CHAPTER 3shodhganga.inflibnet.ac.in/bitstream/10603/38449/8/chapter 3.pdfCell lines: Hela, MCF 7 and the PC 12 cancer cell lines, were from American Type Culture Collection (ATCC),

CHAPTER 3

Materials and

methods

“Evaluation of ethno pharmacologically important plants - Catunaregum spinosa Thunb and Pavonia zeylanica Cav. for antioxidant and anticancer activity”

Manasagangotri, University of Mysore, Mysore – 570006 49

3. MATERIALS AND METHODS

3.1 MATERIALS

3.1.1 PLANT MATERIAL

The selection of the plant species for the present study was mainly based on the

traditional uses of these species for the treatment of various diseases.

The plants shortlisted for the present studies are as follows:

i. Pavonia zeylanica, Cav.

ii. Catunaregum spinosa, Thunb.

i. Pavonia zeylanica Cav.

It belongs to the Family – Malvaceae. It is commonly known as the Ceylon Swamp Mallow

and in Kannada it is called as Topala, Balarakshasi, and Antutogari. The Ceylon Swamp

Mallow is a profusely branched, bristly, large herb, growing up to 1-1.5 m tall. The Plant

extract is used for vomiting, vermifuge, oliguraia, tumours and fever.

ii. Catunaregum spinosa Thunb.

It belongs to the Family- Rubiaceae. It is commonly known as the emetic nut and mountain

promogranate and in Kannada it is called as Kaarekaayi-gida. Mountain Pomegranate is an

armed shrub or small tree. Fruits and Bark of the plant pacifies vitiated pitta, kapha, cough,

skin diseases, ulcers, cancer, asthma, flatulence, colic, and is widely used as a medicine for

emesis therapy in ayurveda.

The plants were collected from the forests of Nanjangud area, Mysore district, Karnataka,

voucher specimens were collected, identified properly referring a flora and the same has

been authorized by the taxonomist..



The chemicals used in the present investigation are of analytical grade, they are:-

Preliminary phytochemical Analysis

Picric acid, α- naphthol, Benedict‟s reagent, 5%Ferric chloride, 1%gelatin, 10% sodium

hydroxide, Alcohol, Biuret‟s reagent, Ninhydrine reagent, Lead acetate, NaOH,

Conc.H2SO4, Dragondroffs reagent, Mayer‟s reagent, Hager‟s reagent, Anthrone, Molishs

reagent , Fehling‟s solution A&B etc., All the chemicals used including the solvents, were

of analytical grade and were from Merck Chemical Supplies.

Antioxidant activity

1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2, 4, 6-tripyridyl-s-triazine (TPTZ), potassium

ferricyanide; ascorbic acid and FeCl3 were purchased from Sigma Chemical Co. and Folin-

Ciocalteus‟s phenol reagent and sodium carbonate were from Merck Chemical Supplies.



PLATE No.1

Fig.9 (a): Catunaregum spinosa Thunb. – A Habit and with flower

Fig.9(b): Pavonia zeylanica Cav. – A Habit and with flower



Anticancer activity

Cell lines: Hela, MCF 7 and the PC 12 cancer cell lines, were from American Type Culture

Collection (ATCC), the global bioresource centre, USA. The local distributors in India are

LGC Promochem India Pvt. Ltd. Peenya, Bangalore - 560058, India. The morphology of

the cells and the assays are shown in PLATE No.1

3.1.2 CANCER CELL LINES

HeLa (Cervical cancer cells)

Growth Properties: Adherent

Organism: Homo sapiens (human)

Morphology: epithelial

Source: Organ: cervix

Disease: adeno carcinoma

Cell Type: epithelial

The cells are positive for keratin by immunoperoxidase staining.

HeLa cells have been reported to contain human papilloma virus 18 (HPV-18)

sequences. P53 expression was reported to be low, and normal levels of pRB

(retinoblastoma suppressor) were found.

MCF-7 (Breast cancer cells)

Growth Properties: adherent

Organism: Homo sapiens (human)

Morphology Epithelial

Source:

Organ: mammary gland; breast

Disease: adenocarcinoma

Derived from metastatic site: pleural effusion

Cell Type: epithelial

Doubling Time: 29 hrs



PLATE No.2

MORPHOLOGY OF THE CANCER CELLS AND THE ASSAYS

HeLa Cells MCF-7

Trypan blue



Hoechst assay

MTT assay



The MCF7 line retains several characteristics of differentiated mammary epithelium

including ability to process estradiol via cytoplasmic estrogen receptors and the capability

of forming domes. The cells express the WNT7B oncogene [PubMed: 8168088]. Growth of

MCF7 cells is inhibited by tumor necrosis factor alpha (TNF alpha). Secretion of IGFBP's

can be modulated by treatment with anti-estrogens.

PC-12 (Adrenal gland cancer)

Growth Properties: Floating clusters; few scattered lightly attached cells.

Organism: Rattus norvegicus (rat)

Morphology: small irregularly shaped cells

Source: Organ: adrenal gland

Disease: pheochromocytoma

Doubling Time: 48 hrs

The PC-12 cell line was derived from a transplantable rat pheochromocytoma.

The cells respond reversibly to NGF by induction of the neuronal phenotype when plated

on Collagen IV coated culture flasks. The cells do not synthesize epinephrine.

3.1.3 CHEMICALS AND REAGENTS

1. Fetal Bovine Serum (FBS): Fetal bovine serum ( Fetal calf serum) is the portion of

plasma remaining after coagulation of blood, during which process the plasma protein

fibrinogen is converted to fibrin and remains behind in the clot. Fetal bovine serum

comes from the blood drawn from the unborn bovine foetus. Fetal bovine serum is the

most widely used serum due to being low in antibodies and containing more growth

factors, allowing for versatility in many different applications.



The globular protein bovine serum albumin (BSA), is a major component of fetal

bovine serum. The rich variety of proteins in fetal bovine serum maintains cultured

cells in a medium in which they can survive, grow, and divide.

2. Saline (0.9%NaCl):0.9%NaCl is normal saline. Saline is used mainly during

trypisinization of the cultures

3. Trypsin-EDTA: Tissue culture media contains calcium and magnesium ions, fetal calf

serum contains proteins that are trypsin inhibitors. Both Mg2+

/ Ca2+

inhibit trypsin. EDTA

is a calcium chelator which will remove the divalent cations. If trypsin is allowed to stay in

contact with the cells for too long a time, cell viability will reduce.

TABLE 2: Composition of trypsin-EDTA

Component mg/litre Molecular weight Mol (mM)

Inorganic salts

EDTA 2 Na.2H2O 372.20 372.2 1.00

Potassium Chloride 400.00 74.55 5.37

Potassium Phosphate Monobasic 60.00 136.09 0.44

Sodium Bicarbonate 350.00 84.01 4.17

Sodium Chloride 8000.00 58.44 136.89

Other

Dextrose 1000.00 180.2 5.55

Phenol red Sodium Salt 17.00 376.4 0.05

Trypsin1:250 2500.00 N/A N/A



Media: DMEM (Dulbecco‟s modified Eagle‟s medium) is used for the growth of MCF-7 cells.

TABLE 3: Composition of DMEM

Component Composition(gm/1000ml)

Amino acids

L-arginine 0.840

L-cystine 0.480

Glycine 0.300

L-histidine 0.420

L-isoleucine 0.1050

L-lysine HCl 0.1460

L-methionine 0.300

L-phenylalanine 0.660

L-serine 0.420

L-threonine 0.950

L-tyrosine 0.720

L-tryptophan 0.160

L-valine 0.940

Vitamins

Choline chloride 0.40

Folic acid 0.40

Inositol 0.72

Nicotinamide 0.40

D-calcium pantothenate 0.40

Pyridoxal HCl 0.40

Riboflavin 0.40

Thiamine HCl 0.40



Inorganic salts

Cacl2 0.2

KCL 0.4

MgSo47 H2O 0.2

NaCl 6.4

NaHCo2 3.7

NaH2Po4 0.14

Trace Elements

Fe(NO2)3.9 H2O 0.01

Energy metabolites

D-glucose 4.5

Sodium pyruvate 0.16

Lipids

Linoleic acid

Other components

Phenol red 0.010

Gas phase

Co2 5%

3. Other reagents:

a) Trypan blue: dissolve 400mg trypan blue in double distilled water.

b) DMSO (Di –methyl sulphoxide): used to cryopreserve the cells in the liquid nitrogen

at final concentration at 10%v/v of cell suspension.

c) Hoechst dye: It is fluorescent stain used for labelling DNA in florescent microscopy

d) MTT reagent: MTT, (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tertasodium bromide)

used to evaluate cell viability.



e) Antibiotics: In common usage an antibiotic is a substance or compound (also called

chemotherapeutic agent) that kills or inhibits the growth of bacteria. Antibiotics belong

to the group of antimicrobial compounds used to treat infections caused by microbes

including fungi and protozoa. The plate or dishes are coated with antibiotics before use

to reduce the contamination. Examples penicillin, neomycin etc.

f) Double distilled water: Double distilled water also abbreviated ddH2O is a water

prepared by double distillation. Double distilled water is used often in laboratory when

single distillation of water is not of sufficient purity for some research applications.

3.1.4 GLASSWARES AND INSTRUMENTS

Instruments used:

1) Centrifuge: a centrifuge is a piece of equipment, generally driven by a motor that puts

an object in rotation around a fixed axis, applying a force perpendicular to the axis.

Centrifuge works using the sedimentation principle, where the centripetal acceleration

is used to evenly distribute substances (usually present in a solution for small scale

applications) of greater and lesser density.

2) CO2 incubator: It is used to maintain the cell cultures. CO2 is used to maintain the pH of

the cell cultures. CO2 levels within the chamber are established with a set point and are

controlled to maintain that set point. When the door is open CO2 escapes from the

chamber and a CO2 sensor detects a drop in the level of CO2 is automatically injected to

raise the level to the set point.



3) Haemocytometer: It is a device originally designed for counting of the blood cells. It is

now also used to count other types of cells as well as other microscopic particles.

4) Fluorescent microscopy: Fluorescent microscope is alight microscope used to study the

properties of organic and inorganic substances using the phenomena of fluorescent and

phosphorescence instead of or in addition to, reflection and absorption.

5) Optical microscope: Optical microscope, often referred to as the “light microscope”, is

a type of microscope which uses visible light and a system of lenses to magnify images

of small samples.

6) Vertical laminar air flow: The Vertical laminar air flow station comes complete with

transparent acrylic side panels that restrict the laminar flow and ensure an effective

wash of HEPA (high efficiency particulate air) filtered air over the entire work surface.

7) Autoclave: it is a device to sterilize equipments and supplies by subjecting them to high

pressure steam at 1210c or more.

8) Cyclomixer: I t is a variable speed mixer to eliminate time consuming and mixing.

Holding tube against vibrating rubber cup does rapid mixing of contents. Speed

regulator controls the degree of vibration. A unique touch feature operates the unit

when tube is pressed on the rubber cup.

9) Weighing machine: It is a measuring instrument for measuring the weight or mass of

the object.

10) Media filter: it is a unit used to filter the media. The prepared media is poured in the

upper container and it is brought down by creating vacuum pressure. The filter is

present in between the two containers.



11) Slow cooling device: used for cryopreservation of the cells and is filled with liquid

nitrogen.

12) Plate reader: Micro plate readers are laboratory instruments designed to detect

biological, chemical or physical events of sample in micro titre plates. Sample reactions

can be (assayed) in 6-1536 well formed micro titre plates. In most cases, a high

intensity lamp passes light to the microtitre well and the light emitted by the reaction

happening in the microplate well is quantified by a detector. Common detection modes

for microplate assays are absorbance, fluorescence intensity, luminescence, time-

resolver fluorescence and fluorescence polarisation.

Consumables:

1) Micropipettes: these are used to accurately measure and dispense small volumes of

liquid. The capacity of a micropipette can range from less than 2 µL to 1000 µL, while

macropipettes can measure volumes greater than 1ml.

2) Eppendorf tubes: these are polypropylene tubes. Their capacity ranges from micro

centrifuge Eppendorf tubes of 1.5µL to tubes of 2mL.

3) Cryovials: it is designed for storing biological material at temperature as low as -1900C

but should be used only in the gas phase of liquid nitrogen. Both the cap and tube are

manufactured of polypropylene and have the same coefficient of expansion, which

ensures an equally secure seal all temperatures. The silicon seal between the cap and the

tube ensures a positive, leak proof seal. Tubes have a white marking area and the cap

can be colour coded with a cap insert. They are sterilized by gamma radiations and



PLATE No. 3

Fig.12: Figure showing A. ELISA plate reader, B. Florescence

microscope and C. Deep Freezer with CO2 cylinder

A B

C



packed in unique tamper proof bags of 100 vials per bag.

4) Cellulose acetate membrane filter: composed of cellulose di- and triacetate, these filters

exhibit low static charge and high strength. May be sterilized repeatedly without loss of

integrity. Good resistance to heat and low molecular weight alcohols.

5) Syringe filter: these are useful for small volume filtration of liquids. They are made

with wide variety of membrane filters with polypropylene housing using the most

advanced methods and design features available today.

6) Filter paper: it is used to remove the coarse and unwanted particles from a solution.

7) 96 well plates: a microtiter plate or micro plate is a flat plate with multiple wells used as

small test tubes. The microtiter plate has become a standard tool on analytical research

and clinical diagnostic testing laboratories. A very common usage is in the ELISA.



3.2 METHODOLOGY

3.2.1 Plant material Extraction

The leaves of the two species were taken for the extraction process using a series of

polar to non-polar solvents like, Distilled water, Ethanol, Methanol, Acetone, Chloroform;

Petroleum ether and Benzene. The leaves of Catunaregum spinosa and Pavonia zeylanica

are shade dried and powdered. These powdered plant materials were subjected to extraction

(soxhlet amd maceration method) where in, 8gm of powdered plant material is extracted by

the maceration method using 250ml of various solvents (WHO, 2007). The macerated

extracts were selected for further analysis. The macerated extract was kept on the shaker for

24 hours, later it was centrifuged and the supernatant was taken for the excess solvent

evaporation in laboratory conditions. After evaporation of excess solvent the crude extract

was stored in refrigerator till further analysis.

3.2.2 Preliminary phytochemical analysis:

Preliminary phytochemical tests for the identification of amino acids, carbohydrates,

saponins, tannins, phytosterols, alkaloids, proteins, glycosides, flavanoids and phenolic

compounds were carried out for all the extracts by the methods described by Harborne

(1998). The present investigation was planned with an objective to establish

Pharmacognostic standards and to evaluate preliminary phytochemical data‟s on the two

plant species that can facilitate the authentification and the isolation of the desired

constituent from the correct extract.



Successive Solvent extraction

About 50g of the air dried powdered plant material was extracted successively with

petroleum ether (60-80 ), followed by benzene, chloroform, acetone, methanol, ethanol

(95%) and water by maceration method. The extracts were filtered, the solvent was

evaporated and accurate weight of the extracts was taken. The extractive value (%) was

calculated with reference to air dried drug. The color and consistency of the extracts were

noted.

Detection of chemical constituents

Detection of alkaloids

Small portions of solvent free chloroform, alcohol and water extracts were stirred

separately with a few drops of dilute hydrochloric acid and filtered. The filtrate was tested

with various alkaloid reagents.

Mayer’s test

The filtrates were treated with Potassium mercuric iodide (Mayer‟s reagent) and the

formation of white colored precipitate indicates the presence of alkaloids.

Dragendorff’s test

The filtrates were treated with Potassium bismuth iodide (Dragendorff‟s reagent) and

the formation of orange colored precipitate indicates the presence of alkaloids.

Hager’s test

The filtrates were treated with saturated solution of Picric acid (Hager‟s reagent) and

the formation of yellow precipitate indicates the presence of alkaloids.



PLATE No. 4

Fig.10: Soxhlet Apparatus set up for extraction



PLATE No. 5

Fig.11: Figure showing excess solvent removal using heating

mantle at 37 – 400C

Leaves of Catunareum spinosa in ethanol Leaves of Pavonia zeylanica in ethanol



Detection of carbohydrates and glycosides

Small quantity of alcohol and aqueous extracts were dissolved separately in distilled

water and filtered. The filtrate was subjected to various tests to detect the presence of

different carbohydrates.

Molisch’s test

The filtrates were treated with solution of -Naphthol in alcohol (Molisch reagent) and

a few drops of conc. sulphuric acid was added through the sides of the test tube. The

formation of violet ring at the junction of the liquids indicates the presence of

carbohydrates.

Fehling’s test

The filtrates were treated with a few ml of dilute hydrochloric acid and heated on a

water bath for 30 minutes. After hydrolysis the solutions were neutralized with sodium

hydroxide solution. To the neutralized solutions, equal quantities of Fehling‟s A &

Fehling‟s B solutions were added and heated on a water bath for a few minutes. Formation

of red-orange precipitate indicates the presence of reducing sugars.

Detection of phytosterols

The petroleum ether, benzene, acetone and alcohol extracts were refluxed separately

with solution of alcoholic potassium hydroxide till complete saponification took place. The

saponified mixtures were diluted with distilled water and extracted with solvent ether. The

ethereal extract was evaporated to dryness and the residue subjected to Liebermann-

Burchard‟s tests.



Liebermann-Burchard’s test

The ethereal residues were treated with a few drops of acetic anhydride, boiled and

cooled. 1 ml of sulphuric acid was added through the sides of the test tube. Formation of a

brown ring at the junction of two liquids and green color in the upper layer indicate

presence of steroids and triterpenoids.

Salkowski Test

In chloroform solution of a sterol is shaken with an equal volume of conc. sulphuric acid;

when the liquids have separated, the chloroform layer is seen to be brownish-yellow, and

the sulphuric acid layer to be yellow-brown with a green fluorescence; on allowing the test-

tube to stand for several hours, the sulphuric acid layer becomes deeper and redder in

colour and more strongly fluorescent, while the chloroform layer assumes, if the sterol was

present in sufficient amount, a cherry-red or purple colour.

Detection of fixed oils and fats

Spot test

Small quantities of petroleum ether and benzene extracts were pressed separately

between two filter papers. The formation of oil stains on the filter paper indicates the

presence of fixed oil.

Detection of saponins

Foam test

About 1 ml of alcohol and aqueous extracts were diluted separately with distilled

water to 20 ml and shaken in a graduated cylinder for 15 minutes. The formation of any

froth above the surface indicates the presence of saponins.



Detection of phenolic compounds and tannins

Small quantities of alcohol and aqueous extracts were diluted separately in water and

were tested for the presence of phenolic compounds and tannins.

Ferric chloride test

To the test solutions, a few drops of 5% ferric chloride solution were added. Formation

of a blue-black or green-black color indicates the presence of phenolic compounds and

tannins.

Lead acetate test

To the test solution, a few drops of 10% lead acetate solution was added. The test

solution was tested with solution of NaOH containing gelatin. The formation of white

precipitate was observed for the presence of tannins. Formation of a yellow precipitate

indicates the presence of flavonoids.

Detection of proteins and free amino acids

Small quantities of alcohol and aqueous extracts were diluted separately in water and

tested for the presence of proteins and free amino acids by subjecting the extracts to various

tests.

Millon’s test

To 2 ml of the test solutions, 2 ml of Millon‟s reagent was added and heated. Formation

of white precipitate that gradually turns red indicates for the presence of proteins and amino

acids.



Biuret’s test

To the test solutions, a few drops of 0.7 % copper sulphate solution was added.

Formation of a purplish violet color indicates the presence of amino acids.

Ninhydrin test

To the test solutions, a few drops of Ninhydrin solution were added. Formation of a

bluish color indicates the presence of amino acids.

Detection of gums & mucilage

About 10 ml of aqueous extract was added to 25 ml of absolute ethanol with constant

stirring. The precipitate was examined for its swelling properties and for the presence of

carbohydrates.

Detection of flavonoids

The aqueous and 95 % alcohol extracts were subjected to the following additional tests.

Shinodaw’s test

To the test solutions, a few fragments of magnesium metal were added along with

concentrated hydrochloric acid. This was heated. Formation of red color indicates the

presence of flavonoids.

Alkaline reagent test

To the test solutions a few drops of sodium hydroxide solution was added. Formation

of an intense yellow color that turns less intense on addition of acid indicates the presence

of flavonoids.



TABLE 4: Preliminary phytochemical tests.

Sl.

No.

Name of the

test Procedure Observation

1 Alkaloids

Drug +

Dragondroffs reagent

Mayer‟s reagent

Hager‟s reagent

Orange color

White ppt.

Yellow ppt.

2 Glycosides Anthrone + H2SO4+ Heat Purple or green

3 Carbohydrates

Drug + Molishs reagent+

conc.H2SO4

Fehling‟s solution A&B

Purple color

Brick red color

4 Phytosterols/triterpenoids

Liebermann Test

Salkowski Test

Noller‟s test

Bluish green

Red & fluorescent

Pink color

5 Proteins & Amino acids

Biuret test

Xanthoprotein test

Millon‟s reagent test

Lead acetate test

Ninhydrin test

Violet color

Orange color

White ppt

White ppt

Blue color

6 Saponins Drug + water + shaking Formation of honey comb

like froth

7 Flavonoids Shinodaw‟s Test

Zn-HCl acid reduction Test

Red color

Magenta color

8 Fixed oils & Fats Spot test Stains appear after drying

9 Gums/Mucilage Drug + water

No thickening of the

substance

10 Volatile oil Spot test ------

11 Phenolics/Tannins Fecl3

Drug + lead acetate + water

Intense color

Formation of white ppt



3.2.3 Antioxidant activity

The antioxidant activity was carried by the evaluating the Radical scavenging

activity by the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) method and the total antioxidant

activity by using the ferric ion reducing antioxidant power (FRAP) method.

DPPH radical scavenging assay

The effect of the extracts on DPPH radical was estimated using the method of

Liyana- Pathiranan and Shahidi. A solution of 0.135 mM DPPH in methanol was prepared

and 1.0 ml of this solution was mixed with 1.0 ml of extract in ethanol containing 0.02–0.1

mg of the extract. The reaction mixture left in the dark at room temperature for 30 min. The

absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid

was used as reference. The ability to scavenge DPPH radical was calculated by the

following equation:

DPPH radical scavenging activity (%) = [(Abs control – Abs sample)]/ (Abs control)] x 100

where Abs control is the absorbance of DPPH radical + methanol;

Abs sample is the absorbance of DPPH radical + sample extract /standard.

Reducing ability (FRAP assay)

The determination of the total antioxidant activity (FRAP assay) in the extract is a

modified method of Benzie and Strain. The stock solutions included 300 mM acetate

buffer (3.1 g C2H3NaO2·3H2O and 16 ml C2H4O2), pH 3.6, 10 mM TPTZ (2, 4, 6-

tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl3·6H2O solution.



The fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ, and

2.5 ml FeCl3·6H2O. The temperature of the solution was raised to 37 °C before use. Plant

extracts (150 μL) were allowed to react with 2850 μl of the FRAP solution for 30 min in

the dark condition. Readings of the colored product (ferrous tri-pyridyl-triazine complex)

were taken at 593 nm. The standard curve was linear between 200 and 1000 μM FeSO4.

Results are expressed in μM Fe (II)/g dry mass and compared with that of ascorbic acid.

The total antioxidant activity of FRAP is calculated by the following equation:

FRAP value of Sample (µM) = (Change in absorbance of sample from 0 to 4 minute /Change in

absorbance of standard from 0 to 4 minute) X FRAP value of

standard (1000 µM)



3.2.4 In- Vitro Anticancer activity

The ethanolic leaf extracts of Catunaregum spinosa and Pavonia zeylanica were

tested for in-vitro anticancer activity against Hela cells, Mammarian cancer cells (MCF-7)

and PC-12 cell line. This work was carried out at Sri Raghavendra Biotechnologies Ltd.

Bangalore. These cancer cell lines were treated against different concentrations of the

ethanolic leaf extracts of the plants for studying their viability, apoptosis/ necrotic and the

cytotoxic nature.

The cancer cell lines were trypsinized, centrifuged and a cell count was taken with

the help of haemocytometer and then seeded into the 96 well plates. These plates were than

incubated for 24 hrs at 37oC at 5% CO2. The Tryphan Blue staining Hoechst‟s staining and

MTT Assay were carried out for all the three cancer cell lines.

Methods used:

Preparation of media:

Media is available as ready to use 1X media or as lyophilized powder. Ready to use

media has a shelf life of one month at 40C

. Lyophilized powder can be reconstituted by

dissolving the contents in double distilled water. Procedure for preparation of media:

1. Weigh 10 gm of media that is available in powder form.

2. Dissolve in 600-700ml of sterile double distilled water and make up the volume to

1000ml.

3. Add 3.7 gm of sodium bicarbonate as buffering agent which reacts with the phenolic

compounds present in the media and gives a pink colour to the media.



4. This pink colour indicates the normal pH of 6.8-7.0. To maintain the normal pH for

longer time, CO2 incubator is used.

5. The reconstituted media is filter sterilized through 0.22 medium membrane filters

using a filter assembly.

6. Media has to be tested for sterility before use.

Preparation of the sample extract:

The sample extract is syringe filtered to avoid contamination.

Thawing of the cells

It involves bringing the freezed ampoule to room temperature by slow agitation i.e

by slow thawing or by rapid agitation in water bath i.e. rapid thawing. It is vital to thaw the

cells correctly in order to maintain the viability of the culture and enable the culture to

recover more quickly. Cryoprotectants help in preserving the cells from damage during

freezing. Some cryoprotectants such as DMSO are toxic above 40C. Therefore it is essential

that cultures are thawed quickly and diluted in culture medium to minimise the toxic

effects.

Procedure:

1. The freezed cryovials plunged into the water bath (370C).

2. The sample is rapidly thawed until it gets liquefied.

3. The solution is transferred to a 15ml centrifuge tube and saline is added and

centrifuged at 1500rpm for 10 minutes. Centrifugation is done to remove the DMSO

as it becomes toxic when it reaches room temperature.



4. The saline is discarded and minimum amount of DMEM is added to it.

5. Some aliquot is taken for cell counting and for cell viability.

Trypisination and sub culturing of the cells:

After the cells are grown they adhere to the culture dish and make a monolayer.

Trypsin is added to the culture dish before they reach their confluent stage. Trypsin, an

enzyme commonly found in the digestive tract, can be used to “digest” the proteins that

facilitate adhesion to the container and between the cells, this is known as trypsinisatiion.

Thus the detached cells from the culture dish are subcultured with the fresh medium. The

subculture of these cells from one vessel to another usually involves the sub-division of a

proliferating cell population.

Procedure:

1. Discard the media from the T- flask using the sterile micropipette.

2. Wash the inner surface of the flask with sterile saline.

3. Add 500µl of trypsin-EDTA into the flask.

4. Rotate the flask for uniform mixing and wait for 3-5 minutes.

5. Add 1ml of 20% FBS+DMEM media into the flask and mix it well.

6. Transfer 500µl of cell suspension into another sterile flask.

7. Add fresh DMEM media of 1.5ml to both the flaks.

8. Incubate the flasks at 370C in 5%CO2incubator for 24 hrs.



Total cell count

It determines the number of cells in a cellular suspension. This is performed by using a

Haemocytometer. To calculate the total number of cells in the cell suspensions following

steps are used:

1. Take 10µl of the sample in an Eppendorf and add 20 µl of trypan blue to it and load

on the haemocytometer.

2. Four cells on the boundaries, only cells intersect two of the cells are counted.

3. Total number of cells is calculated by:

Total number of cells = Average count x dilution factor x 104 x final volume.

Cell viability

To determine the percentage of cells living.

Procedure:

1. Take 10µl of cell suspension and mix with 20µl of trypan blue in an Eppendorf tube.

2. Clean the surface of the glass slide and semi silver area of the haemocytometer using

alcohol.

3. Mix the sample thoroughly and load 10µl into the haemocytometer using a

micropipette.

4. Focus the slide under the microscope and count the dead (attained) cells and living

(unstained) cells.



5. Calculate the percentage viability by following formulae:

% viability = Number of living cells 100

Number of total cells (dead + living)

Cryopreservation:

It is a process where the cells are presented by cooling to low sub-zero temperatures

such as 77K or -1960C (the boiling point of liquid nitrogen). At these low temperatures any

biological activity, including the biochemical reactions that would lead to cell death is

effectively stopped.

Cryoprotectants or cryoprotective agents are used for this purpose. Cryoprotectants

are like anti-freeze, they lower the freezing temperature and they also increase the

viscosity. Water is the main component of all living cells and it must be available for

chemical processes of life to occur. Cellular metabolism stops when all water in the system

is converted to ice.

A basic principle of cryobiology is that the extent of freezing damage depends on

the amount of free water in the system and the ability of the water to crystallize during

freezing. Few examples of cryoprotectants are DMSO, glycerol etc. Procedure for

cryopreservation is as follows:

1. Remove the culture from the dish and collect in a tube.

2. Centrifuge at 1500rpm for 10 minutes.

3. Dissolve the pellet in minimum volume of the media.

4. Prepare freezing mixture (containing 70% media, 20% serum, 10% DMSO)

in ice bath.



5. Add freezing mixture to the cell suspension slowly in the ice bath.

6. Keep the cryovial in the slow cooling device.

7. Transfer the sample to long term storage device.

Drug dilutions

Dilution is a process of reducing the concentration by adding of a solution such as

water. The crude extracts were diluted according to the requirements. Before using the

extracts they were syringe flittered to avoid contamination.

Trypan Blue assay

Trypan blue is a vital stain used to selectively color dead tissues or the cells blue.

This assay is used to determine the dead cell count as well as the living cell count. The

living cells will have an intact membrane which does not allow the dye to pass since the

cells are very selective in compounds. The dead cell does not process an intact membrane

and takes up the stain.

Hoechst stain assay

This assay is done to check that the cell death has occurred due to apoptosis (in

which cells suffer a major insult, resulting in a loss of membrane integrity, swelling and

disruption of the cells) or not.

MTT assay

This is a colorimetric assay that measures the reduction of yellow 3- (4, 4-

dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate

dehydrogenase. The MTT enters the cell and passes into the mitochondria where



it is reduced to an insoluble, colored (dark purple) formazan product. The cells are then

solubilised with a solvent (DMSO) and the released solubilised formazan reagent is

measured spectrophotometrically. Since reduction of MTT can only occur in metabolically

active cells the level of activity is a measure of the viability if the cells.

MTT reagent preparation

Dissolve the MTT powder 5mg/ml in Hank‟s balanced salt solution (e.g. 100 mg

MTT/20 ml Hank‟s). When not in use protect the reconstituted MTT reagent from the light

and store at 40C. (Can be stored for several months). Stop mix solution: 20% SDS in 50%

Dimethyl formamide. Toxic: store at room temperature in a fume hood.

Protocol

Aseptic technique and good cell culture practice:

To ensure all cell culture procedures are performed to a standard that will prevent

contamination from the bacteria, fungi and mycoplasma and cross contamination with the

other cell lines.

Procedure:

1. Sanitize the cabinet and gloves using 70% ethanol and allow to air dry for 30

seconds before commencing the work.

2. Put all materials and equipments to the cabinet after sanitization.

3. Discard the gloves after all cell culture procedures and after handling contaminated

cultures.

4. All equipment taken into the cabinet must be washed with 70%ethanol.



5. Speech, sneezing and coughing must be directed outside the cabinet

6. After completing work disinfectant all equipment and material before removing

from the cabinet. Spray the work surfaces inside the cabinet with 70%ethanol and

wipe dry. Disposal of tissue should be done after autoclaving.

Plating of cell lines:

All the three cancer cell lines procured were in the lyophilized form in the ampoule which

was thawed, trypsinized and later sub cultured in the fresh media.

Procedure:

1. The flask in which the cells have reached the confluent stage is selected. The medium

is discarded and the cells are washed twice with the saline. The saline should be

discarded properly from the flask as saline inhibits the action of trypsin.

2. Add 500µl of trypsin-EDTA to the flask and wash the inner surface properly with

trypsin. Wait for 1-5 minutes until the cells get detached from the surface.

3. Add 2 ml of fresh complete media and aspirate and collect the complete solution in a

sterile centrifuge tube and centrifuge at 1500rpm for 10 minutes.

4. Discard the supernatant and add 1ml of fresh media and do the total cell count.

5. Pipette out 0.1 to 0.2 million cells and make up the volume upto 6ml.

6. Add 10µl of cell suspension to each well in the 96 well plates. The cell suspension

should be contamination. The cell suspension that is left over should be cryopreserved

7. The plate is kept in 5%CO2 incubator for 24hrs.



Drug addition

Procedure:

1. After 24 hrs of incubation of the cells, discard the spent media from all the well\s.

2. The 2nd

and 3rd

column was added with media and vehicle (10%distilled water made

in media) respectively. The first and second column was taken as control for

evaluating the extracts.

3. Add 100µl of diluted drugs to each well in the order of the lowest dilution to the

highest dilution.

4. The plate is incubated for 24 hrs in a CO2 incubator.

Trypan Blue assay

Procedure:

1. After 24hrs of addition of the drug, discard the spent media and drug.

2. Add 50µl of trypan blue is added to each of the ells from which media and drug has

been discarded. The plate is kept undisturbed for 1 minute.

3. The trypan blue is then discarded from the wells properly with the help of pipette.

4. The plate was observed under the microscope.

5. Repeat the same procedure after 48 hrs of addition of the drug.



Hoechst assay

Procedure:

1. This assay is performed after 48hrs of addition of the drug.

2. Add 100µl of Hoechst dye to the wells and incubate in dark for 10 minutes.

3. Switch on the fluorescent microscope and wait for 10 minutes to allow the microscope

to warm and emit the fluorescent light.

4. Observe the plate under the microscope and note down the observations.

MTT Assay

Procedure:

1. After 24 /48 hrs of addition of the drug this colorimetric assay is performed.

2. Add 20µl of MTT reagent to wells already containing the media as well as the drug.

3. Incubate the plate in the incubator for 3 hrs

4. After 3 hrs discard the MTT reagent along with the media and the drug and add 100µl of

DMSO to stop the reaction of MTT.

5. Keep the plate for incubation for 1 hr.

6. After incubation pipette out the suspension from each well into the plate reader.

7. Read the plate on a plate reader using 550nM as test wavelength and 630nM as the

reference wavelength.

8. Record the data and tabulate columns.

Documents

CHAPTER 3shodhganga.inflibnet.ac.in/bitstream/10603/38449/8/chapter 3.pdfCell lines: Hela, MCF 7 and the PC 12 cancer cell lines, were from American Type Culture Collection (ATCC),