Preparation of AHE
Fresh A. hookeri root (100 kg) was purchased from the Hanam farm located in Sunchang-gun (Jeonbuk, Republic of Korea). A. hookeri was harvested on November 2014 and identified by the Agricultural Technology Research Institute. A voucher specimen (RDAAH15) has been kept in the Rural Development Administration. Root of A. hookeri were hot air-dried for 12 h at 60 °C. The dried roots (12 kg) were extracted with distilled water at 100 °C for 4 h. A. hookeri root extract (AHE) was concentrated under reduced pressure at 60 °C for 12 h to obtain concentrated one (7.2 kg). AHE was freeze dried at − 66 °C for 72 h under approximately 5–10 mmHg in a vacuum freeze-drying apparatus (4.5 kg). For standardization, the marker compound was cycloallin, and the sample was extracted with methanol and analyzed using LC-MS/MS under following condition: Column Acquity BEH Amide 1.7 μm (2.1 × 150 mm); mobile phase, solvent A: 10 mM CH5NO2, solvent B: 0.1% formic acid; flow rate 0.2 mL/min; temperature 25 °C. Standardized AHE containing 0.65–0.98 mg/g cycloallin was encapsulated to contain 243 mg of AHE and diluting ingredients. The placebo contains microstallin cellulose, hydroxypropyl metylcellulose, garlic flavor, and caramel flavor, so the flavor and appearance were not distinguished from AHE (Table 1).
The study was designed as an eight-week, randomized, double-blind, and placebo-controlled crossover trial. Random allocation sequence was generated using a computer program, and was concealed from all subjects, investigators, and staff until the end of the study. The trial protocol and informed consent form were approved by the Institutional Review Board of Chonbuk National University Hospital (IRB No.: 2015–02-038) and trial was conducted from 2015 to 2017. This trial adhered to CONSORT guidelines and included CONSORT checklist as an Additional file 1. Prior to the trial, written consent was obtained from all subjects, and the entire process of the trial was conducted in accordance with the Helsinki Declaration, International Conference on Harmonization (ICH-GCP), and trial protocol. Subjects were randomly assigned to receive either AHE or placebo in the first period. Based on the crossover design, subjects received the opposite investigational product (IP) after a four-week washout period (Fig. 1). During the trial period, subjects were instructed to take the IP twice daily and maintained their usual diet, except for taking functional foods or dietary supplements. Compliance was calculated by collecting the remaining IP consumed for 8 weeks from the prescribed IP, and subjects with compliance less than 70% were excluded from the analysis. The subjects also reported to the researchers the adverse events and lifestyle changes. Efficacy and safety were assessed before and after taking the investigational products.
The study subjects were recruited from the Clinical Trial Center for Functional Foods at Chonbuk National University Hospital. Prediabetes subjects (100 ≤ fasting plasma glucose (FPG) < 126 mg/dL) who had not been diagnosed with any disease and met the inclusion criteria were recruited for this study. Exclusion criteria for the study were as follows: (1) significant variation in body weight (> 10%) in the past 3 months; (2) diabetes; (3) total cholesterol ≥260 mg/dL and/or LDL-cholesterol ≥180 mg/dL; (4) familial combined hyperlipidemia; (5) systolic blood pressure > 160 mmHg and/or diastolic blood pressure > 100 mmHg; (6) use of corticosteroids within the past 4 weeks; (7) use of anti-obesity, hypolipidemic, or hypoglycemic drugs within the past 6 weeks; (8) use of obesity-, blood lipid-, or blood glucose-related functional foods within the past 2 weeks; (9) cardiovascular disease, heart failure, or myocardial infarction; (10) local or systemic inflammatory disease such as rheumatoid arthritis or autoimmune disease; (11) history of alcohol or substance abuse; (12) history of a disease that could interfere with the test products or impede their absorption, such as gastrointestinal disease or gastrointestinal surgery; (13) allergic or hypersensitivity; (14) participation in any other clinical trials within the past 2 months; (15) a laboratory test or medical or psychological conditions deemed by the investigators to interfere with successful participation in the study; (16) pregnancy or breast feeding.
Efficacy outcome measures
At every visit, all subjects underwent blood tests and oral glucose tolerance test (OGTT) after an overnight fast. After a fasting blood sample was collected, glucose load of 75 g was ingested within 5 min. Blood samples were collected at 30, 60, 90, and 120 min after the glucose load. Blood glucose concentrations were measured using the ADVIA® 2400 chemistry system (Siemens, Bayern, Germany). Total glucose incremental area under the curve (iAUC) during OGTT was determined using the trapezoidal method . Insulin concentration was measured using a Cobas e 601 module (Hitachi High-Technologies Corporation, Tokyo, Japan). C-peptide concentration was measured using a Cobas 8000 chemistry autoanalyzer (Roche Diagnostics System, Basel, Switzerland). Hemoglobin A1c (HbA1c) concentration was measured using an ADAMS A1C HA-8180 (Arkray Factory, Kyoto, Japan). Total cholesterol, triglyceride, HDL-cholesterol, and LDL-cholesterol concentrations were measured using a Hitachi 7600–110 analyzer (Hitachi, Tokyo, Japan).
Safety outcome measures
At each visit, subjects were examined for electrocardiogram, vital signs (blood pressure and pulse rate), and laboratory tests (WBC, RBC, Hb, Hct, platelet count, ALP, γ-GT, AST, ALT, total bilirubin, total protein, albumin, BUN, and creatinine) for safety evaluation. WBC, RBC, Hb, Hct, and platelet count were measured using a System XE-5000TM (Sysmex, Kobe, Japan). ALP, γ-GT, AST, ALT, total bilirubin, total protein, albumin, BUN, and creatinine were measured using the ADVIA® 2400 chemistry system (Siemens, Munich, Germany).
Evaluation of diet and physical activity
Dietary intake survey and physical activity survey were used to determine subjects’ lifestyle changes during the trial period. Dietary intakes investigated by the 3-day dietary record method were analyzed using the CAN-pro 4.0 program (The Korean Nutrition Society, Seoul, Korea). Physical activity was investigated and analyzed by Global Physical Activity Questionnaire (GPAQ).
All statistical analyses were conducted using SAS version 9.4 (SAS Institute, Charlotte, NC, USA). Sample size was calculated to detect a 1.0 mg/dL (SD = 2.3 mg/dL) based on a power of 0.8 and an α levels of 0.05 . Therefore, the calculated number of subjects was 21 for each group considering the dropout rate of 30%. Analysis was performed on a per protocol (PP) approach. Data are shown as the mean value and standard deviation (SD). To determine the differences between the groups, the categorized variables were analyzed using the chi-square test and continuous variables were analyzed using the t-test. A p-value less than 0.05 was considered statistically significant.