International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491
Vol 9, Issue 2, 2017
NEUROPROTECTIVE EFFECTS OF POMEGRANATE JUICE ON LEAD INDUCED NEUROTOXICITY IN MICE LEILA GADOUCHE, NOUREDDINE DJEBLI, KHAYRA ZERROUKI Pharmacognosy Api Phytotherapy Laboratory (LPAP), Mostaganem University, Algeria Email: [email protected]
Received: 21 Oct 2016 Revised and Accepted: 21 Dec 2016 ABSTRACT Objective: This study evaluates the potential neuroprotective of the pomegranate juice against chronic intoxication with lead acetate for 3 months. Methods: Twenty-one female Swiss mice divided into 3 groups were employed in the present investigation. Control group: received drinking water for 90 days, neurotoxic group were exposed to 1000 ppm of lead acetate in the drinking water for 12 weeks, and neurotoxic treated group represents the mice received treatment with juice pomegranate diluted with distilled water (v/v) orally for 4 h / day followed by lead acetate at a dose of 1000 ppm orally for 20 h / day for 90 days. After cessation of treatment, neurobehavioral studies using the open field test, black and white test box and swimming test were made. In the next phase, brain injury was assessed histologically with hematoxylin-eosin staining. Results: Chronic exposure to lead led to significant increase in the level of anxiety, depression and the locomotor activity (P < 0.05). It was confirmed by histopathological alterations in many areas of the cerebral cortex and hippocampus including neuronal degeneration and decrease cell density. Treatment with the juice significantly improve the level of depression, locomotor function (P < 005) and anxiety (P > 0.05) in mice exposed to lead as well as restored the histological structure in cerebral cortex and hippocampus of mice. The total phenolic and flavonoids content in juice of pomegranate was found to be 3809. 8±29.404 mg GAE/l; 2109. 57±18.936 mg QE /l of juice. Conclusion: This finding suggests that phenolic compounds found in pomegranate juice provide a neuroprotective effect on behavioural impairments and histopathological change induced by lead. Keywords: Pomegranate juice, Neurotoxicity, Neurobehavioral, Histopathology, Cerebral cortex, Hippocampus © 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ijpps.2017v9i2.15806
INTRODUCTION Heavy metals occur as natural constituents of the earth crust, and are persistent environmental contaminants since they cannot be degraded or destroyed . Their multiple industrial, domestic, agricultural, medical and technological applications have led to their wide distribution in the environment; raising concerns over their potential effects on human health and the environment . Lead (Pb) is a highly neurotoxic agent that particularly affects the developing central nervous system . In fact, cerebral damage induced by lead occurs preferentially in the cerebral cortex, cerebellum, and hippocampus . In the brain, the cerebellum was found to be most severely affected  accompanied by areas of focal cortical necrosis. A significant decrease in spine density  and reduction in the maximum width of the hippocampus  have also been reported. Consistent neuropsychological research over the years has revealed that Pb exposure can result in declines in intelligence, memory, processing speed, comprehension and reading, visuospatial skills, motor skills, and, to a probable lesser extent, executive skills. Among the cognitive deficits induced by Pb toxicity, visuospatial deficits appear to be notably prominent. Anxiety, depression, and phobia can also occur . Although chelating therapy is currently an available treatment of Pb neurotoxicity, it is observed to have many adverse effects such as divalent metal ion imbalance and it is also ineffective in improving previous nerve injury induced by Pb. Currently, no efficient drugs are available for treating chronic lead-induced cognitive deficits . Calcium Disodium EDTA is a traditional synthetic lead expellant, but it is toxic to the liver and kidneys. The amelioration of neurotoxicity induced by certain heavy metals by herbal principles is of great significance as they are cost-effective and highly active and lead to no side effects . Therefore recently there have been many
studies on the use of natural products such as vitamins, and herbal drugs to expel lead . Polyphenols represent a wide variety of naturally occurring compounds which are present in high amounts in fruits, vegetables, and natural products. Polyphenols have been proposed to exert a multiplicity of neuroprotective actions within the brain, including a potential to protect neurons against injury induced by neurotoxins  ability to suppress neuroinflammation , and the potential to promote memory, learning, and cognitive function . Pomegranates contain very high levels of polyphenols compared with other fruits and vegetables. Punica granatum is a good source of natural compounds which might have benefits for health [15,16]. Dietary supplementation of pregnant mice with pomegranate juice was shown to protect against neurodegeneration in neonatal mice subjected to hypoxic–ischemic brain injury . Previous studies reported that pomegranate extract showed neuroprotective effects against Alzheimer’s disease and depression . This work aims to investigate the neuroprotective effect of pomegranate juice against damage caused by lead acetate in mice brain. Evaluation will be done through behavioural and histopathological assay. MATERIALS AND METHODS Chemicals Lead acetate (Pb (C2H3O2) 2), folin-ciocalteu’s phenol reagent, gallic acid, sodium carbonate (Na2CO3), methanol, quercetin, aluminum chloride (AlCl3) were purchased from Sigma-Aldrich Co. Juice preparation The Sefri variety of Punica granatum L was collected from the locality of Oued sly in the western region of Algeria and has been identified and confirmed at The National School of Agronomy "ENSA". The species exist
Gadouche et al. Int J Pharm Pharm Sci, Vol 9, Issue 2, 207-212 in the package number 35 at the herbarium of North Africa and voucher specimen was deposited in the Department of Botany. To prepare pomegranate juice, the fruit was individually cut and peels are covering the arils removed. Arils juice were extracted using food processor after removing the seeds. Arils juice obtained were centrifuged at 10000 rpm for 15 min at 4 °C and supernatants were collected immediately diluted with distilled water (v/v) , stored at-40 °C.
dark water bottles (renewed every 1 hour) for 4 h/day followed by lead acetate at a dose of 1000 ppm orally for 20 h/day for 90 d . Neurobehavioral studies Open-field test
Mice were tested in acrylic cages (32 x 32 cm) divided into 16 equal squares. The number of crossed squares was recorded for each mouse per time of 5 min for 20 min investigated.
Determination of total phenolic compounds
Black and white test box
The total phenolic content of pomegranate juice was determined according to the method of Mavi et al. . The reaction mixture contained 250 µl of folin–ciocalteu reagent and 50 µl of pomegranate juice. Afterwards, 500 µl of 20% water solution of Na2CO3 was added to the mixture. Mixtures were vortexed and completed with water to 5 ml followed by incubation in the dark at ambient temperature for 30 min. Absorbance against blank was measured at 760 nm. The results were expressed as mg gallic acid equivalents (GAE) per l of juice .
As described , this model permits simple and quick evaluation of the anxious behaviour and its modification compared between an illuminated compartment and a dark one. The test boxes consist of two compartments, the first of these, coloured matt black and the other one, matt white. Both compartments are separated by a wall with a 70 x70 mm opening in its base. Total time spent in each compartment was recorded for 20 min with sample intervals of 5 min. Forced swimming test
Determination of total flavonoids compounds The total flavonoid content in juices was determined spectrophotometrically according to the method of Lamaison and Carnat , using a method based on the formation of a complex flavonoidaluminum, having the absorptivity maximum at 430 nm. Quercetin was used to make the calibration curve. The sample (1 ml) was separately mixed with 1 ml of 2% aluminium chloride methanolic solution. After incubation at room temperature for 15 min, the absorbance of the reaction mixture was measured at 430 nm and the flavonoid content was expressed as mg of Quercetin equivalent per l of juice .
Mice were placed in a Plexiglas cylinder (10 cm internal diameter, 50 cm high) filled with 25-26 °C water (10 cm height). Duration of the experiment was 5 min. The immobility time was measured, a mouse was judged to be immobile when it remained floating in the water, making only those movements necessary to keep its head above the water . Histological studies
The brains of mice were removed right after sacrifices. Removed brains were fixed in 10% buffered formalin and were processed for paraffin sectioning. Sections of about 5 µm thickness were stained with haematoxylin and eosin (HE) to study the histology .
Induction of neurotoxicity and treatment
Twenty-one healthy female Swiss mice weighing from 18-22 g were obtained from Pasteur Institute of Algiers were randomly divided into three experimental groups of seven mice each:
Values represent the means of 5 experiments. The differences between experimental groups were assessed by analysis of variance followed by Student Test using SPSS statistic (Version 22.0). The difference was considered significant at P