Parkinson's disease (PD) is a chronic neurodegenerative disorder in which dopaminergic neurons progressively degenerate, which causes depletion of dopamine (DA) levels in the striatum. The physiology of cholinergic neurons and dopaminergic neurons in striatum are out of balance leading to PD. The disease is characterized by features such as postural imbalance, tremor, dystonia, myotonia, bradykinesia and dyskinesia, etc.[1]. It is considered to be the second most severe neurodegenerative disorder after Alzheimer’s disease and is reported in about 2-3?ter 65 years of age. The pathological study depicts that the brain of PD patients show the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain, the aggregation of protein called Lewy bodies, and accumulation of cytoplasmic inclusions containing insoluble ?-synuclein[2]. In the recent past it has been studied that oxidative stress, impaired mitochondrial function, inflammation, apoptosis, dysfunction of proteolysis, and loss of neurotrophic factors also contribute to the pathogenesis of PD [3]. Herbal medicine, also referred to as Herbalism, Botanical medicine or Herbology deals with the use of plants, in various forms for their therapeutic value. Herbs produce and possess a wide variety of chemical compounds that act upon the body and are used to prevent or treat disease or promote health and wellbeing. Herbal drugs are widely used throughout the world since last few decades which is evident by rapidly growing global and national markets of herbal drugs. India is the second largest exporter of medicinal plants. With advanced and detailed study on Pharmacological and Phytochemical investigations provide extensive knowledge and information for the medicinal use of herbal plants by demonstrating the presence of active principles in them and their therapeutic effect on living beings. Hence, it has become of utmost importance for screening and collecting plants for exploration of new and safe medicinal agents [4]. With the implication of side effects from synthetic drugs more stress and importance is being laid on medicines from natural origin. Plant extracts and the phytoconstituents present in them are good source of antioxidants. The antioxidant property of the plant extracts is due to the presence of secondary active constituents such as flavonoids, tannins and alkaloids. Flavonoids mainly serve as potent antioxidant and play a crucial role in neuroprotective effect [5-8]. The present study utilized the leaves from 3 different plants namely, Allium fistulosum, Jacquemontia caerulea and Tabernaemontana divaricata which were then shade dried and soaked in ethanol by Soxhlet extraction. The Ethanolic Extract of Allium fistulosum, Jacquemontia caerulea and Tabernaemontana divaricata was then obtained after distillation. The extract thus obtained was subjected to Phytochemical Screening and Acute Toxicity Study. All these plants exhibit potent antioxidant property and hence were used to prove Anti-Parkinsonian effect which was assessed by using different models such as Hole Board Test, Locomotor activity, Catalepsy or Catatonia and Grip Strength. 

MATERIALS & METHODS

COLLECTION, IDENTIFICATION, AND EXTRACTION OF PLANT MATERIAL:

 The leaves of Allium fistulosum, Jacquemontia caerulea and Tabernaemontana divaricata were obtained from an authorized plant supplier and underwent authentication by the Department of Botany at Sri Venkateshwara University in Tirupati-517 502, Andhra Pradesh, India. The leaves were air-dried in the shade for a duration of 15 days and were coarsely powdered using a blender. The plant material was taken up for the extraction using ethanol and water in 70:30 ratio. The extraction was carried out by soxhlet extraction followed by distillation and subsequently stored in the refrigerator.[9]

PREPARATION OF EXTRACT:

300g of leaves of Allium fistulosum, Jacquemontia caerulea and Tabernaemontana divaricata were collected, washed thoroughly and dried in shade. Leaves were made into coarse powder. The powder of the leaves was extracted in a soxhlet extractor with successive ethanol solvent. The extract was then subjected to distillation and heated on water bath for semisolid consistency and then placed in the refrigerator.[10]

Fig 1. Powdered Leaves Of Allium Fistulosum, Jacquemontia Caerulea And Tabernaemontana Divaricata

Fig.2.  Soxhlet Extraction

PRELIMINARY PHYTOCHEMICAL SCREENING

The preliminary Phytochemical Screening of Ethanolic Extract of Allium fistulosum, Jacquemontia caerulea and Tabernaemontana divaricate (EEAJT) was assessed for the presence of steroids, flavonoids, saponins, glycosides, tannins, carbohydrates, oils, phenols and fats. The phytochemical screening tests for active principles was carried out by using standard procedures [11-13].

TEST FOR CARBOHYDRATES AND GLYCOSIDES 

 A small quantity of extract was dissolved in 4ml of distilled water and filtered. The filtrate was subjected to the following tests to detect the presence of carbohydrates and glycosides.  

MOLISCH'S TEST:

The filtrate was treated with 2-3 drops of 1% alcoholic alpha-naphthol and 2ml of concentrated Sulphuric acid was added along the sides of the test tubes. Appearance of brown ring at the junction of two liquids shows the presence of carbohydrates.  

FEHLING'S TEST:

The filtrate was treated with 1ml of Fehling's A and B and heated on a water bath. A reddish precipitate was obtained shows the presence of carbohydrates. Another portion of extract was hydrolyzed with dilute hydrochloric acid for few hours on a water bath and the hydrolysate was subjected to the following tests to detect the presence of glycosides. 

LEGAL'S TEST:

To the hydrolysate 1ml of pyridine and few drops of sodium nitroprusside solution were added and then it was made alkaline with sodium hydroxide solution. Appearance of pink to red colour shows the presence of glycosides. 

BORNTRAGER'S TEST:

Hydrolysate was treated with chloroform and the chloroform layer was separated. To this equal volume of dilute ammonia solution was added. Ammonia layer acquires pink colour shows the presence of glucosides. 

DETECTION OF FIXED OILS AND FATS 

FILTER PAPER TEST:

Small quantity of extract was pressed between the papers. Appearance of oil stain on the paper indicates the presence of fixed oils. 

SAPONIFICATION TEST:

Few drops of 0.5 N alcoholic potassium hydroxide were added to small quantity of extract along with a drop phenolphthalein. The mixture was heated on water bath for 1-2 hours. Formation of soap indicates the presence of fixed oils and fats. 

DETECTION OF PROTEINS AND FREE

AMINOACIDS:

A small quantity of extract was dissolved in few ml of water and they were subjected to the following tests. 

MILLON'S TEST:

The above-prepared extract was treated with Millon's reagent. Red colour was formed shows the presence of proteins and frees amino acids. 

BIURET TEST:

To the above prepared extract equal volume of 5 % sodium hydroxide and 1% copper sulphate solution were added. Violet was produced which shows the presence of proteins and amino acid. 

NINHYDRIN TEST:

The extract was treated with ninhydrin reagent. Purple colour was produced on heating which shows the presence of proteins and free amino acids.  

DETECTION OF TANNINS AND PHENOLIC COMPOUNDS:

 Small quantity of each extract was taken separately in water and test for the presence of phenolic compounds and tannins was carried out with the following reagents. 5 ?rric chloride and 1 % Solution of gelatin containing-Violet Colour 10 %Sodium Chloride-White Precipitate 10 % Lead acetate solution -White Precipitate Above findings shows the presence of phenolic compounds and tannins. 

 DETECTION OF PHYTOSTEROLS:

 Small quantity of each extract was dissolved in 5 ml of chloroform separately. Then this chloroform solution was subjected to the following tests to detect the presence of phytosterols.  

SALKOWSKI TEST:

To 1 ml of above prepared chloroform solution, a few drops concentrated sulphuric acid was added. Brown colour was produced shows the presence of phytosterols. 

LIBERMANN BURCHARD TEST:

The above prepared chloroform solution was treated with a few drops of concentrated sulphuric acid followed by the few drops of diluted acetic acid,3 ml of acetic anhydride. A bluish green colour was appeared indicates the presence of phytosterols.  

DETECTION OF GUMS AND MUCILAGES: A small quantity of each extract was added separately to 25ml of absolute alcohol with constant stirring and filtered. The precipitate was dried in air and examined for its swelling properties. No swelling was observed indicates the absence gums and mucilages.

DETECTION OF FLAVONOIDS: 

1. Small quantity of each extract was dissolved separately in aqueous sodium hydroxide. Appearance of yellow colour indicates the presence of flavonoids. 
2. To the small portion of each extract, concentrated sulphuric acid was added. Yellow orange colour was obtained shows the presence of flavonoid.                                                          

SHINODA TEST: 

Small quantity of each extract was dissolved in alcohol. To those pieces of magnesium followed by concentrated hydrochloric acid was added drop wise and heated.  Appearance of magenta colour shows the presence of flavonoid. 

EXPERIMENTAL ANIMALS USED 

Albino mice of both genders weighing around 25-30 grams were obtained from the animal facility at Shadan College of Pharmacy in Peerancheruvu, Hyderabad. The mice were acclimatized to standard environmental conditions, including ambient temperature, relative humidity, and a 12/12-hour light-dark cycle, for one week before the commencement of the experiment. Throughout this period and the experiment, the animals had unrestricted access to standard pellet diet and water. All procedures involving animals adhered to the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Additionally, the experimental protocol received approval from the Institutional Animal Ethical Committee.

ACUTE TOXICITY STUDY (OECD GUIDELINE 425)

Acute oral toxicity study was performed on mice in accordance with the OECD guidelines 425. The animals were kept on fasting for 24 hours. Firstly, the limit test was done at a dose of 2500 mg/kg bw., p.o and were continuously noticed for 3-4 hours for any changes in their general behavior, morbidity and finally death during a period of 24 hours. When there were no marks of harmfulness at a dose of 2500mg/kg three dosage levels were selected in such a manner that, the middle dose (250 mg/kg) was approximately one tenth of the determined dose throughout acute toxicity studies and a low dose (125 mg/kg) which was half of the one tenth of the determined dose and a high dose (500 mg/kg) which was twice of the one tenth dose of the maximum dose. For common behavior, neural, autonomic profiles and to find out proportion of death remarks were charted according to Irwin's table [14].

EVALUATION OF ANTI-PARKINSON’S ACTIVITY OF EEAJT IN INVIVO MODELS

CATALEPSY 

       
           
       
    Fig.3. Catalepsy Model

  Principle: 

Catalepsy, well-defined as a reduction in the capability to start motion and letdown to precise unusual position, which was examined by means of the block examination. For the examination mice were placed so that their hind legs were on the workbench, and their forelimbs relaxed on a 1 cm width flat block, 6-9 cm beyond the workbench. The length of time that animal maintained this position was recorded by stopwatch to a maximum of 180s (mean of three consecutive trials; interval: 1 min). Animals would determine judge to be cataleptic if they maintain this position for 30s or more. 

Instrument used:

Horizontal Bar

Test drug used:

Haloperidol 

Standard drug used:

Levodopa Carbidopa

Procedure: 

The mice were distributed into 6 groups having 6 mice in every group. 

Group 1-

was treated as normal with 10ml/kg normal saline. 

Group 2-

was treated with Haloperidol 1 mg/kg i.p 

Group 3-

was treated with Levodopa + Carbidopa (30mg/kg, p.o.) along with Haloperidol. was given 30 mins before the haloperidol administration for 08 days of investigational time. 

Group 4-

was treated with Herbal formulation (100mg/kg, orally) along with Haloperidol 

Group 5- was treated with Herbal formulation (200 mg/kg, orally) along with Haloperidol. 

HOLE BOARD TEST 

       
           
       
    Fig.4. Hole Board Model

 Principle: 

Head dipping is an exploratory behavior of the animals in the hole board test which is deliberate rated to be an indicator of anxiety. Animals were located in a box (50 x 50 cm, walls 30 cm high) with 16 equally spaced holes (2.5 cm diameter, 10 cm separately after every holler) in the floor and the box was raised to a height of 25cms from the ground. A mouse was positioned in the middle of the hole-board and is permitted to easily travel in the tool for 5 min. The total no. of lines crossed and the sum of head dipping were recorded. A skull dip was counted if both the eyes gone into the hole. 

LOCOMOTOR ACTIVITY 

Principle: This trial processes the consideration and the controlled movement inside an enclosed Motor action was attained by using an actophotometer. The mice were inside an enclosed X 30cm dark metallic compartment with a shade bottom and a light-tight cover one into of light were focused 2cm above the floor. Every ray disruption was recorded as an episode. The light beam breaks were counted for 5mins. 

       
           
       
    Fig.5. Actophotometer Model

ROTAROD TEST 

Principle: The rotarod machine comprises of a motor rod with a drum of 7.0 cm diameter. It was adjusted to a speed of 25rpm during the test session. Swiss mice (20-30g body weight) undergo a pre-test on the apparatus. Thirty minutes afterwards intraperitoneal management of test/standard drug, the mice were positioned on the rotarod for 3 min.   The latency to fall in trial period of 180s was taken as a degree of motor coordination. 

       
           
       
    Fig.6. Rotarod Test Model

  STATISTICAL ANALYSIS  

The results for electrically haloperidol induced Parkinson’s disease were shown as Mean ±

Standard Error of Mean. Paired Student's t-test was used to analyze the level of significance. A P value of <0>

PRELIMINARY PHYTOCHEMICAL SCREEING RESULTS : 

Table 1.:  Note: + indicates presence and- indicates absence of phytoconstituents

EEAJT was proved to be harmless at the maximum dose of 2500mg/kg body weight by oral route. After 24 hours, animals were found well tolerated. No signs of harmfulness and no death was observed. General behaviour. neurological and autonomic profiles were normal. The effects have been tabulated in. 

Values are stated as mean ± SEM (number of animals, n=6), one-way ANOVA after Dunnette's multiple comparisons test. *indicates P<0>

       
           
       
    Graph 3 Anti-Parkinsonian Effect Of Eeajt On Locomotor Activity By Using Actophotometer In Haloperidol Treated Mice

GRAPH 4 ANTI-PARKINSONIAN EFFECT OF EEAJT ON ROTAROD APPARATUS MODEL IN HALOPERIDOL TREATED MICE

DISCUSSION