Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/jep 7 8 9 Research Paper 10 11 12 13 14 15 Muhammad Shahdaat Bin Sayeed a,b,n, Tahiatul Shams b,c, Sarder Fahim Hossain c, 16 Md. Rezowanur Rahman c, AGM Mostofa a,b, Mohammad Fahim Kadir b,c, 17 d b,c 18 Q1 Sharif Mahmood , Md. Asaduzzaman a 19 Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka-1000, Bangladesh b 20 Department of Pharmacy, University of Asia Pacific, Dhaka-1209, Bangladesh c Department of Pharmaceutical Technology, University of Dhaka, Dhaka, Bangladesh 21 d Institute of Statistical Research and Training, University of Dhaka, Bangladesh 22 23 24 art ic l e i nf o a b s t r a c t 25 26 Article history: Ethnopharmacological relevance: Previous studies conducted on animals linked consumption of Nigella 27 Received 13 September 2013 sativa L. seeds (NS) to decreased anxiety and improved memory. The present study, which was carried 28 Received in revised form out at a boarding school in Bangladesh, was designed to examine probable effect of NS on mood, anxiety 29 17 December 2013 and cognition in adolescent human males. Accepted 28 December 2013 30 Materials and methods: Forty-eight healthy adolescent human males aged between 14 to 17 years were 31 randomly recruited as volunteers and were randomly split into two groups: A (n ¼24) and B (n ¼ 24). The 32Q3 Keywords: treatment procedure for group A and B were one capsule of 500 mg placebo and 500 mg NS respectively 33 Nigella sativa L. seeds once daily for four weeks. All the volunteers were assessed for cognition with modified California verbal Adolescents 34 learning test-II (CVLT-II), mood with Bond–Lader scale and anxiety with State–Trait Anxiety Inventory State–Trait Anxiety Inventory (STAI) at the beginning and after four weeks of either NS or placebo ingestion. 35 Modified California verbal learning test-II Results and discussion: No parameter showed statistically significant variation between A and B in 36 Bond–Lader scale measurements in the beginning, but after 4 weeks of one capsule of NS 500 mg intake, there was 37 Mood statistically significant variation of mood within group B but there was not statistically significant Anxiety 38 variation between group A and B. No significant variation was found in state anxiety within groups and Memory 39 between group A and B but in case of trait anxiety, significant variation was found within group B but not 40 between group A and B. In case of CVLT II, there was significant variation within B in immediate short41 term recall at trial 4 and 5 whereas this difference was found only in case of trial 5 between group A and B. 42 Within group B, short term-free recall, long-term free recall and long-term cued recall had statistical 43 difference whereas between group A and B long-term free recall and long-term cued recall had statistical 44 difference. No parameters had significant variation within group A after placebo intake for 4 weeks. 45 Conclusions: Over the 4 weeks study period, the use of NS as a nutritional supplement been observed tostabilize mood, decrease anxiety and modulate cognition positively. However, long term study is suggested 46 before using NS extensively. 47 & 2014 Published by Elsevier Ireland Ltd. 48 49 50 51 52 1. Introduction 53 54 Nigella sativa L. (Family: Ranunculaceae), commonly known as 55 Abbreviations: NS, Nigella sativa L. seeds; CVLT-II, modified California verbal black cumin, is an annual herb. It is native to south west Asia and 56 learning test-II; STAI, State–Trait Anxiety Inventory; ADHD, attention deficit cultivated in countries like Middle Eastern Mediterranean region, 57 hyperactivity disorder; CONSORT, consolidated standards of reporting trials; 5-HT, South Europe, Syria, Turkey, Saudi Arabia, Pakistan, India (Zaman 5-hydroxytryptamine; GABA, gamma-aminobutyric acid; 5-HIAA, 58 et al., 2004). The oil and seed constituents have potential medi5-hydroxyindoleacetic acid; NO, nitric oxide; SDCR, short delay cued recall; SDFR, 59 short delay free recall; LDCR, long delay cued recall; LDFR, long delay free recall cinal properties (Salem, 2005) and have been traditionally used in 60 n Corresponding author at: Department of Clinical Pharmacy and Pharmacology, folk medicine for the treatment and prevention of a number of 61 University of Dhaka, Dhaka 1000, Bangladesh. Tel.: þ 880 2 9661900x8156; diseases and conditions including asthma, diarrhea, dyslipidaemia, Mobile: þ 880 1713459747; fax: þ880 2 8612069, þ880 2 8615583. 62 kidney, liver dysfunction and memory impairments (Butt and E-mail addresses: [email protected], 63 Sultan, 2010; Sharrif, 2011). [email protected] (M.S. Bin Sayeed). 64 65 0378-8741/$ - see front matter & 2014 Published by Elsevier Ireland Ltd. 66 http://dx.doi.org/10.1016/j.jep.2013.12.050

Journal of Ethnopharmacology

Nigella sativa L. seeds modulate mood, anxiety and cognition in healthy adolescent males

Please cite this article as: Bin Sayeed, M.S., et al., Nigella sativa L. seeds modulate mood, anxiety and cognition in healthy adolescent males. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2013.12.050i

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M.S. Bin Sayeed et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Recent studies have demonstrated that the pathogenesis of anxiety is linked to oxidative stress and decreased antioxidant capacity (Gingrich, 2005; Halliwell, 2006; Valko et al., 2007; Ng et al., 2008). Oxidative stress induced anxiety-like behavior in rodents can be reversed by using appropriate antioxidant that acts via different mechanisms (Urushitani et al., 2000; Masood et al., 2008; Salim et al., 2010). Some studies previously reported that the seeds of Nigella sativa L. (NS) have a potent antioxidant activity (Atta and Imaizumi, 1998; El Shenawy et al., 2008; Yoruk et al., 2010; Panahi et al., 2011). The principles in NS are thymoquinone, carvacrol, t-anethol and 4-terpineol. They possess antioxidant activities (Burris and Bucar, 2000), prevent oxidative injuries (Ilaiyaraja and Khanum, 2010; Sharrif, 2011), reduce oxidative stress through free radical scavenging activity (Butt and Sultan, 2010) and improve the antioxidant defense system (Khan et al., 2003; Turkdogan et al., 2003; Kanter, 2008). Multiple studies have shown that NS has neuroprotective (Kanter et al., 2006; Ezz et al., 2011; Azzubaidi et al., 2012), nephroprotective (Bayrak et al., 2008; Uz et al., 2008; Yaman and Balikci, 2010), lung protective (Hossein et al., 2008; Tayman et al., 2013), cardioprotective (Ebru et al., 2008) and hepatoprotective (Kanter et al., 2005; Sogut et al., 2008; Yildiz et al., 2008) activity in animals. At first, Raza et al. (2006) reported about anxiolytic effect of NS in rats. Later Perveen et al. (2008) showed that long term administration of NS improves learning and memory in rats. Jalali and Roghani (2009) reported enhancement of the consolidation and recall capability of stored information and spatial memory in rats followed by NS administration. Again, studies showed that NS protects hippocampal neurodegeneration (Kanter, 2008), has positive modulation effects on aged rats with memory impairments (Azzubaidi et al., 2011a), has preventive effect against hippocampal pyramidal cell loss (Azzubaidi et al., 2011b) and protective effect against spatial cognitive functions of rats that suffered global cerebrovascular hypoperfusion (Azzubaidi et al., 2012). Safety profile of NS is well established through research conducted both on rodents (Jalali and Roghani, 2009; Tauseef Sultan et al., 2009) and on human (Dehkordi and Kamkhah, 2008; Qidwai et al., 2009; Tissera et al., 1997a, 2000b). Recent studies by Bin Sayeed et al. (2013) showed positive modulating impact of NS on memory, attention and cognition. This investigation also showed safety status of NS consumption through studying different parameters of heart, liver and kidney functions. Thus NS could be considered as a useful and safe choice for the treatment of anxiety. The aim of the present study was to investigate the effect of NS intake on anxiety, mood and cognition in adolescents, as well as to examine whether there is any relation between anti-anxiety and cognition enhancement in adolescents due to NS intake. This study will be helpful for indicating NS as food supplement in maintaining sound psychological health in adolescence especially those from developing and under-developed countries.

2. Materials and method

Association Declaration of Helsinki, 1964) and was approved by the authority of the boarding school from where students were recruited. Written informed consent was obtained from all partici- Q4 pants and their guardians before the study. The participants were introduced to a complete set of medical health questions prior to selection for evaluation of their physical and mental health conditions and to determine their suitability for the study. 2.2. Preparation of capsules In current investigation, NS was taken as it is used traditionally i.e. without any modifications either by fractionation of the seeds or by adding any other substances. A representative of the study team purchased the seeds from a known quality vendor under supervision of a registered pharmacist. NS seeds were crushed with mortar and pestle for convenient capsule filling. The crushed seeds were passed through a stainless steel screen (mesh size #30) and filled into empty hard gelatin capsule shells (size #0) using a manual capsule filling machine. The quality of NS and crushed NS was vigorously monitored under the direct supervision of a registered pharmacist. Crushed NS were filled into capsules in a local GMP compliant company. It was ensured that each of the capsule contained 500 mg powdered NS. The quality of seeds was ensured by direct observation of a registered pharmacist. Husk of Isabgol (Psyllium seed husk) was used as placebo and capsules were prepared in similar fashion. 2.3. Treatment and design The study consolidated standards of reporting trials (CONSORT) flow diagrams (Schulz et al., 2010) (Fig. 1) was conducted over 4 weeks and the participants were assigned random code numbers generated by computer. All the participants were assessed for baseline data to measure cognition, mood and anxiety level before administering the first dose. The tests were taken in Bengali language (native language of the subjects) and all the terminologies used in the Bond–Lader scale and state–trait anxiety questionnaire were explained to all the volunteers before conducting the study. Each participant from group B received one capsule of NS 500 mg and each participant from group A received one placebo capsule once daily after dinner for 4 weeks. Placebo and NS capsules had the same color, texture, size and shape. All investigators (except the principal investigator who was involved in designing the experiment, analyzing final data and writing the manuscript but neither involved in the administration of capsules nor in documenting the data) and the participants were kept blind about NS or placebo. The code numbers and the group allocation were only revealed after the last participant had completed the entire follow up. All the participants were assessed again for cognitive performance, anxiety level and mood status at the end of 4 weeks. The sequence of administration of the study was same for each subject and all the tests for participants were completed during 0700 to 0930 h. All the participants were instructed to call the study-center in case of any adverse effect felt during the study. They had the opportunity to withdraw from the study at any time.

2.1. Participants 2.4. Mood A total of 57 healthy adolescent males, age ranging between 14 and 17 years participated in the present study. Participants were recruited from a boarding school located at Rangpur in Bangladesh. Subjects having previous history of attention deficit hyperactivity disorder (ADHD) and frequent sickness were excluded from this investigation. Thus nine subjects were excluded and finally there were 48 volunteers who took part in this investigation. The study (Code number: RSBS042012) was carried out in accordance with the Declaration of Helsinki and subsequent revisions (World Medical

The Bond–Lader scale (Bond and Lader, 1974) used for assessing mood (Wesnes et al., 2003; Smith and Foster, 2008) was made up of 16  100 mm visual analogue scales with the end-points anchored by the antonyms: alert-drowsy, calm-excited, strong-feeble, clearheaded-fuzzy, well coordinated-clumsy, energetic-lethargic, contented-discontented, tranquil-troubled, quick witted-mentally slow, relaxed-tense, attentive-dreamy, proficient-incompetent, happy-sad, friendly-antagonistic, interested-bored and sociable-withdrawn.

Please cite this article as: Bin Sayeed, M.S., et al., Nigella sativa L. seeds modulate mood, anxiety and cognition in healthy adolescent males. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2013.12.050i

M.S. Bin Sayeed et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Fig. 1. Study flowchart.

Participants were presented with a sheet of paper containing all the scales and instructed to mark their current mood state on each line. These were combined as recommended by the authors (Bond and Lader, 1974) to form three mood factors: ‘alert’, ‘calm’ and ‘contented’ with scores on each ranging from 0 to 100. Scores in the lower range indicates positive mood. 2.5. State–Trait Anxiety Inventory (STAI) The STAI (Laux et al., 1981; Spielberger, 1983) comprises of two scales (i) state anxiety (current) and (ii) trait anxiety (general). Each of these scales contains 20 statements (e.g. ‘I feel calm’). Participants are required to rate each statement on a four-point scale (1, not at all/almost never; 2, somewhat/sometimes; 3, mood rarely so/often; 4, very much so/almost always). Aggregate ratings of each of the two scales (state and trait anxiety) can range from 20 to 80, with higher rating indicating more anxiety.

was repeated four times (Trials 2–5), accumulating to five trials total. Following the list-learning procedure in trials 2 to 5, participants heard a second list of words (List B) and subsequently tried to name as many of these words as possible (Immediate Free Recall: List B). Then, the participants were asked to name as many words as possible from the first list again (short-delay free recall: List A). Following this free recall task, participants were given a cued recall task, in which they were asked to name all the words from the first list that belonged to each of the four categories (short-delay cued recall). With an interval of approximately 20 to 30 min they were asked to recall words from the first list that was read to them (long-delay free recall). Participants were not expecting the last free recall condition, as they were not informed about any further tests with the word lists. Categorization similar to earlier with provided cues were done at the end (long-delay cued recall). 2.7. Safety profile study

2.6. Modified California verbal learning test-II (CVLT-II) The CVLT-II is a test of immediate, short delay and long delay episodic memory for a 16-item word list (Delis et al., 2008). It comprises of a standard form and an alternate form that can be used for a repeat testing session. In the present study, one form was administered in the first session, and the complementary form was administered in the second session, in a counter balanced order. The word lists used in the CVLT-II were made up of 16 words from four different categories. List A comprised of names of four fruits, four vegetables, four musical instruments, and four ways of traveling. List B included names of four fruits, four vegetables, four parts of a house and four animals. Two of the four categories (fruits and vegetables) were kept common on both the lists. The experimenter read a list of words (List A), and participants were asked to recall as many of the words as they could, in any order (Immediate Free Recall: List A). This procedure

Systolic–diastolic blood pressure as well as the weight and height of the volunteers were measured twice: before and after treatment with either placebo or NS. 2.8. Statistical analysis Results were analyzed independently for each test. Poisson regression was employed to identify whether groups had any significant differences over the time. To estimate the parameters of the model, we employed generalized estimating equations as over the time the responses are associated by employing R. To find the difference between placebo and treatment group we checked normality assumption and employed statistical tests that was appropriate. Repeated measure ANOVA was employed to observe between and within effect by using IBM Statistics 19. Repeated measure multivariate analysis was performed to find the effect of

Please cite this article as: Bin Sayeed, M.S., et al., Nigella sativa L. seeds modulate mood, anxiety and cognition in healthy adolescent males. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2013.12.050i

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NS over four week time period. Chi-square test was performed for demographic data between groups. p o0.05 was considered statistically significant.

3. Result 3.1. Mood Variation of mood was statistically significant (summation of score of all the factors) within group B (p o0.05) but variation between group A and B (p 40.05) was not statistically significant. Repeated measure multivariate analysis shows that there was significant difference between group A and group B on mood over the four weeks study period, F(3, 44) ¼ 9.48, p ¼0.000, η2 ¼0.393. Univariate tests also indicated that there was NS effect on all of the tests of mood; F(1, 44) ¼6.880, p ¼0.012, η2 ¼ 0.130 for alertness, F(1, 44) ¼13.808, p ¼0.001, η2 ¼ 0.231 for contendness, F(1, 44) ¼ 5.718, p ¼0.021, η2 ¼ 0.111 for calmness (Fig. 2). 3.2. State–Trait Anxiety Inventory (STAI) The intervention effect was analyzed using repeated measures ANOVA. There was statistically significant difference between intervention and control over time four weeks study period on state anxiety score, F(1, 46) ¼7.2, p ¼0.01, η2 ¼7.221. Repeated measure multivariate analysis shows that there was significant difference between group A and group B on state anxiety over time four weeks study period, F(19, 28) ¼ 4.975, p ¼ 0.000, η2 ¼ 0.771. However, univariate tests indicated there was not NS effect on all of the items in state questionnaire except items 1: ‘I feel calm’ [F(1, 46) ¼ 22.490, p ¼0.000, η2 ¼ 0.328], Items: 3 ‘I feel tense’[F(1, 46) ¼34.677, p ¼0.000, η2 ¼0.430], and Items: 6 ‘I feel upset’ [F(1, 46) ¼33.891, p ¼0.000, η2 ¼0.424]. On the other hand, there was statistically significant difference between intervention and control over time four weeks study period on trait anxiety score [F(1, 46)¼ 16.233, p ¼0.000, η2 ¼0.261]. Repeated measure multivariate analysis shows that there was significant difference between group A and group B on trait anxiety over time four weeks study period, F(19, 28) ¼ 7.660, p ¼0.000, η2 ¼0.850. However, univariate tests indicated there was not NS effect on all of the

Fig. 2. The score of Bond–Lader scale (alertness, calmness and contentedness). Repeated measure multivariate analysis was performed to find the effect of NS on “Alertness”, “Calmness”, “Contentedness” over four weeks time period.

items in trait questionnaire except in the trait Items 2: ‘I feel nervous and restless’ [F(1, 46) ¼12.760, p ¼0.000, η2 ¼0.666], Items: 6 ‘I feel rested’ [F(1, 46) ¼7.193, p¼ 0.000, η2 ¼0.135], Items: 16 ‘I feel content’ [F(1, 46) ¼27.046, p ¼0.000, η2 ¼ 0.370], Item: 18 ‘I take disappointments so keenly that I can0 t put them out of my mind’ [F(1, 46) ¼27.806, p ¼0.000, η2 ¼0.377], Item: 20 ‘I get in a state of tension or turmoil over my recent concerns and interests’ F (1, 46) ¼24.383, p ¼0.000, η2 ¼0.346 (Fig. 3). 3.3. Modified California verbal learning test-II (CVLT-II) 3.3.1. Immediate free recall Repeated measure multivariate analysis shows that there was significant of NS over time four weeks study period on immediate free recall of CVLT-II over time four weeks study period, F(5, 42) ¼ 4.489, p ¼0.002, η2 ¼0.348 (Fig. 4). Univariate tests indicated there was not NS effect over time four weeks study period on trial 1 [F(1, 46)¼ 2.706, p ¼0.107, η2 ¼0.056], trial 2 [F(1, 46) ¼0.044, p ¼0.835, η2 ¼0.001] and trial 3 [F(1, 46) ¼0.020, p ¼0.888, η2 ¼ 0.000] but have effect on trial 4 [F(1, 46) ¼ 5.343, p ¼0.025, η2 ¼ 0.104], trial 5 [F(1, 46) ¼11.792, p ¼0.001, η2 ¼ 0.204 and summation of all trials [F(1, 46) ¼12.611, p ¼0.001, η2 ¼0.215]. 3.3.2. Delayed recall In case of delayed recall of CVLT-II, repeated measure multivariate analysis shows that there was significant effect of NS over time four weeks study period [F(4, 43) ¼ 5.955, p ¼0.001, η2 ¼0.356] (Fig. 5). Univariate tests indicate that NS does not have effect on short delay free recall [F(1, 46) ¼3.010, p ¼0.089, η2 ¼ 0.061] but affect short delay cued recall [F(1, 46) ¼2.259, p¼ 0.140, η2 ¼0.047], long delay free recall [F(1, 46) ¼8.697, p ¼0.005, η2 ¼0.159] and long delay cued recall [F(1, 46) ¼ 11.058, p ¼0.002, η2 ¼0.194]. 3.4. Safety profile assessment Blood pressure, body weight and body mass index (BMI) of the volunteers did not change significantly (p 40.05) during the study period (Table 1). Repeated measure multivariate analysis also shows that there were not significant effect of NS over time four weeks study period [F(5, 42) ¼ 1.363, p ¼0.258, η2 ¼0.140]. Univariate tests indicated that NS does not have effect on systolic blood pressure [F(1, 46) ¼ 0.004, p¼ 0.950, η2 ¼0.000], diastolic

Fig. 3. The scores of State and Trait Anxiety Inventory (STAI). Repeated measure multivariate analysis was performed to find the effect of NS on “State Anxiety” and “Trait Anxiety” over four weeks time period.

Please cite this article as: Bin Sayeed, M.S., et al., Nigella sativa L. seeds modulate mood, anxiety and cognition in healthy adolescent males. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2013.12.050i

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blood pressure [F(1, 46)¼ 0.133, p ¼0.717, η2 ¼ 0.003], weight [F(1, 46) ¼0.811, p ¼ 0.373, η2 ¼ 0.017], height [F(1, 46) ¼0.945, p¼ 0.336, η2 ¼0.020], BMI [F(1, 46) ¼0.392, p ¼0.534, η2 ¼0.008].

4. Discussion

Fig. 4. Immediate free recall on modified California verbal learning test-II (CVLT-II). The Repeated measure analysis of variance was performed to assess the effect of NS on trials of CVLT-II over four weeks time period.

Fig. 5. Delayed recall on modified California verbal learning test-II (CVLT-II). Here: SDCR¼ short delay cued recall, SDFR¼ short delay free recall, LDCR¼ long delay cued recall, LDFR ¼ long delay free recall. Repeated measure multivariate analysis was performed to find the effect of NS on “SDCR”, “SDFR”, “LDCR” and “LDFR” over four weeks time period.

This study was designed to investigate the effects of NS on anxiety, mood and cognition in male healthy adolescents. The link between anxiolytic property and cognitive function was also studied. Results from this study suggest that the ingestion of NS by the participants may have reduced anxiety and improved mood and cognition. Before starting the treatment all the participants were assessed for baseline data to measure cognition and status of mood and anxiety level. None of the observed baseline data at that stage showed statistically significant difference in between group A and B. Chi-squared test indicated that there were not significant variation in their demographic parameters such as age (p ¼0.967), weight (p ¼0.688), height (p ¼0.119) and BMI (p ¼0.500). The results of Bond–Lader scale showed that there was decrease and increase in alertness in group A and B respectivelybut none of them was statistically significant. Again the difference in the measure for alertness between group A and B after four weeks was statistically significant. Calmness was significantly different within group B, but such difference was not evident in between observations from groups A and B. The STAI test was conducted to compare the anxiety levels of the two groups. The anxiety levels observed were lower in group A and B compared to baseline but none of them were statistically significant. However, some of the items showed statistically significant difference in scores of group B, but such difference was not found in case of group A. There might have some decrease of anxiety due to the effect of NS on several neurotransmitters like 5-hydroxy tryptamine (5-HT) and gamma amino butyrate (GABA). According to Perveen et al. (2009) NS increases the level of 5-HT and thus decrease anxiety. TQ in NS might also decrease NO and reverse decreased brain GABA content and give anxiolytic effect (Gilhotra and Dhingra, 2011). Lower number of sample and short period of study could be responsible the lower strength of the study and for exhibiting such results. Participants exhibited superior performance on the short delay free recall, long delay free recall and long delay cued recall phases of the modified version of the CVLT-II after receiving NS. The rate of learning was also shown to be faster in group A compared to group B, with participants of group B demonstrating significantly enhanced performance on the fourth and fifth trials of the immediate free recall phase of the modified CVLT-II subsequent to NS ingestion. This could be due to antioxidant, anticholinesterase activities of NS (Atta and Imaizumi, 1998; El Shenawy et al., 2008; Yoruk et al., 2010; Panahi et al., 2011).

Table 1 Parameters of Health status indicators. Parameter

Health status indicators Systolic BP (mmHg) Distolic BP (mmHg) Weight (kg) Height (m) BMI (kg/m2)a a

Group A

Group B

p-Value

Baseline

After four weeks

Baseline

After four weeks

133.707 2.628 84.50 7 1.063 55.58 7 3.670 1.569 7 0.051 1.5687 0.0511

134.047 2.255 84.29 7 0.858 56.047 3.568 1.5717 0.050 1.5717 0.0500

134.08 7 2.602 84.177 1.167 56.667 3.607 1.58 7 0.067 1.581 7 0.0670

134.487 2.903 83.79 7 0.858 56.95 7 3.482 1.581 7 0.066 1.582 7 0.0658

0.950 0.717 0.373 0.336 0.534

Body mass index¼ mass/height2.

Please cite this article as: Bin Sayeed, M.S., et al., Nigella sativa L. seeds modulate mood, anxiety and cognition in healthy adolescent males. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2013.12.050i

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Anxiety is generally considered as impediment to cognition (Eysenck et al., 2007; Derakshan et al., 2009; Cornwell et al., 2012). The negative effects of anxiety on cognitive-behavioral performances have been explained by the cognitive models of anxiety (Eysenck and Calvo, 1992; Bishop, 2007; Eysenck et al., 2007). Highly anxious human subjects showed disturbances in goal directed control of attention leading to poor cognitive behavioral performances (Bishop et al., 2007). Some neurotransmitters play role in the modulation of anxiety e.g. 5-HT, GABA (Handley and McBlane, 1993; Streeter et al., 2010). Increased 5-HT and decreased 5-hydroxyindoleacetic acid (5HIAA) levels are related to reversal of anxiety (Collinge et al., 1983). Increase in thalamic GABA level is associated with improved mood and decreased anxiety (Streeter et al., 2010). GABA is also reported to attenuate stress-induced nitric oxide (NO) release and thus preventing NO induced increase in anxiety (Harvey et al., 2004; Sevgi et al., 2006). NS might show the positive modulating effect on cognition because of decrement of anxiety which is because of interaction of NS with these neurotransmitters. Multiple studies have shown that oxidative stress is associated with anxiety and cognitive defects in both human and animal models which could be reversed by treatment with ROS scavengers (Liu et al., 2003; Sklan et al., 2004). Enhancement of cognition and improvement of memory in group B might be due to activation of the cholinergic neuronal system of hippocampus that plays an important role in learning and memory (Nabeshima 1993). We propose further investigations in animals with different fractions of NS or with different compounds found in NS in different ratios and doses.

Acknowledgement

5. Conclusion

References

The observed effect of NS on anxiety as measured by STAI was statistically significant, on calmness of mood as measured by Bond–Lader scale and on cognition as measured by CVLT-II was also statistically significant. This demonstrates that there might have been improvement of cognition via affecting anxiety and mood. Therefore NS might be a potential source for developing new drugs for treating anxiety and stabilizing mood and improving cognition in adolescents. It is known that NS composition includes nutritional components such as carbohydrates (glucose, xylose, rhamnose, and arabinose), vitamins as thiamine, riboflavin, pyridoxine, niacin and folic acid (Khan, 1999; El-Naggar et al., 2010), which might act synergistically for positive modulating effect on cognition and mood. Exhibition of these activities in human is also justified from their traditional use (Sharrif, 2011). On the other hand, smaller sample size and short period of study might be considered as limitation of the study. Conducting the study with adolescence with anxiety, mood disorders and impaired cognition could have been a better study design for claiming the effect of NS on anxiety, mood and cognition. The study did not measure any serological parameters before and after NS intake. This was not feasible both financially as well as due to lack of having monitoring system. However, further long term study is recommended with adolescent with anxiety, mood disorders and impaired cognition as well as extensive study about the mechanism of the effect of NS on memory and cognition. A thorough safety and toxicological study of NS in human model is recommended before using NS extensively. On the other hand, the use of NS as nutritional supplement for the purposes as mentioned earlier could be justified after further toxicological investigations in animals and human. Q2 Uncited references

Ansari et al. (2008), Jazbec et al. (2005), Saad (1975).

The first and corresponding author, Muhammad Shahdaat Bin Sayeed, got travel award to present partial result of this study in the 33rd Annual Meeting of the Australian Neuroscience Society, Melbourne, Australia held from 3rd to 6th February 2013. The authors appreciate the cooperation of Mohammad Mizanur Rahman, Headmaster of Sathibari Madrasa,for conducting the study. The Nigella sativa L. seeds were bought and quality of the seeds was assured by Md. Asaduzzaman, Asst. Prof. in the Department of Pharmacy, University of Asia Pacific, Dhaka, Bangladesh. The seeds were identified by Botanist Mr. Manzur-ul-Kadir Mia, Principal Scientific Officer and Consultant of Bangladesh National Herbarium, Dhaka. The capsules were prepared by Md. Rezowanur Rahman, Senior Executive Officer, R & D F, Incepta Pharmaceuticals Ltd, Dewan Idris Road, Bara Rangamatia, Zirabo, Savar, Dhaka-1341, Bangladesh. However, Incepta Pharmaceuticals did not have any involvement with this project. Senior psychologist Dr. Monowara Parveen Jahangiri of Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh gave important suggestions regarding preparation and administration of different modules of Neuropsychological tests used in this experiment. We are grateful to Khondker Galib Bin Mohiuddin, Lecturer, North South University, Bangladesh for giving very thoughtful suggestions in manuscript writing. We took generous help from Faez Mahbub Chowdhury, Department of psychology, University of Dhaka regarding psychological issues of the volunteers during the investigations. Finally we appreciate Sharif Mahmood, Associate professor, ISRT, university of Dhaka for helping us in analyzing statistical data.

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Please cite this article as: Bin Sayeed, M.S., et al., Nigella sativa L. seeds modulate mood, anxiety and cognition in healthy adolescent males. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2013.12.050i