Eighty six volunteers aged 20-60 years were recruited from the local community in Daegu (population 4.1 million), Republic of Korea. Inclusion criteria were overweight individuals with BMI > 23 and < 29. We used a lower BMI cut-off for overweight individuals compared to the World Health Organization overweight BMI criteria, because some reports suggest the prevalence of obesity related co-morbidities is higher in Asian cohort studies at a lower BMI cut-off . Exclusion criteria included: pregnancy, smoking, serious illness, current treatment with any medications for the control of blood glucose levels, clinical or biochemical evidence of acute or chronic infection, hepatic dysfunction, chronic alcohol consumption, regular functional food supplement consumption which may affect the outcome of this study or any major surgery in the 6 months prior to the study. In addition, no subjects were actively using any other methods for weight reduction or control of blood lipids, including hypocaloric diet, anorexic drugs or lipid-lowering drugs. The study was performed in accordance with the Declaration of Helsinki. All subjects provided written informed consent prior to participating in this study. The study protocol was approved by the Kyungpook National University Human Research Committee (No. 2010-2).
The study design was a randomized, double-blind, placebo-controlled trial to determine the effectiveness of extract of Glycine max leaves or Garcinia cambogia extract supplementation in overweight subjects to alter body composition, plasma cholesterol, lipids, adipocytokine or antioxidant levels. The primary outcome measures were %body fat and plasma total cholesterol. The secondary outcome measures were plasma TG, HDL-C, non-HDL-C, %HTR, atherosclerosis index, FFA, phospholipid, Apo A-1, Apo B, Apo B/Apo A-1, antioxidant enzyme activity and adipocytokine concentrations. Subjects were randomly assigned into three nutritional intervention groups, extract of Glycine max leaves (EGML; n = 28), Garcinia cambogia extract (GCE; n = 29) and placebo (n = 29). Assignment of subjects to nutritional intervention groups was conducted using randomly generated numbers. Both subjects and investigators were blinded to the nutritional intervention allocated. Subjects were instructed to maintain their routine food intake and physical activity throughout the course of the study.
At baseline and after the 10 week nutritional intervention subjects attended the Science Research Center laboratory at Kyungpook National University between 08:00 and 11:00 h after a 12-14 h overnight fast. Blood samples were drawn into heparin coated tubes, then centrifuged at 3000× g for 15 min at 4°C and stored at -70°C until plasma lipid, adipocytokine, antioxidant and toxicology analysis.
Waist girth was measured at the minimum circumference between the iliac crest and the rib cage. Hip girth was measured at the maximum width over the greater trochanters. Waist-to-hip ratio (WHR) was calculated as waist girth divided by hip girth. Systolic and diastolic blood pressure (BP) was measured using an automatic blood pressure monitor (Omron, Japan).
Body composition was determined with the X-Scan plus II body composition analyzer (Jawon Medical Company, Republic of Korea). The X-Scan plus II uses a 4-point tactile electrode system that measures the total and segmental impedance and phase angle of alternating electric current at 8-12 different frequencies. The X-Scan plus II software automatically calculates body composition based on tetrapolar bioelectrical impedance  with a proprietary formula: Total body water = A × Height2/Impedance + B × Weight + C × Age + D × Gender + E. Constants were derived from a validation study using the isotope dilution method. Before the body composition assessment, the subjects rested for 10 min, and then were asked to remove outer clothing (coats, sweaters), shoes and socks. Body weight was measured with a digital scale to the nearest 0.1 kg. Height was assessed with a stadiometer to the nearest 0.1 cm. Subjects were instructed to stand on the instrument's foot platforms and hold the palm-and-thumb electrodes, with their arms not touching their torso, while body composition was determined. Day-to-day reproducibility of bioelectrical impedance based body composition monitors to determine %body fat is reported to be 3.5-5% . Body mass index (BMI) was calculated as weight(kg)/height(m2).
Food intake was recorded before and during the nutritional intervention trial using a 24 h dietary recall. Subjects were instructed to recall and describe the foods and beverages consumed over the previous 24 h. Subjects were asked to estimate portion sizes using common household bowls, cups and spoons. The 24 h dietary record for each subject was coded, and standard reference tables were used to convert estimated food portions to weight in grams. Nutritional analysis was performed using CAN-Pro 3.0 software (The Korean Nutrition Society), which provides a comprehensive database of the nutritional content of general foods and specialty Korean foods. The 24 h diet recall method has been reported to be a reliable and valid method to determine daily nutritional intake, which has been used in previous clinical trials [25, 26].
Subjects consumed either four capsules containing EGML (2000 mg/day; Yuyu Health Care Co., Korea), eight capsules containing GCE (2000 mg/day; 60% hydroxyl citric acid; Newtree Inc., Korea) or four capsules containing placebo (2000 mg/day starch) in the morning and evening daily for 10 weeks. The total polyphenol content of EGML determined by HPLC (Shimadzu Corp., Japan) was 44.5 ± 2.1 mg gallic acid equivalents/g of EGML. The compliance of subjects to the nutritional intervention was regularly monitored every second day by telephone during the entire study period, all subjects reported consuming the supplement capsules as instructed. No serious adverse effects were reported by subjects consuming EGML, GCE or placebo supplements. There were no subject withdrawals from the study, therefore intention to treat analysis was not necessary.
Plasma lipid and apolipoprotein analyses
Plasma total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C), free fatty acid (FFA) and phospholipid (PL) concentrations were determined using commercially available kits based on enzymatic methods (Asan Pharm. Co., Korea). The ratio of HDL cholesterol to total cholesterol (HTR) was calculated as [(HDL-C/Total-Cholesterol × 100)]. Atherogenic index (AI) was calculated by [(Total-C - HDL-C)/HDL-C], Non-HDL-C was determined by [Total-C - HDL-C]. The apolipoprotin A-1 (Apo A-1) and apolipoprotein B (Apo B) were measured using commercial assay kits (ALerCHEK. Inc, USA).
Plasma toxicology analysis
Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were measured to assess toxicity of the EGML, GCE and placebo supplements. Plasma GOT and GPT activities were determined based on enzymatic methods using commercially available kits (Asan Pharm. Co., Korea).
Oxidative stress and antioxidant analysis
To determine whether EGML and GCE supplementation provided protection against oxidative stress, the activity of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured in erythrocytes. To separate erythrocytes, EDTA treated blood samples were centrifuged at 3000× g for 15 min at 4°C. Erythrocytes were washed three times in 0.9% NaCl, lysed and mixed to produce a hemolysate. The hemoglobin concentration of the hemolysate was then estimated using a commercial assay kit (Asan Pharm. Co. Korea). SOD activity was measured according to the method of Marklund and Marklund . SOD activity was expressed in unit/g hemoglobin. The CAT activity was measured using previously published methods . A molar extinction coefficient of 0.041 (mM-1cm-1) was used to determine the CAT activity. CAT activity was defined as the reduction in μmol H2O2/min/g hemoglobin. GSH-Px activity was measured using the method described by Paglia and Valentine . GSH-Px activity was expressed as oxidized μmol NADPH/min/g hemoglobin. As a marker of lipid peroxide production, the plasma thiobarbituric acid-reactive substances (TBARS) concentration was measured using the method of Tarladgis et al. .
Plasma adipocytokine analysis
Adiponection, adipsin, leptin, resistin, TNF-α and IL-6 were measured in plasma samples, using multiplex detection kits (Bio-Rad, Hercules, USA). Data analyses were performed using the Bio-Plex Manager software version 4.1.1 (Bio-Rad, Hercules, USA).
All data are presented as mean ± S.E. Statistical analysis was performed using SPSS software (version 11.5). Significant within group changes in body composition, plasma lipids, adipocytokine and antioxidant parameters between baseline and 10 weeks were assessed using paired Student's t-test. Significant differences between EGML, GCE and placebo supplemented groups at baseline and at 10 weeks were analyzed using one-way ANOVA. Post-hoc Duncan's multiple range tests were conducted when appropriate to further examine any significant between group differences at 10 weeks. Statistically significant differences were accepted at p < 0.05.