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CHAPTER 3

MATERIALS AND METHODS

3.1 GENERAL

Established laboratory procedures as recommended by Purvis et al

(1966), Tuite (1969) were followed for the preparation of media, inoculation

and maintenance of cultures.

3.2 GLASSWARES

All the glasswares (Borosil or Corning) were immersed in cleaning

solution for a few hours. Then, the glasswares were washed thoroughly with

water, followed by detergent solution and finally rinsed with distilled water.

The cleaned glasswares were dried in hot air oven and stored.

Cleaning solution (Mahadevan and Sridhar, 1996)

Potassium dichromate - 60 g

Conc H2SO4 - 60 mL

Distilled water - 1000 mL

Potassium dichromate was dissolved in warm water, cooled and

sulphuric acid was added slowly. It was mixed thoroughly and used for

cleaning glasswares.

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3.2.1 Sterilization

Dried glassware and media were sterilized in an autoclave for 20

min at 15 psi pressure.

3.3 CHEMICALS

Analytical grade chemicals supplied by Loba, Hi-Media, S.D.Fine

Chemicals, E-Merck and Sigma Chemicals (USA) were used.

3.4 SENNA SIAMEA (Lam) PLANT SEED COLLECTION

Senna siamea plant is easily available in medium size tree to 15-20

cm tall, with a straight trunk up to 30 cm in diameter, bole short, crown

usually dense and rounded at first, later become irregular and spreading with

dropping branches. Plant seeds collected in Anna university campus, Chennai,

were sterilized and used for further processing

3.4.1 Preparation of Plant Seed Aqueous Extract

Collected seeds of Senna siamea plant (Fig 3.1) was washed

several times with deionized water. Fresh 50grams seeds of Senna siamea

plants were collected and washed with double distilled water. These were

then cut into small pieces, transferred to 500 mL beaker and boiled with

300 mL of distilled water for 10 min up to 700C temperature using magnetic

stirrer. Obtained mixture was cooled and filtered through Whatman No. 1

filter paper. The boiled extract was refrigerated and used for experimental

procedures.

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Figure 3.1 Synthesis of plant seed aqueous extract

3.4.2 Synthesis of Silver Nanoparticles

1 mM (1×10-3

) solution of silver nitrate (AgNO3) stock solution

was prepared using sterile deionized triple distilled water. A series of

different volumes (2-10 mL) of plant seed extract was added to 5 mL of

1×10-3

M aqueous silver nitrate (AgNO3) solution followed by addition of

distilled water to acquire a volume of 15 mL. After 3 hours of incubation

period at ambient conditions, color change from transparent light yellow to

brown color indicating the formation of silver nanoparticles. The occurrence

of brown color is a primary indication of the formation of silver nanoparticles

(AgNPs).

3.4.3 Synthesis of Gold Nanoparticles

1 mM (1×10-3

) solution of gold chloride (HAuCl4) stock solution

was prepared using sterile deionized triple distilled water. A series of

different volumes (2-10 mL) of plant seed extract was added to 5 mL of

1×10-3

M aqueous HAuCl4 solution followed by a addition of disttiled water

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being added to acquire a volume of 15 mL. After 3 hours incubation period at

ambient conditions, color change from transparent light yellow to violet color

indicating the formation of gold nanoparticles. The occurrence of violet color

is a primary indication of the formation of gold nanoparticles (AuNPs).

3.5 IN VITRO ANTIBACTERIAL SENSITIVITY

DETERMINATION TEST

The in vitro activities of the antibiotics by the disc diffusion

method, (as recommended by the National Committee for Clinical Laboratory

Standards) and test compounds were determined by modified well diffusion

method as proposed by Magaldi (1997) were determined

3.5.1 Preparation of Test Extracts

The stock solution of different concentrations were prepared. The

seed aqueous extracts, silver and gold nanoparticles from the stock solution

with 50 μL, 100 μL and 200 μL concentrations were immediately dispensed

into each agar wells of culture inoculated in muller hinton agar (MHA) plates

using sterilized micropipette.

Group: 1 Test group: consisted of the organism plus different

concentrations of the plant seed aqueous extract, silver and gold nano

particles (this group determined whether the extract was effective as

antibacterial agents).

Group: 2 Positive controls: organism plus a known antibiotic (This

ensured that utilized organisms are susceptible to common chemotherapeutics

and not resistant strains).

Group: 3 Pure cultures: Only the organism in the absence of

antibiotics or plant seed aqueous extract, silver and gold nano particles. This

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is to ensure that the organism was growing properly under the defined

laboratory conditions. This was necessary to distinguish poor growth from

inhibition of growth.

Group: 4 Negative controls: Organism plus AgNO3 and HAuCl4

solution (this was necessary to prove that the extraction solvent had no

inhibitory action of its own).

All quantitative data determinations were carried out in duplicate

with error < ±0.1.

3.5.2 Microbial Type Culture Collection (MTCC) Cultures

Five MTCC cultures of clinically important bacterial pathogens

which were obtained from Microbial culture collection, Chandigarh, India

were obtained as follows: Two-gram positive (Staphylcoccus aureus and

Bacillus subtilis) and four gram negative (Pseudomonas aeruginosa,

Klebsiella pneumoniae and Escherichia coli) were used. All strains were

recently purchased, collected between 2011 and 2012. The cultures were

stored frozen in skimmed milk 50% glycerol at -70ºC.

3.5.3 Inoculum Preparation

Bacterial inoculums were prepared using gram positive and gram-

negative bacterial pathogens from a 24 hours old culture on brain heart

infusion agar. With a sterile loop, the tops of four to five colonies were

transferred to a tube containing 5 mL of Mueller Hinton broth or Brain Heart

infusion broth. The tube was incubated at 35ºC for 24 hours. The turbidity of

the culture suspension was adjusted with broth or a sterile saline solution

(0.85 – 0.9%). The density of this culture was adjusted with 0.5 McFarland

standard and final inoculum size was approximately of 5x 105 CFU/mL.

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3.5.4 Turbidity standards for inoculum preparation

To standardize the inoculum density for a susceptibility test, a BaSO4

turbidity standard, equivalent to a 0.5 McFarland standard or its optical

equivalent (e.g., latex particle suspension), should be used. A BaSO4 0.5

McFarland standard may be prepared as follows:

1. A 0.5-mL aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2

.2H2O) was added to 99.5 mL of 0.18 mol/L H2SO4 (1% v/v)

with constant stirring to maintain a suspension.

2. The correct density of the turbidity standard was verified by

using a spectrophotometer with a 1-cm light path and matched

cuvette to determine the absorbance. The absorbance at 625 nm

was 0.008 to 0.10 for the 0.5 McFarland standard.

3. The Barium sulfate suspension was transferred in 4 to 6 ml

aliquots into screw-cap tubes of the same size as those used in

growing or diluting the bacterial inoculum.

4. These tubes were tightly sealed and stored in the dark at room

temperature.

5. The barium sulfate turbidity standard was vigorously agitated

on a mechanical vortex mixer before each use and inspected for

a uniformly turbid appearance. If large particles appeared, the

standard was replaced. Latex particle suspensions were mixed

by inverting gently, not on a vortex mixer

6. The barium sulfate standards were be replaced or their densities

verified monthly.

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3.5.5 Test Medium

The well diffusion method was performed using Mueller Hinton

Agar (MHA) medium: Casein acid hydrolysate: 17.5 g; Beef Heart infusion: 2

g; Starch soluble 1.5 g; (PH

7.3 ± 0.2), Agar 17 g; H2O 1000 mL was used.

3.5.6 Well diffusion method

The well diffusion test (Bennet et al 1966, Janssen et al 1987,

Magaldi et al 2004) was performed using MHA. The medium was prepared

and autoclaved at 15 lbs pressure (121ºC) for 15 min followed by cooling in a

50-55ºC water bath after removal from the autoclave. The cooled medium

was poured into sterile petri plates to a uniform depth of 4 mm; this is

equivalent to approximately 25 mL in a 90 mm plate. Once the medium had

solidified, then the culture was inoculated on the medium. Within 15 min of

adjusting the density of the inoculum, a sterile cotton swab was dipped into

the standardized bacterial suspension or inoculated with 1 mL of the organism

suspension. The sterile swab was used to streak on the surface of the MHA

medium to ensure an even distribution of the inoculum. The plates were left

to undisturbed for 3 to 5 min to absorb the excess moisture. Sterilized 10 mm

cork borer was used to made agar wells and 50 μL, 100 μL and 200 μL of the

stocks solutions were placed into each wells. The plates were incubated at 35-

37ºC for 24 hours. However NCCLS disc diffusion and MIC standard

breakpoints were used for the interpretative results. Streptomycin antibiotic

used as standard for these studies. Streptomycin is an antibiotic that inhibits

both Gram-positive and Gram-negative bacteria and is therefore a useful

broad-spectrum antibiotic (Jan-Thorsten Schantz 2004).

The percentage of inhibition was calculated by the formula,

I (Diameter of the inhibition zone)

% of inhibition = × 100

90 (Diameter of the petri-plate in mm)

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3.6 ANTIOXIDANT ACTIVITY

3.6.1 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical Scavenging

assay

Various concentrations of the stock solutions (concentrations of

100 to 500 µL) were mixed with 0.25 mM DPPH in ethanol, to produce a

final DPPH concentration of 0.1 mM. The mixture was vigorously shaken and

left to stand for 10 min in the dark and its absorbance was measured at

517 nm. L-Ascorbic acid was used as the control (McCune and Johns, 2002).

The radical-scavenging activities of samples, expressed as percentage

inhibition of DPPH, were calculated according to the formula:

Absorbance of Control – Absorbamce of sample

% of inhibition = × 100

Absorbance of control

3.7 IN VITRO ANTICANCER STUDY

3.7.1 Cell Line and Culture

A549 (lung carcinoma) cell line was obtained from National centre

for cell sciences Pune (NCCS). The cells were maintained in Minimal

Essential Media supplemented with 10% FBS, penicillin (100 U/ml), and

streptomycin (100 μg/ml) in a humidified atmosphere of 50 μg/ml CO2

at 37 °C.

3.7.2 Reagents

Minimal Essential Media (MEM) was purchased from Hi Media

Laboratories, Fetal bovine serum (FBS) was purchased from Cistron

laboratories and Trypsin, methylthiazolyl diphenyl- tetrazolium bromide

(MTT), and Dimethyl sulfoxide (DMSO) were purchased from Sisco research

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laboratory chemicals Mumbai. All other chemicals and reagents were

procured from Sigma Aldrich Mumbai.

3.7.3 Minimal Essential Media Preparation

Media is defined as a complex source of nutritional supplement

which is vital for the growth proliferation and maintenance of cells in vitro.

The MEM vial is dissolved in the pre sterilized Millipore distilled water and

mixed thoroughly, closed and sterilized at 15lbs, 121ºC for 15mins.

Depending on the concentration of foetal calf serum (2% or 10%) the

ingredients are allowed to mix well by shaking. Adequate care should be

taken to avoid spills.Then pass CO2 using sterile pipette, shake the bottle,

check pH and adjust to 7.2 to 7.4. The MEM bottles are kept for 2 days at

37ºC and checked for sterility, pH drop and floating particles. The contenets

are then transferred to the refrigerator.

Table 3.1 List of ingredients used in minimal essential media preparation

INGREDIENTS

10%

GROWTH

MEDIA

2%GROWTH

MEDIA

MAINTANCE

MEDIA

WITHOUT

FCS

MEM 857 ml 937 ml 957 ml

Penicillin and

streptomycin 1 ml 1ml 1ml

Phenol red 1 ml 1 ml 1 ml

Amphotericin B 1 ml 1 ml 1 ml

3% L-glutamine 10 ml 10 ml 10 ml

Foetal calf serum 100 ml 20 ml nil

7.5%NaHCO3 30 ml 30 ml 30 ml

Total volume 1000 ml 1000 ml 1000 ml

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3.7.4 Preparation of Ingredient

a) Penicillin and streptomycin: (concentration 100 IU of

penicillin and 100 µg of streptomycin)

Dissolve both antibiotics in sterile Millipore distilled water, so

as to give a final concentration 100 IU of penicillin and 100

µg of streptomycin/ml. Mix well and distribute in 1ml

aliquots. Store at -20ºC and check sterility.

b) Fungizone (amphotericin B) (20µg/ml)

Dissolve in sterile Millipore distilled water so as to give a

final concentration of 20 µg/ml and distribute in 1ml aliquots

in vials. Store at -20ºC. Check sterility before using.

c) L-glutamine (3%)

Weigh 3g of l-glutamine accurately and dissolve in 100 ml

sterile Millipore distilled water and mix well. Filter through

Millipore membrane filter 0.22µ and distribute in 5ml aliquots

in vials. Store at -20ºC. Check sterility.

d) 5% sodium-bi-carbonate

Weigh requisite quantity of sodium-bi-carbonate (to give

7.5% solution) accurately and dissolve in 100 ml of sterile

Millipore distilled water. Filter through What man filter paper

No.4, distribute into bottles at 121ºC, 15lbs, 15mins. Cool and

store at 4ºC.

e) Foetal calf serum

Bring FCS at room temperature. Inactivate at 56ºC in water

bath for½ hour and cool at room temperature. If floating

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particles are seen filter through Seitz filter. Distribute in

100 ml, 50 ml, and 20 ml quantities in sterile bottles. Store at -

20ºC.

3.7.5 Trypsin, PBS, Versene, Glucose Solution (TPVG)

a) 2% trypsin: 100 ml

Weigh 2g of trypsin accurately; dissolve in 100 ml sterile

Millipore distilled water with magnetic stirrer for ½ hour.

Filter through membrane filter. Store at -20ºC

b) 0.2%EDTA (versene)

Weigh 200mg of EDTA accurately. Dissolve in 100 ml of

sterile Millipore distilled water and autoclave at

15 lbs/15mins.

c) 10% glucose-100 ml

Weigh 1g of glucose accurately. Dissolve in 100 ml of sterile

Millipore distilled water and filter through whatmann filter

paper and autoclave at 15lbs/15mins.

d) TPVG-100 ml

PBS - 840 ml

% trypsin - 50 ml

0.2% EDTA - 100 ml

10% glucose - 5 ml

Penicillin & streptomycin - 5ml

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Mix all ingredients and adjust the pH to 7.4 with 0.1 N HCl or

0.1 N NaOH. Distribute in 100 ml aliquots. Store at -20ºC.

3.7.6 Subculturing and Maintenance of Cell Line

• Bring the medium and TPVG to room temperature.

• Observe the tissue culture bottles for growth, cell

degeneration, pH and turbidity.

• Select the bottles for splitting.

The following procedure is followed in sequence.

• Wipe the mouth of the bottle with cotton soaked in spirit.

• Remove the growth medium using 10 ml pipette.

• Then the cells in the bottle were gently rinsed with MEM

without FCS. The dead cells and FCS are washed out and then

the medium is discarded.

• 4-5 ml of TPVG was added over the cells. Allow TPVG to act

for 3-5 minutes and it was pipetted out.

• Incubate at 37oC for 3-5 minutes. The cells become individual

in nature as suspension.

• Add 5ml of 10% MEM with FCS by using serological pipette.

• Release carefully by using serological pipette and add 20 ml

of MEM and homogenize.

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3.7.7 Seeding of Cells

After homogenization pour 4-5 ml in to 24 well plates. In each well

add 1ml of the suspension to the 24 well plates and keep in a dessicator under

5% CO2 atmosphere. After 2 days of incubation period observe the cells in

inverted microscope. If the cells became 80% confluent, then it is used for the

Cytotoxicity studies.

3.7.8 MTT assay for in vitro cytotoxicity study

MTT assay is a calorimetric assay for measuring cell viability, for

cellular proliferation and activation. It is also used to determine the

cytotoxicity of potential medical agents and other toxic materials.

a) Principle

MTT was first described by Mosmann in 1983. Yellow MTT (3-(4,

5-dimethyl thiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazol) is

reduced to purple formazan in the mitochondria of the living cells. A

solubilization solution is added to dissolve the insoluble purple formazan

product into a colored solution. The absorbance can be quantified by

measuring at a certain wavelength (usually between 500 and 600 nm) by a

spectrophotometer.

The reduction takes place only when mitochondrial dehydrogenase

enzyme is active and therefore conversion is directly related to number of

viable cells. When the amount of purple formazan produced by cells treated

with an agent is compared with the amount of formazan produced by

untreated control cells, effectiveness of the agent causing death of cells can be

deduced

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After the addition of the drug, cell death and cell viability were

estimated. The result is confirmed by additional metabolic intervention

experiment such as MTT assay.

b) Procedure

• After incubation, remove the medium from the wells for

MTT assay.

• In each well wash with MEM (w/o) FCS. And add 200µl of

MTT concentration of (5mg/ml).

• Incubate for 6-7hrs in 5% CO2 incubator.

• After incubation, 1ml of DMSO was added in each well and

mixed by pipette and leave for 45 sec and it shows the

purple color formation.

• The suspension is transferred in to the cuvette of

spectrophotometer and O.D values are read at 595 nm

In order to study the antitumor activity of a drug, it is important to

determine the cytotoxic concentration of the drug. Cytotoxicity tests define

the upper limit of the extract concentration, which is non-toxic to the cell line.

The concentration which is nontoxic to the cells is chosen for antitumor

assay.The Cytotoxicity of samples on A549 was determined by the MTT

assay (Mosmann et al 1983). Cells (1 × 105/well) were plated in 1ml of

medium/well in 24-well plates (Costar Corning, Rochester, NY). After 48

hours incubation, the cell reaches the confluence. Then, cells were incubated

in the presence of various concentrations of the samples in 0.1% DMSO for

48h at 37°C.

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After removal of the sample solution and washing with phosphate-

buffered saline (pH 7.4), 200µl/well (5 mg/ml) of 0.5% 3-(4,5-dimethyl-2-

thiazolyl)-2,5-diphenyl--tetrazolium bromide cells(MTT) phosphate- buffered

saline solution was added. After 4h incubation, 0.04M HCl/ isopropanol was

added. Viable cells were determined by the absorbance at 570 nm.

Measurements were performed and the concentration required for a 50%

inhibition of viability (IC50) was determined graphically. The absorbance at

570 nm was measured with a UV-Spectrophotometer using wells without

sample containing cells as blanks. The effect of the samples on the

proliferation of A549 was expressed as the % cell viability, using the

following formula:

% cell viability = A549 of treated cells / A549 of control cells × 100%.

3.7.9 Drug Dilution

a) Stock drug concentration

1. 5 ml of extract was prepared with a concentration of

10 mg/ml.

2. 500 µl of MEM without FCS was taken in about 9 eppendroff

tubes.

3. 500 µl of the working concentration was added to the first

eppendroff tube, then same was transferred from first to last

tube by serial dilution to obtain the desired concentration of

the drug.

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b) Sampling

• 48h monolayer culture of Vero cell line at a concentration of

one lakh /ml /well (10 cells / ml / well) was seeded in 24 well

titer plate.

• The plates were microscopically examined for confluent

monolayer, turbidity and toxicity.

• The growth medium (MEM) was removed using pipette. Care

was taken so that the tip of the pipette did not touch the cell

sheet.

• The cell monolayer was washed twice with MEM without

FCS.

• To the washed cell sheet, 1ml of the medium (without FCS)

containing defined concentration of the drug was added. .

• Then each dilution of the drug ranges from 1:1 to 1:256 and

they were added to the respective wells of the 24 well titer

plates.

• To the cell control wells add 1ml MEM (w/o) FCS control.

The plates were incubated at 37ºC in 5% CO2 environment and

observed for cytotoxicity using inverted microscope.