The effect of Nigella sativa on TAC and MDA in obese and overweight women: secondary analysis of a crossover, double blind, randomized clinical trial

Abstract

Purpose

Since obesity is a risk factor for various diseases and is associated with increased oxidative stress conditions, some herbs are considered to be effective in reducing obesity and its complications.

Methods

This secondary analysis investigates the effect of Nigella sativa (N.S) oil supplement on total antioxidant capacity (TAC) and malondialdehyde (MDA) levels in obese/overweight women. Obese and overweight healthy women were randomized to receive 2,000 mg/d of N.S supplement and placebo. The intervention periods lasted 8 weeks and were separated by a 4-week washout period. Also, each participant was given an iso-calorie diet. Baseline characteristics and TAC and MDA levels were measured. Pkcross analysis was performed for statistical analysis using Stata software. Also, Cohen’s d was estimated as effect size for all results to assess the magnitude of the effects.

Results

39 women completed the study. N.S oil supplementation at a dose of 2000 mg/d significantly increased serum TAC (P effect = 0.017, Cohen’s d = 1.81) and reduced serum MDA (P effect < 0/001, Cohen’s d =  − 0.32).

Conclusion

Based on our findings taking N.S supplementation for 8 weeks can improve antioxidant conditions in obese and overweight adults. However, more studies with a larger population and the presence of both genders need to be done to confirm the results.

Registration number: IRCT20180430039475N1

Introduction

The outbreak of overweight and obesity has multiplied over double since 1980 and nearly a 3rd of the world's population is categorized as obese or overweight [1]. The World Health Organization (WHO) defines overweight and obesity as an abnormal or excessive accumulation of fat, which is considered a health hazard and is diagnosed at a BMI of 25 to 29.9 kg/m² and a BMI ≥ 30 kg/m² respectively [2]. Obesity- principally abdominal obesity- is one in every of the foremost common causes of chronic diseases such as type 2 diabetes, high blood pressure, insulin resistance, dyslipidemia, and metabolic syndrome [3]. As per the evidence, obesity is a condition of chronic oxidative stress which induces it through various biochemical mechanisms [4, 5]. Oxidative stress is an imbalance among the overproduction of free radicals, reactive oxygen species, and antioxidant defenses of body [6]. In recent years, plasma malondialdehyde (MDA) and total antioxidant capacity (TAC) have been used as the most considerable parameters in oxidative stress [7]. Furthermore, previous data indicate that the level of plasma TAC would change in oxidative stress and obesity [8]. Also, plasma MDA concentration is higher in obese or overweight women than in normal-weight women [9].

Today, various methods are used for fat reduction and obesity treatment [10]. Lifestyle modification, diet change, and inactivity reduction are three important factors in improving obesity and overweight [11]. As well as, another method is the use of complementary and alternative therapies, which include herbs and their active ingredients [12]. The use of medicinal plants is attracting a lot of attention as an obesity treatment [13]. Some plant extracts can be used as anti-obesity agents. In addition, natural herbal supplements can help you lose weight by neutralizing ROS. Herbal supplements are a source of many active chemicals with antioxidant properties, including polyphenols, carotenoids, flavonoids, isoflavones, catechins, resveratrol, curcumin, quercetin, ferulic acid, phloretin, and thymoquinone [11]. Prabhakar et al. found that the aqueous extract of the bark of the stem of Boswellia ovalifoliolata has significant antioxidant activity [14]. Furthermore, another study showed that the aqueous extract of S. paniculatum fruit had a potential effect on the control of oxidative stress and the activity of antioxidant enzymes in diabetic rats by improving insulin secretion via restoration capacity of beta cells [15]. Plant-derived chemicals may modify adipose tissue status in obese and overweight individuals by decreasing intracellular oxidative stress (OS) [16].

One such plant is Nigella sativa L (N.S) [17]. N.S is an annual herb from the botanical family of Ranunculaceae. It is commonly called black caraway seeds (USA), black cumin (English), kalajira (Bangali), and shonaiz (Persian) [18]. N.S has multiple therapeutic properties including, anti-inflammatory and analgesic, antioxidant, hypoglycemic and, antimicrobial [19]. According to previous studies, most of the antioxidant and inflammatory properties of N.S are due to its major active ingredient, thymoquinone (TQ) [18, 20]. In this regard, Nikkhah et al. found that N.S consumption in patients with ulcerative colitis causes a significant decrease in MDA levels but has no significant effect on the level of TAC [21]. However, Kaatabi et al. revealed that N.S supplementation with a dosage of 2 g/day significantly increased TAC in patients with type 2 diabetes [22].

Although some studies have assayed the effect of N.S consumption on oxidative stress markers among individuals with certain diseases [22–24], few studies have been conducted on healthy obese populations [25].

The aim of this study is to determine the effect of N.S intervention on oxidative stress markers including MDA and TAC in obese and overweight women in a double-blind cross-over clinical trial.

Methods

Study protocol registration

The present study protocol was approved by the Ethics Committee of Shahid Sadoughi University of Medical Sciences on December 15th, 2018 (IR.SSU.SPH.REC.1397.150). The recruitment of eligible individuals was initiated on April 21st, 2019. After providing the related information about the study procedure for participants, they were requested to sign an informed consent form. This is a secondary analysis and previous articles have been published on Anthropometric and Cardiovascular Factors [26, 27]. This study was registered in the Iranian Registry of Clinical Trials on February 15th, 2019 (www.irct.ir) with the code No. IRCT20180430039475N1.

Participants

The present study was conducted among eligible participants who were recruited from the obesity clinic at Shahid Sadoughi University of Medical Sciences.

Inclusion criteria were as followed:

1. Adult women aged between 25–55-years old

2. Body mass index (BMI) between 25 to 35 kg/m 2

3. Fasting serum total cholesterol (TC) levels < 250 mg/dl

4. Blood pressure measures < 139/89 mmHg

Participants were excluded if they were diagnosed with hepatic, cardiovascular diseases; having allergies to N. sativa or any of its compounds; pancreatic, renal or, thyroid problems, as well as alcohol consumption, smoking, reporting a history of having a weight-loss program or diet for at least 6 months before enrolling in the study. In addition, consumption of anti-inflammatory drugs, anti-obesity drugs, lipid-lowering drugs, platelet aggregation inhibitors such as vitamin K, aspirin, vitamin E, and warfarin, the uses of any herbal substance or antioxidant supplements for 6 months before the study. However, using routine medications such as blood pressure drugs or lipid-lowering medicines was permitted unless their dosages needed to change during the study.

Study design

The protocol for the exact method of this study has been published [28]. This randomized, double-blind crossover, placebo-controlled study was composed of two intervention periods of eight weeks separated by a four-week washout phase.

Moreover, a person outside the research team allocated participants into the two groups (1:1) using stratified block randomization based on age (25–40 and 40–55 years) through computer-generated random numbers. Based on this randomization the participants were randomized for the first phase of the study and the intervention was altered for them in the second phase.

Sealed envelopes were used for allocation concealment. Not all participants and supervisors were aware of the contents of the bottles, the method of intervention and treatment, and their allocation until the end of the analysis. Furthermore, an outsider to the research team packed placebo and N.S capsules in perfectly identical bottles and encoded them in A and B letters to blind the participants and researcher. Participants were randomly assigned to periods of treatment (2000 mg/day N.S oil capsule or paraffin oil capsule as placebo). The N.S oil and placebo capsules were the exact shape and size, smelled, and tasted the same. Each participant was given 2 bottles of 56 capsules for each stage of the intervention (one bottle at the beginning of the first week and the other at the end of the fourth week of each intervention period). Participants were asked to take two capsules daily (one capsule before lunch and another before dinner). Moreover, each individual was given a specific iso-calorie diet during the intervention period to match the amount of micronutrients and calories received. The compliance rate of the treatment protocol was evaluated daily by phone interviews as well as face-to-face interviews once a week. The participants were asked to bring back the remaining capsules at the end of each phase of the intervention, and then they were given the second bottle of capsules. If the remaining capsules in each bottle exceeded 10% of the total prescribed capsules (12 capsules), that person was classified as non-adherent. Also, participants were asked about possible side effects after taking supplements at the end of each intervention phase. Based on previous research, therapeutic doses of 2,000 mg of N.S per day were considered safe [29, 30]. Detailed information about the design, method, and intervention, including gas chromatography-mass spectrometry (GC / MS) test, preparation of N.S oil and placebo capsules, and iso-calorie diet, has been previously reported [28].

Measurements of TAC, MDA and blood pressure

A total of 12 ml of fasting blood samples of venous blood were collected from each participant. Serum levels of TAC were determined by enzyme-linked immunosorbent assay (ELISA) procedure using a commercially kit (Zellbio, Germany) with inter-and intra-assays < 3.4 and < 4.2%, respectively. Also, serum levels of MDA were measured by applying the ELISA kit (Zellbio, Germany) with inter-and intra-assays 5.8 and 7.6%, respectively.

Moreover, blood pressure was measured with a validated automated digital blood pressure monitor according to the guidelines of the American Heart Association. further information on important aspects of the potential changes in other biochemical parameters of interest in overweight/obese patients, such as lipid profile, liver enzymes, etc. is described in detail in the study by Razmpoosh et al. [26].

Anthropometric measurements

The height and weight of the participants in the study were measured through a stadiometer (Seca, Germany, model number: 222) and body composition analyzer (Inbody, USA, model number: 770) with an accuracy of 0.1 cm and 0.1 kg, respectively, while they were barefoot and wore the least amount of clothing. Then, BMI was calculated according to the following formula: weight (kg)/ height (m²).

The exact method of anthropometric measurements and the effect of N.S on them have been discussed in detail in a study by Safi et al. [27].

Dietary and physical activity assessment

To quantify dietary intake used two 3-day food records (at baseline and after the intervention phase). Then, dietary intakes were changed to g/day by applying household measures. Data were calculated by nutritionist IV software (First Databank, San Bruno, CA) adjusted for Iranian foods.

To determine the physical activity level, metabolic equivalent (MET) was calculated through a validated questionnaire at the beginning and the end of every intervention phase [31]. This questionnaire contained nine categories of information on physical activity with various types and intensities. The total MET-hours/day was computed applying the summation of the average hours spent daily on each physical activity time based on its MET value.

Sample size calculation and statistical analysis

This study was a part of a previous study in which the method used to calculate sample size is presented in detail [28]. We calculated the sample size of 35 people based on the population variance of 0.01 and the significance level of 0.05 with a power of 80%. Considering the dropout rate of 15%, a final sample size of 40 was considered for the present study. Continuous variables were presented as mean and standard deviation. A paired-samples Student t-test was used to compare baseline characteristics. Skewness and Kurtosis tests were used to assess the normality of the data. The Box-Cox transform formula was used to normalize the data that were not normal (P Skewness < 0.05). We used the pkcross test to analyze the crossover design. In addition, analysis of variance was done to assess the differences in intra personal responses between interventions for each response variable. We also used the Cohen’s d effect size to measure the size of the effects. Cohen's d is defined by calculating the means after the intervention and then dividing the result by the pooled standard deviation. Effect sizes were determined by Cohen’s d as large, d = 0.8, medium, d = 0.5, and small, d = 0.8. If Cohen’s d was zero, it would mean that there was no mean difference between the intervention and control groups, and 50% of outcomes in the control group were less than in the mean of intervention group. Therefore, Cohen’s d values of 0.8, 0.5, and 0.2 were in the 79^th, 69^th, and 58^th percentiles of the distribution of the control group, respectively [32]. We used the usual method to analyze data of both periods if the carryover test was not significant. However, if the carryover effect (residual P-value < 0.05) was significant, the results of the first period were analyzed by applying covariance analysis, and the results of the second period were discarded. It is noteworthy that the baseline BMI and age of participants were adjusted as covariates. We performed all the analyzes using Stata software version 13.0 (Stata Corp LLC, College Station, TX).

Results

In the present study, 46 obese and overweight women enrolled, and 39 participants completed the study according to the set plan. The presence of various diseases, pregnancy, and dissatisfaction to participate were the main reasons for exclusion from the study. (Fig. 1).

Fig. 1.

CONSORT flow diagram of the study

The height, BMI, and age of participants were calculated to be 159 ± 6.4 cm, 32 ± 5 kg/m², and 36 ± 10 years, respectively.

Table 1 indicated the baseline characteristics of the participants. No significant differences were observed between participants consuming N.S or placebo regarding their baseline characteristics, physical activity, dietary intakes, or drug intakes in this regard all analyses were carried out compared with pooled values. Except for 12 participants who reported approximately heavy menstrual bleeding, no significant side effects were reported. The phyto-estrogenic properties of N.S may have caused them to menstrual bleeding profusely.

Table 1.

Participants’ baseline characteristics

Variable	Overall (n = 39)	Standard deviation ( n = 39)
Age (years) †	36	10
Height (cm) †	159	6.4
BMI (kg/m2) †	32	5
Physical activity (MET-h/day) †	36	3
Menopause status, (%)	36	3
Mild fatty liver, (%)	17	43.5
High normal blood pressure, (%)	7	18
Family history of obesity, (%)	10	26
Dietary intakes †
Energy intakes (kcal)	1784	187.5
Carbohydrate (gr)	223	34
Protein (gr)	51	8
Fat (gr)	84	12
Fiber (gr)	13	3
Cholesterol (mg)	145	92
PUFA (gr)	25	5
MUFA (gr)	34	6
SFA (gr)	15	3
Vitamin E (α-tocopherol) (IU)	26	6
Vitamin C (mg)	59	27
Biochemical factors a
TAC (mmol/l)	6.08	2.78
MDA (µmol/l)	0.43	0.09

^† Data are shown in mean ± SD

Abbreviations: N.S, Nigella sativa; BMI, body mass index; MET, metabolic equivalent; PUFA, polyunsaturated fatty acids; MUFA, monounsaturated fatty acids; SFA, saturated fatty acids; TAC, total antioxidant capacity; MDA, malondialdehyde

As shown in Table 2, N.S supplementation results in a significant increase in TAC levels compared with placebo. Cohen’s d was also reported for TAC levels very large between two periods, which shows that approximately 90% of the mean TAC levels in the control period is lower than the mean in the intervention period (P carryover effect = 0.15 and P effect = 0.017, Cohen’s d = 1.81). In addition, the level of MDA also decreased significantly after the N.S period compared to the placebo period. Cohen’s d, on the other hand, was considered near-medium (P carryover effect = 0.1; P effect < 0/001, Cohen’s d =  − 0.32) (Fig. 2).

Table 2.

Results after N.S intervention versus placebo in participants at the end of the study (n = 39)

Outcome †	Treatment effect ‡						Period effect (P value)	Carryover effect (P value)
	Placebo period	N.S intervention period	Within- individual difference	Effect size (Cohen’s d) §	P value*	F
TAC (mmol/l)	7.24 (3.29)	6.00 (2.29)	-1.23 (3.73)	-0.32	< 0.001	8.12	0.91	0.1
MDA (µmol/l)	0.428 (0.1)	0.573 (0.1)	0.145 (0.08)	1.81	0.017	5.91	0.24	0.15

^† Data are shown in Mean ± SD

^‡ The Cohen’s d values were defined as the difference between the means at post-intervention, divided by the pool SD. Cohen’s d of 0.2, 0.5 and 0.8 locate at 58th, 69th and 79th percentile of the distribution of the control group, respectively

^§ Pkcross procedure was used for crossover analysis

^* P treatment effect

Abbreviations: TAC, total antioxidant capacity; MDA, malondialdehyde; N.S, Nigella sativa

Fig. 2.

Mean measures serum TAC and MDA at baseline and after Nigella sativa intervention or placebo periods

Discussion

The wide range of N.S therapeutic effects such as antioxidant, anti-inflammatory, immunomodulatory properties and etc., has made it a miraculous plant [29].

The present study is the first placebo-controlled, double-blind, clinical trial that examines the effect of N.S supplementation on MDA and TAC levels in overweight and obese women with a crossover design. In this study, we observed a significant decrease in serum MDA levels and a significant increase in serum TAC levels after the consumption of N.S supplement for 8 weeks. Several studies have been performed to investigate the antioxidant and anti-inflammatory effects of N.S in humans.

Similar to the results of our study, Hadi et al. observed that patients with type 2 diabetes significantly reduced serum MDA levels by intaking 1000 mg/d of N.S capsules [33]. Furthermore, Kataabi et al. found that 2000 mg/d of N.S in patients with type 2 diabetes for one year can significantly increase TAC levels. On the other hand, Nikkhah et al. evaluated the intake of 2000 mg/d of N.S powder in patients with ulcerative colitis for 6 weeks. They observed a significant decrease in the level of MDA, whereas there was no report of significant change in the TAC levels. Also, in a recent meta-analysis article including 12 trials, it was found that N.S supplementation significantly increased TAC and decreased MDA, which was consistent with our results [34]. However, contrary to our results, some previous trials have reported a non-significant effect of N.S supplementation on MDA and TAC parameters [33, 35, 36]. The findings of some animal and in vitro studies have been in line with our findings and have also exhibited that N.S may improve inflammatory conditions and increase the level of antioxidants [37–39].

According to previous studies, it can be suggested that the antioxidant properties of N.S are due to the main bioactive substance of its volatile oil, TQ [40, 41]. Several mechanisms have been proposed for these properties of N.S: It has been suggested that TQ may be involved in anti-inflammatory processes by inhibiting the production of eicosanoids such as thromboxane B2 and leukotriene B4 by suppressing COX and 5-lipoxygenase [42].

Regarding the animal study on rats, it has been found that TQ could reduce the production of nitrite, a substance used to synthesize NO, as well as gene expression and synthesis levels of the iNOS protein, which, in turn, leads to a reduction in the formation of free radicals and oxidative stress [43].

Another mechanism that may explain the simultaneous effect of N.S on inflammatory conditions and oxidative stress is the inhibition of NF-κB. TQ inhibits nuclear expression of the N65-κB p65 subunit and induces binding of NF-κB p50 to the TNFα promoter in vitro and in vivo [44]. N.S also has three other main compounds except for TQ (carvacrol, t-anethole, 4-terpineol) with antioxidant properties [45].

Kanter et al. found that the anti-eicosanoid and antioxidant effects of N.S oil, which contains TQ and its derivatives, were greater than those of TQ alone [46]. TQ has a non-enzymatic reaction with GSH, NADH, and NADPH which generates glutathione Glutathionelate-dihydro-thymoquinone and dihydro-thymoquinone. These products are known for their antioxidant activity and have even more potent radical scavenging power than TQ [47]. Finally, it prevents lipid peroxidation by substituting other antioxidants such as GSH and SOD. Furthermore, chemical suppression in phagocytes is another potential mechanism of antioxidant activity of N.S components [46]. According to another mechanism, TQ increases the plasma antioxidant capacity by up-regulating antioxidant enzyme genes, which is approved by enhancing levels of liver antioxidant enzymes [48].

Therefore, TAC, which is a parameter for evaluating the cumulative of body antioxidants, also increases [49].

To the best of our knowledge, the present study is the first trial to investigate the effect of N.S against placebo in a crossover design. Based on the available evidence, crossover design is more efficient than parallel RCTs and economically includes twice as many measurements per participant [50]. In addition, we performed a reasonable calculation of the sample size to establish the main hypothesis based on the exact characteristics of the test power in a crossover trial [51].

We also estimated Cohens’d effect sizes to measure the magnitude of the effects. Also, the data were analyzed considering the carryover effect to validate the results. Furthermore, due to the longer intervention period, the findings are more reliable and accurate [50]. A noteworthy point in this study was the standardization of participants based on their macronutrient and energy intake by giving a personalized iso-caloric diet. According to previous studies, we used the maximum safe dose of N.S oil in the form of capsules because it has been reported to have more advantageous effects than the powder form [29, 30, 52]. We only used N.S supplementation to see its effects alone. But unlike our study, some former studies examined the effects of concurrent use of N.S with other ingredients such as garlic [25], honey [53, 54], or other plant extracts [55]. Furthermore, women with obesity and overweight were included in the study to homogenize.

However, there were some limitations in our study. We did not measure N.S or TQ metabolites in urine and blood samples, we only included female participants but due to the phytoestrogenic properties of N.S, it might behave differently in the male population.

Considering an additional group without any intervention and more sample size could provide more accurate results. It is recommended that future studies be performed with higher doses and longer durations and different types of N.S supplements in both sexes.

Conclusion

In conclusion, the present study showed the beneficial effects of N.S oil supplementation on TAC and MDA factors. Further studies with longer intervention and follow-up periods and a larger number of participants are recommended to elucidate the exact effects of N.S as complementary medicine for the treatment of inflammatory diseases and oxidative stress.

Authors’ contributions

Nooshin Abdollahi: Writing—Original Draft, Conceptualization, Software, Methodology,Investigation, Visualization. Azadeh Nadjarzadeh: Supervision, Conceptualization, Methodology, Validation, Resources, Writing—Review & Editing, Project administration, Funding acquisition. Amin Salehi-Abargouei: Conceptualization, Methodology, Software, Resources. Hossein Fahhahzadeh: Methodology, Software.Elham Razmpoosh: Conceptualization, Software, Methodology, Investigation. Elnaz Lorzaedeh: Writing—Review & Editing. Sara Safi: Investigation, Data Curation.

Funding

Shahid Sadoughi University of Medical Sciences funded this trial (No. 961020). Also, Irans National Science Foundation (INSF) has supplied educational grant funding for this study (No. 96012986).

Data availability

Available.

Code availability

Not applicable.

Declarations

The authors have no financial or proprietary interests in any material discussed in this article.

Authors are responsible for correctness of the statements provided in the manuscript.

Ethical approval

This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures were approved by the Ethics Committee of Shahid Sadoughi University of Medical Sciences on December 15th, 2018 (IR.SSU.SPH.REC.1397.150). Written informed consent was obtained from all subjects/patients.

Consent to participate

Written informed consent was obtained from all study participants.

Consent for publication

The manuscript has been read and approved by all the authors, and each author believes that the manuscript represents honest work.

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.