Prevention and treatment of brain damage in streptozotocin induced diabetic rats with Metformin, Nigella sativa, Zingiber officinale, and Punica granatum

Introduction: Diabetes mellitus (DM) is well-known metabolic disorder, which causes serious effects on human healthwith its complications. Manymechanisms has been postulated to cause DM related complications. Oneof themain complications is neuronal damage, forwhich noproper preventive and curative therapies are available. Methods: In this study the effects of Ginger, Nigella sativa, Punica granatum and Metformin were seen on the prevention and treatment of brain damage caused by diabetes mellitus in streptozotocin (STZ)induced diabetes in rats. 50 adult Wistar albinomale rats were used in the study, the rats were divided in 10 study groups. The body weight, serum glucose levels were measured, and histopathological examination was performed. Results: In comparison to the diabetic control rats, significant increase in weight was found in animals of all the studied groups. Serum glucose levels reduced significantly in comparison to the STZ induced diabetic rats in all the animals. Histopathological examination showed prevention from brain damage and repair of the neuronal tissues by Ginger, Nigella sativa, Punica granatum and Metformin. Conclusion: The studied substances were observed to possess preventive and curative effects on the brain damage caused by diabetes mellitus.


INTRODUCTION
Diabetes mellitus (DM) is a metabolic disorder that is characterized by an increase in blood glucose (BG) and excretion of glucose in urine. DM is one of the major global health hazards of the recent years and also an emerging threat to public health in Kingdom of Saudi Arabia. The endemic Saudi population appears to possess a special genetic trend to develop Type II DM, that is complicated by an increment in obesity statistics, and the presence of other causes of the insulin resistance 1 . The main pathology associated with Type II DM is insulin resistance, which causes insulin deficiency in the hepatic and peripheral tissues causing hyperglycemia 2,3 . In type1 and type II diabetes mellitus, glucose uptake gets disturbed, especially in muscle and adipose tissues, resulting in hyperglycemia 4 . The neurons start taking up glucose without insulin, therefore, in hyperglycemic diabetes mellitus, hyperglycemia causes an up to four-fold increase in neuronal cell glucose uptake. Increased cytosolic glucose concentration and metabolism leads to neuron damage 2,5 .
In previous human and animal studies, DM has been found to be associated with pathological changes in the central nervous system, causing cognitive function decline, behavior disorders and an increased risk of vascular abnormalities in the brain 2,6,7 . It has been suggested that long-term DM augments the risk of brain shrinkage, lacunar infarcts and white matter (WM) abrasions. The functional and behavioral consequences of diabetic brain abnormalities include cognitive decline and movement disorders. Many structural brain changes have been described such as increased hippocampal astrocyte reactivity, abnormal synaptic plasticity, vascular alterations, decreased dendritic complexity and disturbed neurotransmission [8][9][10] . In the pathogenesis of diabetes related brain damage, disturbed coupled electron transport systems are caused by damaged mitochondria, which is the main foci for reactive oxygen species (ROS) in the neurons. Therefore, oxidative stress is recognized as a main mediating process in the pathogenesis of diabetic complications, due to increased production of free radicals and abnormal antioxidant defenses 11,12 . Many of herbal substances have been found to possess antioxidants and protect brain from damages. Nigella Sativa is known to possess antioxidants and antihyperglycemic effects and prevents neuronal damage 13,14 . Because of the chronic symptoms, the development of new treatment strategies is requiredto improve the effectiveness in treatments 10 . Ginger has also been found to possess anti-oxidative properties and has been shown to perfrom neuroprotection in diabetic rats and protective and therapeutic effect on Alzheimer's disease [15][16][17] . Pomegranate or Punica granatum, is a small tree or shrub found in the Mediterranean region 18 . It possess several biological effects such as antitumor and antibacterial functions, which have been reported with the extracts from different parts of P. Granatum 19,20 . It has been shown that Punica granatum can alleviate brain oxidative stress in diabetic rats via the regulation of antioxidant defense mechanisms, which can ameliorate the abnormality in learning and memory performances in diabetic rats 21,22 . Metformin is a standard antidiabetic drug, belonging to the Biguanide group, which was shown to attenuate stroke-induced nitrative signaling in GK rats 23 . Previous studies showed that metformin can significantly reduce Neuro-inflammation, decrease the loss of neurons in the hippocampus of diabetic animals, and prevent diabetes-induced memory loss in rats 24,25 .
The main objective of this study was to ascertain the prophylactic and therapeutic potential of Zingeber officinale, Nigella sativa, Punica granatum, and Metformin in diabetes mellitus associated brain damage and to discover cost effective treatment of the ailment.

Plant materials
Seeds of Nigella sativa, Ginger (Zingiber officinale), and Punica granatum were acquired from the regional market.

Preparation of Ginger ( Zingiber Officinale ) Extracts
Fresh ginger root was purchased from the local market in Rafha Municipal Council, Northern Border Region of Saudi Arabia. The roots were identified and authenticated by the Department of Pharmacognosy, in the Faculty of Pharmacy, Northern Border University, Rafha, Saudi Arabia. Preparation was done according to the modified method used by (Kebe et al., 2015) 26 . Cleaning of 2.5 kg of fresh ginger rhizome was done with running tap water. It was shredded into small pieces and air dried for 2 weeks, then crushed into powder form with electric blender. 2000 g of this powdered Zingiber officinale was soaked in 5000 ml of 99.9% ethanol and rattled vigorously. It was left for 48 hours at room temperature and was stirred at different times. The dissolved ginger in ethanol was filtered with a mesh with small pores after 48 hours. Then, it was filtered using No1 Whatmann paper (filter paper) and funnel. The percolate was assembled in a tray and was air dried for 5 days to ensure complete evaporation of the ethanol used. The ginger paste was collected from the tray with a spatula into a container and was weighed using an electric scale. 50g of ginger paste was collected and then dissolved in extra virgin olive oil 100ml (to serve as a vehicle). The extract was then kept in a dry place at 37 • C.

Preparation of Black Seed (Nigella sativa) Extract
This method was adopted from Shahraki et al. 27 .
To obtain a hydroalcholic extract, powder was made from 100 g of dried N. sativa seeds. After that, it was macerated in a solution of 70% alcohol and 30% DW for 72 hours. To prepare the fractions, 10 g of the extract was mixed with 100 ml of ethanol and decanted by funnel. The n-hexane solvent was added to the funnel, and the n-hexane fraction was then extracted. Then, the remaining solvent in the decanter funnel was mixed with dichloromethane solvent, followed by extraction of the dichloromethane fraction. Finally, the remaining solvent from the previous steps was mixed with ethyl acetate, and the ethyl acetate fraction was taken out. The total extract, n-hexane and ethyl acetate fractions were prepared after the removal of the solvent.

Preparation of Punica granatum peels extract
The preparation was performed as done by Anibal et al. 28 . Fresh fruit, separated into skin, coverings and seeds, pericarp, and the whole fruit was submitted to ethanolic extraction (70% ethanol) at 37 • C by maceration. The extract was filtered, the solvents was eliminated under vacuum and lyophilized to get the crude extract. Crude extracts from all parts of the fruit were monitored by Thin Layer Chromatography. Animals 50 adults Wistar albino male rats, of 8 weeks old and weighing 250±10g, were obtained from the animal facility of faculty of pharmacy, Northern Border University. The experimental animals were kept in temperature controlled rooms (25 • C), with uniform humidity (40-70%) and 12h/light-12h/dark cycle before the experiment. All the animals were treated in consensus with the Principles of Laboratory Animal Care. The research protocol approval was taken from Deanship of Scientific Research at Northern Border University, in conformity with the guidelines for the care and use of experimental animals. All rats were fed a proper feed and aqua. The daily intake of animal water was checked once a week before beginning of treatments to determine the amount of water needed per experimental rat.

Histological examination
Anesthetized rats were perfused trans-cardially with normal saline and 4% paraformaldehyde in phosphate-buffered solution. The brains of the sacrificed animals were removed immediately and post fixed in the same fixative at 4 • C, until being sectioned on a cryostat (Leica, Germany). Coronal brain sections of 10 µm measurement were obtained and stored at -20 • C until used. Assessment of neuronal damage in the cortex was done with Nissl staining. Incubation of the brain sections was done with a 5% toluidine blue solution at room temperature for fifteen minutes. The brain sections were dehydrated and mounted following rinses water. The axons and neutrophil, morphology and integrity were assessed with Bielschowsky's sliver (BS) staining.

Statistical Analysis
To analyze the data, Statistical Package for Social Science (SPSS) version 20 was applied. The data were expressed as means +/-standard deviation (SD). Comparison of variables between groups were performed using One Way ANOVA test (LSD). Statistical significance was considered at P-value ≤ 0.05.

RESULTS
The body weight in G4 significantly decreased compared to G1, G2, and G3 on the    The control group showed that most vital neurons (cortical, hippocampal and cerebellar Purkinje cells) have active large vesicular lightly stained nuclei figure 7. STZ induced Type 2 diabetic rat showed an increase in dark degenerated neurons compared to cells with highly active stained nuclei, the cortex and striatum of the diabetic animals were characterized by demyelination and axonal degradation (Figures 7 and 8).
In all the treated groups, brain tissue sections show cerebral cortex with distinct gray and white matter areas. Cortex shows normal neuronal cells with scattered glial cells and neutrophils in background (Figure 9).

DISCUSSION
Diabetes mellitus type II is a common prevailing metabolic disorder emerging as global health hazard and it is associated with various complications such as micro-angiopathy, nephropathy, retinopathy, and neuropathy. Dating back to 1922, it has been noticed that diabetes mellitus may even lead to central nervous system disorder 30 . Long-standing hyperglycemia DM affects the brain and manifests anatomical, structural, neurophysiological, and neuropsychological changes. Various pathophysiological factors are found to be involved in the development of the cerebral dysfunction in diabetes mellitus, such as the hypoglycemic bouts, cerebrovascular changes, insulin's role in the brain and associated structures, and the mechanisms of hyperglycemia induced alterations 31 . Diabetes mellitus type II is known to cause a decrease in different areas of cognitive functioning. The risk of cognitive dysfunction is higher for the patients, who suffer from diabetes mellitus, prediabetes and from metabolic syndrome, characterized by dyslipidemia, central abdominal obesity and hyperglycemia 32,33 . Hyperglycemia decreases antioxidant levels and at the same time, causes an increase in the production of free radicals. These effects add to the deleterious effects on tissues, facilitating the complications/tissue damage in DM, leading to changes in the redox potential of the cells with consequent activation of redox-sensitive genes 34 . Neurons are especially sensitive to oxidative stress, and because of that, reactive oxygen species (ROS) cause several neural degenerative processes in diabetes 35-37 . In the current study, it was discovered that longstanding hyperglycemia in rats causes significant damage in different areas of the brain. Treatment with different substances containing antioxidants prevented the harmful effects of diabetes mellitus on neurological tissues. In the previous studies [38][39][40] , it has been found that metformin prevents the brain damage by reducing oxidative stress. The results of our study are in line with these studies, and significant prevention of the neuronal tissue damage was found in all areas of the brain of the animals.
Studies conducted by [41][42][43] revealed that Nigella sativa, which is rich in antioxidants, reduces neuroinflammatory damage and improves cognitive functions. The results in this study are in conformity with the previous studies regarding these findings. The previous research suggests that ginger shows a neuroprotective effect by speeding up the processes of brain antioxidant defense and down-regulating the N-MDA levels to the normal range in diabetic rats [44][45][46] . The current research results also verify the observations found in previous studies regarding the decrease, prevention and improvement by Ginger in the damage caused by DM in the brain. In previous studies, Pomegranate (Punica granatum), has been found to be effective in reducing oxidative stress and damage caused by oxidative stress. It has also been found effective in reducing the blood glucose levels [47][48][49] . In the current study, it was found to possess neuro-protective effects. The results of the current study show promising preventive and repairing effects of brain damage caused by DM.

CONCLUSION
DM is notorious to cause irreversible complications including peripheral neuropathy and brain damage, leading to the early occurrence of dementia     (Alzheimer's disease). The substances used in the study, including Metformin, Nigella sativa, Ginger and Punica granatum, effectively prevented the brain damage and repaired the damaged neurons. These substances can be used as adjuvant therapy to prevent DM related complications. It is highly suggested that the active ingredients of these substances should be studied for their effects on oxidative stress parameters, and composition to pinpoint exact neuroprotective mechanism.