF2α concentrations were expressed as pmol/mmol of creatinine. The intra-assay and inter-assay coefficients of variance were 4.32% and 9.57%, respectively.
Statistical analyses were performed with SPSS version 12.0 for Windows (Statistical Package for the Social Sciences (SPSS) Inc., Chicago, IL, USA). We used the Wilcoxon signed-rank test to determine within-group differences of biochemical parameters before (baseline) and after the intervention. The nonparametric Kruskal-Wallis test and analysis of covariance (ANCOVA) using the general linear model (GLM) with adjustments for age, sex, BMI, smoking, drinking, systolic BP, diastolic BP, and baseline values were used to compare the three groups. Spearman’s correlation coefficients were used to determine relationships between variables. After determining whether data was normally distributed by the Kolmogorov-Smirnov and Shapiro-Wilk W tests, a logarithmic transformation was applied to non-normal data before statistical analysis. For descriptive purposes, the results are expressed as untransformed and unadjusted mean values. Continuous variables are expressed as mean ± SEM, and categorical variables are expressed as absolute numbers and percentages. A two-tailed value of P less than 0.05 was considered significant.