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Antioxidant and Neuroprotective Efficacy of Hydroalcoholic Extracts of Pomegranate Peel

Satish Kumar, Girish Chandran, R P Singh

Satish Kumar, Girish Chandran, R P Singh, Department of Biochemistry & Nutrition, CSIR-Central Food Technological Research Institute Mysore-570020, India

Conflict-of-interest statement: The author(s) declare(s) that there is no conflict of interest regarding the publication of this paper.

Correspondence to: Satish Kumar, Department of Biochemistry & Nutrition, CSIR-Central Food Technological Research Institute Mysore-570020, India.
Email: satskumar55555@gmail.com

Received: May 16, 2016
Revised: June 27, 2016
Accepted: June 30, 2016
Published online: December 16, 2016

ABSTRACT

Free radical mediated mechanisms are known to play a key role in many neurodegenaerative diseases which could be delayed by the presence of appropriate antioxidant defense. Pomegranate (Punica granatum L., family Punicaceae) peel possesses higher antioxidant activity compared to the pulp extract, owing to the presence of flavonoids and tannins. An attempt was made to study the neuroprotective potential of pomegranate peel. The hydroalcoholic extract of pomegranate peel was evaluated for antioxidant potential against 2, 2’-azobis (2-amidinopropane) (AAPH) induced lipid peroxidation in vitro in mice synaptosomes and neuroprotective efficacy against rotenone-induced mortality, motor deficits and oxidative stress in Drosophila melanogaster. Extract showed high radical scavenging activity by DPPH and ABTS methods and inhibitory effect against AAPH induced lipid peroxidation in mice brain homogenate and synaptosomes. The extract also showed inhibition of acetylcholinestrase (AChE) activity in the brain homogenate (IC50 =56.2 µg/mL). The extract provided significant protection against rotenone, as determined by percentage mortality and escape tests in D. melanogaster. The extract rendered protection on head and body regions of D. melanogaster, as determined by the contents of thiols and hydroperoxides and the levels of catalase, superoxide dismutase (SOD), thioredoxin reductase (TR), glutathione-S-transferase (GST) and AChE. The results indicate the antioxidant and neuomodulatory potential of hydroalcoholic extract pomegranate peel.

Key words: Pomegranate; DPPH; AAPH; Synaptosomes; Acetylcholinesterase; Drosophila melanogaster

© 2016 The Author(s). Published by ACT Publishing Group Ltd. This is an open access article under the CC BY-NC-ND license (http: //creativecommons.org/licenses/by-nc-nd/4.

Kumar S, Chandran G, Singh RP. Antioxidant and Neuroprotective Efficacy of Hydroalcoholic Extracts of Pomegranate Peel. International Journal of Neurology Research 2016; 2(3-4): -9 Available from: URL: http://www.ghrnet.org/index.php/jnr/article/view/1716

INTRODUCTION

Oxidative stress mediated through free radicals has been shown to be one of the major factors in the development of neurodegenerative diseases like Parkinson’s disease (PD), Alzheimer’s disease (AD), Multiple Sclerosis (MS) and amyotrophic lateral sclerosis ALS[1-3]. The natural antioxidant system present in biological systems is responsible for combating the damage caused by prooxidants and any impairment of this system favours accumulation of free radicals[4].

Pomegranate is used in folkloric medicine for the treatment of various diseases[5] and has become popular owing to its health-promoting phytonutrient content[6]). Its consumption has been reported to reduce platelet aggregation, oxidative stress and protection of LDL against atherogenic modifications[7]). Pomegranate possesses strong antioxidant and anti-inflammatory properties, and anti-cancer activity against several human cancers[8,9]. Several studies report the ability of pomegranate to fight obesity[10], cancer and other human diseases[11]. Pomegranate peel, which is inadvertently generated from the processing industries, has been shown to possess higher antioxidant activity than the pulp owing to the abundance of flavonoids and tannins[5]. Different parts of pomegranate have been reported as a reservoir of bioactive compounds with potential biological activities. Pomegranate, especially the leaves and peel of pomegranate, decreased the dyslipidemia of obesity and cardiovascular risk factors[12].

By virtue of presence of compounds possessing high antioxidant potential in the pomegranate peel, studies were conducted to evaluate the antioxidant profile of hydroalcoholic extract of peel using various in vitro methods; protection offered against AAPH induced oxidative challenge in mice brain synaptosomes and neuromodulatory efficacy against rotenone induced oxidative stress in the head and body regions of adult Drosophila melanogaster.

MATERIALS AND METHODS

Raw material and Chemicals Pomegranate fruits (Punica granatum) were purchased from the local market. The fruits were washed, cut and the peel, mesocarp, and capillary membranes were manually separated. The peel was cut into pieces of approximately 1 cm × 1 cm size and were dried in hot air cabinet drier for 5-6 h at 55-60°C. Dried peel was ground to moderate coarse powder using mixer-grinder and the peel powder was packed in polythene covers and stored in desiccators at room temperature.

Thiobarbituric acid (TBA), rotenone (ROT), α, α-diphenyl-β-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and other fine chemicals were procured from M/s Sigma Chemical Co., St. Louis, USA. Nicotinamide adenine dinucleotide reduced (NADPH), 1, 1-dithio nitro-bi-benzoic acid (DTNB), 1-chloro 2, 4-dinitro benzene (CDNB) and all other chemicals used were of analytical grade and procured from Sisco Research Laboratory Chemicals, Mumbai, India.

Preparation of pomegranate peel extract (PPE)

PPE was prepared by extracting pomegranate peel in ethanol: water (1:1 v/v) as described by[13]. 1g of peel powder was subjected to overnight extraction at room temperature in 10 mL of extractant with intermittent shaking, centrifuged at 3000 rpm for 5 min and filtered through Whatman filter 2[14].

Radical scavenging activity (RSA) and polyphenols

DPPH assay: 0.2 mL methanol and 5 mL of 100 μM methanolic DPPH (100 uM) was used as control. Different aliquots (0.1 and 0.2 mL) of PPE were taken for analysis. 5 mL of DPPH was added to all the samples, vortexed and incubated at room temperature for 20 minutes under dark. Absorbance was read at 517nm using UV –visible spectrometer, UV-1800 (Shimadzu, Japan) and recorded[13]. The percentage of antioxidant activity was determined using following formula:

ABTS assay: ABTS+ radical cation was produced by reacting 7 mM aqueous ABTS with 2.6 mM potassium persulfate in equal quantity and keeping it in dark at room temperature for 16 h. Blue–green ABTS+ radical solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm using Shimadzu UV-visible spectrometer UV-1800 (Re et al, 1999). As described in DPPH assay, 0.1 and 0.2 mL PPE were taken in separate tubes and to these, 5 mL ABTS+ was added and incubated at room temperature for 20 min. The OD was taken at 734 nm and the percentage RSA was determined using following formula[14].

Estimation of total polyphenols: Different aliquots of standard tannic acid solution and PPE in a total volume of 1.0 mL were taken and made up to 10 mL with water. 0.5 mL of Folin Ciocalteau reagent was added to all the tubes, followed by the addition of 1.0 mL of saturated sodium carbonate solution. The reaction mixture was vortexed and incubated at room temperature for 30 minutes. A blank was prepared without tannic acid solution. The absorbance was read at 760 nm and the total polyphenol content of the samples were estimated and expressed as Tannic Acid equivalents[15].

Biological activity in in vitro cell free systems

Modulatory effect of PPE against AAPH-induced lipid peroxidation in striatum: Adult (8-9 weeks old) Swiss albino male mice (35.1 ± 3.2 g) were drawn from the stock colony of the Institute’s Animal Facility and were housed in a controlled atmosphere with a 12 h light/dark cycle. These were provided commercial diet and water ad libitum. All the experiments were conducted strictly in accordance with approved guidelines by the Institute Animal Ethical Committee (IAEC, Registration number: 49/1999/CPCSEA), regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social justice and Empowerment, Government of India, India. Handling and care of animals was carried out strictly according to the standard guidelines laid by the IAEC.

Mice brain striatum was collected by sacrificing using general anesthesia and brain was dissected to separate striata (St) at 4°C. These were homogenized (10% w/v) in ice cold phosphate buffered saline (PBS, 0.1M, pH 7.4, 0.8% NaCl) and centrifuged at 4,000 rpm for 10 min at 4°C to obtain post nuclear supernatant. The supernatant was separated and recentrifuged (10,000 rpm × 10 min. at 4°C) to obtain crude synaptosomal pellet. The synaptosomes were washed in HEPES buffer and resuspended in the same. Freshly prepared synaptosomes and homogenates were used for experiments. Striata homogenates / synaptosomes were challenged with varying concentrations of 2,2’-azobis (2-amidinopropane) (AAPH) (50, 100, 200 and 500 μM) in vitro (30 hr, 37°C) in Krebs-Ringer bicarbonate (KRB) solution to obtain a concentration-related response. To measure the efficacy of PPE, St-homogenates/ synaptosomes were treated with different concentrations (50, 100 and 200 μg/mL) of PPE and incubated with or without AAPH (200 μM) for 1hr. The amount of malondialdehyde was measured by quantifying thiobarbituric acid reactive substances TBARS[16].

Acetylcholinesterase (AChE) inhibitory potency of PPE: Effect on of PPE on AChE activity was measured using the method of[17]. 50 μL of 0.1 M sodium phosphate buffer (pH 8.0), 25 μL of AChE solution, different aliquots of PPE and 125 μL of DTNB (2.5 mM) were added in a 96-well microplate and incubated for 15 min at 25ºC. The reaction was initiated with the addition of 25 μL of acetylthiocholine iodide and its hydrolysis was monitored by the formation of the yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, at 412 nm. The concentration of the extracts which caused 50% inhibition of AChE (IC50) was calculated by nonlinear regression analysis. The %inhibition was calculated from (1 - S/E) × 100, where E and S were the respective enzyme activities without and with the test sample, respectively.

Neuroprotective efficacy of PPE in Drosophila

Drosophila culture and maintenance

D. melanogaster, wild (Oregon K) obtained from the fly laboratory of our institute which were maintained under standard conditions, were used in this study. The flies were maintained at 22 ± 1°C and 70-80% relative humidity and fed on a standard wheat flour– agar diet with yeast granules[18]. 9-10 Days old age synchronized adult male flies (50/replicate; 3 replicates/group) were introduced into glass vials with 2 mL medium.

Reconstitution of PPE

5 mL PPE was evaporated to dryness (0.5 g dry extract) and was resuspended in 2 mL water (250 mg/mL) for using in the Drosophila experiments.

Experimental design

The adult male flies were maintained on medium containing 0.05, 0.1, 0.2 and 0.4% PPE to determine its modulatory effect on the levels of endogenous oxidative markers in head and body regions of flies (n = 50/ replicate; 2 replicates per group). The propensity of PPE to modulate Rotenone-induced mortality, locomotor dysfunction, oxidative stress and neurotoxicity was determined in independent experiments employing a co-exposure paradigm. The concentration of Rotenone (500 μM) was selected, as described by[19]. The flies were regularly monitored for mortality and locomotor deficits.

Locomotor deficits - negative geotaxis assay

The motor function of the flies was assessed using a negative geotaxis assay, as described by[20] Feany and Bender (2000). The flies were introduced into a graduated flat bottom glass tube (l-25 cm; d-2 cm).The tubes were gently tapped at the bottom and observed for 60 sec for the climbing activity (25 flies/trial; 3 trials/replicate). Locomotor behavior was expressed as percent flies escaped beyond a minimum distance of 10 cm in 20 s.

Biochemical determinations

Preparation of head / body homogenates: After the treatment, the flies were anaesthetized and their heads were separated immediately. Both head and body regions were homogenized in Phosphate buffer (0.1 M, pH 7.4) and centrifuged at 4000 rpm for 10 min at 4˚C. The supernatants were used for the assays. The amount of protein in the head and body homogenates was estimated[21] by incubating an aliquot of the sample with Folin–Ciocalteau’s reagent in an alkaline medium for 30 min and measuring the optical density at 750 nm in a UV–visible Spectrophotometer (Shimadzu). The amount of protein was quantified using a bovine serum albumin standard curve.

Hydroperoxides assay: Hydroperoxides were determined based on ferrous ions mediated oxidation of xylenol orange[22]. Aliquots of head and body homogenates of the flies (20 mg protein) were allowed to react with FOX1 reagent (xylenol orange 100 mM, ferrous ammonium sulphate 250 mM and sorbitol 100 mM) prepared in 25 mM H2SO4 for 30 min at room temperature. The color developed was quantified at 560 nm (ε – 2.2 × 105/M/cm) and expressed as nmol hydroperoxides[23] (HP)/mg protein.

Glutathione/total thiols: The thiol content in head and body homogenate of flies was estimated using the method of[24,25] by incubating an aliquot of the sample (0.05 mg protein) with DTNB (0.02 mM) in phosphate buffer (0.1 M, pH 8.2) for 20 min and measuring the absorbance at 412 nm. Thiol levels were calculated (ε - 13.6/mM/cm) and expressed as nmol thiols/mg protein.

Antioxidant enzymes

Catalase assay: Catalase activity was estimated by the method of[26] by adding an aliquot of homogenates (0.25 mg protein) to phosphate buffer (0.1 M, pH7.4, containing H2O2 10 mM). The decomposition of H2O2 was monitored at 240 nm and activity was expressed as nmol substrate/min/mg protein (ε – 44.2/mM/cm).

Superoxide dismutase (SOD) assay: SOD activity in the homogenates of flies was measured indirectly by monitoring the inhibition of quercetin autooxidation[27]. Reaction mixture consisted of phosphate buffer (16 mM, pH 7.8) containing TEMED (4 mM) and EDTA (0.04 mM). Quercetin (100 mM) was added and the rate of its autoxidation was monitored at 406 nm for 3 min. The ability of the test sample to inhibit quercetin oxidation by 50% was defined as one unit of the enzyme and activity was expressed as units/mg protein.

Thioredoxin reductase (TR) assay: TR activity of the homogenates from head and body of Drosophila was measured by the method of[28] by monitoring the reduction of DTNB (in phosphate buffer 0.1 M, pH 7.0, containing EDTA 10mM and NADPH 0.2 mM) at 412 nm. The activity was expressed as nmol of DTNB reduced/min/mg protein (ε – 13.6/mM/ cm).

Glutathione reductase (GR): GR activity of the homogenates was measured using the method of[29] by the addition of aliquots of homogenates (0.2 mg protein) to the phosphate buffer (0.2 M, pH 7.0 containing EDTA 2 mM, oxidized glutathione 20 mM and NADPH 2 mM). The decrease in the absorbance at 340 nm due to oxidation of NADPH was monitored for 3 min and the activity was expressed as nmol NADPH oxidized/min/ mg protein (ε – 6.22/mM/cm).

Glutathione-S-transferase (GST): GST activity in head and body homogenates of the flies was assayed using the method of[30] by monitoring the conjugation of glutathione to CDNB at 340 nm. The reaction was initiated by aliquots of homogenate (0.01 mg protein) to the phosphate buffer (0.1 M, pH containing EDTA 0.5mM, CDNB 0.075 mM and GSH 0.05 mM). The increase in the absorbance at 340 nm was recorded for 3 min and the activity was expressed as nmol of conjugate formed/min/mg protein (ε – 9.6/mM/cm).

Acetylcholinesterase (AChE) assay: AChE activity in homogenates was estimated as described by[24,25]. The reaction was initiated by the addition of acetylthiocholine iodide (ATCI, 1.95 mM) to the phosphate buffer (0.1 M, pH 8.0) containing DTNB (2.5 mM) and homogenates of head and body (0.01 mg protein). The change in absorbance was monitored at 412 nm for 3 min. The enzyme activity was expressed as nmol substrate hydrolysed/min/mg protein.

AChE inhibitory activities were measured as described in section 2.4.2[17].

Statistical analysis

Data are expressed as mean ± SE (Standard Error) and analyzed by non-parametric one-way ANOVA followed by post hoc Turkey’s test.

RESULTS

Extraction and antioxidant profiling of PPE

PPE was made by extracting peel powder with water: ethanol (1: 1) at room temperature, as this composition has been shown to provide maximum yield of the extract with high content of polyphenol (data under publication). The extraction protocol not only avoids the use of solvents which may have the economic and residual solvent concerns but also utilizes minimum energy and the extract can be easily reconstituted in water. The extract showed RSA of 27.23 ± 0.14% (with DPPH) and 37.71 ± 0.34 % (with ABTS) with 0.1 mL of 1: 200 and 1: 500 dilutions, respectively. The polyphenol content of the extract was estimated to be 32.48 ± 0.63 mg/mL extract.

Effect of PPE on AAPH-induced in vitro lipid peroxidation

The propensity of PPE to inhibit AAPH induced lipid peroxidation in brain homogenates and synaptosomes was determined. AAPH exposure (100-1000 μM) resulted in a concentration-related formation of malondialdehyde (MDA, 100 μM-18%, 200 μM-51%, 500 μM-130% and 1000 μM-215% MDA was produced, data not shown). Figure 1 shows the effect of PPE on reduction of MDA formation in native and AAPH induced homogenate and synaptosome. PPE per se not only reduced the formation of MDA, but also significantly inhibited the AAPH (200 μM) induced lipid peroxide formation in the homogenates (Figure 1A) as well as in synaptosomes (Figure 1B) in a concentration dependent manner. However, due to some unknown and unexplainable reasons, PPE pretreatment of synaptosomal preparation causes increase in lipid peroxidation but not in the homogenate.

PPE also exhibited a concentration dependent inhibitory effect against commercial AChE with an IC50 value of 56.82 ± 0.73 μg/mL (Figure 2).

Figure 1A Inhibitory effect of hydroalcoholic extract pomegranate peel (PPE) on malondialdehyde levels in vitro in mice brain straitum homogenate. (* P ≤0.05, as compared to saline control; # P ≤0.05, as compared to AAPH alone).

Figure 1B Inhibitory effect of hydroalcoholic extract of pomegranate peel (PPE) on malondialdehyde levels in vitro in mice brain striatum synaptosomes. (* P ≤ 0.05, as compared to saline control ; # P ≤0.05, as compared to AAPH alone).

Figure 2 Acetylcholinesterase inhibition by hydroalcoholic extract of pomegranate peel (PPE).

Neuroprotective efficacy of PPE against Rotenone (ROT) in Drosophila Rotenone (at 500 μM dose) induced lethality was evident from 3rd day onwards and increased with duration of exposure and terminally on 6th day, the cumulative mortality was 55%. Coexposure with PPE resulted in significant protection against Rotenone–induced mortality in a dose dependent manner, as depicted in Figure 3. PPE at low concentration of 0.05% provided only a moderate (15%) protection while robust protection was rendered at higher concentrations (0.1%-60%; 0.2%-70% and 0.4%-95%). Rotenone (500 μM) treated flies also exhibited severe (70%) impairment of locomotor activity by day 6, as evident by the decreased number of flies escaping the 10cm height of the glass column. Interestingly, co–exposure with PPE resulted in concentration–dependent improvement in locomotor deficits, suggesting robust protection offered by PPE (Figure 3B).

Figure 3 Protective efficacy of hydroalcoholic extract of pomegranate peel (PPE) against ROT-induced (A) Mortality and (B)motor deficits in adult Drosophila melanogaster. (* P < 0.05, as compared to control ; # P < 0.05, as compared to ROT alone). ROT 500μM - Rotenone; PPE - Pomegranate Peel Extract; PPE1 - 0.05%; PPE2 - 0.1%; PPE3 - 0.2%; PPE4 - 0.4%.

Efficacy of PPE to attenuate ROT induced oxidative stress

While Rotenone exposure (500 uM) enhanced the levels in hydroperoxides in head (20%) and body regions (43%), coexposure with PPE significantly diminished the levels of hydroperoxides in a concentration dependent manner (Figure 4). Though Rotenone did not exert very pronounced effect on the levels of thiol content in head and body regions, coexposure with PPE at higher concentrations elevated thiol levels in both head and body regions of the flies.

Figure 4 Protective efficacy of hydroalcoholic extract of pomegranate peel (PPE) against ROT-induced alterations in Hydroperoxides levels (A-head; B-body) and Thiols content (C-head; D-body) head and body regions of adult Drosophila melanogaster. (* P < 0.05, as compared to control, # P < 0.05, as compared to ROT alone). ROT 500 μM - Rotenone; PPE- Pomegranate Peel Extract; PPE1 - 0.05%; PPE2 - 0.1%; PPE3 - 0.2%; PPE4 - 0.4%.

Rotenone exposure reduced the catalase activity levels significantly in the head (80%, Figure 5A) and body regions (20%, Figure 5B) of flies, while coexposure with PPE caused enhancement in the levels of catalase in both body and head regions, thus attenuating the Rotenone induced effect. The activity of TR was reduced with Rotenone exposure in head (20%, Figure 5C) and body region (16%, Figure 5D), PPE supplements significantly restored the TR activity levels in a concentration dependent manner. Rotenone also caused significant diminution (61%, Figure 5E) in the activity of SOD in the body region, which was attenuated by PPE at lower concentration while higher concentrations of PPE did not alter the Rotenone induced effect in a concentration dependent manner.

Figure 5 Protective efficacy of hydroalcoholic extract of pomegranate peel (PPE) on the perturbations in the activity levels of catalase (A-head; B-body), Thioredoxin reductase (C-head; D-body) and Superoxide dismutase (E-body) among adult Drosophila melanogaster exposed to rotenone. (* P < 0.05, as compared to control ; # P < 0.05, as compared to ROT alone). ROT 500 μM - Rotenone; PPE - Pomegranate Peel Extract; PPE1 - 0.05%; PPE2 - 0.1%; PPE3 - 0.2%; PPE4 - 0.4%.

GST levels were differentially affected in head and body regions of Rotenone fed flies (Figure 6). There was a significant decrease (18%, Figure 6A) activity levels in head regions while the levels were increased (16%, Figure 6B) in body regions. The activity levels of GST were significantly restored in head regions by PPE in a concentration dependent manner but the effect was not pronounced in the body regions of the flies.

Rotenone was shown to significantly elevate the activity levels of AChE in head (22%, Figure 6C) and body (13%, Figure 6D) regions of rotenone treated flies, while the levels were restored to normalcy by PPE in head region but in the body region, PPE does not show the restoration of the activity, as expected.

Figure 6 Modulatory propensity of hydroalcoholic extract of pomegranate peel (PPE) on the alteration in the activity levels of Glutathione-S-transferase (GST, A-head; B-body) and acetylcholinesterase (AChE, C-head; D-body) of adult Drosophila melanogaster exposed to ROT (500μM). (* P<0.05, as compared to control; # P<0.05, as compared to ROT alone). ROT 500μM - Rotenone; PPE- Pomegranate Peel Extract; PPE1 - 0.05%; PPE2 - 0.1%; PPE3 - 0.2%; PPE4 - 0.4%.

DISCUSSION

Free radical generation is primarily due to aerobic metabolism and their accumulation may cause neurodegeneration in specific areas of brain, which may lead to the onset of degenerative diseases, including Alzheimer’s and Parkinson’s disease[1,2,31]. Thus, an approach to treat these oxidative stress mediated neurodegenerative disorders[32] would be through the molecule(s) which could help in attenuating both redox balance and antioxidant defenses. Extracts of pomegranate peel were shown to possess strong antioxidant potential with methanol extract showed the strongest antioxidant potential[13] and rendered protection against CCl4 challenged rats by restoring the levels of various antioxidant enzymes and liver architecture[33]. To avoid solvents, the pomegranate peel extract was prepared in an eco-friendly manner, and was examined against AAPH induced oxidative challenge in mice and rotenone induced oxidative challenge in Drosophila. The extract was shown to provide protection against AAPH induced oxidative challenge by modulating the lipid peroxidation, both in straita and synaptosomes of mice brain, thus could be helpful in attenuating the symptoms of neurodegenerative diseases.

Alzheimer’s disease is characterized by memory dysfunction and the reduction of acetylcholine levels in the brain is the most notable biochemical change observed in it[34]. The application of the agents which could restore the levels of acetylcholine through inhibition of cholinesterases[35] would be a precise strategy to attenuate the symptoms of Alzheimer’s disease. The commercial preparations are known to possess adverse effects[36-38]. Therefore, search for safe and active AChE inhibitors, particularly from natural products has become the focus in the recent time.

In our study, PPE exhibited a concentration dependent anti-AChE effect with an IC50 value of 56.82 ± 0.73 μg/mL. The results suggest the presence of compounds in the peel extract which possess AChE inhibitory property, hence showing their efficacy to modulate cholinergic system of neurotransmission. The extract was further studied for its neuroprotective efficacy against rotenone (redox disruptor) induced oxidative challenge in Drosophila. Rotenone has been well established to induce lethality and locomotor deficits in Drosophila model[18] via oxidative stress mechanisms[39] and due to the specific sensitivity of dopaminergic neurons to oxidative damage[40,41]. The exposure of flies to rotenone caused severe lethality and locomotor deficits along with severe oxidative imbalance, as evident by the elevated levels of oxidative markers (hydroperoxides) and significant alterations in the activity levels of antioxidant enzymes which are in concurrence with the observations of[19].

The lower concentrations of PPE did not seem to be very effective in overcoming the rotenone induced lethality and locomotor deficits but the higher concentrations were more effective and could significantly revert the rotenone effects. This could probably be by protecting the dopaminergic neurons against the oxidative insult caused by rotenone[39,42]. The protection rendered by PPE strongly suggests its primary role in modulating the rotenone induced oxidative burst, both in head and body regions of Drosophila. Rotenone does not cause any significant alteration in the levels of reduced glutathione but the increase in its levels at higher concentrations of PPE with Rotenone in both the regions may indicate its de novo synthesis for its involvement in the mechanism for redox maintenance for neuromodulation[43].

PPE caused restoration of the rotenone-induced reduction in the activity of the TR in both head and body regions which indicates its modulation of in vivo redox system which is not dependent on glutathione. The effect was more pronounced in body than in head region. TR protects the cells against oxidative damages and plays important role in maintaining the redox balance in biological systems via glutathione and thioredoxin systems[44-46]. In addition, PPE also improved the levels of the rotenone induced reduction of GST in head region but not in body region. The increased TR and GST activities by PPE may indicate its possible neuroprotective role. The thioredoxin system generally functions in coordination with glutathione system and in the absence of glutathione reductase in Drosophila, TR provides additional reduced thioredoxin moities for the non enzymatic reduction of glutathione disulfide, thus maintaining redox balance[47]. The reduction in TR activity by rotenone and concentration dependent restoration of activity by coexposure with PPE (especially in head region) points its neuroprotective role.

PPE has been shown to revert back the rotenone induced increase in AChE level, particularly in the head region of the flies, which gives an indication of its modulating role in cholinergic neurotransmission. The effect was not so pronounced in the body region, which as such showed lower activity profile than in head region, indicating the lower importance of AChE in the body region. The inhibition of cholinergic neurotransmission via AChE is an approach to combat the cognitive symptoms of Alzheimer’s disease[48]. Thus phytochemicals with effective AChE inhibitory properties could be a safer approach to attenuate Alzheimer disease[49].

PPE restored the rotenone induced reduction of catalase and SOD activities which are comparable with the control values. This shows the protection provided by PPE in maintaining the levels of catalase and SOD which was reduced by rotenone treatment. The effect of rotenone on these enzymes could probably be due to enzyme inactivation during the catalytic cycle. In these conditions, the pomegranate extract containing a variety of antioxidants (ellagic acid, gallic acid etc.) could act by reducing the levels of hydrogen peroxide and superoxide anion and, consequently, lipid peroxidation and enzyme inactivation, restoring enzyme activity. This may also point towards the possible de novo synthesis of these enzymes induced by the components of pomegranate peel extract[1,2,31,33,49].

The findings of this study clearly suggest that PPE supplements not only significantly modulated the endogenous levels of oxidative markers in mice brain and flies, but also offset rotenone-induced oxidative stress. Furthermore, improved motor function among flies maintained on PPE-enriched diet provide clear evidence of its neuromodulatory propensity. Although, these protective effects could not be precisely attributed to any specific bioactives from PPE, high concentration of Ellagitannis (Punicalagin and ellagic acid) present in the peel could be partly responsible. Nevertheless, the data suggest the need to further understand the mechanisms underlying the neuromodulatory property of PPE.

Acknowledgements

The authors are thankful to Head, Biochemistry and Nutrition department and Director, CSIR-CFTRI for their support and encouragement during the course of the work.

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Peer reviewer: Olga Galkina

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