Neuroprotective effect of Indian propolis in β-amyloid induced memory deficit: Impact on behavioral and biochemical parameters in rats

Biomedicine & Pharmacotherapy 93 (2017) 543–553

Available online at

ScienceDirect www.sciencedirect.com

Original article

Neuroprotective effect of Indian propolis in b-amyloid induced memory deficit: Impact on behavioral and biochemical parameters in rats Sadhana Nanawarea , Madhuri Shelara , Arulmozhi Sinnathambib , K.R. Mahadika , Sathiyanarayanan Lohidasana,* a b

Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune, 411038, India Department of Pharmacology, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune, 411038, India

A R T I C L E I N F O

Article history: Received 4 April 2017 Received in revised form 27 May 2017 Accepted 20 June 2017 Keywords: Indian propolis Neuroprotective activity Behavioural model Acetylcholinesterase Brain-derived neurotropic factor Oxidative stress

A B S T R A C T

The study aimed at the investigation of neuroprotective activity of macerated ethanolic extract of Indian propolis (MEEP) against b-Amyloid 25–35 (Ab25-35) induced memory impairment in Alzheimer’s disease. MEEP was administrated orally to Wistar rats at doses of 100, 200 and 300 mg/kg. Behavioral performances were evaluated using morris water maze and radial arm maze. At the end of behavioral study, the brains were removed and antioxidant parameters and brain monoamines were estimated. Further acetylcholinesterase (AchE) inhibition and brain-derived neurotropic factor (BDNF) were evaluated. In addition hematological parameters and histopathological tests were also carried out. In behavioral models, MEEP significantly (P < 0.05) reversed the cognitive impairment of b amyloidinduced rats. The antioxidant potential was significantly increased (P < 0.05) after administration of MEEP. Malondialdehyde levels were significantly (P < 0.01) decreased in brain homogenate after treatment with MEEP extract as compared with diseased control group (group III). MEEP showed dosedependent AChE inhibition and increased the levels of brain monoamines (P < 0.05) as compared with group III. MEEP improved memory deficits by increasing BDNF in plasma (P < 0.05). The study concludes that MEEP has anti-Alzheimer potential in rats through multiple mechanisms and further studies are ongoing for fractionation and biological screening. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction Alzheimer’s disease (AD) is the chronic, progressive, neurodegenerative disease of central nervous system. As per the recent

Abbreviations: MEEP, macerated ethanolic extract of propolis; AD, Alzheimer’s disease; Ab25-35, beta amyloid 25–35 protein; CAPE, Caffeic acid phenethyl ester; GAL, Galangin; PINO, Pinocembrin; RP-HPLC PDA, reverse phase-high performance liquid chromatography with photodiode array; MWM, Morris water maze; RAM, radial arm maze; BDNF, brain derived neurotrophic factor; ELISA, sandwich enzyme-linked ImmunoSorbent assay; AchE, acetylcholinesterase; ICV, intracerebroventricular route; SOD, superoxide dismutase; GSH, Glutathione; NO, nitric oxide; CAT, catalase; MDA, malondialdehyde; TBA, thiobarbituric acid; NE, norepinephrine; DA, dopamine; IP, isoprotrenol; 5-HT, 5-hydroxy tryptamine; p. o., per os. * Corresponding author at: Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane, Pune, 411038, India. E-mail address: [email protected] (S. Lohidasan). http://dx.doi.org/10.1016/j.biopha.2017.06.072 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

report an estimated 5.2 million people of all ages have AD in U.S. which includes an estimated 5 million people age of 65 and older and approximately 200,000 individuals under age of 65 who have younger-onset of Alzheimer’s [1]. The pathological features of AD are mainly accumulation of amyloid-b (Ab) peptide with senile plaques and intraneuronal deposits of hyperphosphorylated tau protein produce neurofibrillary tangles [2]. Extracellular deposition of b-amyloid plaque is the main component of b-amyloid protein, which is a small polypeptide formed from a b-amyloid precursor protein (APP), while intracellular neurofibrillary tangles are composed of tau protein which is paired helical filaments of microtubules [3,4]. Oxidative stress is considered as one of the major causes of neurotoxicity in AD [5]. The defining feature of AD is the deposition of b-amyloid (Ab) peptide in neuritic plaques and the level of Ab well associated with the extent of cognitive impairment [6]. Several studies have demonstrated that oxidative stress is involved in Ab induced neurotoxicity and progression of AD [7,8]. Ab25-35 seems to be toxic

544

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

and occurs oxidative stress-mediated changes in hippocampal long-term potentiation [9], induction of nitric oxide synthase [10], protein nitration and protein oxidation leads to brain damage [11,12]. Many naturally occurring compounds or extracts have been recommended as potential therapies for treating various ailments and also to treat or prevent Alzheimer’s disease by acting as an antioxidant and by anti amyloid action [1,13]. One of such medicinally important natural products is propolis, a resinous and strongly adhesive material collected by honeybees [14]. Propolis is reported to contain at least 200 compounds which include phenolic acids and esters, substituted phenolic esters, flavonoids, steroids, aromatic aldehydes and alcohols etc. [15]. Propolis has remarkable therapeutic qualities, such as antibacterial [16], anti-inflammatory [17,18], antiatherosclerotic [14], antioxidant [19,20] and anticholinesterase [21,22] activities. Some of the propolis from different geographical sources such as Brazilian propolis has been reported to have neuroprotective activity [23]. In addition, some of the chemical constituents of propolis such as Caffeic acid phenethyl ester, Pinocembrin, and Galangin have been reported to have their potential in neuroprotection [24–26]. However, Indian propolis is not yet reported for neuroprotective activity besides its vast chemical constituents. The complexity of chemical constituents of any natural products is always a challenge for standardization. Even though, few studies have been reported for the chemical profile of Indian propolis, the standardization with respect to marker compounds targeting neuroprotective activity is not achieved. Hence the aim of this study is to standardize and investigate the neuroprotective effect of ethanolic extract of Indian propolis against Ab25-35 induced memory impairment in rats and to investigate the underlying mechanism for the activity. 2. Materials and methods 2.1. Material and chemicals A beta amyloid 25–35 (Ab25-35) was procured from Sigma Aldrich, Delhi, India and stored at 20  C. Anaesthetizing agent’s ketamine and xylazine were supplied from the local market. Indian propolis was collected from local beekeeper Bharatpur, Rajasthan, India, in the month of December and was authenticated from Central Bee Research and training Institute, Pune, Maharashtra, India (Authentication no: CBRTI/BOT/01/2015-16/335). Standard AChE enzyme was procured from Sigma Aldrich, Delhi, India. The pure Caffeic acid phenethyl ester (CAPE) was procured from Sigma Aldrich, Bangalore, Karnataka, India. Galangin (GAL) was procured from Natural remedies Pvt. Ltd., Bangalore, Karnataka, India, and Pinocembrin (PINO) was procured from Mira Biotechnology, China. All stock solutions of standard markers were prepared in ethanol individually. All reagents used in this study were of analytical grade, HPLC grade, and high purity. Standard drug used for treatment Donepezil was supplied from Microlabs, Mumbai, Maharashtra, India. 2.2. Macerated ethanolic extract of propolis (MEEP) Accurately weighed quantity of 150 g of crude propolis was mixed with 450 mL of ethanol and was kept for 10–15 days in a dark place at a room temperature with the replacement of solvent after 2 days. The extract was filtered to remove insoluble matter and kept in the deep freezer for overnight at 20  C to remove wax using Whatmann filter paper No. 1. Evaporation of filtrate was done by rotary evaporator at 40  C at 180 rpm. Obtained extract was designated as Macerated Ethanolic Extract of Propolis (MEEP) [27].

2.3. Standardisation of extract by using HPLC-PDA High performance liquid chromatographic method development and validation was carried out on a Jasco Inc. (Easton MD) Model PU 2089 plus intelligent LC pump with manual injector with a loop of 20 mL injection volume with Jasco PDA detector Model MD 2010 plus (Jasco International Co. Ltd. Japan). Recording and processing of chromatographic data was carried out using the Jasco ChromPass version 1.8 LC-Net II/ADC system. The column used was Syncronis C18 (5 mm, 250 mm  4.6 mm i.d.) and C18 guard column 4  3 mm i.d. (Phenomenex). The mobile phase was a mixture of methanol and water (65:35 v/v) used in gradient mode with a flow rate 1.2 mL/min at ambient temperature. Prior to use mobile phase was passed through 0.45 mm membrane filter and degassed under sonicator. The sample solutions of extract were passed through 0.22 mm filters. The run time was kept 20 min, and the injection volume was 20 mL. Detection was performed between 200 and 400 nm and chromatograms were extracted at respective lmax of each marker compound for improved sensitivity. The retention times (Rt) and UV spectra of marker compound from the sample solution were compared with that of the standards and quantification was done using calibration curves of the standard solutions. In addition, the extract was analyzed for polyphenolic content by Folin-Ciocalteu method and flavonoid content by aluminium chloride method [28]. 2.4. Animals Adult male Wistar rats weighing 250–300 g were obtained from National Institute of Biosciences, Pune (India). Rats were housed separately in a polypropylene cages and fed standard pellet diet (Pranav Agro Industries Ltd., Sangli, India), kept under hygienic conditions. Rats were kept on a 12 h light and dark cycles with free access to water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) (1703/POIC/13/CPCSEA) of Poona college of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, India. 2.5. Intracerebroventricular (ICV) administration of Ab25-35 peptide Aggregates of Ab25-35 was prepared in sterile water at a concentration of 1 mg/mL and stored at 20  C. For ICV injection of Ab25-35 through Hamilton syringe, the rats were anesthetized intraperitoneally with ketamine (90 mg/kg) and xylazine (10 mg/ kg). The animals were then stereotaxically injected at coordinates (posterior 3.6 mm; lateral  2.3 mm; dorsal 3 mm) according to Atlas Paxinos and Watson [29]. Single injection of Ab25-35 with total concentration was injected 10 mg/10 mL bilaterally so that 5 mg/5 mL was injected on both sides at the rate of 1 mL/min. The injection needle was left in place for additional 2 min to prevent reflux of the solution. After induction of Ab25-35 peptide, animals were kept for 14 days for recovery. 2.6. Drugs and treatments MEEP was administered by suspending with Tween 80 in three different doses of 100, 200, 300 mg/kg administered by oral route (p.o.). Donepezil as a standard was administered by (0.75 mg/kg) p.o. The animals were divided into seven groups of 8 animals each. The Ab25-35 solution of 10 mg/rat was bilaterally injected into the region through ICV for group III–VII except group I and II. After 14 days recovery of overhead injury, the animals were treated for next 21 days. The MEEP was firstly suspended in (10% v/v) Tween 80 and the resultant solution was completed to the required volume with distilled water. Group I: group with no

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

treatment and were non-operated. Group II: group received 10 mL of the vehicle (Double distilled water) through ICV and treatment for 21 days with 0.1% of Tween 80 administered p.o. (1 mL/kg), Group III: group with no treatment received but operated, Group IV: group received Donepezil (0.75 mg/kg) p.o., Group V: Test group received MEEP (100 mg/kg) p.o., Group VI: Test group received MEEP (200 mg/kg) p.o., Group VII: Test group received MEEP (300 mg/kg) p.o. 2.7. Postoperative care Recovery of anesthesia took approximately 4–5 h. At the time of animals gained full consciousness, they were kept in a wellventilated room at 25  3  C in individual cages. Then animals were housed together in groups of 4 animals per cage. For easy access to animals, without physical trauma due to overhead injury, food and water were kept inside the cages for the first week. Animals were then treated normally with food and water. The bedding of the cages was changed twice per week as usual. Rats were subjected to morris water maze (MWM) and radial arm maze (RAM) activity conducted on 5th, 10th, 15th and 20th day after treatment. The day after MWM and RAM activity, rats were sacrificed for further study. 2.8. Acute oral toxicity tests for MEEP Acute oral toxicity test was performed for the MEEP in Wistar male rats according to the OECD guidelines-425 [30]. The animals were fasted overnight and divided into groups of 5 animals each. MEEP was administered p. o. at different dose levels of 55 mg/kg, 175 mg/kg, 550 mg/kg, 1750 mg/kg, and 2000 mg/kg body weight. The rats were observed continuously for behavioral changes, respiratory, restlessness, tremors, salivation, and diarrhea or any signs of toxicity or mortality from 2 h to 48 h and next to 14 days.

545

01R). Experiments were carried out every day between 9 am and 5 pm. Rats were trained in groups and always taken into experiments at the same time of day with the same order. During the daily sessions, rats were individually placed on the central platform facing different directions and allowed to orient themselves. The alternate four arms were located by various extra-maze visual cues placed at the same position during the complete study and at the end of four remaining alternate arms, baits were placed. The rats were allowed to freely explore the maze for 10 min per day. The maze was cleaned with 70% ethanol after each trial. The animals were trained one session per day for five consecutive days before stereotaxic surgery. The variables commonly used for analysis of the performance are (a) the number of errors in each session (entering an arm that has been visited previously counted as an error) (b) the number of correct choices in the initial 8 chosen arms at the fifth day of training before surgery and at 5th, 10th, 15th and 20th day (4 trials per day) after treatment. An error was defined as a re-entry into an already visited arm. Training was performed at 24-h intervals [32,33]. 2.10. Blood collection At the end of experiment whole blood samples were collected in EDTA tubes and one part has been forwarded for analysis of haematological parameters. The other part of blood samples were centrifuged at 1500  g for 10–15 min and clear plasma/serum was removed carefully to avoid contamination with platelets. Plasma/ serum was stored at 80  C until it could be used for analysis. 2.11. Hematological parameters White blood cell, red blood cell, hemoglobin count, erythrocyte sedimentation rate, platelet count and packed cell volume were analyzed using a diatron diagnostic Abacus Junior automatic hematology analyzer.

2.9. Behavioral assessment 2.12. Biochemical analysis 2.9.1. MWM The MWM activity was performed following the method reported earlier by Morris [31]. It is a black circular pool (Diameter 210 cm, height 50 cm, Model No. VJMWM-01R), used for the assessment of spatial learning and memory ability. The pool was divided into four quadrants (I–IV) with four different visual cues and it was filled to a depth of 30 cm with water containing milk powder. A circular platform (6 cm in diameter, 2 cm below the surface of the water) was then placed in one of the pool quadrants. During the five subsequent days, the rats were given four trial sessions per day with the hidden platform. The time interval between each trial sessions was 30 min. For four trial sessions, rats were placed in the water facing the pool wall in one of the pool quadrant. The entry of each rat was changed in a different order each day. Rat searched the platform and then was permitted to remain on it for 10 s. If the rat did not search the platform within 120 s, it was placed on the platform for 10 s. The animals were returned to its home cage and were allowed to dry up under an infrared lamp after each trial. During each trial session, the mean platform retrieval times and searching distance at the fifth day of training before surgery (5 consecutive days) and at 5th, 10th, 15th and 20th day (4 trials, 120 s per trial per day) after treatment, were calculated for each group and used for statistical analysis. 2.9.2. RAM The radial eight-arm maze (Automated tracking system MazeMaster, V.J. instruments, Washim, Maharashtra, India) used in the present study consisted of 8 arms, made up of black plexiglass, numbered from 1 to 8 (Dimensions: 50  10 cm, Model No. VJRM-

The glucose, triglyceride, cholesterol, HDL-C, LDL-C, total protein, albumin, AST, ALT, Alk-P, in plasma or serum were analyzed using an Accurex biomedical kit (Mumbai). 2.13. Preparation of brain homogenate After the experimental period of 21 days, all the animals were fasted overnight, subjected to anesthesia with diethyl ether and sacrificed. Brains were removed and rinsed with ice-cold isotonic saline. The respective brain tissue homogenates were stored at 80  C and used for further biochemical analysis, that includes oxidative stress (SOD, GSH, NO, CAT and MDA), brain monoamines (NE, DA and 5-HT) and cholinergic marker (AChE). The brain from each group was fixed in 10% formalin for histological investigation. 2.14. Histological study of brain tissues For histological investigation the brains were fixed in formalin (10% v/v) solution and later embedded in paraffin. After which serial sections were taken in 6 mm thickness. Congo red was used for staining of b- amyloid plaque in the regions of the brain [34]. 2.15. Oxidative stress The brain was weighed and homogenized with ice-cold 0.1 mmol/L phosphate buffer (pH 7.4). This homogenate was used for oxidative parameter and AChE analysis.

546

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

2.15.1. Superoxide dismutase (SOD) activity in brain Equal volumes of brain tissue homogenate (supernatant) and distilled water were mixed, to which 0.25 mL of ice-cold ethanol and 0.15 mL of ice-cold chloroform added. The mixture was mixed well using cyclomixer and centrifuged at 600  g at 4  C for 15 min. To this 0.5 mL of EDTA solution was added. The reaction was initiated by the addition of 0.4 mL of epinephrine and the change in optical density/min was measured at 480 nm against reagent blank [35]. 2.15.2. Glutathione (GSH) activity in brain Equal volumes of brain tissue homogenate (supernatant) and 20% TCA were mixed. The precipitated fraction was centrifuged at 600  g at 4  C for 15 min and 2 mL of DTNB reagent was added to 0.25 mL of supernatant. The final volume was adjusted up to 3.0 mL with phosphate buffer. The color developed was read at 412 nm against reagent blank [36].

The supernatant was filtered through a 0.22 mm membrane filter and 20 mL of the filtrate was injected onto HPLC with autosampler AS-1555, JASCO, Japan, JASCO was used. A guard column: C-18, guard column 4  3 mm i.d. (Phenomenex), and analytical column RP-18, 250  4.6 mm (5 mm) Thermosil was used at an ambient temperature. Borwin chromatographic software was used for recording peaks and data integration. Mobile phase was composed of sodium acetate (0.02 M), methanol (16%), heptane sulphonic acid (0.055%), EDTA (0.2 M), and dibutyl amine (0.01% v/v) with adjusted pH 3.92 with o-phosphoric acid. The flow rate was kept at 0.9 mL/min. Detection was done with a Fluorescent detector, JASCO, Japan. After separation, noradrenaline (NE), dopamine (DA), isoprotrenol (IP) and 5-hydroxy tryptamine (5-HT) were detected at the excitation wavelength of 280 nm and an emission wavelength of 315 nm [40].

2.17. Assay for acetylcholinesterase (AChE) inhibition 2.15.3. Nitric oxide (NO) levels in brain Different concentrations of sample solutions of brain tissue homogenate were prepared in 100 mL volumetric flasks. To this 0.1489 g of sodium nitroprusside (final concentration 5 mM) was added and kept for incubation. At different time intervals, 5.6 mL was taken, to which add 0.2 mL of Griess reagent A was added and kept for incubation at 30  C for 10 min. After incubation 0.2 mL of Griess reagent B was added and kept for incubation 30  C for 10 min and absorbance was measured at 542 nm against blank [37]. 2.15.4. Catalase (CAT) levels in brain CAT activity was assessed by using principle based on the ability of CAT to induce the disappearance of hydrogen peroxide, followed the method of Beers and Sizer [38]. One unit of CAT is equivalent to the amount of enzyme to induce decomposition of 1 mmol of peroxide per min. The 1960 mL of substrate (Mixture containing 1800 mL of 10 mM H2O2 and 100 mL of Tris–HCl buffer and 60 mL of D/W) was added to 40 mL of the supernatant of brain tissue homogenate. Activity was determined by measuring the exponential disappearance of H2O2 at 240 nm at 25  C.

2.15.5. Malondialdehyde (MDA) levels in brain A volume of 75 mL of brain homogenate was mixed with 25 mL  of standard and 25 mL of NaOH (3N) and incubated at 60 C for 30 min in a water bath with occasional shaking. After this 125 mL of H3PO4 (6%) and 125 mL of thiobarbituric acid (TBA, 0.8%) were added and the mixture was heated at 90  C for 45 min. After cooling of the mixture, 50 mL of sodium dodecyl sulfate (SDS, 10%) was added. By using vortex mixer, above mixture was extracted with 300 mL of n- butanol and centrifuged at 3000  g for 10 min 20 mL of butanol layer was injected into HPLC with visible detector, and detection was carried out at 532 nm wavelength and using mobile phase water and methanol (50:50, v/v) with flow rate at 0.6 mL/ min. The column was used Thermosil C18 and run was 8 min. The amount of MDA was found in plasma was calculated by calibration plot of standard MDA. For calibration of MDA, six different concentrations (1–6 mM) were prepared from 3 mM stock solution [39]. 2.16. Brain monoamines Rats were sacrificed by decapitation in the end of an experiment. Brains were put into ice-chilled petri dish. Tissue were weighed and homogenized in 2 mL of 0.1 M perchloric acid with addition of internal standard (isoprotrenol) at the concentration of 30 ng/mL and centrifuged at 14,000  g for 15 min at 4  C.

AChE activity was assessed by Ellman’s method. The assay was carried out by taking 0.05 mL of supernatant, and to this 3 mL of sodium phosphate buffer (pH 8), 0.1 mL of acetylthiocholine iodide, and 0.1 mL of DTNB (Ellman reagent) were added. The absorbance of the solution was recorded for 2 min at 30 s interval at 412 nm. The percentage inhibition of the enzyme was calculated using the formula [41]: % inhibition = [(Absorbance of control- Absorbance of sample)/ Absorbance of control]*100 2.18. Brain-derived neurotropic factor (BDNF) sandwich enzymelinked ImmunoSorbent assay (ELISA) Plasma BDNF levels were measured using a BDNF Sandwich ELISA Kit, according to the manufacturer’s instructions (Elabscience, USA, Cat. No. E-EL-R1235). The micro-ELISA plate provided in this kit has been pre-coated with an antibody specific to BDNF. 100 mL of standards or samples were added to the appropriate micro-ELISA plate wells and combined with the specific antibody. Then 100 mL of biotinylated detection antibody specific for BDNF was added immediately and washing of each well was carried. Avidine-Horseradish Peroxidase (HRP) conjugate was added to each microplate well successively and incubated for 30 min free components were washed away. The 90 mL of substrate solution was added to each well. Only those wells that contained BDNF, biotinylated detection antibody, and Avidin-HRP conjugate were appeared blue in color. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the color was turned yellow. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm  2 nm. The OD value was proportional to the concentration of BDNF. A typical range of standard curve was 31.25–2000 pg/mL. 70 samples in 96 well plates were processed in one BDNF- ELISA. The concentration of BDNF in the samples was calculated by comparing the OD of the samples to the standard curve.

2.19. Statistical analysis Data was expressed as means  SEM. The behavioral study results were subjected to two–way analysis of variance (ANOVA) followed by Bonferroni post test. The antioxidants parameters, such as GSH, NO, SOD, CAT, lipid peroxidation, AChE activity and brain monoamines i.e. NE, DA, 5-HT, BDNF results were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test.

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

3. Results 3.1. Standardization of macerated ethanolic extract of Indian propolis MEEP was standardized by using standard markers such as CAPE, PINO and GAL with the help of high-performance liquid chromatography-photodiode array (HPLC-PDA). The chromatograms are shown in Fig. 1. It was stated that ethanolic extract of Indian propolis was showed the presence of CAPE, PINO and GAL with polyphenolic content (20.86  0.63 mg GAE/g) and flavonoid content (8.39  0.047 mg QE/g). It was further used for illustration of neuroprotective activity. 3.2. Acute oral toxicity test There were no animal deaths in the rats receiving 2000 mg/kg of MEEP of Indian propolis. No sign of toxicity was observed in the wellness parameters and mortality during the 14 days of the observation period. 3.3. Behavioral assessment 3.3.1. MWM The effect of MEEP on spatial learning was assessed using the MWM task. When compared escape latency (s) and distance traveled (cm) of the group I and II, b amyloid-induced disease group III took significantly more time and distance (p < 0.001) on all days (Fig. 2). This indicated that b amyloid-induced memory impairment in group III. The non-significant relationship in all groups related to escape latency and distance traveled when compared with group III at the 5th day of training (P > 0.05, except group V). Significantly decreased in escape latency of group IV (p < 0.05, p < 0.01, p < 0.001) when compared with group III. Also results showed significantly decreased escape latency (p < 0.05, p < 0.001) after administration of MEEP (100, 200 and 300 mg/kg) on 5th, 10th, 15th and 20th day trial after treatment (Fig. 2A). It was observed that MEEP at the dose of 100 mg/kg (group V) significantly increased (p < 0.01, p < 0.001) escape latency as compared to dose of 200 mg/kg (group VI) and 300 mg/kg (group VII) on 10th, 15th and 20th day. MEEP at the dose of 200 mg/kg significantly decreased (p < 0.05) escape latency as compared with 300 mg/kg on 10th day only. Distance traveled by animals significantly decreased in group I, group II (p < 0.001) and group IV (p < 0.01, p < 0.001) except on the 5th day of training after treatment (p > 0.05) as compared with group III (Fig. 2B). After oral administration of MEEP (100, 200 and 300 mg/kg) on 5th, 10th, 15th and 20th day trial after treatment

547

showed significantly decreased distance travelled (p < 0.05, p < 0.01, p < 0.001) except groups IV–VII on 5th day training after treatment (p > 0.05). In the internal group comparison at three doses of MEEP non-significantly decreased searching distance (p > 0.05) was observed. 3.3.2. RAM To explore whether the macerated extract of Indian propolis (100, 200 and 300 mg/kg) affects the formation of spatial memory, rats were further evaluated in the RAM task. For the number of errors in each session, two-way ANOVA, Bonferroni’s post test revealed non-significant relationship between disease control group and other groups on 5th day trial before surgery (p > 0.05) (Fig. 3A), but significantly decreased in number of errors in group I, group II (p < 0.001), group IV (p < 0.05, p < 0.01, p < 0.001) and treatment groups V to VII (p < 0.05, p < 0.01, p < 0.001) on 5th day, 10th day, 15th day and 20th day trial after treatment as compared with group III except in groups V and VI on 15th trial day of treatment (p > 0.05). In the internal group comparison at three doses of MEEP non-significantly decreased (p > 0.05) in number of errors was observed except on 5th day after treatment. The number of correct choices in the initial 8 chosen arms, twoway ANOVA, Bonferroni’s post test revealed non-significant differences between group III and treatment groups V–VII on 5th day of training (p > 0.05) (Fig. 3B), but significantly increased in number of correct choices in group IV, treatment groups V (p < 0.001), VI, VII (p < 0.01, p < 0.001) on 5th, 10th, 15th and 20th day trial after treatment as compared with group III. Internal group comparison in between three doses of MEEP showed nonsignificantly increased (p > 0.05) in number of correct choices in RAM. 3.4. Hematological parameters No significant differences (p > 0.05) were found in packed cell volume in test sample groups V–VII, group IV, group II, and group III as compared with a group I of rats. But significantly increased (p < 0.05, p < 0.01) white blood cell and erythrocyte sedimentation rate. Also significantly decreased (p < 0.01) red blood cells, platelet count and hemoglobin content as compared with group I. Treatment with MEEP reversed this to normal range. 3.5. Biochemical analysis No significant differences were found in cholesterol, triglycerides, glucose, HDL-C, LDL-C, albumin, AST, total protein, and Alk-P between the group I–III and other treatment groups V–VII compared with group IV.

Fig. 1. CAPE, PINO and GAL in (A) Standard, (B) MEEP.

548

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

Fig. 2. Effect of MEEP on A) the escape latency and B) distance to find hidden platform (Searching Distance) in the MWM test. MEEP was administered p.o. daily for 21 days and observations were done four times daily. Group I: group with no treatment and were non-operated. Group II: group received 10 mL of the vehicle (Double distilled water)) through ICV and treatment for 21 days with 0.1% of Tween 80 administered p.o. (1 mL/kg), Group III: group with no treatment received but operated, Group IV: group received Donepezil (0.75 mg/kg) p.o., Group V: Test group received MEEP (100 mg/kg) p.o., Group VI: Test group received MEEP (200 mg/kg) p.o., Group VII: Test group received MEEP (300 mg/kg) p.o. Statistical analysis analyzed by two-way ANOVA followed by Bonferroni’s post-test.

But showed a significant increase (p < 0.001) in ALT ranges in group III animal as compared with group I of animals. Treatment with MEEP reversed this to normal range (Table 1). 3.6. Histological brain tissues Formation of plaque in the Ab25-35- treated brain tissues was stained by Congo red staining method. Amyloid plaque could be detected in different areas of the brain, including the hippocampus. In the present study, hippocampus area was observed regards with plaque formation. For the rats administered with treatment groups, there was decreased size of amyloid plaque as compared with group III. In the group II, no amyloid plaque was observed (Fig. 4).

3.7. Effect of MEEP on Ab25-35 induced rats changes in levels of oxidative stress parameters (SOD, GSH, CAT, NO, MDA) 3.7.1. SOD Fig. 5 showed the activities of antioxidant parameters such as SOD, GSH, and CAT with NO, and MDA estimation in the brain of control and experimental animals. Data represents mean  S.E.M. (n = 8). Ab25-35 induced rats group III significantly decreased (17.51  0.8436) the SOD levels in brain homogenate when compared with group I (p < 0.01, 33.68  1.949) and group II rats (p < 0.001, 32.66  1.012), means to produced oxidative stress. Group IV significantly increased (p < 0.01, 29.84  1.427) SOD activity as compared with group III. The treatment groups V–VII showed significantly dose-dependent increased (p < 0.05, 22.30  1.242; p < 0.05, 23.86  1.204; p < 0.01, 25.44  1.286

Fig. 3. Effect of MEEP on A) the number of errors and B) a number of correct choices in the RAM test. MEEP was administered daily for 21 days through p. o. route and observations were done once a daily. Group I: group with no treatment and were non-operated. Group II: group received 10 mL of the vehicle (Double distilled water)) through ICV and treatment for 21 days with 0.1% of Tween 80 administered p.o. (1 mL/kg), Group III: group with no treatment received but operated, Group IV: group received Donepezil (0.75 mg/kg) p.o., Group V: Test group received MEEP (100 mg/kg) p.o., Group VI: Test group received MEEP (200 mg/kg) p.o., Group VII: Test group received MEEP (300 mg/kg) p.o. Statistical analysis analyzed by two-way ANOVA followed by Bonferroni’s post-test. Asterisks indicate a significant change from the group III. (P < 0.05: *, P < 0.01: **, P < 0.001: ***) and P < 0.001: # indicate a significant change from normal group (Group I).

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

549

Table 1 Effect of MEEP on plasma/serum Enzymes, lipid, and proteins. Groups Treatments

Group I

Group II

Group III

Group IV

Group V

Group VI

Group VII

Glu (mg%) TP (g/dl) ALB (g/dl) GLB (g/dl) Chol (mg/dl) TG (mg%) HDL-C (mg/dl) LDL-C (mg/dl) Alk P (g/dl) AST (U/L) ALT (U/L)

92.89  4.567 6.652  0.423 4.251  0.382 2.968  0.195 143.5  12.82 137.9  12.68 51.03  8.125 99.54  4.426 137.8  20.72 19.19  4.629 25.68  3.546

109.3  6.539 7.534  0.426 4.966  0.394 2.931  0.181 136.4  11.83 124.3  10.27 47.58  5.662 102.5  2.980 171.9  14.37 18.43  4.117 24.56  2.450

80.89  8.952 7.463  0.415 4.619  0.341 3.010  0.160 153.6  7.297 109.5  10.69 66.33  4.816 105.2  5.520 167.6  31.42 20.49  4.939 53.49  4.064***

93.95  6.967 7.316  0.476 4.261  0.399 2.905  0.236 137.4  7.583 107.1  15.92 53.77  6.268 103.5  5.629 163.9  21.90 20.43  5.567 27.24  2.385

100.5  4.547 7.124  0.549 4.612  0.345 2.546  0.154 141.7  10.13 100.4  15.01 63.49  5.491 101.5  6.372 148.4  28.27 20.41  3.198 24.52  3.478

94.21  5.128 7.421  0.361 4.416  0.457 2.463  0.170 136.6  12.60 129.12  19.43 52.66  7.411 104.2  4.121 159.4  22.09 21.46  3.412 26.78  1.402

96.84  5.909 7.518  0.394 4.648  0.391 3.091  0.165 142.7  15.59 127.99  16.33 58.11  7.744 104.2  5.276 165.1  25.18 22.74  6.187 21.57  1.286

Group I: group with no treatment and were non-operated. Group II: group received 10 mL of the vehicle (Double distilled water)) through ICV and treatment for 21 days with 0.1% of Tween 80 administered p.o. (1 mL/kg), Group III: group with no treatment received but operated, Group IV: group received Donepezil (0.75 mg/kg) p.o., Group V: Test group received MEEP (100 mg/kg) p.o., Group VI: Test group received MEEP (200 mg/kg) p.o., Group VII: Test group received MEEP (300 mg/kg) p.o. Glu: Glucose, TP: Total protein, ALB: Albumin, GLB: Globulin, Chol: Cholesterol, TG: Triglyceride, HDL-C: High-density lipoprotein-cholesterol, LDL-C: Low-density lipoprotein-cholesterol, Alk P: Alkaline phosphatase, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase. All values are expressed as mean  SEM for 8 animals in each group. *** Values are significantly different from group I at P < 0.001.

respectively) in SOD activity when compared with group III (Fig. 5A). 3.7.2. GSH Ab25-35 induced rats group III were observed with astonished diminish (8.007  3.63) in the GSH level when compared with group I rats (p < 0.0001, 62.01 1.923) and group II (p < 0.001, 44.99  2.64). Treatment with group IV prevented the decrease in GSH level as compared with group III (p < 0.001, 48.67  3.39). Groups V–VII of treatment dose-dependently increased the GSH level as compared with group III animals (p < 0.001, 37.54  2.41; p < 0.0001, 41.25  1.46; p < 0.0001, 43.87  1.46 respectively) (Fig. 5B).

3.7.3. CAT activity In group III animals significant decreased (2.173  0.3751) the catalase activity in brain homogenate when made comparison with group I (p < 0.001, 13.68  1.398) and group II (p < 0.0001, 12.66  0.4878). The effect of MEEP on catalase activity was found to be significant dose-dependently increased for the doses 100 mg/ kg, 200 mg/kg and 300 mg/kg (p < 0.05, 3.618  0.3635; p < 0.05, 4.029  0.3398; p < 0.01, 4.937  0.3009 respectively) compared with the group III. In case of group IV was also found to be remarkably increased (p < 0.05, 6.174  0.7830) (Fig. 5C) when compared with group III rats.

Fig. 4. Typical photographs correspond to the hippocampus region of the brain with a b-amyloid plaque using Congo red staining method indicating with an arrow with magnification of 40. Group I: group with no treatment and were non-operated, no b- amyloid plaque; Group II: group received 10 mL of the vehicle (Double distilled water)) through ICV and treatment for 21 days with 0.1% of Tween 80 administered p.o. (1 mL/kg), no b- amyloid plaque; Group III: group with no treatment received but operated, arrow indicate position where Ab plaque accumulation has occurred; Group IV: group received Donepezil (0.75 mg/kg) p.o. where arrow indicate reduction in Ab plaque; Group V: Test group received MEEP (100 mg/kg) p.o. arrow indicate lesser reduction in Ab plaque as compared with group III; Group VI: Test group received MEEP (200 mg/kg) p.o. arrow indicate lesser reduction in Ab plaque as compared with group III and V; Group VII: Test group received MEEP (300 mg/kg) p.o. arrow indicate lesser reduction in Ab plaque as compared with group III and VI.

550

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

Fig. 5. Effect of MEEP on A] Superoxide dismutase (SOD) level B] Glutathione level (GSH) C] Catalase (CAT) level D] Nitric oxide (NO) level E] Malondialdehyde (MDA) level (Lipid peroxidation). Group I: group with no treatment and were non-operated. Group II: group received 10 mL of the vehicle (Double distilled water) through ICV and treatment for 21 days with 0.1% of Tween 80 administered p.o. (1 mL/kg), Group III: group with no treatment received but operated, Group IV: group received Donepezil (0.75 mg/kg) p.o., Group V: Test group received MEEP (100 mg/kg) p.o., Group VI: Test group received MEEP (200 mg/kg) p.o., Group VII: Test group received MEEP (300 mg/kg) p.o. Asterisks indicate a significant change from the group III (P < 0.05: *, P < 0.01: **, P < 0.001: ***, P < 0.0001: ****) and (P < 0.05: #, P < 0.01: # #, P < 0.001: # # #, P < 0.0001: # # # #) indicate a significant change from normal group (Group I).

3.7.4. NO activity The Ab25-35 induced group III showed prominent decreased (734  37.51) in nitric oxide level in brain homogenate when made a comparison with group I (p < 0.05, 1020  70.94) and group II (p < 0.05, 918.8  44.12). The effect of MEEP on NO level was found to be significantly increased for the doses 100 mg/kg, 200 mg/kg and 300 mg/kg (p < 0.05, 940.5  40.53; p < 0.01 1060  43.13; p < 0.001, 1194  32.11 respectively) compared with the group III. In case of the group IV was also found to be remarkably increased (p < 0.001, 1262  50.30) (Fig. 5D) after comparison with group III. 3.7.5. MDA To determine whether lipid peroxidation is involved in the ameliorating effect of the MEEP in Ab25-35 induced rats. We examined the effect of MEEP on the levels of MDA in the brain by using HPLC. A significant increased (p < 0.001) in MDA level in Ab25-35 induced group III of rat (162.49  33.49) as compared with the levels in group II (102.0  7.45) and group I (99.69  7.29). Treatment with group IV and V to VII in three different doses significantly dose- dependently prevented (p < 0.001, p < 0.01, Fig. 5E) the increased level of MDA in the whole brain of Ab25-35 treated rats. 3.8. Brain monoamines The levels of biogenic amines in the brain of rats was evaluated and showed in Fig. 6. The concentration of NE in the brain of group III rat was observed 3300  211.14 which was found to be decreased. Group II, group I and group IV showed NE level 5365  195.7, 7091  241.3, 5070  147.7 respectively. After administration of MEEP (100 mg/kg, 200 mg/kg, 300 mg/kg), NE level was

significantly dose-dependent increased as compared with group III and showed 4153  221.1 (p < 0.05), 4481  279.0 (p < 0.05) and 5286  147.3 (p < 0.001) respectively (Fig. 6A). The brain DA concentration in Ab25-35 induced group was found to be decreased in the concentration of 66.11  2.158. The dopamine level in group II, group I and group IV was showed with significant increase level as compared with group III, 102.6  6.876 (p < 0.05), 170.9  3.701 (p < 0.0001), 102.5  5.415 (p < 0.01) respectively. No sharp changes in the DA level in the brain after administration of MEEP. MEEP treated animals showed significant dose-dependent increase in DA levels and found to be 94.34  6.792 (p < 0.05), 94.36  6.647 (p < 0.05), 118.5  1.216 (p < 0.0001) as compared to group III (Fig. 6B). The brain serotonin (5-HT) concentration in group III was found to be 370.4  51.24. Treatment groups V–VII showed significant dose-dependent increase in 5-HT concentration and showed values 768  100 (p < 0.05), 875.4  83.66 (p < 0.01), 1015  28.92 (p < 0.0001), as compared with group III (Fig. 6C). 3.9. Acetylcholinesterase (AChE) inhibition The AChE inhibition absorbance reading were stabilized after 2 min of incubation after observation up to 10 min. The absorbance at 2 min was taken as a final result for calculation. As shown in Fig. 7, % inhibition of AChE in three doses of MEEP was found to be (Group V (100 mg/kg): 38.86%  2.207; Group VI (200 mg/kg): 49.69  1.988; Group VII (300 mg/kg): 60.54  3.100). The significant difference was found in between group IV and group VII (p < 0.01) and non significant difference was found in group IV and group V, VI (p > 0.05). Results showed dose-dependent increase in % inhibition of AChE activity.

Fig. 6. Brain monoamines A] Norepinephrine (NE), B] Dopamine (DA), C] 5- Hydroxytryptamine (5-HT) estimation in brain homogenate. Group I: group with no treatment and were non-operated. Group II: group received 10 mL of the vehicle (Double distilled water)) through ICV and treatment for 21 days with 0.1% of Tween 80 administered p.o. (1 mL/kg), Group III: group with no treatment received but operated, Group IV: group received Donepezil (0.75 mg/kg) p.o., Group V: Test group received MEEP (100 mg/kg) p. o., Group VI: Test group received MEEP (200 mg/kg) p.o., Group VII: Test group received MEEP (300 mg/kg) p.o. Data represent the mean  S.E.M (n = 6) and were analyzed by one-way ANOVA followed by Dunette’s multiplication test. Asterisks indicate significant change from the group III (P < 0.05: *, P < 0.01: **, P < 0.001: ***, P < 0.0001:****) and P < 0.001: #, P < 0.0001: # # indicate a significant change from normal group (Group I).

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

Fig. 7. Acetylcholinesterase enzyme inhibition activity of positive control (group IV) and different doses of Indian propolis. Group IV: group received Donepezil (0.75 mg/kg) p.o., as a positive control, Group V: Test group received MEEP (100 mg/ kg) p.o., Group VI: Test group received MEEP (200 mg/kg) p.o., Group VII: Test group received MEEP (300 mg/kg) p.o. Data represent the Mean  SEM (n = 6) and were analyzed by one-way ANOVA followed by Dunnett’s test. Asterisks indicate significant change from the group IV (P < 0.01: **).

3.10. BDNF In group III BDNF level (279.4  44.53) was found significantly decreased as compared with group I (p < 0.001, 1251  60.30) and group II (p < 0.01, 950.9  71.74). The BDNF concentration was found to be 1037  53.20, 574.4  71.05, 500.8  31.82 and 952.0  84.62 in group IV, V, VI and VII respectively (Fig. 8). After treatment with group IV donepezil in dose 0.75 mg/kg and treatment groups V–VII, BDNF level was significantly doseindependent increased as compared with group III. 4. Discussion In the present study, we examined the effect of MEEP on the memory impairment induced by Ab25-35 in Wistar rats. To the best of our knowledge, this is the first study to show the neuroprotective effect of Indian propolis extract through multi-targeted mechanisms such as inhibition of AChE, BDNF enhancement and regulating oxidative stress. Ab protein accumulation is highly toxic to primary and other cell lines [42,43]. Ab25–35 is the most toxic Ab fragment that has

Fig. 8. BDNF level in rat plasma in terms concentration in pg/ml. Group I: group with no treatment and were non-operated. Group II: group received 10 mL of the vehicle (Double distilled water)) through ICV and treatment for 21 days with 0.1% of Tween 80 administered p.o. (1 mL/kg), Group III: group with no treatment received but operated, Group IV: group received Donepezil (0.75 mg/kg) p.o., Group V: Test group received MEEP (100 mg/kg) p.o., Group VI: Test group received MEEP (200 mg/kg) p.o., Group VII: Test group received MEEP (300 mg/kg) p.o. Data was expressed in mean  S.E.M (n = 6) and were analyzed by one-way ANOVA followed by Dunette’s test (P < 0.05: *, P < 0.01: **, P < 0.001: ***) and P < 0.001: # compared with normal group (group I).

551

been detected in the brain of AD patients with neurotoxic properties such as learning and memory impairment, morphological alterations and cholinergic dysfunction similar as like fulllength Ab [10,44–46]. Therefore effect of MEEP was evaluated in neurotoxicity induced by Ab25-35 in rat. Intrahippocampal or ICV injection of Ab25-35 is reported to produce changes in histological, biochemical, learning deficits [12] and dysfunction of the cholinergic system in animal experiments. Thus, the Ab25-35 animal model is useful for understanding the pathogenesis, progression of AD and evaluating new therapeutic agents for AD treatment [12]. Our study revealed, Ab25–35 injected rat showed memory impairment in behavioral models like MWM and RAM. These results are steady with previous findings that cognitive impairment after ICV injection of Ab25–35 [12]. In MWM test, the escape latency and searching distance were evaluated. The group III (Ab25–35 induced disease control group) significantly increased escape latency and searching distance indicating the memory impairment. The significant decrease in escape latency and searching distance exhibited by MEEP indicate its improved spatial reversal learning and memory ability [1]. In RAM model behavioral aspects with respect to a number of errors and number of correct choices in arms were evaluated. A higher number of errors and lower correct choices in arms suggested impairment of memory in Ab25–35 induced group of rats [47]. A significant decrease in the number of errors and dose-dependent increase in number of correct choices indicated an elevation in retention and recall aspects of learning and memory after administration of MEEP. Any type of diseased condition may affect the haematology. The significant decrease in RBCs, platelets and Hb content and significant increase in WBCs, ESR and ALT in Ab25–35 induced rats, revealed the disease progression. However MEEP administration reversed all parameters to normal range indicates its ability to modify the diseased conditions. The memory deficit observed through behavioural studies has been further confirmed by histopathological results. Beta amyloid plaque showed as red to pink colour. Formation of amyloid plaque confirmed the memory deficits followed by induction of Alzheimer’s disease. Reduced size of plaque confirmed the memory improvement after treatment with MEEP. It has been suggested that oxidative stress plays a vital role in the development of AD [48]. In the central nervous system, GSH is the one of the most abundant intracellular non-protein thiols. It plays a major antioxidant role within both neurons and nonneuronal cells [49]. In the present study, Ab25–35 decreased the GSH level in the hippocampus, which is consistent with reports of depletion of GSH in the brain of AD patients. Furthermore, the Ab25–35-induced decrease in the level of GSH was prevented by treatment with MEEP indicating that the protective effect of MEEP in cognitive impairment involves activation of antioxidative defenses. SOD also plays important role in detoxifying superoxide anions which damages the macromolecules and cell membranes. Catalase has the capability to detoxify H2O2 radicals. The release of H2O2 promotes the production of other oxidant species, which contributes for oxidative stress leads to the pathogenesis of AD. Ab25–35 induced rats showed a significant reduction in activity of SOD, NO, and CAT [50]. After administration of MEEP, significant dose-dependent increase in SOD, NO and CAT activities were observed. Lipid peroxidation is one of the most outcomes of free radical mediated injury which directly affects membranes and generates many secondary products including aldehydes such as MDA and 4- hydroxyl-2-nonenal (HNE), ketones etc. [51]. An increase in lipid peroxidation products in hippocampus, amygdala and parahippocampal gyrus of AD brains are observed [52]. MDA can be considered as a marker of lipid peroxidation and is a measure of free radical generation [53]. Ab25–35 induced rats

552

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553

showed extensive increased lipid peroxidation as evidenced by elevated MDA levels. So, in order to evaluate the effect of MEEP on lipid peroxidation, MDA level in the brain was assessed. MDA level was increased in control group and MEEP showed significant dosedependent decreased MDA level, signifies reduced peroxidation of lipids. It has been demonstrated that brain monoamines have a key role in the memory and learning process. The complexity of chemical transmission in normal neuronal communication and in various neurodegenerative disorders (like Alzheimer’s disease) affecting learning and memory have suggested the functional significance of specific neurotransmitters. Amongst more than 60 neurotransmitters speculated, central cholinergic system and biogenic amines especially noradrenaline, dopamine, serotonin have been found to be more important [54]. NE is responsible for the various functions like attention in learning, memory and motor process. The decrease in NE concentration in the brain leads to depression, diminished alertness and dementia [55]. MEEP showed significant dose-dependent increase in NE levels, indicated the increase in memory and learning process. Dopaminergic neurons arise from mesolimbic, corpus striatum and mesocortical areas of the brain which are responsible for the behavioural pathway including motivational behavior with response selection and habit learning in rats. Thus, it can be concerned with plural process promoting learning and memory [56,57]. Stahl [58] has reported that reduced dopamine level in pre-frontal cortex and medial striatum leads to impairments in acquisition process. In the present investigation, Ab25–35 induced rats increased the DA level, suggested memory deficits. Administration of MEEP significantly decreased DA levels in dose-dependent manner, indicated increase in learning process. In the brain, 5- HT is located in the thalamus, hypothalamus, and midbrain. The role of 5-HT is important in the regulation of memory. Serotonin has been unveiled to be linked to emotional behavior in the rat. It has been reported that diminished levels of 5-HT leads to anxiety and memory impairment which was observed in Ab25-35 induced rats [59]. Increasing level of 5- HT after administration of MEEP, was observed indicated the reversal of above process. Further, acetylcholinesterase enzyme is an acetylcholine hydrolyzing enzyme which is responsible for the termination of cholinergic response [60]. AChE hydrolyzes and inactivates ACh. Increased AChE activity leads to a lack of ACh and thus memory deficits, observed in the brains of AD patients [61,62]. In the present study Ab25-35 induced rats showed increased AChE activity indicate the memory impairment, whereas MEEP exhibited AChE inhibitory effect. Present study results indicated that MEEP reverses cognitive impairment by increasing cholinergic potential through dose-dependent inhibition of AChE activity. Moreover, the role of BDNF in memory is also important. BDNF as a potential biomarker supports the diagnosis in brain disorder, promotes the growth of neurons during brain development and is associated with learning and memory [63]. Changes in BDNF levels are well related to cognitive impairment. Reported studies analyzing Lewy body dementia (LBD), frontotemporal dementia (FTD), vascular dementia (VAD) and AD found a decrease in BDNF levels when compared to controls [64]. Our study showed, Ab25-35 induced rats significantly decreased the BDNF level, whereas MEEP administration increased the BDNF level thus increasing memory and learning ability. MEEP consists of number of polyphenols and flavonoids in addition to CAPE, PINO, and GAL. These markers have been reported to have neuroprotection with different mechanisms in AD. The study results suggest that various chemical constituents of the MEEP act through different mechanisms, thus exhibiting the overall neuroprotection. The advantage of natural products is the multiple constituents with multiple mechanisms contribute

the overall activity in addition to synergistic activity. Thus the present study results suggest the neuroprotective role of MEEP with multiple mechanisms. Further study is needed to fractionate and isolate compounds responsible for the neuroprotective activity of Indian propolis which is under process in our laboratory.

5. Conclusion The present study has shown that macerated extract of Indian propolis ameliorates Ab25-35 induced memory deficits in rats using Morris water maze and radial arm maze tasks. MEEP increased brain catecholamines (NE, DA, 5-HT) concentration in the brain to improve memory. It improved antioxidant defense system with diminishing malondialdehyde in the brain, which is an essential factor in Alzheimer’s disease, inhibited AChE activity and activated BDNF potential which is essential for neuroregeneration in AD. Further studies of fractionation and biological screening are under process. Conflict of interests Authors have no conflict of interests. Acknowledgement We gratefully acknowledge the All India Council for Technical Education (AICTE), Govt of India, New Delhi for providing financial assistance for the work, through the scheme quality improvement programme. References [1] R. Kaur, S. Parveen, S. Mehan, D. Khanna, S. Kalra, Neuroprotective effect of ellagic acid against chronically scopolamine-induced Alzheimer’s type memory and cognitive dysfunctions: possible behavioural and biochemical evidences, Int. J. Prev. Med. Res. 1 (2015) 45–64. [2] K. Blennow, M.J. De Leon, H. Zetterberg, Alzheimer’s disease, Lancet 368 (2006) 387–403. [3] C.L. Masters, G. Simms, N.A. Weinman, G. Multhaup, B.L. McDonald, K. Beyreuther, Amyloid plaque core protein in Alzheimer’s disease and Down syndrome, Proc. Natl. Acad. Sci. 82 (1985) 4245–4249. [4] J. Kang, H.G. Lemaire, A. Unterbeck, J.M. Salbaum, C.L. Masters, K.H. Grzeschik, G. Multhaup, K. Beyreuther, B. Muller-Hill, The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor, Nature 325 (1987) 19–25. [5] M. Aslam, A.A. Sial, Neuroprotective effect of ethanol extract of leaves of Malva parviflora against Amyloid-b-(A b ) mediated Alzheimer’s disease, Int. Sch. Res. Not. (2014) 1–6. [6] P. Lu, T. Mamiya, L.L. Lu, A. Mouri, L.B. Zou, T. Nagai, M. Hiramatsu, T. Ikejim, T. Nabeshima, Silibinin prevents amyloid b peptide-induced memory impairment and oxidative stress in mice, Br. J. Pharmacol. 157 (2009) 1270– 1277. [7] D. Schubert, C. Behl, R. Lesley, A. Brack, R. Dargusch, Y. Sagara, et al., Amyloid peptides are toxic via a common oxidative mechanism, Proc. Natl. Acad. Sci. 92 (1995) 1989–1993. [8] B.A. Yankner, Mechanisms of neuronal degeneration in Alzheimer’s disease, Neuron 16 (1996) 921–932. [9] V.V. Trubetskaya, M.Y. Stepanichev, M.V. Onufriev, N.A. Lazareva, V.A. Markevich, N.V. Gulyaeva, Administration of aggregated beta-amyloid peptide 25–35 induces changes in long-term potentiation in the hippocampus in vivo, Neurosci. Behav. Physiol. 33 (2003) 95–98. [10] M.H. Tran, K. Yamada, A. Olariu, M. Mizuno, X.H. Ren, T. Nabeshima, Amyloid beta-peptide induces nitric oxide production in rat hippocampus: association with cholinergic dysfunction and amelioration by inducible nitric oxide synthase inhibitors, FASEB J. 15 (2001) 1407–1409. [11] J. Choi, C.A. Malakowsky, J.M. Talent, C.C. Conrad, C.A. Carroll, S.T. Weintraub, et al., Anti-apoptotic proteins are oxidized by A beta 25–35 in Alzheimer’s fibroblasts, Biochim. Biophys. Acta 1637 (2003) 135–141. [12] T. Maurice, B.P. Lockhart, A. Privat, Amnesia induced in mice by centrally administered b-amyloid peptides involves cholinergic dysfunction, Brain Res. 706 (1996) 181–193. [13] J. Chen, Y. Long, M. Han, T. Wang, Q. Chen, R. Wang, Water-soluble derivative of propolis mitigates scopolamine-induced learning and memory impairment in mice, Pharmacol. Biochem. Behav. 90 (2008) 441–446.

S. Nanaware et al. / Biomedicine & Pharmacotherapy 93 (2017) 543–553 [14] A. Purohit, K. Joshi, B. Kotru, S. Kotru, H. Ram, Histological study of antiatherosclerotic effect of propolis in induced hypercholestrolemic male albino rabbits, Indian J. Fundam. Appl. Life Sci. 2 (2012) 384–390. [15] A. Purohit, K. Joshi, B. Kotru, S. Kotru, Effect of Indian propolis on haematological parameters in experimentally induced hyperlipidemic male albino rabbits, Asian J. Pharm. Clin. Res. 6 (2013) 17–19. [16] P. Kalia, N.R. Kumar, K. Harjai, Phytochemical screening and antibacterial activity of different extracts of Propolis, Int. J. Pharm. Biol. Res. 3 (2013) 219– 222. [17] D.G. Naik, A.M. Mujumdar, H.S. Vaidya, Anti-inflammatory activity of propolis from Maharashtra, India, J. Apic. Res. 52 (2013) 35–43. [18] R. Ambardekar, S. Gilda, K. Mahadik, A. Harsulkar, A. Paradkar, Free radical scavenging and anti-inflammatory activity of Indian Propolis, Pharmacologyonline 3 (2009) 991–1002. [19] D.G. Naik, H.S. Vaidya, Antioxidant properties of volatile oil of Indian Propolis, J. Apiprod. Apimed. Sci. 3 (2011) 89–93. [20] A. Ramadan, G. Soliman, S.S. Mahmoud, S.M. Nofal, R.F. Abdel-Rahman, Evaluation of the safety and antioxidant activities of Crocus sativus and Propolis ethanolic extracts, J. Saudi Chem. Soc. 16 (2012) 13–21. [21] M.D.G. Miguel, O. Doughmi, S. Aazza, D. Antunes, B. Lyoussi, Antioxidant, antiinflammatory and acetylcholinesterase inhibitory activities of propolis from different regions of Morocco, Food Sci. Biotechnol. 23 (2014) 312–322. [22] F.K.A. El-Hady, A.M.A. Souleman, Z.A. El-Shahid, Antiacetylcholinesterase and cytotoxic activities of Egyptian propolis with correlation to its GC/MS and HPLC analysis, Int. J. Pharm. Sci. 34 (2015) 32–42. [23] M. Shimazawa, S. Chikamatsu, N. Morimoto, S. Mishima, H. Nagai, H. Hara, Neuroprotection by Brazilian green propolis against In vitro and In vivo ischemic neuronal damage, eCAM 2 (2005) 201–207. [24] A. Ilhan, M. Iraz, A. Gurel, F. Amrutcu, O. Akyol, Caffeic acid phenethyl ester exerts a neuroprotective effect on CNS against pentylenetetrazol-induced seizures in mice, Neurochem. Res. 29 (2004) 2287–2292. [25] R. Liu, C.X. Wu, D. Zhou, F. Yang, S. Tian, L. Zhang, T.T. Zhang, G.H. Du, Pinocembrin protects against b-amyloid-induced toxicity in neurons through inhibiting receptor for advanced glycation end products (RAGE)-independent signaling pathways and regulating mitochondrion-mediated apoptosis, BMC Med. 10 (2012) 1–21. [26] Y. Lei, J. Chen, W. Zhang, W. Fu, G. Wu, H. Wei, Q. Wang, J. Raun Raun, In vivo investigation of the potential of galangin, kaempferol and myricetin for the protection of D-galactose-induced cognitive impairment, Food Chem. 135 (2012) 2702–2707. [27] B. Trusheva, D. Trunkova, V. Bankova, Different extraction methods of biologically active components from propolis: a preliminary study, Chem. Cent. J. 7 (2007) 1–13. [28] D. Marinova, F. Ribarova, M. Atanassova, Total phenolics and total flavonoids in Bulgarian fruits and vegetables, J. Univ. Chem. Technol. Metall. 40 (2005) 255– 260. [29] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 6th edition, Academic Press, 2006. [30] OECD/OCDE 425, Acute Oral Toxicity. Guideline for the Testing of Chemicals, Revised Draft Guideline, vol. 425, (2001) 5–6. [31] C.V. Vorhees, M.T. Williams, Morris water maze: procedures for assessing spatial and related forms of learning and memory, Nat. Protoc. 1 (2006) 848– 858. [32] L. Hritcu, O. Cioanca, M. Hancianu, Effects of lavender oil inhalation on improving scopolamine-induced spatial memory impairment in laboratory rats, Phytomedicine 19 (2012) 529–534. [33] D.S. Olton, R.J. Samuelson, Remembrance of places passed: spatial memory in rats, J. Exp. Psychol. Anim. B 2 (1976) 97–116. [34] D.M. Wilcock, M.N. Gordon, D. Morgan, Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain, Nat. Protoc. 1 (2006) 1591–1595. [35] H.P. Misra, I. Fridovich, The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase, J. Biol. Chem. 247 (1972) 3170–3175. [36] M.S. Moron, J.W. Depierre, B. Mannervik, Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver, Biochim. Biophys. Acta 582 (1979) 67–78. [37] S. Rao, Nitric oxide scavenging by curcuminoids, J. Pharm. Pharmacol. 49 (1997) 105–107. [38] R.F. Beers Jr., I.W. Sizer, A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, J. Biol. Chem. 195 (1952) 133– 140. [39] D. Gratto, L.D. Santa maria, S. Boeira, J. Valentini, M.F. Charao, A.M. Moro, P.C. Nascimento, V.J. Pomblum, S.C. Garcia, Rapid quantification of malondialdehyde in plasma by high-performance liquid chromatographyvisible detection, J. Pharm. Biomed. Anal. 43 (2007) 619–624.

553

[40] M.K. Lakshmana, T.R. Raju, An isocratic assay for norepinephrine, dopamine, and 5-hydroxytryptamine using their native fluorescence by highperformance liquid chromatography with fluorescence detection in discrete brain areas of rat, Anal. Biochem. 246 (1997) 166–170. [41] A. Kumar, A. Prakash, S. Dogra, Centella asiatica attenuates d-galactoseinduced cognitive impairment, oxidative and mitochondrial dysfunction in mice, Int. J. Alzheimers Dis. (2011) 1–9. [42] H.J. Kim, K.W. Lee, H.J. Lee, Protective effects of piceatannol against betaamyloid-induced neuronal cell death, Ann. N. Y. Acad. Sci. 1095 (2007) 473– 482. [43] B.M. Nie, X.Y. Jiang, J.X. Cai, S.L. Fu, L.M. Yang, L. Lin, et al., Panaxydol and panaxynol protect cultured cortical neurons against Abeta25–35-induced toxicity, Neuropharmacology 54 (2008) 845–853. [44] C.J. Pike, A.J. Walencewicz-Wasserman, J. Kosmoski, D.H. Cribbs, C.G. Glabe, C. W. Cotman, Structure-activity analyses of b-Amyloid peptides: contributions of the b25-35 region to aggregation and neurotoxicity, J. Neurochem. 64 (1995) 253–265. [45] T. Kubo, S. Nishimura, Y. Kumagae, I. Kaneko, In vivo conversion of racemized beta-amyloid ([D-Ser 26]Abeta 1–40) to truncated and toxic fragments ([D-Ser 26]Abeta 25–35/40) and fragment presence in the brains of Alzheimer’s patients, J. Neurosci. Res. 70 (2002) 474–483. [46] T. Alkam, A. Nitta, H. Mizoguchi, K. Saito, M. Seshima, A. Itoh, et al., Restraining tumor necrosis factor-alpha by thalidomide prevents the Amyloid b-induced impairment of recognition memory in mice, Behav. Brain Res. 189 (2008) 100– 106. [47] F. Pu, K. Mishima, K. Irie, K. Motohashi, Y. Tanaka, K. Orito, T. Egawa, Y. Kitamura, N. Egashira, K. Iwasaki, M. Fujiwara, Neuroprotective effects of quercetin and rutin on spatial memory impairment in an 8-arm radial maze task and neuronal death induced by repeated cerebral ischemia in rats, J. Pharmacol. Sci. 104 (2007) 329–334. [48] M.A. Smith, G. Perry, P.L. Richey, L.M. Sayre, V.E. Anderson, M.F. Beal, et al., Oxidative damage in Alzheimer’s, Nature 382 (1996) 120–121. [49] M.Y. Aksenov, W.R. Markesbery, Changes in thiol content and expression of glutathione redox system genes in the hippocampus and cerebellum in Alzheimer’s disease, Neurosci. Lett. 302 (2001) 141–145. [50] H. Mann, M.T. McCoy, J. Subramaniam, H. Van Remmen, J.L. Cadet, Overexpression of superoxide dismutase and catalase in immortalized neural cells: toxic effects of hydrogenperoxide, Brain Res. 770 (1997) 163–168. [51] T.F. Slater, Overview of the methods used for detecting lipid peroxidation, Methods Enzymol. 105 (1984) 283–293. [52] W.R. Markesbery, M.A. Lovell, Four-hydroxynonenal, a product of lipid peroxidation, is increased in the brain in Alzheimer’s disease, Neurobiol. Aging 19 (1998) 33–36. [53] A. Skoumalova, J. Hort, Blood markers of oxidative stress in Alzheimer’s disease, J. Cell. Mol. Med. 16 (2012) 2291–2300. [54] S.C. Lee, P.T. Linh, Z. Jing, S.Y. Ryu, C.S. Myung, Y.H. Kim, J.S. Kang, Effects of repeated administration of Uncaria hooks on the acquisition and central neuronal activities in ethanol-treated mice, J. Ethanopharmacol. 94 (2004) 123–128. [55] W.F. Ganong, Neural basis of instinctual behaviour and emotions, in: J. Dolan, C. Langan (Eds.), Review of Medical Physiology, Prentice-Hall International Inc., USA, 1991, pp. 239–242. [56] D.M. Eagle, T. Humby, S.B. Dunnet, T.W. Robbins, Effect of regional striatal lesions on motor, motivational and executive aspects of progressive ratio performance in rats, Behav. Neurosci. 113 (1999) 718–731. [57] M. Trond, Neurotransmitter systems involved in learning and memory in the rat: a meta-analysis based on studies of four behavioural tasks, Brain Res. Rev. 41 (2003) 268–287. [58] S.M. Stahl, Neurotransmission of cognition, part3 Mechanism of action of selective NRIs: both dopamine and norepinephrine increase in pre frontal cortex, J. Clin. Psychiatry 64 (2003) 230–231. [59] R. Ganguly, D. Guha, Alteration of brain monoamines & EEG wave pattern in rat model of Alzheimer’s disease & protection by Moringa oleifera, Ind. J. Med. Res. 128 (2008) 744–751. [60] D. Milatovic, R.C. Gupta, M. Aschner, Anticholinesterase toxicity and oxidative stress, Sci. World J. 6 (2006) 295–310. [61] P.R. Lewis, C.C.D. Shute, Confirmation from choline acetylase analyses of massive cholinergic innervations to the hippocampus, J. Physiol. 172 (1964) 9– 10. [62] A. Blokland, Acetylcholine: a neurotransmitter for learning and memory? Brain Res. Rev. 21 (1995) 285–300. [63] J.L. Yang, Y.T. Lin, P.C. Chuang, V.A. Bohr, M.P. Mattson, BDNF and Exercise enhance neuronal DNA repair by stimulating CREB-mediated production of Apurinic/apyrimidinic endonuclease 1, Neuromol. Med. 16 (2014) 161–174. [64] J. Budni, T. Bellettini-Santos, F. Mina, M.L. Garcez, A.I. Zugno, The involvement of BDNF: NGF and GDNF in aging and Alzheimer’s disease, Aging Dis. 6 (2016) 331–341.