Cognitive enhancement and neuroprotection by catechin-rich oil palm leaf extract supplement

Research ArticleReceived: 5 March 2012 Revised: 3 June 2012 Accepted: 13 June 2012 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5802

Cognitive enhancement and neuroprotectionby catechin-rich oil palm leaf extractsupplementSuhaila Mohamed,a,b∗ Tan Lee Minga and Juliana Md Jaffria,c

Abstract

BACKGROUND: Catechin-rich oil palm (Elaeis guineensis) leaf extract (OPLE) has good cardiovascular and phytoestrogenicproperties. The OPLE (0.5 g day−1) was supplemented to young, healthy, adult human volunteers, and their cognitive learningabilities were compared to placebo-controlled groups (N = 15). Their short-term memories, spatial visualisations, processingspeeds, and language skills, were assessed over 2 months by cognitive tests computer programs.

RESULTS: Relative to the controls, volunteers taking OPLE had improved (P < 0.05) short-term memory, after 1 month ofintervention which became highly significant (P < 0.005) after 2 months. The spatial visualisation ability and processing speedimproved (P < 0.05) after 2 months consumption. The dietary OPLE showed neuroprotection in nitric oxide-deficient rats. Themechanisms involved systemic and cellular modulations that eventually enhance neuron survival. The longer the duration ofOPLE consumption, the more significant was the enhancement, as shown for short-term memory.

CONCLUSION: This is the first report on the cognitive-enhancing effects of dietary OPLE in humans. The computer-assistedcognitive tests were simple, low in cost, errors and man hours, and hence are better than conventional cognitive test methods.In rats, the equivalent OPLE dose showed brain antioxidant enzymes modulating properties and neuroprotection under nitricoxide deficiency, with possibly neurogenesis in normal rats. This supported the effects in humans.c© 2012 Society of Chemical Industry

Keywords: catechins; oil palm leaf; memory; neuroprotection; rats; humans

INTRODUCTIONAlcoholic extracts of oil palm (Elaeis guineensis) leaves (OPLE)are rich in antioxidative catechins1 and have good cardiovas-cular as well as phytoestrogenic properties.2 Palm leaves areabundant, under-utilised, by-products of the palm oil industry intropical countries such as Indonesia, Thailand, Malaysia, as wellas countries in Africa and South America. The health benefits ofcatechins for the prevention of inflammation, oxidative stress,3

and neurodegeneration,4 are well documented.5 epigallocatechingallate (EGCG), the most active component of catechins, actsas an antioxidant in biological systems and is rapidly absorbedand distributed mainly into the mucous membranes of the smallintestine and the liver, and can cross the blood–brain barrier.4

Oxidative stress-induced neuronal apoptosis was prevented byEGCG treatment of neuronal cells.3 A reduction in hippocampallipid peroxides (LPOs) improved spatial cognition learning mem-ory in aged rats,6 and an elevation in antioxidative activity in thehippocampus prevents and ameliorates the impairment of learn-ing ability in the animal. Oxidative stress causes serious functionalimpairments such as cognitive decline, due to cellular enzyme de-terioration, and consequently exacerbates the neurodegenerativeprocess. Neurological mechanisms of polyphenols in Alzheimer’sand Parkinson’s diseases have been proposed.7 The OPLE contain24.3 mg gallic acid equivalent (GAE) g−1 dry weight of non-toxic,antioxidative phenolic compounds at a much higher level thangreen tea (22.5 mg GAE g−1 dry weight).8 In South East Asia it

is common for the palm leaves to be woven into pouches forboiling rice, and the polar components from these leaves diffuseinto the boiled rice to give it a pleasant flavour and aroma. TheOPLE showed stronger inhibition of Cu2+-mediated low densitylipid (LDL) oxidation as compared to many edible plant extracts.9

Palm leaves have been used for decades as ruminant feed withoutany report of toxicity.

Demand for functional food in improving cognitive ability in hu-mans is relevant. Food components affect cognitive performancebecause the central nervous system depends heavily on a con-stant supply of almost all of the essential nutrients and glucose,as well as oxygen via the blood supply, for efficient functioning.Any affordable and convenient alternative that is effective withminimal unwanted side effect is a choice. The discovery of thein vivo antioxidative cardiovascular properties of OPLE1 and theirsafety,10 initiated this investigation on their cognitive effects in

∗ Correspondence to: Suhaila Mohamed, Institute BioScience, Universiti PutraMalaysia, 43400, Serdang Malaysia. E-mail: [email protected]

a Faculty of Food Science and Technology, Universiti Putra Malaysia

b Institute BioScience, Universiti Putra Malaysia, 43400, Serdang Malaysia

c Kulliyyah of Pharmacy, International Islamic University Malaysia, Kuantan,Malaysia

J Sci Food Agric (2012) www.soci.org c© 2012 Society of Chemical Industry

www.soci.org S Mohamed, TL Ming, JM Jaffri

adult humans. The scarcity of reports on the effects of catechins(especially from the under-utilised oil palm leaves) on the in vivocognitive functions of healthy, young, human adults warrants in-vestigation. The total OPLE was used because pure compoundshave been known not to exert similar effects to those of the wholeextract.

EXPERIMENTALOil palm (Elaeis guineensis) leaves from the Johore plantation, werethoroughly washed, coarsely chopped and dried in a 40 ◦C ovenfor 24 h. The dried material was milled, and extracted with 50%ethanol, under continuous agitation (turbo extractor) at roomtemperature for 2 h. After filtration, the solvent was completelyremoved using a spray dryer at an outlet temperature of 80 ◦C (byCEPP, Universiti Teknologi Malaysia, Johor, Malaysia). For humanherbal supplement studies the OPLE was packed into 500 mg hardgelatine capsules under GMP by Toko Tenaga Keluarga Sdn. Bhd,in Johor Bahru, Malaysia, and stored at 4 ◦C before distributing tothe volunteers. The OPLE for animal studies was stored in glassjars, flushed with nitrogen at −20 ◦C.

Pyrogallol, reduced glutathione (GSH) and dithio-bis(2-nitrobenzoic acid) were from Merck (Kuala Lumpur, Malaysia). Cap-topril, N-ω-nitro-L-arginine methyl ester (L-NAME), thiobarbituricacid and 1,1,3,3-tetraethoxypropane were from Sigma-Aldrich(Kuala Lumpur, Malaysia). All chemicals used were of analyticalgrade.

Cognitive pilot testBefore the data collection, the cognitive tests were validatedby eight volunteers, to identify the irrelevant or inappropriatequestions, which need to be omitted or modified, to improveaccuracy, clarity and to prevent respondents becoming fatiguedin the actual trials. The instrument reliability was eventuallyconfirmed by the Cronbach Alpha reliability score of 0.77(above 0.7).

A pilot test was then conducted to familiarise the 30 volunteers(men and women of mixed races, aged 22–24 years) from theuniversity campus, to the cognitive tests, followed by the maindata collection. The human ethics committee of the universitywas informed of the study, which did not involve any invasiveprocedures, and subsequently the OPLE capsules were categorisedas herbal functional food supplements (not drugs). The volunteerswere briefed on the objectives, their rights and procedures, andagreed to the terms and conditions by signing the consent form.The volunteers were divided into two groups (N = 15) to consumeone capsule (500 mg) each day of either (A) OPLE (each capsule isequivalent to 2.5 cups of tea) or (B) placebo (control), for 2 months.

Cognitive testThe same cognitive tests were conducted to evaluate thevolunteers at zero, 1 and 2 months intervention, with a calendarto record the starting and final date, as well the days (if any)they forgot to consume the supplement. The cognitive testswhich took about 30 min to complete, evaluated four functionsthat affect learning, namely (1) short-term memory, (2) processingspeed, (3) spatial visualisation ability and language skills.

Short-term memory abilityVolunteers were asked to concentrate, remember the picturesand match the correct pairs from http://www.intelligencetest.

com/mindgames/concentration.htm (last accessed on 11 February2011), in the shortest possible time. The scores were recorded afterthe task was completed.

Processing speed abilityVolunteers were asked to perform two tests. First, they wereasked to match pictures and words (letter matching task),using arrow keys on the keyboard, to measure the time todecide (reaction) which objects matched (http://cognitivelabs.com/cognitive freetest3.htm) (last accessed on 11 February 2011).Second, the volunteers were shown pictures of items in the paired-associative recall task, to test the processing speed. Three similarobjects were given simultaneously, but one out of the three objectswas slightly different from the others. Volunteers were required toidentify the different object with the fastest speed (measured howquickly and accurately the volunteers reacted from their memory),(http://www.onlinegamingzone.com/whackadifferencegame.html) (last accessed on 11 February 2011).

Spatial visualisation learning abilitySince spatial visualisation is the ability to imagine rotations ofobjects folding or unfolding in three-dimensional space, the ‘CardRotations’ test was used. It reflected the ability to perceive anobject from different rotating and reflecting positions, togetherwith pattern recognition. The objects with different colours wereshown for 5 s, and then the positions were changed. Volunteerswere required to clearly identify the colour, pattern and the exactposition of the objects shown previously on the screen, and thescores were recorded (website last accessed on 11 February 2011:http://www.onlinegamingzone.com/memory3.html).

Rat studyTo test the hypothesis that OPLE has beneficial effects on theneurons, the effects of OPLE on normal and nitric oxide (NO)-deficient rats were evaluated. Sixteen-week-old male Wistar Kyoto(WKY) rats were divided into six groups (n = 8): (1) normal controlrats; (2) normal rats given OPLE; (3) normal rats given captopril;(4) L-NAME-induced NO-deficient rats and vehicle; (5) L-NAME-induced NO-deficient rats and OPLE; and (6) L-NAME-inducedNO-deficient rats and captopril. Captopril was used as the positivecontrol because of its vasodilation and antioxidant properties tocounteract the vasoconstriction induced by NO deficiency. Therats were maintained on distilled water and a standard rat chow(Gold Coin Sdn. Bhd, Klang, Malaysia) for 14 days before the study,and kept in well-ventilated room with a 12 h dark/light cycle.Adequate measures were taken to minimise pain or discomfort,and experiments were carried out with the approval of the AnimalEthics Committee of the University of Putra Malaysia. The OPLEwere analysed using high-performance liquid chromatographyand the main flavonoids were epigallocatechin (800 mg kg−1),catechin (3g kg−1), epicatechin (100 mg kg−1), epigallocatechingallate (2.8 g kg−1), epicatechin gallate (500 mg kg−1) and theirglucosides.

Soy oil was used as the vehicle for dissolving and dispensing100 mg OPLE mL−1 (dosage: 500 mg OPLE kg−1 day−1 by oralgavage). This is equivalent to a 500 mg dose per day for anadult, since the metabolic rates of rats are much higher thanhumans. Water-soluble L-NAME (60 mg L−1) was administeredin the drinking water. Water soluble captopril (100 mg kg−1

day−1) was also given in the drinking water. The L-NAME andcaptopril were co-administered in the same drinking water for

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the appropriate group. The vehicle was administered to all rats atsimilar amounts and by the same method as to rats receiving OPLE.

After 12 weeks, the rats were sacrificed before noon by anoverdose of diethyl ether and the brains removed, weighed andwashed with ice-cold saline. A sagittal cut was made in the middleof each brain to form two halves. The right hemispheres were fixedimmediately in 10% neutral buffered formalin for histology studiesand the remaining halves were immediately frozen at −80 ◦C forup to 4 months. For enzyme and malondialdehyde (MDA) analyses,the frozen tissue was thawed and minced with surgical scissors,before homogenising using a Silverson tabletop homogeniser(model L4RT; Chesham Bucks, United kingdom) set in ice-cold KCl0.15 mol L−1 (4 mL for every 1 g tissue). The homogenate wasfreed from cellular debris and nuclei by centrifugation at 5000 × gat 4 ◦C for 20 min. Homogenate protein content was determinedand calibrated with bovine serum albumin.1

Tissue sections (5 µm) were deparaffinised and processed forhaematoxylin–eosin (H&E) staining. Hippocampal neuron damagewas determined by using a light microscope (Olympus BH2;Olympus, New York, USA) under × 400 magnification, from thenumber of viable pyramidal cells in the CA1, CA3 and granule cellsin the dentate gyrus (DG) sub-fields. Neurons were consideredviable if a cell membrane and nucleus were identified and thecytoplasm was not deeply eosinophilic. Viable pyramidal cells inthe grids were counted manually in CA1 and CA3. The grids forcounting were selected in a random and systematic sampling. Thepercentages of viable neurons were calculated in the respectivenerve cell rows. All images were captured using a PixeLINK 1.3megapixel camera (PixeLINK, Ontario, Canada) mounted on LeicaDME light microscope (Leica Microsystems, Buffalo Grove, USA).The average number of viable cells in a grid was obtained and thepercentage calculated.

Superoxide dismutase (SOD) activity was determined on 500 µLbrain homogenate.1 The absorbance of four preparations of blankcontrol was averaged and used to calculate the enzyme activity.One enzyme unit is the amount which inhibits 50% pyrogallolreaction. Catalase activities were determined1 on 20 µL brainhomogenate diluted to 10 mL with 50 mmol L−1 phosphatebuffer pH 7.0. The enzymatic decomposition of H2O2 in thismethod follows a first order reaction, and the rate constant (k)was used as a direct measure of the catalase concentration.Glutathione peroxidase activities were determined1 on 400 µLbrain homogenate. One unit of enzyme activity is the decreasein the log(GSH) of 0.001 min−1 after subtraction of the decreasein log(GSH) min−1 for the non-enzymatic reaction. MDA levelswere determined1 on 200 µL of brain homogenate. A SecomamAnthelie Advance UV–visible spectrophotometer (ALES Cedex,France) with a 1 cm quartz cell was used for all absorbancemeasurements.

Statistical analysisFor human studies, SPSS (Statistical Package for the Social Sciences,IBM Corporation, USA) for repeated-measures analysis in generallinear model, paired sample t-test analysis, independent t-testand χ2 analysis were used to analyse the data. Mean scores withP < 0.05 showed significant differences.

For animal studies, all statistical analysis were done usingMinitab 13 statistical software (State College PA, USA) and valueswere expressed as mean ± SEM of n = 8. Significant differences(P < 0.05) between groups were analysed using a one-way analysisof variance, followed by Tukey’s pair-wise comparison post hoctest.

RESULTSHuman studiesBefore the start of the experiment, there was no significantdifference in the computer scores between the supplemented andcontrol groups in all the four cognitive categories. However, short-term memory significantly improved (P < 0.05) after 1 monthof OPLE consumption. The differences were highly significant(P < 0.001) after 2 months OPLE consumption compared tobaseline and control groups (Fig. 1). These results were supportedby repeated measure analysis showing improvements in thesetwo sections.

In the second month of supplementation, significant improve-ments (P < 0.05) were observed between OPLE group and controlgroup in two other cognitive functions namely (1) spatial visual-isation ability and (2) processing speed. There was no difference(P < 0.05) between genders on cognitive tests. The results indi-cated that the OPLE had similar effects between men and women.

Animal studiesThe neuro-protective or neurogenesis effects of catechin-richOPLE and captopril were evaluated in normal and NO-deficientrats by assessing neuron viability in three sub-fields of thehippocampus; CA1, CA3 and DG (Fig. 2 and Fig. 3). After chronicNO deficiency, only a few normal neurons were seen with roundcell bodies and clear nuclei and nucleoli. Damaged cells areshrunken and distorted, with small dense nuclear remnants.In the control NO-deficient group, the pyramidal neuronsshowed pyknosis, eosinophilia, karyorrhexia, and chromosomecondensation. Chronic NO deficiency destroyed 53% of the viablepyramidal cells in CA1 (Fig. 2a). Oral OPLE treatment significantly(P < 0.001) prevented this neuro-degeneration by retaining 74%of the viable neurons of the normal control rats (P < 0.001).Captopril showed less effective neuroprotection in this region asthe viable pyramidal cell count was lower than that of OPLE.

The CA3 region was observed to be more affected by NOdeficiency than CA1 region leaving only 20% viable neurons(Fig. 2b). The OPLE or captopril treatment significantly protectedCA3 pyramidal cells and maintained viability at 72% and 47%,respectively (P < 0.001) of the neurons from normal control rats.The neurodegeneration in DG of NO-deficient rats was also quiteextensive, leaving only 32% viable granule cells (Fig. 2c) and, again,the neuro-protective properties of OPLE and captopril significantlyretained 76% and 56% of the neurons, respectively (P < 0.001).

The L-NAME significantly (P < 0.001) decreased SOD activitiesand MDA levels (Fig. 4a and d, respectively), since under NOdeficiency, fewer NO radicals are formed, and consequentlyresulted in reduced oxidative status. However, the OPLE andcaptopril treatments significantly (P < 0.001) attenuated theseSOD activity decreases, under NO deficiency. All normal rats hadsimilar brain SOD activities. glutathione peroxidase (GPx) activitieswere not significantly affected by the NO deficiency (Fig. 4b).

Catalase activities in the brain were significantly (P < 0.05)reduced by both OPLE and captopril treatments in normalrats (Fig. 4c) indicating their antioxidant properties. Under NOdeficiency, both the SOD and catalase activities were reduced.The dietary OPLE slightly up-regulated the brain catalase activitiesin NO-deficient rats by 24% to near normal levels, showing itsenzyme homeostatic effects, and this was not seen in the captopriltreatment.

Neuron losses may occur with age, and OPLE supplementationincreased the viable pyramidal cell count compared to control

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Figure 1. The effects of palm leaf extract consumption on cognitive functions in humans: processing speed, short-term memory, spatial visual ability andlanguage skills. ∗Significant at P < 0.05; ∗∗significant at P < 0.005. N = 15.

rats after 12 weeks. Captopril was less effective than OPLE inimproving the cell count. However, the difference in viable cellcounts between all three normal rat groups was statisticallyinsignificant.

DISCUSSIONThe adult human hippocampus is capable of neurogenesis, andnewly generated neurons mature into functional cells to par-ticipate in hippocampal-dependent memory formation.11 Thehippocampal neurogenesis was required for the behavioural ef-fects of antidepressants11 and some volunteers reported feelingcheerful and energetic during the course of the OPLE supplementa-tion. These feedbacks together with the improved cognitive scoressuggest that the positive effects of OPLE may be mediated by neu-rogenesis induction in the hippocampus. Although the cognitiveprocess involved (1) information absorption, (2) initial perception,(3) input recording, (4) content analysis, (5) learning and memorycreation, (6) retention for later use, and finally (7) retrieval theability to create and retrieve memories is essential to all cogni-tion functions.12 Short-term or immediate memory span lasts for15–30 s, and receives the present conscious attention, with limited

storage capacity.12 It is often impaired by distraction, such as noiseor divided attention tasks. Long-term memory is relatively moreresilient with greater capacity.12 The visual memory is affectedby duration and intervals between images presentation, hencecomputer-assisted programs will reduce human facilitator errorrelated to this task.

Besides short-term memory, cognitive functions include work-ing memory, long-term retrieval, executive processes, cognitiveefficiency, delayed recall, processing speed and visual–spatialthinking. The working memory is the ability to hold information inimmediate awareness while performing a mental operation on theinformation. Long-term retrieval is the ability to store informationand fluently retrieve it later in the process of thinking. Delayedrecall is the ability to both recall and re-learn connections. Exec-utive process involves strategic planning, proactive interferencecontrol, and the ability to shift repeatedly the mental setting. Cog-nitive efficiency represents the capacity to process informationautomatically.

The processing speed, which is measured by the reaction timeto completely recognise, process and make decisions after a visualstimulus,13 is linked with other cognitive functions (including long-and short-term memory), thus took longer to show significant



(a)

(b)

(c)

Figure 2. The effects of oil palm leaf extract (OPLE) consumption in normaland NO-deficient rats on the (a) hippocampal CA1 pyramidal cells; (b) CA3pyramidal cells in normal and NO-deficient rats; and (c) hippocampal DGgranule cells in normal and NO-deficient rats. OPFME, oil palm fronds/leavesalcoholic extract. ∗P < 0.05 vs. control; +P < 0.05 vs. L-NAME.

effects. It was measured under pressure to maintain focusedattention. Visual and spatial thinking is the ability to perceive,analyse, synthesise and think with visual patterns, including theability to store and recall visual representations, together withinformation processing which uses various sensory and cognitiveabilities. It also measures the sensitivity to different visual stimuli(different visible wavelengths), and the thinking process. Theeffects by catechin-rich OPLE for visual–spatial working memorywere observed after 2 months of treatment (P < 0.05), whichwas 1 month earlier than previous studies on other flavonoids.14

Attention is important in the recognition and recollection ofimages, and information is easier from photographs rather thandrawings, because they contain more organisational and thematicinformation. Since this program used drawings, they were moredifficult for the volunteers, and the insignificant improvementafter 1 month was due to the large standard deviations betweentheir performances.

Language measures a high cognitive ability, because itis associated with learning and understanding, experienceand knowledge, long-term memory, thinking and reasoning,transforming and modification, before shaping and synthesis.12 Anew word requires remembering its meaning and pronunciationsbefore applying it in a sentence,12 to express thoughts, experiencesand to communicate with others. The long-term memory system

(c) (d)

(a) (b)

Figure 3. Photomicrographs of the hippocampus CA3 sub-field of differentexperimental rat groups to show the effects of OPLE consumption in normaland NO-deficient rats. (a) Normal control; (H&E, × 400); (b) normal + OPLE(H&E, × 400). In (a) and (b) dark and shrunken neurons are scatteredoccasionally as normal ageing occurred. (c) L-NAME (NO deficient). Neuronloss was prominent and the number of normal neurons in the row of nervecells was decreased. (d) L-NAME+OPLE (H&E, × 400). OPLE and captopriltreatments in NO deficiency effectively increased neuron survival from thelow-NO state.

is used in the language ability to obtain information and makesense of future messages.12

There is much evidence indicating that NO is neuroprotectiveto the hippocampus and is important for learning and memory.15

NO also helps regulate blood flow, thrombogenesis and neuronalactivities. Nitric oxide synthase (NOS) inhibitors such as L-NAMEhave been shown to impair learning in rats.16 The relativelysmall brain uses 20% of basal oxygen consumption and is rich inoxidisable polyunsaturated fatty acids, yet is not highly equippedwith antioxidant enzyme defences (very low level of catalaseand moderate amounts of SOD and GPx). Oxidative stress andmitochondrial dysfunction may lead to neuron death. Memory andcognitive loss occurs during normal brain ageing. Biomarkers ofageing are correlated with decreased hippocampal functions andother neurodegenerative disorders, such as Alzheimer’s disease.Here, the 12-week L-NAME administration dramatically reducedthe viable neurons in the CA1, CA2 and DG of the hippocampus,and co-administration with OPLE attenuated this effect moreeffectively than the standard drug, captopril. The neuron loss withNO deficiency reportedly impaired memory in various tasks.15,16

The normal rats supplemented with OPLE showed slightly higherviable neuron counts which either indicated neurogenesis, orprotection against natural neuron losses that occur with age, after12 weeks. It also reflects the absence of neurotoxicity by the OPLE.

The CA3 is differentiated into repeated network of closelyinterconnected pyramidal cells and the CA1 has a feed-forwardnetwork with almost no intrinsic excitatory connections. Nitricoxide deficiency affect hippocampal long-term potentiation,because NO is a retrograde messenger after post-synaptic N-methyl-D-aspartate (NMDA) receptor activation, which is a non-specific cation channel and directly contributes to excitatorysynaptic transmission by depolarising the postsynaptic cell.When pre- and post-synaptic cell are simultaneously active,



(a) (b)

(c) (d)

Figure 4. The effects of oil palm leaf extract (OPLE) in normal and NO-deficient rat on (a) brain SOD activities, (b) brain GPx expression, (c) brain catalaseexpression, and (d) brain MDA levels. OPFME, oil palm fronds/leaves alcoholic extract. ∗P < 0.05 vs. control; +P < 0.05 vs. L-NAME.

NMDA receptors become unblocked and allow calcium ionsto enter the post-synaptic cell, converting electrical signal intobiochemical signal that trigger synaptic plasticity.17 Activation ofNMDA receptors, which is also a glutamate receptor, stimulatesNO production in neurons, which either facilitate or inhibitdopaminergic transmission, depending upon the brain area underinvestigation.15 The major neurotransmitter in the pathwaysconnecting DG to CA3 and to CA1 is glutamate, which regulatesthe release of dopamine in the central vervous system.15

The NO deficiency caused significant MDA reduction whichwas accompanied by decreases in SOD and catalase activities.The diminishing NO in the brain reduced the formation of per-oxynitrite radical, which is more reactive than NO or superoxide.The normal brain ageing is associated with elevated levels ofneuro-inflammation, and enhanced ROS production,18 which sub-sequently increase signal transduction to up-regulate inducibleNOS (iNOS) and NO production. The hypothalamus and otherbrain regions of aged rats have increased iNOS expression. Ex-cessive brain endogenous NO induces cytotoxic and mutageniceffects, especially during inflammation.18 At the end of this studythe rats were 7 months old, thus despite having higher MDA thanthe L-NAME group, they still had a high viable neuron count.Although the untreated L-NAME rat brain had low MDA levels, thenumber of viable neurons was also low, suggesting that the extentof NOS inhibition resulted in a NO levels below the physiologicalrequirement for neurons survival.

The OPLE and captopril showed brain SOD up-regulatingactivities which helped prevent residual NO degradation, andsubsequently protected the neurons. Captopril also inhibits an-giotensin converting enzyme (ACE) which retards nicotine adeninedinucleotide phosphate (NADPH) oxidase that generates reaciveoxygen species (ROS).19 Polyphenols enhance NO availability ei-ther by inducing constitutive NOS expression or by protectingNO against oxidative destruction. Up-regulations of SOD-1 (Cu/ZnSOD) or EC-SOD (extracellular SOD) in aged mice improve hip-pocampal functions and spatial memory.20

Mechanism by which catechins protect the neurons in-clude (1) antioxidant actions,3 (2) fatty acid sequestration andprocessing,6 (3) peroxisome proliferator-activated receptor (PPAR)activation,21 (4) phytoestrogenic effects,22 (5) regulating inflam-mation which reduces NO toxicity, by selectively inhibit-ing iNOS expression,21 (6) modulating endothelial apoptosis,21

(7) enhancing vascular NO production,21 (8) maintaining en-dothelial functions and vascular homeostasis,23 (9) preventingstriatal dopamine and tyrosine hydroxylase protein depletion,24

(10) adjusting brain SOD and catalase activities,4 (11) chelat-ing iron, (12) activating the extracellular signal-regulated ki-nase (ERK1/2), the protein kinase B (PKB/Akt) signalling path-ways and the cAMP response element-binding protein (CREB),to elevate the expression of various neurotrophins for mem-ory improvement,21 and (13) inhibiting various intracellular sig-naling transduction.25 Such inhibitions protect the neurons



further by suppressing (1) angiotensin II- and pressure-overload;(2) NADPH oxidase over-expressions; (3) nuclear factor kappa B(NF-κB) and activator protein 1 activation, thus reducing iNOSexpression;26 (4) ROS-dependent p38 and c-Jun N-terminal ki-nase (JNK) signalling pathways; (5) epidermal growth factor re-ceptor (EGFR) transactivation; (6) extracellular signal-regulatedkinase (ERK)/PI3K/Akt/mTOR/p70(S6K) (PI3K is phosphoinositide3-kinase, Akt is gene encoding a particular protein, and mTOR isa signalling pathway); (7) reactivation of ANP and BNP (atrial andbrain natriuretic peptide);25 and (8) inhibit ACE activity.21 Long-term administration of catechins improved animals’ cognitiveperformance and lipid peroxides levels in the hippocampus.6,27

Additional mechanisms by which polyphenols, in gen-eral, improve cognitive functions include (1) decreasing acetyl-cholinesterase activity;28 (2) elevating protein kinase activities,29

which regulates synaptic plasticity and modulates the formationof short, long-term and spatial memory; (3) protecting cell sig-nalling for processing speed;21,30 (4) improving the brain corticalblood flow and cardiovascular function,21 which consequentlyenhance cerebrovascular function, especially in the hippocampusfor memory; and (5) elevating brain dopamine levels29 for effi-cient memory, attention, problem-solving function and smooth,controlled movements.

The OPLE showed contrasting modulating effects in normaland NO-deficient rat brains, under the two different physiologicalconditions of the rat models. The blood–brain barrier penetrating,antioxidant and iron-chelating properties of catechins makethem important for the treatment of oxidative stress associatedneurodegeneration and possibly encourage neurogenesis.

Polyphenols consumption can improve cognition and process-ing speed of humans,30 and delay or even reverse age-relatedcognitive deficits in animals. They reverse age-related cognitivedecline by enhancing neuronal signalling, controlling synapticplasticity, growth and connectivity, increasing dendritic spinedensity, inducing vascular changes to encourage neurogenesisand help the functional integration of old and new neurons.29 An-tioxidative nutrients must be consumed continuously to maintainthe efficacy on different cognitive functions, to help prevent cog-nitive decline during ageing and neurodegenerative disease. Thecognitive executive function decline is detectable by the inabilityto master new types of tasks in new situations, a lack of flexibilityand an inability to trouble shoot, react appropriately or quickly indangerous or technically difficult situations.

POLYPHENOLS AND NEURODEGENERATIVEDISEASESNeurodegenerative diseases is a growing problem in today’s agingpopulation. An estimated 15% of the elderly above 65 suffer fromAlzheimer’s disease and 1% by Parkinson’s disease. This doesnot include the other types of dementia caused by ischemicinjury.31 These diseases are related to oxidative stress, in whichthe brain tissues are most susceptable,32 hence antioxidants helptheir prevention.31 Aging rats fed with polyphenol-rich plantextracts showed improved cognitive functions and neuronalsignal transductions.33,34 Anthocyanins-rich blueberries were mosteffective and the effects were not caused by the sparing of vitaminsE and C in the brain.35

Intravenous injection of epicatechin or catechin to rodents im-proved the memory impairment caused by cerebral ischemia.36

Dietary grape polyphenols reduced ethanol-induced neurode-generatiion, and improved the synaptic function.37 Dietary EGCG

also restored the dopaminergic neurotransmission, SOD andcatalase levels in parkinsonian syndrome-induced rats.38 Ferulicacid or cucumin consumption protected rodents from β-amyloidpeptide-induced Alzheimer’s disease,39,40 possibly via a transitoryactivation of hippocampal astrocytes.

Catechins dose dependently improved cultured neuronal cellssurvival in vitro when challenged by a β-amyloid peptide, 6-hydroxydopamine, or oxidized LDL.41 – 43 It is probably mediatedby protein kinase C activity restoration, or NF-κB translocationinhibition, related to cell proliferation and apoptosis. Low dosesof epigallocatechin gallate (0.1–10 µM), protected neuronal cellsagainst oxidative damage and improved cell survival, whereashigher doses (50 µM), were seemingly pro-oxidant and toxic.41

Thus, low polyphenol concentrations may be more effectivein preventing neurodegenerative diseases. Dietary genistein,naringin or quercetin concentrations were much lower in thebrain (0.04 nmol/g tissue) than in the plasma (2 µmol/L) andother tissues.44 – 46 The epicatechin glucuronide conjugate did notprotect cortical neurons against H2O2-induced oxidative stress,47

due to the poor blood-brain barrier permeability of anionicconjugates,44,48 which may help explain the observed beneficiallow levels in brain tissues.

The dietary polyphenol inverse associations with neurode-generative disease risk were reported in some epidemiologicalstudies.49 – 51 Cognitive impairment probability were increased inheavy alcohol drinkers.52 The intake of flavonols and flavones wereinversely related with dementia risks in a French cohort report.53

CONCLUSIONThis is the first report on the cognitive enhancing effects of dietaryOPLE in humans, and the neuroprotection under NO deficiencyin mammals. The mechanisms involved systemic and cellularmodulations that eventually enhance neuron survival. Apparently,the longer the duration of OPLE consumption, the more significantwas the enhancement, as shown for short-term memory. Furtherinvestigations on middle-age adults using computer-assistedcognitive tests may provide interesting results. The computer-assisted cognitive tests reduced cost, errors and man hours. Futureresearch may compare this technique to standard, well-validatedtests for use by neuropsychologists.

ACKNOWLEDGEMENTThe authors thank the Government of Malaysia for providinggrants for this research, and all staff of Universiti Putra Malaysiawho have facilitated this research.

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Cognitive enhancement and neuroprotection by catechin-rich oil palm leaf extract supplement

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