Journal of Ethnopharmacology 196 (2017) 186–192

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Journal of Ethnopharmacology

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Squill Oxymel, a traditional formulation from Maritima (L.) Stearn, MARK as an add-on treatment in patients with moderate to severe persistent asthma: A pilot, triple-blind, randomized clinical trial ⁎ Fatemeh Nejatbakhsha, Hossein Karegar-Borzia, , Gholamreza Aminb, Alireza Eslaminejadc, ⁎ Mostafa Hosseinid, Mahbubeh Bozorgie, Mehrnaz Asadi Gharabaghif, a Department of Iranian Traditional Medicine, School of Traditional Medicine, Tehran University of Medical Sciences, Tehran, Iran b Department of Pharmacognosy, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran c Department of Pulmonary Medicine, Masih Daneshvari Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran d Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran e Department of Traditional Pharmacy, Faculty of Traditional Medicine, Tehran University of Medical Sciences, Tehran, Iran f Department of Pulmonary Medicine, Imam Khomeini Hospital, Keshavarz Boulevard, Medical Sciences/Tehran University, Tehran, Iran

ARTICLE INFO ABSTRACT

Keywords: Ethnopharmacological relevance: In Traditional Iranian Medicine (TIM), Squill ( (L.) Asthma Stearn) Oxymel was utilized in the treatment of asthma. Squill has been reported to exert anti-inflammatory, Drimia maritima (L.) Stearn anti-oxidant, anti-cholinergic, and mucus secretion modulating effects. Squill Oxymel Objective: This study aimed to make a preliminary evaluation of the efficacy and safety of an add-on Squill Traditional medicine Oxymel treatment in patients with moderate to severe persistent asthma. Airway resistance Methods: In a 6-week, triple-blind, randomized, placebo-controlled trial, 60 patients with stable moderate to severe persistent asthma were randomly allocated to receive either 10 ml syrup of Squill Oxymel, simple oxymel, or a placebo 2 times a day, as an add-on to their routine treatment (inhaled corticosteroids and β2 agonists). Spirometry and plethysmography were performed on patients to evaluate the effect of the treatment at baseline and end of intervention. Forced Expiratory Volume in first second (FEV1) was considered the primary outcome. St. George's respiratory questionnaire (SGRQ) was also used for the subjective evaluation of patients' responses. Results: Fifty-four patients completed the study. The results showed significant improvement in spirometry parameters, especially FEV1 (1.54 ± .38 vs. 2.11 ± .49 l), in the Squill Oxymel group compared with the other groups. The increases in FEV1 liter, FEV1%, FEV1/FVC%, and MEF 25–75% during the intervention were significantly higher in the Squill Oxymel group than in the other groups (p < .001). However, the improvement of plethysmographic parameters showed no significant difference between the study groups (p>.05). The SGRQ scores (symptoms, activity, and total score) were significantly improved after intervention in both the Squill Oxymel and the simple honey oxymel groups (p < .001), but not in the placebo group. Nausea and vomiting was reported in 5 patients in Squill oxymel and simple oxymel groups. No other serious adverse event was observed. Conclusions: The results of the current study show preliminary evidence for the efficacy and safety of the add- on treatment of Squill Oxymel in patients with moderate to severe persistent asthma.

1. Introduction expected that the number of asthmatic patients in the world will increase by another 100 million by 2025 (Masoli et al., 2004). In the Asthma is among the most common chronic diseases worldwide. It United States alone, the annual cost of asthma is estimated at 56 billion affects about 300 million people (Longo et al., 2012) resulting in dollars (Barnett and Nurmagambetov, 2011). approximately 250,000 deaths annually (Bousquet et al., 2010). It is A cascade of inflammatory mediators plays a central role in the

Abbreviations: TIM, Traditional Iranian medicine; FEV1, forced expiratory volume in first second; FVC, forced vital capacity; MEF25-75%, forced expiratory flow between 25% and 75%; PEF, peak expiratory flow; TLC, total lung capacity; RV, residual volume; RAW (ex, in), airway resistance (inspiratory, expiratory); TGV, thoracic gas volume; IC, inspiratory capacity; sRaw, specific airway resistance ⁎ Corresponding authors. E-mail address: [email protected] (M.A. Gharabaghi). http://dx.doi.org/10.1016/j.jep.2016.12.032 Received 6 August 2016; Received in revised form 12 December 2016; Accepted 16 December 2016 Available online 18 December 2016 0378-8741/ © 2016 Elsevier Ireland Ltd. All rights reserved. F. Nejatbakhsh et al. Journal of Ethnopharmacology 196 (2017) 186–192 pathophysiology of asthma. Currently, inhaled corticosteroids (ICS) are persistent asthma. The study protocol was reviewed and approved by the mainstay of asthma treatment. Despite their positive effect on the Research Ethics Committee of Tehran University of Medical asthma control, evidence shows their incomplete efficacy in asthmatic Sciences (ethical code: 126326) and conducted in accordance with patients (Nomellini and Chen, 2012). ICS are not completely safe, and the Declaration of Helsinki (Association, 2009). The study was fully reports of their various side effects are increasing. Complications such supported financially by the Research Department of Tehran University as posterior sub-capsular and nuclear cataracts (Cumming et al., 1997), of Medical Sciences (Grant No. 9021309004) and registered in IRCT ocular hypertension or open-angle glaucoma (Garbe et al., 1997), bone (ID: IRCT2014111119912N1). All patients provided written informed loss (Richy et al., 2003), and mild adrenal and growth suppression in consent before participating in the study. children have been reported for ICS (Kannisto et al., 2000). Therefore, it is reasonable to look for controlling drugs with low adverse effect 2.2. Sample size calculation capable of reducing the dosage of administered steroid. Practitioners of traditional Iranian medicine (TIM) such as Rhazes Pre-calculated tables for analysis of variance were used to deter- (d. 925 CE) (Heydari et al., 2015), Haly Abbas (d. 982 CE) (Heydari mine sample size (Neter et al., 1996). Based on previous studies and et al., 2014), and Avicenna (d. 1037 CE) (Mosavat et al., 2015) used the mentioned tables, a sample size of 17 patients per group was multiple herbal formulations to treat asthma (Amirghofran, 2010; calculated for at least 10% improvement in forced expiratory volume in Boskabady et al., 2010; Qasemzadeh et al., 2015). Squill, in the first second (FEV1). After adding a possible dropout rate of 20%, the formulation of Squill Oxymel, was the most commonly used drug in sample size of 20 participants in each group was selected. the treatment of asthma (Aghili, 2009; Ibn Sina, 2005). 2.3. Patients 1.1. Squill: (Drimia maritima (L.) Stearn) Patients with moderate to severe persistent asthma with asthma Drimia maritima (L.) Stearn (Squill) is native to the Mediterranean control test (ACT) score less than 20 who referred to the outpatient region, Africa, and India (Stedje, 1987) (12). This and its pulmonary clinic of Imam Khomeini hospital, affiliated with Tehran formulations like Squill Oxymel were widely used by TIM practitioners University of Medical Sciences, Tehran, Iran, were recruited for this to treat different disorders (Aghili, 2009; Ibn Sina, 2005), including study between January and July 2015. melancholia, paralysis, dyspepsia, flatulence, jaundice, ascites, sciatica, The diagnosis criteria for moderate to severe persistent asthma and arthritis. However, Squill Oxymel was most popularly used for were based on the Global Initiative for Asthma (GINA) guidelines respiratory disorders such as hoarseness, pneumonia, chronic bron- (Bateman et al., 2008) (25). All patients were being actively treated chitis, chroniccough, and asthma (Aghili, 2009; Ibn Sina, 2005; with high-dose combination inhaled steroids (up to 1000 mg flutica- Karegar-Borzi et al., 2015). sone or 1600 µg budesonide on a daily basis) and long acting beta Squill has been reported to exert anti-inflammatory, anti-oxidant, -agonists for at least 6 months. anti-bacterial, anti-cholinergic, and mucus secretion modulating ef- Inclusion criteria were patients aged 18–70 years, affected with fects. It is one of the origins of bufadienolide glycosides such as moderate to severe persistent asthma, FEV1 < 80% of the expected Scillaren A, scillirubroside, , scillarenin, and proscillaridin A value, lack of pregnancy, lack of breastfeeding, not taking other herbal (Iizuka et al., 2001). Terness et al. demonstrated that bufadienolides medicines, lack of history of active peptic ulcer, cardiovascular disease such as proscillaridin A have a potent T-cell suppressive activity. This (including heart block), or renal disease, no concomitant use of an suppressive activity is more than 16,000 times stronger than cortisol anticoagulant, and non-smoking. Exclusion criteria were patient's use and 250 times stronger than cyclosporine A or tacrolimus. of drugs that interfere with asthma treatment such as NSAIDs, recent Proscillaridin A is a bufadienolide that acts like immune-regulatory infections leading to hospitalization, or the patient's unwillingness to biomolecules (T cell regulatory substances) (Terness et al., 2001). In continue treatment. addition, some Drimia species such as D. Robusta and D. Delagoensis have anti-inflammatory activities (Du Toit et al., 2005; Stafford et al., 2.4. Preparation of D. maritima 2005). Leaf and tuber extracts of D. maritima have been demonstrated as D. Maritima was collected in September 2013 from Chenarshaijan, having strong antioxidant activity (Mammadov et al., 2010). There is Kazerun city, Fars province, Iran at an altitude of approximately also proof of Squill's antibacterial (Sathiyamoorthy et al., 2008) and 1050 m. The plant was identified and authenticated by Dr. G. Amin antiviral activities (Sato and Muro, 1974). (Department of Pharmacognosy, Faculty of Pharmacy, Tehran Crude extract of Indian Squill () has a bronchodilator University of Medical Sciences), and a voucher specimen (No. 6622- (broncho-relaxant) effect similar to dicyclomine, probably mediated TEH) was deposited in the herbarium of the Faculty of Pharmacy. through anticholinergic and ca2+ antagonistic activities. Also Indian Squill reducesthe mucus secretion of the airway via muscarinic (m3 2.5. Plant analysis receptors) antagonist activity (Bashir et al., 2013). Based on the traditional use of Squill for asthma and its aforemen- A validated High Performance Liquid Chromatography (HPLC) tioned pharmacologic effects in the respiratory system, a randomized method was approved for determining proscillaridin as one of the main clinical trial was designed to evaluate the effects of add-on Squill bufadienolide compounds of D. maritima in a previous study Oxymel on the symptoms and the spirometry and plethysmography (Kasebzade, 2015). Proscillaridin was determined to be 1 mg per each parameters of patients with moderate to severe persistent asthma and gram of plant powder. evaluate its safety in these patients. 2.6. Preparation of syrup and placebo 2. Materials and methods Based on the method used by traditional practitioners for preparing 2.1. Design Squill vinegar, fresh Squill were cleaned thoroughly to remove any soil or debris. Bulbs were cut into small pieces and then boiled in The study was designed as a randomized, triple-blind, placebo- vinegar. After a sufficient time, honey was added to the product. This controlled clinical trial. It was conducted to determine the effects of syrup (Squill Oxymel) was prepared under the supervision of Professor Squill Oxymel in treating 60 patients with moderate to severe Nazem E. of the Traditional Pharmacy Department of the Faculty of

187 F. Nejatbakhsh et al. Journal of Ethnopharmacology 196 (2017) 186–192

Fig. 1. The CONSORT oriented flow diagram.

Table 1 blocks with a block size of four (Matts and Lachin, 1988) (27). All Basic demographic and clinical characteristics of patients in three groups of study. patients, researchers, and evaluators were blinded to the assigned intervention. Group P. value After being enrolled in the study, either Squill Oxymel, simple Squill oxymel Simple honey placebo oxymel, or a placebo was added to the treatment regimen of patients oxymel with a dose of 10 ml twice daily as adjuvant for 6 weeks according to the group allocation. The drugs were delivered to the patients by an Age (year) 40 ± 9.74 49.78 ± 9.2 48.5 ± 16.53 .047 individual nurse who was also blinded to the study. The Pharmacist Sex (female/male) 11/7 10/8 9/9 .8 BMI 27.83 ± 6.83 28.48 ± 5.33 27.9 ± 5.1 .78 who produced the drugs was the only person informed of the type of FEV1% ˂ 60% 10 (55.6) 9 (50) 7 (38.9) .6 syrups labeled as A, B, or C. (n) It was mandatory for patients not to change their administered – 80 60% 8 (44.4) 9 (50) 11 (61.1) medications (combination of full-dose inhaled corticosteroids and (n) β ACT score 15.61 ± 1.79 16.67 ± 2 15.67 ± 1.7 .19 long-acting 2 agonists) during the study. The patients were visited Flut 1000/ salm 18 18 18 NA at 1, 3, and 6 weeks after the study. During each visit, patients were 100 mg or bude evaluated for adjuvant medication tolerance and any possible side 1600 µg /form effects. 18 µg/day

Flut: fluticasone; salm: salmeterol; bude: budesonide; form: formoterol; NA: not 2.8. Lung function tests (Spirometry and the body box) applicable. Spirometry (Ganshorn Medizin Electronic GmbH, Germany) and Traditional Medicine, Tehran University of Medical Sciences, Tehran, body plethysmography were performed once at the beginning and once Iran. Each 5 ml of syrup contained .25 mg of Squill. Acetic acid content again at the end of the study by one trained respiratory technician was determined as 2.24–2.4% w/v which is within the acceptable range according to the American Thoracic Society standards for lung function that described by British pharmacopeia (Commission, 2011). The tests. In addition, the spirometry system was evaluated using a simple oxymel was prepared in the same way, with a base of oxymel computer-driven mechanical syringe most days prior to testing syrup without D. maritima. The placebo was made of simple honey (Miller et al., 1995). boiled in water. The Squill Oxymel syrup, the simple oxymel, and the The considered data measured by the spirometer included: FEV1 placebo had similar packaging. liter, FEV1%, FEV1/FVC%, MEF 25–75%, and the actual PEF. The main study variable was FEV1. Plethysmographic measurements 2.7. Protocol included the actual TLC, RV, RV/TLC, RAW total, RAW ex, RAW in, TGV, TGV/TLC, IC, and sRaw (specific airway resistance). The present study was a triple-blind, randomized, controlled trial. Eligible subjects were randomly divided into 3 treatment groups, the 2.9. St. George's respiratory questionnaire (SGRQ) control group (arm A), Intervention Group 1 (simple oxymel=arm B), and Intervention Group 2 (Squill Oxymel=arm C). Subjects were Health-related quality of life was assessed in all patients by St allocated to 1 of these 3 treatment arms by randomized permuted George's respiratory questionnaire (SGRQ) total score at baseline and 6

188 F. Nejatbakhsh et al. Journal of Ethnopharmacology 196 (2017) 186–192

Table 2 Spirometry parameters of patients in three groups of study before and after intervention.

Parameter Time Time×Group effect

Before After

FEV1 liter Squill oxymel 1.54 ± .38 2.11 ± .49 .001 Simple oxymel 1.59 ± .59 1.74 ± .75 Placebo 1.84 ± 0.63 1.81 ± .55 FEV1% Squill oxymel 5.5 ± 14.16 71.44 ± 21.86 < .0001 Simple oxymel 56.5 ± 15 61.11 ± 15.7 Placebo 65.1 ± 12.47 61.67 ± 14.2 FEV1/FVC% Squill oxymel 65.78 ± 1.57 73.78 ± 13.08 .003 Simple oxymel 66.44 ± 11.64 68.94 ± 9.40 Placebo 71.33 ± 12.36 68.83 ± 13.20 MEF 25–75% Squill oxymel 27.00 ± 12.77 48.22 ± 24.94 < .0001 Simple oxymel 32.22 ± 14.62 36.56 ± 19.94 Placebo 41.89 ± 18.33 38.61 ± 20.76 PEF Squill oxymel 3.68 ± 1.22 4.49 ± 1.19 .12 Simple oxymel 3.86 ± 1.77 4.13 ± 1.77 Placebo 4.30 ± 1.57 4.31 ± 1.48

FEV1: forced expiratory volume in first second; FVC: forced vital capacity; MEF25-75%: forced expiratory flow between 25% and 75%; PEF: peak expiratory flow.

Table 3 Plethysmography parameters of patients in three groups of study before and after intervention.

Parameter Time Time ×Group effect Before After

TLC Squill oxymel 5.39 ± 1.29 5.56 ± 1.27 .95 Simple 5.34 ± 1.21 5.44 ± 1.11 oxymel Placebo 5.03 ± 1.07 5.18 ± 1.12 Fig. 2. Mean changes in percent of Forced Expiratory Volume (FEV1%) in study groups. RV Squill oxymel 2.95 ± 1.04 2.70 ± .90 .63 Simple 2.96 ± 1.12 2.90 ± 1.00 weeks after beginning the study. The SGRQ is a standardized, self- oxymel Placebo 2.37 ± .87 2.43 ± .76 administered questionnaire for the assessment of quality of life (QOL) RV/TLC Squill oxymel 53.89 ± 10.86 48.06 ± 9.28 .36 in patients with chronic airway diseases, including asthma. The total Simple 54.72 ± 13.70 52.89 ± 12.97 score of the SGRQ addresses frequency and severity of respiratory oxymel symptoms, activity limits by breathlessness, and impacts due to Placebo 47.17 ± 13.23 47.39 ± 11.03 disturbances in social and psychological functioning. Thus, the total RAW total Squill oxymel .96 ± .52 .80 ± .37 .25 ff Simple 1.11 ± .57 1.09 ± .51 score summarizes the e ect of the disease on overall health status. It is oxymel easily understood by patients. Higher scores are interpreted as a lower Placebo .90 ± .58 1.00 ± .74 health quality of life (Jones et al., 1992). The validity and reliability of RAW ex Squill oxymel 1.02 ± .56 .83 ± .54 .99 the Persian version of SGRQ in an Iranian population of asthmatic Simple 1.41 ± 1.10 1.2 ± 1.11 patients had been previously evaluated (Tafti et al., 2011). oxymel Placebo 1.06 ± 1.01 .92 ± .55 RAW in Squill oxymel .64 ± .25 .51 ± .22 .27 2.10. Statistical analysis Simple .67 ± .38 .65 ± .23 oxymel Placebo .56 ± .29 .59 ± .38 Descriptive baseline characteristics for comparisons of the three TGV Squill oxymel 4.00 ± 1.06 3.86 ± 1.01 .32 groups were tabulated as means and SD or as percentages. All analyses Simple 3.95 ± 1.15 3.76 ± .93 comparing the efficacy of the outcomes (SGRQ scores, spirometry and oxymel plethysmography parameters) were by intention-to-treat principles. Placebo 3.55 ± .99 3.65 ± .99 Using the General Linear Model (GLM), the score of those parameters TGV/TLC Squill oxymel 74.00 ± 5.14 69.28 ± 7.59 .16 Simple 73.78 ± 9.98 69.22 ± 11.20 among the three groups were compared by repeated measurement oxymel ANOVA test. The compound symmetry assumption was tested using Placebo 69.89 ± 8.67 70.06 ± 8.16 Mauchley's sphericity test. The time groups cross-product (interaction IC Squill oxymel 1.38 ± .37 1.69 ± .54 .22 term) was considered as group differences in their response over time Simple 1.39 ± .57 1.68 ± .77 with the baseline values (age) as a covariate in this model. A oxymel Placebo 1.48 ± .47 1.53 ± .45 fi signi cance level of 5% (alpha=.05) was used for all statistical tests. sRaw Squill oxymel 3.92 ± 2.26 3.13 ± 1.65 .19 Simple 4.38 ± 2.55 4.32 ± 2.74 3. Results oxymel Placebo 3.22 ± 2.42 3.81 ± 3.37

3.1. Flow of participants TLC: total lung capacity; RV: residual volume; RAW (ex, in): airway resistance (inspiratory, expiratory); TGV: thoracic gas volume; IC: inspiratory capacity; sRaw: A total of 83 patients were referred to the study. Of these, 23 specific airway resistance.

189 F. Nejatbakhsh et al. Journal of Ethnopharmacology 196 (2017) 186–192

Table 4 intervention plethysmography parameters of each group. After match- St. George's respiratory questionnaire parameters of patients before and after ing the effects of the possible confounding variables (age), no sig- intervention. nificant differences in TLC, RV, RV/TLC, RAW total, RAW ex, RAW in,

Parameter Time Time×Group TGV, TGV/TLC, IC, and sRaw between study groups were seen (p effect > .05). Before After 3.5. St. George's respiratory questionnaire parameters Activity Squill 76.7 (15 ± .02) 36.25 (22 ± .10) < .001 score oxymel Simple 73.25 (15 ± .36) 45.55 (23 ± .57) Table 4 shows the mean and SD values of the pre- and post- oxymel intervention SGRQ (symptoms, activity, impact, and total) parameters Placebo 59.15 (18 ± .30) 55.75 (25 ± .83) of each group. After matching the effects of the possible confounding Impact Squill 63.37 (18 ± .05) 19.45 (18 ± .70) 0. 059 variables (age), the improvements in symptoms, activity, and total score oxymel fi Simple 58.21 (18 ± .63) 24.91 (17 ± .82) score were signi cantly more in the Squill Oxymel and simple oxymel oxymel groups compared with the placebo group (p < .001) (Fig. 3). Placebo 47.82 (20 ± .76) 32.63 (22 ± .32) Symptoms Squill 73.07 (14 ± .04) 31.58 (21 ± .98) 0. 001 3.6. Adverse events score oxymel Simple 73.42 (16 ± .62) 33.66 (15 ± .21) oxymel Two female patients in Squill oxymel group and three patients in Placebo 61.37 (17 ± .42) 47.98 (22 ± .77) simple oxymel group (2 females) developed nausea and vomiting, Total score Squill 69.26 (14 ± .60) 26.76 (18 ± .15) 0. 004 respectively. The symptoms were mild and resolved after first week and oxymel did not result in patient dropout from the study. No serious adverse Simple 65.53 (14 ± .38) 32.84 (18 ± .23) oxymel event was reported during the study. Placebo 53.67 (17 ± .58) 41.97 (20 ± .53) 4. Discussion

The results of the present study showed improvement in lung function tests in the Squill Oxymel group compared with the control group. Those receiving Squill Oxymel had a significant improvement in absolute values of FEV1 compared with the simple honey oxymel and control groups. Mean FEV1 in the Squill Oxymel group increased > 20%. The demonstrated beneficial effects might be attributed to the anti-inflammatory and anticholinergic activities of Squill. Bufadienolides are the main ingredients of Squill that have immune- modulatory effects. They exert this role by suppressing peripheral T– cell activation. These active glycosides inhibit Na+/K+-ATPase on lymphocytes and suppress their activity. They are many times stronger than steroids and usual immunosuppressive drugs such as steroids, Fig. 3. Mean changes in percent of St. George's respiratory questionnaire (SGRQ) score cyclosporine, and tacrolimus (16,384 times stronger than cortisol and in study groups. 256 times stronger than cyclosporine A or tacrolimus) (Terness et al., 2001). patients did not meet the inclusion criteria, leaving 60 patients eligible Of these, proscillaridin A is a well-recognized immune modulator to participate in the study. They were allocated into three groups. Two biomolecule. Therefore, it is assumed that the increase in FEV1 is due patients from each group were dropped from the study during follow- in part to the immunoregulatory effect of Squill on bronchial asthma. up. Detailed descriptions of patients' enrollment, randomization, and Target cells other than T cells are involved in asthma pathogenesis, so outcomes are shown in the CONSORT oriented flow diagram (Fig. 1). further studies are needed to evaluate possible anti-inflammatory effects of Squill other than T-cell suppressing activities (Akbari et al., 3.2. Baseline characteristics 2003). Dysregulated oxidative stress is present in asthma, but anti- oxidant drugs are not routinely recommended in asthma treatment. Basic demographic and clinical characteristics of the participants Yet, there are studies reporting the oxidative stress suppressing ff are presented in Table 1. Results show that the age of patients di ered features of common asthma medications such as leukotriene inhibitors fi ff signi cantly between groups (p < .05), but di erences in other para- (Riedl and Nel, 2008). In the study performed by Mammadov et al., the fi meters (BMI, sex ratio and severity of disease) were not signi cant (p antioxidant activity of Squill was verified (Mammadov et al., 2010). It is > .05). assumed that the increase in FEV1 is due to both its anti-inflammatory and its bronchodilator effects. Bashir et al. reported that Indian Squill 3.3. Spirometry parameters has smooth muscle relaxant activity in the respiratory tract with the dual effects of calcium influx blockade and anticholinergic effects Table 2 shows the mean and SD values of the pre- and post- (Bashir et al., 2013). The patients were not followed long after the intervention spirometry parameters of each group. After matching the end of study. However, anecdotal data collected from the majority of effects of the possible confounding variables (age), significantly more the patients showed that the subjective beneficial effects of Squill improvement in FEV1, FEV1%, FEV1/FVC%, and MEF 25–75% was continued many weeks after the end of the study. Another study is observed in the Squill Oxymel group (p < .001). Mean FEV1 in the currently being performed by the authors to examine the hypothesis by Squill Oxymel group increased > 20% (Fig. 2). asking patients to fill out the same questionnaires. Therefore, the respiratory effects of Squill are attributable to its anti-inflammatory 3.4. Plethysmography parameters features rather than its bronchodilator effects. No serious adverse event was reported during the study. On regular Table 3 shows the mean and SD values of the pre- and post- outpatient follow-up after the end of study, no patient developed

190 F. Nejatbakhsh et al. Journal of Ethnopharmacology 196 (2017) 186–192 symptoms suggestive of adrenal insufficiency. The findings suggest that all authors. FN, HK and MA made the primary hypothesis and provided Squill does not have any steroid activity or side effects. the proposal. MA, AE and HK visited the patients, enrolled them, and In traditional Iranian medicine (TIM), it is believed that the disease evaluated them. MH carried out the statistical analysis. HK, MH and develops once the balance between body temperaments is disrupted. MA interpreted the results and wrote the first draft of the article. All For instance, asthmatic patients suffer from an overwhelming increase authors approved the final manuscript. in phlegm temperament (Ibn Sina, 2005). Based on TIM, Squill is recognized as one of the best modifiers of phlegm temperament (Aghili, Acknowledgments 2009). Body box parameters, especially the resistance parameters in the The authors thank all the asthmatic patients who participated in the Squill Oxymel group, declined, but the differences were not statistically study and their families. We also acknowledge all the donors in the significant. Also, sRaw decreased in the Squill Oxymel group, but the implementation of this study, especially the technician's breath test of change was not statistically significant. The observed non-significant Imam Khomeini Hospital. This study was a part of Ph.D. dissertation changes were attributed to inadequate sample size. by Dr. Hossein Karegar-Borzi was supported financially by the Based on the results, patients receiving Squill Oxymel and those Research Department of Tehran University of Medical Sciences receiving a simple honey oxymel had a statistically significant improve- (Grant no. 6021309004). ment in their quality-of-life scores (SGRQ scores) from baseline including the SGRQ symptom score, activity score, and total score References compared with the control group. However, this improvement was not statistically significant between the two non-placebo groups. Although Aghili, M., 2009. Makhzan-al-Advia. Tehran University of Medical Sciences, Tehran, 146. the SGRQ impact score in the Squill Oxymel group improved, the Akbari, O., Stock, P., DeKruyff, R.H., Umetsu, D.T., 2003. Role of regulatory T cells in – fi allergy and asthma. Curr. Opin. Immunol. 15, 627 633. change was not statistically signi cant (p value=.059). There is a weak Amirghofran, Z., 2010. Medicinal as immunosuppressive agents in traditional correlation between FEV1 and the quality of life in chronic airway Iranian medicine. Iran. J. Immunol. 7, 65. disease patients (Jones, 2001); in the present study, SGRQ results were Association, W.M., 2009. Declaration of Helsinki. Ethical principles for medical research involving human subjects. consistent with improvements in FEV1 in the Squill Oxymel group but Barnett, S.B.L., Nurmagambetov, T.A., 2011. Costs of asthma in the United States: 2002– not in the simple honey oxymel group. This finding is accordant to TIM 2007. J. Allergy Clin. Immunol. 127, 145–152. notions that both simple honey oxymel and Squill Oxymel are effective Bashir, S., Abbas, S., Khan, A., Gilani, A.H., 2013. Studies on bronchodilator and cardiac – in the treatment of asthmatics, but Squill Oxymel is an enriched and stimulant activities of Urginea indica. Bangladesh J. Pharmacol. 8, 249 254. Bateman, E., Hurd, S., Barnes, P., Bousquet, J., Drazen, J., FitzGerald, M., Gibson, P., more potent formula. Based on TIM, this positive effect is attributed to Ohta, K., O'byrne, P., Pedersen, S., 2008. Global strategy for asthma management the temperament modifying nature of simple honey oxymel in the and prevention: gina executive summary. Eur. Respir. J. 31, 143–178. ff respiratory system. Boskabady, M., Mohsenpoor, N., Takaloo, L., 2010. Antiasthmatic e ect of Nigella sativa in airways of asthmatic patients. Phytomedicine 17, 707–713. Bousquet, J., Mantzouranis, E., Cruz, A.A., Aït-Khaled, N., Baena-Cagnani, C.E., 4.1. Study limitations Bleecker, E.R., Brightling, C.E., Burney, P., Bush, A., Busse, W.W., 2010. Uniform definition of asthma severity, control, and exacerbations: document presented for the World Health Organization Consultation on Severe Asthma. J. Allergy Clin. There are limits to the present study. The sample size was not large Immunol. 126, 926–938. enough; patients were not followed for a long time after the study's Commission, B.P., 2011. British Pharmacopoeia2009. Squill and squill preparation completion; and adjuvant drug safety was based only on clinical monographs, London. Cumming, R.G., Mitchell, P., Leeder, S.R., 1997. Use of inhaled corticosteroids and the assumptions rather than paraclinical parameters such as renal and risk of cataracts. New Engl. J. Med. 337, 8–14. liver function tests. Du Toit, K., Elgorashi, E.E., Malan, S.F., Drewes, S.E., van Staden, J., Crouch, N.R., Mulholland, D.A., 2005. Anti-inflammatory activity and QSAR studies of compounds isolated from Hyacinthaceae species and Tachiadenus longiflorus Griseb. 4.2. Further studies (Gentianaceae). Bioorganic Med. Chem. 13, 2561–2568. Garbe, E., LeLorier, J., Boivin, J.-F., Suissa, S., 1997. Inhaled and nasal glucocorticoids More studies involving more patients and with longer durations are and the risks of ocular hypertension or open-angle glaucoma. Jama 277, 722–727. needed to discover other therapeutic effects, the mechanism of action, Heydari, M., Dalfardi, B., Golzari, S.E., Mosavat, S.H., 2014. Haly Abbas and the early description of obstructive jaundice. Iran J. Public Health 43, 1161–1162. and the effective ingredient(s) of Squill on the respiratory system. Heydari, M., Shams, M., Hashempur, M.H., Zargaran, A., Dalfardi, B., Borhani-Haghighi, According to TIM, asthmatic patients with more phlegm production are A., 2015. The origin of the concept of neuropathic pain in early medieval Persia (9th−12th century CE). Acta Med. Hist. Adriat. 13, 19–22. eligible for Squill administration; however, it is not known which fi fi Ibn Sina, A., 2005. al-Qanun al-tibb 3. Alamy Le-Al-Matbooat institute, Lebanon, asthma phenotype in modern medicine might get more bene t. Further 499–509. studies are needed to evaluate the effects of Squill on total cell Iizuka, M., Warashina, T., Noro, T., 2001. Bufadienolides and a New Lignan from the population of sputum/blood eosinophils or exhaled biomarkers of Bulbs of Urginea maritima. Chem. Pharm. Bull. 49, 282–286. fl Jones, P.W., 2001. Health status measurement in chronic obstructive pulmonary disease. airway in ammation such as FENO (fraction of exhaled NO). Thorax 56, 880–887. Jones, P.W., Quirk, F.H., Baveystock, C.M., Littlejohns, P., 1992. A self-complete 5. Conclusion measure of health status for chronic airflow limitation: the St. George's respiratory questionnaire. Am. Rev. Respir. Dis. 145, 1321–1327. Kannisto, S., Korppi, M., Remes, K., Voutilainen, R., 2000. Adrenal suppression, The results of the current study show preliminary evidence for the evaluated by a low dose adrenocorticotropin test, and growth in asthmatic children efficacy and safety of an add-on treatment of Squill Oxymel in patients treated with inhaled steroids 1. J. Clin. Endocrinol. Metab. 85, 652–657. Karegar-Borzi, H., Salehi, M., Rahimi, R., 2015. Laūq A sustained-release dosage form with moderate to severe persistent asthma. More studies with larger for respiratory disorders in traditional persian medicine. J. Evid. Based sample sizes and longer durations of follow-up are needed for the Complement. Altern. Med., (2156587215587417). confirmation of results. Kasebzade, S., 2015. A validated method for analysis of proscillaridin in Drimia maritima (L.) stearn by high performance liquid chromatography. Islamic Azad University, pharmaceutical sciences branch, Tehran. Conflict of interest statement Longo, D.L., Fauci, A.S., Kasper, D.L., Hauser, S.L., Jameson, J.L., Loscalzo, J., 2012. 18eHarrison's Principles of Internal Medicine 2. EB. McGraw Hill Professional. The authors state that they have no conflicts of interest. Mammadov, R., Makasci-Afacan, A., Uysal-Demir, D., Gork, C., 2010. Determination of antioxidant activities of different Urginea maritima (L.) baker plant extracts. Iran J. Chem. Chem. Eng., 29. Author Contributions Masoli, M., Fabian, D., Holt, S., Beasley, R., 2004. The global burden of asthma: executive summary of the GINA dissemination Committee report. Allergy 59, 469–478. The work presented in this paper is the result of the participation of

191 F. Nejatbakhsh et al. Journal of Ethnopharmacology 196 (2017) 186–192

Matts, J.P., Lachin, J.M., 1988. Properties of permuted-block randomization in clinical 179–190. trials. Control. Clin. Trials 9, 327–344. Riedl, M.A., Nel, A.E., 2008. Importance of oxidative stress in the pathogenesis and Miller, M., Hankinson, J., Brusasco, V., Burgos, F., Casaburi, R., Coates, A., Crapo, R., treatment of asthma. Curr. Opin. Allergy Clin. Immunol. 8, 49–56. Enright, P., van der Grinten, C., Gustafsson, P., 1995. Standardization of spirometry, Sathiyamoorthy, P., Lugasi-Evgi, H., Schlesinger, P., Kedar, I., Gopas, J., Pollack, Y., 1994 update. American thoracic society. Am. J. Respir. Crit. Care Med. 152, Golan-Goldhirsh, A., 2008. Screening for cytotoxic and antimalarial activities in 1107–1136. desert plants of the Negev and Bedouin market plant products. Pharm. Biol.. Mosavat, S.H., Ghahramani, L., Rahmanian Haghighi, E., Rostami Chaijan, M., Sato, N., Muro, T., 1974. Antiviral activity of scillarenin, a plant bufadienolide. Jpn. J. Hashempur, M.H., Heydari, M., 2015. Anorectal Diseases in Avicenna's "Canon of Microbiol. 18, 441–448. Medicine". Acta Med.-Hist. Adriat. 13, 103–114. Stafford, G., Jäger, A., Van Staden, J., 2005. Effect of storage on the chemical Neter, J., Kutner, M.H., Nachtsheim, C.J., Wasserman, W., 1996. Applied linear composition and biological activity of several popular South African medicinal statistical models.Irwin Chicago. plants. J. Ethnopharmacol. 97, 107–115. Nomellini, V., Chen, H., 2012. Murray and Nadel's Textbook of Respiratory Medicine. Stedje, B., 1987. A revision of the Drimia (Hyacinthaceae) in East Africa. Nord. J. Academic Press. Bot. 7, 655–666. Qasemzadeh, M.J., Sharifi, H., Hamedanian, M., Gharehbeglou, M., Heydari, M., Sardari, Tafti, S.F., Cheraghvandi, A., Mokri, B., Talischi, F., 2011. Validity and specificity of the M., Akhlaghdoust, M., Minae, M.B., 2015. The Effect of Viola odorata flower Syrup Persian version of the Saint George Respiratory Questionnaire. J. Asthma 48, on the cough of children With asthma A Double-blind, randomized controlled trial. J. 589–592. Evid. Based Complement. Altern. Med. 20, 287–291. Terness, P., Navolan, D., Dufter, C., Kopp, B., Opelz, G., 2001. The T-cell suppressive Richy, F., Bousquet, J., Ehrlich, G.E., Meunier, P.J., Israel, E., Morii, H., Devogelaer, J.- effect of bufadienolides: structural requirements for their immunoregulatory activity. P., Peel, N., Haim, M., Bruyere, O., 2003. Inhaled corticosteroids effects on bone in Int. Immunopharmacol. 1, 119–134. asthmatic and COPD patients: a quantitative systematic review. Osteoporos. Int. 14,

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