Anti-Parkinson Activity of Petroleum Ether Extract of Ficus religiosa (L.) Leaves

In the present study, we evaluated anti-Parkinson's activity of petroleum ether extract of Ficus religiosa (PEFRE) leaves in haloperidol and 6 hydroxydopamine (6-OHDA) induced experimental animal models. In this study, effects of Ficus religiosa (100, 200, and 400 mg/kg, p.o.) were studied using in vivo behavioral parameters like catalepsy, muscle rigidity, and locomotor activity and its effects on neurochemical parameters (MDA, CAT, SOD, and GSH) in rats. The experiment was designed by giving haloperidol to induce catalepsy and 6-OHDA to induce Parkinson's disease-like symptoms. The increased cataleptic scores (induced by haloperidol) were significantly (p < 0.001) found to be reduced, with the PEFRE at a dose of 200 and 400 mg/kg (p.o.). 6-OHDA significantly induced motor dysfunction (muscle rigidity and hypolocomotion). 6-OHDA administration showed significant increase in lipid peroxidation level and depleted superoxide dismutase, catalase, and reduced glutathione level. Daily administration of PEFRE (400 mg/kg) significantly improved motor performance and also significantly attenuated oxidative damage. Thus, the study proved that Ficus religiosa treatment significantly attenuated the motor defects and also protected the brain from oxidative stress.


Introduction
Parkinson's disease (PD) is caused by the gradual and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) [1,2]. PD produces bradykinesia, muscular rigidity, rest tremor, and loss of postural balance along with some secondary manifestations like dementia, sialorrhoea [3], soft speech, and difficulty in swallowing due to uncoordinated movements of mouth and throat [4]. PD occurs due to inhibition of mitochondrial complex-1 [5,6], different mechanisms of cell damage like excitotoxicity, calcium homeostasis, inflammation, apoptosis, distressed energy metabolism, and protein aggregation [7], and interaction between genetic and environmental factors [8].
Oxidative stress interferes with dopamine metabolism leading to Parkinson's disease. This oxidative damage leads to formation of reactive oxygen species (ROS) leading to neuronal death [9,10]. This was evidenced by reduced level of endogenous antioxidant compounds. These findings introduced the requirement of using antioxidants as a therapeutic intervention in PD in addition to other protective agents.
The current available drug treatments for PD possess various side effects. Therefore, herbal therapies should be considered as alternative/complementary medicines for therapeutic approach.
Since ancient times, plants have been an ideal source of medicine. Plants have played a noteworthy role in maintaining human health and improving the quality of life for thousands of years and have served humans as well, as valuable components of medicines, seasonings, beverages, cosmetics, and dyes. In modern times, focus on plant research has increased all over the world and a large body of evidence has been collected to demonstrate immense potential of medicinal plants used in various traditional systems. Ficus religiosa Linn. (Moraceae) commonly known as "Pimpala" or "Pipal" tree is a large widely deciduous tree, heart-shaped without aerial roots from the branches, with spreading branches and grey bark [11][12][13]. The tree is held sacred by Hindus and Buddhists. In India it is known by several vernacular names, the most commonly used ones being Asvatthah (Sanskrit), Sacred fig (Bengali), Peepal (Hindi),

Collection of Plant Material.
Fresh leaves of Ficus religiosa were collected from local area of Ahmedabad district, Gujarat, India, during July-September. This plant was identified and authenticated by Dr. A. Benniamin, Scientist D, Botanical Survey of India, Pune. Voucher specimens number BSI/WC/Tech./2015/JOB-1 have been kept in Botanical Survey of India, Pune, Maharashtra, India.

Animals.
Adult male Wistar rats, weighing 180-220 g, and albino mice of either sex weighing 25-30 g were used and acclimatized to laboratory condition for one week. All animals were housed in well-ventilated polypropylene cages at 12 : 12 h light/dark schedule at 25 ± 2 ∘ C and 55-65% RH. The rats were fed with commercial pelleted rats chow and water ad libitum as a standard diet. Institutional Animal Ethics Committee approved the experimental protocol in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Preparation of Leaf
Extract. The leaves were collected and dried in shade and ground. Coarsely powdered plant material (1000 g) was weighed and extracted with 5 lit of solvents like petroleum ether (60-80 ∘ C), ethyl acetate, and ethanol by successive extraction in a Soxhlet apparatus for 72 h. After each extraction, the solvent was distilled off and concentrated extract was transferred to previously weighed petri dish and evaporated to dryness at room temperature (45-50 ∘ C) to obtain dried extracts. The dried extract was weighed and the percentage yield of the extracts was calculated as follows: Weight of dried extract Weight of dried leaves powder × 100. 3) of -naphthol solution in alcohol were added to 2-3 mL of solution of extract and shaken for few minutes and then 0.5 mL of conc. sulfuric acid was added from the side of test tube. The formation of violet ring at the junction of two solutions indicated presence of carbohydrates.

Test for Glycosides
(1) Legal's Test. To the extract, 1 mL of pyridine and 1 mL of sodium nitroprusside were added. Change in color to pink or red indicated presence of cardiac glycosides.
(2) Keller-Kiliani Test. Glacial acetic acid (3-5 drops), one drop of 5% ferric chloride, and concentrated sulfuric acid was added to the test tube containing 2 mL of solution of extract. Appearance of reddish-brown color at the junction of two layers and bluish green in the upper layer indicated presence of cardiac glycosides.
(3) Borntrager's Test. Dilute sulfuric acid was added to 2 mL of solution of extract, boiled for few minutes, and filtered. To the filtrate, 2 mL of benzene or chloroform was added and shaken well. The organic layer was separated and ammonia was added. The change in color of ammoniacal layer to pink red indicated presence of anthraquinone glycosides. Haloperidol, an antipsychotic drug, blocks central dopamine receptor in striatum. It also produces a behavioral state in animals like mice and rats in which they fail to correct externally imposed postures (called catalepsy); thus, keeping the above fact in mind, the haloperidol induced catalepsy model was selected. The method described by Elliott and Close in 1990 [20] was followed for the anticataleptic activity. The animals were divided into five groups ( = 6). Group I served as vehicle control, Group II served as standard, Levodopa (6 mg/kg, p.o.), and Groups III-V served as test group treated with PEFRE (100, 200, and 400 mg/kg, p.o.), respectively. Standard bar test was used to measure the catalepsy. Catalepsy was induced by haloperidol (1 mg/kg, i.p.) and examined at every 30 min interval for 210 min. The duration for which the rat retains the forepaws extended and resting on the elevated bar was considered as cataleptic score. A cut-off time of 5 min was applied.

Induction of Parkinsonism by 6-OHDA.
The rats were anesthetized with an intraperitoneal injection of 50 mg/kg of sodium pentobarbital and were fixed in a stereotaxic apparatus [21,22]. A stainless steel needle (0.28 mm o.d) was inserted unilaterally into the substantia nigra with the following coordinates: anterior/posterior: −4.8 mm; medial/lateral: −2.2 mm; ventral/dorsal: −7.2 mm-3.5 mm from bregma, and injection of 6-OHDA (20 g of 6-OHDA hydrobromide in 4 L 0.9% saline with 0.02 g/mL ascorbic acid) was then made over 5 min and the needle was left in place for a further 5 min. Then the skull was secured with stainless metallic screws and the wound area was covered by dental cement. Each rat was housed individually following the surgical procedure. Sham operated animals were also treated in the same manner, but they received equivalent volumes of normal saline instead of 6-OHDA.

Experimental
Design. Animals were divided into six groups of 6 rats in each group. Group I served as sham operated animals and received normal saline (10 mL/kg, p.o.); Groups II to VI were induced with parkinsonism by 6-OHDA as follows: Group II served as a 6-OHDA control and received normal saline (10 mL/kg), Group III served as a L DOPA ( where Abs 532 is absorbance, is total volume of mixture (4 mL), 1.56 × 10 5 is molar extinction coefficient, is weight of dissected brain (1 g), and is aliquot volume (1 mL).

Superoxide Dismutase (SOD)
Level. SOD activity was determined according to the method described by Beyer and Fridovich in 1987 [26]. 0.1 mL of supernatant was mixed with 0.1 mL EDTA (1 × 10 −4 M), 0.5 mL of carbonate buffer (pH 9.7), and 1 mL of epinephrine (1 mM). The optical density of formed adrenochrome was read at 480 nm for 3 min on spectrophotometer. The enzyme activity was expressed in terms of U/min/mg. One unit of enzyme activity is defined as the concentration required for the inhibition of the chromogen production by 50% in one minute under the defined assay conditions. CAT activity where O.D. is change in absorbance/minute; is extinction coefficient of hydrogen peroxide (0.071 mmol cm −1 ).

GSH Level (Reduced Glutathione).
For the estimation of reduced glutathione, the 1 mL of tissue homogenate was precipitated with 1 mL of 10% TCA. To an aliquot of the supernatant, 4 mL of phosphate solution and 0.5 mL of 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) reagent were added and absorbance was taken at 412 nm [28]. The values were expressed as nM of reduced glutathione per mg of protein: where is Abs 412 of tissue homogenate, is dilution factor (1), is brain tissue homogenate (1 mL), and is aliquot volume (1 mL).

Histopathological
Studies. The brains from control and experimental groups were fixed with 10% formalin and embedded in paraffin wax and cut into longitudinal section of 5 m thickness. The sections were stained with hemotoxylin and eosin dye for histopathological observation.

Statistical Analysis.
All the values were expressed as mean ± SEM. Statistical evaluation of the data was done by one-way ANOVA (between control and drug treatments) followed by Dunnett's -test for multiple comparisons and twoway ANOVA followed by Bonferroni's multiple comparison test, with the level of significance chosen at < 0.001 using Graph-Pad Prism 5 (San Diego, CA) software. Table 1 showed the phytochemical screening of the different extract of F. religiosa.

The Effects of PEFRE on Haloperidol Induced Catalepsy.
Chronic oral administration of higher doses of PEFRE (200 and 400 mg/kg) showed significant ( < 0.001) reduction in cataleptic score from 60 min to 210 min as compared to vehicle treated animals. Administration of PEFRE (100 mg/kg) did not show significant activity. Treatment with Levodopa (6 mg/kg) significantly ( < 0.001) reduced duration of catalepsy as compared to vehicle treated group (Figure 1).

The Effects of PEFRE on 6-OHDA Induced Parkinson's
Disease in the Locomotor Activity. Total locomotor activity of rats in 6-OHDA treated group was significantly ( < 0.001) reduced as compared to vehicle treated group. Oral administration of PEFRE of different doses (200 and 400 mg/kg) showed significant ( < 0.001) increase in the locomotor activity from day 20 to 55 as compared to 6-OHDA treated control animals. Administration of PEFRE (100 mg/kg) did not show significant activity. Levodopa (6 mg/kg) significantly ( < 0.001) increased locomotor activity (Figure 2).

The Effects of PEFRE on 6-OHDA Induced Parkinson's Disease in MDA, CAT, SOD, and GSH Level.
Administration of 6-OHDA resulted in significant changes in biochemical parameters when compared to the vehicle control animals. The inoculation of 6-OHDA induced oxidative stress, as indicated by increased MDA level, and decreased CAT, SOD, and GSH levels when compared to vehicle control animals in brain levels. The treatment with pet. ether extract of FRE (400 mg/kg, p.o.) showed significant ( < 0.001) decrease in MDA level compared to OHDA rats. Similarly, daily administration of PEFRE (400 mg/kg) attenuated the increase in SOD and CAT activity with 6-OHDA treated group. Pretreatment with PEFRE (400 mg/kg) significantly ( < 0.001) increased GSH levels in the brain as compared to 6-OHDA treated animals, thus preventing the reduction in GSH induced by 6-OHDA (Table 2).

Effect of PEFRE on Histopathological Changes in the Brain of Normal and 6-OHDA Treated Animals.
The histopathological study showed that neurotoxins, that is, 6-OHDA, caused marked hypertrophic changes, increased intracellular space, infiltration of neutrophils, decreased density of cells, alterations of architecture, hemorrhage, and neuronal damage and even cell death. Furthermore, many neurons were shrunken, pyknotic, and darkly stained with small nuclei (Figure 4(b)) compared with normal vehicle treated rats (Figure 4(a)). There is significant reversal of neuronal damage or neuronal alterations observed in Levodopa (6 mg/kg) treated rats (Figure 4(c)) and PEFRE treated rats at doses of 200 (Figure 4(e)) and 400 mg/kg (Figure 4(f)). Treatment with PEFRE (100 mg/kg) did not show significant recovery of neuronal damage (Figure 4(d)).

Discussion
Parkinson's disease is a chronic neurodegenerative disorder characterized by loss of dopamine neurons of the SNpc. The pathogenesis of PD includes oxidative stress, protein accumulation like a-synuclein, mitochondrial dysfunction, apoptosis, and neuronal excitotoxicity. Among all, oxidative stress is a crucial pathological mechanism for PD. SNpc is more vulnerable to reactive oxygen species as it contains more amount of dopamine.
In the present study, we evaluated the effect of pet. ether extract of F. religiosa in neurotoxins (haloperidol and 6-OHDA) induced Parkinson disease in experimental animals.
Haloperidol induced catalepsy is a widely accepted animal model of PD. Haloperidol (nonselective D 2 dopamine antagonists) provides a pharmacological model of parkinsonism by interfering with the storage of catecholamine's intracellularly, resulting in dopamine depletion in nerve endings. In the present study, haloperidol (1 mg/kg, i.p.) induced significant catalepsy in rats as evidenced by a significant increase in the time spent on the block as compared to vehicle treated animals. Treatment with F. religiosa significantly reduced the catalepsy in haloperidol treated rats in dose dependent manner. The PEFRE at doses of 200 and 400 mg/kg showed protective effect against haloperidol induced catalepsy indicated that this plant has an ability to protect dopaminergic neurotransmission in striatum.
There are well-known pharmacological PD models in mammalian systems including the classical and highly selective neurotoxin 6-hydroxydopamine (6-OHDA), as well as 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its Advances in Pharmacological Sciences metabolite, MPP+ (1-methyl-4-phenylpyridinium ion). These toxins cause decreased ATP production, increased ROS production, and increased apoptosis of DAergic cells [29]. The efficacy of F. religiosa in 6-OHDA induced PD has not been well established. In the present study, 6-OHDA administration to rats caused a significant decrease in locomotor activity and muscle activity. Lack of motor coordination and maintenance of normal limb posture has been reported in PD condition. The evaluated data suggested damage to the dopaminergic neurons and progression of Parkinson's disease like behavioral abnormalities in rats exposed to 6-OHDA. Pretreatment of rats with PEFRE at the doses of 200 and 400 mg/kg exhibited significant increase in locomotor activity and increase in muscle activity and thus could be proved with possible action on CNS.
Oxidative stress generated as a result of mitochondrial dysfunction particularly mitochondrial complex-1 impairment plays an important role in the pathogenesis of PD. The oxidative stress was measured through determination of levels of malondialdehyde, catalase, superoxide dismutase, and reduced glutathione in the brain tissue.
6-OHDA generates an increase in the production of hydrogen peroxide and free radicals [30,31]. These reactive oxygen species are generated through the nonenzymatic breakdown of 6-OHDA or direct inhibition of complex-I and complex-IV of the mitochondrial electron transport chain [31][32][33]. The resulting ROS production from 6-OHDA breakdown leads to lipid peroxidation, protein denaturation, and increases in glutathione, which are found in PD patients [34]. 8

Advances in Pharmacological Sciences
Lipid peroxidation, a sensitive marker of oxidative stress, was estimated by measuring the levels of thiobarbituric acid. Lipid peroxidation occurs due to attack by radicals on double bond of unsaturated fatty acid and arachidonic acid which generate lipid peroxyl radicals and that initiate a chain reaction of further attacks on other unsaturated fatty acid. As we know, lipid peroxidation is the process of oxidative degradation of polyunsaturated fatty acids and its occurrence in biological membranes causes impaired membrane function, impaired structural integrity [35], decreased fluidity, and inactivation of number of membrane bound enzymes. Increased levels of the lipid peroxidation product have been found in the substantia nigra of PD patient. In the present investigation, similar results were observed in the brain homogenate of 6-OHDA treated control animals.
Catalase is an antioxidant which helps in neutralizing the toxic effects of hydrogen peroxide. Hydrogen peroxide is converted by the catalase enzyme to form water and nonreactive oxygen species, thus preventing the accumulation of precursor to free radical biosynthesis. Oxidative stress results in decrease in catalase level. 6-OHDA inoculation in rats induced oxidative stress, as indicated by a decrease in the catalase levels.
Superoxide dismutase (known as SOD) is an enzyme which acts as a catalyst in the process of dismutation of superoxide into nonreactive oxygen species and hydrogen peroxide. It is therefore a critical antioxidant defense which is present in nearly all cells which are exposed to oxygen [36,37]. Superoxide dismutase helps in neutralizing the toxic effects of free radicals [38,39]. 6-OHDA treated control group showed a decrease in the level of SOD in the brain of animals, thus indicative of production of oxidative stress.
GSH, potent enzymes, are an important factor in etiology of PD [40]. The depletion of reduced glutathione in the substantia nigra in Parkinson's disease could be the result of neuronal loss. As a matter of fact, the positive correlation has been found to exist between the extent of neuronal loss and depletion of glutathione [41]. A decrease in the availability of reduced glutathione would impair the capacity of neurons to detoxify hydrogen peroxide and increase the risk of free radical formation and lipid peroxidation. A reduction in GSH levels was evident in 6-OHDA treated control animals.
Thus, the 6-OHDA per se group showed a significant increase in the levels of thiobarbituric acid (which is an indication of extent of lipid peroxidation) and decrease in the levels of SOD and GSH in the brain as compared to the vehicle treated control animals. All these indicate an increase in the oxidative stress in the brain of animals treated with 6-OHDA. Pretreatment with higher dose of petroleum ether extract of F. religiosa (400 mg/kg) resulted in a decrease in MDA level and increase in the levels of SOD, catalase, and GSH, indicating its antioxidant effect in the brain of 6-OHDA treated animals.
Histopathological findings showed that pet. ether extract of Ficus religiosa treated animals had decreased infiltration of neutrophils, reduced intracellular space, increased density of cells, and regained normal architecture and moderate necrosis in striatum region of brain.

Conclusion
In view of the above facts, we are concluding that petroleum ether extract of Ficus religiosa plant showed to be an antioxidant and showed a promising effect in animals with Parkinson's disease. And we appreciate further detailed molecular studies with this drug in anti-Parkinson's pharmacology and toxicology and also characterization of active constituents responsible for neuroprotective effect.