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Biomedicine & Pharmacotherapy 93 (2017) 837–851
Original article
Therapeutic impact of grape leaves polyphenols on certain biochemicaland neurological markers in AlCl3-induced Alzheimer’s disease
Ibrahim H. Boraia, Magda K. Ezza, Maha Z. Rizkb, Hanan F. Alyb, Mahmoud El-Sherbinyb,Azza A. Matloubc, Ghadha I. Fouadb,*aBiochemistry Department, Faculty of Science, Ain-Shams University, Cairo, Egyptb Therapeutical Chemistry Department, National Research Center, Dokki, Cairo, Egyptc Pharmacognosy Department, National Research Center, Dokki, Cairo, Egypt
A R T I C L E I N F O
Article history:Received 14 April 2017Received in revised form 27 June 2017Accepted 6 July 2017
Keywords:Vitis viniferaPolyphenolsAluminumAlzheimer’s diseaseAD-ratsT-maze
A B S T R A C T
Alzheimer’s disease (AD) is a grave and prevailing neurodegenerative disease, characterized by slow andprogressive neurodegeneration in different brain regions. Aluminum (Al) is a potent and widelydistributed neurotoxic metal, implicated in the neuropathogenesis of AD. This study aimed to evaluatethe possible neurorestorative potential of Vitis vinifera Leaves Polyphenolic (VLP) extract in alleviatingaluminum chloride (AlCl3)-induced neurotoxicity in male rats.AlCl3 neurotoxicity induced a significant decrease in brain/serum acetylcholine (ACh) contents and
serum dopamine (DA) levels, along with a significant increment of brain/serum acetylcholinesterase(AChE) activities. In addition, Al treatment resulted in significantly decreased serum levels of both totalantioxidant capacity (TAC) and brain-derived neurotrophic factor (BDNF), and significantly increasedserum levels of both interleukin-6 (IL-6) and total homocysteine (tHcy), as compared to control.Behavioral alterations, assessed by the T-maze test, showed impaired cognitive function. Furthermore,AD-brains revealed an increase in DNA fragmentation as evidenced by comet assay. AlCl3 induction alsocaused histopathological alterations in AD-brain. Treatment of AD-rats with VLP extract (100 mg/kg bodyweight/day) improved neurobehavioral changes, as evidenced by the improvement in brain function, aswell as, modulation of most biochemical markers, and confirmed by T-maze test, the histopathologicalstudy of the brain and comet assay. The current work indicates that the VLP extract has neuroprotective,antioxidative, anti-inflammatory, and anti-amnesic activities against AlCl3-induced cerebral damagesand neurocognitive dysfunction.
© 2017 Elsevier Masson SAS. All rights reserved.
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ScienceDirectwww.sciencedirect.com
Abbreviations: VLP, Vitis vinifera leaves polyphenols; ACh, acetylcholine; DA,dopamine; AChE, acetylcholinesterase; TAC, total antioxidant capacities; BDNF,brain-derived neurotrophic factor; IL-6, interleukin-6; Ab, amyloid-b; NFTs,neurofibrillary tangles; AD, Alzheimer’s disease; NMR, Nuclear magnetic reso-nance; RIVA, rivastigmine; EDTA, ethylene-diamine-tetra-acetic acid; SCGE, singlecell gel electrophoresis; CTL, comet tail length; TM, Tail moment; TE, Troloxequivalent; CE, catechin equivalent; GAE, gallic acid equivalent; DPPH, 2,2-diphenyl-1-picrylhydrazyl; MAO, monoamine oxidase.* Corresponding author at: Assistant Researcher in Therapeutical Chemistry
Department, National Research Center (NRC), 33 El-Bohouth Street, P.O. 12622,Dokki, Cairo, Egypt.
E-mail addresses: [email protected] (I.H. Borai),[email protected] (M.K. Ezz), [email protected] (M.Z. Rizk),[email protected] (H.F. Aly), [email protected](M. El-Sherbiny), [email protected] (A.A. Matloub),[email protected] (G.I. Fouad).
http://dx.doi.org/10.1016/j.biopha.2017.07.0380753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
Alzheimer disease (AD) is the most common neurodegenerativedisorder affecting the elderly with cumulative neurocognitivedecline and memory impairment (dementia). Multiple pathogenicfactors of AD involve amyloid-b (Ab) plaques, neurofibrillarytangles (NFTs), cholinergic dysfunction and oxidative stress [1].Accounting for about 60 to 80% of dementia cases in the elderlypopulations; AD has become one of the major global healthchallenges of the century [2].
During ageing, humans and animals show a decline in motorand cognitive functions, that could be attributed to increasedsusceptibility to the cumulative effects of oxidative stress andinflammation [3]. The glutamatergic pyramidal neurons in brainare highly vulnerable to deterioration in both age-associated andAD-induced cognitive impairment [4].
838 I.H. Borai et al. / Biomedicine & Pharmacotherapy 93 (2017) 837–851
The current symptomatic treatment of mild-to-moderate ADpatients is based on drugs such as donepezil, rivastigmine, galant-amine and memantine, which help in alleviating the clinicalsymptoms of AD, but are associated with side effects and showlittle potential for AD treatment [5]. AD is a typical example of acomplex multifactorial disease; that means, the ‘one change, onedisease, one drug’ strategy is no more applicable [6]. There is a highdemand for search for new drugs of natural origin, directed forprotection from this neurodegenerative disease, or even forprevention, slowing down and/or halting the disease progressionand deterioration in its early stages, which may reduce the sideeffects of clinically used drugs and hence increase healthy ageing [7].
Grape-derived compounds have a beneficial potential inchronic diseases, including cardiovascular disease, neurodegener-ative disorders, ageing and cancer [8]. Grape (Vitis vinifera) leavescontain a wide range of polyphenols; such as anthocyanins,flavonoids and organic acids, mainly malic acid, oxalic acid andtartaric acid [9].
Polyphenols have important biological activities, they arepowerful anti-oxidants that inhibit the production of free radicals,thus limiting the risk of developing oxidative stress-induceddegenerative disorders such as in ischemia, Parkinson’s disease orAlzheimer’s disease [10]. Several mechanisms, underlying thepotency of polyphenols to improve neurological health, includetheir interaction with neuronal and glial signaling pathways,decreasing neurotoxins-mediated neuronal damage and loss orneuroinflammation, diminishing reactive oxygen species (ROS)production, and attenuating the accumulation of neuropathologi-cal markers, such as amyloid-b (Ab) and Tau protein [11]. Due totheir favorable safety profile, availability and cheap price,supplementation of polyphenols in diets is a promising nutraceu-tical or pharmaceutical tool to halt AD progression [12], providingnew protective and/or therapeutic strategies for the prevention ordelay of neurocognitive impairment in brain disorders [11].
Aluminium (Al) represents an important risk factor for severalage-associated neurodegenerative disorders [13], including AD[14]. Aluminium chloride (AlCl3) is a neurotoxicant that accumu-lates in the brain and negatively affects ionic, cholinergic, anddopaminergic neurotransmission [15].
The present study aimed to highlight the possible beneficialand therapeutic effects of Vitis leaves polyphenols (VLP) extract inregression of the neurodegenerative features of Alzheimer’sdementia in Al-intoxicated rat model, this may provide simple,noninvasive, inexpensive, reliable, and reproducible blood-basedpanel of circulating diagnostic biomarkers, to pave the way forpossible early therapeutic applications.
2. Materials and methods
2.1. Plant (Vitis vinifera) leaves samples
Red grape (Vitis vinifera L.) -table grape- cv. Flame Seedless,belonging to the family Vitaceae. Vitis leaves were collectedfrom a local farm in Egypt during the lush vegetation period inMay 2015. The collected samples of Vitis leaves were cleaned,dried at room temperature under shady conditions, powdered forextraction and stored in polyethylene plastic bags in a dry place.Herbarium specimens of the plant were identified by Dr. AzzaMatloub, Pharmacognosy Dept., National Research Center.
2.2. Chemicals
� All chemicals and reagents were of analytical grade and werepurchased from Biodiagnostic Company for diagnostic andresearch reagents (Cairo, Egypt).
� Aluminium Chloride anhydrous (AlCl3) was purchased fromSigma Co. USA. Its M.Wt. is 133.34.
� All phenolic and flavonoid compounds were purchased fromSigma-Aldrich Co. USA.
� Reference drug (Exelon -Rivastigmine- 1.5 mg) was purchasedfrom NOVARTIS Pharmaceuticals (Cairo, Egypt).
2.3. Phytochemical study of Vitis vinifera leaves
2.3.1. Preparation of Vitis vinifera leaves polyphenols (VLP) extractDried Vitis vinifera leaves were defatted first using petroleum
ether (in a ratio of 1:10 w/v, 40–60 �C). The defatted powder wassoaked in dark flasks separately for 30 min using acetone/watersolvent mixture (80–20, v/v%). The extraction process wasrepeated three times and the filtrate was combined and thenwas evaporated under vacuum in the rotary evaporator at 37 �C;the remaining water solution was lyophilized. This was doneaccording to Amarowicz et al. [16].
2.3.2. Estimation of total phenolic contentThe total phenolic content was determined spectrophotomet-
rically by Folin-Ciocalteu methods and expressed as mg of Gallicacid equivalent (GAE) per g extract [17]. Briefly, the extract(500 ml) was mixed with 250 ml of Folin-Cicalteu reagent. After5 min, the mixture was neutralized with 1.25 ml 20% aqueousNa2CO3 solution. After 40 min, the absorbance of the mixture wasmeasured at 725 nm against the solvent blank. The total phenoliccontent was determined by means of a calibration curve preparedwith gallic acid and expressed as mg of Gallic acid equivalent(GAE) per g extract. The coefficient of determination wasr2 = 0.9992.
2.3.3. Estimation of total flavonoid contentThe total flavonoid content was determined by the aluminium
chloride colorimetric method and expressed as mg of catechinequivalent (CE) per g of extract [17]. The extract (500 ml) wasmixed with 250 ml of NaNo2. After 6 min, 2.5 ml of 10% AlCl3solution were added. After 7 min 1.25 ml NaOH were added and themixture was centrifuged at 5000g for 10 min. The absorbance ofthe supernatant was measured at 510 nm against the solvent blank.The total flavonoid content was determined by means of acalibration curve prepared with catechin and expressed as mg ofcatechin equivalent (CE) per g of extract. The coefficient ofdetermination was r2 = 0.998.
2.3.4. Determination of radical DPPH� scavenging activityFree radical scavenging capacity was determined spectropho-
tometrically using stable 2,2-diphenyl-1-picrylhydrazyl (DPPH�),according to Hwang and Thi [18]. The absorbance of thesupernatant was measured at 517 nm against a blank of puremethanol. Inhibition % of DPPH free radical was calculated by thefollowing equation:
Inhibition (%) = 100 � (Acontrol� Asample)/Acontrol
Where: A control is the absorbance of the control reaction(containing all reagents except the test compound). Asample isthe absorbance of the test extracts and test fractions.
The antioxidant activity was determined by means of acalibration curve prepared with Trolox, and expressed as mg ofTrolox equivalent (TE) per g of extract and/or fraction. Thecoefficient of determination was r2 = 0.9992.
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2.4. In vivo biological study
2.4.1. Animals and ethicsThe current study was conducted on 125 adult male Wistar rats
(12–15 weeks) weighing 250–300 g, obtained from the AnimalHouse Colony of the National Research Centre, (NRC); Egypt. Theanimals were maintained on a standard laboratory diet and waterad libitum. After two weeks of acclimation period, the animalswere housed in polypropylene cages in a temperature controlled(25 �1 �C) and artificially illuminated (12 h dark/light cycle) room,free from any source of chemical contamination. All animalsreceived humane care and handling, according to the guidelines ofthe Medical Ethical Committee of the National Research Centre inEgypt (Approval number: 15-042).
Induction of Alzheimer’s disease (AD) in rats (AD- Rat Model)The rat model mimicking AD was obtained by administering
AlCl3 orally at a dose of 17 mg/kg body weight daily for 4 successiveweeks, according to Krasovskiñ et al. [19] and Ahmed et al. [20].
2.4.2. Experimental designThe animals used were classified into five groups (25 rats/
group), and treated orally for 21 days as follows:
� Group (1): Normal, healthy rats served as negative controls� Group (2): Normal, healthy rats receiving (VLP)� Group (3): AD-induced rats (positive control rats)� Group (4): AD-induced rats treated daily orally with (VLP)extract for 21 consecutive days at a dose 100 mg/kg body weight,according to Pari and Suresh [21].
� Group (5): AD-induced rats treated orally with Rivastigmine(RIVA) aqueous infusion (0.3 mg/kg body weight/Day) asreference drug, daily for 21 consecutive days (after stoppingAlCl3 administration) [22].
2.4.3. Behavioral assessment: assessment of cognitive abilities usingrewarded T-maze test
The neurocognitive function of rats was estimated by T-mazetest (constructed in the NRC, Egypt) according to Deacon andRawlins [23]. Before performing this experiment, the animals wereleft without food for 24 h, with only water to drink. The five groupswere subjected to the rewarded T-maze test which was donethrice: at zero time before starting oral induction with AlCl3, afterAD-induction period by 24 h, and 24 h after the last oral treatmentwith the tested materials. Behavioral observations were recordedbefore and at the end of the experiment.
2.4.4. Blood sampling and brain tissue sample collection
Collection of blood sampleThe day after assessing T-maze tests, rats were fasted overnight,
with free access to water; blood samples were collected from allgroups just before sacrificing the rats (by cervical decapitation, toavoid the possible biochemical changes because of ischemia),under light anesthesia with diethyl ether. The blood was collected,before decapitation, from the sublingual vein of random rats ineach group, the blood samples were left to clot in clean, dry testtubes for 30 min at room temperature and then centrifuged at4000 RPM for 10 min. The clear supernatant serum was then frozenat �20 �C for biochemical analysis (ACh, AChE, DA, TAC, tHcy, IL-6and BDNF).
Brain tissue homogenate sampling and preparationRats of each group were decapitated, with the head moved
onto the dry ice, the whole brain was rapidly dissected on an ice-
cooled glass plate, thoroughly washed with isotonic saline, dried[24] and sagittally divided into two portions. The first portion washomogenized using an electrical homogenizer (Remi 8000 RPM),to give 10% (w/v) homogenate in ice-cold medium in 9 volumes(1: 9 w/v) of a 50 mM phosphate buffered saline (PBS) pH 7.0containing 0.1 mmol/L ethylene-diamine-tetra-acetic acid(EDTA). The unbroken cells and cell debris were removed bycentrifugation at 4000 RPM for 30 min at 4 �C to prepare clearsupernatants (10%) for acetylcholine (ACh) and acetylcholines-terase (AChE) assays [25]. The second portion of the brain wasused for histopathological investigation. Finally, whole brains ofthree rats from each group were removed simultaneously, chilledin an ice-cold saline solution and kept for the comet assay.
2.4.5. Biochemical assays and analysesSerum Total antioxidant capacity (TAC) was assayed colori-
metrically according to the method of Koracevic et al. [26]. Brain/serum acetylcholine (ACh) levels, brain/Serum acetylcholinester-ase (AChE) levels, serum dopamine (DA) levels, serum IL-6 levels,serum BDNF levels were all measured by ELISA (a sandwichenzyme Immunoassay) [27–31], respectively. Serum total Homo-cysteine (tHcy) levels were determined by High-PerformanceLiquid Chromatography (HPLC) according to Feussner et al. [32].
2.4.6. Histopathological investigationsSections of brain tissues were fixed in 4% buffered-saline
formalin, dehydrated in graded ethanol and embedded in paraffinusing standard procedures. Sections of 4 mm thickness werestained with hematoxylin and eosin (H&E) for histopathologicalexamination, using a light microscope [33].
2.4.7. Comet assay (single gel electrophoresis)Genotoxicity testing was performed using single cell gel
electrophoresis (SCGE) or Comet assay according to the methodused by Singh et al. [34] .The comet tail length (CTL) was valuedfrom the middle of the nucleus to the tail end with 40� increase inthe count. DNA migration length and the migrated DNA% weremeasured, to estimate the quantitative and the qualitative scope ofDNA damage in the cells, and tail moment (TM) was calculated.
2.5. Statistical analysis
Data were analyzed using computer software, StatisticalPackage for the Social Sciences (SPSS) version 16 (SPSS Inc.Released 2007, SPSS for Windows, and Version 16.0. Chicago, SPSSInc.). Simple one-way analysis of variance (ANOVA) and Duncan’smultiple range tests were used. All data were expressed asMean � SE (Standard Error), for n = 10 rats of each group.
3. Results
3.1. Phytochemical study
Extraction yield, total phenolic content, total flavonoid contentand antioxidant capacity (DPPH�) of Vitis vinifera leavespolyphenols (VLP) acetone extract
The yield, total phenolic content, total flavonoid content andDPPH� scavenging activity of 80% acetone extract were 19.25 w/w%,217.53 mg GAE/g extract, 189.97 mg CE/g extract, and 6496.99 mgTE/g extract, respectively.
3.2. In vivo biological study
3.2.1. Rewarded alternation T-Maze test (behavior stress maze)Our results demonstrated a significant increase in time (in
seconds) taken by rats to reach the food in the T-maze, for AlCl3-
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neurotoxicated rats (AD-group); denoting deteriorated neuro-cognitive function. Whereas treated AD-groups with either VLP(100 mg/kg body weight/day) or RIVA (0.3 mg/kg body weight/day)showed a significant decrease in time, taken by rats to reach food inthe T-Maze, in comparison to AD-induced group; indicatingimproved cognitive abilities. Additionally, the VLP-treated ratsshowed a significant decrease in time to achieve the task, ascompared to the negative control group (Table 1).
3.2.2. Effect of treatment on acetylcholine (ACh) levels andacetylcholinesterase (AChE) activities in serum and/or brain tissue inAD-induced rats
An insignificant change was detected in ACh/AChE levels inhealthy negative control rats administrated VLP extracts, ascompared to control rats, Figs. 1 and 2. In AD-induced rats, theserum and brain levels of ACh were significantly lower by 54.23and 51.29% respectively, as compared to negative controls. Amarked increase in ACh was shown in both serum and brain levelsafter treatment with either VLP extract (34.71 and 34.26%,respectively) or with RIVA (48.70 and 49.76%, respectively), ascompared to AD.
On the other hand, by comparison to the control group, serum/brain AChE activities in AlCl3-treated rats were found to be twicehigher than those of the control group. However, treatment of AD-induced rats with RIVA or VLP resulted in a significant inhibition inthe brain and serum AChE activities (31.50, 22.89 for VLP and 23.16,28.37%, for RIVA), as compared to AD-rats.
3.2.3. Effect of treatment on serum dopamine (DA) levels in AD-induced rats
Serum DA level was significantly decreased by (72.41%)following Al treatment, as compared to normal control rats,Fig. 3, however, it increased upon VLP and RIVA administration(38.77 and 46.01%, respectively).
Compared to AD-rats, AD-VLP rats and AD-RIVA rats showed amarked elevation in DA with percentage increase of 95.66 and121.90%, respectively.
3.2.4. Effect of treatment on serum total antioxidant capacities (TAC)levels in AD-induced rats
An insignificant change was detected in TAC levels in negativecontrol rats treated with VLP extract. While, In AD-induced rats,there was a significant depletion (p � 0.001) in TAC serum levels(53.51%), as compared to normal control, Fig. 4.
Treatment of AD-rats with either VLP or RIVA, resulted in asignificant increase in TAC levels (112.80 and 82.56%, respectively),as compared to AD-induced rats.
Table 1Therapeutic effects of VLP extract and RIVA drug on the transit time spent in the T-ma
Groups/trials 1st trial (sec.) Baseli
Negative control Mean � SE 13.12a� 2.12
Negative VLP Mean � SE 12.73a� 0.94
% Change to Control �18.22
AD Mean � SE 18.92d� 3.91
% Change to Control 44.21
AD-RIVA Mean � SE 15.27a� 2.09
% Change to Control 16.39
% Change to AD �19.29
AD-VLP Mean � SE 14.33a� 1.50
% Change to Control 9.22
% Change to AD �24.26
Data are presented as mean � SE (n = 10). Mean with different superscripts (a, b, c, d, e
3.2.5. Effect of treatment on serum total homocysteine (tHcy) levels inAD-induced rats
A significant decrease was detected in serum tHcy levels incontrol rats treated with VLP extract (15.84%), as compared tonegative control rats. While, Al-intoxicated rats showed asignificant elevation in serum tHcy levels (121.17%).
As compared to negative control rats, AD-rats treated with VLPextract and RIVA drug showed significant decrements in tHcylevels, in comparison with control rats by 62.47 and 54.66%,respectively, Fig. 5.
As compared to AD-induced rats, treatment with VLP extractand RIVA showed marked reduction in tHcy levels with apercentage decrease of 26.21and 29.75%, respectively.
3.2.6. Effect of treatment on serum interleukin-6 (IL-6) levels in AD-induced rats
From Fig. 6, it can be easily demonstrated that IL-6 levels wereinsignificantly changed in healthy negative control rats treatedwith VLP extract. On the other hand, AD-induced rats showed anincrement in IL-6 by (440.12%). Treatment of AD-rats with both VLPextract and RIVA drug showed significant decreases in IL-6 (341.46and 321.93%, respectively), as compared to control.
With regard to AD-induced rats, treatment of AD-rats with VLPextract and RIVA showed a significant decrease in IL-6 by 18.27 and21.88%, respectively (P � 0.05).
3.2.7. Effect of treatment on serum brain-derived neurotrophic factor(BDNF) levels in AD-induced rats
An insignificant change was detected in BDNF levels in healthynegative control rats treated with VLP extract, Fig. 7. Serum BDNFlevels reduced significantly in AD-induced rats by 20.78%.Treatment of AD-rats with VLP extract, as well as, RIVA showedinsignificant changes in BDNF levels, rendering BDNF nearly to itsnormal levels in both treatments, as compared to control rats.While both treatments showed, a significant increase in BDNFlevels, by 18.32 and17.82%, respectively, for VLP extract and RIVA,as compared to AD-induced rats.
3.2.8. Histopathological investigationsRegarding the histopathological study of the cerebellum, Fig. 8,
the sections of control rats showed normal histological featureswith the well-organized three cortical cell layers, large Purkinjecells, and the dense layer of granular cell layer in the cerebellum(Fig. 8a). The same normal intact histological features wereobserved in brains of VLP-treated rats (Fig. 8b). Al-treated ratsshowed marked histological changes and showed decreasednumber of cells in the granular and Purkinje layers, indicatingcellular degeneration and atrophy. Sections of AD-brains showedsome histological changes, including thin, irregular reduction in
ze by different rat groups.
ne 2nd trial (sec.) Induction 3rd trial (sec.) Treatment
16.57c� 4.70 14.88a� 1.1414.45c� 0.98 15.00a� 2.77�12.79 0.8125.39e � 3.12 23.36e� 4.6653.23 56.9920.00b� 1.57 18.43c� 3.8220.70 23.86�18 �21.1021.83b� 1.83 19.76c� 1.3631.74 32.80�14.02 �15.41
) are significant at p � 0.05.
Fig. 1. Therapeutic effects of VLP extract and RIVA drug on serum and brain levels of acetylcholine (ACh) in AD-induced rats. Data are presented as mean � SE, (n = 10). Meanwith different superscripts (a–c) are significant at p � 0.05.
Fig. 2. Therapeutic effects of VLP extract and RIVA drug on serum and brain levels of acetylcholine esterase (AChE) in AD-induced rats. Data are presented as mean � SE,(n = 10). Mean with different superscripts (a–d) are significant at p � 0.05.
I.H. Borai et al. / Biomedicine & Pharmacotherapy 93 (2017) 837–851 841
Fig. 3. Therapeutic effects of VLP extract and RIVA drug on serum levels of dopamine (DA) in AD-induced rats. Data are presented as mean � SE, (n = 10). Mean with differentsuperscripts (a–c) are significant at p � 0.05.
Fig. 4. Therapeutic effects of VLP extract and RIVA drug on serum levels of total antioxidant capacity (TAC) in AD-induced rats. Data are presented as mean � SE, (n = 10). Meanwith different superscripts (a–c) are significant at p � 0.05.
Fig. 5. Therapeutic effects of VLP extract and RIVA drug on serum levels of total homocysteine (tHcy) in AD-induced rats. Data are presented as mean � SE, (n = 10). Mean withdifferent superscripts (a–d) are significant at p � 0.05.
842 I.H. Borai et al. / Biomedicine & Pharmacotherapy 93 (2017) 837–851
cell size of the molecular layer, distortion of the granular cell layerand scattered, sparse cell distribution of Purkinje layers (Fig. 8c).Both RIVA and VLP treatments (Fig. 8e and d) alleviatedneurodegenerative brain changes, resulted in almost normalhistological features, and demonstrated normal cytoarchitecturewith increased number of cells in the granular and Purkinje layers.
Concerning the histopathological study of cerebral cortices,Fig. 9), negative control rats showed the normal intact histopath-ological architecture of cerebral cortices (Fig. 9a). In addition, thecerebral cortices of VLP-treated control rats showed no histopath-ological changes (Fig. 9b), as compared to normal control rats.Meanwhile, cerebral cortices of AD-rats showed neurodegenera-tion, diffuse gliosis, congestion of cerebral blood vessel, as well as,amyloid (Ab) plaques deposition and formation (Fig. 9c).
Examined sections from rats treated with either RIVA or VLPrevealed almost normal histopathologic structure of the cerebralcortices (Fig. 9d and e), indicating neurorestorative effect oftreatments.
3.2.9. Comet assayTable 2, Figs. 10 and 11 demonstrated AlCl3-induced DNA
damage in the brain tissue of treated rats. They were significantdifferences in three comet parameters; comet tail length (CTL), tailmoment (TM) and DNA%, in samples of single brain cells ofdifferent groups.
The Comet assay showed representative photomicrographs offluorescent images of damaged DNA. The mean tail length ofcomets measured in normal brain cells was 2.13 � 0.16, with the
Fig. 6. Therapeutic effects of VLP extract and RIVA drug on serum levels of interleukin 6 (IL-6) in AD-induced rats. Data are presented as mean � SE, (n = 10). Mean withdifferent superscripts (a–c) are significant at p � 0.05.
Fig. 7. Therapeutic effects of VLP extract and RIVA drug on serum levels of brain-derived neurotrophic factor (BDNF) in AD-induced rats. Data are presented as mean � SE,(n = 10). Mean with different superscripts (a, b) are significant at p � 0.05.
I.H. Borai et al. / Biomedicine & Pharmacotherapy 93 (2017) 837–851 843
corresponding tail intensity of 1.86 � 0.18 and TM of 4.01 �0.70.Genotoxicity with AlCl3 induced a significant increase in CTL(4.03 � 0.04) with the corresponding tail intensity of 3.99 � 0.07and TM of 16.10 � 0.15. As compared to negative control rats, VLP-rats showed a marked decrease of CTL by 45.07%, accompanied by adecrease of DNA intensity by 36.02 and 65.09% for TM. AD-inducedrats showed a marked increase of CTL by 89.20%, accompanied by114.52% for DNA%, and 301.50 for TM. Treatment of AD-rats withVLP extract and RIVA showed, marked reduction in TL, DNA% andTM levels, with a percentage decrease of 70.89, 100, and 237.91%,respectively for VLP, and by 46.48, 68.82, and 144.39%, respectivelyfor RIVA.
Compared to genotoxic AD-rats; AD-VLP rats showed, markeddecrement in CTL, % DNA and TM levels, with percentage decrease9.68, 6.77, and 15.84%, respectively, for VLP and by 22.58, 21.30, and39.13%, respectively, for RIVA. DNA strand breaks (fragmentation)caused by AlCl3, analyzed through comet assay in brain cells,revealed an increase by 89.20%, 114.52%, and 301.50% in TL, DNA%,and TM. Administration of VLP and RIVA decreased the level of DNAdamage as expressed by the mean length of comets observed in9.68%, 6.77% and 15.84% in CTL, DNA% and TM, respectively, and22.58%, 21.30% and 39.13% in CTL, DNA%, and TM, respectively, ascompared to Al-intoxicated rats. RIVA treatment demonstratedgreater impact than VLP in alleviating DNA damage (p < 0.05).
4. Discussion
Sporadic AD (SAD) accounts for more than 90% of AD patients,the rest accounts for familial AD (FAD) that is caused by mutationsin certain genes; the majority of SAD cases is ageing-dependent [6].
Relatively little work has been done on the neuroprotective effectof the polyphenolic extract of grape leaves against aluminium-induced dementia in rats. This study aimed to investigate thepotential therapeutic neuromodulating impacts of VLP in AD-induced rats.
AlCl3 is cholinotoxin (neurotoxin) that provokes functionalalterations in the cholinergic, dopaminergic and noradrenergicneurotransmission; therefore, it has the propensity to causeimpaired cholinergic transmission by affecting the synthesis andrelease of neurotransmitters [35]. Impaired cholinergic transmis-sion occurs in two ways: First, it occurs either due to decline in AChrelease or due to decreased choline acetyltransferase activity,which results in the scarcity of ACh. Second, elevated AChE activityfurther adds to scarcity of ACh at the synapse by acceleratingdecomposition of available ACh, this degradation of ACh isabolished by effective RIVA (AChE-inhibitor) [36]. Moreover, acetylCo-A synthesis relies on pyruvate formation through an energy-dependent glycolysis, which was also found to be altered andjustified the deterioration in ACh levels and AChE activity [37].
Furthermore, Al-induced oxidative disruption in membranefluidity/composition can also affect the membrane-bound AChEactivity; thus also corroborated the decreased AChE activity [38].Our data showed that administration of AlCl3 induced cholinergicimpairment in AD-induced rats; represented by a marked decreasein brain and serum ACh levels and a significant increase brain andserum AChE activities, as compared to control group. These resultsrun in accordance with those of Mohamd et al. [39] and Mahdyet al. [40] who stated that AlCl3 administration produced asignificant elevation of AChE activity in the brain, as compared tothe neurologically normal control rats.
Fig. 8. Histopathological investigation of brain sections of AD-induced rats and different treated groups with VLP extract and RIVA drug. (a): Negative Control group showedintact histological structure of cerebellum with well-defined molecular (black arrow), granular (yellow arrow), and Purkinje layers (red arrow), and presence of numerousclosely packed small cells in the granular layer as well a large Purkinje cells in the Purkinje cell layer (H&E stain, �100, �400). (b): VLP- treated normal rats showed normalcerebellum with well-defined molecular (black arrow), granular (yellow arrow) and Purkinje layers (red arrow) (H&E stain, �100, �400). No recorded histopathologicalfindings were found. (c): AD-rats showed the abnormal cerebellum with thin, irregular reduction in cellular size of the molecular layer (black arrow), distortion of granularcell layer (red arrow) and scattered, sparse cell distribution of Purkinje layers (yellow arrow) (H&E stain, �100, �400). (d): AD-rat treated with RIVA showed almost normalcerebellum with well-defined molecular (black arrow), granular (red arrow), and Purkinje layers (yellow arrow) (H&E stain, �100, �400). (e): AD-rat treated with VLP showedcerebellum that appear more or less as normal one, with well-defined molecular (black arrow), granular (yellow arrow) and Purkinje layers (red arrow, notice the darkneurons with hyperchromatic nuclear chromatin) (H&E stain, �100, �400).
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Fig. 9. Histopathological investigation of cerebral cortices (neocortices) of AD-induced rats and different treated groups with VLP extract and RIVA drug. (a): Negative Controlgroup demonstrated normal intact histological structure of the cerebral cortex (neocortex). Several pyramidal cells, with large nuclei, are present within perineuronal spaces(red arrow), in addition to the presence of small glial cells (black arrow) (H&E, �400). (b): VLP-treated normal group showed intact cerebral cortex (neocortex) and healthypyramidal neurons with large nuclei (red arrow), in addition to the presence of small glial cells (yellow arrow) (H&E, �400). (c): AD-rats demonstrated deposition of amyloidplaques of different sizes (black arrow) and showed an extensive neuronal loss and congestion with perivascular oedema (yellow arrow) (H&E stain, �400). (d): AD-rat treatedwith RIVA showed almost normal cerebral cortex with numerous pyramidal cells (yellow arrow) and glial cells (red arrow) (H&E stain, �400). (e): AD-rat treated with VLPrevealed almost normal cerebral cortex (neocortex) with several neurons, that appear more or less like normal, within perineuronal spaces (red arrow) and glial cells (yellowarrow) (H&E stain, �400).
Table 2Oxidative DNA damage observed by comet assay, in the brains of different therapeutic groups.
Groups Tailed% Untailed% CTL (mm) Tail DNA% TM
Negative control Mean � SE 5.00a� 0.58 95.00d� 0.58 2.13b� 0.16 1.86b� 0.18 4.01b� 0.70Negative VLP Mean � SE 4.33a� 0.67 95.67d� 0.67 1.17a� 0.08 1.19a� 0.02 1.40a� 0.095
% Change to Control �13.4 0.705 �45.07 �36.02 �65.09AD Mean � SE 17.67d� 1.45 82.33a� 1.45 4.03e� 0.04 3.99d� 0.07 16.10e� 0.15
% Change to Control 71.70 �13.34 89.20 114.52 301.50AD-RIVA Mean � SE 10.00b� 0.58 90.00c� 0.58 3.12c� 0.09 3.14c� 0.06 9.80c� 0.40
% Change to Control 100 �5.26 46.48 68.82 144.39% Change to AD �43.41 9.32 �22.58 �21.30 �39.13
AD-VLP Mean � SE 13.33c� 0.67 86.67b� 0.67 3.64d� 0.06 3.72d� 0.05 13.55d� 0.37% Change to Control 166.6 �8.77 70.89 100 237.91% Change to AD �24.56 5.27 �9.68 �6.77 �15.84
(CTL): comet tail legnth, (TM): tail moment. Tail Moment (unit) = Tail length � % total DNA. Data are presented as mean � SE (n = 10). Mean with different superscripts (a, b, c, d)are significant at p � 0.05.
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Our results declared that the activities of serum/brain AChEwere significantly inhibited whereas; serum/brain ACh contentswere markedly increased when AlCl3-intoxicated rats received VLP
treatment; suggesting the potency of VLP to regulate cholinergicfunction, possibly by decreasing AChE activity and inhibiting thebreakdown of brain ACh. This finding could be attributed to the
Fig. 10. AlCl3-induced oxidative DNA damage in the brain tissue of treated rats. Slides 1st row (1, 2, and 3): The negative control rats showed insignificant DNA damage, Slides2nd row (4, 5, and 6): AD-group revealed a remarkable DNA damage. Slides 3rd row (1, 2, and 3): The VLP-alone group showed insignificant DNA damage, Slides 4th row (4, 5,and 6): AD-RIVA group demonstrated lesser percent of DNA damage, as compared to AD-induced group, Slides 5th row (7, 8, and 9): AD-VLP group showed significantreduction of oxidative DNA damage.
Fig. 11. Percentage change in Comet Tail Length (CTL), tail DNA% and Tail Moment (TM) in different groups, as compared to the control group.
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high polyphenolic content of VLP extract, suggesting its possiblepotential role in the clinical treatment of AD and proving the abilityof Vitis polyphenols to reach the brain by crossing the gastrointes-tinal tract (GIS) and blood-brain barrier (BBB), as well as, exertingmemory-enhancing effect. These results were supported by aprevious study by Pervin et al. [41], which suggested that grapes-derived polyphenols could offer AChE inhibitory activity andantioxidant effects, therefore ameliorating damaged membraneintegrity. Grape-originated polyphenols might exert the same
anticholinergic effect as sesamol (SML) -a component of sesameoil- [35].
In addition, aluminium (Al) exposure to rats induced significantdecrease in dopamine (DA) level, this runs in parallel with Singlaand Dhawan [42], who indicated the negative impact of Al onmemory function. The adverse effects of Al on cognitive behavior inrats and mice were observed [43]. This may be due to interventionof Al with the dopaminergic system [42], and also to its ability toinduce oxidative stress. Oxidative stress and inflammation cause
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deficiency of several major neurotransmitters, including ACh andDA [44]. Furthermore, the significant alterations of brain neuro-transmitters in Al-exposed rats may be related to increasedformation of O2
� and H2O2,and aggregation of Lewy bodies in thebrain; thus increasing the risk for neurodegenerative diseases [45].Further, DA oxidation is enhanced by the increased concentrationof iron in Al-exposed rats, leading to the production of DAquinones, that covalently interact with cysteine residues ofglutathione (GSH) enzymes inhibiting its antioxidative activity[46].
Moreover, Al promotes the aggregation of a-synuclein anddecreases DA-binding receptors (D1 and D2) in the brain cortexand striatum of Al-exposed subjects, as well as, it exerts inhibitingactivity on different levels; including inhibition of DA b-hydroxy-lase (responsible for the conversion of DA into norepinephrine) andinhibition of tryptophan decarboxylase activity (responsible for DAformation) [47]. The decreased level of DA and altered cholinergicfunction might also be attributed to increased monoamine oxidase(MAO) activity, that led to increased degradation of dopamine [42].Collectively; neurotransmission is negatively modified by Al;either by directly inhibiting the enzymes involved in neurotrans-mitter synthesis and/or utilization or by affecting the structuralproperties of synaptic membranes that could affect the releaseand/or uptake of these molecules [43].
Supplementations of VLP extract, as well as, RIVA drugincreased DA levels. The DA recovery in AD-VLP rats could beascribed to the potential role of polyphenols in restoring theactivity of enzymes involved in the synthesis of neurotransmitters.However, VLP extract displayed recovery level that is stillsignificantly lower than that of normal control rats. A study byShukitt-Hale et al. [48] showed that rats that drank 10% grape juiceshowed improvement in DA release from brain striatal and incognitive performance of the Morris Water Maze.
Concerning oxidative stress implication in Alzheimer's demen-tia, the present study showed a significant decrease in TAC level,suggesting that Al intake promoted oxidative injury by decreasingthe activity of free radical scavenging enzymes and enhancing ROSlevels in the brain which is characterized by high lipid content andoxygen turnover and low mitotic rate [49]. Lukyanenko et al.,Newairy et al., and Shrivastava reported similar findings [50–52].Additionally, the other inhibitory mechanism of TAC may berelated to Al-induced decreased mRNA expression of endogenousantioxidants [53].
Herein, VLP- treated rats showed significantly enhancedactivity of TAC bringing it to almost normal levels; suggestingthat polyphenols can protect against peroxidative and free radicalsmediated oxidative injury, as previously reported by Schaffer et al.[54].
Total Homocysteine (tHcy) is one of the important markers ofendothelial dysfunction [55]. Therefore, tHcy could directly harmthe brain or its vasculature under some conditions [56].Noteworthy, serum levels of tHcy and TAC are inverselycorrelated, as reported by Besler and Como�glu [57] and Ismailet al. [58]. This runs in agreement with our results of declined TACand elevated tHcy serum levels in AD-induced rats. Hyper-homocysteinemia produce structural and functional change ofcerebral blood vessels along with increased vascular oxidativestress [59], leading to cerebral vascular endothelial dysfunctionthat could enhance progression of AD [60]. This could beexplained that during inflammatory status, BBB becomesdysfunctional and permeable; enabling proteins, only foundnormally in serum, to enter cerebrospinal fluid (CSF) [61]. tHcycan cross BBB through carrier receptor-mediated transport,hence; the brain tHcy concentration should be similar to thatof blood, elevated blood tHcy compromises the integrity of theBBB [62]. Treatment of AD-rats by either VLP or RIVA showed a
significant decrease in tHcy levels, proving the antioxidativeactivities of polyphenols against AlCl3-induced endothelialdysfunction.
AD-induced rats showed a marked increment of the proin-flammatory cytokine (IL-6), as compared to negative control. Thisresult agrees with Waetzig et al. [63], and demonstrates that AlCl3-induced neurotoxicity is due to accumulation of abnormal proteinaggregates, like Ab-42 and free radicals (NO, ROS and RNS), thattrigger cellular stress and neuroinflammation by activation of thebrain’s innate immune system involving microglia and astrocytes[35].
This rise in IL-6 was counteracted by treatment of AD-rats byeither VLP or RIVA, through increasing ACh level and acting onnicotinic a7 receptors, this peripheral increase in ACh mightdecrease the inflammatory signal to the brain by suppressing theproduction of proinflammatory cytokines by macrophages [64].We could suggest that, VLP showed anti-inflammatory, antioxi-dant, and immunomodulatory activities to prevent neuroinflam-mation, by down regulating the inflammatory activation ofimmune cells, and subsequently inhibiting the release of proin-flammatory IL-6, and finally correcting the cognitive dysfunction.
BDNF is involved in the regulation of working memory andbehavioral processes, as it controls synaptic plasticity, promotesthe survival of striatal dopaminergic neurons and plays a criticalrole in maintaining normal prefrontal cortex function [65].Furthermore; it was revealed that decreased expression of BDNFmight be strongly implicated in the pathophysiology of AD [66].Therefore, serum BDNF level may represent a biomarker ofneurocognitive correction in AD-subjects.
Aluminum intoxication was found to cause a synergisticincrease in proinflammatory cytokine (IL-6) production, as wellas, a dramatic decrease in BDNF; proving the reciprocal relation-ship between pro-inflammatory cytokines and neurotrophicfactors in CNS [67]. Herein, serum BDNF levels were significantlydecreased in AlCl3-treated rats, as compared to negative controls,this change in serum BDNF levels might contribute to shrinkage ofthe hippocampus that is correlated with age-associated neuro-cognitive decline in late adulthood [68]. Moreover, Al inducesformation of Ab-plaques, which is linked to decreased brain BDNFlevel and function [69], as well as, it reduces BDNF signaling byimpairing axonal retrograde transport, and ultimately contributesto the synaptic dysfunction in AD [70].
Treatment of AD-rats with either VLP or RIVA produced asignificant increase in serum BDNF levels, restoring it to its normallevel. Therefore, treated AD-rats showed an inhibitory effect oninflammatory cascade, followed by the decrement in production ofproinflammatory cytokine that leads to stimulated release ofneurotrophic factors and subsequently BDNF production. Hence,Vitis polyphenols might act as AChE inhibitor, elevating ACh and IL-6, inhibiting AChE and consequently increasing BDNF level.Moreover, the anti-inflammatory effect of the bioactive constit-uents of this polyphenolic extract could pool to this assumption.Rendeiro et al. [71] estimated that increase in BDNF was associatedwith enhancement of memory and the underlying molecularmechanisms in rats administrated pure flavanols; thus confirmingthe therapeutic value of polyphenol extracts in cognitiveimprovement.
Assessment of cognitive ability is a requirement for evaluatingany possible CNS depressant/stimulant effect of interventions onrats. In this study, we demonstrated that AD group showedincreased elapsed (transit) time to receive the reward �afterovernight fasting- displaying significant cognitive deficits anddecreased working memory and learning that finally points to theneurocognitive decline. This runs in agreement with Umesalma[72] who reported that the performance of the AlCl3- treated rats,in contrast to neurologically normal control rats, did not progress
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with repetitive trials. Furthermore, our findings also agreed withYassin et al. [73], who observed that AlCl3-treated rats showed anincrease in the length of time taken to attain the food in the T-mazetest, establishing that aluminum is a neurotoxic agent.
Interestingly, treatment of AD-rats with VLP demonstrated asignificant reduced transit time to achieve the task and suppressedthe neurotoxic effect of AlCl3, as compared to AD- rats. VLPameliorated AlCl3-induced memory impairment probably bymodulating synaptic functioning and enhancing neuronal survival.This runs in agreement with previously published data [11].
Biochemically, VLP administration to AD-rats showed a markedamelioration in neurocognitive abilities through increasing AChlevels, and decreasing AChE levels, as well as, restoring TAC level.Thus, the memory-improving activity of VLP might be attributed totheir anticholinesterase, antiamnesic, pro-cholinergic, anti-in-flammatory, antiapoptotic, and antioxidative activities. Polyphe-nols, as antioxidants, could lead to the inhibition of neuronalapoptosis induced by neurotoxicants (such as AlCl3) and to thepromotion of neuronal survival and synaptic plasticity [74]. Inaddition, most polyphenols or at least key metabolites, can crossBBB in sufficient concentrations, reach the brain and exert an effecton cognition by having the potential to inhibit brain/serum AChEactivities of the cholinergic system that are involved in thememory retention process [11].
Furthermore, Al-induction caused a remarkable DNA damage,assessed by agarose gel electrophoresis, as indicated by theincreased DNA fragmentation and the number of observedcomets, demonstrating the genotoxicity in the Al-induced cellas compared to control cell. DNA fragmentation and increasedcomets had been previously reported as a consequence of Al-neurotoxicity [75]. Moreover, the present finding is in agreementwith Sumathi et al. [76], who showed that Al neurotoxicity canlead to faster neural apoptosis, as seen in the micrographs whichclearly showed DNA damage and disruption of cells. Thisoxidative DNA damage induced by Al might be ascribed to itschemical nature of being a trivalent cation with a high affinity fornegatively charged groups such as phosphates and phosphory-lated proteins in nucleic acids. Therefore, Al may bind to DNA andRNA, inhibit the activity of enzymes, enhance the peroxidativedamage of lipids and decrease the antioxidant status of the ratbrain [77]. Normally, DNA in the brain may be protected fromoxidation by a key mechanism implied in the dendriticremodeling subsequent to stress, which is glucocorticoid-inducedglutamatergic excitotoxicity [78].
On the other hand, VLP or RIVA treated AD-rats displayed areduction in the DNA damage, as compared to AD rats. AD-VLP ratsrevealed neurorestorative effects, as demonstrated by decreasedoxidative DNA damage in brain tissue; possibly by increasingantioxidants by donating electrons to unstable, reactive species,rendering them more stable and unreactive; thus preventingdegradation of nucleic acids in AD-rats, thus maintaining theproper functioning of the brain.
These findings imply the ability of VLP extract to correct the Al-mediated neuronal, as well as, behavioral changes. Therefore, itcould be regarded as an alternative to the chemically synthesizedRIVA drug. The possible underlying mechanisms of neurorestor-ative activity could include: direct effects on signaling to enhanceneuronal communication, increased neurogenesis and suppressedneural apoptosis, alterations in neuronal structure, enhancementof neuroprotective stress, shock proteins, alteration of inflamma-tory gene expression and protection against neurodegenerationafter excitotoxic stress, and reduction of microbial and astrologicalactivation [79].
The aforementioned results, either biochemical or behavioral,were confirmed by histopathological examination of brain sectionsof rats, as Al-treated rats revealed altered behavioral alterations
within four weeks, and showed that Al neurotoxicity causedmarked alterations such as heavy loss of cortical neurons, ghostcells and vacuolated cytoplasm in the cerebellum [77]. Concerningcerebral cortices of AD-rats, Ab-plaques are defined as extracellu-lar deposits of Ab abundant in the cortices of AD subjects [80]. Ourresults run in agreement with Ahmed et al. [20] who demonstratedthat Al-neurotoxicity induces the formation and deposition of Ab-plaques that appeared with a dark center, neuronal damage, anddegeneration in the cerebral cortex. On the other hand, aremarkable amelioration of brain architecture was observed inboth AD-VLP and AD-RIVA groups since the brain cells appearedmore or less similar to the cells of the control group, accompaniedby the disappearance of most of amyloid plaques in the cerebralcortex. These results are in agreement with those of Bihaqi et al.[81] who revealed that RIVA reversed/normalized Al-inducedhistopathological alterations. Virtually, AD-VLP rats revealedreduced brain damage by reducing AlCl3-induced modificationsand improving the architecture histology, and finally amelioratingfunctional outcome, as observed in T-maze test, this indicates thestrong potential of polyphenols to limit or delay neurodegenera-tion, and to prevent or reverse the Al-induced cognitive deteriora-tion.
Finally, acetone was chosen as a solvent for extraction ofpolyphenols in this study, as it was found that using 80% acetone inthe crude extraction of phenolic compounds from grapevine leavesresulted in a higher content of total phenolics, than using 80%methanol [16]. In addition, Tatiya et al. [82] demonstrated thatusing acetone as an extraction solvent showed the highestpolyphenol content and highest antioxidant activity. Moreover,aqueous acetone is a good extraction solvent for polar antioxidantsand its use demonstrated higher extraction efficiency than othersolvents [83]. According to Pinelo et al. [84]; acetone is the bestsolvent for flavonols and flavan-3-ols.
VLP acetone extract is characterized by the highest polyphe-nolic content mainly composed of flavonoidal compounds. Thebehavior of VLP extract as a potent antioxidant, that amelioratedAl-induced AD, might be due to its high flavonoidal content. Thebehavior of VLP extract as a potent antioxidant, that amelioratedAl-induced AD, might be due to its high flavonoidal content. Itwas revealed that flavonoid structure; the number and location ofthe hydroxyl moieties, the presence of 2,3-double bond inconjugation with a 4-Oxo function in ring C, 3- and 5-OH, 3,5,7-tri-hydroxy, ortho-catechol group (30,40-OH), in addition to theglycosylation model (C-glycosides or O-glycosides) and position,played a critical role in antioxidant activity [85]. According to theirantioxidative effect, the potential mechanism of action offlavonoids is their interaction with neuronal signaling cascades,leading to decreased apoptosis and enhanced neuronal survival[86].
Additionally, brain bioavailable polyphenols are considered asAD-modifying agents that could halt or prevent the onset ofdifferent neural disorders before the onset of clinical symptoms,including AD-dementia. It was found that that multiple polyphenolmetabolites are able to cross BBB and penetrate the brain atpharmacologically relevant concentration, for example, quercetin-3-O-glucoside is one of brain-penetrating polyphenol metabolitesthat could modulate AD neurodegenerative pathways [87].
Virtually; nutritional intervention studies must be carried outto confirm that polyphenols can modulate brain health andfunction, and thus could provide nature-based strategies aimed atdelaying or preventing age-associated cognitive decline and thedevelopment of neural diseases, as well as, sustaining the idea ofhealthy brain ageing [11]. Therefore, several studies, concernedwith neurotrophic activity, neurobehavioral, and neurodegenera-tive disorders, are interested in the design of alternativeimmunomodulatory strategies based on dietary interventions.
I.H. Borai et al. / Biomedicine & Pharmacotherapy 93 (2017) 837–851 849
Conclusion
In AlCl3-induced Alzheimer’s disease, a model of oxidativestress-mediated neurodegeneration, grape leaves polyphenols(VLP) extract was effective in reversing the aluminium-inducedneurotoxicity in AD-rats, by reducing brain damage and amelio-rating the functional outcome, as shown in behavioral T-maze testand confirmed by comet assay and histopathological investiga-tions. This neuromodulatory effect of VLP extract was achievedthrough neurorestorative, antiapoptotic, anti-inflammatory, anti-cholinesterase, antioxidative, and antiamnesic activities againstAlCl3-induced cerebral damages. Overall, this study showed thatpolyphenols-based treatment could represent a beneficial thera-peutic approach against neurobehavioral and neurochemicalchanges associated with Alzheimer's dementia.
Future studiesFurther work on the molecular level is needed to illustrate the
mechanisms underlying the therapeutic neuroprotective effects ofVLP against AlCl3-induced behavioral and biochemical changes.Furthermore, it is needed to estimate the most neuroactiveformulations, whether through the diet or supplement, tosubsequently design and perform informative clinical trials.
Conflict of interest
The authors declare that no conflict of interest.
Acknowledgments
The authors would like to acknowledge Dr. Dalia Osama,Researcher at Pharmacology department, Medical research divi-sion, National Research Center (NRC), for supplying us with T-mazeinstrument. The corresponding author would like to thankreviewers for their in-depth comments that improved themanuscript.
FundingThis work was supported, under number (2-2-10), by National
Research Center (NRC), Egypt.
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