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RJPS Vol No: 14 Issue No: 3 eISSN: pISSN:2249-2208

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Original Article

Hiba Gafoor, Ananya Bhattacharjee*, Muhammed Muzammil Mohammed Ali, A. Ramakrishna Shabaraya

Srinivas College of Pharmacy, Valachil, Farangipete (Post), Mangalore, Karnataka, India– 574143

Corresponding author:

*Ms. Ananya Bhattacharjee, Assistant Professor, Department of Pharmacology, Srinivas College of Pharmacy Valachil, Farangipete (post), Mangalore- 574143, E-mail: mouroland@gmail.com

Received Date: 05/01/2020 Accepted Date : 25/03/2020 

Year: 2020, Volume: 10, Issue: 2, Page no. 14-22,
Views: 795, Downloads: 17
Licensing Information:
CC BY NC 4.0 ICON
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0.
Abstract

Aim of the study: Free radicals are one of the major reasons for the development of Parkinson disease where antioxidant from herbal origin can play beneficial role. Ficus carica reported wide spread of pharmacological activities including its beneficial effects against different CNS disorders majorly due to presence of polyphenols. This present study was designed to investigate the antiparkinsonian potential of acute and chronic treatment of ethanolic extract of Ficus carica fruits (EEFCF) against haloperidol induced parkinsonism.

Materials and methods: Rats were divided into five groups and treated with saline, combination of levodopa+ carbidopa and low (100 mg/kg, p.o.) and high dose (200mg/kg, p.o.). In case of acute model all treatments were given for single dose where as in case of chronic model all treatments were given for 14 days. Apart from normal control, all other groups received haloperidol (1mg/kg, i.p.). In both the models effects of different treatments were evaluated by estimation of motor co-ordination, behavioral assessment, biochemical estimation and histological studies.

Results and conclusions: Both the lower and higher dose of ethanolic extract of Ficus carica fruit showed dose dependent significant increase in anti-parkinsonian activity when compared with control. The finding of the present study provides the evidence that the ethanolic extract of Ficus carica fruit reported dose dependent beneficial effect against Parkinson’s disease.

<p>Aim of the study: Free radicals are one of the major reasons for the development of Parkinson disease where antioxidant from herbal origin can play beneficial role. <em>Ficus carica</em> reported wide spread of pharmacological activities including its beneficial effects against different CNS disorders majorly due to presence of polyphenols. This present study was designed to investigate the antiparkinsonian potential of acute and chronic treatment of ethanolic extract of <em>Ficus carica</em> fruits (EEFCF) against haloperidol induced parkinsonism.</p> <p>Materials and methods: Rats were divided into five groups and treated with saline, combination of levodopa+ carbidopa and low (100 mg/kg, p.o.) and high dose (200mg/kg, p.o.). In case of acute model all treatments were given for single dose where as in case of chronic model all treatments were given for 14 days. Apart from normal control, all other groups received haloperidol (1mg/kg, i.p.). In both the models effects of different treatments were evaluated by estimation of motor co-ordination, behavioral assessment, biochemical estimation and histological studies.</p> <p>Results and conclusions: Both the lower and higher dose of ethanolic extract of <em>Ficus carica </em>fruit showed dose dependent significant increase in anti-parkinsonian activity when compared with control. The finding of the present study provides the evidence that the ethanolic extract of <em>Ficus carica </em>fruit reported dose dependent beneficial effect against Parkinson&rsquo;s disease.</p>
Keywords
Antiparkinsonian, Ficus carica, neuroprotective.
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Introduction

Neurodegenerative disease like Parkinson’s disease (PD) poses a significant threat to human health. This deterioration can affect body movement and brain function, causing dementia. These diseases are one of the most important medical and socio-economic problems of our time, affecting people of all ages.1,2 Free radical generation is responsible for generation of excessive oxidative damage which is associated with dopamine auto-oxidation, α-synuclein aggregation, mitochondrial dysfunction, alterations in calcium signaling, glial cell activation, and excess free iron. With the advancing of age reduced counteracting mechanism and alterations in transcription of various pathways such as glycogen synthase kinase 3β, nuclear factor kappa B, nuclear factor erythroid 2-related factor 2, mitogen activated protein kinase, reduced activity of superoxide dismutase, catalase and glutathione are majorly responsible for increased incidence of PD.3

There has been an unprecedented increase in the herbal medicine in recent years, and these medicines are gaining popularity in both developing as well as developed countries due to their natural origin and less side effects. Most traditional therapeutic drugs in use come from medicinal plants, organic matter and minerals.4

Treatment based on modern medicine is expensive and also shows adverse complications either in short term or long term. Popularity of alternative medicine and natural products is increasing worldwide due to inexpensive and health promoting activities. In this vista, Ficus carica, commonly known as Fig, is a small deciduous tree and has a wide disease control activity due to rich in antioxidant. Experiments based on different types of extracts have confirmed their function in curing diseases such as, leaves of figs traditionally used in the disease management such as vitiligo, diabetes, coughs, asthma, constipation, and gingivitis. Moreover, roots used in the treatment of leukoderma and ringworms and fruits also shows role as antipyretic, purgative, aphrodisiac properties, and have shown to be valuable in inflammations and paralysis. Latex and its derivatives have been revealed to suppress the growth of transplanted and spontaneous tumors.5

Ficus Carica act as reducing agents, hydrogen donators, free radical scavengers, singlet oxygen quenchers and exhibit potential antioxidant activity. Presence of polyphenols, flavonoids and anthocyanins are majorly responsible for antioxidant activities. Ficus carica witnessed beneficial effects against different CNS orders. Further it has been reported treatment with Ficus carica fruit was responsible for increased dopamine level which can play beneficial role against PD and related disorders.6,7 Till now no study has been performed to evaluate effect of Ficus carica fruit against PD. So the present study has been designed to evaluate acute and chronic treatment of Ficus Carica against haloperidol induced PD.

Materials and Methods

Experimental animals:

Wister albino rats of 150-200g wight of either sex were procured from Srinivas College of Pharmacy animal house. They were maintained under standard conditions (temperature 22 ± 2 ℃, relative humidity 50±5% and 12 hrs. light/dark cycle). The animals were housed in sanitized polypropylene cages with sterile paddy husk as bedding. They had free access to standard pellet diet and water. The Institutional Animal Ethics Committee approved the experimental protocol. All the animals were given human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the “National Academy of Sciences” and published by the “National Institute of Health”. All the procedures were performed as per Institutional Animal ethics committee constituted as per the direction of the CPCSEA, under ministry of animal welfare division, Government of India, New Delhi, India. 

Ethical considerations:

All efforts were made to minimize animal suffering and to reduce the number of animals used. The animals received humane care; the experiments were conducted strictly in accordance with the approved guidelines by the IAEC regulated by the CPCSEA according to Government of India accepted principles for lab animals use and care. The study protocol was approved by IAEC, Srinivas College of Pharmacy, Mangalore (Ref. no.SCP/IAEC/F150/P140/2018). Drugs and chemicals: Neurotoxicant Haloperidol was obtained from yarrow chem. (Mumbai, Maharashtra). All other chemicals used in the study were obtained from Agape Diagnostics LTD, Kochi, and Hi media Laboratories and were of analytical grade.

Collection and authentication of plant material:

The Ficus carica fruits were procured from local region of Kerala was authenticated by botanist Dr Siddaraju M N M.Sc. PhD Assistant Professor, Department of Botany, University College, Mangalore.

Preparation of Ficus carica fruit extract:

The extraction was carried out using the electrical shaker by the process of maceration using 95% of ethanol as a solvent.200g of Finely grounded powder of Ficus carica fruit was mixed and agitated with 250ml of ethanol and subjected to maceration with intermittent shaking for 24 hrs. The obtained extract was collected by filtration using the Whatman’s filter paper. The solvent was recovered by distillation process from flash rotary evaporator, and was concentrated to a thick mass in the water bath.8 The dried extract was weighed and the percentage yield of the extracts was calculated with the help of following formula

% of extractive yield(w/w)

= weight of dried extract/ weight of dried fruit powder x 100

= 3.5 (w/w)

The obtained semisolid extract was weighed and stored in refrigerator in an airtight container and used for the current studies.

Phytochemical screening of Ficus carica fruit:

Phytochemical screening of EEFCF for the presence of alkaloids, carbohydrate, flavonoids, glycosides, steroids, tannins, proteins were carried out as per the procedure previously described.9

Acute toxicity study in mice

(acute toxicity study determination)

The acute toxic study was performed for the ethanolic extract of Ficus carica fruit (EEFCF) as per the OECD guideline no. 423 (2001) for Acute Toxic Classic Method. Three female swiss albino mice were used for each step. The animals were kept on fasting for overnight only on water, after which the extracts were administrated intragastrically at different doses (50 and 300mg/kg). Animals were observed for initial 4 hours after the administration and then once daily for 14 days to observe the mortality. As mortality did not occur the procedure was repeated for further higher dose i.e.2000mg/kg. 1/10th and 1/20th of maximum tolerated dose of EEFCF (2000mg/kg) for acute toxicity did not indicate mortality. Hence 200mg/kg and 100 mg/kg doses are selected for the evaluation of Anti-Parkinson potential of EEFCF.10

Experimental design:

Rats weighing about 150–200g were selected and randomly divided in to five groups (n = 6 in each). Group I & II received vehicle (distilled water), Group III received L-dopa and carbidopa (syndopa 10 mg/kgp.o.), Group IV & V received Ficus carica high (200mg/kg, p.o.) and low dose (100mg/kg, p.o.)

Acute Study:

Haloperidol was given to all groups except Group I at a dosage 1 mg/kg,(i.p.) i.e. after 60 min of the extracts/drug administration. The mean catalepsy score was noted at an interval of 30 min for a period of 4 hrs. 

Haloperidol induced catalepsy:

Behavioral assessment (Metal bar test):

After 30 min of administration of haloperidol (1 mg/kg, i.p.), the duration of catalepsy was measured for 5 min at an interval of 30, 60, 90, and 120 min. Duration of catalepsy was determined by placing an animal on horizontal metal bar at a height of 6 cm in such a way that the fore-limbs of the animals should be on the horizontal bar while the hind limbs touche the surface.11

Chronic study

Experimental grouping was same as earlier model. All treatments were given for a period of 14 days. Except group I, all other groups received Haloperidol through intra peritoneal route (1 mg/kg). After 30 mins of last treatment locomotor activity, motor coordination, behavioral assessment studies were carried out.

Locomotor activity: (Actophotometer)

The exploration and the voluntary movement within an enclosed area is estimated by this test. The spontaneous motor activity was evaluated using a photoactometer (INCO Ltd., India). The animals were placed individually into a 30 cm × 30 cm chamber with a seive floor and a tight lid. Beams of red light were focused above the floor into the photocells on the other side. Beam interruption was recorded on the external counter. The count of interruption in light beam were performed for 5 min.12

Motor co-ordination: (Rota-rod)

The major clinical symptoms of PD is the muscle rigidity. This was evaluated in an animal model by rotarod. The apparatus consists of a 70 cm long rod with diameter 3 cm placed at a height of 50 cm and divided into 4 sections. Five trials were taken before the main readings to all the groups by adjusting the rate of rotation at 30 rpm.13

Behavioral assessment (Metal bar test)

Catalepsy is reduced ability to move and to correct abnormal posture; it was estimated by bar test. For this test, animals were placed in such a way that their hindquarters were on the bench, while their forelimbs were on a 1 cm diameter horizontal bar, placed 6–9 cm above the bench. The duration the animal maintained this posture was estimated by a stopwatch to a maximum of 180 sec (mean of three consecutive trials; interval: 1 min). Animals were said to be cataleptic if they maintained this posture for a period of 30 sec or more.12 

Preparation of tissue homogenate:

For the rats of chronic model, brain tissue homogenate was prepared and various oxidative parameters in brain tissue homogenate for the estimation of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and malondialdehyde (MDA) were estimated as per the reported protocols.12 At the end of experimental period, rats were sacrificed by decapitation under mild anesthesia. The brains were immediately removed, from that forebrain was removed, cerebellum was disposed off.

Brains were kept on ice, washed in ice-cold isotonic saline so that blood is removed. 0.1 M phosphate buffer (pH 7.4) was used to prepare 10% (w/v) tissue homogenate. The homogenate was centrifuged (Remi Industries Ltd. Mumbai, India) at a speed of 10,000 rpm for 15 min and the supernatant was used for biochemical estimation.14

Catalase activity:

The catalase activity was evaluated by the method of Aebi in 1974.The assay mixture consists of 0.05 mL of tissue homogenate supernatant (10%) and 1.95 mL of 50 mM phosphate buffer (pH 7.0) in 3 mL cuvette. 1 mL of 30 mM hydrogen peroxide (H2O2 ) was added. The absorbance differences were checked for 30 sec at 240 nm at 15 sec intervals. The catalase activity was estimated by millimolar extinction coefficient of H2O2 (0.071 mmol cm−1), and it was expressed as micromoles of H2O2 oxidized per minute per milligram protein.14

Superoxide dismutase activity (SOD):

Superoxide dismutase (SOD) activity was assayed according to the method of Kono, 1978 wherein the reduction of nitrobluetetrazolium (NBT) wasinhibited by the SOD and was measured spectrophotometrically at 560 nm. Briefly, the reaction was initiated by the addition of the hydroxylamine hydrochloride to the mixture containing NBT and the post nuclear fraction of the homogenate (10%). The result was expressed as unit per milligram of protein, with one unit of enzyme defined as the amount of SOD required to inhibit the rate of reaction by 50 %.15

Malondialdehyde (MDA):

The malondialdehyde was used as an indirect estimation of lipid peroxidation and was evaluated by reaction with thiobarbituric acid (TBA). Briefly, in test tubes 1 mL of aliquots of supernatant was taken, 3 mL of TBA reagent: TBA 0.38% (w/w), 0.25 M hydrochloric acid (HCl), and trichloroacetic acid (TCA 15%) were added. Then the solution was shaken well and kept for 15 min, which was followed by cooling in an ice bath. After this, the solution was centrifuged for 10 min to 3500 g. From this, the upper layer of the solution was taken to assess with a spectrophotometer at 532 nm. All the parameters determination were done in triplicate. The results were shown as nanomoles per mg of protein.14

Reduced glutathione (GSH):

For the estimation of reduced glutathione, 1mL of tissue homogenate was precipitated with 1mL of 10% TCA. To an aliquot of the supernatant, 4mL of phosphate solution and 0.5mL of 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent were added and absorbance was taken at 412nm.14

Histopathological studies:

The control and experimental groups brain samples were fixed with 10% formalin and embedded in paraffin wax, longitudinal section of 5 μm thickness were cut. The sections were stained with hematoxylin and eosin dye for the histopathological observation.14

Statistical analysis:

Results were expressed as mean ± SEM. Statistical significance was estimated by using One-way Analysis of variance (ANOVA) followed by Tukey-Kramermultiple comparisons tests. p<0.05 was considered significant. 

Results

Phytochemical screening:

Phytochemical screening of EEFCF revealed the presence of alkaloids, carbohydrate, flavonoids, glycosides, steroids and tannins.

Acute toxicity studies:

EEFCF were found to be safe at all the doses used and there was no mortality found up to the dose of 2000 mg/kg of EEFCF when administered orally. Therefore, 1/10th (200 mg/kg) and 1/20th (100mg/kg) of this dose were selected.

Behavioral Assessment (Acute Study):

The effects of EEFCF on haloperidol induced catalepsy:

Haloperidol control group showed extremely significant (p<0.001) increase in all time intervals in the cataleptic behaviour when compared to vehicle control group. In duration of catalepsy there was extremely significant decrease with time (p<0.001) in standard group compared with haloperidol control. Pre-treatment with EEFCF 100 mg/kg showed moderately significant (p<0.01) whereas 200 mg/kg showed extremely significant (p<0.001) reduction in the duration of catalepsy as compared to haloperidol control group. (Table 1)

All the values are Mean±SEM, n=6 ***P<0.001, when compared with vehicle control group. ^^^P<0.001, ^^P<0.01 when compared with haloperidol control group. Low dose EEFCF= low dose ethanolic extract of Ficus carica fruit, High dose EEFCF= high dose of ethanolic extract of Ficus carica fruit.

Behavioral Assessment (Chronic Study):

The effects of EEFCF on haloperidol induced catalepsy

Haloperidol control group showed extremely significant (p<0.001) increase in the cataleptic behaviour when compared to vehicle control group. Standard drug showed an extremely significant (p<0.001) decrease in the cataleptic behaviour, EEFCF 100 mg/kg showed moderately significant (p<0.01) whereas 200 mg/kg showed extremely significant (p<0.001) reduction in the cataleptic behaviour when compared to the haloperidol control group (Table 2).

All the values are Mean±SEM, n=6 ***P<0.001 when compared with vehicle controlgroup. ^^^P<0.001, ^^P<0.01 when compared with haloperidol control group. Low dose EEFCF= low dose ethanolic extract of Ficus carica fruit, High dose EEFCF= high dose of ethanolic extract of Ficus carica fruit

Locomotor activity test:

Haloperidol control group showed extremely significant (p<0.001) reduction in locomotor activity compared to vehicle control group. Standard and high dose of EEFCF showed extremely significant (p<0.001) whereas low dose showed moderately significant (p<0.01) increase in the locomotor activity as compared to haloperidol control group (Table 2).

Motor co-ordination activity test:

Haloperidol control group showed extremely significant (p< 0.001) reduction in motor coordination activity compared to vehicle control group. Standard and high dose of EEFCF showed significant (p<0.001), whereas low dose of EEFCF showed moderately significant (p<0.01) increase in the fall of time when compared to haloperidol control group (Table 2).

Biochemical parameters:

The Effects of EEFCF on haloperidol Induced Parkinson’s Disease in Malondialdehyde (MDA) Level.

In the haloperidol control group, extremely increase in brain MDA levels (p<0.001) was seen as compared to the control group as administration of haloperidol induced oxidative stress. The treatment with high dose of EEFCF (200 mg/kg, p.o.) showed extremely significant (p<0.001), and low dose of EEFCF (100 mg/kg, p.o.) showed moderately significant (p<0.01) decrease in MDA level compared to haloperidol control group.

The Effects of EEFCF on haloperidol Induced Parkinson’s Disease in Catalase (CA), Superoxide dismutase (SOD), and Reduced glutathione (GSH). Administration of haloperidol resulted in significant changes in biochemical parameters when compared to the vehicle control animals. The administration of haloperidol induced oxidative stress, as indicated by decreased CA, SOD, and GSH levels (p<0.001) in brain levels when compared to vehicle control animals. Daily administration of EEFCF high dose (200mg/kg, p.o.) showed extremely significant (p<0.001) and low dose (100mg/kg, p.o) showed moderately significant (p<0.01) increase in SOD, CA and GSH levels in the brain significantly as compared to haloperidol control group.

Histopathological Study:

Effect of EEFCF on histopathological changes in brain of vehicle and haloperidol control treated animals. The histopathological study exhibited that haloperidol caused marked hypertrophic changes, neutrophils infiltration, increased intracellular space, decreased cell density, changes of architecture, congestion, hemorrhage due to decrease the number of neural cells in SNpc in brain tissue and neuronal damage and cell death. It was evident that many neurons were pyknotic, shrunken, and darkly stained with small nuclei. (Figure 1(b)) compared to vehicle treated rats (Figure 1(a)). Significant restoration of neuronal damage or neuronal alterations were observed in standard (10mg/kg) treated rats (Figure 1(c)) and EEFCF treated rats at doses of 100 (Figure 1(d)) and 200mg/kg (Figure 1(e)).

Discussion

In this present study we investigated acute and chronic treatment of ethanolic extract of Ficus carica fruit (EEFCF) against haloperidol induced Parkinson’s disease (PD) conditions. PD is a commonly diagnosed neurodegenerative disorder, characterized by degeneration of dopamine producing neurons in the substantia nigra leading to resting tremor, bradykinesia, shuffling gait, flexed posture and rigidity. Still, the cause of the degeneration is not well defined. Oxidative stress may play a major role. Oxidative stress may arise from the metabolism of dopamine with the generation of harmful free radicals. Compared to the rest of the brain, the substantia nigra pars compacta are exposed to a higher rate of free radical formation and to increased level of oxidative stress. Various studies have revealed oxidative stress changes evident in the brain of PD patients.15

Haloperidol, a neuroleptic drug, induces catalepsy which is due to a blocking of post synaptic striatal dopamine D2 receptors and many studies have shown reactive oxygen species as a cause of haloperidol induced toxicity. Drugs which attenuate haloperidol-induced motor disorders might reduce the extrapyramidal signs of PD.15 

The important parameter for the selection of ethanolic extract of Ficus carica fruit (EEFCF) as the test drug is because of flavonoids and alkaloids, due to their major antioxidant potential, which are responsible for antiparkinsonian activities. Most of the studies on extract of Ficus carica are based on its richness in antioxidants and other important nutrients.16-19

In the present study, LD50 dose of EEFCF were obtained by performing the acute toxicity study. The safe dose was found to be 1000ml/kg for rats. From the LD50 dose two doses were selected for the present study 100mg/kg and 200mg/kg, respectively. In the present study, we evaluated the effect of EEFCF in haloperidol induced Parkinson disease in experimental animals.

Haloperidol induced catalepsy is a widely accepted animal model of PD. Haloperidol (nonselective D2 dopamine antagonists) gives a pharmacological model of parkinsonism by alteration of intracellular storage of catecholamines, resulting in reduction of dopamine in nerve endings.14 In the present study, anti-parkinson potential of Ficus carica fruit extract were determined by using three different pharmacological models i.e. Metal bar test, locomotor activity and motor co-ordination. Also, acute and chronic studies were carried out. Acute study was done for 24hrs and chronic study continued for 14 days.

In acute study, after 30 min of administration of haloperidol (1 mg/kg, i.p.), the duration of catalepsy was measured for 5 min at an interval of 30, 60, 90, and 120 min. Duration of catalepsy was estimated by placing an animal on the horizontal metal bar at a height of 6 cm. The rats are considered cataleptic if it shows reduced ability to initiate movement and a failure to correct abnormal posture. Pretreatment with EEFCF 100 and 200 mg/kg significantly reduced the duration of catalepsy induced by haloperidol when compared to haloperidol treated group. Duration of catalepsy was also significantly decreased by L dopa carbidopa.

In chronic study, after 14 days of treatment with the vehicle, standard, haloperidol control and Ficus carica high and low doses, haloperidol (1mg/kg, i.p.) caused catalepsy in rats which was evident by a significant increase in the time spent on the block when compared to vehicle treated animals. Ficus carica treatment significantly decreased the catalepsy in haloperidol treated rats in dose dependent manner. The EEFCF at doses of 100 and 200 mg/kg exhibited protective effect against haloperidol induced catalepsy which suggested F. carica has an ability to protect dopaminergic neurotransmission in striatum.

Administration of haloperidol to rats showed a significant decrease in locomotor activity and muscle co-ordination activity. Test drug is compared with normal, haloperidol control and standard drug. In rotarod test, the group which received only haloperidol, significantly, decreases fall off time as compared to the normal group. In standard treated group, a significant increase in fall off time as compared to the haloperidol treated group. In extract treated group, a significant increase in fall off time as compared to the haloperidol treated group.

In locomotor, the group which received only haloperidol, significantly, decreases locomotor activity compared to the normal group. In standard treated group an increase in locomotor activity was seen as compared to the haloperidol treated group. In extract treated group a significant increase in locomotor activity was seen as compared to the haloperidol treated group.

In PD abnormality, motor coordination and maintenance of normal limb posture has been reported.14 Damage to the dopaminergic neurons and progression of Parkinson’s disease like behavioral abnormalities in rats exposed to haloperidol were evident. Pretreatment of rats with EEFCF at the doses of 100 and 200mg/kg exhibited significant increase in locomotor activity, which indicates possible effect of EEFCF on CNS.

Oxidative stress is due to mitochondrial dysfunction (mitochondrial complex-1 impairment) which possess an important role in the pathogenesis of PD. The oxidative stress was evaluated by determination of malondialdehyde, catalase, superoxide dismutase levels, whereas reduced glutathione in the brain tissue.14

Another antioxidant catalase neutralizes the toxic effects of hydrogen peroxide. Catalase converts hydrogen peroxide to form water and nonreactive oxygen species, and prevents the accumulation of free radical biosynthesis precursors. Oxidative stress causes reduction in catalase level.14

Haloperidol administration in rats was responsible for induction of oxidative stress, indicated by a decrease in the catalase levels.

Another enzyme SOD acts as a catalyst in superoxide dismutation and formation of nonreactive oxygen species and hydrogen peroxide. It is present in nearly all cells which are exposed to oxygen and is a critical antioxidant defence. It helps in neutralizing the toxic effects of free radicals.20 Haloperidol treated control group exhibited a decrease in the SOD level in the brain of animals, which indicate production of oxidative stress.

Another potent enzymes GSH, plays as an important factor in etiology of PD. The depletion of reduced glutathione in Parkinson’s disease specifically in the substantia nigra is thought to be due to neuronal loss. There is evidence of correlation between the extent of neuronal loss and depletion of glutathione. A decreased level of reduced glutathione can be responsible for impairment of detoxifying capacity of neurons to hydrogen peroxide and increase in the risk of free radical formation and lipid peroxidation. Decrease in GSH levels was evident in haloperidol treated control animal.11

The various reactive oxygen species such as hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide, produced during normal cellular metabolic functions, produce oxidative damage in brain. The pro-oxidant/antioxidant balance is crucial in neurodegenerative processes including cell death, motor neuron disease and neuronal injury. The microsomal lipid peroxidation of polyunsaturated fatty acids (PUFA) produced MDA, lipid peroxides and conjugated dienes. The restrained stress enhanced lipid peroxidation, thus increasing malondialdehyde. The extract reduced the rat brain malondialdehyde levels as compared to control; hence the antiparkinsonian action of the extract is due to a decrease in lipid peroxidation in stress.14

Thus, the haloperidol treated animals exhibited a decrease in the levels of SOD and GSH in the brain as compared to the vehicle treated control animals. It indicated increase in the oxidative stress in the animal brains treated with haloperidol. Pretreatment with higher dose of EEFCF (200mg/kg) showed a decrease in MDA level and increase in the levels of SOD, catalase, and GSH, which indicated its antioxidant effect in the brain of haloperidol treated animals.

Histopathological findings revealed haloperidol treated group was having congestion of degenerative changes due to decrease the number of neural cells in SNpc in brain tissue. Pretreatment with EEFCF showed regenerative changes in SNpc, decreased infiltration of neutrophils, increased density of cells, reduced intracellular space, and regained normal architecture and moderate necrosis in striatum region of brain. It further substantiates the neuroprotective activity against haloperidol induced disease model.

The above pharmacological, biochemical and histopathological results suggest that Ficus carica has the ability to improve symptoms of parkinsonism, in part, by the restoring the level of dopamine, and by the regulation of the antioxidant system. Thus, antioxidant and neuroprotective activities may be responsible for anti-parkinson’s effect. Hence, Ficus carica may be useful as a neuroprotective agent in the treatment of PD. The above observed beneficial effects of Ficus carica may be attributed to diverse chemical components namely flavonoids, alkaloids, saponins, and tannins.

Conclusion

This present investigation can be concluded with the fact that the higher and lower dose of ethanolic extract of Ficus carica fruit (EEFCF) (100 and 200mg/kg, p.o) reflected significant beneficial effect in haloperidol induced parkinson and also conclusively showed that Ficus carica has anti-oxidant activity and neuroprotective role in haloperidol experimental model of PD. The anti‑oxidative properties of Ficus carica was also found to be effective in increasing locomotor activity, Rota rod performance and decreasing catatonic response. Hence, the neuromodulator effect of Ficus carica on behavioral, oxidative stress may be due to its neuroprotective and antioxidant properties. The anti‑oxidant activity of EEFCF could be possibly due to the scavenging of the superoxide radicals by the various flavonoids known to be present in the EEFCF. From the results, it can be concluded that EEFCF exhibited dose dependent protection in the treatment of drug‑induced EPS and related disorders. Future studies can be done to establish the fact clinically.

Acknowledgements

The authors are thankful to Srinivas College of Pharmacy, Mangalore, Karnataka, for providing necessary facilities for the present research work. The authors are also thankful to Dr Siddaraju M N, M.Sc. PhD Assistant Professor, Department of Botany, University College, Mangalore for authentication of the Fruit.

Declaration of Interest

None.

Supporting File
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