1. INTRODUCTION:

The brain is a very important and critical organ in humans where neurons are the functional unit of the nervous system. The nervous system controls and coordinates almost all functions of our body. Any malfunction in the nervous tissue leads to the disease condition. The destruction of neurons in a different part of the nervous system causes neurological disorders. As neuronal cells cannot divide, damage to the nervous system remains permanent.

Most of the neurological disorders progress during the late phase of life. The old age neurological disorders are the result of many mutations which occurs in the neuronal cells. Apart from neuronal cells, certain non-neuronal cells like glial cells are also involved in the progression of the disease. Neurological diseases are incurable to date as a complete mechanism for any of the disorder is unknown. Most of the neurological disorders show common characteristics like loss of synaptic connections, decrease in brain mass, impairment of neurons, chronic and progressive nature, and increased prevalence with age (Shrada and Kumar, 2015; Soto and Pritzkow, 2018). There are many age-dependent neurological disorders such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, and spinocerebellar ataxias. Due to the complex nature of genetic inheritance and development in humans, it is very difficult to study the nature of the disease, pathways involved in disease progression, genes and proteins responsible for disease condition (Antony et al., 2010; Moloney 2010: Moore et al., 2005). Also, the lack of sufficient genetic data and genetic heterogeneity in the human population make it troublesome to analyze such disease in a proper manner.

So in order to study these lethal diseases, a model system is needed which can recapitulate the disease features and symptoms. Various model systems are adopted by the scientists, working on neurodegenerative disorders such as cell lines, worms (Caenorhabditis elegans), D. melanogaster (fruit fly) and mice (Mus musculus) to provide any insight into disease mechanism (Reddy et al., 2018). Studies on these model system revealed that most of these diseases are caused by aggregation of misfolded proteins, expansion of tri-nucleotide repeats. These misfolded proteins forms aggregates known as amyloid. Some proteins which take part in aggregate formation have been well reported by the scientific community. These proteins include amyloid-beta (Aβ) and tau in Alzheimer, alpha-synuclein (α -Syn) in Parkinson (Himadri and Muniyan, 2018). Lewy bodies in multiple system atrophy, and dementia, TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis and frontotemporal dementia; and prion proteins in Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy, and scrapie (Chan and Bonini, 2000; Cummings and Zoghbi, 2000; Chen and Feany, 2015). Further identification and characterization of additional detrimental pathways and protein associated with these disorders will provide better and efficient targets to treat these disorders.

Paraquat (PQ) is a highly toxic herbicide which induces oxidative stress in flies on its exposure. PQ generated oxidative stress damages the DNA and cause mutations resulting in cell death. On exposure to high PQ concentration, the number of dopaminergic neurons reduced resulting in a low level of dopamine. The reduced level of dopamine in flies causes neurodegeneration and locomotor dysfunction (Shukla et al., 2014). In various neurological diseases, it was observed that these molecular chaperones suppress neurodegeneration. Studies conducted on Drosophila and other model organisms show that heat shock proteins (Hsp 70 and Hsp 40) reduce protein aggregation and toxicity of PolyQ proteins (Muchowski, 2002). Tinospora cordifolia (giloy) is a herb and known for its medicinal property. The leaves of giloy contain many antioxidants like phenols, flavinoids and, terpinoids. The antioxidant ability of giloy is used to treat oxidative stress-induced disorder like Parkinson’s disorder (Reddy and Reddy, 2015). Tinospora cordifolia contains many diterpenoid, alkaloids, glycosides, sesquiterpenoids, phenolics, polysaccharides and aliphatic compounds. Tinospora cordifolia reported as nontoxic in various toxicity studies. Tinospora cordifolia is known for its many medicinal properties which are being used for the treatment of various diseases and disorders. Various pharmacological studies have reported anti-diabetic, anti-cancerous, anti-microbial, anti-toxic, anti-oxidant and immunomodulatory property (Srivastava, 2011). The Tinospora cordifolia stem-methanol extract is known to enhance the catalase and lipid peroxide activity in the erythrocyte membrane. Tinospora cordifolia have been extensively studied for its potent antioxidant activity and free radical scavenging property. It reduces the activity of superoxide dismutase (SOD) and glutathione peroxidase enzymes thus reducing the oxidative stress within the cells (Premanath and Lakshmidevi, 2010). It is had been shown that the crude powder of Tinospora cordifolia reduces the toxic effect of L-DOPA therapy for Parkinson’s disorder (Antony et al., 2010; Reddy and Reddy, 2015).

The present study, therefore, aims to examine phenolic content, free radical scavenging capacity of Tinospora cordifolia through in vitro methods and also to investigate paraquat-induced neurodegeneration in Drosophila melanogaster and an amelioration through Tinospora cordifolia via locomotor, memory assay and also role of giloy on the logevity of fruit flies.

2. Material and method:

Fly stock: Wild type Drosophila melanogaster were procured from Nitte University, Mangalore, kindly gifted by Dr. Anurag Sharma and reared in the lab on corn, sugar meal prepared according to Singh et al. (2009) and maintained at standard culture conditions i.e 24+1^oC.

2.1 Preparation of Giloy extract:

For the preparation of giloy extract (Tinospora cordifolia), 5.0 gram of fresh giloy leaf was taken, cleaned and chopped into small pieces. The leaf pieces were then boiled in 50 ml of distilled water for 10 min, the extract was filtered with filter paper (Whattman). The extract was stored at 4ºC for further use.

2.2 2,2´-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay:

The antioxidant property of giloy leaf was measured on the basis of the scavenging activity of 2,2´-diphenyl-1-picrylhydrazyl (DPPH) protocol given by Mensor et al. (2001). Briefly, for DPPH test, 11mg of DPPH was added in 50ml of methanol. The solution was left for an hour at room temperature under dark conditions until completely dissolve the DPPH in methanol. To check the antioxidant properties of Tinospora cordifolia leaf, ascorbic acid (Vitamin C) was taken as the standard. The standard optical density of 0.90 was taken for the DPPH solution. The different concentration of giloy extract was prepared and 2.0ml of DPPH was added. For different concentrations of giloy leaf extract, optical density (OD) was measured using the spectrophotometer and experiment was carried out in the triplicates. Percent scavenging activity of giloy leaf was calculated for each dilution on the basis of the absorbance.

2.3 Total phenolic content:

The total phenolic content was estimated by Folin-Ciocalteu’s phenol reagent using the spectrophotometric method. First, 1.0ml of giloy extract was added to 5.0ml of 75% methanol. The mixture was centrifuged at 10000 rpm for 20 minutes at room temperature (24^oC±1), the supernatant was again centrifuged at the same speed for 10 min. The supernatant was transferred to a new tube for further use. We followed method given by Malik and Singh, 1980. Total phenolic content was estimated by extrapolating values on the calibration curve drafted against gallic acid.

2.4 Treatment method of paraquat and paraquat with giloy extract:

To check the effect of paraquat and giloy on development of Drosophila, two separate sets of treated food were prepared. The first set of food medium contained paraquat (1mM in one vial set and 2mM in another vial set) while the second set of food medium was supplement with paraquat and giloy extract. Next, the newly emerged first instar larvae were transferred to the treated food vials (20 larvae in each vial). The control set contained normal food with first instar larvae. The first instar larvae were allowed to feed on treated food for different time periods (24 h and 48 h). After treatment, the surviving larvae were transferred to the regular food medium. The development of treated larvae was observed and recorded. The experiment was done in triplicates.

2.5 Locomotory Assay:

To study the effect of paraquat-treated and paraquat-giloy treated flies on the locomotion, a set of flies were treated with paraquat (10 flies per vial) while the other set were treated with the paraquat-giloy extract (10 flies per vial) for two time period 24 and 48 hours. Different concentrations, 1.0mM and 2.0mM of paraquat were used for the experiment. After the treatment period, each set of flies were tested for climbing ability. A measuring cylinder marked at 10cm distance and flies from each set was transferred to the cylinder. The flies which crossed 10cm mark in 10 seconds were observed and readings were noted (Shukla et al., 2014).

2.6 Memory assay:

For memory assay we followed given by Shukla et al. (2014), briefly, the flies were given a conditioning phase in which flies, both untreated and treated set of flies were put in a T-maze. T-maze consists of a column in the centre and two chambers, a dark and lighted chamber. The light chamber contained a light source while the dark chamber was without any light source and is covered with the aluminum foil. In the conditioning phase, food for flies was only provided in the presence of light so that flies can learn this behaviour. This process was repeated for 4 h which made flies to acclimatize to such conditions. Next, the food was placed in the light chamber of T-maze and PQ treated flies were put into the T-maze. The number of flies moving towards light chamber in 10 seconds was observed and recorded. In the same manner, the PQ+giloy treated flies were put in T-maze and number of flies moving towards the light source in 10 sec was recorded. The process was repeated five times. The Average, SD and SEM were calculated and the graph was plotted.

2.7 Data interpretation and statistical analysis:

The data were recorded from each assay. The standard deviation (SD) and Standard error±mean (SEM) was calculated and data were represented in the graphical form. The differences between the untreated and treated set of flies were then determined by applying student t-test.

3. RESULTS:

3.1 Antioxidant activity of giloy extract:

The total antioxidant property of giloy extract was obtained by calculating percent scavenging capacity. For giloy extract, at 100mg concentration showed minimum OD (0.276) and maximum percent scavenging activity (69.73%). The percent scavenging activity of giloy leaf was compared with the standard antioxidant ascorbic acid. For ascorbic acid, maximum (97.74) percent scavenging activity was observed at 0.1mg/ml.

3.2 Phenolic content in giloy extract:

The total phenolic content calculated in 0.01gm of giloy extract was 0.024±0.003 mg GAE/gm.

3.3 Role of giloy on the survival of the flies after 24 and 48 h:

The flies which were treated with different concentrations of PQ (1.0mM and 2.0mM) for 24 h show high mortality rate while the flies which were treated with giloy extract and PQ show improved the survival time. The control flies show the maximum survival. The calculated mean value for survival was highest for the control group followed by PQ plus (1.0mM and 2.0mM) giloy extract (0.1%) and PQ treated group. The least survival mean value was obtained for PQ (1.0mM and 2.0mM) treated group.

Figure 1: Survival rate of the flies after 24 and 48 h treatment of 1.0. 2.0mM of PQ and PQ along with giloy (0.1%). Date represent mean+SD of three groups and n=20 used in each group. Arrow represent a significance attenuation in PQ+giloy treated groups as compared to PQ alone treated flies.

The survival rate decreases rapidly after 48 h of exposure to the PQ. The flies treated with the PQ+giloy extract show reduction in mortality. The glioy extract treated flies show improved lifespan after 48 h of PQ exposure indicating its property to reduce the mortality induced by PQ. The highest lifespan after 48 h was observed in case of control flies followed by PQ+giloy treated flies. The least lifespan was observed in case of PQ treated flies.

Figure 2: Locomotor assay of D. melanogaster exposed to control, paraquat alone (1.0 and 2.0mM) and paraquat cotreated (1.0 and 2.0mM) with giloy extract (01%) after 24 h (A) and 48 h (B) respectively. Data represents mean+SD of three groups (n=20). Significance ascribed *P<0.01 as compared to control, ^#P<0.01 as compared to PQ treated groups.

Modulation in locomotor activities in PQ treated Drosophila via giloy after 24 and 48 h respectively: The locomotor assay was employed to detect dopaminergic neuron-linked movement dysfunction. The flies treated with PQ (1mM and 2mM) exhibited reduced locomotor function as there was a decline in the climbing rate of PQ treated flies in comparison to the control flies whereas the flies which were treated with PQ+giloy show better performance in locomotor behaviour as an effect of PQ was minimized by giloy extract. The results show a significant difference (p<0.01) between the control, PQ treated and PQ+giloy treated group. The data show dose-dependent loss of locomotor activity (as shown in fig. A). After 48 h, flies which were treated with PQ show highly decreased climbing potential as flies tend to remain at the bottom of vials. The major decline in locomotor ability was seen in flies which were treated with 2mM PQ as most of the flies were falling backward and showed trembling movements in comparison to the control flies. The flies which were treated with the PQ+giloy show improved climbing behaviour after 48 h. The number of flies which crossed the 10 cm distance increased after treatment with the giloy extract indicating that the effect of PQ was mitigated by the giloy extract. The 48 h treatment results also show a significant difference (p<0.01) between the control, PQ treated and PQ+giloy treated group. In both 24 and 48 h of treatment, data show dose-dependent loss of locomotor activity by PQ. PQ treated flies show motor disability and impairment and show no sign of recovery even after 48 h. It was observed that in PQ treated flies the PQ effect was irreversible. However, the giloy treated flies were able to recover significantly and locomotor function was restored. The result also demonstrates that locomotor dysfunction induced by paraquat can be reversed to some extent by treating flies with the giloy extract (Figure 2).

Figure 3: Memory and learning assay of wildtype Drosophila following treatment of PQ and and PQ (1.0 and 2.0mM)+giloy (01%) after 24 h (A) and 48 h (B), respectively. Data represents mean+SD of three groups (n=20). Significance ascribed *P<0.01 as compared to control. ^#P<0.01 as compared to PQ treated groups.

3.4 An improved memory status was evident after giloy treatment in PQ challenged fruitflies:

In memory assay, it was observed that control flies show movement towards the light source while very few flies show movement towards the dark chamber. Fewer flies from the PQ (1.0mM and 2.0mM) treated group show movement towards the light chamber as most of the flies remain in the dark chamber. The result revealed the effect of PQ on the learning behaviour of flies. The PQ+giloy treated flies show movement towards the light source. The response of flies seems dose-dependent as in case of flies treated with 2mM PQ +giloy, a number of flies move towards the light was less in comparison with the flies treated with 1.0mM PQ + 01.% giloy. Whereas, after 48 h, a similar trend was evident as observed of 24 h treated group. The flies treated with the PQ (1.0mM and 2.0mM) show little response towards the light source. The flies treated with PQ+giloy show much-improved learning skills as most of the flies move towards the light source (Figure 3 A and B). These results illustrate that giloy extract might be promoting flies to overcome PQ stress by boosting neuro-protective pathways or mechanisms.

4. Discussion:

In this study, we studied the effect of paraquat on neurodegenration in an in vivo model organism and an amelioration in chemical induced neurotoxicity by Tinospora cordifolia (giloy) (Ragavee et al., 2018 and Sahu et a., 2018) in wildtype Drosophila melanogaster (Oregon R⁺). Paraquat is toxic because it produces free radicals and superoxide anions. Continuous exposure to PQ induces oxidative stress within the cells of the organism. Accumulation of free radicals generates reactive active oxygen (ROS) which further damages the proteins, lipids and DNA resulting in the death of the cells (Hosamani and Muralidhara, 2013; Pramila and Julius, 2019). In recent years PQ exposure has been shown to cause the death of dopaminergic neurons which leads to movement disability and reduction in learning skills. Drosophila melanogaster is being used as a model system to study the effect of PQ on the lifespan, mobility and learning behaviour. On exposure to paraquat, flies show features of neurodegeneration. There are several reports which suggest that PQ induces Parkinson’s disease by affecting dopaminergic neurons of the substantia nigra (McCormack et al., 2005). Many reports suggest that herbal medicinal plants contain anti-oxidative properties as they contain numerous constituents like polyphenols, flavonoids, and tannins within their extract (Aqil et al., 2006; Al asad et al., 2017; Pranabesh and Ramanna, 2017; Chitra et a., 2017 and Jain et al., 2018). In our study, we used Tinospora cordifolia an herbal plant with known antioxidant potential. Tinospora cordifolia extract was used to cure the PQ treated flies.

In our study, we find that on treating flies with different concentrations of PQ (1.0mM and 2.0mM) the lifespan, locomotor ability and learning efficiency of flies were significantly reduced in comparison to the control flies. These results indicate that PQ triggers the oxidative stress in the flies which causes neuromotor dysfunction. Similar results were also known from other studies (McCarthy et al., 2004). However, in order to investigate the antioxidant potential of Tinospora cordifolia on PQ treated flies were fed with its extract. Our results revealed that after feeding the giloy+PQ treated food flies show improved survival rates in comparison to the PQ treated flies. It was also observed that PQ effect on flies was dose-dependent as high PQ concentration (2mM) causes more lethal effects on the survival of flies. The giloy treatment significantly enhanced survival rate. Our experiments also demonstrated that giloy extract was able to reverse the effect of PQ to some extent as giloy+PQ treated flies show improved locomotor function and memory skills. Our findings show that a high level of antioxidants such as polyphenols and flavonoid content in giloy extract prevents or reduces the deleterious effects of ROS by their strong antioxidant property. Various studies on different plant extracts show that the polyphenols induce intracellular signaling pathways which are coupled with cell survival and proliferation (Kratchanova et al., 2010). The enhanced locomotor performance flies can be correlated with the fact that anti-oxidative property might prevent the reduction of dopamine level. It has been seen that a reduced level of dopamine is responsible for locomotor dysfunction (Hosamani, 2009). Our data also indicate that flies treated with giloy+PQ augment learning skills and memory performance.

5. CONCLUSION:

From our study, we conclude that paraquat is a good agent to study oxidative stress response in Drosophila. Our study also indicates that giloy extract provides neuroprotective function due to the presence of phenolic and other compounds in the extract. Drosophila is a good model to study these disorders as large-scale genetic screening of mutants is possible, the anticipation of novel cellular mechanisms, testing of small molecule and compounds that can be used to reduce disease pathogenesis. The giloy extract (0.1%) can be used as therapeutic compound for neurodegereative diseases like AD and PD as our data showed significant amelioration in fruit flies but further validation in rodent models is warranted and need to be investigated.

6. CONFLICT OF INTEREST:

Authors report no conflict of interest and authors are responsible for the content this review paper.

7. ACKNOWLEDGEMENT:

We hereby acknowledge Lovely Professionals University for providing infrastructure and lab facilitlies for prelimenary experiments.

8. FUNDING:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Received on 03.07.2019 Modified on 29.02.2020

Research J. Pharm. and Tech. 2021; 14(6):3056-3062.

DOI: 10.52711/0974-360X.2021.00534