Trial design
This was a randomized, double blind, and placebo-controlled clinical trial with a parallel design. The sample size was calculated based on the comparison of ratios formula with α = 0.05 and β = 0.2. As there is no similar study to our trial, we used the data of our pilot study and by р1 = 0.231 and p2 = 0.0, the sample size of 29 subjects in each group was calculated. Considering the probability of filling 25% of the samples, the final sample size of 39 subjects in each group (78 subjects) was calculated. The sample size calculation was based on the asthma severity change.
The comparison of ratios formula \( n=\frac{{\left({Z}_{1-a/2}+{Z}_{1-\beta}\right)}^{2\ast}\left[{p}_1\left(1-{p}_1\right)+{p}_2\left(1-{p}_2\right)\right]}{{\left({p}_{1-}{p}_2\right)}^2} \)
Considering the length of the treatment, 40 subjects were finally enrolled in each group (1: 1 ratio) according to a computer-generated blocked randomization list with a block size of 6. To decrease the probable bias, we used an allocation concealment technique. A researcher, who did not have any clinical participation in the study placed the saffron and placebo capsules into numbered bottles based on a random list. Another researcher, who was not aware of the random sequences and was not involved in the trial, assigned the numbered bottles to the patients. After the allocation was done, the participants were asked to fill in all the questionnaires during the baseline visit. The blood samples were then collected in the next day and the participants were accordingly allowed to start having treatment capsules. Filling the questionnaires and taking blood samples were repeated again at the end of the study. Every two weeks all the participants were contacted to remind them about taking the treatment capsules and also ask them to report possible side effects of the treatment. Anthropometric measurements including weight and height were determined pre- and post-intervention. Disease history, demographic data, history of using medications and supplements, history of smoking and physical activity level (assessed by the short form of the International Physical Activity Questionnaire) were also assessed. All the participants, researchers, and the physicians were blind to the allocations using the random codes until the statistical analyses were completed. In the eight-week study period, all the participants were given dietary advices such as avoiding consumption of saffron (from food), fast foods, sausage, smoked and canned foods. Patients were also asked not to change their energy intake and physical activity during the study. To control the energy intake, we used the 24-h recalls of 3 days (2 work days and one weekend day). According to the doctor recommendations, patients were asked not to go out when the air was polluted and use filter masks in emergencies. The lung specialist used the same drugs for the patients in this study. Compliance was monitored during a weekly call, assessing compliance by asking the number of capsules consumed. Those subjects who did not take their capsules regularly or were intolerant to the medication were excluded from the study. Our patients had no disease except asthma and consumed no drug except asthma drugs. Personal information of participants collected, shared and maintained in order to protect confidentiality before, during, and after the trial by coding the patients.
Participants
Participants included 80 men and women between the ages of 18 and 65 years. According to the Global Initiative for Asthma (GINA) diagnostic criteria for asthma, 80 patients with mild and moderate allergic asthma were examined by an attendant pulmonologist in May and October 2017, and were then recruited from the outpatient clinic at Imam Khomeini Hospital, Ahvaz, Iran. After that, the participants were provided with information about the study by both verbal explanation and written information sheets. They were asked to take the capsules regularly, provide adequate tracking information, and complete the questionnaires and the treatment procedure until the end of the study. To collect information on their socio-demographic status, occupation, smoking behavior, medical history and medication, all patients were also interviewed. The inclusion criteria were an age range of 18–65 years, with mild and moderate persistent allergic asthma according to the GINA criteria and body mass index (BMI) up to 27 kg/m2. The exclusion criteria included smoking, pregnancy, lactation, diabetes, autoimmune disease, malignancy and other pulmonary diseases. The chemical drugs which our patients consumed were not significantly different between saffron and placebo groups (p = 0.66). All the patients provided written informed consent, and the whole protocol met the requirements of Ahvaz Jundishapur University of Medical Sciences Ethics Committee.
Intervention
During the intervention, all the patients received either two oral capsules including 50 mg dries saffron (Crocus sativus L.) stigma or a placebo (all the capsules were made in the Faculty of Pharmacy at Ahvaz Jundishapure University of Medical Sciences). The placebo capsules contained edible colors similar in color, shape, size, and package to the saffron ones. Dried saffron stigma was taken from Estahban, Fars province, Iran (Herbarium code: JPS018118). It was formulated as a capsule containing 50 mg of dried saffron stigma and starch (as fillers). The placebo capsules contained starch and permitted food colors to trace amount (E104, E110 and E122). The patients received the capsules at the start of the trial in the pulmonary clinic of Imam Khomeini hospital.
Endpoints
The primary outcomes were to measure asthma clinical symptoms (i.e., shortness of breath during the day and night time, activity limitation, frequency of salbutamol spray consumption and waking up due to asthma symptoms) and asthma severity at the baseline and post-intervention. Secondary outcomes were to measure eosinophils, basophils, blood pressure and lipid profiles pre- and post-intervention.
Asthma clinical symptoms and severity score
Diagnosis of the allergic asthma was done through measuring the serum levels of immunoglobulin E (IgE) total by IgE enzyme-linked immunosorbent assay (ELISA) kit (DiaMetra DKO060 ELISA Kit, Italy) and the values over than 30 international units are known as allergic patients [2]. Clinical symptoms of the patients were assessed based on the symptoms used as criteria for asthma control [2]. Asthma severity was diagnosed according to the forced expiratory volume in 1 s (FEV1) and clinical symptoms. Those patients with moderate asthma had FEV1 between 60 to 80%, while the ones with mild asthma had FEV1 ≥ 80% and standard clinical symptoms according to GINA [2]. Asthma severity and its clinical symptoms were evaluated at the baseline and endpoint of the trial.
Collection of blood samples
Blood samples were collected at the baseline and at the end of the study. Each time, 5 mL of blood sample was drawn. The serum samples were frozen at − 20 °C immediately, and were then stored at − 80 °C until further laboratory analyses were done. Blood samples of each patient were collected in the morning after a 12-h fasting, and then the haemolyzed samples were excluded from each analysis and in these cases blood sampling were repeated.
Laboratory analyses
We used ELISA, as a validated method, (BIO TEK ELX 800 (microplate reader), Highland Park Winooski, VT 05404–0998, USA) for measuring IgE total. During each laboratory analysis, the technicians were blind to the saffron–placebo status. Eosinophils and basophils were evaluated in the results of Complete Blood Count (CBC)-diff. For CBC analysis, 2 ml whole blood in the tubes containing anticoagulant EDTA were placed in the automatic counting machine (Convergy® X5 made in Germany). Lipid profiles (triglyceride, total cholesterol, LDL-C and HDL-C) were measured by enzymatic method kit (Pars Azmoon Co, Tehran, Iran). All laboratory tests were done in the Central Laboratory at Ahvaz Jundishapur University of Medical Sciences.
Dietary analysis and blood pressure
We assessed the intake of calorie and the percentage of calorie from carbohydrate, protein and fat in all the patients at the baseline and at the endpoint of the trial by 24-h food recalls for two work days and one weekend. The diets were analyzed by the modified nutritionist 4 (NUT4) software. We assessed the calori intake because can affect weight gain and weight gain can affect asthma symptoms and percentage of calori intake from fat is important in respiratory disease because fats have lower work load on respiratory system. Blood pressure was measured in standard method with mercury barometer and after 15 min rest.
High performance liquid chromatography (HPLC)
The amount of crocin in saffron samples was measured via high performance liquid chromatography (HPLC) method [13]. Following this procedure, 10 mg saffron and 10 ml ethanol 80% were extracted over 15 min sonication in darkness. After that, the samples were centrifuged and, then the supernatant was used for HPLC.
HPLC instrument was done using Agilent technology (1260 infinity II, a multiple wavelength Uv-visible, PDA 1260 infinity model). The column used for separation was ZORBAX Eclipse plus C 18 (Rapid Resolution 4.6 × 100 mm 3.5-Microns). All the experiments were done at room temperature, and the mobile phase composition included a mixture of formic acid (0.2%), acetonitrile and methanol. Linear gradient solution system was used as 76.5% formic acid (0.2%), 13.5% acetonitrile and 10% methanol which changed into 100% methanol by 60 min. The detector was adjusted at different maximum wavelengths including 250 nm, 308 nm and 440 nm. The amount of crocin in the saffron samples was found to be 304.3 mg/kg.
Statistical analysis
All analyses were done on the intention-to-treat population corresponding to the subjects having consumed at least one dose of the treatment. The normal distribution of the data was assessed using a Kolmogorov–Smirnov test. Blood pressure, lipid profiles, eosinophil and basophil count and dietary factors were analyzed by paired t-test or Wilcoxon paired rank test (when data were not normally distributed) for the within-group comparisons (pre-and post-intervention values in each group). On the other hand, comparisons of changes (endpoint minus baseline) after 8 weeks of intervention between the two groups were done by independent t-test or Mann–Whitney U-test (for non-normal distribution data). Data were reported as mean ± SD or median (25th, 75th percentile) for parametric and non-parametric data, respectively. Chi-squared test, or Fisher’s exact test were used for clinical symptoms of asthma and its severity and also for the baseline characteristic data. All statistical analyses were carried out using SPSS version 16 statistical software (SPSS Inc., Chicago, IL, USA). The most important outcomes were presented at 95% confidence intervals (CI). For all the tests, two-sided p-values < 0.05 were considered statistically significant.
