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This trial took place in Daejeon Oriental Hospital of Daejeon University after approval of the Institutional Review Board of Daejeon Oriental Medical Centre (authorisation number: DJOMC-51) and was registered on Clinical Research Information Service in Rep. of Korea (KCT0000048). It was conducted in accordance with the Helsinki Declaration of 1975 (as sixth revision in 2008) and the Guidelines for Good Clinical Practice approved from Korea FDA since Aug. 2005 (Approval No. 110). From June, 1st, 2010 to January, 31st, 2011, total 90 patients were informed preoperatively about the study via a standardized leaflet and provided written consent for their participation. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1.
Adults (age from 20 to 65 years) who had experienced chronic fatigue for longer than 6 months were recruited for the study. A physician and radiologist examined potential candidates and thus excluded those who had any haematological or radiological test abnormalities related to their fatigue. Using the Korean version of the Beck Depression Inventory (BDI) and the Korean translation of the State–Trait Anxiety Inventory (STAI), subjects with a history of psychological disorders or currently experiencing severe depression (BDI score >29) or anxiety (STAI score >70) were excluded [24], [25]. Subjects who worked at night, used alcohol, smoked, took medication or were severely overweight (body mass index >30) were excluded. In this study, the sample size was calculated based on a two-tailed alpha level of 0.05, a power level of 0.90 and 10% drop-out, respectively. The minimal detectable effect size was r = 0.53 and the target sample size were 90. Only patients with ICF were selected, while those patients who met the criteria for CFS were excluded. When diagnosing patients with ICF, the criteria of CFS is typically used [26]. CFS is the unexplained chronic fatigue which has concurrently 4 or more of 8 symptoms (post-exertion malaise lasting more than 24 hours; multi-joint pain without swelling or redness; unrefreshing sleep; headaches of a new type, pattern, or severity; significant impairment of short-term memory or concentration; tender cervical or axillary lymph nodes; muscle pain; a sore throat that is frequent or recurring). Patients were diagnosed as ICF when they had only 3 or less of 8 above symptoms. Total 101 patients were recruited for pre-screening, while 90 subjects (21 men and 69 women) were finally enrolled (median age: 39.5 years; range: 20–60 years; mean body weight: 56.7±9.1 kg, mean height 161.5±7.1 cm, Table 1 and Figure 1).
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0061271.g001 Flow diagram of subject progress through the phases of the RCT. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0061271.t001 Physical baseline characters of subjects. No significant difference of distribution was observed among groups. Values are means ± SD for body weight and height.
This study was designed as a randomised, double-blind, controlled clinical trial. The randomized allocation was performed with computer-generated block randomization (three, six and nine of block size), performed by a statistical professor from Daejeon University, who was not involved in the data collection and analysis. Ninety of subjects were randomly assigned by a medical staff to three groups as following; a placebo group, 1 g P. ginseng group or 2 g P. ginseng group. Each subject ingested four capsules (250 mg each) of P. ginseng extract (1 or 2 g total per day, respectively) or the placebo two times a day (09∶00 and 19∶00 h) for 4 weeks. The decision of dosage and duration for administration with P. ginseng were based on the previous studies [21], [27]. Assessment of fatigue severity was performed using a numerical self-rating scale (NRS) and a visual analogue scale (VAS) at 0, 2 and 4 weeks. When subjects visited to assess the fatigue severity, they returned the remaining drugs. After the left pill count, staffs offered next pills to each subject. The serum levels of biomarkers associated with oxidative stress and antioxidants were measured at 0 and 4 weeks. Every subject was subjected to peripheral blood sampling in the Daejeon University Hospital laboratory after an 8-h fast, the morning 1 day before the first and 1 day after the final administration of P. ginseng or placebo.
Preparation and Compositional Analysis of Ginseng Extract and Placebo: A 20% ethanol extract of P. ginseng was prepared by the Guryoung Pharmaceutical Company, Ltd. (Cheorwon, South Korea) according to over-the-counter Korean monographs. Briefly, 4-year-old ginseng roots (100 kg) were broken into pieces (100 mesh) and then boiled at 105°C for 6 h. The extract solution was condensed into 20 Brix, and vacuum-freeze-dried. The final extract yield was 13.2% (w/w). The compositional analysis was performed using high-performance liquid chromatography (HPLC), and the results are summarised in Table 2. A placebo was made by mixing 99.36% cornstarch (Daesang Co., Seoul, South Korea), 0.3% P. ginseng flavouring (Hanbit Flavor & Fragrance Co., Eumseong-gun, South Korea) and 0.02% caramel colouring (Nam Young Food Co., Busan, South Korea). The 100% P. ginseng extract (2 g P. ginseng), mixture of 50% P. ginseng extract with 50% placebo (1 g P. ginseng) and the placebo (control) were put into 250 mg soft capsules.
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0061271.t002 Compositional analysis of P. ginseng by HPLC. P. ginseng (1 g) was dissolved in 90% methanol and subjected to HPLC. The column was eluted with solvents A (18% acetonitrile) and B (80% acetonitrile) at a flow rate of 1.6 ml min–1. The following solutions were used: 100% A and 0% B changed over 32 min, 80% A and 20% B to 80 min, 0% A and 100% B to 100 min and 100% A and 0% B to 110 min.
Assessment of Fatigue Severity using Numerical Rating Scale (NRS) and Visual Analogue Scale (VAS): The primary endpoint of this study was the change in fatigue severity assessed by measuring NRS and VAS. NRS was used with the Korean-translated Chalder fatigue severity questionnaire [28]. The survey consisted of seven physical health-related questions (1st to 7th) and four mental health-related questions (8th to 11th) as following: (1) How tired do you feel? (2) How strongly do you currently feel the need to rest? (3) How sleepy or drowsy do you feel? (4) Do you have problems starting things? (5) Are you lacking energy? (6) Do you have less strength in your muscles? (7) Do you feel weak? (8) Do you have difficulty concentrating? (9) Do you have problems thinking clearly? (10) Do you make slips of the tongue when speaking? (11) How is your memory? All subjects scored each item on a 10-point scale (0 = not at all to 9 = unbearably severe fatigue condition). Additionally, patients were asked to indicate their feeling of general fatigue by drawing a vertical line on a 10-cm visual analogue scale (VAS, 0 cm = not at all to 10 cm = unbearably severe fatigue condition).
Determination of Total Reactive Oxygen Species: The total reactive oxygen species (ROS) level in serum was determined according to Hayashi’s method [29]. Briefly, hydrogen peroxide (H2O2) was used to generate the standard calibration curve. N,N-Diethyl-para-phenylenediamine (DEPPD) and ferrous sulphate solutions were prepared beforehand. Five microlitres of standard solution or serum was added to 140 µl of 0.1 M sodium acetate buffer (pH 4.8) in 96-well plates and incubated at 37°C for 5 min. One hundred microlitres of DEPPD and ferrous mixture solution were added to each well, and the amount of ROS was determined at 505 nm using a spectrophotometer.
Determination of Lipid Peroxide as Malondialdehyde: Serum lipid peroxide levels were determined using thiobarbituric acid reactive substances (TBARS), as described by Kamal [30]. TBARS concentration was expressed as µM malondialdehyde (MDA) in serum. Briefly, 250 µl of serum or standard solution was added to 2.5 ml of 20% trichloroacetic acid (TCA) and then mixed with 1 ml of 0.67% thiobarbituric acid (TBA), followed by heating at 100°C for 30 min, cooling on ice and vigorous vortexing with 4 ml n-butanol. After centrifugation at 3000×g for 20 min, the absorbance of the upper organic layer was measured at 535 nm with a spectrophotometer and compared with a 1,1,3,3-tetraethoxypropane (TEP) standard curve.
Determination of Total Antioxidant Capacity: Total antioxidant capacity (TAC) was determined according to Kambayashi [31]. Ninety microlitres of 10 mM phosphate-buffered saline (PBS; pH 7.2), 50 µl of 18 µM myogloblin solution and 20 µl of 3 mM 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) solution were mixed with 20 µl of diluted serum samples or various concentrations of gallic acid in a 96-well microplate at 25°C for 3 min. Then, 20 µl of H2O2 was added to each well and plates incubated for 5 min. The absorbance was measured using a plate reader (Molecular Device Corp., Sunnyvale, CA, USA) at 600 nm. TAC was expressed as gallic acid equivalent antioxidant capacity (GEAC).
Determination of Superoxide Dismutase and Catalase Activity: Serum superoxide dismutase (SOD) activity was determined using a SOD assay kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. Bovine erythrocyte SOD (Sigma, St. Louis, MO, USA) was used as a standard. Serum catalase activity was determined using the method of Beers and Siezer [32]. Briefly, 100 µl of diluted serum or standard solution was mixed with 2.9 ml of substrate solution (0.0036% [w/w] H2O2 in 50 mM potassium phosphate), followed by absorbance readings at 240 nm after 5 min.
Determination of Total Glutathione Contents and Glutathione-peroxidase and Glutathione-reductase Activities: Total GSH content was determined according to a previously described method [33], [34], with slight modifications. Briefly, 50 µl of diluted serum (in PBS 10 mM, pH 7.2) or total GSH standard was combined with 80 µl of DTNB/NADPH mixture (10 µl of 4 mM DTNB and 70 µl of 0.3 mM NADPH) in a 96-well microplate. Next, 20 µl (0.06 U) of GSH-reductase (GSH-Rd) solution was added to each well, and the absorbance at 405 nm was measured using a plate reader (Molecular Devices, Sunnyvale, CA, USA). GSH-peroxidase (GSH-Px) activity was determined according to the method of Paglia [35]. Briefly, 50 µl of NADPH reagent (5 mM NADPH, 42 mM GSH, 10 units ml–1 of GSH-Rd in 1.25 ml of distilled water) was added to 890 µl of GSH-Px buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA). Then, 50 µl of serum and 10 µl of 30 mM tert-butyl hydroperoxide solution were added to the mixture. The final absorbance was measured at 340 nm using a UV-visible spectrophotometer (Varian; Agilent Technologies, Santa Clara, CA, USA). GSH-Rd activity was determined according to the method of Worthington, with slight modifications [36]. Briefly, 150 µl of GSSG with 30 µl of GSH-Rd assay buffer (100 mM potassium phosphate buffer, pH 7.5, with 1 mM EDTA) was added to 30 µl of serum sample and diluted with GSH-Rd dilution buffer (100 mM potassium phosphate buffer, pH 7.5, with 1 mM EDTA and 1 mg ml–1 bovine serum albumin). Then, 75 µl of DTNB and 2 mM NADPH were added, and the absorbance at 412 nm was read.
Based on a power calculation, total 90 subjects were enrolled and randomly allocated into three groups (Placebo, P. ginseng 1 g and 2 g) for this trial. Only those subjects who completed the trial were included in the statistical analysis: 30 subjects in the placebo (control), 29 subjects in the 1 g P. ginseng and 29 subjects in the 2 g P. ginseng groups, respectively. Linear mixed models with compound symmetric covariance [37] were used to compare the combined effects on fatigue symptoms at two time points (2 and 4 weeks). Subgroup analysis was respectively carried out for physical and mental fatigue. Multiple comparisons among groups were performed using the Duncan method when differences among the treatment effects were significant (P<0.05). Changes in other biological parameters were examined using one-way ANOVA with PASW Statistics ver. 17 (SPSS, Inc., Chicago, IL, USA).
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