Study design and patient population
The effects of linalool on antioxidative activity in patients with carpal-tunnel syndrome were assessed using a pretest-posttest control group study design. Healthy adults with no underlying disease (non-CTS group) were recruited. In addition, patients with CTS diagnosed by electrodiagnostic tests (CTS group) who were scheduled for outpatient surgery at the Center for Plastic and Reconstructive Surgery in Seoul, Korea, were recruited. Each subject was randomly assigned to inhale linalool or carrier oil. Adequate sample size was determined using the G-Power program. Based on a statistical power of 0.50, an effect size of 0.40, and a significance level of 0.05, the minimum number of total patients required to compare differences between the experimental groups was estimated to be 41 patients.
After the study protocol was approved by the Ethical Review Committee of the Korea University Medical Center’s approval (Code: ED12257), subjects were recruited for 18 months, starting in June 2013. In addition to providing written informed consent for study participation, all subjects were required to have no history of psychiatric illness, no disturbances of olfactory acuity, no experience with aromatherapy and to be free of allergies to linalool. Patients with CTS were diagnosed by a registered physician and had not undergone surgery. After exclusion of participants who dropped out of the study, 37 individuals completed the study (14/37 non-CTS group with linalool; 6/37 non-CTS group with carrier; 11/37 CTS group with linalool; and 6/37 CTS group with carrier).
Linalool was purchased from Sigma (St. Louis, MO, USA) and almond oil was obtained from Aromarant Co. Ltd. (Rottingen, Germany). Linalool was dissolved in almond oil at a concentration of 1 %. A 0.5 mL aliquot of 1 % linalool in almond oil (or almond oil alone) was placed onto a gauze pad (3 × 2 cm2), and the pad was positioned five centimeters from the nose of each subject for 10 min, with the subject in a sitting position after deep breathing. Before the intervention, general characteristics, blood pressure and heart rates were measured. After the intervention, blood samples were collected and blood pressure and heart rates were measured.
Blood pressure and pulse rate measurements
Blood pressure and heart rate as indicators of the response of the autonomic nervous system were measured before and after linalool or carrier oil inhalation. Blood pressure was measured in the right brachial artery after a 10-min rest in a sitting position. Heart rate was measured at the radial artery for 1 min.
Assay of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
Plasma concentrations of the radical scavenger 1,1-diphenyl-2-picrylhydrazyl (DPPH) were measured. Blood samples (3 mL) were obtained from each participant after inhalation and centrifuged at 3500 rpm for 10 min at 4 °C to separate the plasma. The samples were stored at −80 °C until assayed. Proteins were removed by mixing plasma and acetonitrile at a 1:1 ratio, incubating the samples at room temperature for 2 min, and centrifuging the samples at 9500 × g for 10 min at 4 °C. The resultant supernatants were diluted five-fold with ethanol. DPPH in anhydrous ethanol was mixed 1:1 with Tris HCL and added to 96 well plates, to which were added plasma supernatants. Ascorbic acid was used as the standard antioxidant. The 96 well plates were incubated in the dark at 37 °C for 30 min, and the absorbances of the solutions were measured spectrophotometrically at 517 nM using a microplate reader. The percentage of scavenging activity was compared with that obtained in the sample of ascorbic acid.
DPPH free radical scavenging activity (%) was defined as (1-sample absorbance/control absorbance)*100.
All statistical analyses were performed using SPSS software (version 12.0). Participants’ characteristics at baseline were explored using Fisher’s exact test. A Shapiro-Wilk test was applied to evaluate data normality, and differences among groups were tested by the Kruskal-Wallis test or one-way analysis of variance (ANOVA). Within group differences in scores before and after interventions were compared using the Wilcoxon’s rank-sum test. All data were reported as mean ± standard deviation (SD), with P-values < .05 considered statistically significant.