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The effect of a mixture of Nigella sativa and Thymus vulgaris extracts in children with refractory epilepsies: A randomized trial
Accepted Manuscript Title: The effect of a mixture of Nigella sativa and Thymus vulgaris extracts in children with refractory epilepsies; A randomized trial Authors: Ali Akbar Momen, Ali Asghar Hemati, Gholamreza Hoshmand, Maryam Heydar Azadzadeh, Reza Azizi Malamiri PII: DOI: Reference:
S2210-8033(18)30052-6 https://doi.org/10.1016/j.hermed.2018.10.007 HERMED 242
To appear in: Received date: Accepted date:
23-2-2018 25-10-2018
Please cite this article as: Momen AA, Hemati AA, Hoshmand G, Azadzadeh MH, Malamiri RA, The effect of a mixture of Nigella sativa and Thymus vulgaris extracts in children with refractory epilepsies; A randomized trial, Journal of Herbal Medicine (2018), https://doi.org/10.1016/j.hermed.2018.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The effect of a mixture of Nigella sativa and Thymus vulgaris extracts in children with refractory epilepsies; A randomized trial
Ali Akbar Momena, Ali Asghar Hematib, Gholamreza Hoshmandc, Maryam Heydar
Department of Paediatric Neurology, Golestan Medical, Educational, and Research Center,
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Azadzadeha, Reza Azizi Malamiria,*
Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran b
Department of Pharmacology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz,
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Department of Pharmacology, Mazandaran University of Medical Sciences, Sari, Iran
author: Reza Azizi Malamiri, Department of Paediatric Neurology, Golestan
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*Corresponding
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c
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Iran
Medical, Educational, and Research Center, Ahvaz Jundishapur University of Medical Sciences, # 90, Golestan Blvd., P.O. Box 6135733118, Ahvaz, IRAN, Tel/Fax: +98 613
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3743063,
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email; [email protected]; [email protected]
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Abstract More than 25% of seizures in children are refractory to anti-seizure medications and need other modalities to control. Recently, herbal agents with anticonvulsive effects on seizure models in animals have received significant attention, but studies that show their impact on
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seizures in children are scarce. In a randomized, double-blind placebo-controlled cross-over design, we tried to demonstrate the efficacy of a mixture of Nigella sativa and Thymus
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vulgaris extracts (Epistop) on seizures in a group of children with refractory epilepsies.
Twenty-two children with refractory epilepsies were randomly assigned into two sequences
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to receive Epistop or placebo in a cross-over design. Neither Epistop nor placebo had effects
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on the seizure frequency and duration over the study period. Only four children experienced
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clinically substantial seizure frequency (more than 50%) reduction after Epistop
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administration. These results indicate that a mixture of Nigella sativa and Thymus vulgaris extracts does not affect the seizure frequency and duration in children with refractory
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seizures. However, a minority of highly selected children with refractory seizures might benefit from the effects of a mixture of these agents’ extracts. Keywords: Children; Refractory Epilepsies; Nigella sativa; Thymus vulgaris; Randomized
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cross-over trial
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1. Introduction Epilepsy and epileptic syndromes are common neurologic disorders in children. Many of epilepsies and epileptic syndromes could be treated successfully with anti-seizure medications. However, about 25% percent of seizures in children are resistant to three appropriately selected anti-seizure medications and need other modalities to control including ketogenic diet and
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epilepsy surgery. These types of epilepsies and epileptic syndromes have been called
refractory. Some children tolerate the ketogenic diet for a while, but the long-term use of this
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diet has been shown to be extremely hard to continue. A few children with refractory epilepsies could be good candidates for epilepsy surgery, but epilepsy surgery has its limitations and
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preparing a child for epilepsy surgery needs substantial work-intensive tasks to complete.
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(Chapell et al., 2003; Hallbook et al., 2007; Hallbook et al., 2005; Hallbook et al., 2013;
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Hossain et al., 2013; Majkowska-Zwolinska et al., 2012)
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Recently, herbal agents that are effective in controlling seizures have received attention. Among these agents, the most notable are Nigella sativa and Thymus vulgaris and the primary
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active chemical agents found in their extracts are thymoquinone (Nigella sativa) and thymol (Thymus vulgaris). Some studies showed the controlling effects of these agents on animal
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models of seizures. Moreover, a few studies showed the applications of these agents in children with refractory epilepsies (Abdel-Rahman, 2013; Akhondian et al., 2011; Beyazcicek et al.,
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2016; Gilhotra and Dhingra, 2011; Hawkes, 2017; Hosseinzadeh and Parvardeh, 2004; Hosseinzadeh et al., 2007; Hosseinzadeh et al., 2005; Ilhan et al., 2005; Komaki et al., 2016;
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Priestley et al., 2003; Sancheti et al., 2014). Both thymol and thymoquinone have anti-seizure effects in animal models of maximal
electroshock and pentylenetetrazole-induced seizures. Molecular studies showed that the anticonvulsive mechanism of thymol in these models could be explained by the effects of thymol on neuronal sodium channels and on gamma-amino butyric acid (GABA) receptors. 3
Thymol blocked neuronal voltage-gated sodium channels. Thymol also showed antioxidant properties, and a few studies suggest that this role could explain the anti-epileptogenic activity shown by this agent. Thymoquinone showed antiepileptic activity via its effects on GABA receptors, and its anti-epileptogenic activity has also been demonstrated via penicillin-induced epileptiform activity in rats (Abdel-Rahman, 2013; Al-Naggar et al., 2003; Beyazcicek et al.,
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2016; Gilhotra and Dhingra, 2011; Hosseinzadeh and Parvardeh, 2004; Hosseinzadeh et al., 2007; Hosseinzadeh et al., 2005; Ilhan et al., 2005; Komaki et al., 2016; Priestley et al., 2003;
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Sancheti et al., 2014). A few studies have also shown the anticonvulsive effects of thymoquinone in children with refractory seizures (Akhondian et al., 2011).
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To our knowledge, none of the previous studies evaluated the impact of combined extracts
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of Nigella sativa and Thymus vulgaris in children with refractory seizures. Moreover, a
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monitored solution of a combination of extracts of these herbal agents is accessible in Iran via
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herbal drug stores at reasonable prices compared with the new-generation antiepileptic medications, which are expensive. Therefore, we conducted a pilot trial to assess the effects of
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the combined extracts of Nigella sativa and Thymus vulgaris on the frequency and duration of seizures in children with refractory epilepsies.
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2. Methods
2.1. Study design, aims, and setting
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The authors conducted a pilot double-blind, placebo-controlled, randomized, crossover trial
to assess the efficacy and safety of a solution that was a mixture of Nigella sativa and Thymus
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vulgaris extract called Epistop in children with refractory seizures. The study was conducted at Golestan Hospital, a university-affiliated medical, educational and research center. This center is the main child neurology tertiary referral center in the southwest of Iran; almost all children with refractory seizures are referred to this center for further evaluation and seizure control. The study was conducted between January 2016 and July 2017. The study protocol 4
was
approved
by
the
ethical
board
committee
of
the
medical
center
(IR.AJUMS.REC.1394.141). This trial is registered with the Iranian Registry of Clinical Trials, (registration number IRCT2016052428043N1). Written informed consent was obtained from all parents before enrollment.
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2.2. Patients, inclusion and exclusion criteria Eligible children were those who were aged between 2 and 18 years and diagnosed with
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refractory seizure or refractory epileptic syndrome with at least two seizure events per week
while taking three or more appropriately selected anticonvulsants. From these eligible children with refractory seizures, all those were included who had cooperative parents to register the
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frequency and duration of seizures properly. Those children with a history of significant
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adverse reaction to Nigella sativa and Thymus vulgaris extracts were excluded.
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2.3. Monitoring period before administering the herbal agents
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In this period, all those children who were selected to be included in the trial were visited by a child neurologist who took a proper history about the frequency and duration of seizures in each child and examined all the children. In this visit, all the parents were educated to register
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the frequency and duration of seizures properly in predefined charts on a daily basis for 4
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weeks. We asked all the parents to continue anticonvulsive medication during this period and along the trial without any change. We acknowledged that all the parents have rights to abort
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prolonged breakthrough seizures using Diazepam rectal tubes, but we asked them to register all these events accurately. This monitoring period allowed us to have an estimate of the frequency and duration of seizures in the children before the herbal agent administration.
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2.4. Randomization, blinding, crossing over, and follow up Since refractory seizures have no cure and can only be controlled, we decided to carry out a randomized cross-over trial. Because of the particular design, these trials need small sample sizes and merely remove the confounders. These trials are also the best design for complaints that have no cure. All the eligible children who met the inclusion criteria were randomized to
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two sequences to receive Epistop or placebo. Children were randomized using a random
number table to allocate consecutive patients into two groups with blocks of four. After
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randomization, all the children received the Epistop or the placebo for four weeks along with
their medication. After this four weeks, either Epistop or placebo was discontinued for two
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weeks as a washout period. After washout period the medication group was changed, and the
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children in the Epistop group switched to the placebo treatment, and the children in the placebo
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group switched to the Epistop treatment, and the trial was continued for another 4 weeks. All
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parents were asked to register the frequency and duration of seizures during these periods of the trial accurately. All the parents, the children who were included, and the staff who
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administered either Epistop or placebo were blinded to the study groups over the study periods.
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2.5. Herbal agent administration and the placebo
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After 4 weeks of the monitoring period, all the children took a solution of the herbal agents called Epistop composed of Nigella sativa and Thymus vulgaris extracts. This solution
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was made by herbal pharmacologists of the University. Based on the previous studies (Akhondian et al., 2011), the solution needs to have 25 mg/ml of each of thymoquinone and thymol. We used the following procedure to prepare this solution which is called Epistop syrup. Nigella sativa seeds and Thymus vulgaris branches and leaves were obtained from the local herbal garden of the pharmacology department in Ahvaz, Iran. Herbal pharmacologist
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from the pharmacology department identified seeds of the Nigella sativa and Thymus vulgaris branches and leaves, and voucher samples (Nigella sativa voucher no. A14017009P and Thymus vulgaris voucher no. C12014174P) were preserved for reference in the herbarium of the pharmacology department, Ahvaz Jundishapur University of Medical Sciences, Iran. We dried them at room temperature then ground them into a fine powder using an electric
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grinder. One kilogram of dry powdered Nigella sativa was added to a solution of 7 liters of 70% ethanol and 3 liters of distilled water. The same procedure was also performed for
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Thymus vulgaris. This mixture provided 200 gr extract of each herbal agent after standard
vaporization. To prepare the Epistop solution, 1.125 gr of each extract of Nigella sativa and
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Thymus vulgaris was added to a solution of 40 gr of sucrose, 15 ml sorbitol 70%, 3 ml
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glycerin, sodium benzoate (0.2% from the whole volume), citric acid (0.1% from the whole
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volume) and distilled water was added to receive to a volume of 100 ml. The herbal
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pharmacologist of the university analyzed the phytochemical content of the extract mixture using a Shimadzu QP5050 GC/MS with the DB-5 capillary column. This analysis showed
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that this mixture has 25 mg/ml of each thymoquinone and thymol. Based on a previous study in children, we administered the equivalent dose of 0.5 mg/kg/dose of thymoquinone every 12 hours (Akhondian et al., 2011). The placebo was made precisely like Epistop but without
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herbal extracts and was packaged in similar bottles.
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2.6. Outcome measures and adverse effects The main outcome measure was the efficacy of Epistop on the frequency and duration of
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seizures during the periods of the trial. The proper efficacy was defined by a seizure frequency reduction of more than 50% after the medication administration compared with the monitoring period. We also monitored the adverse effects of the Epistop on the children. We asked parents to register the frequency of breakthrough seizures that needed rectal diazepam gel.
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2.7. Statistics Due to the special design of cross-over trials and based on a previous study of the effects of thymoquinone in children with refractory seizures and by considering alpha= 0.05 and Beta=0.2, the sample size was calculated to provide at least ten patients in each treatment group (Akhondian et al., 2011). For each patient randomized to receive either Epistop or placebo, the
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median and the mean frequency, and duration of seizures at the end of each 4-week period was determined. The analysis of the results of this trial was performed based on the statistic tests
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that are appropriate for the cross-over design in clinical trials. In this kind of trial, the authors need to consider some assumptions. To perform a cross-over trial, it is necessary to know about
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the period effect and the carryover effect. If these effects show a significant difference before
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and after the washout period, then we could not perform cross-over statistics. After the
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declaration of the presence of these assumptions, the cross-over statistics were conducted
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(Wellek and Blettner, 2012). To perform the statistical analysis, we used the NCSS 11 statistical software (NCSS, LLC 329 North 1000 East Kaysville, Utah 84037 USA), which is
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specifically designed to conduct cross-over statistics. In all statistical evaluations, P < 0.05 was considered significant.
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3. Results
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3.1. Baseline characteristics The authors enrolled 22 children with refractory seizures into the study and randomly
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assigned them to be in the sequence one (which was administered Epistop) or two (which was administered placebo) of the study. Of these, 14 were girls (63.6%) and 8 (36.4%) were boys. The age range was between 4 and 17 years. The median age was 7 years old (Fig 1. Consort flow diagram). The main seizure types were generalized tonic-clonic (GTC), absences and GTCs, tonic, and myoclonic. The minimum number of anti-seizure medications was three, and
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one of the children received seven anti-seizure medications during the study. In 18 (81.8%) children, the interIctal electroencephalography showed epileptiform abnormalities. In 10 (45.5%) children, brain MRI showed structural abnormalities. The development was delayed in 13 (59.1%) of the children. Six of the patients had developmental regression because of the uncontrolled seizures. Before randomization, the range of seizure frequency was between 2 and
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140 events per week, and the median seizure frequency was 35 events per week. Before
randomization, the range of seizure duration was 5 and 600 seconds, and the median seizure
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duration was 30 seconds.
3.2. Effects of Epistop on the frequency and duration of seizures
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No difference was found between two groups regarding seizure frequency or seizure
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duration during the monitoring period and after administering Epistop and placebo. The results
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showed that neither Epistop nor placebo reduced the seizure frequency and the seizure duration
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in the patients (Cross-Over analysis using T-tests; T value= 2.06, P= 0.0528 for the frequency
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of seizures and T value= 1.7, P= 0.1043 for the duration of seizures). In one patient, the seizure frequency was increased by 150% (from 70 to 150 per week) by administering Epistop. In only four patients, seizures frequency reduced more than 60%
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(clinically significant) after Epistop administration. In these four patients, seizure frequency increased significantly after the washout period and during placebo administration.
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Interestingly, all of these children had normal development, normal interIctal EEGs, normal MRI, and the median seizure frequency of fewer than 10 events per week. In only five patients,
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the seizure duration significantly reduced during Epistop administration (more than 50%). 3.3. Adverse effects None of the patients reported significant adverse effects during Epistop or placebo administration. Only three patients, two in Epistop group and one in the placebo group reported 9
lethargy and diarrhea, but none of these patients discontinued the trial because of this adverse effect. The frequency of diazepam rectal gels for controlling breakthrough seizures in the two groups was the same during the study. 4. Discussion
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In this study, we have shown that a 2-month course of either Epistop (a mixture of Nigella sativa and Thymus vulgaris extracts) or placebo does not affect the seizure frequency and
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duration in a group of children with refractory seizures. However, some children with
refractory seizures may benefit from using Epistop as we have shown in this study that seizure frequency in four children with refractory seizures was significantly reduced by Epistop syrup
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administration. As mentioned previously in the results section, all of these children had normal
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development, normal Interictal EEGs, normal MRI, and the median seizure frequency of fewer
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than 10 events per week. Extrapolation of these results to a population of children with
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refractory seizure is difficult. However, administering Epistop might be indicated in children
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with refractory seizures who have normal development, normal Interictal EEGs, normal MRI, and those who have a low seizure frequency. Almost all the previous studies showed that refractory epilepsies are a subgroup of seizures
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that are intractable to nearly all the treatment modalities and need ketogenic diet and epilepsy surgery in addition to the anti-seizure medications in most patients. However, after all these
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efforts, these seizures still occur in patients and have a substantial negative impact on the families' and the patient's lives. Therefore, introducing a new controlling modality could be
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very helpful to improve the quality of life in children with refractory seizures. In recent years, herbal agents with anticonvulsive effects have received significant attention and studies in animals showed their effects in different seizure models (Abdel-Rahman, 2013; Al-Naggar et al., 2003; Beyazcicek et al., 2016; Gilhotra and Dhingra, 2011; Hawkes, 2017; Hosseinzadeh and Parvardeh, 2004; Hosseinzadeh et al., 2007; Hosseinzadeh et al., 2005; Ilhan et al., 2005; 10
Komaki et al., 2016; Priestley et al., 2003; Sancheti et al., 2014). However, human studies that demonstrate their effects on the refractory seizures in children are scarce (Akhondian et al., 2011). To our knowledge, only one study showed that a 28-day administration of thymoquinone in a group of children with refractory seizures had a significant reducing effect on the seizure
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frequency. However, the results of our study did not duplicate the results of that study. The authors could explain the different results of our study and the mentioned study by the
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following evidence:-
In the study performed by Akhondian et al., (Akhondian et al., 2011) they administered
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only the solution of thymoquinone in a group of children with refractory seizures. When the
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authors looked at the exclusion criteria in the above mentioned study, it was understood that
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they excluded children with epilepsy secondary to a progressive cerebral disease or any
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progressive neurodegenerative disease and children with severe exacerbation of seizures during the study. The authors had different inclusion and exclusion criteria in this study. They also
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designed a cross-over trial. Cross-over trials are appropriate when there is no cure, and just reduction of symptoms can be found after administering the agent, but it is noticeable that these trials need particular attention during statistical analysis. After a critical analysis of the
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Akhondian et al. study, we could not find the cross-over analysis in the statistics and the result
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section. Based on this evidence, the authors were able to explain the differences in the two studies.
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Almost all previous studies in animals that have explored the effects of Nigella sativa and
Thymus vulgaris on seizure models showed the anticonvulsive effects of these agents (AbdelRahman, 2013; Al-Naggar et al., 2003; Beyazcicek et al., 2016; Gilhotra and Dhingra, 2011; Hosseinzadeh and Parvardeh, 2004; Hosseinzadeh et al., 2007; Hosseinzadeh et al., 2005; Ilhan et al., 2005; Komaki et al., 2016; Priestley et al., 2003; Sancheti et al., 2014). However, without
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trials that show the effects of these agents on seizures in humans, administering these agents in humans could not be recommended. The results of our study indicated that a solution of a mixture of Nigella sativa and Thymus vulgaris extracts might be effective in reducing seizure frequency and duration just in a minority of children with refractory seizures. These results also indicate that the children showed no significant adverse effects after
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taking the mixture of Nigella sativa and Thymus vulgaris and none of the children discontinued this agent, as previous studies have shown.
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The results of this study should be interpreted with certain limits. Unfortunately, no definite dose of the solution of Nigella sativa and Thymus vulgaris could be found in the human
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literature. Hence, experimental dosages based on the animal studies and just one human study
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were attempted because ethical issues could not let us try higher dosages of these agents in the
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patients. Refractory seizures have no definite cure. Therefore, the authors could not use trials
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with parallel designs. Complaints such as refractory seizures can only be controlled by medication and not be cured. Thus, studies on refractory seizures need cross-over designs, but
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it should be emphasized that cross-over designs have difficulties especially for concealment and should have strict statistics carried out.
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5. Conclusion
In conclusion, the results of this study indicate that a solution of a mixture of Nigella
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sativa and Thymus vulgaris extracts does not affect the seizure frequency and duration in children with refractory seizures. However, a minority of highly selected children with
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refractory seizures might benefit from the effects of a mixture of these agents’ extracts. In the authors opinion administering the extracts of these agents in every child with refractory seizures should not be carried out, however, children with refractory seizures who have normal development, normal EEG, normal MRI, and those who have a low seizure frequency fewer than 10 events per week might be profit from administering Epistop. 12
Conflicts of interest: none
Acknowledgments The Research Deputy of Ahvaz Jundishapur University provided financial and logistic support
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for this study, but had no role in study design; data collection, analysis, or interpretation;
writing the report; or in the decision to submit the article for publication. The authors are
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grateful to all parents and patients for their participation in this study. We are incredibly indebted to the authorities of the Research Deputy of Ahvaz Jundishapur University of Medical
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Sciences for their financial and logistic support.
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Funding: This work was supported by the Research Deputy of Ahvaz Jundishapur University
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of Medical Sciences (number: MPRC 9404)
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Figure captions
Enrollment
Assessed for eligibility (n= 27)
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Fig 1 CONSORT Flow Diagram
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Excluded (n= 5) Not meeting inclusion criteria (n=0 ) Declined to participate (n=5 ) Other reasons (n=0 )
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Randomized (n=22)
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Allocation
Allocated to Epistop (4 weeks) (n= 12) Received allocated Epistop (n= 12) Did not receive allocated Epistop (give reasons) (n= 0 ) Then 2 weeks of washout period Then 4 weeks switch to other sequence
Allocated to Placebo (4 weeks) (n= 10) Received allocated Placebo (n= 10) Did not receive allocated Placebo (give reasons) (n= 0 ) Then 2 weeks of washout period Then 4 weeks switch to other sequence
Follow-Up Lost to follow-up (give reasons) (n=0)
Discontinued intervention (give reasons) (n= 0)
Discontinued intervention (give reasons) (n= 0)
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Lost to follow-up (give reasons) (n= 0)
Analysis Analysed (n= 10) Excluded from analysis (give reasons) (n= 0)
Analysed (n= 12) Excluded from analysis (give reasons) (n= 0)
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S2210-8033(18)30052-6 https://doi.org/10.1016/j.hermed.2018.10.007 HERMED 242
To appear in: Received date: Accepted date:
23-2-2018 25-10-2018
Please cite this article as: Momen AA, Hemati AA, Hoshmand G, Azadzadeh MH, Malamiri RA, The effect of a mixture of Nigella sativa and Thymus vulgaris extracts in children with refractory epilepsies; A randomized trial, Journal of Herbal Medicine (2018), https://doi.org/10.1016/j.hermed.2018.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The effect of a mixture of Nigella sativa and Thymus vulgaris extracts in children with refractory epilepsies; A randomized trial
Ali Akbar Momena, Ali Asghar Hematib, Gholamreza Hoshmandc, Maryam Heydar
Department of Paediatric Neurology, Golestan Medical, Educational, and Research Center,
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Azadzadeha, Reza Azizi Malamiria,*
Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran b
Department of Pharmacology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz,
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Department of Pharmacology, Mazandaran University of Medical Sciences, Sari, Iran
author: Reza Azizi Malamiri, Department of Paediatric Neurology, Golestan
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*Corresponding
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c
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Iran
Medical, Educational, and Research Center, Ahvaz Jundishapur University of Medical Sciences, # 90, Golestan Blvd., P.O. Box 6135733118, Ahvaz, IRAN, Tel/Fax: +98 613
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3743063,
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email; [email protected]; [email protected]
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Abstract More than 25% of seizures in children are refractory to anti-seizure medications and need other modalities to control. Recently, herbal agents with anticonvulsive effects on seizure models in animals have received significant attention, but studies that show their impact on
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seizures in children are scarce. In a randomized, double-blind placebo-controlled cross-over design, we tried to demonstrate the efficacy of a mixture of Nigella sativa and Thymus
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vulgaris extracts (Epistop) on seizures in a group of children with refractory epilepsies.
Twenty-two children with refractory epilepsies were randomly assigned into two sequences
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to receive Epistop or placebo in a cross-over design. Neither Epistop nor placebo had effects
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on the seizure frequency and duration over the study period. Only four children experienced
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clinically substantial seizure frequency (more than 50%) reduction after Epistop
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administration. These results indicate that a mixture of Nigella sativa and Thymus vulgaris extracts does not affect the seizure frequency and duration in children with refractory
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seizures. However, a minority of highly selected children with refractory seizures might benefit from the effects of a mixture of these agents’ extracts. Keywords: Children; Refractory Epilepsies; Nigella sativa; Thymus vulgaris; Randomized
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cross-over trial
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1. Introduction Epilepsy and epileptic syndromes are common neurologic disorders in children. Many of epilepsies and epileptic syndromes could be treated successfully with anti-seizure medications. However, about 25% percent of seizures in children are resistant to three appropriately selected anti-seizure medications and need other modalities to control including ketogenic diet and
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epilepsy surgery. These types of epilepsies and epileptic syndromes have been called
refractory. Some children tolerate the ketogenic diet for a while, but the long-term use of this
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diet has been shown to be extremely hard to continue. A few children with refractory epilepsies could be good candidates for epilepsy surgery, but epilepsy surgery has its limitations and
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preparing a child for epilepsy surgery needs substantial work-intensive tasks to complete.
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(Chapell et al., 2003; Hallbook et al., 2007; Hallbook et al., 2005; Hallbook et al., 2013;
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Hossain et al., 2013; Majkowska-Zwolinska et al., 2012)
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Recently, herbal agents that are effective in controlling seizures have received attention. Among these agents, the most notable are Nigella sativa and Thymus vulgaris and the primary
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active chemical agents found in their extracts are thymoquinone (Nigella sativa) and thymol (Thymus vulgaris). Some studies showed the controlling effects of these agents on animal
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models of seizures. Moreover, a few studies showed the applications of these agents in children with refractory epilepsies (Abdel-Rahman, 2013; Akhondian et al., 2011; Beyazcicek et al.,
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2016; Gilhotra and Dhingra, 2011; Hawkes, 2017; Hosseinzadeh and Parvardeh, 2004; Hosseinzadeh et al., 2007; Hosseinzadeh et al., 2005; Ilhan et al., 2005; Komaki et al., 2016;
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Priestley et al., 2003; Sancheti et al., 2014). Both thymol and thymoquinone have anti-seizure effects in animal models of maximal
electroshock and pentylenetetrazole-induced seizures. Molecular studies showed that the anticonvulsive mechanism of thymol in these models could be explained by the effects of thymol on neuronal sodium channels and on gamma-amino butyric acid (GABA) receptors. 3
Thymol blocked neuronal voltage-gated sodium channels. Thymol also showed antioxidant properties, and a few studies suggest that this role could explain the anti-epileptogenic activity shown by this agent. Thymoquinone showed antiepileptic activity via its effects on GABA receptors, and its anti-epileptogenic activity has also been demonstrated via penicillin-induced epileptiform activity in rats (Abdel-Rahman, 2013; Al-Naggar et al., 2003; Beyazcicek et al.,
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2016; Gilhotra and Dhingra, 2011; Hosseinzadeh and Parvardeh, 2004; Hosseinzadeh et al., 2007; Hosseinzadeh et al., 2005; Ilhan et al., 2005; Komaki et al., 2016; Priestley et al., 2003;
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Sancheti et al., 2014). A few studies have also shown the anticonvulsive effects of thymoquinone in children with refractory seizures (Akhondian et al., 2011).
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To our knowledge, none of the previous studies evaluated the impact of combined extracts
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of Nigella sativa and Thymus vulgaris in children with refractory seizures. Moreover, a
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monitored solution of a combination of extracts of these herbal agents is accessible in Iran via
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herbal drug stores at reasonable prices compared with the new-generation antiepileptic medications, which are expensive. Therefore, we conducted a pilot trial to assess the effects of
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the combined extracts of Nigella sativa and Thymus vulgaris on the frequency and duration of seizures in children with refractory epilepsies.
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2. Methods
2.1. Study design, aims, and setting
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The authors conducted a pilot double-blind, placebo-controlled, randomized, crossover trial
to assess the efficacy and safety of a solution that was a mixture of Nigella sativa and Thymus
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vulgaris extract called Epistop in children with refractory seizures. The study was conducted at Golestan Hospital, a university-affiliated medical, educational and research center. This center is the main child neurology tertiary referral center in the southwest of Iran; almost all children with refractory seizures are referred to this center for further evaluation and seizure control. The study was conducted between January 2016 and July 2017. The study protocol 4
was
approved
by
the
ethical
board
committee
of
the
medical
center
(IR.AJUMS.REC.1394.141). This trial is registered with the Iranian Registry of Clinical Trials, (registration number IRCT2016052428043N1). Written informed consent was obtained from all parents before enrollment.
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2.2. Patients, inclusion and exclusion criteria Eligible children were those who were aged between 2 and 18 years and diagnosed with
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refractory seizure or refractory epileptic syndrome with at least two seizure events per week
while taking three or more appropriately selected anticonvulsants. From these eligible children with refractory seizures, all those were included who had cooperative parents to register the
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frequency and duration of seizures properly. Those children with a history of significant
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adverse reaction to Nigella sativa and Thymus vulgaris extracts were excluded.
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2.3. Monitoring period before administering the herbal agents
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In this period, all those children who were selected to be included in the trial were visited by a child neurologist who took a proper history about the frequency and duration of seizures in each child and examined all the children. In this visit, all the parents were educated to register
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the frequency and duration of seizures properly in predefined charts on a daily basis for 4
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weeks. We asked all the parents to continue anticonvulsive medication during this period and along the trial without any change. We acknowledged that all the parents have rights to abort
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prolonged breakthrough seizures using Diazepam rectal tubes, but we asked them to register all these events accurately. This monitoring period allowed us to have an estimate of the frequency and duration of seizures in the children before the herbal agent administration.
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2.4. Randomization, blinding, crossing over, and follow up Since refractory seizures have no cure and can only be controlled, we decided to carry out a randomized cross-over trial. Because of the particular design, these trials need small sample sizes and merely remove the confounders. These trials are also the best design for complaints that have no cure. All the eligible children who met the inclusion criteria were randomized to
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two sequences to receive Epistop or placebo. Children were randomized using a random
number table to allocate consecutive patients into two groups with blocks of four. After
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randomization, all the children received the Epistop or the placebo for four weeks along with
their medication. After this four weeks, either Epistop or placebo was discontinued for two
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weeks as a washout period. After washout period the medication group was changed, and the
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children in the Epistop group switched to the placebo treatment, and the children in the placebo
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group switched to the Epistop treatment, and the trial was continued for another 4 weeks. All
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parents were asked to register the frequency and duration of seizures during these periods of the trial accurately. All the parents, the children who were included, and the staff who
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administered either Epistop or placebo were blinded to the study groups over the study periods.
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2.5. Herbal agent administration and the placebo
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After 4 weeks of the monitoring period, all the children took a solution of the herbal agents called Epistop composed of Nigella sativa and Thymus vulgaris extracts. This solution
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was made by herbal pharmacologists of the University. Based on the previous studies (Akhondian et al., 2011), the solution needs to have 25 mg/ml of each of thymoquinone and thymol. We used the following procedure to prepare this solution which is called Epistop syrup. Nigella sativa seeds and Thymus vulgaris branches and leaves were obtained from the local herbal garden of the pharmacology department in Ahvaz, Iran. Herbal pharmacologist
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from the pharmacology department identified seeds of the Nigella sativa and Thymus vulgaris branches and leaves, and voucher samples (Nigella sativa voucher no. A14017009P and Thymus vulgaris voucher no. C12014174P) were preserved for reference in the herbarium of the pharmacology department, Ahvaz Jundishapur University of Medical Sciences, Iran. We dried them at room temperature then ground them into a fine powder using an electric
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grinder. One kilogram of dry powdered Nigella sativa was added to a solution of 7 liters of 70% ethanol and 3 liters of distilled water. The same procedure was also performed for
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Thymus vulgaris. This mixture provided 200 gr extract of each herbal agent after standard
vaporization. To prepare the Epistop solution, 1.125 gr of each extract of Nigella sativa and
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Thymus vulgaris was added to a solution of 40 gr of sucrose, 15 ml sorbitol 70%, 3 ml
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glycerin, sodium benzoate (0.2% from the whole volume), citric acid (0.1% from the whole
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volume) and distilled water was added to receive to a volume of 100 ml. The herbal
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pharmacologist of the university analyzed the phytochemical content of the extract mixture using a Shimadzu QP5050 GC/MS with the DB-5 capillary column. This analysis showed
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that this mixture has 25 mg/ml of each thymoquinone and thymol. Based on a previous study in children, we administered the equivalent dose of 0.5 mg/kg/dose of thymoquinone every 12 hours (Akhondian et al., 2011). The placebo was made precisely like Epistop but without
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herbal extracts and was packaged in similar bottles.
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2.6. Outcome measures and adverse effects The main outcome measure was the efficacy of Epistop on the frequency and duration of
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seizures during the periods of the trial. The proper efficacy was defined by a seizure frequency reduction of more than 50% after the medication administration compared with the monitoring period. We also monitored the adverse effects of the Epistop on the children. We asked parents to register the frequency of breakthrough seizures that needed rectal diazepam gel.
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2.7. Statistics Due to the special design of cross-over trials and based on a previous study of the effects of thymoquinone in children with refractory seizures and by considering alpha= 0.05 and Beta=0.2, the sample size was calculated to provide at least ten patients in each treatment group (Akhondian et al., 2011). For each patient randomized to receive either Epistop or placebo, the
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median and the mean frequency, and duration of seizures at the end of each 4-week period was determined. The analysis of the results of this trial was performed based on the statistic tests
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that are appropriate for the cross-over design in clinical trials. In this kind of trial, the authors need to consider some assumptions. To perform a cross-over trial, it is necessary to know about
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the period effect and the carryover effect. If these effects show a significant difference before
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and after the washout period, then we could not perform cross-over statistics. After the
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declaration of the presence of these assumptions, the cross-over statistics were conducted
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(Wellek and Blettner, 2012). To perform the statistical analysis, we used the NCSS 11 statistical software (NCSS, LLC 329 North 1000 East Kaysville, Utah 84037 USA), which is
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specifically designed to conduct cross-over statistics. In all statistical evaluations, P < 0.05 was considered significant.
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3. Results
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3.1. Baseline characteristics The authors enrolled 22 children with refractory seizures into the study and randomly
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assigned them to be in the sequence one (which was administered Epistop) or two (which was administered placebo) of the study. Of these, 14 were girls (63.6%) and 8 (36.4%) were boys. The age range was between 4 and 17 years. The median age was 7 years old (Fig 1. Consort flow diagram). The main seizure types were generalized tonic-clonic (GTC), absences and GTCs, tonic, and myoclonic. The minimum number of anti-seizure medications was three, and
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one of the children received seven anti-seizure medications during the study. In 18 (81.8%) children, the interIctal electroencephalography showed epileptiform abnormalities. In 10 (45.5%) children, brain MRI showed structural abnormalities. The development was delayed in 13 (59.1%) of the children. Six of the patients had developmental regression because of the uncontrolled seizures. Before randomization, the range of seizure frequency was between 2 and
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140 events per week, and the median seizure frequency was 35 events per week. Before
randomization, the range of seizure duration was 5 and 600 seconds, and the median seizure
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duration was 30 seconds.
3.2. Effects of Epistop on the frequency and duration of seizures
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No difference was found between two groups regarding seizure frequency or seizure
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duration during the monitoring period and after administering Epistop and placebo. The results
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showed that neither Epistop nor placebo reduced the seizure frequency and the seizure duration
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in the patients (Cross-Over analysis using T-tests; T value= 2.06, P= 0.0528 for the frequency
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of seizures and T value= 1.7, P= 0.1043 for the duration of seizures). In one patient, the seizure frequency was increased by 150% (from 70 to 150 per week) by administering Epistop. In only four patients, seizures frequency reduced more than 60%
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(clinically significant) after Epistop administration. In these four patients, seizure frequency increased significantly after the washout period and during placebo administration.
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Interestingly, all of these children had normal development, normal interIctal EEGs, normal MRI, and the median seizure frequency of fewer than 10 events per week. In only five patients,
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the seizure duration significantly reduced during Epistop administration (more than 50%). 3.3. Adverse effects None of the patients reported significant adverse effects during Epistop or placebo administration. Only three patients, two in Epistop group and one in the placebo group reported 9
lethargy and diarrhea, but none of these patients discontinued the trial because of this adverse effect. The frequency of diazepam rectal gels for controlling breakthrough seizures in the two groups was the same during the study. 4. Discussion
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In this study, we have shown that a 2-month course of either Epistop (a mixture of Nigella sativa and Thymus vulgaris extracts) or placebo does not affect the seizure frequency and
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duration in a group of children with refractory seizures. However, some children with
refractory seizures may benefit from using Epistop as we have shown in this study that seizure frequency in four children with refractory seizures was significantly reduced by Epistop syrup
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administration. As mentioned previously in the results section, all of these children had normal
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development, normal Interictal EEGs, normal MRI, and the median seizure frequency of fewer
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than 10 events per week. Extrapolation of these results to a population of children with
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refractory seizure is difficult. However, administering Epistop might be indicated in children
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with refractory seizures who have normal development, normal Interictal EEGs, normal MRI, and those who have a low seizure frequency. Almost all the previous studies showed that refractory epilepsies are a subgroup of seizures
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that are intractable to nearly all the treatment modalities and need ketogenic diet and epilepsy surgery in addition to the anti-seizure medications in most patients. However, after all these
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efforts, these seizures still occur in patients and have a substantial negative impact on the families' and the patient's lives. Therefore, introducing a new controlling modality could be
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very helpful to improve the quality of life in children with refractory seizures. In recent years, herbal agents with anticonvulsive effects have received significant attention and studies in animals showed their effects in different seizure models (Abdel-Rahman, 2013; Al-Naggar et al., 2003; Beyazcicek et al., 2016; Gilhotra and Dhingra, 2011; Hawkes, 2017; Hosseinzadeh and Parvardeh, 2004; Hosseinzadeh et al., 2007; Hosseinzadeh et al., 2005; Ilhan et al., 2005; 10
Komaki et al., 2016; Priestley et al., 2003; Sancheti et al., 2014). However, human studies that demonstrate their effects on the refractory seizures in children are scarce (Akhondian et al., 2011). To our knowledge, only one study showed that a 28-day administration of thymoquinone in a group of children with refractory seizures had a significant reducing effect on the seizure
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frequency. However, the results of our study did not duplicate the results of that study. The authors could explain the different results of our study and the mentioned study by the
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following evidence:-
In the study performed by Akhondian et al., (Akhondian et al., 2011) they administered
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only the solution of thymoquinone in a group of children with refractory seizures. When the
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authors looked at the exclusion criteria in the above mentioned study, it was understood that
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they excluded children with epilepsy secondary to a progressive cerebral disease or any
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progressive neurodegenerative disease and children with severe exacerbation of seizures during the study. The authors had different inclusion and exclusion criteria in this study. They also
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designed a cross-over trial. Cross-over trials are appropriate when there is no cure, and just reduction of symptoms can be found after administering the agent, but it is noticeable that these trials need particular attention during statistical analysis. After a critical analysis of the
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Akhondian et al. study, we could not find the cross-over analysis in the statistics and the result
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section. Based on this evidence, the authors were able to explain the differences in the two studies.
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Almost all previous studies in animals that have explored the effects of Nigella sativa and
Thymus vulgaris on seizure models showed the anticonvulsive effects of these agents (AbdelRahman, 2013; Al-Naggar et al., 2003; Beyazcicek et al., 2016; Gilhotra and Dhingra, 2011; Hosseinzadeh and Parvardeh, 2004; Hosseinzadeh et al., 2007; Hosseinzadeh et al., 2005; Ilhan et al., 2005; Komaki et al., 2016; Priestley et al., 2003; Sancheti et al., 2014). However, without
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trials that show the effects of these agents on seizures in humans, administering these agents in humans could not be recommended. The results of our study indicated that a solution of a mixture of Nigella sativa and Thymus vulgaris extracts might be effective in reducing seizure frequency and duration just in a minority of children with refractory seizures. These results also indicate that the children showed no significant adverse effects after
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taking the mixture of Nigella sativa and Thymus vulgaris and none of the children discontinued this agent, as previous studies have shown.
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The results of this study should be interpreted with certain limits. Unfortunately, no definite dose of the solution of Nigella sativa and Thymus vulgaris could be found in the human
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literature. Hence, experimental dosages based on the animal studies and just one human study
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were attempted because ethical issues could not let us try higher dosages of these agents in the
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patients. Refractory seizures have no definite cure. Therefore, the authors could not use trials
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with parallel designs. Complaints such as refractory seizures can only be controlled by medication and not be cured. Thus, studies on refractory seizures need cross-over designs, but
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it should be emphasized that cross-over designs have difficulties especially for concealment and should have strict statistics carried out.
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5. Conclusion
In conclusion, the results of this study indicate that a solution of a mixture of Nigella
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sativa and Thymus vulgaris extracts does not affect the seizure frequency and duration in children with refractory seizures. However, a minority of highly selected children with
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refractory seizures might benefit from the effects of a mixture of these agents’ extracts. In the authors opinion administering the extracts of these agents in every child with refractory seizures should not be carried out, however, children with refractory seizures who have normal development, normal EEG, normal MRI, and those who have a low seizure frequency fewer than 10 events per week might be profit from administering Epistop. 12
Conflicts of interest: none
Acknowledgments The Research Deputy of Ahvaz Jundishapur University provided financial and logistic support
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for this study, but had no role in study design; data collection, analysis, or interpretation;
writing the report; or in the decision to submit the article for publication. The authors are
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grateful to all parents and patients for their participation in this study. We are incredibly indebted to the authorities of the Research Deputy of Ahvaz Jundishapur University of Medical
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Sciences for their financial and logistic support.
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Funding: This work was supported by the Research Deputy of Ahvaz Jundishapur University
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of Medical Sciences (number: MPRC 9404)
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Figure captions
Enrollment
Assessed for eligibility (n= 27)
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Fig 1 CONSORT Flow Diagram
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Excluded (n= 5) Not meeting inclusion criteria (n=0 ) Declined to participate (n=5 ) Other reasons (n=0 )
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Randomized (n=22)
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Allocation
Allocated to Epistop (4 weeks) (n= 12) Received allocated Epistop (n= 12) Did not receive allocated Epistop (give reasons) (n= 0 ) Then 2 weeks of washout period Then 4 weeks switch to other sequence
Allocated to Placebo (4 weeks) (n= 10) Received allocated Placebo (n= 10) Did not receive allocated Placebo (give reasons) (n= 0 ) Then 2 weeks of washout period Then 4 weeks switch to other sequence
Follow-Up Lost to follow-up (give reasons) (n=0)
Discontinued intervention (give reasons) (n= 0)
Discontinued intervention (give reasons) (n= 0)
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Lost to follow-up (give reasons) (n= 0)
Analysis Analysed (n= 10) Excluded from analysis (give reasons) (n= 0)
Analysed (n= 12) Excluded from analysis (give reasons) (n= 0)
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