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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article   Research Article

Therapeutic importance of hydrolic fraction of Celtis integrifolia roots for prevention of brain oxidative stress, increase of acetyl cholinesterase activity and tissues damage in monosodium glutamate-induced neurodegenerative-like diseases in mice

Bih Belta Lilian Fubi , Kada Sanda Antoine , Tangu Patience Neng , Mumbi Laurentine Ngenteh , Ndifor Rose Nchang , Nsah Bertrand Kiafonm , Oumar Mahamat 

Department of Zoology, Faculty of Science, The University of Bamenda, Cameroon

 

 

BACKGROUND

Neurodegeneration is a complicated process that occurs when the functions of specific cells (nerve cells) in the brain subside; it is often connected to aging ¹. The pathological mechanism associated with neurodegenerative diseases depends on the biological factors associated resulting in nervous cell death ². These factors include proteins aggregation, inflammation, energy deficiency, oxidative stress and DNA damage. Globally, now it is recognized that oxidative stress causes changes in the biochemical and biomolecular parts of the body, leading to diseases ^2-4. However, it is important to note that small amounts of ROS and RNS are essential for signaling in the brain. They help with communication between cells and memory. However, too many of these reactive species can harm parts of cells, such as DNA, proteins, and fats ^5,6. This can cause stress and lead to nerve cells dying. 

The current drugs against neurodegenerative diseases have side effects and are sometimes ineffective in preventing or stopping the progression of the illness ⁷. The inadequate access to modern medicine and physicians, the high cost of actual therapies, and the side effects of modern drugs have prompted patients to search for alternative therapies. The use of plants particularly in low developing countries has gained interest as supported by their availability and less side effects ⁸. Celtis integrifolia a plant belonging to the family Ulmaceace commonly found in Northern parts of Cameroon. A report by Muazu and Kaita ⁹ indicated that C. integrifolia is one of the components of a polyherbal formulation for the treatment of epilepsy and mental problems in Northern Nigeria. C. integrifolia is reported to be used in preventing neurodegenerative diseases. The phytochemistry analysis of the plant also revealed the presence of saponins, flavonoids ¹⁰, which are components that have proven potential in treatments of these neurodegenerative diseases ^11,12. Therefore, the study of the protective potential of C. integrifolia in prevention of neurodegeneration may offer a great interest. In this study, the hydrolic fraction of C. integrifolia was evaluated whether it affects the cholinergic activity and oxidative status in brain and whether it prevents neurological damage in mice exposed to MSG.

MATERIALS AND METHODS

Plant Collection and Identification

A specimen of Celtis integrifolia was collected in Logone et Chari Division, Far North region of Cameroon. The plant material collected was identified by a botanist in the University of Maroua and confirmed in comparison with the voucher specimen of Letouzey 6513 of the National Herbarium, Yaounde-Cameroon (9032 SRF Cam). Roots of C. integrifolia were therefore collected, washed and dried at 50⁰ C in thermostat oven (DHG-9101-1SA PEC). Dried root were crushed into powder. The fine powder was used for the extraction. 

Preparation of Plant Extract 

The hydrolic extract was obtained in sequential extraction as previously described ¹³ with slight modifications. The powdered plant materials (500 g) were subjected to exhaustive sequential solvent defection with varying polarities as follows, n-hexane, followed by acetone and lastly ethanol. Then the residue was macerated in 3 L of distilled water and procedure repeated thrice and evaporated at 40⁰ C to obtain the hydrolic extract (16.11 g) given a yield of 6.7%. 

Experimental Animals

Thirty-five (35) Swiss mice weighing 25±5 g age two months old were used in this experiment. These animals were bought from a breeder in Bamenda, Northwest Region, Cameroon. They were then acclimatized for two weeks in the animal house of Faculty of Science at the University of Bamenda. Mice were fed with a diet consisting of (50% corn flour, 10% fish powder, 10% bone powder, 10% soya bean flour, 3% salt, 5% oil, and 2% water), given access to water ad libitum and maintained in a temperature and light-controlled room (25±2°C, natural day/night cycle). Animals were handled according to the guidelines of the Cameroon Bioethics Committee (reg. no. FWA -IRB00001954). 

Animals Grouping and Treatment

Mice were distributed into seven groups (n=5): a normal control group (distilled water only 5 mg/L); negative control group was receiving MSG (2 g/kg/day). Three test groups were given the C. integrifolia hydrolic extract at 200, 400, 800 mg/kg followed by MSG. Two other groups were taken as positive control groups (1 group received the Vitamin C (300 mg/kg/day) + MSG and the second was given Donepezil (3 mg/kg) + MSG. Treatment was done orally (p.o) by gavage for 21 days. 

Collection and Preparation of Biospecimens

After 21 days of treatment, on day 22 of the experiment, all mice were anesthetized (using sodium pentobarbital, 100 mg/kg b.w., i.p.) and quickly decapitated. The entire brain was harvested and weighed and some placed in 10% formalin for histological studies. While some of the brain tissue samples were homogenized with 0.1 M phosphate buffer (pH 7.4), centrifuged (3000 rpm for 15 minutes at 4°C), and the supernatant was collected and stored at -20°C for biochemical assay.

Evaluation of brain oxidative status

The evaluation of the enzymatic activities of superoxide dismutase (SOD) was done based on the ability of SOD to inhibit the reduction of nitroblue tetrazolium dye as described by ¹⁴ with little modifications. Moreover, catalase (CAT) activities was measured based on the breakdown rate of H₂O₂ according to the method of ¹⁵. The determination of reduced glutathione (GSH) was assessed according to the method of ¹⁶. Lipid peroxidation was assessed in terms of malondialdehyde (MDA) according the method mentioned by ¹⁷. Moreover, nitric oxide (NO) level was measured in the brain homogenate through dye formation after adding the Griess reagent at 540 nm, as previously described ¹⁸.

Acetylcholinesterase activity in the brain

The level of acetyl cholinesterase was estimated by the method described by ¹⁶ with slight modifications. For the estimation of acetylcholinesterase, 20 μL of buffer Tris-HCl 0.1 M (pH 8.0) and 3mL Ellman reagent were introduced into all tubes (test and blank vials) and then 100μL homogenate followed by 100μL Tris buffer (HCl 50 mM; KCl 150 mM; pH 7.4) in the blank tube. Subsequently, 20 μL of 30 mM acetylthiocholine iodide was added to all the tubes. After a rapid homogenization of the mixture at room temperature, the absorbance was read at 412nm after 30s and 90s against the blank.

Histopathological analysis

Brain specimens were fixed in neutral buffered formalin (10%), dehydrated, embedded in paraffin wax and cut into sections (3-4 μm in thickness). Next, tissue sections were deparaffinized, and stained with hematoxylin and eosin and observed under a light microscope (OLYMPUS BX51) 100X magnification.

Statistical Analysis

All results were expressed as mean ± standard error mean (SEM) and analyzed using Graph Pad Prism version 8.01 software. Data analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey post-test. A significant difference was considered at p<0.05. 

RESULTS                                                                                       

Effect of C. integrifolia Extract on brain levels of malondialdehyde, catalase, superoxide dismutase and reduced glutathione in mice exposed to monosodium glutamate

Administration of monosodium glutamate significantly (p<0.001) caused an increase in the level of lipid peroxidation (or MDA) and decrease in level of SOD, GSH and CAT in brain homogenate compared to that in normal mice. The administration of the doses of hydrolic fraction of C. integrigolia (200, 400, and 800 mg/kg) increased significantly the brain catalase levels (p<0.01, p<0.01 and p<0.001), superoxide dismutase (p<0.01, p<0.001 and p<0.001) and the reduced glutathione levels (p<0.01, p<0.01 and p<0.001) of MSG-treated mice as compared to the negative group. It also decreased the level of lipid peroxidation (p<0.05, p<0.01 and p<0.001) of MSG-treated mice as compared to the negative group (Table 1).

 

 

Table 1: Brain homogenate levels of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and reduced glutathione (GSH) after administration of Celtis integrifolia roots hydrolic extract in mice.

	Controls			Extract (mg/kg)			ANOVA sig. (F, p-value)
Test parameter	Normal	Negative	Vit C	200	400	800
Catalase (U/g of tissue)	3.79±0.02***	1.07±0.04	2.26±0.03**	0.93±0.03*	1.12±0.002*	1.44±0.02***	F (978) ; P<0.001
SOD (%I)	36.4±0.62***	14.3±0.06	20.4±0.53***	14.9±0.05**	21.5±0.09**	28.5±0.26***	F (275) ; P<0.001
MDA (µmol/L)	0.24±0.01***	1.22±0.001	0.3±0.01***	1.01±0.01	0.76±0.01**	0.39±0.01b ***	F(1457); P<0.001
GSH (µmol/L)	0.16±0.002***	0.07±0.001	0.1±0.001**	0.08±0.001**	0.08±0.003**	0.13±0.001***	F (556) ; P<0.001

Values represent Mean ± SEM (n=5). *p<0.05; ** p < 0.01; ***p < 0.001 indicate the difference compared to the Negative control. Vit. C: Vitamin C. HCi: hydrolic fraction of Celtis integrifolia. Normal control: normal mice. Negative control: group of mice taking Monosodium glutamate only

 

Effect of C. integrifolia root hydrolic extract on brain level of nitric oxide (NO) in mice exposed to monosodium glutamate 

Brain nitric oxide (NO) level was significantly higher (p<0.001) in mice exposed MSG than in normal mice. Administration of extract at doses of 200, 400 and 800 mg/kg prior to the MSG exposure reduced significantly (p<0.01; p<0.01; p<0.001) the NO level compared to the negative group (Figure 1)

 

Figure 1: Serum level of nitric oxide following the administration of monosodium glutamate and C. integrifolia roots hydrolic extract. Values represent Mean ± SEM (n=5). *p<0.05; ** p < 0.01; ***p < 0.001 indicate the difference compared to the Negative control. ns indicates not significant at p > 0.05 compared to the Negative control. Don: Donepezil. HCi: hydrolic fraction of Celtis integrifolia. Normal control: normal rats. Negative control: group of rats taken Monosodium glutamate only

Effect of C. integrifolia Extract on brain acetylcholinesterase activity in mice exposed to monosodium glutamate

Exposure of mice to MSG caused a significant increase (p < 0.01) in brain acetylcholinesterase activity as compared to normal mice. Administration of the extract doses prior to exposure to MSG significantly (p < 0.01; p < 0.001; p < 0.001) decreased acetylcholinesterase activity in mice compared to the negative control (Figure 2).

 

Figure 2: Serum level of brain Acetylcholinesterase activity following the administration of monosodium glutamate and C. integrifolia roots hydrolic extract. Values represent Mean ± SEM (n=5). *p<0.05; ** p < 0.01; ***p < 0.001 indicate the difference compared to the Negative control. ns indicates not significant at p > 0.05 compared to the Negative control. Don: Donepezil. HCi: hydrolic fraction of Celtis integrifolia. Normal control: normal rats. Negative control: group of rats taken Monosodium glutamate only

 

Effect of hydrolic extract of C. integrifolia root on the histoarchitecture of the cortex of mice exposed to monosodium glutamate 

Plate 1 shows the effect of hydrolic extract of Celtis integrifolia on the histoarchitecture of cortical slices: group (Neg) revealed the presence of chromatilysis, pyknotic nucleus amid associated vacuolations or necrosis, and hyperchromatic neuron with overall decrease in neuronal density. Groups (C.I 200, 400, 800) showed restored neuronal density, numerous granular cells restored in a dose dependent manner. 

Plate 1: Effect of hydrolic extract of Celtis integrifolia on the histoarchitecture of cortical slices. NC: normal, Neg: Negative (group exposed to MSG the monosoduim glutate), HC.I 200, 400 and 800: Groups taken exposed to MSG and receiving doses of extract, VitC: group exposed to MSG and receiving vitamine C, while DPZ: group exposed to MSG and receiving Donepezil. MSG: monosoduim glutate. The green arrow identifies normal neuronal cell, the blue arrow identifies chromatilysis, the yellow arrow identifies pyknotic nucleus amid associated vacuolations or necrosis, while the orange identifies hyperchromatic neuron. C.I (Celtis integrifolia); VitC (Vitamin C); DPZ) NC (Normal control).

Effect of hydrolic extract of C. integrifolia root on the histarchitecture of CA1 region of hippocampus of mice exposed to monosodium glutamate 

Plate 2 shows the effect of hydrolic extract of C. integrifolia on the histoarchitecture of CA1 region of the hippocampus: group (Neg) identifies damage area (abnormal cells indicating inflammation and tissue damage), eosinophilic necrosis with overall decrease in neuronal density. Groups (C.I 200, 400, 800) showed restored neuronal density, numerous granular cells restored in a dose dependent manner. 

Plate 2. Effect of hydrolic extract of Celtis integrifolia on the histoarchitecture CA1 area of hippocampus. NC: normal, Neg: Negative (group exposed to MSG the monosoduim glutate), HC.I 200, 400 and 800: Groups taken exposed to MSG and receiving doses of extract, VitC: group exposed to MSG and receiving vitamine C, while DPZ: group exposed to MSG and receiving Donepezil. MSG: monosoduim glutate. The red arrow identifies the damage area (abnormal cells indicating inflammation and tissue damage), the black arrow identifies eosinophilic necrosis, while the green arrow identifies relatively normal neuronal cell. 

 

Effect of hydrolic extract of C. integrifolia root on the histarchitecture of CA3 region of hippocampus of mice exposed to monosodium glutamate 

Plate 3 shows the effect of hydrolic extract of C. integrifolia on the histoarchitecture of CA3 region of the hippocampus: group (Neg) identifies eosinophilic necrosis, abnormal cells with pyknotic nucleus, with chromatolysis and overall decrease in neuronal density. Groups (C.I 200, 400, 800) showed restored neuronal density, numerous granular cells were restored in a dose dependent manner. 

Plate 3: Effect of hydrolic extract of Celtis integrifolia on the histoarchitecture of CA3 area of hippocampus. NC: normal, Neg: Negative (group exposed to MSG the monosoduim glutate), HC.I 200, 400 and 800: Groups taken exposed to MSG and receiving doses of extract, VitC: group exposed to MSG and receiving vitamine C, while DPZ: group exposed to MSG and receiving Donepezil. MSG: monosoduim glutate. CA:cornu ammonis. The black arrow identifies eosinophilic necrosis, abnormal cells with pyknotic nucleus, while the red arrow identifies chromatolysis, while the green arrow identifies relatively normal neurons.

Effect of hydrolic extract of C. integrifolia root on the histarchitecture of dentate gyrus of mice exposed to monosodium glutamate 

Plate 4 shows hydrolic extract of C. integrifolia on the histoarchitecture of gyrus: Group (Neg) showed loss of neuronal density, loss of hilum of the DG vacuolations, neuronal dispersal, neuronal atrophy, presence of necrosis. Groups (C.I 200, 400, 800) showed restored neuronal density, preserved DG hilum, numerous granular cells were restored in a dose dependent manner.

Effect of hydrolic extract of C. integrifolia root on the histarchitecture of amygdala of mice exposed to monosodium glutamate 

Plate 5 shows the effect of hydrolic extract of C. integrifolia on the histoarchitecture of amygdala: group (Neg) identifies eosinophilic necrosis, abnormal cells with pyknotic nucleus, with chromatolysis and overall decrease in neuronal density. Groups (C.I 200, 400, 800) showed restored neuronal density, numerous granular cells restored in a dose dependent manner.

Plate 4: Effect of hydrolic extract of Celtis integrifolia on the histoarchitecture of gyrus. NC: normal, Neg: Negative (group exposed to MSG the monosoduim glutate), HC.I 200, 400 and 800: Groups taken exposed to MSG and receiving doses of extract, VitC: group exposed to MSG and receiving vitamine C, while DPZ: group exposed to MSG and receiving Donepezil. MSG: monosoduim glutate.The red arrow identifies necrosis, the yellow arrow identifies pyknotic nucleus amid associated vacuolations, while the green arrow identifies relatively normal granular neurons with preserved density, the black arrow identifies hilum of the Dentate Gyrus. 

Plate 5: Effect of hydrolic extract of Celtis integrifolia on the histoarchitecture of amygdala. NC: normal, Neg: Negative (group exposed to MSG the monosoduim glutate), C.I 200, 400 and 800: Groups taken exposed to MSG and receiving doses of extract, VitC: group exposed to MSG and receiving vitamine C, while DPZ: group exposed to MSG and receiving Donepezil. MSG: monosoduim glutate. The green arrow identifies normal neuronal cell, the orange arrow identifies chromatilysis, while the yellow arrow identifies pyknotic nucleus amid associated vacuolations or necrosis.

 

DISCUSSION

In experimental model, monosodium glutamate (MSG) causes oxidative stress and activation of inflammatory pathways ¹⁹. In addition, it has been established that chronic administration of MSG results in an excessive accumulation of glutamate, which stimulates the hypothalamus, leading to impairment of the hypothalamic–pituitary–adrenal (HPA) axis. Hence, the underlying pathophysiology common to all forms of neurodegenerative disease seems to involve oxidative stress (OS) ^3,4.

Findings from previous studies have established that MSG induces brain oxidative damage by the over-generation of reactive oxygen species (ROS) that is a possible cause of the excitotoxicity ²⁰. Oxidative injury is a key mechanism of oxidative stress is the consequence of uncontrolled production of ROS that results in antioxidant depletion and imbalance between pro-oxidant and antioxidant status ²¹. The brain tissue vulnerable to free radical effect due to its high oxygen consumption rate, low levels of antioxidant enzymes and the high lipid content (polyunsaturated fatty acids) ²². This results in neuronal injury and subsequent progress of neurodegenerative diseases ²¹. Findings in this study revealed significant decrease in SOD, CAT activities in MSG group. In addition, MSG induced decrease in GSH with resulting increase in MDA levels. Also, MSG-induce lipid peroxidation cause decrease in GSH. As a radical scavenger, GSH maintains the membrane structure by removing the acyl peroxides that resulted from lipid peroxidation ²³. More so, GSH plays a vital role in antioxidant defense system by maintaining the redox homeostasis in neurons ²⁴. The oxidative damage in brain is generally altered histopathological features confirmed with the presence of necrosis and hyperchromatic neuron and an overall decrease in neuronal density in cortex, amygdala, gyrus. The hydrolic fraction C. integrifolia showed antioxidant activities with potent free radical scavenging capacity. This was proved by high levels of CAT, SOD, and GSH with concomitant decrease in MDA levels in mice exposed to MSG. This is in line with previous studies, indicating that flavonoids free radical scavenging property is attributed for the presence of hydroxyl and keto groups in its chemical structure ²⁵. Previous studies revealed that apigenin scavenges free radicals by donating its hydrogen atom and electron to the hydroxyl, peroxyl, peroxynitrite radicals to form stable flavonoid radicals ²⁶.  Therefore, these results suggest that C. integrifolia protect against neurotoxicity by enhancing the cellular antioxidant system. 

NO is an important inflammatory mediator, and its release is controlled by the level of NOS expression in activated macrophages ²⁷. Marked upregulation in iNOS gene expression has been reported to be strongly implicated in neurotoxicity ²¹. Our study revealed high levels of NO in mice treated with MSG only. In contrast, administration of the plant extraction reduced NO concentration in brain mice exposed to MSG. this suggests that C. intergrifolia prevents neurons from cytotoxic levels of NO by decreasing iNOS activation.

Increased AChE activity cause the loss of cholinergic neurotransmissions leading to cognitive impairment ⁴. Previous studies have establish that Inhibition of AChE decrease the hydrolysis of ACh in the brain and increase cholinergic neurotransmissions which might be helpful in targeting cognitive decline ^25,26. In the present study, administration of MSG to mice causes significant increase in AchE activity. This is because suppressed activity of AChE is associated with increased ACh concentration that results in over stimulation of cholinergic, muscarinic and nicotinic receptors ²⁸. Thus, over activation of these receptors results in excess neuronal excitation and paralysis of cholinergic transmission ²⁵. On the contrary, the administration with hydrolic extract of C. integrifolia reduced this effect by decreasing greatly the activity of this enzyme. Indicating that the ability of C. integrifolia to inhibit AchE activity thereby restoring cholinergic functions and allowing more retention of acetylcholine in the brain, which is essential for enhancing cognitive functions especially learning, and memory ^4,29,30.

The results a low density of cells in mice given the MSG evidenced by the presence of necrotic cells observed in histopathology analysis of cerebral cortex CA1, CA3 regions of hippocampus and Gyrus indicate MSG causes the degeneration of different regions of brain. This effect of MSG is similar to that reported in various studies and this could account for high levels of ROS observed in these mice. Therefore, the administration of C. integrifolia prior to the MSG exposure showed that extract has prevented the damage of normal architecture of cortex, CA1, CA3, Gyrus pyramidal cell layer with a high cell density characteristic of nervous cells. These findings demonstrate that C. integrifolia root aqueous extract may prevent neurodegeneration.

CONCLUSIONS

In MSG-induced neurodegenerative like disease, the results revealed that the hydrolic fraction of Celtis integrifolia downregulated the brain AChE activity. Moreover, C. integrifolia extract reduced the brain level of NO, MDA, while increased the CAT, SOD and GSH levels. In addition, it prevented necrosis in different regions of brain. Therefore, C. integrifolia extract may alleviate the neurodegenerative like symptoms by preventing the brain oxidative stress, inflammation, high level of neurotransmitters, dysregulation of the HPA-axis and necrosis.

DECLARATIONS

Ethical clearance

Not applicable

Availability of data and material

The data generated or analyzed during this study are available under request to the corresponding author

 

 

Competing interests

Authors of this manuscript declare that they have no conflicts of interest

Funding 

Not applicable

Abbreviations

CAT: Catalase

SOD: Superoxide dimutase

GSH: Reduced Glutathione

NO: Nitric oxide

MDA: Malondialdehyde

AChE: Acetylcholinesterase

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