Neuroprotective activity of Garcinia morella desr against monosodium glutamate-induced neurotoxicity in rats

The aim of the present study is to evaluate the neuroprotective effect of methanol extract of Garcinia morella Desr leaves in monosodium glutamate (MSG) induced neurotoxicity in rats. The MSG 2 g/kg, i.p. was used to induce the neurotoxicity. The rats were administrated with methanol extract of Garcinia morella Desr leaves (MEGM) 200 and 400 mg/kg, p.o. and Dextromethorphan 30 mg/kg, p.o. after 1 h injection of MSG. The animals were evaluated for its behavioural factors such as muscle grip and locomotor activity. At the end of the study, the antioxidant enzymes levels, neurotransmitter levels, TNFα, ß-amyloid and mineral levels were estimated in brain homogenate. The MEGM treated group shows signi icant improvement of behavioural and locomotor activity and muscle strength against MSG induced neurotoxicity. The glutathione, SOD, catalase and total protein levels were signi icantly increased in MEGM treated group. However, signi icant reduction within the level of varied neurotransmitters like AChE, Dopamine, TNFα, ß-amyloid were observed on treatment with MEGM extract. Further, MEGM also signi icantly decrease the MSG induced toxicity through declined levels of Ca and Na with increased levels of K. In conclusion, the present study suggests that the MEGM has signi icant neuroprotective activity against MSG induced neurotoxic rats.


Antioxidants, Dextromethorphan, Monosodium glutamate, Neurotransmitters
A The aim of the present study is to evaluate the neuroprotective effect of methanol extract of Garcinia morella Desr leaves in monosodium glutamate (MSG) induced neurotoxicity in rats. The MSG 2 g/kg, i.p. was used to induce the neurotoxicity. The rats were administrated with methanol extract of Garcinia morella Desr leaves (MEGM) 200 and 400 mg/kg, p.o. and Dextromethorphan 30 mg/kg, p.o. after 1 h injection of MSG. The animals were evaluated for its behavioural factors such as muscle grip and locomotor activity. At the end of the study, the antioxidant enzymes levels, neurotransmitter levels, TNFα, ß-amyloid and mineral levels were estimated in brain homogenate. The MEGM treated group shows signi icant improvement of behavioural and locomotor activity and muscle strength against MSG induced neurotoxicity. The glutathione, SOD, catalase and total protein levels were signi icantly increased in MEGM treated group. However, signi icant reduction within the level of varied neurotransmitters like AChE, Dopamine, TNFα, ß-amyloid were observed on treatment with MEGM extract. Further, MEGM also signi icantly decrease the MSG induced toxicity through declined levels of Ca +2 and Na + with increased levels of K + . In conclusion, the present study suggests that the MEGM has signi icant neuroprotective activity against MSG induced neurotoxic rats.

INTRODUCTION
Neuroprotection is a treatment which means to shield the rest of the nerve cells from further harm. It is a broadly uncovered treatment for many central nervous system (CNS) illnesses such as stroke, neurodegenerative diseases, spinal cord injury, brain injury, paralysis and neurotoxin consumption. Any substance from natural molecule that has a protective in luence in the nervous system against neurodegenerative disease or brain injury will possess neuroprotective activity (Djaldetti et al., 2003;Ha et al., 2011).Common neurotoxicity mechanisms are elevated levels of oxidative stress, mitochondrial dysfunction, excitotoxicity, in lammatory changes, iron accumulation, and protein aggregation (Seidl and Potashkin, 2011;Andersen, 2004;Zádori et al., 2012).
The world health organization assesses that, 80% of the world's population (more than six billion people) fundamentally rely upon animal and plant based medicine (Alves and Rosa, 2005). Medicinal loras are a best foundation to discover new solutions for neurological diseases. Garcinia morella Desr (GM) is a medicinal plant under the family of Guttiferae. GM is distributed in Asian, African and Polynesian countries and it is locally called as kujithekera. GM is commonly used for the management of stomach disorders, bowel problems and in lammations. Different extracts of Garcinia species has been revealed for its anticancer (Choudhury et al., 2016) , anti-oxidative (Yamaguchi et al., 2000), antifungal (Gopalakrishnan et al., 1997), antibacterial (Rao and Verma, 1951;Sani and Narasimha-Rao, 1969) activities. And also the Garcinia species contains secondary metabolites such as xanthones, lavonoids, lactones, benzophenones and phenolic acids. The Morello lavone, Gambogic acid and guttiferic acid has reported in GM (Karanjgaokar et al., 1967;Chantarasriwong et al., 2010). The fruits of GM are rich in hydroxyl citric acid, garcinol, guttiferones, camboginol and xanthochymol (Hemshekhar et al., 2011). The lavonoids, xanthones, and prenylated benzophenones have been reported for its Neuropharmacological property. But there is no scienti ic validation of GM for neuroprotective activity. Therefore, this research work was aimed to evaluate the neuroprotective activity of GM.

Plant Collection and Identi ication
Raw leaves of GM were acquired in Tirunelveli region of Tamil Nadu, India. The GM was veri ied by the taxonomist Dr.P.Jayaraman, Institute of Herbal Science, Plant Anatomy Research Center, Chennai. The voucher specimen numbers is PARC/2017/4338.

Preparation of Extracts
Unprocessed leaf of GM was completely cleaned, shadow dried and pulverized. Then it was extracted using continuous extraction (Soxhlet extraction) method. The methanol was utilized for extraction for 48 h. The concentrate product was concentrated by using vacuum evaporation at 45 • C. Then the end product was collected in a well closed container for pharmacological screening Gopalasatheeskumar (2018) (Gopalasatheeskumar, 2018).

Laboratory animals
Wistar albino rat (either sex; 125-150 g body weight) were kept in a well-organized room temperature for 12 h. The animals were free admittance to clean food and water. The investigations were led in sound proof research centre. This investigation was done with the approval of the animal ethical com-mittee no. KPCP/2017-2018/CPCSEA/0006/1c.

Experimental protocol
Animal were randomly divided into ive groups 6 animals in each as follows,
The entire group received the treatment for a period of 7 days. After MSG administration the general behavioural changes, were observed for 30 min daily, on 8th day, the rats were evaluated for locomotor and muscle relaxant activity. The locomotors activity was studied using actophotometer. In this, animals of all the groups were placed in actophotometer for 10 min and the score were recorded. Muscle relaxant activity was investigated using rota rod apparatus. Rats were placed on the rotating rods and the time of fall from the rotating rods were recorded (Mahalaxmi et al., 2018)Mahalaxmi et al., 2018.

Biochemical parameters
On 9th day, the experimental rats were sacri iced by cervical dislocation. The brain was isolated carefully and perfused in 0.85% NaCl to eliminate blood. The brain tissue 10% w/v was homogenised in handheld homogeniser using Tris-HCl buffer at pH 7.4 and it is centrifuged at 5000 rpm for 10 min. Supernatant obtained was used for estimation of antioxidant enzymes, minerals and neurotransmitter levels (Hemshekhar et al., 2011).

Estimation of antioxidant enzyme levels
The antioxidant parameters including Superoxide dismutase (SOD), Reduced glutathione (GSH), Catalase (CAT) and Lipid peroxidation (LPO) levels were estimated as per the standard protocols followed by (Hemshekhar et al., 2011;Yuvaraja et al., 2020). The 0.05 ml of brain homogenate was used for the estimation of these parameters.

Estimation of neurotransmitters and minerals
The Acetylcholinesterase (AChE), Glutamate, Serotonin, Dopamine, calcium and sodium levels were estimated from 250 µL of brain homogenate and followed by standard assay methods (Usama et al., 2017;Boopathi and Thangavel, 2020).

Estimation of β-amyloid and tumor necrosis factor-α (TNF-α)
The level of β-amyloid peptide and TNF-α in brain homogenate were estimated by the rat ELISA Kit with a microplate reader as per the ventors protocol (Usama et al., 2017;Silva et al., 2015).

Statistical analysis
Results are communicated as mean±SEM. Results were analysed by one-way analysis of variance (ANOVA) trailed by Dunnet's multiple comparison test utilizing Graph Pad Prism software. <0.05 is re lected as signi icant.

Effect of MEGM on Locomotor and muscle relaxant activity
Treatment with MSG treated group shows signi icant decrease in the time of fall from the rotating rod compared with control group. While on Treatment with MEGM at the both doses and standard drug dextromethorphan it is reversed ( Table 1). The locomotor activity is decreased signi icantly in MSG treated groups as compared with control. On Treatment with MEGM at the dose of 200 and 400 mg/kg as well as standard drug, dextromethorphan locomotor activity score is increased as compared with negative control ( Table 2).

Effect of MEGM anti-oxidant parameters
Signi icant accumulation of lipid peroxides in brain tissue of rat was observed and effect of antioxidant levels including SOD, Catalase and GSH was decreased signi icantly in MSG injected group when it was compared with control, whereas MEGM and dextromethorphan treated rat produce a signi icant reduction in LPO activity and signi icant elevation in SOD, CAT and RGSH in brain tissues as compared with MSG treated rat it is shown in Table 3.

Effect of MEGM on minerals
The MSG treated groups shows the signi icant elevated levels of calcium and sodium when compared to normal control group (, P<0.01). However, potassium levels were signi icantly decreased. These altered levels were signi icantly normalized in MEG and Dextromethorphan treated groups (Table 4).

Effect of MEGM on neurotransmitters, β-amyloid and TNFα
The MSG treated groups shows the signi icant elevated levels of glutamate, AChE, TNFα, dopamine and β-amyloid when compared to normal control group (P<0.01). However, GABA and Serotonin levels were signi icantly decreased. These altered levels were signi icantly normalized in MEG and Dextromethorphan treated groups (Table 5).
MSG a lavouring agent causes neuronal symptoms like numbness, weakness, dizziness and headaches and also it's reported as MSG changes antioxidant levels and lipid peroxidation of mitochondria in brain (Meldrum, 2000). Treatment of MSG in rat may cause severe behavioural abnormalities like increased irritability, hypoactivity, and de icits in spontaneous alternation behaviour (Shivasharan et al., 2013). MSG produces excitotoxicity by activating NMDA receptors, which successively disturb the calcium homeostasis, which can cause glutamateinduced neuronal damage. Earlier report says that there's association between increased glutamate neurotransmission and cognitive dysfunction. Glutamate elicits synaptic responses via metabotropic mGLU and ionotropic NMDA receptors resulting in increased cellular excitation and induce the discharge of Ca +2 from intracellular stores. On the opposite hand activation of ionotropic receptors increases cellular permeability to Ca+2 (Weil et al., 2008).
Acetyl choline is a most vital neurotransmitter of CNS and it plays a major role in behavioural also as learning and memory and neurodegenerative diseases. Our results showed that on treatment with MSG shows that there's an increased AChE level within the brain may lead to a reduction of cholinergic neurotransmission ef iciency due to a decrease in acetyl choline level within the synaptic cleft, thus contributing to progressive cognitive impairment (Jiang and Zhang, 2008). Additionally, MSG causes oxidative stress by increasing lipid peroxides and reactive oxygen species (ROS), which could enhance AChE level. Signi icant alterations in serotonin, dopamine, and β amyloid levels within the brain of MSG-treated rats were recorded in our study. This decrease in serotonin level in brain may cause neurotoxicity. While on treatment with MEGM at the both doses as well as standard drug dextromethorphan signi icantly increased the serotonin level which gives neuroprotection property and therefore the report was supported by a previous report on exposed of MSG (Sylvie et al., 2015). Moreover, the MSG induced rats were raised in Aβ accumulation which cause development of No. of Animals 6, Data were analysed as Mean±SEM, One way ANOVA followed by Dunnett's test, All groups were matched with negative control, *p<0.05, **p<0.01,***p< 0.001 No. of Animals 6, Data were analysed as Mean±SEM, One way ANOVA followed by Dunnett's test, All groups were matched with negative control, *p<0.05, **p<0.01,***p< 0.001   No. of Animals 6, Data were analysed as Mean±SEM, One way ANOVA followed by Dunnett's test, All groups were matched with negative control,*p<0.05, **p<0.01,***p< 0.001 amyloid plaques within the rat hippocampus and induced neurobehavioral abnormalities, this effect was reversed by MEGM and standard drug dextromethorphan. Furthermore, MSG treated group shows increased levels of TNF-α, it con irms the neuro in lammation caused by MSG. While on treatment with MEGM at the both doses as well as standard drug dextromethorphan signi icantly reduced the amount of these in lammatory mediators and mainly it suppress the expression of TNF-α (Jain et al., 2009).

CONCLUSIONS
The results getting from this research clearly revealed the neuroprotective effect of MEGM against MSG induced neurotoxic rats. Since MSG is extremely dangerous its use must be limited. This effect may be due to the presence of antioxidants like Xanthones, lavonoids and prenylated benzophenones present within the extract. Further the isolation of active principles for ind out the exact mechanism of neuroprotective activity.