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Currently, natural products play an important role in the prevention, limitation and treatment of brain diseases due to their ability to scavenge the free radicals. Saffron and turmeric are among the natural products that have neuroprotective effects against D-galactose due to their antioxidant and anti-inflammatory properties.
Saffron is a perennial herbaceous plant (Crocus sativus L.) of the family Iridaceous. Saffron is one of the highly nutritive value spices. It contains several plant-derived chemical compounds that are known to have antioxidant, disease preventing and health promoting properties. Saffron is effective in curing memory impairment and learning disabilities due to its medical properties.
Turmeric is an herbaceous evergreen plant (Curcuma longa L.) in the Zingiberaceae (ginger) family. Turmeric extract is a good source of natural antioxidants, as indicated by their high contents of polyphenols, flavonoids, tannins and ascorbic acid. The bioactive element of turmeric is curcumin which is responsible for the yellow color of turmeric , prevention and treatment of neurodegenerative diseases.
D-galactose is a simple sugar that is found naturally in some foods mostly in dairy foods like cheese and yogurt also found in honey, kiwi fruit and dry figs. It’s used at large concentration as a sweetener in the manufacturing of ice-cream, frozen desserts and soft drinks.
D-galactose can be metabolized if administered within normal concentration range, but the over-supply of D-galactose leads to disruption of metabolic pathway. D-galactose is changed to aldose and hydrogen peroxide by galactose oxidase enzyme. In addition, it is converted to galactitol which accumulates in the cells and leads to osmotic stress and production of reactive oxygen species (ROS) causing deleterious brain effects.
The aim of the present work was to investigate the active chemical constituents and antioxidant capacities of saffron stigmas and turmeric rhizomes extracts (ESE and ETE), respectively. D- galactose deleterious brain effects as well as the role of ESE and ETE supplementation against D-galactose intoxication were evaluated on male rat’s brain.
Saffron and turmeric were chemically analyzed for their total phenolic, flavonoids, anti-oxidant activities and also, the main phenolic constituents of saffron and turmeric were determined using HPLC technique. Ethanolic saffron extract (ESE), ethanolic turmeric extract (ETE), ethanolic saffron and turmeric extracts (ESE+ETE) were separately suspended in distilled water immediately before administration to rats and administered to each rat according to its body weight.
Brain deleterious effects were induced by subcutaneous (S.C) injection with a daily dose of D-galactose (250mg / kg B.W) which was dissolved in saline immediately before administration to rats.
Fifty- five adult male rats (Sprague -Dawely) strains weighing (200-210) g received the commercial diet and water ad libitum for 7 days (acclimatization period) and continued to have the same diet throughout the 6 weeks of the experiment (experimental period). All rats were divided into five groups each of ten rats except D-galactose control group that composed of fifteen rats and divided as the following; group (1) HCG: Healthy control group, rats were given distilled water orally and injected with normal saline subcutaneously (s.c) daily. group (2) DGCG: D-galactose control group, rats were given distilled water daily orally and injected with D-galactose (250mg/kg /day/S.C). group (3) DG+ESE, group (4) DG+ETE, group (5) DG+ESE+ETE : D-galactose intoxicated rats (250mg/kg/day/s.c) supplemented with ethanolic saffron extract (30mg /kg/daily orally), ethanolic turmeric extract (30mg /kg/daily orally) and mixture of saffron extract (15mg /kg/daily orally) and turmeric extract (15mg/kg/daily orally) respectively.
During the experimental period, feed intake was recorded and animals were weighed weekly to calculate body weight change. At the end of the experimental period (6weeks), the experimental animals were sacrificed under sodium barbiturate anesthesia. The brain was removed, washed with saline and weighed. Random brain samples were fixed in 10% neutral formalin solution for histological study and immunohistochemical analysis, the remaining brain samples were prepared to form tissue homogenate for other analysis.
The results of the current study are summarized as follows:
1- Total polyphenols, flavonoids and antioxidant activity of ethanolic saffron and turmeric extracts
The result illustrated that each 1 g of the tested saffron extract contains 39.195 mg as gallic acid equivalent (GAE)of total poly phenols ,2.787 mg as catechin equivalent (CE) of total flavonoids and 8.883mg as trolox equivalent(TE) of total anti-oxidant capacity while each 1 g of the tested turmeric extract contains 1.955 mg as gallic acid equivalent of total poly phenols , 1.023 mg as catechin equivalent of total flavonoids and 2.012 mg as trolox equivalent of total anti-oxidant capacity, from these results. It’s found that saffron ethanolic extract contains higher phenolic, flavonoids and antioxidant activity than turmeric ethanolic extract.
2- The main phenolic compounds of ethanolic saffron and turmeric extracts
These results concluded that phenolic compounds of saffron extract by using HPLC technique were apigenin, chrysin, cateachin, qurecetin, cinnamic acid, sinapic acid, rutin, kaempferol, caffeic , vanillic, rosmarinic acids, apigenin-7-glucoside, p-coumaric , p-hydroxybenzoic and ferulic acids .Apigenin represented the highest concentration (8.26 mg/g) of saffron phenolic components when compared to all other components while ferulic represented the lowest concentration (0.02 mg /g ). Also this result concluded that phenolic compounds of turmeric extract by using HPLC technique were apigenin-7-glucoside, ferulic acid, cateachin , vanillic , p-coumaric acids, qurecetin and p-hydroxybenzoic acid. Apigenin-7-glucoside represented the highest concentration (2.166 mg/g) of turmeric phenolic components when compared to all other components while p-hydroxybenzoic acid represented the lowest concentration (0.01 mg /g) .
3- Body weight change, feed intake and relative brain weight
Administration of D-galactose to rats caused a significant decrease in body weight, while relative brain weight significantly increased and it was noticed that there were not any significant change between groups in feed intake. ESE and ETE served as good sources of phenols, flavonoids and also high free radical scavenging activity which could have great effects on body weight, feed intake and relative brain weight, also reducing the oxidative stress and the inflammation resulted from D-galactose injection, so decreasing the deleterious brain effects induced by D-galactose as showed by marked elevation in body weight with a significant decrease in relative brain weight.
4-Oxidative stress and antioxidant status in brain
Injection with D-galactose remarkably elevated brain oxidative biomarkers including AGEs, CBP, MDA and NO levels and significantly declined brain antioxidant markers as TAC level, brain CAT activity and GSH level as a result of oxidative stress caused by D-galactose. Supplementation with ESE, ETE and ESE+ETE decreased the oxidative stress induced by D-galactose and caused a significant enhancement in the brain TAC level, brain CAT activity and GSH level, where the supplementation with both of ESE and ETE caused the most significant improvement in brain AGEs, MDA, NO, CBP content, TAC level, CAT activity and GSH level followed by (ESE) and finally (ETE) supplemented groups.
5- Inflammatory biomarkers
D-galactose toxicity induced a massive increase in the levels of brain TNF-α and IL-6. Administration of ESE, ETE, ESE+ETE attenuated the inflammatory effects of D-galactose. Supplementation with ESE+ETE caused the most significant improvement in brain TNF-α and IL-6 levels followed by (ESE) and finally (ETE) supplemented groups. These improvements were due to the anti-inflammatory effect of saffron and turmeric extracts.
6- Neural cell markers
Administration of D-galactose to rats caused a state of oxidative stress resulting in increased loss of brain neurotransmitters leading to decreased brain functions as shown by a significant increase in (MTs) and (5-HT) levels with significant decrease (AChE) activity. Administration of ESE, ETE and (ESE+ETE) to intoxicated rats caused improvement in the levels of neural cell markers due to presence of active components in the extracts that restored and attenuated the loss of brain neurotransmitters. Treatment with mixture of (ESE and ETE) caused the most significant improvement in brain AChE activity and MTs, 5-HT levels compared to other treatments as a result of the synergistic effects between saffron and turmeric active constituents.
7- Epigenetic marker
D-galactose toxicity induced a massive increase in the level of P16INK4a . Administration of ESE, ETE, ESE+ETE attenuated the inflammatory effect of D-galactose. Treatment with ESE+ETE caused the most significant improvement in brain P16INK4a level followed by (ESE) and finally (ETE) supplemented groups.
8- Microscopic examination of brain tissues
Microscopic examination for brain tissues from the D-galactose group showed numerous neuropathological alterations which described as congestion of cerebral blood vessel, meningeal infiltration with mononuclear cells, marked necrosis, pyknosis and atrophy of neurons associated with neuronophagia and neurofibrillary tangles. Focal gliosis was also observed in most examined sections meanwhile, administration of saffron and turmeric extracts showed improvement in the histopathological picture and moderate regression in the neuropathic change. The most significant improvements in the microscopic examination of brain tissue were recovered in DG+ESE+ETE group followed by DG+ESE group and finally DG+ETE group respectively.
9- Immunohistochemical analysis of apoptotic and anti-
apoptotic markers in brain tissues
D-galactose activated both extrinsic and intrinsic pathways of apoptosis enhancing the level of the cytochrome complex (cyt C) which stimulated the activation of Caspase-3, Bax levels and reduced anti-apoptotic Bcl -2 expression level in different regions of brain. However, ethanolic saffron and turmeric extract caused significant improvements as indicated by moderate immune reaction as illustrated without brown staining in the cerebral cortex and hippocampus of rats. Both saffron and turmeric extracts reduced apoptotic biomarkers in neurons and prevented neurodegeneration. The anti-apoptotic effects of ESE and ETE shown by the reduction of Bax and increase of Bcl-2 levels in the brain. Both ESE and ETE attenuated Caspase-3 inducing cleavages which induced Bax production and nuclear condensation resulting from neurodegeneration processes. The most significant improvements in the apoptotic and anti-apoptotic markers were noticed in the DG+ESE+ETE group followed by DG+ESE group and finally DG+ ETE group and this attributed to the high antioxidant properties that found in ESE and ETE.