Ginger collection and preparation
The fresh rhizomes of ginger were collected in May, 2015 from Ratchaburi province, Thailand. The voucher specimen (BKF 192198) was deposited by Office of the Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Bangkok, Thailand and was identified by Mr. Sukid Rueangruea, Forestry Technical Operations Investigators Plant Species official, Bangkok Forest Herbarium, Herbarium Department of National Parks, Wildlife and Plant Conservation, Thailand. The ginger rhizomes were cleaned, steamed by autoclave and dried with hot air oven at 50 °C. The quality standards of ginger rhizomes were applied with the following parameters: contamination testing, loss on drying (moisture content), total ash, acid insoluble ash for inorganic contamination, extractive value and heavy metal content [17]. The dried rhizomes were mechanically powdered and extracted by maceration with 95% ethanol (Liquid: Solid ratio: 1:1) for 3 days and filtered. These were repeated twice, the combined filtrates were concentrated under reduced pressure by a rotary evaporator (Rotavapor R-205, Buchi, Switzerland). Biological quality control of ginger extract was conducted by an anti-allergic assay using the inhibitory effect on β-hexosaminidase in which IC50 not more than 30 μg/ml. The high-performance chromatography (HPLC) was also performed to ensure the composition of 6-gingerol and 6-shogaol. HPLC analysis of the study was carried out according to the method of Pattanacharoenchai [18]. Chromatogram of ginger extract and standard compound are shown in Fig.1. From HPLC analysis, the mean contents of 6-gingerol and 6-shogaol in ginger extract were 71.13 and 19.65 mg/g of extract, respectively.
Drug preparation
The ginger extract was weighed and combined with necessary excipients, and then filled into 500 mg capsules (red-black capsules for the morning meal and white-blue capsules for the evening meal) each containing 125 mg of the ginger extract, produced according to Good Manufacturing Practice (GMP) for Traditional Medicine. Ginger extract capsules were packed in aluminium foil complied with the quality standards of Thai Herbal Pharmacopeia, contamination testing, weight variation and dissolution. Loratadine (Clarityne®) tablets containing 10 mg of micronized loratadine were encapsulated in the same size and color as ginger extract. Lactose monohydrate as a placebo was prepared in a 500 mg capsule.
Study design
This study was a prospective randomized, double blind, controlled trial (Phase 2), designed to investigate the efficacy and safety of ginger extract compared with loratadine for treating AR patients at Thammasat University Hospital, Pathumthani, Thailand. Before the commencement of the study, the study protocol and informed consent were approved by the Medical Ethics Committee of the Faculty of Medicine, Thammasat University (registry number MTU-EC-TM-4-077/57) and also was registered at ClinicalTrials.gov (NCT02576808).
Study population and protocol
The sample size determination was calculated from this formula, N (each group) = (r + 1)(Zα/2 + Z1-β)2 σ2/ rd2 [19] where Zα is the normal deviate at a level of significance (Zα is 1.96 for 5%) and Z1-β is the normal deviate at 1-β% power with β% of type II error (0.84 at 80% power. r = n1/n2 is the ratio of sample size required for 2 groups, generally it is one for keeping equal sample size for 2 groups. σ and d are the pooled standard deviation and difference of means of 2 groups.
From conducting a pilot study, the minimal detectable difference means (d) of two group as 0.66 scores of total nasal symptom scale (TNSS) and 1.01 is standard deviation (σ).
Thus, the minimum sample size for each group to detect the mean difference between the two means is 36 persons/group. Lastly, considering 10% of drop-out was count out, so forty patients per each treatment group were required for the study.
Eighty patients from the Department of Ear Nose and Throat, Thammasat University Hospital were between 18 and 70 years old were chosen. The patients had a clinical history of AR symptoms (itching, nasal congestion, watery nasal discharge or runny nose and sneezing) and were diagnosed by doctor with a moderate AR; minimum TNSS scores of 7 points. Patients could stop taking antihistamine or intranasal steroids for 1 week before trial and did not have history of the following disease: heart disease, kidney disease, liver disease, epilepsy, high blood pressure and severe asthma. Exclusion criteria included patients having fever, taking anti-coagulant, anti-platelet aggregation, erythromycin, clarithromycin, ketoconazole, itraconazole and fluconazole, experienced serious side effects from loratadine and ginger allergy. Pregnant and lactating women were also excluded.
Informed consent was obtained from the patients who were eligible for the study. The patients were randomly divided into 2 groups (1:1) by using a computer-generated program ensuring no contact with investigators. The patient received a randomized code number sequentially from a secret random list. Treatment assignment was also concealed from all investigators involving in the trial. The masking was opened in medical emergency or if trial successfully accomplished, opened after data analysis.
All patients were instructed about the same appearance of treatment and to take two capsules two times daily for 6 weeks; the experimental group received ginger extract capsules, or the control group, received loratadine. In this study, all patients were followed up at 3rd week and 6th week for evaluating the efficacy, safety, and patient compliance.
The clinical efficacy evaluation
The efficacy was evaluated by total nasal symptom scores (TNSS) and secondary efficacy variables were measuring the cross-sectional area of the nasal cavity with acoustic rhinometry (ARM) and rhino conjunctivitis quality of life questionnaire (RQLQ).
TNSS score(s), a subjective evaluation as a primary effective tool to measure the intensity symptoms of patients with AR [20], Overall assessment of nose symptoms uses four aspects: runny nose, itchy nose, nasal congestion and sneezing with the score of 4 (0 = no symptoms - 3 = severe symptoms). The total possible score ranged from 0 (no symptoms) to12 (maximum symptom intensity) [3].
ARM is one of the standard diagnostic tools in objective evaluation of nasal patency. ARM can detect minimal cross section area (MCA); narrow points within the nose that may lead to nasal blockage, volume estimates of the nasal cavity (Vol.) and distance from the nostril (Dis.). The reliability of the method is greatest in the anterior nasal cavity, which is the site of the nasal valve [21].
The RQLQ has 28 questions in 7 domains (activity limitation, sleep problems, nose symptoms, eye symptoms, non-nose/eye symptoms, practical problems and emotional function). There are 3 patient-specific questions in the activity domain which furnish patients to choose 3 activities in which they are mostly limited by their rhino conjunctivitis. Patients gave responses to each question on a 7-point scale (0 = not impaired at all - 6 = severely impaired). The overall RQLQ score is the mean of all 28 responses and the individual domain scores are the means of the items in those domains [22].
The safety evaluation
The safety is measured by using blood analysis, measuring blood pressure and questionnaire. All patients had blood analysis is preformed, three times (before treatment, 3rd week and 6th week). A 10 cc. of blood was taken from each patient in the morning at 7:00 to 9:00 am after 8 h of fasting. The blood specimen were analysed by The Bangkok Pathology-Laboratory including; liver function test and renal function test. All patients were requested to immediately contact the investigator if they noticed any kind of adverse reactions.
Statistical analysis
All statistical analyses were performed using the standard statistical software. The independent t-test or Mann-Withney U test was used to compare these mean values between the 2 groups. The repeated measured analysis of variance (ANOVA) or Friedman’s test was used to analyze the changes in the mean values from baseline to 3rd week and 6th week for each group. TNSS score, Total score of RQLQ and ARM values were examined by multivariate regression analyses. Independent variables including treatment with confounders selected demographic and clinical variables (age, gender, body mass index and using steroids). A p-value of < 0.05 was considered to indicate statistical significance.