This study was reviewed and approved by the Institutional Review Board (IRB) (CUH 2017-11-009) of Chonbuk National University Hospital (CBNU). The entire human study was conducted in accordance with the provisions of the Helsinki Declaration and the standards for clinical trial management (IGCP), and the protocol was registered at Clinical Research Information Service of Republic of Korea (https://cris.nih.go.kr/cris/en/: board approval number: KCT0003002). This study was performed from January 2018 to March 2018 as a single-organization, randomized, parallel-group-controlled clinical study.
The subjects in this study were recruited from the Clinic Trial Center for Functional Foods (CTCF2) at CBNU through advertising (brochures, posters) and the CBNU website. Volunteers were evaluated for eligibility after providing written consent. Suitable subjects for this study were selected through screening tests such as medical interviews, medical examinations, and diagnostic medical examinations within four weeks (Day − 28 to Day − 1) of the baseline evaluation date (Day 0). To meet the criteria for selection, subjects needed to be women older than 19 and younger than 49 years at the time of the screening test, with body mass index (BMI) of 23.5 to 30 kg/m2. The subjects received and fully understood the detailed description of this human study, voluntarily decided to participate, and agreed in writing to comply with the precautions.
Subjects who met any of the following criteria were excluded from this study:
1) those who had lost more than 10% of their body weight within three months before the screening test; 2) those who had taken medicines or health supplements related to detoxification or weight loss within one month prior to the screening test; 3) those with clinically significant acute or chronic diseases of the cardio-cerebrovascular system, endocrine system, immune system, respiratory system, hepatobiliary system, kidney and urinary system, nervous system, or musculoskeletal system or with inflammatory diseases or blood tumors; 4) those with a history of gastrointestinal disease (e.g., Crohn’s disease) or gastrointestinal surgery (excluding simple appendectomy or herniotomy) that could affect absorption of the study diet; 5) those with hypersensitivity reactions to the ingredients of the study diet; 6) those who had taken antipsychotic drugs or narcotic analgesics within six months prior to the screening test; 7) those suspected of drug abuse or a history thereof; 8) those drinking alcohol in excess of 21 units/weeks or have a history of alcohol abuse; 9) those with serum AST or ALT level three times greater than the upper limit of the reference range, or serum creatinine level over 2.0 mg/dL in diagnostic examinations; 10) those who had participated in other studies within two months of the screening test; 11) those who were in menopause (for more than 12 months) or perimenopause period (for a continuous period of 3 months or more); 12) those who were pregnant, breastfeeding, or planning to become pregnant during the study; 13) those who did not agree with the use of effective contraception methods (condoms, contraceptives, intrauterine contraceptives, and male partners with vasectomy) during the study period; and 14) those who were deemed unfit for this study by the tester due to diagnostic examination results or other reasons.
Among 61 volunteers who provided written consent to participate in this study, 45 who met the selection and exclusion criteria were selected. Among the 45 selected subjects, 30 were assigned 1:1 to the test group and control group 1 by a single-blind and random arrangement method, and 15 were assigned to control group 2. A total of 45 participants were randomly assigned into one of the study groups (15 subjects each) using a computer-generated random number table by the Randomization program of the version 9.2 SAS® system (SAS Institute, Cary, NC, USA). The subjects were to consume the respective study diets for four weeks (test group, WD; control group 1, CRD; control group 2, MRD). The average daily energy values of the menus provided to the test group and control group 1 are shown in Table 1. All 45 of the registered subjects completed all the procedures and examinations specified in the test plan.
Experimental diet (Wellnessup diet: WD)
The diet of the test group (WD) consisted of organic ingredients produced by smart farms (pesticide-free and pollution-free). The menu consisted of breakfast (shakes), lunch (fresh fruit and vegetable juice), dinner (salads), and snacks (nut bars) and was followed on 14days cycle. The ingredients in the shakes included whole-food materials to promote detoxification while supplementing grain, and fruits powder. The shakes were delivered in powder form in individually packed sticks, which were to be opened, poured into a shake bottle, combined with at least 250 mL of water (the amount of water used is a matter of personal preference), and mixed well before consumption. For lunch, 450 mL of vegetable juice (a form made by grinding various raw fruits and vegetables) was refrigerated and shaken before consumption. For dinner, the salad consisted of about 400 g of organic green vegetables, whole grains, meat, and fruit, along with a salad dressing. The nut bars was made using a variety of dried fruits and roasted nuts. Which was recommended as snacks between lunch and dinner and were to be stored at room temperature (Supplementary Table 1).
Control group 1 (Calorie-restricted diet: CRD)
The diet of control group 1 was similar to the WD in its dietary weight and caloric content. This calorie-restricted diet consisted of a shake for breakfast, fruit and vegetable juice for lunch, salad for dinner, and nut bars for snacks, as in the test group. The CRD was composed of general ingredients purchased from supermarkets. Shake powder products for market sale (grain and yogurt) were used for breakfast, and fruit juice products for market sale were served for lunch. Salads consisting of vegetables, fruit, meat, and whole grains cultivated by general farming were served for dinner, and nut bars were provided as snacks. For breakfast, the shake was prepared through addition of at least 250 mL of water (based on personal preference) in a shake bottle, mixed well before consumption. For lunch, 450 mL of refrigerated vegetable juice was shaken and ingested. For dinner, the salad consisted of about 400 g of green vegetables, whole grains, meat, and fruit, along with a salad dressing. The nut bars were recommended as snacks between lunch and dinner and were to be stored at room temperature.
Control group 2 (Maintaining regular diet: MRD)
The subjects assigned to control group 2 were required to maintain their daily meal patterns without calorie restriction for four weeks and did not receive provided meals. The subjects recorded all foods and beverages consumed for 28 days in detail on a diet record form.
Compliance and safety of the study subjects
We recommended that the subjects eat only the clinical research diet provided during the study period and asked them to maintain the same level of physical activity that they were performing before the study to minimize the impact of lifestyle changes on the test results. To observe changes in diet and physical activity, we assessed the dietary intake, diet compliance, and physical activity levels of the subjects during the study period. For examination of diet, subjects were asked to record the foods they consumed every day in a dietary diary. Subjects were monitored for current drug use, self-reported symptoms or side effects, changes in physical activity, lifestyle habits, and suitability for the chosen diet. A safety test was conducted to evaluate the clinical condition of each subject, including adverse reactions, and the results were recorded in a case report to investigate possible adverse reactions in this human study. In addition, vital signs, blood tests, and chemical tests were reviewed.
Anthropometric measures, harmful elements in hair, and biochemical analysis
All subjects underwent efficacy and safety assessments before and after the four-week study. The primary efficacy evaluation items were harmful elements in hair (29 types of heavy metals), and the secondary efficacy evaluation items were anthropometric indexes (weight, BMI, body fat, body fat percentage, fat-free mass, waist circumference(WC), hip circumference(HC), and waist-to-hip ratio(WHR)), lipid metabolic indexes (total cholesterol, Apo A1, Apo B, triglycerides, HDL-cholesterol, and LDL-cholesterol levels), blood glucose indexes (glucose, insulin, HbA1c), an inflammatory index (hs-CRP), uric acid level, GGT level, and urinary organic acid levels (β-hydroxybutyrate, isocitrate, methylmalonate, α-ketoisocaproate, α-hydroxybutyrate, 3,4-dihydroxyphenylpropionate, and 8-hydroxy-2-deoxyguanosine). As safety measurements, abnormal responses were monitored, a diagnostic examination was performed, vital signs were tested, a physical examination was conducted, and an electrocardiogram was recorded.
Anthropometry And Medical Examination
The anthropometric measures were height, weight, WC, HC, and BMI. Height and weight were measured with a GL-150 (G-Tech Co., Uijeongbu, Korea) while subjects were dressed in light clothing. WC was measured with a tape measure around the middle of the pelvis and the lower part of the ribs while the subject stood with his/her feet 25–30 cm apart and breathed comfortably. Body fat, body fat percentage, and muscle mass were measured with an Inbody 720 (Biospace Co., Seoul, Korea).
Blood pressure measurement
Blood pressure was measured with an HBP-9020 (Omron Healthcare Co., Ltd, Kyoto, Japan) after the subject had arrived at the research site and had rested comfortably for at least 10 min. Three measurements of systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse rate were recorded at intervals of approximately 2 min while the subject was seated, and the average was calculated. The medical team carried out the examination through interviews, ocular inspection, auscultation, percussion, and palpation.
Harmful elements in hair
In total, 29 harmful elements were measured in hair by means of an ICP, Agilent 7800 ICP-MS (Agilent, CA, USA). Twelve carcinogenic heavy metals (arsenic, beryllium, cadmium, lead, mercury, nickel, palladium, rhodium, thallium, tin, thorium, and uranium) and 17 toxic heavy metals (aluminum, antimony, barium, bismuth, cerium, cesium, gadolinium, gallium, gold, indium, platinum, rubidium, silver, tellurium, titanium, tungsten, and zirconium) were identified in hair. For collection, 0.4 g of hair was cut as close to the roots as possible from 5–6 places in the occipital region. Hair was also collected at the nearest point to the scalp (two to three cm from the scalp), and long hairs or long hair ends were not used. It was recommended that the subjects wash their hair with only water on the day of hair sampling (sweaty or dirty hair was not suitable).
Blood was collected after subjects had fasted for more than 12 h overnight. The blood was centrifuged at 3,000 rpm (Hanil Science Industrial Co., Ltd. Seoul, Korea) for 20 min and kept frozen at − 80 °C until analysis. Total cholesterol, neutral blood lipid, and HDL-C levels were analyzed with a Hitachi 7600 − 100 analyzer (Hitachi High Technologies Corporation, Tokyo, Japan), and the LDL-C content was calculated with the Friedewald formula . Glucose, insulin and HbA1c levels were measured to investigate blood glucose control. Lipid metabolic indexes of free fatty acid, apolipoprotein A1, apolipoprotein B, and apolipoprotein E, along with liver enzyme indexes of GGT, ALT, AST and total bilirubin, were analyzed with an ADVIA 2400 chemistry system (SIEMENS, Munich, Germany).
Urinary organic acid test
The intermediate urine of the first urine of the morning (about 10 mL) was collected from each subject, and urinary organic acid tests were conducted. The urine samples were analyzed for levels of β-hydroxybutyrate, isocitrate, α-ketoisocaproate, methylmalonate, and α-hydroxybutyrate (AHB) by GC-MS (Agilent 5977B Series GCC/MSD System, Santa Clara, CA, USA). The 8-hydroxy-2-deoxyguanosine (8-OHdG) level was analyzed by LC-MS (Agilent 5977B Series GCC/MSD System). The water intake of the subjects was limited to one cup (240 mL) after 8 p.m. the day before urine sampling. The subjects were also instructed to refrain from excessive concentrate, drinks, coffee, and alcohol.
Investigation of dietary intake and physical activity
Trained nutritionists explained the dietary diary to each subject and provided instructions for use. The dietary intake surveys were collected at the first (baseline) and second visits, and the results were verified directly through interviews when the data were retrieved. The subjects completed the first dietary intake survey three days prior to the first visit in the dietary diary according to the meal record method. The purpose of the first survey was to determine the total calorie and nutrient composition of the average daily diet before the study. The second dietary intake survey was conducted to determine the intake of the study diet and all the other foods (beverages) for 28 days. The subjects assigned to the MRD group recorded all the foods they ate freely every day in the diary. The randomly assigned subjects in the WD and CRD groups marked whether or not they had eaten the study diet each day in the issued diary. The subjects in these groups also recorded their intake levels of the provided foods and photographed the remaining amounts after they had eaten the meals each day. Other foods and beverages consumed were also recorded in detail on a separate form. Even if the subjects ate meals other than those provided in this study, they were not eliminated. The subjects were taught to accurately record the additional foods that they would inevitably consume, after receiving training to eat only the foods that were provided and those that were added to the served foods. In the analysis of dietary intake data, the average values of the dietary diaries for 28 days were evaluated by the researchers assigned to each group in the Can Pro 4.0 program (The Korean Nutrition Society, Seoul, Korea). Physical activity was evaluated according to a metabolic equivalent task (MET) assessment using the global physical activity questionnaire (GPAQ). The MET value was used for analysis of physical activity or GPAQ data, representing the relative proportion of working metabolic rate to metabolic rate at rest.
All statistical analyses were performed using SAS® version 9.2 (SAS Institute, Cary, North Carolina, USA). The mean ± standard deviation (SD) was used for continuous variables, and the frequency was determined for categorical variables. The Chi-square test and Wilcoxon rank-sum test were used for the homogeneity test and the baseline homogeneity test, respectively, among the study groups. The variation in primary and secondary outcomes efficacy evaluation item after four weeks of diet intake (Δ Week 4 - Week 0) was compared among the WD, CRD, and MRD groups by the Kruskal-Wallis test and the Repeated Measures ANOVA. Control groups 1 and 2 were compared with the test group by the Wilcoxon rank-sum test. For each group (WD, CRD, and MRD group), the changes in intake before and after the four-week study were evaluated by the Wilcoxon signed-rank test. The significance was statistically significant at the level of p < 0.05.
The sample size was statistically calculation for this study was based on the assumption of -2.6 kg variation in measured weight after 7days of Lemon detox diet supplementation, -0.6 kg variation in the control group, and standard deviation of 1.7 kg, a sample size of each group was determined to be 15 participants, allowing for a 20% dropout rate. The groups were equal in size in order to obtain the greatest statistical power. The number of subjects required was calculated as described in Kim et al. (2015)  therefore, a total of 45 participants were randomly assigned into one of the study groups.