Research Article Tualang Honey Attenuates Noise Stress ...downloads.hindawi.com/journals/omcl/2016/1549158.pdfResearch Article Tualang Honey Attenuates Noise Stress-Induced Memory

Research ArticleTualang Honey Attenuates Noise Stress-InducedMemory Deficits in Aged Rats

Khairunnuur Fairuz Azman1 Rahimah Zakaria1

Che Badariah Abdul Aziz1 and Zahiruddin Othman2

1Department of Physiology School of Medical Sciences Universiti Sains MalaysiaKubang Kerian 16150 Kota Bharu Kelantan Malaysia2Department of Psychiatry School of Medical Sciences Universiti Sains MalaysiaKubang Kerian 16150 Kota Bharu Kelantan Malaysia

Correspondence should be addressed to Rahimah Zakaria rahimahusmmy

Received 12 January 2016 Accepted 13 March 2016

Academic Editor Liang-Jun Yan

Copyright copy 2016 Khairunnuur Fairuz Azman et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Ageing and stress exposure may lead to memory impairment while oxidative stress is thought to be one of the underlyingmechanisms involvedThis study aimed to investigate the potential protective effects of Tualang honey supplementation onmemoryperformance in aged rats exposed to noise stress Tualang honey supplementation was given orally 200mgkg body weight for 28days Rats in the stress groupwere subjected to loud noise 100 dB(A) 4 hours daily for 14 days All rats were subjected to novel objectrecognition test for evaluation ofmemory performance It was observed that the rats subjected to noise stress exhibited significantlylower memory performance and higher oxidative stress as evident by elevated malondialdehyde and protein carbonyl levels andreduction of antioxidant enzymes activities compared to the nonstressed rats Tualang honey supplementation was able to improvememory performance decrease oxidative stress levels increase brain-derived neurotrophic factor (BDNF) concentration decreaseacetylcholinesterase activity and enhance neuronal proliferation in the medial prefrontal cortex (mPFC) and hippocampus Inconclusion Tualang honey protects against memory decline due to stress exposure andor ageing via enhancement of mPFC andhippocampal morphology possibly secondary to reduction in brain oxidative stress andor upregulation of BDNF concentrationand cholinergic system

1 Introduction

It is well known that ageing leads to a progressive loss ofcognitive function especially in spatial and workingmemory[1] There is abundant evidence that such age-associatedalterations in cognition are related to decreased functionof cholinergic neurons in the hippocampus and cortex[2ndash4] Survival and functional maintenance of cholinergicneurons are dependent upon nerve growth factors (NGF)including brain-derived neurotrophic factor (BDNF) [5ndash7]In regions such as the hippocampus and cerebral cortexNGF and BDNF supplementation protects neurons againstexperimentally induced damage from procedures [8 9]

Neurotrophin supplementation also appears to have func-tional consequences for learning and memory and effectivelyameliorates memory deficits in aged rats [10ndash12]

Noise exposure exceeding 90 dB has been reported to be asource of stressor [13] Working and reference memory errorincreased significantly following the noise stress exposure100 dB(A)4 h per day for 30 days when compared to controlanimals [14] Acute and chronic exposure to noise canproduce excessive free radicals which may attack proteinnucleic acids and lipid membranes thereby disrupting nor-mal cellular functions [15] Exposure to long-term oxidativestress in brain tissue has been shown to cause reductions inlearning [16 17] and memory [18 19] Several studies have

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 1549158 11 pageshttpdxdoiorg10115520161549158

2 Oxidative Medicine and Cellular Longevity

shown that noise induces hippocampal dependent memorydeficits and volumetric reduction in all layers of hippocampalfield [15ndash17 20]

Malaysia Tualang honey is a wild pure multifloral honeyproduced by Asian rock bee species (Apis dorsata) whichbuilds hives on the branches of Tualang tree (Koompas-sia excelsa) located mainly in the rainforest of northernPeninsular Malaysia Honey contains significant antioxidantactivities as well as choline and acetylcholine which areessential for brain function and as neurotransmitters [21ndash25] Recent studies reported that honey is one of the naturalpreventive therapies of both cognitive decline and dementiaas it possesses antioxidant properties and it enhances thebrainrsquos cholinergic system [26] Correspondingly anotherstudy reported that consumptions of honey may improvespatial memory in middle aged rats compared to those fedsucrose or sugar-free diet [27] In closely related studies it wasdemonstrated that Tualang honey was able to improve mem-ory performance in stressed ovariectomized rats [28] andpostmenopausal women [29] Therefore it is hypothesizedthat Tualang honey could revert oxidative stress by removingthe reactive oxygen species that were produced during thenoise stress and thus prevent oxidative damage in memory-related brain areas particularly in aged rats

2 Material and Methods

21 Experimental Animals A total of 48 male Sprague-Dawley rats (16 months old weighing 600ndash800 g) werepurchased from Sterling Ascent Sdn Bhd (Malaysia)The ratswere maintained in standard polypropylene cages (40 times 25 times16 cm) under a reversed 12-hour lightdark cycle (lights offat 0800 h) at a consistent room temperature of 27 plusmn 1∘Cin the laboratory of Animal Research and Service CentreUniversiti Sains Malaysia The rats received commercial ratchow food pellets (Gold Coin Ltd Malaysia) and waterad libitum Rats were allowed to acclimatize to the hold-ing room for 24 h before the behavioural procedures Theprocedures in this study were approved by Animal EthicsCommittee ofUniversiti SainsMalaysia (USMAnimal EthicsApproval2013(85)(444)) in accordance with the interna-tionally accepted principles for laboratory animal use andcare

22 Honey Supplementation The Tualang honey used wasfrom a single batch honey supplied by Federal AgriculturalMarketing Authorities (FAMA) Malaysia The honey wasfiltered by FAMA to remove solid particles concentrated inan oven at 40∘C and evaporated to achieve a water content ofabout 20 It was then subjected to 120574 irradiation at 25 kGyat Steril Gamma (M) Sdn Bhd (Selangor Malaysia) forsterilization and bottled 230 g per jarThe final concentrationof the bottled Tualang honey was 13 gmL Tualang honeyat 200mgkg body weightday [28 30] was administeredvia oral gavage 14 days prior to stress procedure and thetreatments were continued throughout the 14 days of stressprocedure The Tualang honey was freshly dissolved in 1mLof distilled water prior to administration Control groups

received an identical volume of distilled water as placebo forthe same period of time

23 Experimental Design The animals were randomlyassigned to the following groups (119899 = 12) (i) nonstressedwith placebo (ii) nonstressed with Tualang honey (iii)stressed with placebo and (iv) stressed with Tualanghoney All rats were subjected to novel object recognitiontest for cognitive performance evaluation and killed bydecapitation upon completion of the test Individual bodyweight was recorded weekly using electrical balance Bloodsamples (10mL) were collected immediately All bloodsamples were left to clot for 2 h prior to centrifugation for15min at 4000 rpm Approximately 3mL of serum wascollected and stored at minus20∘C until assay The brain of eachanimal was quickly harvested and weighed The right brainhemisphere was homogenated (10wv) in ice-cold 01Mphosphate-buffered saline at pH 74 The homogenate wasthen centrifuged at 10000timesg for 10 minutes and kept atminus80∘C until being analyzed The left brain hemisphere wasimmediately fixed in 10 formalin for histological study

24 Noise Stress Exposure The animals of the test groups((iii) and (iv)) were exposed to white noise for 4 hours(0900ndash1300 h) daily for 14 days Noise was recorded fromthe generator and amplified by speakers in a separate roomSpeakers were located 30 cm above the cages The noise levelwas set at 100 dB(A) and intensity was measured by a soundlevel meter CENTER 325 (range 80ndash130 dB(A) accuracy+15 dB(A)made inTaiwan) Sound levelswere verified in thecentre of the cage before each exposure and varied by less than1 dB(A) in the space the cage occupiedThe control groups ((i)and (ii)) were kept in the same room for the same period oftime without switching on the noise

25 Novel Object Recognition Test The test was carried outin a separate room that was ventilated soundproofed andmaintained at a constant temperature (27plusmn1∘C)The animalswere brought into the test room 1 hour before the testcommenced to minimize the arousal caused by the trans-ference The test was performed during the active period ofthe animals (dark phase) between the hours of 0900ndash1400All the animals were tested in a random order The trainedobserver remained blind to the treatment group of the ratsuntil scoring was completed

The test employedwas similar to that described elsewhere[31] The test uses the natural preference for novel objectdisplayed by rats and normally used to assess cognitive alter-ations associated with ageing genetic manipulations or drugtreatmentsThe chamberwas an open field apparatus (60times 60times 30 cm) Training sessionswere conducted on two successivedays during which they were allowed to explore the arenafor 10min each day In the training session two identicalsample objects were placed in the field in a symmetricalposition about 10 cm away from the wall After the twosuccessive training sessions testingretention sessions wereconducted The retentions sessions consisted of two sessionsthat is short-termmemory and long-termmemory in which

Oxidative Medicine and Cellular Longevity 3

the retention interval for short-term memory and long-termmemory was 2 hours and 24 hours after the last trainingsession respectively In the retention session rats were placedback in the same field wherein one of the familiar objectsused in the training session was replaced by a novel objectand the rats were allowed to explore for 5 minutes

All objects consisted of plastic toys and had a heightof about 5 cm Objects presented similar textures coloursand sizes but distinctive shapes The location of objects wasalternated with each new animal it was approximately placedin 50 trials in the right side and 50 in the left side of thefield Between tests the objectswere cleanedwith 10 ethanolsolution to mask any olfactory cues Exploration was definedas sniffing or touching the object with the nose Sitting on theobject was not considered as exploration

Total exploration times of the familiar and novel objectswere recorded and used to calculate a discrimination index[time spent with novel object minus time spent with familiarobject][total time exploring both objects] The discrimi-nation index can range from minus1 to 1 wherein minus1 indicatescomplete preference for the familiar object 0 signifies nopreference for either object and 1 indicates complete pref-erence for the novel object Increased preference to novelobject was interpreted as successful memory retention forthe familiar object An absence of any difference in theexploration of the two objects was interpreted as memorydeficit

26 Serum Corticosterone and Adrenocorticotropic Hormone(ACTH) Levels Serum levels of corticosterone and ACTHwere measured by enzyme-linked immunosorbent assay(ELISA) kits using polyclonal antibody specific for corticos-terone (LDN Labor Diagnostika Nord GmbH amp Co KGNordhorn Germany) and monoclonal antibody specific forACTH (Cloud-Clone Corp Houston USA)

27 Brain Oxidative Stress Markers The evaluation of oxida-tive stress in the brain homogenates was performed bymeasuring the levels of plasma malondialdehyde (MDA) andprotein carbonyl (PCO) The concentration of MDA wasanalyzed using commercially available kits from NorthwestLife Sciences SpecialtiesWashington USA whereas the levelof PCO was determined by commercially available kits fromCayman Chemical Michigan USA

28 Brain Antioxidant Enzymes Activities The activities ofsuperoxide dismutase (SOD) glutathione peroxidase (GPx)and glutathione reductase (GR) in the brain homogenateswere measured using commercially available kits fromNorthwest Life Sciences SpecialtiesWashington USA Com-mercially available kits from Bioassay Systems CaliforniaUSA andOxford Biomedical ResearchMichigan USA wereused to determine the activities of catalase (CAT) and thetotal antioxidant capacity respectively

29 Acetylcholinesterase (AChE) Activity MeasurementCommercially available assay kit from Bioassay SystemsCalifornia USA was usedThe assay is based on an improved

Ellmanmethod in which thiocholine produced by the actionof AChE forms a yellow colour with 551015840-dithiobis(2-nitrobenzoic acid) The intensity of the product colourmeasured at 412 nm is proportionate to the enzyme activityin the sample

210 Brain-Derived Neurotrophic Factor (BDNF) Concentra-tion Measurement BDNF concentration was measured byELISA kit from Boster Biological Technology Co CaliforniaUSA Amonoclonal antibody frommouse specific for BDNFand the assay Avidin-Biotin-Peroxidase complex was usedto bind the detection antibody HRP substrate 331015840551015840-tetramethylbenzidine (TMB) was used to visualize HRPenzymatic reaction which produces a blue colour productthat changed into yellow after adding acidic stop solutionThedensity of yellow colour measured at 450 nm is proportion-ate to the BDNF concentration in the sample

211 Protein Concentration Following homogenization analiquot was removed from each brain sample to determineits protein concentration using commercially available kitsfrom Bioassay Systems California USA Briefly proteinconcentration was quantified by comparing the colorimetricintensity of the reaction product from each sample with aseries of protein standards All antioxidant activities andoxidative stress markers levels were normalized to their totalprotein concentration in the sample in order to accountfor possible differences in protein concentrations betweensamples

212 Histopathological Analysis The left brain hemisphereswere embedded in paraffin wax cut into 5 120583m thick coronalsections using a rotary microtome and mounted on slidesfollowed by Nissl staining which was performed accordingto the standard procedure The slides were observed under alight microscope and images were captured to visualize thearrangement of pyramidal neurons in the medial prefrontalcortex (mPFC) and each hippocampal region CA1 CA2CA3 and DG The Nissl-positive cells were counted at dif-ferent magnifications using High Definition Medical ImageAnalysis Program (analySIS docu 50 Munster Germany)The mean of two fields was taken as the number of Nissl-positive cells for each section and the mean of four sectionswas taken as the number of Nissl-positive cells of eachgroup Cells which had a shrunken or unclear body withsurrounding empty spaces were excluded

213 Statistical Analysis All analyses were performed usingIBM SPSS statistics software (version 200) Statistical dataare expressed as mean plusmn SEM and a result was deemed tobe statistically significant if 119875 lt 005 Two-way analyses ofvariance (ANOVA) were utilized to examine the main effectsof stress (nonstressed versus stressed) and honey treatment(placebo versus honey) on the memory performance antiox-idant enzymes activities oxidative stress markers stresshormone levels and the number of Nissl-positive cells inthe mPFC and hippocampal regions After confirming thenormality of data and the homogeneity of variance of data


Table 1 Effects of stress and honey treatment on serum corticosterone and adrenocorticotropic hormone levels

No stress No stress + honey Stress Stress + honeyCorticosterone level (ngmL) 6875 plusmn 288 6568 plusmn 235lowast 7382 plusmn 231 6590 plusmn 184lowastsect

Adrenocorticotropic hormone level (pgmL) 9590 plusmn 410 8243 plusmn 435lowast 11378 plusmn 472curren 9175 plusmn 340lowastsect

The values are expressed as meanplusmn SEM Significant main effects of loud noise stress (119875 lt 005) Significant main effects of honey treatment (lowast119875 lt 005)Significant difference between no stress and stress control (curren119875 lt 005) Significant difference between stress control and stress treated with honey (sect119875 lt 005)

450

500

550

600

650

700

0 1 2 3 4 5

Body

wei

ght (

g)

Week

No stressNo stress + honey

StressStress + honey

(a)

00

05

10

15

20

25

30

35

40

45

No stress Stress

Body

wei

ght c

hang

es (

)

ControlHoney

(b)

Figure 1 Effects of stress and honey treatment on (a) body weight and (b) percentage of body weight changes The values are expressed asmeanplusmn SEM

the significance of the differences between the means of thetest and control studies was established by one-way analysisof variance (ANOVA) coupled with post hoc Tukey HSD test

3 Results

31 Effects of Tualang Honey on Body Weight Mean bodyweights of all groups over five weeks of experimental periodwere illustrated in Figure 1(a) Percentage of body weightchanges was calculated as [(final body weight minus initial bodyweight)initial body weight] times 100There was no significantdifference in the percentage of body weight changes betweenall groups (Figure 1(b))

32 Effects of Tualang Honey on Serum Corticosterone andACTH Levels A significant effect of stress (119865

122= 824

119875 lt 001) was observed on the serum ACTH level whereassignificant effects of honey treatment were observed on theserum corticosterone (119865

120= 393 119875 lt 005) and ACTH

(119865122

= 1182 119875 lt 001) levels (Table 1) There was nosignificant stress-treatment interaction (119865

319= 201 119875 gt

005) in corticosterone level while significant stress-treatmentinteraction (119865

321= 692 119875 lt 001) in ACTH level was

observed Stress exposure significantly (119875 lt 005) increases

ACTH level while honey treatment significantly (119875 lt 005)decreases corticosterone andACTH levels in the stressed rats

33 Effects of Tualang Honey on Memory Performance Two-way ANOVA revealed significant effects of stress on short-term memory (119865

127= 1606 119875 lt 0001) and long-term

memory (119865128

= 365 119875 lt 005) indicating that stresswas associated with memory deficit (Figures 2(a) and 2(b))Interestingly there were significant effects of honey treatmenton short-term memory (119865

127= 5559 119875 lt 0001) and

long-term memory (119865128

= 4729 119875 lt 0001) Stressed ratssupplemented with honey showed significantly (119875 lt 0001)higher mean discrimination index in both short- and long-term memory compared to stressed control rats indicatingbetter memory performance There were significant stress-treatment interactions in short-term memory (119865

326= 3333

119875 lt 0001) and long-term memory (119865327

= 1940 119875 lt 0001)

34 Effects of Tualang Honey on Brain Oxidative StatusThe effects of Tualang honey administration on brain tissuedamage index were evaluated as the content of MDA andPCO and the antioxidative defence systems such as SODGPx GR CAT and total antioxidant capacity (Table 2) Two-way ANOVA revealed significant effects of stress on the levelsof MDA (119865

127= 1252 119875 lt 001) PCO (119865

113= 1066


Table 2 Effects of stress and honey treatment on brain oxidative status

No stress No stress + honey Stress Stress + honeyMDA (120583Mmg protein) 703 plusmn 044 731 plusmn 032 876 plusmn 027curren 788 plusmn 020sect

PCO (nmolmg protein) 237 plusmn 040 315 plusmn 066 517 plusmn 036curren 373 plusmn 022sect

SOD activity (Umg protein) 968 plusmn 187 1239 plusmn 112lowast 483 plusmn 073curren 987 plusmn 068lowastsectsect

GPx activity (mUmg protein) 5271 plusmn 874 5238 plusmn 953 4291 plusmn 715 4384 plusmn 917GR activity (mUmg protein) 8093 plusmn 1229 7582 plusmn 555 5761 plusmn 675 7277 plusmn 663CAT activity (mUmg protein) 466080 plusmn 15659 474884 plusmn 24692 424081 plusmn 22686 414937 plusmn 11638Total antioxidant capacity (120583M Trolox equivalentsmgprotein) 005 plusmn 000 005 plusmn 000 004 plusmn 000 004 plusmn 000

The values are expressed as meanplusmn SEM Significant main effects of loud noise stress (119875 lt 005) Significant main effects of honey treatment (lowast119875 lt 005)Significant difference between no stress and stress control (curren119875 lt 005) Significant difference between stress control and stress treated with honey (sect119875 lt 005sectsect119875 lt 001)

000

010

020

030

040

050

060

070

No stress Stress

Short-term memory discrimination index

sectsect

currencurrenDisc

rimin

atio

n in

dex

ControlHoney

lowastlowast

lowastlowast

(a)

000

010

020

030

040

050

060

070

No stress Stress

Disc

rimin

atio

n in

dex

Long-term memory discrimination index

curren

ControlHoney

lowastlowast sectsectlowastlowast

(b)

Figure 2 Effects of stress and honey treatment on mean discrimination index ratio of (a) short-term memory and (b) long-term memoryThe values are expressed as meanplusmn SEM Significant main effects of loud noise stress (119875 lt 005 119875 lt 001) Significant main effects of honeytreatment (lowast119875 lt 005 lowastlowast119875 lt 001) Significant difference between no stress and stress control (curren119875 lt 005 currencurren119875 lt 001) Significant differencebetween stress control and stress treated with honey (sect119875 lt 005 sectsect119875 lt 001)

119875 lt 001) SOD (119865134

= 960 119875 lt 001) CAT (119865134

= 631119875 lt 005) and total antioxidant capacity (119865

129= 803 119875 lt

001) Stress exposure increases oxidative stress as evidentby elevated levels of MDA and PCO while the activity ofantioxidant enzymes as well as the total antioxidant capacitywas reduced There was significant effect of honey treatmenton the activity of SOD (119865

134= 1022 119875 lt 001) There were

significant stress-treatment interactions in the levels of MDA(119865326

= 576 119875 lt 001) PCO (119865312

= 640 119875 lt 001) SOD(119865337

= 750 119875 lt 001) and total antioxidant capacity (119865328

=323 119875 lt 005) One-way ANOVA confirms that stressedrats treated with honey possessed significantly lower MDA(119875 lt 005) and PCO (119875 lt 005) levels and significantly higher(119875 lt 0001) SOD activity compared to stressed control rats

35 Effects of Tualang Honey on Brain AChE Activity Aninhibitory effect of honey treatment was observed on theAChE activity (119865

120= 393 119875 lt 005) (Figure 3) No

significant effect of stress and stress-treatment interactionwas observed

36 Effects of Tualang Honey on BDNF Concentration Asignificant effect of stress (119865

122= 824 119875 lt 001) and

honey treatment (119865122

= 1182 119875 lt 001) was observed onthe BDNF concentration (Figure 4) Significant interactionbetween stress and treatment (119865

321= 692 119875 lt 001) was

evident Stress exposure significantly (119875 lt 005) decreases


Table 3 Effects of stress and honey treatment on the number of Nissl-positive cells in mPFC and hippocampal regions

No stress No stress + honey Stress Stress + honeymPFC004mm2 1443 plusmn 075 2007 plusmn 188lowast 1413 plusmn 076 1863 plusmn 045lowastsect

CA1 region001mm2 2444 plusmn 106 3111 plusmn 478 2722 plusmn 113 2778 plusmn 213CA2 region001mm2 2356 plusmn 247 3389 plusmn 464lowastlowast 1933 plusmn 260curren 2489 plusmn 413lowastlowastsect

CA3 region001mm2 1022 plusmn 128 1722 plusmn 164lowast 1456 plusmn 080 1556 plusmn 144lowast

DG region001mm2 3456 plusmn 380 3822 plusmn 361lowast 3322 plusmn 215 3911 plusmn 221lowast

The values are expressed as meanplusmn SEM Significant main effects of loud noise stress (119875 lt 005) Significant main effects of honey treatment (lowast119875 lt 005lowastlowast

119875 lt 001) Significant difference between no stress and stress control (curren119875 lt 005) Significant difference between stress control and stress treated with honey(sect119875 lt 005)

0

50

100

150

200

250

No stress Stress

(UL

)

ControlHoney

lowastlowast

Figure 3 Effects of stress and honey treatment on acetyl-cholinesterase activity The values are expressed as meanplusmn SEMSignificant main effects of honey treatment (lowast119875 lt 005)

BDNF concentration whereas honey treatment significantly(119875 lt 005) increases BDNF concentration in the stressed rats

37 Effects of Tualang Honey on the Number of Nissl-PositiveCells in mPFC and Hippocampal Regions Two-way ANOVArevealed significant effect of stress on the number of Nissl-positive cells in CA2 hippocampal region (119865

19= 2014 119875 lt

001) Significant effects of honey treatment were observed onthe number of Nissl-positive cells in mPFC (119865

19= 2294 119875 lt

001) CA2 (11986519

= 2908 119875 lt 0001) CA3 (11986519

= 672 119875 lt005) and DG hippocampal regions (119865

19= 693 119875 lt 005)

(Table 3) There were significant stress-honey interactions onthe number of Nissl-positive cells in mPFC (119865

38= 732 119875 lt

005) and CA2 hippocampal region (11986538

= 2169 119875 lt 0001)One-way ANOVA shows that stressed control rats possessedsignificantly (119875 lt 005) lower number of Nissl-positive cellsin the CA2 hippocampal region than the nonstressed controlStressed rats treated with Tualang honey have a significantly(119875 lt 005) higher number of Nissl-positive cells in the mPFCand CA2 hippocampal region compared to stressed controlrats

0

50

100

150

200

250

300

350

400

450

No stress Stress

(pg

mL)

ControlHoney

sectlowastlowast

curren

Figure 4 Effects of stress and honey treatment on BDNF concen-tration The values are expressed as meanplusmn SEM Significant maineffects of loud noise stress (119875 lt 005) Significant main effects ofhoney treatment (lowast119875 lt 005) Significant difference between nostress and stress control (curren119875 lt 005) Significant difference betweenstress control and stress treated with honey (sect119875 lt 005)

38 Effects of Tualang Honey on Arrangement of Nissl-PositiveCells in mPFC and Hippocampal Regions Stressed controlgroup exhibited moderately shrunken neuronal cell bodieswith cytoplasmic vacuolation (Figures 5(c) and 6(c)) Thearrangement of pyramidal neurons was sparse and the Nisslsubstance was decreasing or dissolving In contrast stressedgroup treated with honey exhibited abundant pyramidalneurons the architecture of these neuronswas preserved andNissl substances in the cytoplasmwere clearly visible (Figures5(d) and 6(d)) Arrangement of pyramidal neurons of thestress group treated with honey appeared quite similar to thenonstressed group

4 Discussion

In the present study no significant differences in the changeof bodyweight between groupswere observed suggesting that


100120583m

(a)

100120583m

(b)

100120583m

(c)

100120583m

(d)

Figure 5 Arrangement of mPFC pyramidal neurons among groups (a) no stress (b) no stress + honey (c) stress and (d) stress + honeyThearrows indicate the cells of interest (Nissl staining times200 scale bar 100 120583m)

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 6 Arrangement of hippocampal CA2 pyramidal neurons among groups (a) no stress (b) no stress + honey (c) stress and (d) stress+ honey The arrows indicate the cells of interest (Nissl staining times400 scale bar 50 120583m)


stress and honey treatment did not cause substantial effect onthe bodyweight In addition we demonstrated that agedmalerats exposed to noise stress exhibited higher corticosteroneand ACTH levels than the nonstressed rats Accumulatingevidence suggests that the hypothalamic-pituitary-adrenal(HPA) axis is altered by ageing [32 33] The pituitaryhypothalamus and hippocampus express glucocorticoidreceptors and may all be putative sites for glucocorticoid-mediated negative feedback on the pituitary-adrenal axis[34] Various studies demonstrated that aged rats exhibit aloss of glucocorticoid receptors in the hippocampus and thusare impaired in their ability to terminate a stress response[35ndash37] resulting in accumulation of stress hormones follow-ing stress exposure

Loss of glucocorticoid receptors in the hippocampuswould obviate glucocorticoid-mediated negative feedbackinhibition of corticotropin-releasing factor (CRF) expressionand secretion resulting in increased HPA activity a furtherdownregulation of glucocorticoid receptors and potentiallya loss of hippocampal neurons [36] Correspondingly wedemonstrated that the stressed rats possessed lower numberof pyramidal neurons in the hippocampus as well as in themPFC compared to the nonstressed rats Similarly Manikan-dan et al [14] reported significant decreases in the dendriticcount in the CA1 and CA3 regions of rat hippocampus afternoise stress exposure 100 dB(A)4 h per day for 30 dayswhile Sharifabad and Sabahi [20] reported that chronic noiseexposure 40 dB(A)1 h per day for 90 days reduces thevolume of CA3 and DG hippocampal subregions

Along with that the stressed rats demonstrated signifi-cantly lower BDNF concentration than the nonstressed ratsPrevious studies reported that stress exposure may causereduction of BDNF expression in the hippocampus andparaventricular nucleus (PVN) [38ndash41] The reduction ofhippocampal BDNF expression induced by stress may alsocontribute to the hippocampal neuronal loss [42] as observedin this studyThe reduced BDNF concentration and neuronalloss in the mPFC and hippocampus may have manifestedtheir effects as memory impairment In the present study wedemonstrated that agedmale rats exposed to noise stress wereimpaired on both short- and long-term memory as assessedin the novel object recognition test It is widely acceptedthat noise exposure is a stressful environmental stimulus andhas been previously shown to impair cognition such as theacquisition of memory consolidation and recall [43] Theobserved adverse effects of noise exposure on recognitionmemory are in agreement with previous studies [14 44]

Interestingly our data showed that supplementation ofTualang honeywas able to improve both short- and long-termmemory in the stressed rats The histopathological resultsrevealed an increase in the number of neuronal cells inmPFCCA2 CA3 and DG hippocampal regions in the groupstreated with Tualang honey which could be interpreted astissue preservation and maintenance of the ability to retaininformation Earlier studies by our research team revealedthat Tualang honey supplementation was able to improvememory performance in young rats exposed to noise stress[45] as well as in ovariectomized rats exposed to socialinstability stress [28] It is suggested that Tualang honey a

phytoestrogen mediated its neuroprotective effects throughits antioxidant properties and via upregulation of BDNFexpression [46] and augmentation of choline acetyltrans-ferase and acetylcholinesterase activities in specific brainareas [47] Honey also modulates ACTH and corticosteronelevels following noise stress either by suppressing HPAmobi-lization in response to stress or by facilitating elevated plasmacorticosterone and ACTH levels back to baseline followingthe termination of the stress Future studies should investigatethese possible antistress properties of honey and alsomeasureglucocorticoid receptors following honey supplementation

In conjunction with that we demonstrated that theaged stressed rats treated with Tualang honey exhibitedsignificantly higher BDNF concentration than the untreatedstressed rats Numerous reports have also demonstratedthat NGF administration improved learning and memoryimpairments in aged rats coupled with a partial reversal ofcholinergic neuron atrophy [10 12] Flavonoids have beendemonstrated to enhance glutamate signaling which leadsto continuous activation of cell survival signaling pathwayssuch as PKC and PI3K in the brain cells triggers gene andprotein expression which most likely is CREB-dependentand then causes a more stable long-term potential [48ndash50]Since BNDF is regulated by CREB it is believed that theflavonoids contents in the honey may have mimicked theabove mechanism thus exhibiting its neuroprotective effect

Our finding on AChE activity following noise stresswas in contrast with earlier report by Manikandan et al[14] which showed significant increase in AChE activity inanimals exposed to noise stress This discrepancy might becontributed to the difference in age of rats used Howeverour result revealed that Tualang honey supplementationwas able to decrease AChE activity in aged rats with orwithout noise stress exposure Decrease in AChE activitymight lead to an increase in acetylcholine in the synapticcleft and a consequent increase in the cholinergic activitywhich in turn leads to memory improvement Numerousother studies have reported the AChE inhibitory effects ofplant extracts for instance Morinda citrifolia L (Noni fruit)[51] Rosmarinus officinalis L (Rosemary leaf) [52] AstragaliRadix and Salviae Miltiorrhizae Radix [53] where all ofthose extracts exhibited memory enhancing abilities Thus itis suggested that the memory enhancing ability of Tualanghoney is partly due to the inhibition of brain AChE activity

Studies have shown that noise exposure causes anincrease in reactive oxygen species (ROS) resulting in oxida-tive stress The negative effects of noise on cell structure andfunction could be at least in part mediated by this increasein ROS [54] For example in an investigation of the effectsof moderate-intensity white noise exposure on learning andmemory of mice the results showed evidence of oxidativedamage in the auditory cortex and hippocampus [55] Inyet another study Manikandan et al [56] found increasedMDA levels in different areas of the brain after 30 days of100 dB white noise exposure Our results are in accordancewith these findings where exposure to noise was shown toinduce oxidative stress as evident by elevated levels of MDAand PCO in the brain of the stressed rats HoweverManikan-dan et al [14 56] reported contradictory results especially


on the SOD level which could possibly be due to betteroxidative stress compensation in adults rats compared to agedrats

In order to protect cells from oxidative damage aerobicmetabolism generally depends on a stringent control ofROS by antioxidants Several antioxidant enzymes withinthe framework of the antioxidant defence system in ourbody are SOD GPx GR and CAT In our study it wasobserved that the levels of SOD CAT and total antioxidantcapacity of the stressed rats were significantly lower thanthe nonstressed rats The results we obtained are similar tothose of other studies [57 58] The decrease in antioxidantenzymes activities might be explained by the consumption ofantioxidant enzymes developed by the increase in MDA andPCO preceded by stress exposure

Phytochemical screening of honey reveals that it containsflavonoids phenolic acids ascorbic acid 120572-tocopherol andcarotenoids [24 59 60] It is widely known that thesecompounds possessed antioxidant activities It was previouslyreported that the total phenolic content of Tualang honeywas 2517 plusmn 79mg gallic acidkg honey and total antioxidantactivity was 3221 plusmn 97 (120583M Fe(II)) whereas the antiradicalactivity was 4130 plusmn 078 ( inhibition) [61] Correspond-ingly in the current study it was observed that Tualang honeysignificantly improved brain oxidative status as shown byelevated antioxidant enzymes activities and reduced oxida-tive markers Other studies have also demonstrated thathoney is able to increase antioxidant enzymes activities andameliorate oxidative stress in plasma and other tissues suchas kidney and pancreas [62ndash66] Thus it is assumed thatthe improvement of memory performance in the stressedrats by Tualang honey is also partly due to its antioxidantcapacity which is attributed to the aforementioned antioxi-dant compounds This is in keeping with studies which havedemonstrated that dietary antioxidants improved cognitiveperformance in clinical studies [67 68] as well as in animals[69ndash71]

5 Conclusion

In conclusion the memory enhancing effects of Tualanghoney could act via enhancement of cholinergic system andmPFC and hippocampal morphology possibly secondaryto reduction in brain oxidative stress andor upregulationof BDNF concentration The presence of several bioactivecompounds in honey particularly flavonoids and phenolicacids might be responsible for these effects Further studieshave to be conducted to evaluate honey biological activity aswell as its toxicity in order to support its potential use as analternative therapy to protect against memory deteriorationdue to stress exposure andor ageing in both males andfemales

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This project was supported by short-term grant of UniversitiSains Malaysia (304PPSP61313056) The financial supportsfrom School of Medical Sciences USM and Ministry ofEducation Malaysia for RLKA fellowship and SLAB schol-arship to Ms Khairunnuur Fairuz Azman are gratefullyacknowledged

References

[1] E S Rosenzweig and C A Barnes ldquoImpact of aging onhippocampal function plasticity network dynamics and cog-nitionrdquo Progress in Neurobiology vol 69 no 3 pp 143ndash1792003

[2] D M Armstrong R Sheffield G Buzsaki et al ldquoMorphologicalterations of choline acetyltransferase-positive neurons in thebasal forebrain of aged behaviorally characterized fisher 344ratsrdquo Neurobiology of Aging vol 14 no 5 pp 457ndash470 1993

[3] R T Bartus R L Dean III B Beer andA S Lippa ldquoThe cholin-ergic hypothesis of geriatric memory dysfunctionrdquo Science vol217 no 4558 pp 408ndash417 1982

[4] R Quirion A Wilson W Rowe et al ldquoFacilitation of acetyl-choline release and cognitive performance by anM

2

-muscarinicreceptor antagonist in agedmemory-impaired ratsrdquoThe Journalof Neuroscience vol 15 no 2 pp 1455ndash1462 1995

[5] A-C Granholm ldquoOestrogen and nerve growth factor-neuroprotection and repair in Alzheimerrsquos diseaserdquo ExpertOpinion on Investigational Drugs vol 9 no 4 pp 685ndash6942000

[6] R Levi-Montalcini ldquoThe nerve growth factor thirty-five yearslaterrdquo Bioscience Reports vol 7 no 9 pp 681ndash699 1987

[7] N J Woolf ldquoCholinergic systems in mammalian brain andspinal cordrdquo Progress in Neurobiology vol 37 no 6 pp 475ndash5241991

[8] C E Dixon P Flinn B Juliang R Venya and R L HayesldquoNerve growth factor attenuates cholinergic deficits followingtraumatic brain injury in ratsrdquo Experimental Neurology vol 146no 2 pp 479ndash490 1997

[9] C RAlmli T J Levy BHHanA R Shah JMGidday andDM Holtzman ldquoBDNF protects against spatial memory deficitsfollowing neonatal hypoxia-ischemiardquo Experimental Neurologyvol 166 no 1 pp 99ndash114 2000

[10] C Backman G M Rose B J Hoffer et al ldquoSystemic admin-istration of a nerve growth factor conjugate reverses age-related cognitive dysfunction and prevents cholinergic neuronatrophyrdquo The Journal of Neuroscience vol 16 no 17 pp 5437ndash5442 1996

[11] W Fischer A Bjorklund K Chen and F H Gage ldquoNGFimproves spatial memory in aged rodents as a function of agerdquoThe Journal of Neuroscience vol 11 no 7 pp 1889ndash1906 1991

[12] M C Gustilo A LMarkowska S J Breckler C A FleischmanD L Price and V E Koliatsos ldquoEvidence that nerve growthfactor influences recent memory through structural changes inseptohippocampal cholinergic neuronsrdquo Journal of ComparativeNeurology vol 405 no 4 pp 491ndash507 1999

[13] R Ravindran R S Devi J Samson and M SenthilvelanldquoNoise-stress-induced brain neurotransmitter changes and theeffect of Ocimum sanctum (Linn) treatment in albino ratsrdquoJournal of Pharmacological Sciences vol 98 no 4 pp 354ndash3602005


[14] S Manikandan M K Padma R Srikumar N JeyaParthasarathy A Muthuvel and R Sheela Devi ldquoEffectsof chronic noise stress on spatial memory of rats in relation toneuronal dendritic alteration and free radical-imbalance inhippocampus and medial prefrontal cortexrdquo NeuroscienceLetters vol 399 no 1-2 pp 17ndash22 2006

[15] M A Lovell andW R Markesbery ldquoOxidative DNA damage inmild cognitive impairment and late-stage Alzheimerrsquos diseaserdquoNucleic Acids Research vol 35 no 22 pp 7497ndash7504 2007

[16] J H Jhoo H-C Kim T Nabeshima et al ldquo120573-Amyloid (1ndash42)-induced learning and memory deficits in mice involvementof oxidative burdens in the hippocampus and cerebral cortexrdquoBehavioural Brain Research vol 155 no 2 pp 185ndash196 2004

[17] A Wu Z Ying and F Gomez-Pinilla ldquoDietary omega-3 fattyacids normalize BDNF levels reduce oxidative damage andcounteract learning disability after traumatic brain injury inratsrdquo Journal of Neurotrauma vol 21 no 10 pp 1457ndash1467 2004

[18] K Fukui K Onodera T Shinkai S Suzuki and S UranoldquoImpairment of learning and memory in rats caused by oxida-tive stress and aging and changes in antioxidative defensesystemsrdquo Annals of the New York Academy of Sciences vol 928pp 168ndash175 2001

[19] C Pieta Dias M N Martins de Lima J Presti-Torres et alldquoMemantine reduces oxidative damage and enhances long-termrecognition memory in aged ratsrdquo Neuroscience vol 146 no 4pp 1719ndash1725 2007

[20] M H Sharifabad and A Sabahi ldquoExposure to chronic noisereduces the volume of hippocampal subregions in ratsrdquo IranianJournal of Basic Medical Sciences vol 11 no 1 p 37 2008

[21] AMAljadi andMY Kamaruddin ldquoEvaluation of the phenoliccontents and antioxidant capacities of two Malaysian floralhoneysrdquo Food Chemistry vol 85 no 4 pp 513ndash518 2004

[22] M Al-Mamary A Al-Meeri and M Al-Habori ldquoAntioxidantactivities and total phenolics of different types of honeyrdquoNutrition Research vol 22 no 9 pp 1041ndash1047 2002

[23] G Beretta M Orioli and R M Facino ldquoAntioxidant andradical scavenging activity of honey in endothelial cell cultures(EAhy926)rdquo Planta Medica vol 73 no 11 pp 1182ndash1189 2007

[24] N Gheldof and N J Engeseth ldquoAntioxidant capacity of honeysfrom various floral sources based on the determination ofoxygen radical absorbance capacity and inhibition of in vitrolipoprotein oxidation in human serum samplesrdquo Journal ofAgricultural and Food Chemistry vol 50 no 10 pp 3050ndash30552002

[25] R K Kishore A S Halim M S N Syazana and K N SSirajudeen ldquoTualang honey has higher phenolic content andgreater radical scavenging activity compared with other honeysourcesrdquo Nutrition Research vol 31 no 4 pp 322ndash325 2011

[26] F A Al-Himyari ldquoThe use of honey as a natural preventivetherapy of cognitive decline and dementia in the middle eastrdquoAlzheimerrsquos amp Dementia vol 5 no 4 p P247 2009

[27] L M Chepulis N J Starkey J R Waas and P C MolanldquoThe effects of long-term honey sucrose or sugar-free diets onmemory and anxiety in ratsrdquo Physiology amp Behavior vol 97 no3-4 pp 359ndash368 2009

[28] B Al-Rahbi R Zakaria Z Othman A Hassan Z IMohd Ismail and S Muthuraju ldquoTualang honey supplementimproves memory performance and hippocampal morphologyin stressed ovariectomized ratsrdquoActa Histochemica vol 116 no1 pp 79ndash88 2014

[29] Z Othman N Shafin R Zakaria N H N Hussain andW MZ W Mohammad ldquoImprovement in immediate memory after

16 weeks of tualang honey (Agro Mas) supplement in healthypostmenopausal womenrdquo Menopause vol 18 no 11 pp 1219ndash1224 2011

[30] S S M Zaid S A Sulaiman K N M Sirajudeen and N HOthman ldquoThe effects of tualang honey on female reproductiveorgans tibia bone and hormonal profile in ovariectomisedratsmdashanimal model for menopauserdquo BMC Complementary andAlternative Medicine vol 10 article 82 2010

[31] A Ennaceur and J Delacour ldquoA new one-trial test for neu-robiological studies of memory in rats 1 behavioral datardquoBehavioural Brain Research vol 31 no 1 pp 47ndash59 1988

[32] M J Meaney D OrsquoDonnell W Rowe et al ldquoIndividualdifferences in hypothalamic-pituitary-adrenal activity in laterlife and hippocampal agingrdquo Experimental Gerontology vol 30no 3-4 pp 229ndash251 1995

[33] P S Wang M-J Lo and M-M Kau ldquoGlucocorticoids andagingrdquo Journal of the FormosanMedical Association vol 96 no10 pp 792ndash801 1997

[34] M M Wilson S E Greer M A Greer and L RobertsldquoHippocampal inhibition of pituitary-adrenocortical functionin female ratsrdquo Brain Research vol 197 no 2 pp 433ndash441 1980

[35] RM Sapolsky L C Krey and B SMcEwen ldquoThe adrenocorti-col stress-response in the agedmale rat impairment of recoveryfrom stressrdquo Experimental Gerontology vol 18 no 1 pp 55ndash641983

[36] R M Sapolsky L C Krey and B S McEwen ldquoProlongedglucocorticoid exposure reduces hippocampal neuron numberimplications for agingrdquo The Journal of Neuroscience vol 5 no5 pp 1222ndash1227 1985

[37] R M Sapolsky L C Krey and B S McEwen ldquoThe neuroen-docrinology of stress and aging the glucocorticoid cascadehypothesisrdquo Science of Aging Knowledge Environment vol 2002no 38 p 21 2002

[38] P A Adlard and C W Cotman ldquoVoluntary exercise protectsagainst stress-induced decreases in brain-derived neurotrophicfactor protein expressionrdquoNeuroscience vol 124 no 4 pp 985ndash992 2004

[39] S Murakami H Imbe Y Morikawa C Kubo and E SenbaldquoChronic stress as well as acute stress reduces BDNF mRNAexpression in the rat hippocampus but less robustlyrdquo Neuro-science Research vol 53 no 2 pp 129ndash139 2005

[40] M Nibuya M Takahashi D S Russell and R S DumanldquoRepeated stress increases catalytic TrkB mRNA in rat hip-pocampusrdquoNeuroscience Letters vol 267 no 2 pp 81ndash84 1999

[41] M A Smith S Makino R Kvetnansky and R M Post ldquoEffectsof stress on neurotrophic factor expression in the rat brainrdquoAnnals of the New York Academy of Sciences vol 771 no 1 pp234ndash239 1995

[42] Y Watanabe E Gould and B S McEwen ldquoStress inducesatrophy of apical dendrites of hippocampal CA3 pyramidalneuronsrdquo Brain Research vol 588 no 2 pp 341ndash345 1992

[43] C Chengzhi T Yan J Xuejun L Xiang Q Youbin and TBaijie ldquoRecovery of chronic noise exposure induced spatiallearning and memory deficits in young male Sprague-Dawleyratsrdquo Journal of Occupational Health vol 53 no 3 pp 157ndash1632011

[44] S Haider F Naqvi Z Batool et al ldquoDecreased hippocampal5-HT and DA levels following sub-chronic exposure to noisestress impairment in both spatial and recognition memory inmale ratsrdquo Scientia Pharmaceutica vol 80 no 4 pp 1001ndash10112012


[45] K F Azman R Zakaria C B Abd Aziz Z Othman and BAl-Rahbi ldquoTualang honey improves memory performance anddecreases depressive-like behavior in rats exposed to loud noisestressrdquo Noise and Health vol 17 no 75 pp 83ndash89 2015

[46] B Al-Rahbi R Zakaria Z Othman A Hassan and A HAhmad ldquoEnhancement of BDNF concentration and restora-tion of the hypothalamic-pituitary-adrenal axis accompanyreduced depressive-like behaviour in stressed ovariectomisedrats treated with either Tualang honey or estrogenrdquo The Scien-tific World Journal vol 2014 Article ID 310821 8 pages 2014

[47] B Al-Rahbi R Zakaria Z Othman A Hassan and AH Ahmad ldquoThe effects of tualang honey supplement onmedial prefrontal cortexmorphology and cholinergic system instressed ovariectomised ratsrdquo International Journal of AppliedResearch in Natural Products vol 7 no 3 pp 28ndash36 2014

[48] Z Fu W Zhang W Zhen et al ldquoGenistein induces pancreatic120573-cell proliferation through activation of multiple signalingpathways and prevents insulin-deficient diabetes in micerdquoEndocrinology vol 151 no 7 pp 3026ndash3037 2010

[49] S-L Hwang and G-C Yen ldquoModulation of Akt JNK and p38activation is involved in citrus flavonoid-mediated cytoprotec-tion of PC12 cells challenged by hydrogen peroxiderdquo Journal ofAgricultural and Food Chemistry vol 57 no 6 pp 2576ndash25822009

[50] S Rainey-Smith L-W Schroetke P Bahia et al ldquoNeuropro-tective effects of hesperetin in mouse primary neurones areindependent of CREB activationrdquoNeuroscience Letters vol 438no 1 pp 29ndash33 2008

[51] S D Pachauri P R P Verma A K Dwivedi et al ldquoAmeliorativeeffect of Noni fruit extract on streptozotocin-induced memoryimpairment in micerdquo Behavioural Pharmacology vol 24 no 4pp 307ndash319 2013

[52] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[53] J-S Lee H-G Kim J-M Han et al ldquoEthanol extract ofAstragali Radix and Salviae Miltiorrhizae Radix Myelophilexerts anti-amnesic effect in a mouse model of scopolamine-induced memory deficitsrdquo Journal of Ethnopharmacology vol153 no 3 pp 782ndash792 2014

[54] P Lenzi G Frenzili M Gesi et al ldquoDNA damage associatedwith ultrastructural alterations in rat myocardium after loudnoise exposurerdquo Environmental Health Perspectives vol 111 no4 pp 467ndash471 2003

[55] L Cheng S-H Wang Q-C Chen and X-M Liao ldquoModeratenoise induced cognition impairment of mice and its underlyingmechanismsrdquo Physiology and Behavior vol 104 no 5 pp 981ndash988 2011

[56] S Manikandan R Srikumar N Jeya Parthasarathy and RSheela Devi ldquoProtective effect of Acorus calamus LINN on freeradical scavengers and lipid peroxidation in discrete regions ofbrain against noise stress exposed ratrdquo Biological and Pharma-ceutical Bulletin vol 28 no 12 pp 2327ndash2330 2005

[57] A Ilhan A Gurel F Armutcu et al ldquoGinkgo biloba preventsmobile phone-induced oxidative stress in rat brainrdquo ClinicaChimica Acta vol 340 no 1-2 pp 153ndash162 2004

[58] R Srikumar N J Parthasarathy S Manikandan G SNarayanan and R Sheeladevi ldquoEffect of Triphala on oxidativestress and on cell-mediated immune response against noisestress in ratsrdquoMolecular and Cellular Biochemistry vol 283 no1-2 pp 67ndash74 2006

[59] M L Al D Daniel A Moise O Bobis L Laslo and S Bog-danov ldquoPhysico-chemical and bioactive properties of differentfloral origin honeys from Romaniardquo Food Chemistry vol 112no 4 pp 863ndash867 2009

[60] I C F R Ferreira E Aires J C M Barreira and L MEstevinho ldquoAntioxidant activity of Portuguese honey sam-ples different contributions of the entire honey and phenolicextractrdquo Food Chemistry vol 114 no 4 pp 1438ndash1443 2009

[61] M Mohamed K N S Sirajudeen M Swamy N S Yaacoband S A Sulaiman ldquoStudies on the antioxidant propertiesof tualang honey of Malaysiardquo African Journal of TraditionalComplementary and Alternative Medicines vol 7 no 1 pp 59ndash63 2010

[62] N S Al-Waili ldquoEffects of daily consumption of honey solutionon hematological indices and blood levels of minerals andenzymes in normal individualsrdquo Journal of Medicinal Food vol6 no 2 pp 135ndash140 2003

[63] O O Erejuwa S A SulaimanM SWahab K N S SirajudeenM S Salleh and S Gurtu ldquoAntioxidant protection ofMalaysiantualang honey in pancreas of normal and streptozotocin-induced diabetic ratsrdquo Annales drsquoEndocrinologie vol 71 no 4pp 291ndash296 2010

[64] O O Erejuwa S A SulaimanM SWahab K N S SirajudeenM S Salleh and S Gurtu ldquoComparison of antioxidant effectsof honey glibenclamide metformin and their combinations inthe kidneys of streptozotocin-induced diabetic ratsrdquo Interna-tional Journal of Molecular Sciences vol 12 no 1 pp 829ndash8432011

[65] N Gheldof X-HWang and N J Engeseth ldquoBuckwheat honeyincreases serum antioxidant capacity in humansrdquo Journal ofAgricultural and Food Chemistry vol 51 no 5 pp 1500ndash15052003

[66] E Perez A J Rodrıguez-Malaver and P Vit ldquoAntioxidantcapacity of Venezuelan honey in wistar rat homogenatesrdquoJournal of Medicinal Food vol 9 no 4 pp 510ndash516 2006

[67] H J Wengreen R G Munger C D Corcoran et al ldquoAntioxi-dant intake and cognitive function of elderly men and womenthe CacheCounty studyrdquo Journal of Nutrition Health andAgingvol 11 no 3 pp 230ndash237 2007

[68] J W Jama L J Launer J C M Witteman et al ldquoDietaryantioxidants and cognitive function in a population-based sam-ple of older persons the Rotterdam studyrdquo American Journal ofEpidemiology vol 144 no 3 pp 275ndash280 1996

[69] R V Abreu E M Silva-Oliveira M F D Moraes G S Pereiraand T Moraes-Santos ldquoChronic coffee and caffeine ingestioneffects on the cognitive function and antioxidant system of ratbrainsrdquo Pharmacology Biochemistry and Behavior vol 99 no 4pp 659ndash664 2011

[70] C W Cotman E Head B A Muggenburg S Zicker andN W Milgram ldquoBrain aging in the canine a diet enrichedin antioxidants reduces cognitive dysfunctionrdquo Neurobiology ofAging vol 23 no 5 pp 809ndash818 2002

[71] M Fahnestock M Marchese E Head et al ldquoBDNF increaseswith behavioral enrichment and an antioxidant diet in the ageddogrdquo Neurobiology of Aging vol 33 no 3 pp 546ndash554 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


shown that noise induces hippocampal dependent memorydeficits and volumetric reduction in all layers of hippocampalfield [15ndash17 20]

Malaysia Tualang honey is a wild pure multifloral honeyproduced by Asian rock bee species (Apis dorsata) whichbuilds hives on the branches of Tualang tree (Koompas-sia excelsa) located mainly in the rainforest of northernPeninsular Malaysia Honey contains significant antioxidantactivities as well as choline and acetylcholine which areessential for brain function and as neurotransmitters [21ndash25] Recent studies reported that honey is one of the naturalpreventive therapies of both cognitive decline and dementiaas it possesses antioxidant properties and it enhances thebrainrsquos cholinergic system [26] Correspondingly anotherstudy reported that consumptions of honey may improvespatial memory in middle aged rats compared to those fedsucrose or sugar-free diet [27] In closely related studies it wasdemonstrated that Tualang honey was able to improve mem-ory performance in stressed ovariectomized rats [28] andpostmenopausal women [29] Therefore it is hypothesizedthat Tualang honey could revert oxidative stress by removingthe reactive oxygen species that were produced during thenoise stress and thus prevent oxidative damage in memory-related brain areas particularly in aged rats

2 Material and Methods

21 Experimental Animals A total of 48 male Sprague-Dawley rats (16 months old weighing 600ndash800 g) werepurchased from Sterling Ascent Sdn Bhd (Malaysia)The ratswere maintained in standard polypropylene cages (40 times 25 times16 cm) under a reversed 12-hour lightdark cycle (lights offat 0800 h) at a consistent room temperature of 27 plusmn 1∘Cin the laboratory of Animal Research and Service CentreUniversiti Sains Malaysia The rats received commercial ratchow food pellets (Gold Coin Ltd Malaysia) and waterad libitum Rats were allowed to acclimatize to the hold-ing room for 24 h before the behavioural procedures Theprocedures in this study were approved by Animal EthicsCommittee ofUniversiti SainsMalaysia (USMAnimal EthicsApproval2013(85)(444)) in accordance with the interna-tionally accepted principles for laboratory animal use andcare

22 Honey Supplementation The Tualang honey used wasfrom a single batch honey supplied by Federal AgriculturalMarketing Authorities (FAMA) Malaysia The honey wasfiltered by FAMA to remove solid particles concentrated inan oven at 40∘C and evaporated to achieve a water content ofabout 20 It was then subjected to 120574 irradiation at 25 kGyat Steril Gamma (M) Sdn Bhd (Selangor Malaysia) forsterilization and bottled 230 g per jarThe final concentrationof the bottled Tualang honey was 13 gmL Tualang honeyat 200mgkg body weightday [28 30] was administeredvia oral gavage 14 days prior to stress procedure and thetreatments were continued throughout the 14 days of stressprocedure The Tualang honey was freshly dissolved in 1mLof distilled water prior to administration Control groups

received an identical volume of distilled water as placebo forthe same period of time

23 Experimental Design The animals were randomlyassigned to the following groups (119899 = 12) (i) nonstressedwith placebo (ii) nonstressed with Tualang honey (iii)stressed with placebo and (iv) stressed with Tualanghoney All rats were subjected to novel object recognitiontest for cognitive performance evaluation and killed bydecapitation upon completion of the test Individual bodyweight was recorded weekly using electrical balance Bloodsamples (10mL) were collected immediately All bloodsamples were left to clot for 2 h prior to centrifugation for15min at 4000 rpm Approximately 3mL of serum wascollected and stored at minus20∘C until assay The brain of eachanimal was quickly harvested and weighed The right brainhemisphere was homogenated (10wv) in ice-cold 01Mphosphate-buffered saline at pH 74 The homogenate wasthen centrifuged at 10000timesg for 10 minutes and kept atminus80∘C until being analyzed The left brain hemisphere wasimmediately fixed in 10 formalin for histological study

24 Noise Stress Exposure The animals of the test groups((iii) and (iv)) were exposed to white noise for 4 hours(0900ndash1300 h) daily for 14 days Noise was recorded fromthe generator and amplified by speakers in a separate roomSpeakers were located 30 cm above the cages The noise levelwas set at 100 dB(A) and intensity was measured by a soundlevel meter CENTER 325 (range 80ndash130 dB(A) accuracy+15 dB(A)made inTaiwan) Sound levelswere verified in thecentre of the cage before each exposure and varied by less than1 dB(A) in the space the cage occupiedThe control groups ((i)and (ii)) were kept in the same room for the same period oftime without switching on the noise

25 Novel Object Recognition Test The test was carried outin a separate room that was ventilated soundproofed andmaintained at a constant temperature (27plusmn1∘C)The animalswere brought into the test room 1 hour before the testcommenced to minimize the arousal caused by the trans-ference The test was performed during the active period ofthe animals (dark phase) between the hours of 0900ndash1400All the animals were tested in a random order The trainedobserver remained blind to the treatment group of the ratsuntil scoring was completed

The test employedwas similar to that described elsewhere[31] The test uses the natural preference for novel objectdisplayed by rats and normally used to assess cognitive alter-ations associated with ageing genetic manipulations or drugtreatmentsThe chamberwas an open field apparatus (60times 60times 30 cm) Training sessionswere conducted on two successivedays during which they were allowed to explore the arenafor 10min each day In the training session two identicalsample objects were placed in the field in a symmetricalposition about 10 cm away from the wall After the twosuccessive training sessions testingretention sessions wereconducted The retentions sessions consisted of two sessionsthat is short-termmemory and long-termmemory in which



















450

500

550

600

650

700

0 1 2 3 4 5

Body

wei

ght (

g)

Week



(a)

00

05

10

15

20

25

30

35

40

45

No stress Stress

Body

wei

ght c

hang

es (

)

ControlHoney

(b)



3 Results



122= 824


120= 393 119875 lt 005) and ACTH

(119865122


319= 201 119875 gt






127= 1606 119875 lt 0001) and long-term

memory (119865128


127= 5559 119875 lt 0001) and



326= 3333


= 1940 119875 lt 0001)


127= 1252 119875 lt 001) PCO (119865

113= 1066








000

010

020

030

040

050

060

070

No stress Stress


sectsect

currencurrenDisc

rimin

atio

n in

dex

ControlHoney

lowastlowast

lowastlowast

(a)

000

010

020

030

040

050

060

070

No stress Stress

Disc

rimin

atio

n in

dex


curren

ControlHoney


(b)


119875 lt 001) SOD (119865134

= 960 119875 lt 001) CAT (119865134


129= 803 119875 lt


134= 1022 119875 lt 001) There were


= 576 119875 lt 001) PCO (119865312

= 640 119875 lt 001) SOD(119865337




120= 393 119875 lt 005) (Figure 3) No



122= 824 119875 lt 001) and



321= 692 119875 lt 001) was










0

50

100

150

200

250

No stress Stress

(UL

)

ControlHoney

lowastlowast




19= 2014 119875 lt


19= 2294 119875 lt

001) CA2 (11986519

= 2908 119875 lt 0001) CA3 (11986519


19= 693 119875 lt 005)


38= 732 119875 lt



0

50

100

150

200

250

300

350

400

450

No stress Stress

(pg

mL)

ControlHoney

sectlowastlowast

curren



4 Discussion



100120583m

(a)

100120583m

(b)

100120583m

(c)

100120583m

(d)


50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)















5 Conclusion


Competing Interests


Acknowledgments


References





2













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































450

500

550

600

650

700

0 1 2 3 4 5

Body

wei

ght (

g)

Week



(a)

00

05

10

15

20

25

30

35

40

45

No stress Stress

Body

wei

ght c

hang

es (

)

ControlHoney

(b)



3 Results



122= 824


120= 393 119875 lt 005) and ACTH

(119865122


319= 201 119875 gt






127= 1606 119875 lt 0001) and long-term

memory (119865128


127= 5559 119875 lt 0001) and



326= 3333


= 1940 119875 lt 0001)


127= 1252 119875 lt 001) PCO (119865

113= 1066








000

010

020

030

040

050

060

070

No stress Stress


sectsect

currencurrenDisc

rimin

atio

n in

dex

ControlHoney

lowastlowast

lowastlowast

(a)

000

010

020

030

040

050

060

070

No stress Stress

Disc

rimin

atio

n in

dex


curren

ControlHoney


(b)


119875 lt 001) SOD (119865134

= 960 119875 lt 001) CAT (119865134


129= 803 119875 lt


134= 1022 119875 lt 001) There were


= 576 119875 lt 001) PCO (119865312

= 640 119875 lt 001) SOD(119865337




120= 393 119875 lt 005) (Figure 3) No



122= 824 119875 lt 001) and



321= 692 119875 lt 001) was










0

50

100

150

200

250

No stress Stress

(UL

)

ControlHoney

lowastlowast




19= 2014 119875 lt


19= 2294 119875 lt

001) CA2 (11986519

= 2908 119875 lt 0001) CA3 (11986519


19= 693 119875 lt 005)


38= 732 119875 lt



0

50

100

150

200

250

300

350

400

450

No stress Stress

(pg

mL)

ControlHoney

sectlowastlowast

curren



4 Discussion



100120583m

(a)

100120583m

(b)

100120583m

(c)

100120583m

(d)


50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)















5 Conclusion


Competing Interests


Acknowledgments


References





2













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















450

500

550

600

650

700

0 1 2 3 4 5

Body

wei

ght (

g)

Week



(a)

00

05

10

15

20

25

30

35

40

45

No stress Stress

Body

wei

ght c

hang

es (

)

ControlHoney

(b)



3 Results



122= 824


120= 393 119875 lt 005) and ACTH

(119865122


319= 201 119875 gt






127= 1606 119875 lt 0001) and long-term

memory (119865128


127= 5559 119875 lt 0001) and



326= 3333


= 1940 119875 lt 0001)


127= 1252 119875 lt 001) PCO (119865

113= 1066








000

010

020

030

040

050

060

070

No stress Stress


sectsect

currencurrenDisc

rimin

atio

n in

dex

ControlHoney

lowastlowast

lowastlowast

(a)

000

010

020

030

040

050

060

070

No stress Stress

Disc

rimin

atio

n in

dex


curren

ControlHoney


(b)


119875 lt 001) SOD (119865134

= 960 119875 lt 001) CAT (119865134


129= 803 119875 lt


134= 1022 119875 lt 001) There were


= 576 119875 lt 001) PCO (119865312

= 640 119875 lt 001) SOD(119865337




120= 393 119875 lt 005) (Figure 3) No



122= 824 119875 lt 001) and



321= 692 119875 lt 001) was










0

50

100

150

200

250

No stress Stress

(UL

)

ControlHoney

lowastlowast




19= 2014 119875 lt


19= 2294 119875 lt

001) CA2 (11986519

= 2908 119875 lt 0001) CA3 (11986519


19= 693 119875 lt 005)


38= 732 119875 lt



0

50

100

150

200

250

300

350

400

450

No stress Stress

(pg

mL)

ControlHoney

sectlowastlowast

curren



4 Discussion



100120583m

(a)

100120583m

(b)

100120583m

(c)

100120583m

(d)


50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)















5 Conclusion


Competing Interests


Acknowledgments


References





2













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























000

010

020

030

040

050

060

070

No stress Stress


sectsect

currencurrenDisc

rimin

atio

n in

dex

ControlHoney

lowastlowast

lowastlowast

(a)

000

010

020

030

040

050

060

070

No stress Stress

Disc

rimin

atio

n in

dex


curren

ControlHoney


(b)


119875 lt 001) SOD (119865134

= 960 119875 lt 001) CAT (119865134


129= 803 119875 lt


134= 1022 119875 lt 001) There were


= 576 119875 lt 001) PCO (119865312

= 640 119875 lt 001) SOD(119865337




120= 393 119875 lt 005) (Figure 3) No



122= 824 119875 lt 001) and



321= 692 119875 lt 001) was










0

50

100

150

200

250

No stress Stress

(UL

)

ControlHoney

lowastlowast




19= 2014 119875 lt


19= 2294 119875 lt

001) CA2 (11986519

= 2908 119875 lt 0001) CA3 (11986519


19= 693 119875 lt 005)


38= 732 119875 lt



0

50

100

150

200

250

300

350

400

450

No stress Stress

(pg

mL)

ControlHoney

sectlowastlowast

curren



4 Discussion



100120583m

(a)

100120583m

(b)

100120583m

(c)

100120583m

(d)


50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)















5 Conclusion


Competing Interests


Acknowledgments


References





2













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























0

50

100

150

200

250

No stress Stress

(UL

)

ControlHoney

lowastlowast




19= 2014 119875 lt


19= 2294 119875 lt

001) CA2 (11986519

= 2908 119875 lt 0001) CA3 (11986519


19= 693 119875 lt 005)


38= 732 119875 lt



0

50

100

150

200

250

300

350

400

450

No stress Stress

(pg

mL)

ControlHoney

sectlowastlowast

curren



4 Discussion



100120583m

(a)

100120583m

(b)

100120583m

(c)

100120583m

(d)


50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)















5 Conclusion


Competing Interests


Acknowledgments


References





2













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100120583m

(a)

100120583m

(b)

100120583m

(c)

100120583m

(d)


50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)















5 Conclusion


Competing Interests


Acknowledgments


References





2













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























5 Conclusion


Competing Interests


Acknowledgments


References





2













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion


Competing Interests


Acknowledgments


References





2













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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