Chapter 3 Materials and

Methods
3.1. Phytochemical screening

For the identification of different phytochemicals in C. polygonoides various qualitative

and quantitative test were carried out.

3.2.1. Qualitative chemical identification tests

Various qualitative chemicals tests were conducted for the identification of the saponins,
tannins, flavonoids, steroids, alkaloids, quinones, anthraquinone and coumarin in the C.
polygonoides.

3.1.1.1. Saponins identification

Frothing test
Qualitative determination of the saponins was carried out by following (Sofowora, 1993)
procedure. About 10 mg of plant was boiled for 5 minutes with 10 mL of dH 2O water
and was then filtered. Again 5 mL filtrate mixed with 5 mL dH 2O and shaken forcefully
until froth formed. Brief 4 drops of olive oil was then supplemented in the froth, shaken
forcefully and allowed for emulsion formation.

3.2.1.2. Tannins determination

Ferric chloride test:

Tannins determination test was carried out by following standard protocol of (Sofowora,
1993) procedure. About 25 mg plant crude extract and 10 mL of dH 2O were boiled and
then filtered. Now 0.1% ferric chloride was added drop by drop in the filtrate and saw for
colour change. Appearance of blue black or brownish green color will indicated the
existence of tannins.

3.2.1.3. Determination of flavonoids

Flavanoids were determined in the plant extracts by following the standard procedure of
(Sofowora, 1993). About 50 mg of plant extract was mixed in 100 mL of dH 2O and then
filtered to obtained filtrate. About 10 mL filtrate was mixed with 5 mL diluted NH 3
aqueous solution. Now conc H2SO4 was added drop by drop to the mixture and allowed
for color change. Appearance of yellow coloration specified the existence of flavonoids.

3.2.1.4. Alkaloids screening

Wagner’s reagent test (Solution of I2 in KI):
Chapter 3 Materials and
Methods
Alkaloids determination test was carried out by following (Harborne, 1973) standard
procedure. About 0.4 gm of plant and 8 mL of 1% HCl were mixed. The mixture was
warmed and then filtered. Few drops of Wagner’s reagent (I2+KI) was added to 2 mL of
filtrate and allowed for coloration. Reddish-brown colored precipitate specified the
alkaloids existence.
3.2.1.5. Coumarins identification
Alkali reagent test:
Coumarins were tested according to standard procedure of (Trease and Evans, 1989).
About 0.3 gm plant extract was taken; concealed with filter paper and then moistened
with 1 N NaOH. The solution was then boiled in water bath for few minutes. After
boiling the tilter paper was removed and then observed by using spectrophotometer
(Ultra violet-2600). Appearance of yellow florescence specified the coumarins existence.
3.2.1.6. Test for steroids
Steroids were detected according to procedure of (Siddiqui et al., 2009). About 5 mL
CH3OH and 100 mg powdered plant were mixed together and then filtered. To the filtrate
2 mL of acetic anhydride was added. To this mixture few drops of conc H 2SO4 was added
and allowed for colored change. Green coloration specified the existence of steroids.
3.2.1.7. Test for anthraquinones

Hydrochloric acid test:

Conformation for the presence of anthraquinones was carried out by following (Trease
and Evans, 1989) procedure. About 20 mg plant and 1 mL of 1% HCl were boiled
together in water bath and filtered. About 5 mL C 6H6 was added to the filtrate and shaken
vigorously. The upper layer was removed and followed by addition of 10% NH 4OH.
Pink, red or violet coloration specified the existence of anthraquinone.

3.2.2. Quantitative analysis

3.2.2.1. Alkaloids determination test
The conformation test for presence of alkaloids was carried out by following (Harborne,
1973) procedure. About 200 mL of 10% CH3COOH in C2H5OH was mixed with 5
mg/mL of plant extract; it was then covered and then reserved for 4 hrs. After 4 hrs the
mixture was filtered and reduced to ¼ of actual volume and followed by addition of
NH4OH (conc) until complete precipitation occurred. The precipitate was then collected,
washed with Ammonium hydroxide (diluted) and then filtered. The residue (alkaloid)
was then dried and weighed. Total alkaloids contents were calculated by using equation;
Chapter 3 Materials and
Methods
Weight of residue
% of total alkaloids = × 100
Weight of sample taken

3.2.2.2. Tannins determination

Tannin determination was carried out by following the procedure of (Van-Burden and
Robinson, 1981). About 500 mg plant extract was mixed with 50 mL dH 2O, shaken in
vigorously for 1 hr and then filtered. About 5 mL of the filtrate was mixed with 2 mL of
0.1 M FeCl3 in 0.1N HCl and 0.008M K4Fe(CN)6 (Potassium ferrocyanide). After 10 min
absorbance was recorded by spectrophotometer at 395 nm.

3.2.2.3. Saponins estimation of the plant extract

In the present study saponins was by following (Obadoni and Ochuko, 2001) method
with slight modification. About 50 mg/mL of plant extract was taken and mixed with 50
mL of 20% methanol. The mixture was then heated at 55 0C for 4 hrs on magnetic stirrer
with constant stirring over hot water bath; filtered and the residue again re-extracted with
50 mL of 20% methanol.The mutual filtrate was then condensed to 40 mL at 90 0C in the
water bath. In separatory funnel the concentrated filtrate was mixed with 10 mL of
C2H5OC2H5 followed by forcefully shaking. The upper C 2H5OC2H5 film was discorded
and the aqueous was recovered; this process was repeated. To the recovered aqueous 30
mL n-C4H9OH was added followed by washing with 5 mL of 5% NaCl aq. In a water bath
the mixture was evaporated, dried in the oven and then weighted.

The saponins contents were deliberated as a percentage of the dried fraction by using the
equation;

Weight of residue
% of total Saponins = × 100
Weight of sample taken

3.2.2.4. Phenolic estimation test

The phenolic contents (TPC) was estimated with the Folin-Ciocalteu reagents by
following a standard procedure as described by (Singleton and Rossi, 1965). About 200
µL (1-5 mg in methanol) concentration of plant was reacted with 130 µL of 1:10 Folin-
Ciocalteu reagent. The solution was incubate for 5 minutes and then followed by the
addition of 2.5 mL of saturated Na 2CO3 (0.115 mg/mL).The absorbance was recorded at
765 nm after 2 hrs of incubation at 25 0C. Gallic acid (1, 2, 3, 4 and 5 mg/mL) was used
as standard compound. The results were expressed as (mg GAE)/g of the dried fraction.
Chapter 3 Materials and
Methods
3.2.2.5. Flavonoid estimation test
The total flavonoids contents (TFC) was determined according to the standard procedure
of (Sakanaka et al., 2005). About 250 µL (1-5 in methanol) concentration of plant and
rutin (1-5 mg in methanol) as standard was mixed with 1250 µL dH 2O and 75 µL of 5%
NaNO2. About 150 µL of 10% (w/v) AlCl 3.6H2O was added after 6 min and again
incubate for 5 min. After 5 min of incubation 500 µL of 1M NaOH added to the solution
mixture and then raised up to 2500 µL with distilled H 2O followed by forcefully shaken.
The absorbance was taken at 510 nm by using spectrophotometer. The results were
expressed as (mg RUE)/g of the dried fraction.

3.6. Bio-assays of silver nano particles

3.6.1. Antioxidant screening

3.6.1.1. Ferric-Reducing Antioxidant Power screening

Ferric chloride/Iron (III) chloride (FeCl3) is a mild oxidizing agent.

3.6.1.1.1. Requirements

Potassium ferricyanide, deionize water, ferric chloride, Trichloroacetic acid, phosphate

buffer (pH 7.4), AgNO3.

3.6.1.1.2. Methodology

The reducing power potential of AgNPs was by following (Oyaizu, 1986) method with a
slight modification. About 2 mL AgNPs, 2 mL of 10 mg/mL potassium ferricyanide and
2 mL of 0.2 phosphate buffer (pH 6.6) were mixed and followed by incubation for 20
min at 50 °C. After incubation 2 mL of 100 mg/mL Trichloroacetic acid was mixed with
solution. About 2 mL of the above solution was mixed with 0.4 mL of 0.1% ferric
chloride and 2 mL deionize H 2O followed by the incubation for 10 min. Absorbance was
observed at 700 nm by spectrophotometer. The %age was determined by using the
formula;

Ac− As
% Scavenging = × 100
Ac

Ac is the control reading of Free radicals + sulphuric acid+phosphate buffer

As is the reading of Phosphomolybdate oxidants + AgNPs/standard Ascorbic

3.6.1.2. Phosphomolybdate assay of AgNPs

Chapter 3 Materials and
Methods
3.6.1.2.1. Requirements

Sulphuric acid, sodium phosphate, Phosphomolybdate, ascorbic acid, AgNPs, deionize

water.

3.6.1.2.2. Methodology

The Phosphomolybdate antioxidant potential of AgNPs was carried by following

(Velavan et al., 2012) procedure. About 1 mL of AgNPs (20-100 µg/mL) concentration
and 9 mL of (28 mM sodium phosphate, 600 mM sulphuric acid and 4 mM
Phosphomolybdate) were mixed together in test tubes. The test tubes were capped with
aluminium foil and followed by the incubation for 90 min at 95 0C in water bath. After
90 min of incubation the mixture was then cool to room temperature and absorbance was
noted at 695 nm by spectrophotometer.

The %age scavenging of Phosphomolybdate was deliberated by using the formula;

Ac− As
% Scavenging = × 100
Ac

Where Ac is the absorbance of control and As is the absorbance of sample

3.6.1.3. DPPH activity

3.6.1.3.1. Solution of DPPH

DPPH stock was prepared by liquefying 3 mg DPPH in 100 mL methanol. DPPH

absorbance was calculated to be 0.765 nm (<1).

3.6.1.3.2. Methodology

1,1-Diphenyl-2-picrylhydrazyl (DPPH) antioxidant potential of AgNPs was carried by

following (Brand-Williams et al., 1995) method. Stock solution of AgNPs was prepared
by dissolving 1 mg/mL AgNPs in deionize water which was further fractioned into (20,
40, 60, 80,100 µg/mL) by using the formula M 1V1 =M2V2. Standard ascorbic acid also
prepared in the similar concentration as AgNPs. About 200 µL from different
concentration of AgNPs and standard was mixed with 800 µL of DPPH (3 mg/50mL)
and then incubated for 30 min in dark at room temperature. Absorbance spectra was
Chapter 3 Materials and
Methods
recorded at 517 nm by using UV spectrophotometer against water as a reference. The
%age scavenging was deliberated by using the equation;

Ac− As
% Scavenging = × 100
Ac

Where Ac is the absorbance of control and As is the absorbance of sample

3.6.1.4. Hydrogen peroxide scavenging

3.6.1.4.1. Requirements

Ascorbic acid, deionize water, AgNPs, phosphate buffer, H2O2.

3.6.1.4.2. Methodology

The H2O2 scavenging activity was analyzed by (Pick and Mizel, 1981) method with
certain modification. About 200 µL from various concentration (20 to100 µg/mL) of
AgNPs in deionize water ,400 µL of 2 mM H2O2 and 400 µL of 50 mM phosphate
buffer ( pH 7.4) were mixed together and followed by the incubation for 20 minutes at
35 0C. The absorbance was recorded by using spectrophotometer at 610 nm against
phosphate buffer as blank.

The %age was determined by using the equation;

Ac− As
% Scavenging = × 100
Ac

Where Ac is the absorbance of control and As is the absorbance of sample

3.6.1.5. ABTS screening assay

3.6.1.5.1. Requirements

ABTS (2, 2 azobis, 3-ethylbenzothiozoline-6-sulphonic acid, potassium persulfate

(K2S2O8), AgNPs, ascorbic acid, deionize water.

3.6.1.5.2. Methodology

The ABTS free radical scavenging activity of AgNPs was accomplished by (Mathew and
Abraham, 2006) procedure with slight modification. About 7 mM of ABTS solution and
2.45 mM of potassium persulfate (K2S2O8) solution was prepared in deionize water.
These two solutions were mixed and allowed for overnight incubation, dark coloration
indicted the existence of ABTS•+ free radicals in the solution. Optical density of the
mixture was determined using spectrophotometer and was brought to 0.700 (± 0.02) by
Chapter 3 Materials and
Methods
addition of more solvent. About 300 μL of AgNPs (20-100 µg/mL) and standard mixed
with 300 μL of (K2(SO4) +ABTS) mixture. The absorbance was recorded immediately
after mixing the solution at 734 nm by using spectrophotometer. The %age scavenging
was deliberated by using the equation;

Ac− As
% Scavenging = × 100
Ac

Where Ac is the absorbance of control and As is the absorbance of sample

3.6.2. Cytotoxic brine shrimp lethality test

3.6.2.1. Methodology

Cytotoxic potential of AgNPs was carried by using (Bibi et al., 2011) standard
procedure. Sea salt solution was prepared by liquefying 5 mg of sea salt in 250 mL
deionize water and shaken vigorously for about 2 hrs. About1mg/mL AgNPs was
prepared in deionize water which fractioned into (25, 50, 75 and 100 µg/mL). The Salt
saline solution was poured into two compartment tray and the brine shrimps eggs were
scattered in dark section of the two section tray and allowed for hatching. After 24 hrs
shrimps were hatched in larvae and were found in the lightened section of tray. These
larvae were then collected from the lightened section by poster pipette. About 5 mL
saline solution and 500 µL from each concentration of AgNPs were taken in bottle. Brine
Shrimps (n=12) were transferred to each bottle and incubated at 25–28 °C for 24 hrs.
After 24 hrs lived shrimps were calculated with 3 x magnifying glasses.

Survivors were calculated by using the equation;

Ac− As
% Death = × 100
Ac

3.6.3. Antifungal assessment of green synthesised silver nano particles

3.6.3.1. Requirement

AgNPs, fungus strains, test tubes, cotton, beaker, tips, DMSO, deionize water, wire loop,
sabourad dextrose agar media, volumetric flask and standard terbinafine was used in this
experiment.

3.6.3.2. Samples preparation

Solution of 5 mg/5mL of AgNPs was prepared in deionize water. It was further diluted to
25, 50, 75 and 100 μg/mL by using (M1V1=M2V2) dilution formula.
Chapter 3 Materials and
Methods
3.6.3.3. Methodology

In the present study (Ruparelia et al., 2008) procedure was followed for measuring the
fungicidal capacities against three fungal strains A. Niger, A. fumigates and A. Flaves.
About 6.2 gm sabourad dextrose agarose (SDA) was weighted and dissolved in 100 mL
deionize water, autoclaved for 15 min at 121 ºC and reserved for cooling at 40-50 ºC.
About 7 mL SDA along with 67 µL (25, 50, 75, 100 µg/mL) AgNPs dissolved in
deionize water were transferred to the test tubes. The negative control test tubes were
treated only with DMSO and positive control received antifungal drug terbinafine. All
the tubes were then placed in slanted position at room temperature inside the laminar
flow hood to solidify. Applied the fungus strains to each tube; were placed in incubator
at 30 ºC with open water up to 9 days and zone of inhibition were studied and expressed
in millimeter (mm).

3.6.4. Antibacterial activity of AgNPs

3.6.4.1. Requirements

AgNPs, six bacterial strains, three Gram-negative E. coli, P. vulgaris and K. pneumonia
and three Gram-positive M. luteus, S. epidermidis and S. aureus, nutrients broth, petri
plates, spatulas, cotton, tips, nutrient agar, DMSO, standard (Levofloxacin).

3.6.4.2. Methodology (Agar well diffusion method)

Bactericidal activity of AgNPs was measured by following (Revati et al., 2013) standard
procedure. Six bacterial strains, three Gram-negative E. coli, P. vulgaris and K.
pneumonia and three Gram-positive M. luteus, S. epidermidis and S. aureus a were used
in this experiment. Bacterial cultures were refreshed in nutrients broth for 24 hrs at 37
0
C. The antibacterial activity equipment’s such as nutrient agar, petri plates, spatulas,
cotton, tips were autoclaved at 121 ºC for about 15 min and then allowed to cooled up to
60 0C. To each petri plate 30 mL media was transferred and allowed for overnight
solidification. The 24 hrs old bacterial cultures were swabbed on nutrients agar plates
with sterile cotton swab in laminar flow aseptically. About 6 wells were perforated on
swabbed plates by using an antiseptic cork borer (3-6 mm). The stock solution 1 mg/mL
of silver nanoparticles was prepared in deionizes water which was further diluted to the
different concentrations (25-100 µg/mL). About 67 µL of AgNPs from various
concentrations (25-100 µg/mL) were loaded in the labeled wells. Standard levofloxacin
(1000 µg/10mL dissolved in DMSO) and DMSO were also loaded into the respectively
Chapter 3 Materials and
Methods
wells. The standard levofloxacin and DMSO served as +ve and -ve control respectively.
The petri plates were then incubated for 24 hrs at 37 ºC and the outcomes were expressed
by determining the zone of inhibition around each well in mm.

3.6.5. In-vitro antidiabetic activity of AgNPs

In the present study antidiabetic effect of AgNPs was determined using β-glucosidase
and α-amylase enzymes.

3.6.5.1. In-vitro β-glucosidase Assay of AgNPs

3.6.5.1.1. Requirements

β-glucosidase, β-D glucopyranoside (pNPGlc), citrate buffer, AgNPs, standard tagipmet,

6N HCl, sodium deionize water.

3.6.5.1.2. Methodology

In-vitro β–glucosidase inhibitory activity of AgNPs was carried using (Krishnaveni et

al., 1984) procedure. About 290 mM solution of β-D glucopyranoside (pNPGlc) was
prepared in 20 mM citrate buffer of pH 5.6. The stock solution of AgNPs and standard
(tagipmet) was prepared at 1 mg/mL concentration in the deionize water which
fractioned into (200,400,600 and 800 µg/mL). About 200 µL was taken from each
concentration of AgNPs and standard (tagipmet) to be mixed with 980 µL of β-D
glucopyranoside and incubated at 37 ºC for 5 min. About 20 µL of a β-glucosidase
enzyme (IU/mL) was added to above mention each mixture and then incubated at 35 ºC
for 40 min. About 200 µL of 6 N-HCl was supplemented to the reaction to terminate the
reaction and the absorbance was recorded at 405 nm by using spectrophotometer. The
experiment was repeated twice to eliminate any mistakes and water taken as a reference.
The %age inhibition was deliberate by using the equation;

Ac− As
% Inhibition = × 100
Ac

Where Ac is enzyme activity of standard and As is enzyme activity of AgNPs;

3.6.5.2. In-vitro α-amylase Assay of AgNPs

3.6.5.2.1. Requirements

AgNPs, potato starch, sodium acetate buffer, sodium potassium tartrate, 3, 5 di nitro
salicylic acid, standard (tagipmet).
Chapter 3 Materials and
Methods
3.6.5.2.2. Methodology

In-vitro α-amylase enzyme inhibitory activity of AgNPs was carried using by following
(Malik and Singh, 1981) protocol with slight modification. To obtained starch solution
(0.1% w/v) 0.1 gm potato starch was dissolved in 100 mL of 16 mM C 2H3NaO2 buffer.
The enzyme was diluted by mixing 300 µL of α-amylase from stock (250 units/mL) with
700 µL deionizes water. Sodium potassium tartrate and 3, 5 di nitro salicylic acid (96
mM) mixture used as calorimetric reagent. The stock solution of AgNPs and standard
(tagipmet) was prepared at 1 mg/mL concentration in the deionize water which
fractioned into (25, 50, 75 and 100 µg/mL). About 250 µL from the four different
concentration of AgNPs or standard tagipmet was mixed with 250 µL potato starch
solution and 250 µL α-amylase and incubate for 5 min at 25 0C (room temperature).
About 250 µL was taken from the combine mixture of 3,5 di nitro salicylic acid and
sodium potassium tartrate and was added to each concentration. The reaction is detected
at 450 nm against water was taken as a blank. The standard drug tagipmet served as a
positive control. The experiment was repeated twice.

The %age inhibition was deliberate by using the equation;

Ac− As
% Inhibition = × 100
Ac

Ac is enzyme activity standard drug and As is enzyme activity of AgNPs

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Chapter 3 Materials and Methods

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