Pharmacological Evaluation of Selected Natural Drugs Promoting The Neuronal Health Against Cuprizone Model Of Multiple Sclerosis In BALB/c Mice

Abstract

Abstract Background: Multiple sclerosis (MS) is a primary progressive demyelinating or chronic inflammatory disease of the central nervous system (CNS). The pathology involves astrocyte degeneration, and change in morphology; demyelination of myelin sheath on neurons; overexpression of p53 factor leading to oligodendrocytes inflammation and apoptosis; increase in pro-inflammatory factors like NF- kappa B, TNF-alpha; increase in reactive oxygen species; improvement in the brain neurotrophic factor; loss in body weight; locomotor, coordination and balance dysfunction; left and right paw coordination loss; and working memory, spatial memory and cognitive dysfunction. The available treatments are not found to give a complete cure for MS, having only narrow spectrum therapy status. Withania somnifera (Ashwagandha), Bacopa monnieri (Brahmi), and Asphaltum (Shilajit) powder contains alkaloids, carbohydrates, steroids and saponins which reported for properties like memory and cognition enhancer, neuroprotectants, anti-inflammatory, and antioxidant linking it to have positive neurological effects on the CNS. Objective: The objectives of the study are to evaluate the neuropharmacological activity and to investigate the mechanism of action of Withania somnifera (Ashwagandha) and Bacopa monnieri (Brahmi) and Asphaltum (Shilajit) against cuprizone induced multiple sclerosis model in balb/c mice. Materials and Methods: The pharmacognostical analysis of the Withania somnifera (Ashwagandha), Bacopa monnieri (Brahmi), and Asphaltum (Shilajit) powder to confirm the presence of reducing sugars, carbohydrates, tannins, steroids, flavonoids, proteins, saponins, phenolic compounds and alkaloids. The pharmacological evaluation was performed for 6 weeks in which Balb/c mice were distributed in ten groups consisting 6 animals in each group: Normal Control, Disease Control (CPZ- 400mg/kg, p.o.), Disease treated groups (Standard-Methylprednisolone p.o., 10 mg/kg from 3rd week and 5 mg/kg from 5th week, Ashwagandha- 100mg/kg, Brahmi- 100 mg/kg, Shilajit- 50 mg/kg from 3rd week), and Normal treated groups (Standard-Methylprednisolone p.o., 10 mg/kg from 3rd week and 5 mg/kg from 5th week, Ashwagandha- 100mg/kg, Brahmi- 100 mg/kg, Shilajit- 50 mg/kg from 3rd week). The animal’s behavior was assessed by Y-maze test, Tail flick test, Morris water maze test and Rotarod test. The biochemical estimation included oxidative and nitrosative Stress parameters, anti- inflammatory parameters (NF- kappa B, TNF-alpha), Brain Neurotrophic Factor (BNGF, Neprilysin), and Apoptotic Factor (p53/Tp53). The histopathological and immunohistochemical examination of the brain was also done.Results: The pharmacognostic analysis revealed presence of Carbohydrates, Steroids, Saponins and Alkaloids in the Withania somnifera (Ashwagandha), Bacopa monnieri (Brahmi), and Asphaltum (Shilajit) powder. All the selected natural drugs, when provided as treatment with in-vivo CPZ toxin induced MS model, showed an insignificant increase in body weight (physical parameter) in the treatment group in comparison to the disease control group. All the treatment groups showed improved results in all the neurobehavioral tests, by decreasing the tail flick latency in tail flick test, by increasing the fall-time in Rota-rod test, by increasing the percentage correct alternation in Y-maze test explaining the working memory, by decreasing the escape latency and increasing the probe counts in Morris Water-maze test explaining the spatial long-term memory, all in comparison to Disease control group. The antioxidant property of these selected natural drugs was proved by insignificant increase in SOD levels and catalase level and significant decrease in nitrosative stress factor (NO levels) in treatment groups in comparison to disease control groups. The brain neurotrophic factors like- BNGF and Neprilysin were increased in all the treatment groups proving them to be the neuroprotectants. The NF-kappa B and TNF-alfa, to check the presence of pro-inflammatory cytokine levels were found to be slightly lower in treatment arms in comparison to disease control groups. The apoptotic marker like p53/Tp53 was observed to have significant decrease in p53/Tp53 levels in all three-treatment groups, explaining the decrease in chances of ODCs apoptosis, when compared with disease control groups. The histopathological examination depicted the presence of inflammation, cell structure disruption and apoptosis in perineural ODCs and in interfascicular ODCs in the Disease control group. In the treatment groups, negligible apoptosis cues, slight disruption of cell structure and perineural leaking was found with some inflamed perineural or interfascicular ODCs. The immunohistochemistry analysis exhibited an increase in astrocytic perinuclear space with partial to complete loss of astrocyte processes with anti-GFAP immunoreactivity in the disease control group while the treatment groups showed an intact astrocyte structure and size, with enclosed perinuclear space. Conclusion: The study showed that Withania somnifera (Ashwagandha), Bacopa monnieri (Brahmi), and Asphaltum (Shilajit) powder in aqueous solution have antioxidant, anti-inflammatory, and nitrosative stress inhibitory activity which can be useful in the treatment and prevention of remitting and progressive types of Multiple sclerosis. They also have found their effects on apoptotic biomarkers like p53/Tp53 which can resist the progression of MS disease by decreasing chances of ODCs apoptosis. They all are suggestive of their neuroprotective activity which can be useful in the treatment of Multiple sclerosis and provides evidence for a broad-spectrum combination therapy as a future prospect.