CHAPTER THREE
METHODS AND MATERIALS
3.1 STUDY AREA
faculty of pharmaceutical sciences, Nnamdi Azikiwe University,Agulu.
3.2 Software application and web pages
personalcomputer,pubchem,pubchem,Linuxoperating
system(Ubuntudesktop18.04),proteindatabank,Microsoftoffice ,Pymol -1 eval-bin-
win32 ,Autodock tools 1.5.6 ,Autodockvina 1.12 on Ubuntu operating system ,Discovery
studio ,snipping tools ,Swiss target prediction .
3.21 APPARATUS AND EQUIPMENTS
Test-tubes(Pyrex)
Beakers( Pyrex)
Analytical weighing balance
Measuring cylinders
Pasteur pipette
Micropipette
Water bath
Scissors
Electric blender
Well ventilated animal cage
Syringes and needles
Capillary tubes
Blood sample tubes
Refrigerator
Whattmann filter paper
Funnel
3.2.2 CHEMICAL REAGENTS
Hager’s reagent
Dragendroff’s reagent
10% sodium hydroxide
10% lead acetate
Conc. Sulfuric acid
Ferric chloride
Hydrogen Chloride
Glacial acetic acid
Absolute ethanol(100%JHD,England)
Distilled water of analytical standard
3.3 Methods
3.3.1 selection of plants
The plant selected were shown to have anti prostrate cancer effect and was obtained from
Litreature.
3.3.2 identification of phytochemicals
Literature was reviewed to identify the phytocompounds found in medicinal
plants and the phytochemicals obtained from the selected plants were isolated and used for
the study .
3.3.3 literature Mining
Litreature was mined to identify a common receptor for the onset of prostrate cancer. This was
done to know the importance of the receptor and target protein in the Pathophysiology of
prostrate cancer .
3.3.4. Selection and preparation of the receptors
After the identification of the target/receptor, literature mining and analysis of the target/receptor; their 3D
structures were obtained from Protein Data Bank (http:/[Link]) with their respective protein data bank
code; 4NKW & 2YHD. The initial preparation of the pdb files to select the needed chains, delete multiple ligands
and non-protein parts were done using PyMOL. The PyMOL tool was employed to gain insight into the
ligand binding to the receptor. The receptors wereprepared for molecular docking simulations
using AutoDockTools. In the preparation, polar hydrogens were added to the receptor, and it was saved
as pdbqt file format. The electrostatic Grid boxes and 3-dimensional affinity of different sizes and centers, as
indicated in the table 1 below were created around the active sites of the protein .
Table 3.1: table of grid box parameters used in the molecular docking simulations
protein code : 4nkw Centers sizes
X 16.176 15
Y 33.881 31
Z 32.058 18
Protein code :2yhd centers sizes
X 16.291 17
Y 34.134 35
Protein code :2yhd centers sizes
Z 31.706 20
[Link] preparation of ligands
The phytochemicals with their reference ligands were prepared for molecular docking simulation.
The reference ligands were identified and gotten from literature. The reference compounds are
shown in table 2 below . The ligands were prepared for molecular docking simulation using the
Autodocktools and all rotatable bonds , torsions and charges were assigned and saved as pdbqt
files. Using
Ubuntu (molecular docking site )and a bash script, the ligands
were prepared for molecular docking simulation; the bash script used is;
Table 3.2 table of reference and existing standards used in the molecular docking simulations.
REFRENCE LIGANDS
Abiraterone acetate
Bicalitamide
Enzalutamide
Fluorouracil
3.3.5. validation of Docking protocol
In order to validate the molecular docking simulations protocol for the 4nkw and 2yhdproteins, the PDB structure
of these proteins in complex with their reference inhibitors were reproduced in-silico. Polar
hydrogen, Kollmancharges, grid box sizes and centers at grid space were determined with MGLTools-1.5.6 (Trott
& Olson, 2009). They were prepared for molecular docking simulations using [Link] hydrogen
bonds
[Link] Molecular docking simulation
The phytochemicals were batched for molecular docking simulations against 4nkw and 2yhd
using virtual screening scripts. The script used for molecular docking was;
Molecular docking simulations were carried out in four replicates on a Linux platform using
Autodockvina and associated tools after validation of the docking protocols (Trott Oleg & Arthur
[Link],2010) . Binding free energy values ( kcal/Mol -SD) were ranked to enable identification of
the frontrunner phytochemicals.
3.3.6 Post-Docking Analysis
The mean binding affinities and SD(standard deviation)after docking were calculated and
recorded on Microsoft excel spread sheet . The binding affinities of the 68 phytochemicals were
compared with the reference compounds binding affinities. The phytochemicals with lower
binding affinities than that of the refrence compounds for each protein were screened out.
3.3.7 SELECTION OF SOLVENT
The selection of the solvent affects the quality of the isolated phytochemical. The solvent to be used was
determined based on the solubility profile of the frontrunner phytochemicals.
3.3.8 ASSESSMENT OF PROTEIN - LIGAND INTERACTION
The frontrunner phytochemicals was observed for protein interaction using
discovery studio visualizer in order to identify the amino acid interactions
and observe the amino acids responsible for the activity of interest in
comparison with the reference compounds.
3.3.9 Swiss target prediction
Swiss Target Prediction was utilized to identify and determine the protein class targeted and bound by the
frontrunner phytochemicals, and also the quantification of the target molecule confirming whether the protein class
falls within the interest group.
3.3.10 Plant identification and collection
After collection of the fresh leaves of the plant the leaves were left to dry at room temperature for 3 days on a
clean surface. The dried leaves were cut into smaller pieces using a scissors to reduce particle size. Prior to
maceration in solvent, the plant materials were weighed using a weighing balance and was recorded.
3.3.11 preparation of crude extract/ Extraction
A 82.85g of Melissa officinalis dried leaves , A 24.45g of moringa oleifera dried leaves and A 78.46g of
Catharanthus roseus dried leaves respectively were weighed out using the weighing [Link]
were each macerated in 100% ethanol solvent for Melissa officinalis and 50% ethanol for
Catharanthus roseus and moringa oleifera .It was allowed to stand for 48 hours placed in the
water bath to undergo hot extraction at 60 [Link] mixture was filtered using whatman filter
0
paper and the filtrate concentrated using the water bath at 60 c for 7days until all the solvent was evapourated.
0
ensure that all the phytochemicals of interest
the water bath was kept at 60 c to 0
were entirely recovered using the hot extraction.
3.3.12 PHYTOCHEMICAL ANALYSIS
The test was carried out according to the procedures outlined by trease and evans (1989) and Habourne(1973). Ten
percent (10%) of the extracts : 20% absolute ethanol were prepared and considered as the test samples. This
preparation containing no test reagents were used as the negative control throughout the phytochemical tests.
Test for the presence of alkaloids
1. Hager’s test
To 2ml of each test samples in different test-tubes,2ml of 1%Hydrochloric acid was added. The mixture was
warmed on a steam bath and allowed to cool.5 drops of Hager’s reagent were added and observed for
a colour change. A yellow coloured precipitate indicates the presence of alkaloids.
2. Dragendoff’s test
To 2ml of each test samples in different testtubes, 2ml of 1%Hydrochloric acid was added. The mixture was
warmed on a steam bath and allowed to cool.5 drops of Dragendoff’s reagent were added and observed for
a colour change. A yellow coloured precipitate indicates the presence of alkaloids.
Test for the presence of saponins (Frothing test)
2 mg of each plant extract was taken in different test-tubes. 2mL of distilled H O was added to the test-tubes. Test-
2
tubes with solution were vigorously shaken for a minute and allowed to stand. The mixture was observed for the
presence of stable froth (foam), which indicates the presence of saponins.
Test for the presence of flavonoids
1. Lead acetate test
To 2ml of each test samples in different test tubes, 2ml of 1%Hydrochloric acid was added. Then 2 ml of 10%lead
acetate was added and observed for a colour change. A white colouredprecipitate indicates the presence of
flavonoids.
2. Sodium hydroxide test
To 2ml of each test samples in different test tubes, 2ml of 1%Hydrochloric acid was added. Then 2 ml of 10%
sodium hydroxide (NaOH) was added and observed for a colour change. A yellow colouration indicates the
presence of flavonoids; the intensity indicates the abundance.
Test for the presence of steroids (Salkowski’s test)
To 2ml of each test samples in different test tubes, 2 ml of concentrated sulfuric acid (H SO ) was added. The
2 4
appearance of a clear reddish brown colour ring at the interface confirms the presence of steroids.
Test for the presence of tannins (Ferric chloride test)
To 2ml of each test sample in different test tubes, 5 drops of freshly prepared ferric chloride (FeCl ) was 3
added. Dark black color precipitation can be observed which gives green black to blue black color on dilution
which indicates the presence of tannins.
Test for cardiac glycosides (Keller-kiliani test)
To 2ml of each plant extract taken in test-tubes, 1 mL of glacial acetic acid was added. After that, 3 drops of liquid
FeCl was added and then 1mL of concentrated H SO was added to the test-tube. Brown ring at the interface
3 2 4
indicates the presence of glycosides.
Test for the presence of phlebotannins (HCl test)
To 2ml of each plant extract taken in different test-tubes, 2mL of 2% HCl was added and colour change was
observed. Formation of red precipitate indicates the presence of phlebatannins.
Test for terpenoids (Salkowski’s test)
To 3ml of each plant extract taken in different test-tubes, 1ml of chloroform was added and the test tubes shaken
gently. Then 2 ml of concentrated sulfuric acid was carefully added to form a layer. A reddish brown ring at the
interface indicates the presence of terpenoids.
3.3.13 ANIMALS STUDIES
3.3.14 EXPERIMENTAL ANIMALS AND HOUSING
3.3.15 ACUTE TOXICITY STUDIES
3.3.16 EXPERIMENTAL DESIGN AND TREATMENT PROTOCOL
3.3.17 STATISTICAL ANALYSIS
The data obtained from the study were analysed using Microsoft excel package 2010. Results were presented using
mean and percentage.