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Efficacy and safety of honey based formulation of Nigella sativa seed oil in functional dyspepsia: A double blind randomized controlled clinical trial
Author’s Accepted Manuscript Efficacy and safety of Honey based formulation of nigella sativa seed oil in functional dyspepsia: A double blind randomized controlled clinical trial Reza Mohtashami, Hasan Fallah Huseini, Mojtaba Heydari, Mohsen Amini, Zainab Sadeqhi, Habib Ghaznavi, Saeed Mehrzadi www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(15)30147-1 http://dx.doi.org/10.1016/j.jep.2015.09.022 JEP9745
To appear in: Journal of Ethnopharmacology Received date: 7 May 2015 Revised date: 31 July 2015 Accepted date: 15 September 2015 Cite this article as: Reza Mohtashami, Hasan Fallah Huseini, Mojtaba Heydari, Mohsen Amini, Zainab Sadeqhi, Habib Ghaznavi and Saeed Mehrzadi, Efficacy and safety of Honey based formulation of nigella sativa seed oil in functional dyspepsia: A double blind randomized controlled clinical trial, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.09.022 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 galley proof before it is published in its final citable 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.
Efficacy and safety of honey based formulation of Nigella sativa seed oil in Functional dyspepsia: A double blind randomized controlled clinical trial
Reza Mohtashami a, Hasan Fallah Huseini b, Mojtaba Heydari c, Mohsen Amini d , Zainab Sadeqhi e, Habib Ghaznavi f, Saeed Mehrzadi g,* a
Medicine Quran and hadith Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran b
Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran
c
Research center for Traditional Medicine and History of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. d
Gastroentrology and Liver diseases Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran e
Educational Administration Department, Kharazmi University, Tehran, Iran
f
Cellular and Molecular Research Center, Zahedan University of Medical Sciences, Zahedan, Iran g Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran *
Corresponding author: Dr. Saeed Mehrzadi, Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran, P.O.Box: 1449614535, Tehran, Iran Tel/Fax: 98-21- 8622523 Email: [email protected] [email protected]
ABSTRACT
Ethnopharmacological relevance: A honey based formulation from Nigella sativa L. (N. sativa) has been used in Traditional Persian Medicine for upper gastrointestinal symptoms. Considering the traditional use of this formulation and its ingredients known pharmacologic effects, this study aimed to evaluate its the efficacy and safety of N. sativa seed oil mixed with honey in treatment of patients with functional dyspepsia. Methods and materials: Seventy patients diagnosed as with functional dyspepsia according to ROME III criteria and confirmed by upper gastrointestinal endoscopy were selected to receive a traditional honey based formulation of Nigella sativa (5 ml N. sativa oil orally daily) or placebo for 8 weeks in a double-blind randomized placebo-controlled clinical trial using a parallel design with a 1:1 allocation ratio. Patients were evaluated prior to and following 8 weeks of the intervention in terms of the Hong Kong index of dyspepsia severity, presence of H. pylori infection based on urease test, scores of in different domains of short form (SF-36) health survey, and any observed adverse events. Results: The mean scores of Hong Kong index of dyspepsia severity sores and the rate of H. pylori infection were significantly lower in the N. sativa group comparing the placebo group after the intervention (P < 0.001). No serious adverse event was reported. Conclusion: This study showed that adjuvant supplementation of honey based formulation of N. sativa can cause significant symptomatic improvement of patients with functional dyspepsia whom received the standard anti-secretory therapy. The results should be more investigated further in studies with longer duration and larger sample size. Keywords: Functional dyspepsia, Nigella sativa, Honey, Traditional Persian Medicine
1.
Introduction
Nigella sativa (Ranunculaceae family), commonly known as kalonji or black cumin in English, Siyah-dane (black seed) in Persian and Shoniz in Arabic language, is an annual flowering plant native to Middle East, Southern Asia and Eastern Europe. The seeds are the most important part of Nigella sativa which are used for medicinal purpose. Different active ingredients such as nigellone, nigellimine, nigellicine, thymoquinone, thymohydroquinone, dithymoquinone, thymol, carvacrol, and alpha-hedrin have been isolated from N. sativa seeds (Paarakh and Padmaa, 2010). Nigella sativa has shown different pharmacologic activity activities including antimicrobial, anti-inflammatory, analgesic, antidiabetic, anticancer, immunomodulator and antioxidant properties (Ahmad et al., 2013). Beside numerous preclinical studies, it has been evaluated in clinical trials for a wide range of health conditions such as hypertension (Dehkordi et al., 2008; Fallah Huseini et al., 2013), hyperlipidemia (Sabzghabaeei et al., 2012), diabetes mellitus (Bamosa et al., 2010), infertility (Kolahdooz et al., 2014), asthma (Boskabady et al., 2008), allergy (Nikakhlagh et al., 2011; Yousefi et al., 2013) and rheumatoid arthritis (Gheita et al., 2012). Its gastro-protective and anti H. pylori activity are activities were also confirmed in previous studies (Al Mofleh et al., 2008; Salem et al., 2010). Honey is also used in a wide range in of gastrointestinal diseases (Boukraâ, 2013). It is known as active antibacterial agent (Yaghoobi et al., 2013; Ahmadi-Motamayel et al., 2013) which showed shows beneficial effects in treatment of Helicobacter pylori infection (Boyanova et al., 2015; al Somal et al., 1994). Previous studies showed its effect on reducing mucosal inflammation by inhibiting nitric oxide and prostaglandin E2 production and coating the of mucus membrane (Math et al., 2013). A honey based formulation from N. sativa has been mentioned in Traditional Persian Medicine (TPM) manuscripts such as Avicenna Canon of Medicine (1037 CE) (Heydari et al., 2013) and Haly Abbas Complete Book of the Medical Art (982 CE) (Heydari et al., 2014), for upper gastrointestinal symptoms such as epigastric pain, flatulence and post-prandial fullness. This hot temperament formulation was thought to be able to dispel the cold and wet humors in the stomach, considered responsible for the mentioned gastrointestinal symptoms.
Considering the traditional use of the honey based formulation from N. sativa and mentioned pharmacologic effects of its ingredients, , this study aimed to evaluate the efficacy and safety of N. sativa seed oil mixed with honey in treatment of patients with functional dyspepsia.
2.
Methods and Materials
N. sativa oil and mineral oil were purchased from local market at the in Tehran, Iran city. N. sativa oil was the product of Barig Essence Company, Kashan city, Iran. N. sativa oil had been prepared by cold press procedure as indicated on in its brochure. The mineral oil was obtained from Rose Polymer Company Ltd. Karaj city, Iran and it was consisted consisting of light alkanes in the range of C15 to C40. Honey was also bought from local market in of Tehran. It was the product of Asale-Khomein Company, Isfahan, Iran. The formulation was made according to traditional product instructions with through mixing the N. sativa oil, honey and water with 1:1:1 ratio. The placebo was made by adding mineral oil to honey and water with the mentioned ratio. In order to give color and taste to placebo, 0.1 ml of a mixture consisting of chlorophyll and red chili pepper extract in equal proportions dissolved in oil was added to 100 ml of mineral oil according to our previous study (Kolahdooz et al., 2014).
2.1. Determination of total phenols The total phenol concentration in of N. sativa oil was measured by the high-performance liquid chromatography (HPLC) method (Dogan et al., 2010). 2.2. Determination of total fatty acids The concentrations of fatty acids in N. sativa oil were measured by the gas–liquid chromatography method (Duchateau et al., 1996). 2.3. Determination of chemical composition of the volatile oil of N.sativa oil N. sativa oil (25 ml) was hydro-distilled for 4 h. The dis-tillate distillate volatile oil was diluted using n-hegxane and analyzed by the GC–MS using Agilent 6890 system (Adams et al., 2007).
2.4. Trial design The study was designed as a two-arm, double-blind randomized placebo-controlled clinical trial using a parallel design with a 1:1 allocation ratio. There was no change in methods after trial commencement 2.5. Participants One hundred and five patients attending the gastroenterology clinic of the Baghiatallah University of Medical Sciences between March 2014 and August 2014 with a clinical diagnosis of functional dyspepsia were evaluated for inclusion in the study. Upper gastrointestinal endoscopy was performed for all patients to rule out any organic causes. Patients with organic causes of dyspepsia, peptic ulcer disease, inflammatory bowel disease or pure gastroesophageal reflux disease were excluded from the study. Patients with a history of taking H. pylori eradicating medication in previous 3 months or any gastrointestinal surgery were also excluded. Clinical history of any underlying disease such as coronary heart disease, diabetes mellitus, asthma, chronic obstructive pulmonary disease, cancer or taking any routine medication such as corticosteroids, Iron or calcium supplements and antibiotics were also considered other exclusion criteria. Serum biochemistry profile including fasting blood sugar, lipid profile, liver and kidney function tests and complete blood count were done before enrolment of patients in the study to exclude any non-diagnosed undiagnosed underlying disease. At last, 70 patients were allocated to the intervention and control groups. The patients were visited in the 2nd, 4th and 8th weeks of intervention and evaluated in terms of outcomes.
2.6. Sample size The sample size was calculated by a statistician using 48% efficacy of famotidine, which was taken by both group, 85% expected efficacy of adding trial drug to famotidine, one-sided significance level of 0.05, a power of 0.80, and a 30% probable drop in the sample; the required sample size was calculated to be 35 participants per group.
2.7. Randomization, blinding and allocation concealment Seventy eligible patients were randomly allocated to two parallel groups (the drug and placebo groups) by the clinic secretary, who had been instructed to use a randomized list. The randomized list was generated using Microsoft Excel with a block randomization method, as previously described (Kim et al., 2014). The physicians, researchers and statisticians were blind to the allocation of patients. Based on the same shape and size of the drug and placebo containers and a similarity in colour and taste, the patients were blind to the drug allocation. 2.8. Ethical considerations
The study protocol was in compliance with the Declaration of Helsinki (1989 revision), and approved by the Local Medical Ethics Committee of Baghiatallah University of Medical Sciences and provided with the following reference number: 22-340/s. The study was designed in a way that both groups received famotidine to avoid any deprivation from the of standard treatment. The trial was registered in the Iranian Registry of Clinical Trials (Irct ID: IRCT201505031157N11).
2.9. Outcomes All patients were evaluated prior to and following 8 weeks of the intervention for dyspepsia severity as a primary outcome and quality of life and presence of H. pylori infection as secondary outcomes measures measurements. Dyspepsia severity was evaluated in terms of the Hong Kong index of dyspepsia severity (Hu et al., 2002). Presence of H. pylori infection was based on urease test and quality of life was evaluated according to scores of different domains of in short form (SF-36) health survey. Different domains of SF-36 health survey including include scores of vitality, general health perceptions, physical functioning, physical role functioning, emotional role functioning, social role functioning, bodily pain and mental health. The number of participants with any observed or reported adverse events were was also registered and compared between groups after the study.
2.10. Statistical methods The descriptive data were was presented by means and standard deviations in case of quantitative and percentage in case of qualitative data. Student t-test, Paired t-test and Chi-square test were used for statistical comparison of primary characteristics and outcomes in each group and also between the drug and placebo groups. Binomial distribution, Chi-square, univariate and multivariate analysis were used to evaluate the effect of study variables on outcomes. A P value of less than 0.05 was considered significant.
3. Results 3.1. Phenols, fatty acids contents and chemical compositions of the volatile oil
Phenols and fatty acids contents of N. sativa oil are presented in Tables 1 and 2. The N. sativa oil contained 1.1% volatile constituents. The chemical compositions of the volatile oil component of N. sativa oil are presented in Table 3. 3.2. Baseline characteristics (demographic and clinical) From May 2012 to August 2013, a total of 103 patients were assessed for eligibility and finally, 70 of them were randomized to receive the trial drug or placebo (35 patients in each group). All patients completed the study. Two patients in the N. sativa group and three patients in the placebo group did not have had not full adherence to the study protocol (taking other medications for their dyspepsia). However, their outcome data were analysed in the data of their allocated groups (intention to treat analysis). Figure 1 is a flowchart that reveals detailed descriptions of patients' enrolment, randomization and outcomes. The baseline characteristics (demographic and clinical) of the patients in the two groups are shown in Table 4. There were no significant differences between the two arms in basic characteristics. 3.3. Hong Kong index of dyspepsia severity In the field of the Hong Kong index of dyspepsia severity score, a significant decrease was observed in the 2nd, 4th and 8th weeks score of both N. sativa and placebo groups when comparing their base scores (Table 5). The mean scores of Hong Kong index of dyspepsia severity were significantly lower in the N. sativa group compared with the placebo group at the in 2nd, 4th and 8th
weeks (P value <
0.001) (Table 5). There was no significant difference between the outcome dyspepsia severity scores in males and females (p= 0.211). There was also no significant correlation between the outcome dyspepsia severity score and patients' age (p= 0.688). 3.4. H. pylori eradication There was no significant difference in basal rate of H. pylori infection between the N. sativa and placebo groups (23 vs. 21 out of 35, respectively, p= 0.621). The rate of H. pylori infection was significantly lower in the N. Sativa group compared with the placebo group (6 vs. 21 out of 35, respectively, p < 0.001). The outcome dyspepsia severity scores in the N. Sativa group was not significantly associated with the presence or absence of H. pylori (15.16 ± 4.16 vs 16.31 ± 8.61, respectively, p= 0.22). 3.5. Quality of life In terms of the quality of life, there were significant differences between drug and placebo scores of short form (SF-36) health survey in all domains after 8 weeks of intervention (Table 6). There was no significant difference in basal scores of these domains (p values ranging from 0.36 to 0.92).
3.7. Adverse events There was no report of serious adverse event in the drug and placebo groups. However, 3 patients in the placebo group and 5 patients in the N. Sativa group experienced mild adverse events including nausea, bloating (both groups), burning sensation (N. sativa group). The difference between the number of these adverse events between the groups was not statistically significant (p= 0.452).
4. Discussion This study showed that 8 weeks of supplementation of traditional honey based formulation of N. Sativa can improve the control of the symptoms of patients with functional dyspepsia. It also improved the rate of H. pylori eradication and quality of life in these patients, without any significant adverse event. The mechanism of the effect of N. Sativa and its ingredients on gastrointestinal system is has been investigated in multiple studies. N. Sativa was found to have gastro-protective activity against ischemia reperfusion animal model of gastric mucosal injury. These effects were considered to be due to the conservation of the gastric mucosal redox state by N. Sativa and its active ingredients (El-Abhar et al., 2003). Another study using this animal model of gastric injury showed inhibiting proton pump, acid secretion and neutrophil infiltration, while enhancing mucin secretion, and nitric oxide production by thymoquinone, one of active constituents of N. Sativa (Magdy et al., 2012). Thymoquinone also showed anti-inflammatory properties in animal models of inflammatory bowel diseases (Lei et al., 2012). On the other hand, increased acid sensitivity, oxidative stress and inflammatory response have shown important roles in the pathophysiology of functional dyspepsia (Li et al., 2010; Suzuki et al., 2012; Bolling-Sternevald et al., 2002). So the mentioned pharmacological effects of N. Sativa can partially justify the observed clinical benefits of this formulation in patients with functional dyspepsia. However, more studies are needed to better further clarify the potential mechanism of action. In addition Additionally Honey also has phenolic compounds and flavonoids which have shown to reduce inflammation by inhibiting nitric oxide and prostaglandin E2 production. It also has coating effect on the mucus membrane which can protect it from the oxidative, acid or other irritant injury injuries (Math et al., 2013).
Treatment of gastrointestinal diseases plays an important role in Traditional Persian Medicine, because many other organ diseases are considered to be originated in gastrointestinal system dysfunction (pasalar et al., 2015; Fazljou et al., 2013; Inaloo et al., 2014). TPM suggests a wide range of pharmacologic and non- pharmacologic therapeutic options for patients with gastrointestinal diseases (Nimrouzi et al., 2014; Nimrouzi and Zarshenas, 2015). Pharmacologic treatments include simple and compound formulations (Pasalar et al., 2015). Honey is the base for most compound formulations of gastrointestinal system in TPM which were named as Jawarish (Zargaran et al., 2012). Jawarish of black seed is a honey based formulation of N. Sativa for treatment of phlegmatic distemperment of stomach in TPM. The current popular use of these traditional treatments (Hashempur et al., 2015; Roozbeh et al., 2013) justifies the importance of their reevaluation with present scientific methods such as controlled trials. The most important limitation of this study is the short duration of patients’ follow up in a clinical trial on functional dyspepsia as a disease with chronic long lasting natural course (Bhatia and Anumeet, 2012). The small sample size can be considered as another limitation. However, these issues may be justifiable in this study as due to being the first and preliminary investigation on this formulation in patients with functional dyspepsia. Absence of a control group with standard therapy for functional dyspepsia, beside the placebo group, prevents us from comparing the observed effects with the current standard protocols for functional dyspepsia. Lack of the histo-pathologic evaluation of patients after the intervention also limits our understanding from the histo-pathologic changes which was associated with the observed clinical benefits. However, this problem seems to be unavoidable, due to ethical limitation for the repeating upper gastrointestinal endoscopy in patients with no organic pathology in the first endoscopy. Another important point is that H2-receptor antagonists should not be administered for long duration because their discontinuation may lead to rebound acid secretion effects (Lerotić et al., 2011). To summarize, this study showed that adjuvant supplementation of honey based formulation of N. sativa can cause significant symptomatic improvement of patients with functional dyspepsia whom received the standard anti-secretory therapy. It also significantly improves the rate of H. pylori eradication and quality of life in these patients. These results should be further investigated in studies with longer duration and larger sample size and comparing the studied drug with standard treatments.
Acknowledgements We are grateful to the Iranian Academic Center for Education, Culture and Research (ACECR) and the Baqiyatallah University of Medical Sciences, Tehran, Iran for sponsoring this study. Conflict of Interest The authors have declared that there is no conflict of interest. References:
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Table 1. The phenols content (mg/1000 g) of N. sativa oil (HPLC method). Phenols
mg/1000g oil
Total polyphenols
825.51
Hydroxytyrosol Tyrosol Vanillic acid Para-coumaric acid o-coumaric acid Decarboxymethyl oleuropein aglycone Tyrosyl acetate Ethyl vanillin
0.65 32.84 0.27 5.93 12.00 21.12 9.09 5.58
Table 2. The fatty acids content (%) of N. sativa oil (gas–liquid chromatography method). Fatty acids
Percentage
Myristic acid
0.2090
Palmitic acid
12.48
Palmitoleic acid
0.2812
Heptadecanoic acid
0.1047
Stearic acid
3.3884
Oleic acid
22.5836
linoleic acid
58.2370
arachidic acid
0.2250
eicosenoic acid
0.3839
Behenic acid
0.0356
Lignoceric acid
0.0414
Table 3. Chemical compositions of volatile oil component of N. sativa oil. Compound
Percentage
α-Thujene
13.95
α-Pinene
3.74
2,4,10,Thujaden
0.08
Camphene
0.05
2-Heptenal(z)
0.18
Sabinene
1.52
β-Pinene
3.27
ρ-Cymene
51.62
Limonene
1.95
1,8-Cineole
0.07
ϒ-Terpinene
0.17
cis-Thujon
0.06
(-)-cis-Sabinol
0.11
4-ol-Terpineol
0.27
Dodecane(n)
0.07
Thymoquinone
14.48
Isobornyl acetate
0.09
Carvacrol
0.96
α-Longipinene
0.28
β-Longipinene
0.82
Table 4. Basic characteristics of patients in the N. sativa and placebo groups Basic characteristics Age Sex (female) Marital status (married) BMI H. pylori (positive)
Groups (Mean±SD) N. sativa Placebo 42.31 ± 13.85 36.31 ± 13.64 18 (51%) 17 (48%) 30 (85%) 24 (68%) 26.26 ± 3.80 24.64 ± 4.34 23 (65%) 21 (60%)
Diff 6 -4.62 -7.54 1.62 -10.51
95% CI Lower Upper -0.558 12.558
-0.322
3.574
P value 0.07† 0.057‡ 0.08‡ 0.10† 0.62‡
† Independent sample t-test, ‡ Chi-square test
Table 5. Hong Kong index of dyspepsia severity in the N. sativa and placebo groups before and after 2, 4 and 8 weeks of the intervention Functional dyspepsia score Base score After 2W After 4W After 8W P-Within groups‡
Groups (Mean±SD) N. sative Placebo 28.08 ± 8.11 29.74 ± 8.05 23.40 ± 7.47 28.02 ± 7.59 19.48 ± 5.19 27.02 ± 8.35 16.11 ± 3.71 26.62 ± 8.46 <0.001 0.006
Diff -1.65 -4.62 -7.54 -10.51
95% CI Lower Upper -5.51 2.19 -8.22 -1.03 -10.85 -4.22 -13.63 -7.39
P value† 0.394 0.012 <0.001 <0.001
† Independent sample t-test, ‡ paired sample t-test (0-8 weeks).
Table 6. Short form (SF-36) health survey scores in the N. sativa and placebo groups before and after the intervention Groups (Mean±SD) N. sative Placebo
SF-36 domains vitality general health perceptions physical functioning physical role functioning emotional role functioning social role functioning bodily pain mental health
Before After Before After Before After Before After Before After Before After Before After Before After
47.86±20.61 62.49±19.41 53.25±19.74 67.43±18.08 76.14±22.07 87.71±17.50 58.93±25.51 72.04±22.44 57.62±28.82 72.62±22.19 58.57±25.32 75.00±18.93 54.14±24.29 74.57±20.61 52.29±19.79 64.86±17.97
50.21±17.90 52.35±16.60 52.00±17.50 52.14±17.25 75.43±25.45 75.86±23.53 57.32±23.80 57.14±23.34 53.57±23.16 53.57±21.13 54.29±23.86 55.43±22.85 48.93±23.43 50.71±21.47 51.86±16.76 52.14±16.68
P value† 0.611 0.022 0.780 0.001 0.901 0.020 0.786 0.008 0.519 <0.001 0.469 <0.001 0.364 <0.001 0.922 0.003 † Independent sample t-test
Table 7. Comparison of side effect complaints of patients between the N. sativa and placebo groups during the study
Placebo
Absent Number Percent 32 91.4 %
N. sativa
30
Total
62
85.7 %
Mild Number Percent 3 8.6 % 5 8
14.2 %
Moderate Number Percent 0 0% 0 0
0%
Sever Number Percent 0 0% 0 0
0%
Very severe Number Percent 0 0% 0 0
P= 0.452, using chi square test.
Graphical abstract
0%
fig 1
PII: DOI: Reference:
S0378-8741(15)30147-1 http://dx.doi.org/10.1016/j.jep.2015.09.022 JEP9745
To appear in: Journal of Ethnopharmacology Received date: 7 May 2015 Revised date: 31 July 2015 Accepted date: 15 September 2015 Cite this article as: Reza Mohtashami, Hasan Fallah Huseini, Mojtaba Heydari, Mohsen Amini, Zainab Sadeqhi, Habib Ghaznavi and Saeed Mehrzadi, Efficacy and safety of Honey based formulation of nigella sativa seed oil in functional dyspepsia: A double blind randomized controlled clinical trial, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.09.022 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 galley proof before it is published in its final citable 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.
Efficacy and safety of honey based formulation of Nigella sativa seed oil in Functional dyspepsia: A double blind randomized controlled clinical trial
Reza Mohtashami a, Hasan Fallah Huseini b, Mojtaba Heydari c, Mohsen Amini d , Zainab Sadeqhi e, Habib Ghaznavi f, Saeed Mehrzadi g,* a
Medicine Quran and hadith Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran b
Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran
c
Research center for Traditional Medicine and History of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. d
Gastroentrology and Liver diseases Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran e
Educational Administration Department, Kharazmi University, Tehran, Iran
f
Cellular and Molecular Research Center, Zahedan University of Medical Sciences, Zahedan, Iran g Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran *
Corresponding author: Dr. Saeed Mehrzadi, Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran, P.O.Box: 1449614535, Tehran, Iran Tel/Fax: 98-21- 8622523 Email: [email protected] [email protected]
ABSTRACT
Ethnopharmacological relevance: A honey based formulation from Nigella sativa L. (N. sativa) has been used in Traditional Persian Medicine for upper gastrointestinal symptoms. Considering the traditional use of this formulation and its ingredients known pharmacologic effects, this study aimed to evaluate its the efficacy and safety of N. sativa seed oil mixed with honey in treatment of patients with functional dyspepsia. Methods and materials: Seventy patients diagnosed as with functional dyspepsia according to ROME III criteria and confirmed by upper gastrointestinal endoscopy were selected to receive a traditional honey based formulation of Nigella sativa (5 ml N. sativa oil orally daily) or placebo for 8 weeks in a double-blind randomized placebo-controlled clinical trial using a parallel design with a 1:1 allocation ratio. Patients were evaluated prior to and following 8 weeks of the intervention in terms of the Hong Kong index of dyspepsia severity, presence of H. pylori infection based on urease test, scores of in different domains of short form (SF-36) health survey, and any observed adverse events. Results: The mean scores of Hong Kong index of dyspepsia severity sores and the rate of H. pylori infection were significantly lower in the N. sativa group comparing the placebo group after the intervention (P < 0.001). No serious adverse event was reported. Conclusion: This study showed that adjuvant supplementation of honey based formulation of N. sativa can cause significant symptomatic improvement of patients with functional dyspepsia whom received the standard anti-secretory therapy. The results should be more investigated further in studies with longer duration and larger sample size. Keywords: Functional dyspepsia, Nigella sativa, Honey, Traditional Persian Medicine
1.
Introduction
Nigella sativa (Ranunculaceae family), commonly known as kalonji or black cumin in English, Siyah-dane (black seed) in Persian and Shoniz in Arabic language, is an annual flowering plant native to Middle East, Southern Asia and Eastern Europe. The seeds are the most important part of Nigella sativa which are used for medicinal purpose. Different active ingredients such as nigellone, nigellimine, nigellicine, thymoquinone, thymohydroquinone, dithymoquinone, thymol, carvacrol, and alpha-hedrin have been isolated from N. sativa seeds (Paarakh and Padmaa, 2010). Nigella sativa has shown different pharmacologic activity activities including antimicrobial, anti-inflammatory, analgesic, antidiabetic, anticancer, immunomodulator and antioxidant properties (Ahmad et al., 2013). Beside numerous preclinical studies, it has been evaluated in clinical trials for a wide range of health conditions such as hypertension (Dehkordi et al., 2008; Fallah Huseini et al., 2013), hyperlipidemia (Sabzghabaeei et al., 2012), diabetes mellitus (Bamosa et al., 2010), infertility (Kolahdooz et al., 2014), asthma (Boskabady et al., 2008), allergy (Nikakhlagh et al., 2011; Yousefi et al., 2013) and rheumatoid arthritis (Gheita et al., 2012). Its gastro-protective and anti H. pylori activity are activities were also confirmed in previous studies (Al Mofleh et al., 2008; Salem et al., 2010). Honey is also used in a wide range in of gastrointestinal diseases (Boukraâ, 2013). It is known as active antibacterial agent (Yaghoobi et al., 2013; Ahmadi-Motamayel et al., 2013) which showed shows beneficial effects in treatment of Helicobacter pylori infection (Boyanova et al., 2015; al Somal et al., 1994). Previous studies showed its effect on reducing mucosal inflammation by inhibiting nitric oxide and prostaglandin E2 production and coating the of mucus membrane (Math et al., 2013). A honey based formulation from N. sativa has been mentioned in Traditional Persian Medicine (TPM) manuscripts such as Avicenna Canon of Medicine (1037 CE) (Heydari et al., 2013) and Haly Abbas Complete Book of the Medical Art (982 CE) (Heydari et al., 2014), for upper gastrointestinal symptoms such as epigastric pain, flatulence and post-prandial fullness. This hot temperament formulation was thought to be able to dispel the cold and wet humors in the stomach, considered responsible for the mentioned gastrointestinal symptoms.
Considering the traditional use of the honey based formulation from N. sativa and mentioned pharmacologic effects of its ingredients, , this study aimed to evaluate the efficacy and safety of N. sativa seed oil mixed with honey in treatment of patients with functional dyspepsia.
2.
Methods and Materials
N. sativa oil and mineral oil were purchased from local market at the in Tehran, Iran city. N. sativa oil was the product of Barig Essence Company, Kashan city, Iran. N. sativa oil had been prepared by cold press procedure as indicated on in its brochure. The mineral oil was obtained from Rose Polymer Company Ltd. Karaj city, Iran and it was consisted consisting of light alkanes in the range of C15 to C40. Honey was also bought from local market in of Tehran. It was the product of Asale-Khomein Company, Isfahan, Iran. The formulation was made according to traditional product instructions with through mixing the N. sativa oil, honey and water with 1:1:1 ratio. The placebo was made by adding mineral oil to honey and water with the mentioned ratio. In order to give color and taste to placebo, 0.1 ml of a mixture consisting of chlorophyll and red chili pepper extract in equal proportions dissolved in oil was added to 100 ml of mineral oil according to our previous study (Kolahdooz et al., 2014).
2.1. Determination of total phenols The total phenol concentration in of N. sativa oil was measured by the high-performance liquid chromatography (HPLC) method (Dogan et al., 2010). 2.2. Determination of total fatty acids The concentrations of fatty acids in N. sativa oil were measured by the gas–liquid chromatography method (Duchateau et al., 1996). 2.3. Determination of chemical composition of the volatile oil of N.sativa oil N. sativa oil (25 ml) was hydro-distilled for 4 h. The dis-tillate distillate volatile oil was diluted using n-hegxane and analyzed by the GC–MS using Agilent 6890 system (Adams et al., 2007).
2.4. Trial design The study was designed as a two-arm, double-blind randomized placebo-controlled clinical trial using a parallel design with a 1:1 allocation ratio. There was no change in methods after trial commencement 2.5. Participants One hundred and five patients attending the gastroenterology clinic of the Baghiatallah University of Medical Sciences between March 2014 and August 2014 with a clinical diagnosis of functional dyspepsia were evaluated for inclusion in the study. Upper gastrointestinal endoscopy was performed for all patients to rule out any organic causes. Patients with organic causes of dyspepsia, peptic ulcer disease, inflammatory bowel disease or pure gastroesophageal reflux disease were excluded from the study. Patients with a history of taking H. pylori eradicating medication in previous 3 months or any gastrointestinal surgery were also excluded. Clinical history of any underlying disease such as coronary heart disease, diabetes mellitus, asthma, chronic obstructive pulmonary disease, cancer or taking any routine medication such as corticosteroids, Iron or calcium supplements and antibiotics were also considered other exclusion criteria. Serum biochemistry profile including fasting blood sugar, lipid profile, liver and kidney function tests and complete blood count were done before enrolment of patients in the study to exclude any non-diagnosed undiagnosed underlying disease. At last, 70 patients were allocated to the intervention and control groups. The patients were visited in the 2nd, 4th and 8th weeks of intervention and evaluated in terms of outcomes.
2.6. Sample size The sample size was calculated by a statistician using 48% efficacy of famotidine, which was taken by both group, 85% expected efficacy of adding trial drug to famotidine, one-sided significance level of 0.05, a power of 0.80, and a 30% probable drop in the sample; the required sample size was calculated to be 35 participants per group.
2.7. Randomization, blinding and allocation concealment Seventy eligible patients were randomly allocated to two parallel groups (the drug and placebo groups) by the clinic secretary, who had been instructed to use a randomized list. The randomized list was generated using Microsoft Excel with a block randomization method, as previously described (Kim et al., 2014). The physicians, researchers and statisticians were blind to the allocation of patients. Based on the same shape and size of the drug and placebo containers and a similarity in colour and taste, the patients were blind to the drug allocation. 2.8. Ethical considerations
The study protocol was in compliance with the Declaration of Helsinki (1989 revision), and approved by the Local Medical Ethics Committee of Baghiatallah University of Medical Sciences and provided with the following reference number: 22-340/s. The study was designed in a way that both groups received famotidine to avoid any deprivation from the of standard treatment. The trial was registered in the Iranian Registry of Clinical Trials (Irct ID: IRCT201505031157N11).
2.9. Outcomes All patients were evaluated prior to and following 8 weeks of the intervention for dyspepsia severity as a primary outcome and quality of life and presence of H. pylori infection as secondary outcomes measures measurements. Dyspepsia severity was evaluated in terms of the Hong Kong index of dyspepsia severity (Hu et al., 2002). Presence of H. pylori infection was based on urease test and quality of life was evaluated according to scores of different domains of in short form (SF-36) health survey. Different domains of SF-36 health survey including include scores of vitality, general health perceptions, physical functioning, physical role functioning, emotional role functioning, social role functioning, bodily pain and mental health. The number of participants with any observed or reported adverse events were was also registered and compared between groups after the study.
2.10. Statistical methods The descriptive data were was presented by means and standard deviations in case of quantitative and percentage in case of qualitative data. Student t-test, Paired t-test and Chi-square test were used for statistical comparison of primary characteristics and outcomes in each group and also between the drug and placebo groups. Binomial distribution, Chi-square, univariate and multivariate analysis were used to evaluate the effect of study variables on outcomes. A P value of less than 0.05 was considered significant.
3. Results 3.1. Phenols, fatty acids contents and chemical compositions of the volatile oil
Phenols and fatty acids contents of N. sativa oil are presented in Tables 1 and 2. The N. sativa oil contained 1.1% volatile constituents. The chemical compositions of the volatile oil component of N. sativa oil are presented in Table 3. 3.2. Baseline characteristics (demographic and clinical) From May 2012 to August 2013, a total of 103 patients were assessed for eligibility and finally, 70 of them were randomized to receive the trial drug or placebo (35 patients in each group). All patients completed the study. Two patients in the N. sativa group and three patients in the placebo group did not have had not full adherence to the study protocol (taking other medications for their dyspepsia). However, their outcome data were analysed in the data of their allocated groups (intention to treat analysis). Figure 1 is a flowchart that reveals detailed descriptions of patients' enrolment, randomization and outcomes. The baseline characteristics (demographic and clinical) of the patients in the two groups are shown in Table 4. There were no significant differences between the two arms in basic characteristics. 3.3. Hong Kong index of dyspepsia severity In the field of the Hong Kong index of dyspepsia severity score, a significant decrease was observed in the 2nd, 4th and 8th weeks score of both N. sativa and placebo groups when comparing their base scores (Table 5). The mean scores of Hong Kong index of dyspepsia severity were significantly lower in the N. sativa group compared with the placebo group at the in 2nd, 4th and 8th
weeks (P value <
0.001) (Table 5). There was no significant difference between the outcome dyspepsia severity scores in males and females (p= 0.211). There was also no significant correlation between the outcome dyspepsia severity score and patients' age (p= 0.688). 3.4. H. pylori eradication There was no significant difference in basal rate of H. pylori infection between the N. sativa and placebo groups (23 vs. 21 out of 35, respectively, p= 0.621). The rate of H. pylori infection was significantly lower in the N. Sativa group compared with the placebo group (6 vs. 21 out of 35, respectively, p < 0.001). The outcome dyspepsia severity scores in the N. Sativa group was not significantly associated with the presence or absence of H. pylori (15.16 ± 4.16 vs 16.31 ± 8.61, respectively, p= 0.22). 3.5. Quality of life In terms of the quality of life, there were significant differences between drug and placebo scores of short form (SF-36) health survey in all domains after 8 weeks of intervention (Table 6). There was no significant difference in basal scores of these domains (p values ranging from 0.36 to 0.92).
3.7. Adverse events There was no report of serious adverse event in the drug and placebo groups. However, 3 patients in the placebo group and 5 patients in the N. Sativa group experienced mild adverse events including nausea, bloating (both groups), burning sensation (N. sativa group). The difference between the number of these adverse events between the groups was not statistically significant (p= 0.452).
4. Discussion This study showed that 8 weeks of supplementation of traditional honey based formulation of N. Sativa can improve the control of the symptoms of patients with functional dyspepsia. It also improved the rate of H. pylori eradication and quality of life in these patients, without any significant adverse event. The mechanism of the effect of N. Sativa and its ingredients on gastrointestinal system is has been investigated in multiple studies. N. Sativa was found to have gastro-protective activity against ischemia reperfusion animal model of gastric mucosal injury. These effects were considered to be due to the conservation of the gastric mucosal redox state by N. Sativa and its active ingredients (El-Abhar et al., 2003). Another study using this animal model of gastric injury showed inhibiting proton pump, acid secretion and neutrophil infiltration, while enhancing mucin secretion, and nitric oxide production by thymoquinone, one of active constituents of N. Sativa (Magdy et al., 2012). Thymoquinone also showed anti-inflammatory properties in animal models of inflammatory bowel diseases (Lei et al., 2012). On the other hand, increased acid sensitivity, oxidative stress and inflammatory response have shown important roles in the pathophysiology of functional dyspepsia (Li et al., 2010; Suzuki et al., 2012; Bolling-Sternevald et al., 2002). So the mentioned pharmacological effects of N. Sativa can partially justify the observed clinical benefits of this formulation in patients with functional dyspepsia. However, more studies are needed to better further clarify the potential mechanism of action. In addition Additionally Honey also has phenolic compounds and flavonoids which have shown to reduce inflammation by inhibiting nitric oxide and prostaglandin E2 production. It also has coating effect on the mucus membrane which can protect it from the oxidative, acid or other irritant injury injuries (Math et al., 2013).
Treatment of gastrointestinal diseases plays an important role in Traditional Persian Medicine, because many other organ diseases are considered to be originated in gastrointestinal system dysfunction (pasalar et al., 2015; Fazljou et al., 2013; Inaloo et al., 2014). TPM suggests a wide range of pharmacologic and non- pharmacologic therapeutic options for patients with gastrointestinal diseases (Nimrouzi et al., 2014; Nimrouzi and Zarshenas, 2015). Pharmacologic treatments include simple and compound formulations (Pasalar et al., 2015). Honey is the base for most compound formulations of gastrointestinal system in TPM which were named as Jawarish (Zargaran et al., 2012). Jawarish of black seed is a honey based formulation of N. Sativa for treatment of phlegmatic distemperment of stomach in TPM. The current popular use of these traditional treatments (Hashempur et al., 2015; Roozbeh et al., 2013) justifies the importance of their reevaluation with present scientific methods such as controlled trials. The most important limitation of this study is the short duration of patients’ follow up in a clinical trial on functional dyspepsia as a disease with chronic long lasting natural course (Bhatia and Anumeet, 2012). The small sample size can be considered as another limitation. However, these issues may be justifiable in this study as due to being the first and preliminary investigation on this formulation in patients with functional dyspepsia. Absence of a control group with standard therapy for functional dyspepsia, beside the placebo group, prevents us from comparing the observed effects with the current standard protocols for functional dyspepsia. Lack of the histo-pathologic evaluation of patients after the intervention also limits our understanding from the histo-pathologic changes which was associated with the observed clinical benefits. However, this problem seems to be unavoidable, due to ethical limitation for the repeating upper gastrointestinal endoscopy in patients with no organic pathology in the first endoscopy. Another important point is that H2-receptor antagonists should not be administered for long duration because their discontinuation may lead to rebound acid secretion effects (Lerotić et al., 2011). To summarize, this study showed that adjuvant supplementation of honey based formulation of N. sativa can cause significant symptomatic improvement of patients with functional dyspepsia whom received the standard anti-secretory therapy. It also significantly improves the rate of H. pylori eradication and quality of life in these patients. These results should be further investigated in studies with longer duration and larger sample size and comparing the studied drug with standard treatments.
Acknowledgements We are grateful to the Iranian Academic Center for Education, Culture and Research (ACECR) and the Baqiyatallah University of Medical Sciences, Tehran, Iran for sponsoring this study. Conflict of Interest The authors have declared that there is no conflict of interest. References:
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Table 1. The phenols content (mg/1000 g) of N. sativa oil (HPLC method). Phenols
mg/1000g oil
Total polyphenols
825.51
Hydroxytyrosol Tyrosol Vanillic acid Para-coumaric acid o-coumaric acid Decarboxymethyl oleuropein aglycone Tyrosyl acetate Ethyl vanillin
0.65 32.84 0.27 5.93 12.00 21.12 9.09 5.58
Table 2. The fatty acids content (%) of N. sativa oil (gas–liquid chromatography method). Fatty acids
Percentage
Myristic acid
0.2090
Palmitic acid
12.48
Palmitoleic acid
0.2812
Heptadecanoic acid
0.1047
Stearic acid
3.3884
Oleic acid
22.5836
linoleic acid
58.2370
arachidic acid
0.2250
eicosenoic acid
0.3839
Behenic acid
0.0356
Lignoceric acid
0.0414
Table 3. Chemical compositions of volatile oil component of N. sativa oil. Compound
Percentage
α-Thujene
13.95
α-Pinene
3.74
2,4,10,Thujaden
0.08
Camphene
0.05
2-Heptenal(z)
0.18
Sabinene
1.52
β-Pinene
3.27
ρ-Cymene
51.62
Limonene
1.95
1,8-Cineole
0.07
ϒ-Terpinene
0.17
cis-Thujon
0.06
(-)-cis-Sabinol
0.11
4-ol-Terpineol
0.27
Dodecane(n)
0.07
Thymoquinone
14.48
Isobornyl acetate
0.09
Carvacrol
0.96
α-Longipinene
0.28
β-Longipinene
0.82
Table 4. Basic characteristics of patients in the N. sativa and placebo groups Basic characteristics Age Sex (female) Marital status (married) BMI H. pylori (positive)
Groups (Mean±SD) N. sativa Placebo 42.31 ± 13.85 36.31 ± 13.64 18 (51%) 17 (48%) 30 (85%) 24 (68%) 26.26 ± 3.80 24.64 ± 4.34 23 (65%) 21 (60%)
Diff 6 -4.62 -7.54 1.62 -10.51
95% CI Lower Upper -0.558 12.558
-0.322
3.574
P value 0.07† 0.057‡ 0.08‡ 0.10† 0.62‡
† Independent sample t-test, ‡ Chi-square test
Table 5. Hong Kong index of dyspepsia severity in the N. sativa and placebo groups before and after 2, 4 and 8 weeks of the intervention Functional dyspepsia score Base score After 2W After 4W After 8W P-Within groups‡
Groups (Mean±SD) N. sative Placebo 28.08 ± 8.11 29.74 ± 8.05 23.40 ± 7.47 28.02 ± 7.59 19.48 ± 5.19 27.02 ± 8.35 16.11 ± 3.71 26.62 ± 8.46 <0.001 0.006
Diff -1.65 -4.62 -7.54 -10.51
95% CI Lower Upper -5.51 2.19 -8.22 -1.03 -10.85 -4.22 -13.63 -7.39
P value† 0.394 0.012 <0.001 <0.001
† Independent sample t-test, ‡ paired sample t-test (0-8 weeks).
Table 6. Short form (SF-36) health survey scores in the N. sativa and placebo groups before and after the intervention Groups (Mean±SD) N. sative Placebo
SF-36 domains vitality general health perceptions physical functioning physical role functioning emotional role functioning social role functioning bodily pain mental health
Before After Before After Before After Before After Before After Before After Before After Before After
47.86±20.61 62.49±19.41 53.25±19.74 67.43±18.08 76.14±22.07 87.71±17.50 58.93±25.51 72.04±22.44 57.62±28.82 72.62±22.19 58.57±25.32 75.00±18.93 54.14±24.29 74.57±20.61 52.29±19.79 64.86±17.97
50.21±17.90 52.35±16.60 52.00±17.50 52.14±17.25 75.43±25.45 75.86±23.53 57.32±23.80 57.14±23.34 53.57±23.16 53.57±21.13 54.29±23.86 55.43±22.85 48.93±23.43 50.71±21.47 51.86±16.76 52.14±16.68
P value† 0.611 0.022 0.780 0.001 0.901 0.020 0.786 0.008 0.519 <0.001 0.469 <0.001 0.364 <0.001 0.922 0.003 † Independent sample t-test
Table 7. Comparison of side effect complaints of patients between the N. sativa and placebo groups during the study
Placebo
Absent Number Percent 32 91.4 %
N. sativa
30
Total
62
85.7 %
Mild Number Percent 3 8.6 % 5 8
14.2 %
Moderate Number Percent 0 0% 0 0
0%
Sever Number Percent 0 0% 0 0
0%
Very severe Number Percent 0 0% 0 0
P= 0.452, using chi square test.
Graphical abstract
0%
fig 1