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

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.

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.

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.

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.

DM was induced by a single intra-peritoneal injection of STZ, (Sigma-Aldrich, St Louis, MO, USA) in 0.1 M citrate buffer (pH 4.0), 55 mg/kg body weight29. Serum glucose concentration and alterations in body weight were checked regularly. Male Wistar rats were divided into ten groups, each group comprised of five rats as follows:

G1: Control rats were given only 5cc saline (0.9% NaCl).

G2: Control rats were given Zingeber officianale (ginger) (100mg/kg/rat) daily).

G3: Control rats were given Nigella sativa (Black seed) (80 mg/kg).

G4: Rats were given Punica granatum (Pomegranate) 400 mg/kg/day.

G5: Rats were given Metformin 150 mg/kg/day

G6: Diabetic control (55 mg/kg, single I/p injection of STZ).

G7: Diabetic group (55 mg/kg, single I/p injection of STZ) received 100mg/kg/day ginger.

G8: Diabetic group (55 mg/kg, single I/p injection of STZ) received Nigella sativa 80 mg/kg/day.

G9: Diabetic group (55 mg/kg, single I/p injection of STZ) received (Pomegranate) 400 mg/rat/day.

G10: Diabetic group (55 mg/kg, single I/p injection of STZ) received Metformin 150 mg/kg/day.

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.

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.

The body weight in G4 significantly decreased compared to G1, G2, and G3 on the 1^st day (142.83±16.36 versus 182.83±16.81, 176.50±20.34 and 174.50±5.68; P =0.001 for all groups), day 15^th (151.17±15.47 versus 183.00±21.23, 183.33±19.97 and 177.33±5.65; P =0.005, P =0.004 and P =0.019, respectively) and on 28^th day (158.33±15.24 versus 190.00±20.36, 195.50±16.53 and 183.83±9.62; P =0.004, P =0.001 and P =0.018, respectively). Body weight in G5 significantly increased compared to G1, G2, G3 and G4 at 1^st (205.33±6.98 versus 182.83±16.81, 176.50±20.34 174.50±5.68 and 142.83±16.36; P =0.011, P =0.001, P =0.001 and P =0.001, respectively), on day 15^th (209.00±12.82 versus 183.00±21.23, 183.33±19.97, 177.33±5.65 and 151.17±15.47; P =0.020, P =0.021, P =0.005 and P =0.001, respectively) and on 28^th day (218.00±16.46 versus 190.00±20.36, 195.50±16.53, 183.83±9.62 and 158.33±15.24; P =0.010, P =0.035, P =0.002 and P =0.001, respectively). In G6, on 1^st day, body weight significantly increased compared to G4 (185.80±11.41 versus 142.83±16.36; P =0.001), but significantly decreased compared to G5 (185.80±11.41 versus 205.33±6.98; P =0.035). While on 15^th day, BW significantly decreased compared to G5 (166.60±15.53 versus 209.00±12.82, P =0.001); on 28^th day, BW significantly decreased compared to G1, G2, G3 and G5 (142.5±8.54 versus 190.00±20.36, 195.50±16.53, 183.83±9.62 and 218.00±16.46; P = 0.001 for all).

In G7, on 1^st day, BW significantly decreased compared to G1, G2, G3, G5 and G6 (148.50±9.03 versus 182.83±16.81, 176.50±20.34, 174.50±5.68, 205.33±6.98, 185.80±11.41; P= 0.001. P =0.00. P =0.004, P =0.001 and P =0.001) at 15^th day, BW significantly increased compared to G4 (181.67±9.07 versus 151.17±15.47; P =0.007) but significantly decreased compared to G5 (181.67±9.07 versus 209.00±12.82; P =0.014); on 28^th day, BW significantly increased compared to G4 and G6 (158.33±15.24 and 194.83±8.08 versus 142.5±8.54; P =0.001 for both) but significantly decreased compared to G5 (194.83±8.08 versus 218.00±16.46; P =0.030). In G8, on 1^st day BW significantly increased than G4 and G7 (185.50±8.89 versus 142.83±16.36 and 148.50±9.03; P =0.001 for both) but significantly decreased than G5 (185.50±8.89 versus 205.33±6.98; P =0.025); on 15^th day, BW significantly increased than G1-7 (247.33±31.19 versus 183.00±21.23, 183.33±19.97, 177.33±5.65, 151.17±15.47, 209.00±12.82, 166.60±15.53, 181.67±9.07; P =0.001 for all); on 28^th day, BW significantly increased than G1, G2, G3, G4, G5, G6, and G7 (263.00±33.93 versus 190.00±20.36, 195.50±16.53, 183.83±9.62, 158.33±15.24, 218.00±16.46, 142.5±8.54, 194.83±8.08; P =0.001 for all).

In G9, on 1^st day, BW significantly decreased compared to G1, G5, G6 and G8 (160.50±28.31 versus 182.83±16.81, 205.33±6.98, 185.80±11.41, 185.50±8.89; P = 0.012, P =0.001, P =0.007 and P =0.005); on 15^th day, BW significantly increased than G4 (175.83±28.58 versus 151.17±15.47; P =0.026) but significantly decreased than G5 (175.83±28.58 versus 209.00±12.82; P =0.003); on 28 days, BW significantly increased than G4, G6 (195.33±25.56 versus 158.33±15.24 and 142.5±8.54; P =0.001 for both) but significantly decreased than G5 and G8 (195.33±25.56 versus 218.00±16.46 and 263.00±33.93; P =0.034 and P =0.001). In G10, BW increased than G4 (173.50±6.80 versus 142.83±16.36; P =0.001) but significantly decreased than G5and G7 173.50±6.80 versus 205.33±6.98 and 148.50±9.03; P =0.001 and P =0.005); on 15^th day, BW significantly increased than G4 (179.83±7.83 versus 151.17±15.47; P =0.010) but significantly decreased than G5 and G8 (179.83±7.83 versus 209.00±12.82 and 247.33±31.19; P =0.009 and P =0.001); on 28^th day, BW significantly increased than G4 and G6 (186.00±8.60 versus 158.33±15.24 and 142.5±8.54; P =0.011 and P =0.001) but significantly decreased than G5 and G8 (186.00±8.60 versus 218.00±16.46 and 263.00±33.93; P =0.003 and P =0.001)Figure 1,Figure 2,Figure 3.

Figure 1

Figure 2

Figure 3

Blood glucose levels in G6 significantly increased compared to G1, G2, G3, G4 and G5 on 1^st (234.80±13.03 versus 130.17±6.46, 126.50±17.00, 116.33±9.05, 121.67±6.95, 122.83±7.33; P =0.001 for all), on 15^th (237.20±12.72 versus 119.17±5.04, 124.83±5.27, 125.83±5.42, 124.33±4.03 and 122.67±9.37; P =0.001 for all) and on 28^th day (227.00±8.37 versus 125.17±3.76, 123.67±6.15, 122.67±2.80, 125.83±8.64 and 124.33±5.47; P =0.001 for all). Blood glucose levels in G7 significantly increased compared to G1, G2, G3, G4 and G5 but significantly decreased compared to G6 on 1^st (195.83±10.03 versus 130.17±6.46, 126.50±17.00, 116.33±9.05, 121.67±6.95, 122.83±7.33; P =0.001 for all and 195.83±10.03 versus 234.80±13.03; P =0.001), on 15^th day (157.00±8.00 versus 119.17±5.04, 124.83±5.27, 125.83±5.42, 124.33±4.03 and 122.67±9.37 and 157.00±8.00 versus 237.20±12.72; P =0.001) and on 28^th day (155.67±7.03 versus 125.17±3.76, 123.67±6.15, 122.67±2.80, 125.83±8.64 and 124.33±5.47; P =0.001 for all; and 155.67±7.03 versus 227.00±8.37, P =0.001).

Blood glucose levels in G8 significantly increased compared to G1, G2, G3, G4 and G5 but significantly decreased compared to G6 on 1^st day (190.67±5.13 versus 130.17±6.46, 126.50±17.00, 116.33±9.05, 121.67±6.95, 122.83±7.33; P =0.001 for all and 190.67±5.13 versus 234.80±13.03; P =0.001), on 15^th day (156.40±6.88 versus 119.17±5.04, 124.83±5.27, 125.83±5.42, 124.33±4.03 and 122.67±9.37 and 156.40±6.88 versus 237.20±12.72; P =0.001) and on 28^th day 143.00±4.00 versus 125.17±3.76, 123.67±6.15, 122.67±2.80, 125.83±8.64 and 124.33±5.47; P =0.001 for all; and 143.00±4.00 versus 227.00±8.37, P =0.001) and significantly decreased compared to G7 on 28^th day (143.00±4.00 versus 155.67±7.03; P =0.009).

Blood glucose levels in G9, on 1^st day significantly increased compared to G1, G2, G3, G4, G5 and G8 (208.67±18.93 versus 130.17±6.46, 126.50±17.00, 116.33±9.05, 121.67±6.95, 122.83±7.33, 190.67±5.13; P =0.001, P =0.001, P =0.001, P =0.001, P =0.001 and P =0.008, respectively) but significantly decreased compared to G6 (208.67±18.93 versus 234.80±13.03; P =0.001), on 15^th day (174.83±12.84 versus 119.17±5.04, 124.83±5.27, 125.83±5.42, 124.33±4.03, 122.67±9.37 and 156.40±6.88; P =0.001 for all) but significantly decreased compared to G6 (174.83±12.84 versus 237.20±12.72; P =0.001) and 28^th days blood glucose levels significantly increased compared to G1, G2, G3, G4 and G5 (144.67±4.97 versus 125.17±3.76, 123.67±6.15, 122.67±2.80, 125.83±8.64 and 124.33±5.47; P =0.001 for all) but significantly decreased compared to G6 and G7 (144.67±4.97 versus 227.00±8.37 and 155.67±7.03; P =0.001 and P =0.017).

Blood glucose levels in G10, on 1^st day significantly increased when compared to G1, G2, G3, G4, G5, G7 and G8 (209.17±10.26 versus 130.17±6.46, 126.50±17.00, 116.33±9.05, 121.67±6.95, 122.83±7.33, 195.83±10.03 and 190.67±5.13; P = 0.001, P =0.001, P =0.001, P =0.001, P =0.001, P =0.046 and P =0.006) but significantly decreased when compared to G6 (209.17±10.26 versus 234.80±13.03; P = 0.001); on 15^th day, blood glucose levels significantly increased compared to G1, G2, G3, G4 and G5 (158.00±10.37 versus 119.17±5.04, 124.83±5.27, 125.83±5.42, 124.33±4.03, 122.67±9.37; P =0.001 for all) but significantly decreased compared to G6 and G9 (158.00±10.37 versus; 237.20±12.72 and 174.83±12.84; P =0.001 for all); on 28^th day, blood glucose levels significantly decreased compared to G6, G7, G8 and G9 (131.17±16.30 versus 227.00±8.37, 155.67±7.03, 143.00±4.00, 144.67±4.97; P= 0.001, P =0.001, P =0.015 and P =0.004, respectively)Figure 4,Figure 5,Figure 6.

Figure 4

Figure 5

Figure 6

Figure 7

Examination of sections from different regions of control rat brain showing the most vital neurons (cortical, hippocampal and cerebellar Purkinje cells) have active large vesicular lightly stained nuclei (arrows). H&E stain, Magnification 100&400.

Figure 8

Figure 9

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 (Figure 7,Figure 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).

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 disorder30.

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 alterations31.

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 hyperglycemia32,33.

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 genes34.

Neurons are especially sensitive to oxidative stress, and because of that, reactive oxygen species (ROS) cause several neural degenerative processes in diabetes35,36,37.

In the current study, it was discovered that long-standing 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 studies38,39,40, 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 by41,42,43 revealed that Nigella sativa, which is rich in antioxidants, reduces neuro-inflammatory 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 rats44,45,46. 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 levels47,48,49. 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.

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 neuro-protective mechanism.

The authors declare that there is no conflict of interest.

Author 1 responsible for research /experimental design, histopathological study and interpretation of results. Author 2 responsible for literature review, compilation of results.

The author wishes to acknowledge the approval and the support of this research study by the grant No (7267-PHM-2017-1-8-F) from the Deanship of Scientific Research in Northern Border University (N.B.U.), Arar, KSA.

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