PHYTOCHEMICAL, IN VITRO AND IN SILICO EVALUATION ON CLITORIA TERNATEA FOR ALZHEIMER’S DISEASE

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OPENING THE RIGID PROTEIN
Grid> Macromolecule> Open: Launches a browser to open an existing PDBQT file.

Grid> Macromolecule> Choose: Chooses a molecule that has been previously read into ADT. Merged non-polar hydrogen atoms and charges, assign aromatic carbons, and prompt the user to write a PDBQT file.

PREPARATION GRID PARAMETER FILE
Select> select from string > Residue> name of active site found by Q site finder> add> dismiss: Select the residue to be flexible.

Display> label> by properties> select name and number> from one or more properties> Ok.

Grid> grid box: Interactive commands were launched for setting the grid dimensions and center. The numbers were entered on the thumb wheel, placed the cursor over the thumb wheel and type the new value. Right clicking on the thumb wheel gave more options. IMPORTANT: when finished, use the “close saving current” option in the “File” menu on the Grid Options Panel. Options in the “Center” menu on the browser provide different methods to choose the center of the grid box.

Grid> Output> save by grid.gpf: Save grid settings as gpf file.

PREPARING DOCKING PARAMETER FILE (.DPF)
Docking> Macromolecules> Set rigid filename (choose receptor.PDBQT): Specifying the rigid molecules.

Docking> Ligand> choose> (choose ligand.PDBQT) > accept: Specifying the ligand molecules.

Docking> Search parameter> Genetic Algorithm>( for first time short number of evaluations and  for other runs choose medium or long> accept: Setting the parameters  chosen for the docking method.

Docking>docking parameters> choose the defaults: Setting docking parameters.

Docking> Output> Lamarckian GA> dock.dpf:  The name was specified on the ligand dpf file to be formed, containing the docking instruction.

RUNNING OF DOCKING
Pwd Enter
Cd folder: Enter
Cd folder (sub folder) Enter
./autogrid4.exe –p grid.gpf –l grid.glg
./autogrid4.exe –p dock.dpf –l dock.dlg
Visualization of docking was done by chimera.

ISOLATION OF BIOACTIVE CONSTITUTENT

DETERMINATION AND OPTIMIZATION OF SOLVENT SYSTEM BY TLC [21, 22, 23, 24]

PREPARATION OF THE THIN LAYER CHROMATOGRAPHY (TLC)
The TLC plate was prepared by suspending silica gel G powder in distilled water. The slurry was poured carefully on to the glass plate to get uniformly coated plates. The plates were air dried and kept in an oven at 100-120°C about 30 minutes for activation.

DETERMINATION OF SOLVENT SYSTEM SOLVENT SYSTEM
Different solvent systems viz Toluene: Ethyl acetate, Toluene: Chloroform: Ethanol, Ethyl acetate: acetic acid and Toluene: Ethyl acetate: Acetic acid were tried out to select the suitable solvent system for the identification of maximum chemical constituents present in aqueous extract

THE COMPOUND ON TLC PLATE
The origin is marked by drawing a thin line across the bottom of the plate with a pencil. The aqueous extract was spotted with a glass capillary tube on to the plate.

RUNNING OF TLC
The spotted plate was placed into the saturated chamber; preventing the sample from dissolving from the plate into the eluent layer.  When the eluent reaches 3/4of the plate, the plate was removed from the chamber.  The point that the eluent had reached is called the eluent front and was immediately marked with a pencil. The plate was dried by allowing the eluent to evaporate form the plate. 

VISUALIZATION OF SPOTS ON TLC PLATE
A developed TLC plate was sprayed with Vanillin sulphuric acid and visualized spots were marked. Distance travelled by solute and solvent was measured to calculate the Retardation factor of solute using the following equation
Rf = Distance travelled by solute front / Distance travelled by solvent front

ISOLATION OF CHEMICAL CONSTITUTENT BY COLUMN CHROMATOGRAPHY
PACKING OF COLUMN

A cylindrical glass column was taken and plugged with a small piece of cotton.
Mounted the column on the stand.
Weighed 20 g of fresh silica gel (for column chromatography 60-120 mesh) into a 250 mL beaker.
Mixed with 100ml of Toluene: Ethyl acetate (8:2) into the beaker was stirred well using a glass rod to made slurry of the silica.
Poured the slurry into the column.
Placed a beaker below the mounted column to drain out the excess solvent.
Closed it when the level of the solvent reached just above the settled silica gel.

PREPARATION OF SAMPLE
5g of aqueous extract was dissolved with eluent and equal amount of silica gel was added to it. Mixed thoroughly by continuous stirring in one direction till solvent got evaporated.

METHOD[25, 26, 27]
For column chromatography sample was loaded on to the column packed with adsorbent silica gel G (100-200 mesh). The mobile phase was added from the top of the column and allowed to flow down by gravity. Elutants was collected from the bottom of the column and was stored as fractions of 10 ml. Each fraction was analyzed by TLC and those with same pattern were pooled. Fifth fraction which gave a single spot and Rf similar to the bioactive constituent, β-sitosterolwas evaporated and subjected to IR, NMR and MASS spectroscopy studies.

RESULT

EXTRACTION YIELDS
Extractive yields for each extract were calculated and Aqueous extract found have more yield than other two extracts.

Table: Extractive Yields of Various Extracts of Clitoria ternatea

S.l:No:

Extracts

Extractive Yield of dry Sample (g)

1

Hexane (HE)

3.20

2

Alcoholic (AE)

2.66

3

Aqueous (AQE)

3.48

Preliminary screening
The preliminary phytochemical screening of hexane extract, alcoholic extract and aqueous extract were done to identify the phytoconstituents and the results obtained were tabulated in the table

Table: Phytochemical Screening

Sl.No:

Chemical Tests

Observation

Inference



Detection of Alkaloids


HE

AE

AQE

Mayer's test

Yellow ppt

-

+

+

Wagner’s test

Reddish ppt

-

+

+

Dragendroff’s test

Red ppt

-

+

+

Hager’s test

Yellow ppt

-

+

+

Detection of carbohydrates


Violet ring at the junction of test tube


-


+


-

Molisch’s test

 


             Benedict’s test

Red ppt

-

+

-

Fehling’s test

Red ppt

-

+

-

Detection of Glycosides


Rose pink colour in

the ammonical layer

-

+

-

Modified Borntrager’s test

Legal’s test

Blood  red colour

-

+

+

Detection of Phytosterols

Golden yellow colour

+

-

+

Salkowski’s test

Libermann Burchard’s’ test


Formation of brown ring at the junction of test tube Yellow ppt

+

-

+

Detection of Phenols

Bluish black colour

-

+

+

Ferric chloride test

Detection of  Flavonoid’s





Alkaline reagent test

Intense yellow colour

-

-

+

Lead acetate

yellow colour

-

+

+

Detection of Tannins





Ferric chloride test

Greenish ppt

-

-

+

Potassium Hydroxide

Dirty white ppt

-

-

+

Detection of Saponins

Formation of 1cm layer foam

-

+

+

Froth test

Foam test

Foam produced persists for ten minutes

-

+

+

Detection of Proteins and amino acids

yellow colour

-

-

-

Xanthoproteic test

ANTI- CHOLINESTERASE ACTIVITY BY ELLMAN’S METHOD
The extracts were tested for their inhibitory effects against acetyl cholinesterase and compared with neostigmine as standard. The results obtained were tabulated in the table

Table: Percentage inhibitions obtained for anti- cholinesterase assay

S.l.No:

Concentration

% Scavenging

1. Standard (Neostigmine)

25

68.53

50

82.64

75

83.25

100

84.88

2. Hexane Extract

25

42.08

50

42.91

75

44.72

100

45.69

3. Alcoholic Extract

25

53.32

50

62.50

75

64.81

100

68.04

4. Aqueous Extract

25

75.69

50

77.63

75

78.55

100

79.86

A graph was plotted taking Concentration on X-axis and percentage scavenging on Y-axis and results were depicted in the figure.

Concentration Vs. Percentage Inhibition

From the graph, IC50 values were calculated for each extract and standard. The values were tabulated in the table and graphically represented in the figure.

Table: Comparisons of IC50 values of various Extracts with Neostigmine

S.l:No:

Extracts

IC50 (µg/ml)

1

Neostigmine (Standard)

10

2

Hexane

100

3

Alcoholic

22

4

Aqueous

12

Graphical representations of IC50 Values of extract with Neostigmine

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