Research Paper

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

Muhammad Shahdaat Bin Sayeed a,b,n, Tahiatul Shams b,c, Sarder Fahim Hossain c,Md. Rezowanur Rahman c, AGM Mostofa a,b, Mohammad Fahim Kadir b,c,Sharif Mahmood d, Md. Asaduzzaman b,c

a Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka-1000, Bangladeshb Department of Pharmacy, University of Asia Pacific, Dhaka-1209, Bangladeshc Department of Pharmaceutical Technology, University of Dhaka, Dhaka, Bangladeshd Institute of Statistical Research and Training, University of Dhaka, Bangladesh

a r t i c l e i n f o

Article history:Received 13 September 2013Received in revised form17 December 2013Accepted 28 December 2013Available online 8 January 2014

Keywords:Nigella sativa L. seedsAdolescentsState–Trait Anxiety InventoryModified California verbal learning test-IIBond–Lader scaleClinical Trial

a b s t r a c t

Ethnopharmacological relevance: Previous studies conducted on animals linked consumption of Nigellasativa L. seeds (NS) to decreased anxiety and improved memory. The present study, which was carriedout at a boarding school in Bangladesh, was designed to examine probable effect of NS on mood, anxietyand cognition in adolescent human males.Materials and methods: Forty-eight healthy adolescent human males aged between 14 to 17 years wererandomly recruited as volunteers and were randomly split into two groups: A (n¼24) and B (n¼24). Thetreatment procedure for group A and B were one capsule of 500 mg placebo and 500 mg NS respectivelyonce daily for four weeks. All the volunteers were assessed for cognition with modified California verballearning test-II (CVLT-II), mood with Bond–Lader scale and anxiety with State–Trait Anxiety Inventory(STAI) at the beginning and after four weeks of either NS or placebo ingestion.Results and discussion: No parameter showed statistically significant variation between A and B inmeasurements in the beginning, but after 4 weeks of one capsule of NS 500 mg intake, there wasstatistically significant variation of mood within group B but there was not statistically significantvariation between group A and B. No significant variation was found in state anxiety within groups andbetween group A and B but in case of trait anxiety, significant variation was found within group B but notbetween group A and B. In case of CVLT II, there was significant variation within B in immediate short-term recall at trial 4 and 5 whereas this difference was found only in case of trial 5 between group A and B.Within group B, short term-free recall, long-term free recall and long-term cued recall had statisticaldifference whereas between group A and B long-term free recall and long-term cued recall had statisticaldifference. No parameters had significant variation within group A after placebo intake for 4 weeks.Conclusions: Over the 4 weeks study period, the use of NS as a nutritional supplement been observed to-stabilize mood, decrease anxiety and modulate cognition positively. However, long term study is suggestedbefore using NS extensively.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Nigella sativa L. (Family: Ranunculaceae), commonly known asblack cumin, is an annual herb. It is native to south west Asia andcultivated in countries like Middle Eastern Mediterranean region,South Europe, Syria, Turkey, Saudi Arabia, Pakistan, India (Zamanet al., 2004). The oil and seed constituents have potential medi-cinal properties (Salem, 2005) and have been traditionally used infolk medicine for the treatment and prevention of a number ofdiseases and conditions including asthma, diarrhea, dyslipidaemia,kidney, liver dysfunction and memory impairments (Butt andSultan, 2010; Sharrif, 2011).

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0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jep.2013.12.050

Abbreviations: NS, Nigella sativa L. seeds; CVLT-II, modified California verballearning test-II; STAI, State–Trait Anxiety Inventory; ADHD, attention deficithyperactivity disorder; CONSORT, consolidated standards of reporting trials; 5-HT,5-hydroxytryptamine; GABA, gamma-aminobutyric acid; 5-HIAA, 5-hydroxyindo-leacetic acid; NO, nitric oxide; SDCR, short delay cued recall; SDFR, short delay freerecall; LDCR, long delay cued recall; LDFR, long delay free recall

n Corresponding author at: Department of Clinical Pharmacy and Pharmacology,University of Dhaka, Dhaka 1000, Bangladesh. Tel.: þ880 2 9661900x8156;Mobile: þ880 1713459747; fax: þ880 2 8612069, þ880 2 8615583.

E-mail addresses: shahdaat2013@yahoo.com,shahdaat2013@du.ac.bd (M.S. Bin Sayeed).

Journal of Ethnopharmacology 152 (2014) 156–162

Recent studies have demonstrated that the pathogenesis ofanxiety is linked to oxidative stress and decreased antioxidantcapacity (Gingrich, 2005; Halliwell, 2006; Valko et al., 2007; Nget al., 2008). Oxidative stress induced anxiety-like behavior inrodents can be reversed by using appropriate antioxidant that actsvia different mechanisms (Urushitani et al., 2000; Masood et al.,2008; Salim et al., 2010).

Some studies previously reported that the seeds of Nigellasativa L. (NS) have a potent antioxidant activity (Atta andImaizumi, 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 antioxidantactivities (Burris and Bucar, 2000), prevent oxidative injuries(Ilaiyaraja and Khanum, 2010; Sharrif, 2011), reduce oxidativestress 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 haveshown 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 (Hosseinet 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 improveslearning and memory in rats. Jalali and Roghani (2009) reportedenhancement of the consolidation and recall capability of storedinformation and spatial memory in rats followed by NS adminis-tration. Again, studies showed that NS protects hippocampalneurodegeneration (Kanter, 2008), has positive modulation effectson 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 spatialcognitive functions of rats that suffered global cerebrovascularhypoperfusion (Azzubaidi et al., 2012). Safety profile of NS is wellestablished through research conducted both on rodents (Jalali andRoghani, 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) showedpositive modulating impact of NS on memory, attention andcognition. This investigation also showed safety status of NSconsumption through studying different parameters of heart, liverand kidney functions. Thus NS could be considered as a useful andsafe choice for the treatment of anxiety.

The aim of the present study was to investigate the effect of NSintake on anxiety, mood and cognition in adolescents, as well as toexamine whether there is any relation between anti-anxiety andcognition enhancement in adolescents due to NS intake. This studywill be helpful for indicating NS as food supplement in maintain-ing sound psychological health in adolescence especially thosefrom developing and under-developed countries.

2. Materials and method

2.1. Participants

A total of 57 healthy adolescent males, age ranging between14 and 17 years participated in the present study. Participants wererecruited from a boarding school located at Rangpur in Bangladesh.Subjects having previous history of attention deficit hyperactivitydisorder (ADHD) and frequent sickness were excluded from thisinvestigation. Thus nine subjects were excluded and finally therewere 48 volunteers who took part in this investigation. The study(Code number: RSBS042012) was carried out in accordance with theDeclaration of Helsinki and subsequent revisions (World Medical

Association Declaration of Helsinki, 1964) and was approved by theauthority of the boarding school from where students wererecruited. Written informed consent was obtained from all partici-pants and their guardians before the study. The participants wereintroduced to a complete set of medical health questions prior toselection for evaluation of their physical and mental health condi-tions and to determine their suitability for the study.

2.2. Preparation of capsules

In current investigation, NS was taken as it is used traditionallyi.e. without any modifications either by fractionation of the seedsor by adding any other substances. A representative of the studyteam purchased the seeds from a known quality vendor undersupervision of a registered pharmacist. NS seeds were crushedwith mortar and pestle for convenient capsule filling. The crushedseeds were passed through a stainless steel screen (mesh size #30)and filled into empty hard gelatin capsule shells (size #0) using amanual capsule filling machine. The quality of NS and crushed NSwas vigorously monitored under the direct supervision of aregistered pharmacist. Crushed NS were filled into capsules in alocal GMP compliant company. It was ensured that each of thecapsule contained 500 mg powdered NS. The quality of seeds wasensured by direct observation of a registered pharmacist. Husk ofIsabgol (Psyllium seed husk) was used as placebo and capsuleswere 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 over4 weeks and the participants were assigned random code numbersgenerated by computer. All the participants were assessed forbaseline data to measure cognition, mood and anxiety level beforeadministering the first dose. The tests were taken in Bengalilanguage (native language of the subjects) and all the termino-logies used in the Bond–Lader scale and state–trait anxietyquestionnaire were explained to all the volunteers before con-ducting the study. Each participant from group B received onecapsule of NS 500 mg and each participant from group A receivedone placebo capsule once daily after dinner for 4 weeks. Placeboand NS capsules had the same color, texture, size and shape. Allinvestigators (except the principal investigator who was involvedin designing the experiment, analyzing final data and writing themanuscript but neither involved in the administration of capsulesnor in documenting the data) and the participants were kept blindabout NS or placebo. The code numbers and the group allocationwere only revealed after the last participant had completed theentire follow up. All the participants were assessed again forcognitive performance, anxiety level and mood status at the endof 4 weeks. The sequence of administration of the study was samefor each subject and all the tests for participants were completedduring 0700 to 0930 h. All the participants were instructed to callthe study-center in case of any adverse effect felt during the study.They had the opportunity to withdraw from the study at any time.

2.4. Mood

The Bond–Lader scale (Bond and Lader, 1974) used for assessingmood (Wesnes et al., 2003; Smith and Foster, 2008) was made up of16�100 mmvisual analogue scales with the end-points anchored bythe antonyms: alert-drowsy, calm-excited, strong-feeble, clear-headed-fuzzy, well coordinated-clumsy, energetic-lethargic, con-tented-discontented, tranquil-troubled, quick witted-mentally slow,relaxed-tense, attentive-dreamy, proficient-incompetent, happy-sad,friendly-antagonistic, interested-bored and sociable-withdrawn.

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Participants were presented with a sheet of paper containing all thescales and instructed to mark their current mood state on each line.These were combined as recommended by the authors (Bond andLader, 1974) to form three mood factors: ‘alert’, ‘calm’ and ‘contented’with scores on each ranging from 0 to 100. Scores in the lower rangeindicates positive mood.

2.5. State–Trait Anxiety Inventory (STAI)

The STAI (Laux et al., 1981; Spielberger, 1983) comprises of twoscales (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-pointscale (1, not at all/almost never; 2, somewhat/sometimes; 3, moodrarely so/often; 4, very much so/almost always). Aggregate ratingsof each of the two scales (state and trait anxiety) can range from20 to 80, with higher rating indicating more anxiety.

2.6. Modified California verbal learning test-II (CVLT-II)

The CVLT-II is a test of immediate, short delay and long delayepisodic memory for a 16-item word list (Delis et al., 2008). Itcomprises of a standard form and an alternate form that can beused for a repeat testing session. In the present study, one formwas administered in the first session, and the complementaryform was administered in the second session, in a counterbalanced order. The word lists used in the CVLT-II were made upof 16 words from four different categories. List A comprised ofnames 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 thefour categories (fruits and vegetables) were kept common on boththe lists. The experimenter read a list of words (List A), andparticipants were asked to recall as many of the words as theycould, in any order (Immediate Free Recall: List A). This procedure

was repeated four times (Trials 2–5), accumulating to five trialstotal. Following the list-learning procedure in trials 2 to 5,participants heard a second list of words (List B) and subsequentlytried to name as many of these words as possible (Immediate FreeRecall: List B). Then, the participants were asked to name as manywords as possible from the first list again (short-delay free recall:List A). Following this free recall task, participants were given acued recall task, in which they were asked to name all the wordsfrom the first list that belonged to each of the four categories(short-delay cued recall). With an interval of approximately 20 to30 min they were asked to recall words from the first list that wasread to them (long-delay free recall). Participants were notexpecting the last free recall condition, as they were not informedabout any further tests with the word lists. Categorization similarto earlier with provided cues were done at the end (long-delaycued recall).

2.7. Safety profile study

Systolic–diastolic blood pressure as well as the weight andheight of the volunteers were measured twice: before and aftertreatment with either placebo or NS.

2.8. Statistical analysis

Results were analyzed independently for each test. Poissonregression was employed to identify whether groups had anysignificant differences over the time. To estimate the parameters ofthe model, we employed generalized estimating equations as overthe time the responses are associated by employing R. To find thedifference between placebo and treatment group we checkednormality assumption and employed statistical tests that wasappropriate. Repeated measure ANOVA was employed to observebetween and within effect by using IBM Statistics 19. Repeatedmeasure multivariate analysis was performed to find the effect of

Fig. 1. Study flowchart.

M.S. Bin Sayeed et al. / Journal of Ethnopharmacology 152 (2014) 156–162158

NS over four week time period. Chi-square test was performed fordemographic data between groups. po0.05 was considered sta-tistically significant.

3. Result

3.1. Mood

Variation of mood was statistically significant (summation ofscore of all the factors) within group B (po0.05) but variationbetween group A and B (p40.05) was not statistically significant.Repeated measure multivariate analysis shows that there wassignificant difference between group A and group B on mood overthe four weeks study period, F(3, 44)¼9.48, p¼0.000, η2¼0.393.Univariate tests also indicated that there was NS effect on all of thetests of mood; F(1, 44)¼6.880, p¼0.012, η2¼0.130 for alertness,F(1, 44)¼13.808, p¼0.001, η2¼0.231 for contendness, F(1, 44)¼5.718, p¼0.021, η2¼0.111 for calmness (Fig. 2).

3.2. State–Trait Anxiety Inventory (STAI)

The intervention effect was analyzed using repeated measuresANOVA. There was statistically significant difference betweenintervention and control over time four weeks study period onstate anxiety score, F(1, 46)¼7.2, p¼0.01, η2¼7.221. Repeatedmeasure multivariate analysis shows that there was significantdifference between group A and group B on state anxiety overtime four weeks study period, F(19, 28)¼4.975, p¼0.000, η2¼0.771. However, univariate tests indicated there was not NS effecton all of the items in state questionnaire except items 1: ‘I feelcalm’ [F(1, 46)¼22.490, p¼0.000, η2¼0.328], Items: 3 ‘I feeltense’[F(1, 46)¼34.677, p¼0.000, η2¼0.430], and Items: 6 ‘I feelupset’ [F(1, 46)¼33.891, p¼0.000, η2¼0.424]. On the other hand,there was statistically significant difference between interventionand control over time four weeks study period on trait anxietyscore [F(1, 46)¼16.233, p¼0.000, η2¼0.261]. Repeated measuremultivariate analysis shows that there was significant differencebetween group A and group B on trait anxiety over time fourweeks study period, F(19, 28)¼7.660, p¼0.000, η2¼0.850. How-ever, univariate tests indicated there was not NS effect on all of the

items in trait questionnaire except in the trait Items 2: ‘I feelnervous and restless’ [F(1, 46)¼12.760, p¼0.000, η2¼0.666],Items: 6 ‘I feel rested’ [F(1, 46)¼7.193, p¼0.000, η2¼0.135], Items:16 ‘I feel content’ [F(1, 46)¼27.046, p¼0.000, η2¼0.370], Item: 18‘I take disappointments so keenly that I can0t put them out of mymind’ [F(1, 46)¼27.806, p¼0.000, η2¼0.377], Item: 20 ‘I get in astate of tension or turmoil over my recent concerns and interests’ F(1, 46)¼24.383, p¼0.000, η2¼0.346 (Fig. 3).

3.3. Modified California verbal learning test-II (CVLT-II)

3.3.1. Immediate free recallRepeated measure multivariate analysis shows that there was

significant of NS over time four weeks study period on immediatefree recall of CVLT-II over time four weeks study period, F(5, 42)¼4.489, p¼0.002, η2¼0.348 (Fig. 4). Univariate tests indicated therewas not NS effect over time four weeks study period on trial 1 [F(1,46)¼2.706, p¼0.107, η2¼0.056], trial 2 [F(1, 46)¼0.044, p¼0.835,η2¼0.001] and trial 3 [F(1, 46)¼0.020, p¼0.888, η2¼0.000] buthave effect on trial 4 [F(1, 46)¼5.343, p¼0.025, η2¼0.104], trial 5[F(1, 46)¼11.792, p¼0.001, η2¼0.204 and summation of all trials[F(1, 46)¼12.611, p¼0.001, η2¼0.215].

3.3.2. Delayed recallIn case of delayed recall of CVLT-II, repeated measure multi-

variate analysis shows that there was significant effect of NS overtime four weeks study period [F(4, 43)¼5.955, p¼0.001,η2¼0.356] (Fig. 5). Univariate tests indicate that NS does not haveeffect on short delay free recall [F(1, 46)¼3.010, p¼0.089, η2¼0.061] but affect short delay cued recall [F(1, 46)¼2.259, p¼0.140,η2¼0.047], long delay free recall [F(1, 46)¼8.697, p¼0.005,η2¼0.159] and long delay cued recall [F(1, 46)¼11.058, p¼0.002,η2¼0.194].

3.4. Safety profile assessment

Blood pressure, body weight and body mass index (BMI) of thevolunteers did not change significantly (p40.05) during the studyperiod (Table 1). Repeated measure multivariate analysis alsoshows that there were not significant effect of NS over time fourweeks study period [F(5, 42)¼1.363, p¼0.258, η2¼0.140].Univariate tests indicated that NS does not have effect on systolicblood pressure [F(1, 46)¼0.004, p¼0.950, η2¼0.000], diastolicblood pressure [F(1, 46)¼0.133, p¼0.717, η2¼0.003], weight

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.

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

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[F(1, 46)¼0.811, p¼0.373, η2¼0.017], height [F(1, 46)¼0.945,p¼0.336, η2¼0.020], BMI [F(1, 46)¼0.392, p¼0.534, η2¼0.008].

4. Discussion

This study was designed to investigate the effects of NS onanxiety, mood and cognition in male healthy adolescents. The linkbetween anxiolytic property and cognitive function was alsostudied. Results from this study suggest that the ingestion of NSby the participants may have reduced anxiety and improved moodand cognition. Before starting the treatment all the participantswere assessed for baseline data to measure cognition and status ofmood and anxiety level. None of the observed baseline data at thatstage showed statistically significant difference in between groupA and B. Chi-squared test indicated that there were not significantvariation in their demographic parameters such as age (p¼0.967),weight (p¼0.688), height (p¼0.119) and BMI (p¼0.500).

The results of Bond–Lader scale showed that there wasdecrease and increase in alertness in group A and B respectively-but none of them was statistically significant. Again the differencein the measure for alertness between group A and B after fourweeks was statistically significant. Calmness was significantlydifferent within group B, but such difference was not evident inbetween observations from groups A and B.

The STAI test was conducted to compare the anxiety levels ofthe two groups. The anxiety levels observed were lower in groupA and B compared to baseline but none of them were statisticallysignificant. However, some of the items showed statisticallysignificant difference in scores of group B, but such differencewas not found in case of group A. There might have some decreaseof anxiety due to the effect of NS on several neurotransmitters like5-hydroxy tryptamine (5-HT) and gamma amino butyrate (GABA).According to Perveen et al. (2009) NS increases the level of 5-HTand thus decrease anxiety. TQ in NS might also decrease NO andreverse decreased brain GABA content and give anxiolytic effect(Gilhotra and Dhingra, 2011). Lower number of sample and shortperiod of study could be responsible the lower strength of thestudy and for exhibiting such results.

Participants exhibited superior performance on the short delayfree recall, long delay free recall and long delay cued recall phasesof the modified version of the CVLT-II after receiving NS. The rateof learning was also shown to be faster in group A compared togroup B, with participants of group B demonstrating significantlyenhanced performance on the fourth and fifth trials of theimmediate free recall phase of the modified CVLT-II subsequentto NS ingestion. This could be due to antioxidant, anti-cholinesterase activities of NS (Atta and Imaizumi, 1998; ElShenawy et al., 2008; Yoruk et al., 2010; Panahi et al., 2011).

Anxiety is generally considered as impediment to cognition(Eysenck et al., 2007; Derakshan et al., 2009; Cornwell et al., 2012).

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 NSon trials of CVLT-II over four weeks time period.

Fig. 5. Delayed recall on modified California verbal learning test-II (CVLT-II). Here:SDCR¼short delay cued recall, SDFR¼short delay free recall, LDCR¼ long delaycued recall, LDFR¼ long delay free recall. Repeated measure multivariate analysiswas performed to find the effect of NS on “SDCR”, “SDFR”, “LDCR” and “LDFR” overfour weeks time period.

Table 1Parameters of Health status indicators.

Parameter Group A Group B p-Value

Baseline After four weeks Baseline After four weeks

Health status indicatorsSystolic BP (mmHg) 133.7072.628 134.0472.255 134.0872.602 134.4872.903 0.950Distolic BP (mmHg) 84.5071.063 84.2970.858 84.1771.167 83.7970.858 0.717Weight (kg) 55.5873.670 56.0473.568 56.6673.607 56.9573.482 0.373Height (m) 1.56970.051 1.57170.050 1.5870.067 1.58170.066 0.336BMI (kg/m2)a 1.56870.0511 1.57170.0500 1.58170.0670 1.58270.0658 0.534

a Body mass index¼mass/height2.

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The negative effects of anxiety on cognitive-behavioral performanceshave been explained by the cognitive models of anxiety (Eysenck andCalvo, 1992; Bishop, 2007; Eysenck et al., 2007). Highly anxioushuman subjects showed disturbances in goal directed control ofattention leading to poor cognitive behavioral performances (Bishopet al., 2007). Some neurotransmitters play role in the modulation ofanxiety e.g. 5-HT, GABA (Handley and McBlane, 1993; Streeter et al.,2010). Increased 5-HT and decreased 5-hydroxyindoleacetic acid(5-HIAA) levels are related to reversal of anxiety (Collinge et al.,1983). Increase in thalamic GABA level is associated with improvedmood and decreased anxiety (Streeter et al., 2010). GABA is alsoreported to attenuate stress-induced nitric oxide (NO) release andthus preventing NO induced increase in anxiety (Harvey et al., 2004;Sevgi et al., 2006). NS might show the positive modulating effect oncognition because of decrement of anxiety which is because ofinteraction of NS with these neurotransmitters.

Multiple studies have shown that oxidative stress is associatedwith anxiety and cognitive defects in both human and animalmodels which could be reversed by treatment with ROS scaven-gers (Liu et al., 2003; Sklan et al., 2004). Enhancement of cognitionand improvement of memory in group B might be due toactivation of the cholinergic neuronal system of hippocampus thatplays an important role in learning and memory (Nabeshima1993). We propose further investigations in animals with differentfractions of NS or with different compounds found in NS indifferent ratios and doses.

5. Conclusion

The observed effect of NS on anxiety as measured by STAI wasstatistically significant, on calmness of mood as measured byBond–Lader scale and on cognition as measured by CVLT-II wasalso statistically significant. This demonstrates that there mighthave been improvement of cognition via affecting anxiety andmood. Therefore NS might be a potential source for developingnew drugs for treating anxiety and stabilizing mood and improv-ing cognition in adolescents. It is known that NS compositionincludes 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 modulatingeffect on cognition and mood. Exhibition of these activities inhuman is also justified from their traditional use (Sharrif, 2011).On the other hand, smaller sample size and short period of studymight be considered as limitation of the study. Conducting thestudy with adolescence with anxiety, mood disorders andimpaired cognition could have been a better study design forclaiming the effect of NS on anxiety, mood and cognition. Thestudy did not measure any serological parameters before and afterNS intake. This was not feasible both financially as well as due tolack of having monitoring system. However, further long termstudy is recommended with adolescent with anxiety, mood dis-orders and impaired cognition as well as extensive study about themechanism of the effect of NS on memory and cognition.A thorough safety and toxicological study of NS in human modelis recommended before using NS extensively. On the other hand,the use of NS as nutritional supplement for the purposes asmentioned earlier could be justified after further toxicologicalinvestigations in animals and human.

Acknowledgement

The first and corresponding author, Muhammad Shahdaat BinSayeed, 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. Theauthors appreciate the cooperation of Mohammad Mizanur Rahman,Headmaster of Sathibari Madrasa,for conducting the study. TheNigella sativa L. seeds were bought and quality of the seeds wasassured by Md. Asaduzzaman, Asst. Prof. in the Department ofPharmacy, University of Asia Pacific, Dhaka, Bangladesh. The seedswere identified by Botanist Mr. Manzur-ul-Kadir Mia, PrincipalScientific 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, DewanIdris Road, Bara Rangamatia, Zirabo, Savar, Dhaka-1341, Bangladesh.However, Incepta Pharmaceuticals did not have any involvement withthis project. Senior psychologist Dr. Monowara Parveen Jahangiri ofDhaka Shishu (Children) Hospital, Dhaka, Bangladesh gave importantsuggestions regarding preparation and administration of differentmodules of Neuropsychological tests used in this experiment. We aregrateful to Khondker Galib Bin Mohiuddin, Lecturer, North SouthUniversity, Bangladesh for giving very thoughtful suggestions inmanuscript writing. We took generous help from Faez MahbubChowdhury, Department of psychology, University of Dhaka regard-ing psychological issues of the volunteers during the investigations.Finally we appreciate Sharif Mahmood, Assistant professor, ISRT,university of Dhaka for helping us in analyzing statistical data.

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