shodhganga.inflibnet.ac.inshodhganga.inflibnet.ac.in/bitstream/10603/29717/10/10... · 2018-07-02 · Pangara (Marathi) or Mandara (Bengali, Sanskrit). It is grown on road side, residential

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In the present investigation, the extracts of stem bark and stem of Erythrina

suberosa Roxb. and extracts of whole plant of Anagallis arvensis L. was studied for their

anticarcinogenic properties using different in vitro assays and in vivo models.

3.1 Plant Material

3.1.1 Erythrina suberosa Roxb.

Division: Magnoliophyta

Class: Magnoliopsida

Order: Fabales

Family: Fabaceae

Sub Family: Faboideae

Tribe: Phaseoleae

Genus: Erythrina

Species: suberosa

Erythrina suberosa Roxb. belongs to the family Fabaceae, is a tall ornamental tree

(Plate 1). It is commonly known as Indian Coral Tree (English), Dhaul dhak (Hindi),

Pangara (Marathi) or Mandara (Bengali, Sanskrit). It is grown on road side, residential

area and public parks etc. due to its red bright flowers (Serrano et al., 2011). The

genus Erythrina is comprised of about 100 species in the tropics and famous for various

types of compounds like alkaloids, flavanoids and terpenes. Plants of this genus are known

to have cytotoxic activity (Balachandran and Govindrajan, 2005). E. suberosa Roxb. has

been used in India as a very important medicinal plant for the treatment of various

ailments. Parts of this plant that are widely used are the roots, leaves, seeds and bark. Its

flower extract with Hibiscus rosa sinesnsis is used as calming drink in summers, as it has

soothing and relaxing effect (Serrano et al., 2011). The ethanol extract of the leaves has

been reported to have antitumor activity (Dhar et al., 1968). Erysodine and erysotrine

present in ethanolic extract from flowers of E. suberosa contributed towards its anti

anxiety activity (Serrano et al., 2011).

The main chemical constituents of E. suberosa Roxb. are waxes, sterols, lectins,

and isoflavanoides (Singh et al., 1970; Bharracharyya et al., 1986; Tanaka et al., 2001).

Singh et al. (1970), also isolated alkaloids i.e. erythralline, erysidine, erysotrine and

hypaphorine from seeds of E. suberosa. They further stated that unsaponifiable matter of

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the seed oil is composed of sitosterol, stigmasterol, campesterol, and cholesterol. The

petroleum ether extract of the bark of E. suberosa was fractionated into wax esters, wax

alcohols and acids, alkyl ferulates, stigmasterol, sitosterol, campesterol and cholesterol

(Singh et al., 1970).

In the present investigation, stem bark and stem of Erythrina suberosa Roxb. was

identified and collected locally in the month of April from the plant near Indian Institute of

Integrative Medicine, Canal Road, Jammu, India. A specimen of the collection was

submitted to the herbarium of Department of Botany, Indian Institute of Integrative

Medicine, Jammu, India (voucher specimen no. 20617).

3.1.2 Anagallis arvensis L.

Division: Magnoliophyta

Class: Magnoliopsida

Order: Primulales

Family: Primulaceae

Genus: Anagallis

Species: arvensis

Anagallis arvensis L. the common scarlet pimpernel belonging to family

primulaceae is a creeping annual herb with simple leaves and solitary axillary bisexual

tubular flowers (Plate 2). It grows preferably on waste lands and sandy grounds. In India it

is popularly known as “jonkmari”. It is a common medicinal plant of Kashmir and

Ladakh, India and known as “Chari Saben” in the region (Kaul, 1997). This plant is also

reported to be used as fodder with a local name “Mangotai” (Zabihullah, 2006). A.

arvensis is known for a variety of medicinal uses. In indigenous system of medicine the

plant is used for treatment of gout, leprosy, and cerebral infections (Kaul, 1997). Its whole

plant is reported to be used for bronchial asthma, as sedative and stimulant (Tiwari, 2008).

Its ethnomedicinal use is as diuretic, expectorant and in lowering fever (Hussain et al.,

2008; Kaul, 1997). In China it is folklore remedy for snake bite, hydrophobia and also has

antiviral activity against herpes simplex virus type and polio virus (Bajaj, 1999). Ethyl

acetate and dichloromethane extracts from A. arvensis has antifungal properties against

Rhizopus stolonifer (Lopez et al., 2008). It has also been used for gall stones, liver

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cirrhosis, lung problems, urinary infections, kidney stones and as detoxifying agents. A.

arvensis is also used as a hepatic therapy in Taiwan (Miro, 1995).

The main chemical constituents of A. arvensis that are reported in literature are

triterpenoids, saponins, arvenins and cucurbitacins from various parts of plant (Amoros

and Girre, 1987; Miro, 1995; Yamada, 1978b). Amoros and Girre, 1987 isolated two novel

saponins 3-O-glucose (1-3 or 4) [arabinose 1-4 or 3]-glucose (1-2)-xyloside of 23-hydroxy

protoprimulagenin-A. The second saponin has an additional glucose. Heitz et al. (1971)

first reported the presence of anagalligene B, a new triterpene which is epoxy-13,28-ceto-

16-oleananedioll-3�,23,2a. Five new triterpene saponins i.e. anagalloside A, B, C and

desglucoanagalloside A and B were reported by Glombitza and Kurth (1987). Shoji et al.

(1994a) isolated six saponnins, anagallosaponin 1-5 and methylanagallosaponin-I. Shoji et

al. (1994b) also isolated four oleanane glycosides, anagallosaponin 6-9 and two artifact

saponins apoanagallosaponin III and IV.

In the present investigation, whole plant of Anagallis arvensis was collected in the

month of June from the field station Bonera of Indian Institute of Integrative Medicine,

Srinagar Branch, Srinagar, India. A voucher specimen (No. IIIM-S010050) was deposited

in the herbarium of the institute after authentication of the plant.

3.2 Preparation of extracts

The plant materials were thoroughly washed with tap water, dried under shade and

ground to fine powder and extracted employing maceration method. The powdered stem

bark, stem of Erythrina suberosa and whole plant of Anagallis arvensis were extracted

with 95% alcohol, 50% aqueous alcohol and water. The extracts were filtered and

concentrated using a rotavapor and evaporated to dryness. A schematic representation of

extraction and fractionation procedure from plants is shown in Flow chart 1.

3.2.1 Preparation of 95% alcoholic extract

Dried plant material (2 kg) was placed in a conical glass percolator, submerged

with 95% ethanol and after standing for 16 h (overnight), the percolate was collected. This

process was repeated four times, which was generally sufficient for exhaustive extraction

of the plant material. However, few drops of the last percolate was placed on a watch glass

and allowed to evaporate, if no residue was left the extraction was considered as

complete, otherwise it was repeated till complete extraction was obtained. The combined

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ethanolic extract was evaporated to dryness under reduced pressure at 50°C. The final

drying was done initially in a vacuum desiccator and finally in lyophilizer. The dried

extract was scrapped off and transferred to a tared wide mouth glass container after

weighing. Nitrogen was blown in the container before capping and stored at -20°C under

desiccation.

3.2.1.1 Preparation of fractions from alcoholic extract (95%)

The alcoholic extract (95%) was subjected to flash chromatography. 3 g of

alcoholic extract (95%) was added to 5 g of silica gel (200-400 mesh) and dissolved in

ethanol, to form slurry. The slurry was dried to fine powder and loaded on ~5 cm silica gel

band (200-400 mesh) in a 150 ml column. The column was eluted sequentially with

petroleum ether 60-80°C (2 x 150 ml), ethyl acetate (3 x 70ml), 95% ethanol (2 x 75 ml)

and 50% ethanol water (2 x 50ml). Thus, in all, 9 fractions were collected and were

concentrated on rotavapour at <45°C, under reduced pressure. Fractions of E. suberosa

were named as ESB-PE1, ESB-PE2, ESB-EA1, ESB-EA2, ESB-EA3, ESB-ET1, ESB-

ET2, ESB-EW1 and ESB-EW2 respectively and that of A. arvensis were named as AAE-

PE1, AAE-PE2, AAE-EA1, AAE-EA2, AAE-EA3, AAE-ET1, AAE-ET2, AAE-EW1 and

AAE-EW2 respectively. These fractions were then tested for their cytotoxic potential.

3.2.2 Preparation of 50 % alcoholic extract

Another lot of the dried plant material (300 g) was extracted in the same manner as

described for 95% alcoholic extract except that 50% aqueous-alcohol was used instead of

alcohol.

3.2.3 Preparation of aqueous extract

The third lot of dried plant material (200 g) was heated with 1.2 � of distilled water

on a steam bath in a flask (corning glass, wide mouth, round bottom) for 4 hrs. The extract

was decanted and filtered. The process was repeated four times using additional amount of

distilled water. The combined extract was evaporated on a steam bath under vacuum. Final

drying was done in a lyophilizer. The dried extract was scrapped off and transferred to a

tared wide mouth glass container after weighing. Nitrogen was blown in the container

before capping and stored at -20°C under dessication.

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3.3 HPLC Analysis

3.3.1 HPLC profile of Erythrina suberosa extract

Extract profile of E. suberosa was generated using Waters HPLC which included a

binary pump (Waters 515 HPLC pumps), in line vacuum degasser, thermostatic column

compartment, a UV- detector (Waters 2996 PDA) and autosampler (Waters 717 plus

autosampler). Samples were injected onto a Li chrospher RP-18 (250 mm x 4.0 mm, 5µm,

Merck) column with a mobile phase containing acetonitrile (mobile phase A) and water

(mobile phase B) with the following gradient profile: in the first 15 min from 50-65% B,

then a linear rise to 100% of B in next 15 min, followed by 100-50% of B in last 15 min,

flow rate: 1ml/min; detection wavelength 280nm; injection volume: 10µl.

3.3.2 HPLC profile of Anagallis arvensis extracts

A Shimadzu HPLC system equipped with CBM-20A controller, LC-20AT tandem

piston pump, SIL-20AC autosampler, SPD-M20A photodiode array detector and CTO-

10ASVP column thermostat was used. Each sample to be tested was dissolved in HPLC

grade methanol at a stock concentration of 1mg/ml and filtered using 0.2 µM filters. A

method was developed using a LiChrospher®

100 RP-18e (250 mm × 4.0 mm, 5 �m)

column with a mobile phase containing HPLC grade acetonitrile (mobile phase A) and

HPLC grade water (mobile phase B) with the following gradient profile was finalised: In

the first 45 min from 100–0% B, followed by 0% of B in last 5 min. Flow rate was

1ml/min; injection volume: 10µl; detection wavelength: 265 nm and run time was 50 min.

3.4 Chemicals and Reagents

3.4.1 Chemicals Required

Following chemicals were used in the present study: 5-Fluorouracil, Dimethyl

sulfoxide (DMSO), Doxorubicin, Fetal bovine serum (FBS), Gentamycin, Minimum

Essential Medium (MEM), Mitomycin C, Paclitaxel, Penicillin, Phosphate Buffer Saline

(PBS), Roswell Park Memorial Institute-1640 (RPMI-1640) medium, Streptomycin,

Sulphorhodamine B (SRB), MTT and Trypsin were of cell culture grade and procured

from Sigma Chem. Co., USA. Ethylenediamine tetra acetic acid disodium salt (EDTA),

Glacial acetic acid, Isopropyl alcohol, Trichloroacetic acid, Tris buffer and Sodium

bicarbonate were procured from HiMedia Laboratories Pvt. Ltd., Mumbai or Sisco

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Research Laboratories. Pvt. Ltd., Mumbai. Rest all the chemicals were procured locally

and were of analytical grade.

3.4.2 Reagents prepared

3.4.2.1 Media

The contents of the vials of RPMI-1640 with 2 mM L-glutamine, DMEM or MEM

as supplied, were dissolved in double distilled water as per supplier’s instructions.

Streptomycin (100 mg/�) and sodium bicarbonate (1.2 g/�) were also added to medium. The

pH of medium was adjusted to 7.2 and it was sterilized by filtering through 0.2 µM filters

in laminar flow under sterile conditions. This is incomplete media and was stored in

refrigerator (2-8°C). For the complete growth medium, the medium was supplemented

with 10% FBS and penicillin (100 IU/ml, before use). For cryopreservation of adherent

cell lines, incomplete media was supplemented with 10% DMSO and 20% FBS; for

suspension cell lines 10% DMSO in FBS was used.

3.4.2.2 Phosphate buffer saline

The contents of a vial of PBS were dissolved in double distilled water, diluted upto

1 � and filtered with 0.2 µM filters under sterile condition.

3.4.2.3 Trypsin-EDTA

50 mg Trypsin (0.05%) and 20 mg EDTA, disodium salt (0.02%) were dissolved in

PBS, diluted upto 100 ml and filtered with 0.2 µM filter under sterile condition.

3.4.2.4 Trichloro acetic acid

50% (w/v) TCA solution was prepared in double distilled water.

3.4.2.5 Acetic acid

Glacial acetic acid was diluted to 1% with double distilled water.

3.4.2.6 SRB Dye

400 mg of SRB dye was dissolved in 1% acetic acid and diluted upto 100 ml

(0.4%w/v).

3.4.2.7 Tris-buffer

1.21 g of Tris was dissolved in 950 ml distilled water; pH was adjusted to 10.5 and

diluted upto 1 �, to make 10 mM Tris-buffer.

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3.4.3 Preparation of test material

Dimethyl sulphoxide (DMSO) is often used as a solvent to dissolve test materials.

It is cytotoxic at high concentrations but has been found to have no effect in the cells

below 1% w/v. The solvents used for dried 95% alcoholic, 50% alcoholic and aqueous

extracts were DMSO, 50% aqueous-DMSO and distilled water respectively. A stock

solution of 20 mg/ml of each was prepared and were serially diluted with complete growth

medium containing 50 µg/ml of gentamycin to obtain working test solutions, as per the

requirement.

3.4.4 Preparation of Positive controls

A stock solution of 2x10-2 M concentration of the positive control was prepared.

The solvent used for Paclitaxel and Doxorubicin was DMSO while 5-fluorouracil and

Mitomycin C were dissolved in distilled water. Aliquots of the stocks were stored at -

20°C.

3.5 Cell lines

3.5.1 Sources of cell lines

Original stock of Human cancer cell lines was received in frozen state (in dry ice)

in cryovials, from National Cancer Institute (Fredrick, USA), American Type Culture

Collection (Virginia, USA) and some cell lines were obtained from National Center for

Cell Science (Pune, India), in culture flasks.

3.5.2 Cell lines studied

The following cancerous cell lines: MCF-7, T-47D (human breast

adenocarcinoma), SF-295 (human central nervous system), COLO 205, HCT-15, SW-620,

502713 (human colorectal adenocarcinoma) , HEP-2 (human liver adenocarcinoma), A-

549, HOP-62 (human lung carcinoma), IMR-32 (human neuroblastoma), OVCAR-5, IGR-

OV-1 (human ovary adenocarcinoma), DU-145, PC-3 (human prostate adenocarcinoma),

HL-60 (human leukemia) and 786-0 (human renal cell adenocarcinoma) were used in the

present study. Two normal human lung fibroblast cell lines: MRC-5, WI-38 (human lung

fibroblast) and CV-1 (monkey kidney normal) cell line, were also used. The cell lines

were grown on RPMI-1640 or MEM growth medium depending on cell type, as

recommended. Specific growth media, cell density and positive controls used for the cell

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lines, has been summarized in the Table 3. Most of the cell lines are included in the panel

recommended by NCI, USA (Monks et al., 1991).

3.5.3 Handling of cell line on arrival and revival

The stock of human cancer cell lines received in frozen state was transferred to

liquid nitrogen immediately upon arrival and were used as per requirement. For reviving,

cryovials containing cells were removed from the liquid nitrogen container and thawed

quickly by shaking in water bath at 37°C. Cryovials were wiped with 70% alcohol to

avoid contamination and transferred to Bio-safety cabinet. The contents of the vial were

transferred into a sterile centrifuge tube containing 10 ml complete growth medium and

centrifuged at 500g for 5 min. Supernatant was discarded and the cells were suspended in

fresh complete medium. Cells were mixed properly to ensure uniform distribution in the

medium. Cells were transferred asceptically to the tissue culture flask containing 7 ml of

complete growth medium aseptically and incubated in CO2 incubator at 37°C, 5% CO2

and 95% relative humidity (RH). Cells were daily checked for proper growth under

inverted phase contrast microscope. The medium of the cells was changed when the color

turned yellow due to change in pH. The medium was discarded by aspirating and replaced

with the fresh medium (5-7 ml in case of TCF-25) under sterile conditions. Depending on

the mass doubling time of cells, sub-culturing of cells was done, when they were at sub-

confluent stage.

3.5.4 Subculturing of the cell lines

It involves detachment of the cells from the surface (substratum) of the culture

flask and reinoculation of the cells into fresh medium in new culture flask i.e TCF-25,

TCF-75 or TCF-150, depending on the quantity of cells, for adherent cell lines. The

medium of the flask at sub-confluent growth was changed one day in advance. The entire

medium from the flask was discarded and cells were washed with PBS. The minimum

quantity of Trypsin-EDTA (pre warmed at 37°C) was added just enough to make a thin

layer and incubated for approximately 5 min at 37°C. After detachment, cell suspension

was made with complete growth medium. An aliquot was taken out and cells were counted

with haemocytometer and checked for viability with trypan blue. Cell stock with more

than 98% cell viability was accepted for determination of in vitro cytotoxicity. The cell

density was adjusted according to requirement by the addition of complete growth

medium and inoculated in to fresh TCF-75 or TCF-150 and incubated in CO2 incubator to

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continue the culture. For suspension cell line change of medium was done by

centrifugation at 500g for 5 min. After centrifugation, the supernatant was discarded and

the pellet of cells was resuspended in fresh complete medium in a fresh and sterile TCF,

for continuation, after checking the viability by trypan blue. The density of the freshly

counted cells were adjusted, depending on cell line, according to their cell density

required, and seeded in sterile 96 well or 6 well plates, for experimental purpose.

3.5.5 Stages of cell growth during culturing

The cells were observed under the microscope, which appears roughly round in

shape, when seeded in the culture flask. The adherent cell lines attach to the surface of the

TCF and grow in a monolayer. Different stages of cell growth during culturing, of few cell

lines, are shown in Fig. 2 A, B, C.

3.5.5.1 Attachment Stage

Within 24 h of incubation, after seeding, the cells get attached to the base of tissue

culture flask.

3.5.5.2 Sub-confluent Stage

It is a stage of rapid growth of cells. At this stage, the cells are in log phase of their

growth and spaces remain between the growing cells. Cells in this stage can be used for

experimental purpose, sub-cultured or cryopreserved.

3.5.5.3 Confluent Stage

The cells form a complete monolayer, leaving no space between the cells.

3.5.6 Suspension cell line and its maintainence

Human leukemia HL-60 cell line was used for the mechanistic studies. HL-60 cells

were seeded and grown in tissue culture flasks in complete growth medium (RPMI-1640)

at 37ºC in an atmosphere of 5% CO2 and 95% RH in a carbon dioxide incubator. Cells

were checked daily for proper growth. Medium was changed when its color became

yellow. The contents of the flask were transferred to a 50ml centrifuge tube aseptically and

centrifuged at 1000 rpm for 10 minutes, discarded the supernatant and resuspended the

pellet in fresh RPMI-1640 medium in a fresh TCF-75 flask.

3.5.7 Preparation of cell suspension

The adherent cells at sub-confluent stage, healthy and with no sign of

contamination are used for in vitro experiments. An aliquot was taken and the cells were

counted by using haemocytometer to measure the cell density of the original suspension.

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The cells were harvested from the TCF after trypsinization and single cell suspension was

prepared. The cell suspension was diluted with appropriate growth medium to obtain the

cell densities (5x104 to 1x10

6 cells/ml) depending on the cell line and experiment.

3.5.8 Cryopreservation

In order to minimize genetic drift in cell lines, senescence or transformation in

infinite cell lines and guard against accidental loss by contamination or otherwise, it is

common practice to cryopreserve aliquots of cells in liquid nitrogen vapours. Briefly, in

adherent cell lines, cells were trypsinised and centrifuged at 500g. Cells with more than

98% viability, as determined by trypan blue exclusion technique were cryopreserved. The

pellet was re-suspended in cryopreservation medium (20% FBS, 10% DMSO, 70%

incomplete medium). Similarly, the suspension cell lines were suspended in freezing

medium containing 90% FBS and 10% DMSO. The aliquots of 1.0 ml were transferred

into cryovials. The temperature of the vials was brought down to -80ºC by using a

specially designed box in a deep freezer overnight. Later, the cryovials are transferred into

liquid nitrogen container.

3.6 In vitro assays

Since the major approach in searching the potential anticancer agents over the last

50 years has been based on selective cytotoxic effects on mammalian cancer cell lines,

therefore, cell-based methods for cytotoxicity are used. Cells are cultured in a 96 well flat

bottom microtitre plate and the rate of multiplication and growth is measured indirectly by

formation of a colour. The intensity of colour is directly proportional to the number of

cells present. A variety of experiments can be used and the most basic is to compare the

rate of proliferation of a cancer cell lines in the presence and absence of the test substance,

usually after a specified time. Ideally, different cancer cell lines can be used so that

selectivity can be assessed and the addition of normal cell lines to the battery enables

selectivity between cancer cell lines and normal cell lines.

3.6.1 Sulphorhodamine-B assay

Principle: Sulphorhodamine-B is a rapid, sensitive, and inexpensive method for

measuring the cellular protein content of adherent and suspension cultures in 96-well

microtiter plates. This assay provides a colorimetric end point that is nondestructive,

indefinitely stable, and visible to the naked eye. SRB is a bright pink aminoxanthene dye

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with two sulfonic groups. Under mild acidic conditions, it binds to protein basic amino

acid residues in TCA-fixed cells to provide a sensitive index of cellular protein content

(Monks et al., 1991; Skehan et al., 1990; Houghton et al., 2007).

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Reagents:

S.No. Chemicals or Reagents Molecular Wt. Concentration used

1. Sulphorhodamine-B 580.65 0.4 % (w/v)

2. Trichloro-acetic acid 163.39 50 % (w/v)

3. Acetic acid 60.05 1% (v/v)

4. Tris buffer 121.14 10 mM

Procedure: To each well, 100µl of cell suspension was added. After 24 hours, when a

partial monolayer was formed, 100 �l of different concentrations of test samples were

added to the cells in cell culture plates. The plates were then incubated at 37oC for 24, 48

or 72 hours in a CO2 incubator (37°C, 5% CO2, 95% RH). After incubation, 50 �l of

chilled 50% trichloroacetic acid was added to the wells gently, in such a manner that it

forms a thin layer over the contents of the well, to form effective concentration of 10%.

The plates were incubated at 4oC for one hour. The plates were flicked and washed five

times with tap water to remove traces of medium, sample and serum, and were then air-

dried. The air-dried plates were stained with 100 �l SRB (0.4%) and kept for 30 minutes at

room temperature. The unbound dye was removed by rapidly washing four times with 1%

acetic acid. The plates were then air-dried. 100 µl of 10mM Tris buffer was then added to

the wells to solubilize the dye. The plates were shaken vigorously for 5 minutes. The

absorbance was measured using microplate reader at a wavelength of 540nm (Fig. 3). The

percentage growth inhibition was calculated using following formula:

% Growth inhibition = 100 – [OD(test sample) – OD(blank)/ OD(control) – OD(blank)] x 100

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3.6.2 MTT colorimetric assay

Principle: This is a colorimetric assay that measures the reduction of 3-(4, 5-

dimethylthiasol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) by mitochondrial succinate

dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is

reduced to an insoluble, colored, formazan product. The cells are then solubilised with an

organic solvent (DMSO or isopropanol) and the released, solubilized formazan reagent is

measured spectrophotometrically (Yedjou et al., 2006).

Reaction:

Reagents:

S.No. Chemicals or Reagents Molecular wt. Concentration used

1. 3-(4, 5-dimethylthiasol-2-yl)-2,

5,- diphenyltetrazolium bromide

(MTT)

414.32 2.5 mg/ml

2. Dimethyl sulphoxide (DMSO) 78.13 100 %

Procedure: Following the procedure followed by Ka et al. (2003), different

concentrations of sample were added to the wells already containing the HL-60 cells (4 x

104) and incubated in a time dependent manner, in a CO2 incubator. 2-3 h prior the

required incubation 20 µl of MTT (2.5 mg/ml) was added and the plates were incubated to

complete the incubation period. After this plate was centrifuged at 1000g for 10 min,

followed by discarding the medium. The formazan blue crystals, formed, were dissolved

with DMSO (100 µl/ well). The optical density was measured at 540 nm (Fig. 4). The

percentage viability was calculated using following formula:

% Viability = 100 x [OD (test sample) – OD (blank)/ OD (control) – OD (blank)]

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3.6.3 BrdU Cell Proliferation Assay

Principle: The BrdU cell proliferation assay involves incorporation of BrdU into cells

cultured in plates and BrdU immunolabeling using the cell layer as the solid phase. During

the final 2 to 24 h of culture, BrdU is added to wells of the plate. BrdU will be

incorporated into the DNA of dividing cells. To enable antibody binding to the

incorporated BrdU cells must be fixed, permeabilized and the DNA denatured. This is all

done in one step by treatment with Fixative/Denaturing Solution. Detector anti-BrdU

monoclonal antibody is pipetted into the wells and allowed to incubate for 1 h, during

which time it binds to any incorporated BrdU. Unbound antibody is washed away and

horseradish peroxidase-conjugated goat anti-mouse is added, which binds to the detector

antibody. The horseradish peroxidase catalyzes the conversion of the chromogenic

substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or

yellow after the addition of stopping reagent), the intensity of which is proportional to the

amount of incorporated BrdU in the cells. The colored reaction product is quantified using

a spectrophotometer.

Procedure: The method of Tura et al., 2007 was followed, with some modifications.

Briefly, cells (1.5x105) were seeded in 96 well microtiter plate and treated with test

material. Before the end of desired time of drug exposure, 20 µL of 5-bromo-20-

deoxyuridine (BrdU, 10 mM) was added to each well and incubated for 3 h at 37°C after

removing content of well. The wells were then washed and per-oxidase goat anti mouse

IgG HRPconjugate was added and incubated the plate for 30 min at RT. The substrate was

added and plates were kept in dark for 15 min. Finally the stop solution was added and

reading was taken at 450 nm to calculate the percent BrdU incorporation.

3.7 In vivo Anti-cancer studies

The samples which were found highly active in in vitro cytotoxic assay i.e. SRB

dye assay, against a panel of human cancer cell lines, were further tested for their in vivo

anticancer activity system using various murine tumor models (Plate 3).

3.7.1 In vivo Anti-cancer activity against Ehlrich Ascites Carcinoma (EAC)

In vivo studies for anticancer activity in mouse models were conducted as per the

guidelines of the National Cancer Institute (NCI), USA. (Geran et al., 1972). Ehrlich

ascites carcinoma (EAC) cells were collected from the peritoneal cavity of the Swiss mice

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harboring 8– 10 days old ascitic tumor. 1×107 EAC cells were injected i.p. on day 0. On

next day, animals were randomized and divided into different groups. The treatment

groups contained 7 animals each and control group contained 10 animals. Treatment

groups were treated with different doses of extract i.p. from day 1 to 9. One treatment

group received 5-fluorouracil (20 mg/kg, i.p) and it served as positive control. The tumor

bearing control group was similarly administered normal saline (0.2 ml, i.p.). On day 12,

animals were sacrificed and ascitic fluid was collected from the peritoneal cavity of each

mouse for the evaluation of tumor growth. Percent tumor growth inhibition was calculated

based on the total number of tumor cells present in the peritoneal cavity as on 12th day of

the experiment, using the following formula:

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3.7.2 In vivo Anti-cancer activity in Sarcoma-180 (Ascites)

Sarcoma-180 (Ascites) cells were collected from the peritoneal cavity of the

BALB/c mice harbouring 8-10 days old ascitic tumor. 1x107 EAC cells were injected i.p.

in BALB/c mice selected for the experiment on day 0. The next day, animals were

randomized and divided into different groups. The treatment groups contained 7 animals

each and control group contained 10 animals. Two treatment groups were treated with

drug from day 1-9. Another treatment group received 5-fluorouracil (20 mg/kg, i.p) and it

served as positive control. The tumor bearing control group was similarly administered

normal saline (0.2 ml, i.p.). On 12th day, animals were sacrificed and ascitic fluid was

collected from peritoneal cavity of each mouse for the evaluation of tumor growth.

Percent tumor growth inhibition was calculated based on the total number of tumor cells

present in the peritoneal cavity as on 12th day of the experiment using the following

formula:

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��

��

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41

3.7.3 In vivo Anti-cancer activity in Sarcoma-180 (Solid)

Sarcoma 180 cells were collected from the peritoneal cavity of the BALB/c mice

harbouring 8-10 days old ascitic tumor. 1x107 cells were injected intramuscularly in right

thigh of swiss male mice selected for the experiment on day 0. The next day, animals

were randomized and divided into groups. The treatment groups contained 7 animals each

and one control group contained 10 animals. The third treatment group was treated with

5-fluorouracil (22 mg/kg, i.p) from day 1-9 and it served as positive control. The control

group was similarly administered normal saline (0.2 ml, i.p.) from day 1-9. On day 9 &

13, tumor bearing thigh of each animal was shaved and longest and shortest diameters of

the tumor were measured with the help of vernier caliper. Tumor weight of each animal

was calculated using the following formula.

Length (mm) x [width(mm)]2

Tumor weight (mg) = -------------------------------------

2

The percent tumor growth inhibition was calculated on 13th

day by comparing the

average values of treated groups with that of control group. Tumor growth in saline treated

control animals was taken to be 100%.

3.7.4 In vivo Anti-cancer activity against Lymphoid leukemia model

L1210 lymphoid leukemia cells grown in the peritoneal cavity of DBA/2 female

mice were collected from the animal harboring 6–7 days old ascites. For testing, CDF1

males were used. Cells (5 x 105) were injected i.p. in CDF1 males weighing 18–23 g on

day 0. The next day, animals were randomized and divided into groups, containing seven

animals each. Treatment groups were treated with required doses of extract i.p. from day 1

to 9. One group received 20 mg/kg (i.p.) of 5-fluorouracil as positive control. Another

group served as control and it received 0.2 ml normal saline (i.p.) for nine consecutive

days. The animals in each group were observed for mortality up to day 18 and the median

survival time of animals in each group was calculated and %T/C values were obtained

using the following formula:

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��$�� %��

�� &�� !��"&#�

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42

3.8 In vitro apoptotic Mechanistic studies

The main goal of this study was to gain information about the mode of cell death

caused by test samples. Induction of apoptosis in cancer cells has been a novel approach

for innovative mechanism - based anticancer drug discovery (Fisher, 1994; Workman,

1996). It is thus important to screen apoptotic inducers from plants, either in the form of

crude extracts or as components isolated from them. To know apoptosis in cancer cells,

mechanistic mulitiparamartic assay were done such as detection of morphological changes

in cancer cell lines caused by tested compounds through fluorescence microscopy,

scanning electron microscopy, Cell cycle analysis, Annexin V, changes in mitochondrial

membrane potential through flow cytometry, Caspase cascade and DNA fragmentation

(Table 4).

3.8.1 Microscopic Analysis

3.8.1.1 Light microscopy

Following the method described by Baskic et al. (2006), the cells after treatment

were centrifuged and a thin smear was made on a glass slide and fixed for one minute in

methanol and stained for 10 minutes with modified Giemsa stain. The morphology was

studied under Olympus Research Microscope and the photography was done using an

Olympus Digital Camera (C4000).

3.8.1.2 Fluorescence microscopy

Cells (5x105 cells/ml) were grown in six wells plate and allowed to adhere for 24 h

in case of adherent cells. The different concentrations of the test sample was added and

incubated for required time intervals.Treated cells were removed from the six well plate

(by trypsinization, in case of adherent cells), centrifuged and re-suspended in PBS to fix

the concentration of cells i.e. 1 x105 cells/ml. The cells were then stained and observed

under a fluorescent microscope as described below.

3.8.1.2.1 Hoechst 33258 staining

Principle: Hoechst 33258 is a membrane-permeable, fluorescent DNA stain with that

intercalate in A-T regions of DNA. Nuclear integrity can be visualized by staining cells

with Hoechst 33258, which penetrates nuclei and binds to DNA (Al-Molawi et al., 2003).

Normal nuclei exhibit blue chromatin with organized structure and apoptotic nuclei exhibit

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43

bright fluorescent blue chromatin which is highly condensed or fragmented when stained

with Hoechst.

Reagents:


1. bisBenzimide H 33258 533.88 1 mg/ml in PBS

2. Formaldehyde 30.03 4% (v/v)

Hoechst 33258

Procedure: The morphology of cells exposed to test material was observed firstly under

inverted microscope. Then treated and untreated cells were fixed on a glass slide in 4%

paraformaldehyde for 20 min, washed with PBS and stained with Hoechst 33258 (1 mg/ml

in PBS) for 10 min (10). Stained cells were washed twice with PBS. The changes in nuclei

were observed with a fluorescent microscope (Olympus IX70, USA) through UV-filter.

3.8.1.2.2 Acridine orange/Ethidium bromide dual staining

Principle: Acridine orange is taken up by both viable and nonviable cells and emits green

fluorescence if intercalated into double stranded nucleic acid (DNA) or red fluorescence if

bound to single stranded nucleic acid (RNA). Ethidium bromide is taken up only by

nonviable cells and emits red fluorescence by intercalation into DNA. Four types of cells

according to the fluorescence emission and the morphological aspect of chromatin

condensation in the stained nuclei were distinguished: (1) Viable cells have uniform bright

green nuclei with organized structure (PMNCs also have orange cytoplasm). (2) Early

apoptotic cells (which still have intact membranes but have started to undergo DNA

cleavage) have green nuclei, but perinuclear chromatin condensation is visible as bright

green patches or fragments. (3) Late apoptotic cells have orange to red nuclei with

condensed or fragmented chromatin. (4) Necrotic cells have a uniformly orange to red

nuclei with organized structure (Baskic et al., 2006).

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Reagents:


1. Acridine orange 265.35 100 µg/ml

2. Ethidium bromide 394.31 100 µg/ml

Procedure: Fluorescent dyes, ethidium bromide (100 µg/ml) and acridine orange (100

µg/ml) were added and the cells were incubated for 10 min at room temperature in dark.

Cell suspension (40 µl) was placed on a microscope slide, observed and photographed

under an inverted fluorescence microscope (Olympus 1X70, magnification 30X and 20X,

NB filter) using UV excitation (Kania et al. 2007)

3.8.1.3 Scanning Electron Microscopy

Cells were grown on plastic cover slips in case of adherent cells in a 6 well plate

and allowed to adhere for 24 hours in a humidified chamber RH 95% containing 5 % CO2

at 37 ºC. After 24 hours the growth medium was replaced with fresh medium containing

test material. For suspension cell line, cells were grown directly in six wells plate. The

samples for SEM were prepared following standard techniques (Rello et al., 2005), which

required fixing of cells in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at 4˚C.

After washing with buffer, the cells were post fixed in 1% osmium tetraoxide in the same

buffer at 4˚C, followed by dehydration with ethanol/acetone, cleared in amyl acetate and

critical point-dried using CO2, followed by coating with carbon and gold. The samples

then were examined by JEOL 100CXII Electron Microscope with ASID at 40KV.

3.8.2 Cell cycle analysis

Principle: The most commonly used dye for DNA content/cell cycle analysis is

propidium iodide (PI). It can be used to stain whole cells or isolated nuclei. The PI

intercalates into the major groove of double-stranded DNA and produces a highly

fluorescent adduct that can be excited at 488 nm with a broad emission centered around

600 nm. The histogram data from flow cytometric analysis will yield the percentage of

cells in the G1 (2n DNA content), S (between 2n and 4n DNA content), and G2/M (4n

DNA content) phases of the cell cycle.

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45

Reagents:


1. Propidium iodide 668.39 1 mg/ml

2. Ethanol 46.07 70% in PBS

Procedure: Cells (1x106 cells/ml) in exponential phase were grown in six well plate and

allowed to adhere for 24 h in case of adherent cells. The different concentrations of test

sample was added to the cells and incubated for required time intervals. Cells were

(trypsnised in case of adherent cells), centrifuged and washed with PBS. Cells were fixed

in 70% ethanol, washed with PBS and then incubated with PI (1 mg/ml) with simultaneous

treatment of RNase at 37° C for 30 min. The percentage of cells having the sub-G0

population were measured using BD-LSR flow cytometer equipped with electronic

doublet discrimination capability using blue (488 nm) excitation from argon laser. Data

was collected in list mode on 10,000 events for FL2-A vs. FL2-W (Park et al., 2007).

3.8.3 Annexin V/PI assay

Principle: Phosphatidylserine occurs almost exclusively on the inner leaflet of the plasma

membrane in viable cells but becomes exposed on the outer leaflet usually during the

early stages of apoptosis and before the loss of membrane integrity. The loss of lipid

asymmetry and exposure of phosphatidylserine can be detected using fluorochrome-

conjugated annexin V (a Ca++-dependent phospholipidbinding protein), but because

phosphatidylserine exposure also occurs during necrosis, simultaneous staining for

membrane permeability (e.g., with PI) enables the two states to be distinguished.

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46

Procedure: Cells (1x106 /ml) were incubated with the different doses of test samples for

required time intervals. The harvested cells were washed with chilled PBS and suspended

in binding buffer (10 mM Hepes/ NaOH [pH 7.4], 140 mM NaCl, 2.5 mM CaCl2) at a

concentration of 1x 106 cells/ml. Then, 5 µl of annexin V-FITC (Axxora) and 10 µl of PI

(20 µg/ml) were added to these cells, which were analyzed with a BD-LSR flow cytometer

using quadrant statistics for apoptotic and necrotic cell population (Vermes et al., 1995).

3.8.4 Bcl-2 level change

The treated and untreated cells were centrifuged and pellet of cells was collected.

The cells were lysed in 1 ml of resuspension buffer (50 mM Tris, containing 5 mM EDTA,

0.2 mM PMSF, 1 �g/ml pepstatin, and 0.5 �g/ml leupeptin adjusted to pH 7.4) according

to the protocol (Calbiochem, Germany). Antigen extraction agent was added to the lysates,

as suggested, and the cells were incubated on ice for 30 min. Thereafter, the lysates were

transferred to fresh microcentrifuge tubes and centrifuged for 5 min. Clear lysates were

transferred to clean microfuge tubes. 50 �l of the Detector Antibody was added into each

well provided with kit. Lysates (50 µl) were added into appropriate wells. Wells were

covered and incubated at room temperature for 2 h. The plate was then washed with

washing buffer to remove unbound enzyme labeled antibody. 100 �l conjugate 1X was

added into each well and incubated at room temperature for 30 min. Wells were washed 3

times with 1X Wash Buffer. After incubation with tetramethylbenzidine substrate solution

for 30 min, the reaction was ceased by addition of a stop reagent. Subsequently, the plate

was read at dual wavelengths of 450/540 nm (Wang et al., 2003) and % decrease in Bcl-2

level was calculated.

3.8.5 To measure mitochondrial membrane potential

Principle: Mitochondria are essential to cell life as they produce ATP, the source of

metabolic energy. Mitochondria play an essential role in the propagation of apoptosis. The

mitochondrial transmembrane potential can be disrupted early in the death process, which

suppresses the electrochemical gradient required for ATP synthetase activity in the

oxidative phosphorylation pathway. Most importantly, the outer membrane becomes

permeable to soluble proteins, leading to the release of molecules from the inter-

membrane space into the cytosol, and to increased production of reactive oxygen species,

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which in turn oxidize the inner membrane cardiolipin. These events can be analyzed to

identify and quantify mitochondrial alterations associated with apoptosis. The

electrochemical gradient across the mitochondrial membrane, referred to as the

mitochondrial membrane potential, drives lipophilic cation accumulation in the

mitochondrial matrix. Such probes include those which exhibit optical and fluorescence

activity after accumulation into energized systems, such as 3,3’-

diehexiloxadicarbocyanine iodide [DiOC6 (3)], nonylacridine orange (NAO), safranine O,

rhodamine-123 (Rh123) etc.

Reagents:


1. Rhodamine - 123 380.82 5 µg/ml

Procedure: Cells (1 x 106/ml) were incubated with the different concentrations of test

material for required time intervals. Cells were washed twice with PBS and incubated with

complete medium containing Rhodamine - 123 (5 µg/ml) at 37 0C for 30 min. Cells were

resuspended in PBS. Rh -123 staining intensity was determined from 10,000 events,

analyzed in FL-1 channel on flow cytometer. Intensity of Rh 123 is directly related to

mitochondrial membrane potential (Wu et al., 2004).

3.8.6 Cytochrome c level estimation

3.8.6.1 Subcellular fractionation: It was done according to manufacture’s protocol

(Calbiochem, USA). Briefly, treated cells (5 x 107) were collected by centrifugation at 600

x g for 5 minutes at 4oC and washed with 10 ml of ice-cold PBS. Pellet was resuspended

in 1.0 ml of 1X Cytosol Extraction Buffer Mix containing DTT and Protease Inhibitors.

Incubated on ice for 10 minutes and centrifuged at 700 x g for 10minutes at 4oC. The

supernatant was collected into a fresh 1.5-ml tube, and centrifuged at 10,000 x g for 30

minutes at 4oC. Supernatant was collected as Cytosolic fraction.

3.8.6.2 Cytosolic Cytochrome c level estimation:

To each well provided with cytochrome c assay kit, 100 µl of calibrater diluents

RDP5 was added (Calbiochem, USA) followed by 100 µl of cytosolic fractions. Wells

were covered and incubated for 2 h and then washing was done with washing buffer. 200

µl of cytochrome c conjugate was added to each well and incubated again for 2 h. After

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48

adding 200 µl substrate, it was incubated for 30 min and then reaction was stopped by

adding 50 µl stop solution. Reading was taken at 450 nm. For each sample within a given

96-well plate, the absorbance at 450 nm (a measure of cytochrome c concentration)

was

corrected by subtracting the background reading at 540 nm. Cytochrome c concentration

was calculated by interpolating these values on a standard curve constructed for each plate.

3.8.7 Nitric oxide (NO) production

Nitric oxide (NO) plays an important role in neurotransmission, vascular

regulation, immune response and apoptosis. NO is rapidly oxidized to nitrite and nitrate

which are used to quantitate NO production. BioVision’s Nitric Oxide Colorimetric Assay

Kit (CA, USA) was used for Nitric oxide estimation. After treatment 80 µl of sample was

added to each well. To this 10 µl of the enzyme cofactor mixture and 10 µl of the nitrate

reductase mixture was added. Plate was covered and incubated at room temperature for 1-

4 hrs. 50 µl each of Griess Reagent (R1) and Griess Reagent (R2) was added to each well.

Absorbance was read at 540 nm using the plate reader and % change in NO production

was calculated. Wells containing assay buffer alone were treated as blank.

3.8.8 Detection of Reactive Oxygen Species (ROS)

The production of intracellular reactive oxygen species (ROS) was measured by

DCFH oxidation (Chakrabarti et al., 2007). The DCFH-DA (Dichlorofluorescin diacetate)

(Sigma Chemical Co., USA) reagent passively enters cell, where it is de-acetylated by

esterase to nonfluorescent DCFH. Inside the cell, DCFH reacts with ROS to form DCF

(Dichlorofluorescin), the fluorescent product. For this assay, treated and untreated HL-60

cells seeded (1x105) in black 96-well plates were exposed to DCFH-DA (5µM) for 1 hr.

After incubation, the fluorescence was read at the 485 nm excitation and 530 nm emission

on a fluorescence plate reader and fold change in ROS generation was recorded.

3.8.9 Changes in Caspase level

3.8.9.1 Caspase-9 Activity

Caspase 9 colorimetic kit (Biovision, CA, USA) provides a simple and convenient

means for assaying the activity of caspases that recognise the sequence LEHD. The assay

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49

is based on the spectrophotometric detection of chromophore p-nitroanilide (pNA) after

cleavage from the labelled substrate LEHD-pNA.

HL-60 cells (1 x107) were incubated with samples at various concentrations in

TCF-25 for different time interval. Cells were centrifuged at 600g for 5 min. Pellet was

resuspended in the lysis buffer (Added 25 �l of chilled cell lysis buffer per 1 x 106 cells)

and was incubated on ice for 10 min. followed by centrifugetion at 10,000g for 1 min. This

supernatant was collected as cell lysate into fresh tube and kept on ice. 50 �l of cell lysate

was then added into a flat bottom 96 well plate. To it 50 �l of 2X Reaction Buffer and 5 �l

of 4mM caspase-9 colorimetric substrate Ac-Leu-Glu-His-Asp-pNA (LEHD-pNA) were

added. Plate was incubated at 37°C for 1 - 2 h. Optical density was taken at 405 nm using

a microplate reader and change in activity was recorded as fold change.


This assay (Sigma, Saint Louis, USA) is based on the hydrolysis of the peptide

substrate Acetyl-lle-Glu-Thr-Asp p-nitroaniline (Ac-IETD-pNA) by caspase 8 resulting in

the release of a p-Nitroaniline (pNA).

HL-60 cells (1 x107) were incubated with samples at different concentrations in

TCF-25 for different time interval. After treatment, cells were centrifuged at 600g for 5

min. Pellet was resuspended in the lysis buffer (Added 100 �l of cold lysis buffer per 1 x

107cells) and was incubated on ice for 15 min., followed by centrifugation at 16,000g for

15 min. This supernatant was collected as cell lysate into fresh tube and kept on ice. 10 �l

of cell lysate was added in a 96 well flat bottom microtitreplate. 90 �l of 1X Assay Buffer

and 10 �l of Caspase-8 colorimetric substrate (Ac-IETD-pNA) were added to lysate. Plate

was incubated at 37°C for 1-2 h. Optical density was taken at 405 nm using a microplate

reader and change in activity was recorded as fold change.

3.8.9.3 Caspase 3 Activity

Caspase 3 is a member of CED-3 subfamily of caspases and is one of the critical

enzymes of apoptosis. The caspase 3 colorimetric assay (Sigma, Saint Louis, USA) is

based on the hydrolysis of peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-

DEVD-pNA) by caspase 3, resulting in the release of p-nitroaniline (pNA), which has high

absorbance at 405 nm.


TCF-25 for different time interval. After treatment, cells were centrifuged at 600g for 5

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50

min. Pellet was resuspended in the lysis buffer (Added 100 �l of cold lysis buffer per 1 x

107cells) and was incubated on ice for 15 min. followed by centrifugation at 16,000g for

15 min. This supernatant was collected as cell lysate into fresh tube and kept on ice. In a

96 well flat bottom microtitre plate 10 �l of cell lysate was added. 90 �l of 1X Assay

Buffer and 10 �l of caspase-3 colorimetric substrate (Ac-DEVD-pNA) were added to

lysate. Plate was incubated at 37°C for 1 - 2 h. Optical density was taken at 405 nm using

a microplate reader and change in activity was recorded as fold change.


The assay is based on spectrophotometric detection of the chromophore p-

nitroanilide (pNA) after cleavage from the labeled substrate Ac-Val-Glu-lle-Asp-pNA

(VEID-pNA) (Biovision, CA, USA). The pNA light emission can be quantified using a

spectrophotometer or a microtiter plate reader at 400- or 405-nm. Comparison of the

absorbance of pNA from an apoptotic sample with an uninduced control allows

determination of the fold increase in caspase-6 activity.


TCF-25 for different time intervals. Cells were centrifuged at 600g for 5 min. Pellet was

resuspended in the lysis buffer (Added 25 �l of chilled cell Lysis Buffer per 1 x 106 cells)

and was incubated on ice for 10 min. followed by centrifugetion at 10,000g for 1 min. This

supernatant was collected as cell lysate into new tube and kept on ice. To a 96 well flat

bottom microplate 50 �l of cell lysate was added. To it 50 �l of 2X Reaction Buffer and 5

�l of 4mM caspase-6 colorimetric substrate (VEID-pNA) were added. Plate was incubated

at 37°C for 1 - 2 h. Optical density was taken at 405 nm using a microplate reader and

change in activity was recorded as fold change.

3.8.10 DNA Ladder Assay

Principle: DNA laddering is one of the hallmark events that confirm apoptosis. Caspase-

activated DNase (CAD), one of the enzymes that cleave DNA during apoptosis, remains in

complex (ICAD) with its inhibitor until it is activated by effector caspase-3.

Internucleosomal DNA fragmentation by CAD leads to formation of DNA fragments of

the discrete size of mono (~200 bp) and oligo-nucleosomes (Huang et al., 2005). The

electrophoresis of DNA in ethidium bromide-stained agarose gels identifies

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internucleosomal DNA fragmentation as a ladder pattern (in contrast, random DNA

cleavage in necrotic cells has a smear appearance).

Adopted from Huang et al. (2005)

Procedure: The method of DNA laddering was done as proposed by Tong et al. (2004)

with slight modifications. In this method, cells (1x106/ml) after treatment with different

concentrations were centrifuged at 500g for 10 min, and washed with PBS. The resultant

pellet was suspended in 250 �l of lysis buffer (10 mM EDTA, 50 mM Tris–HCl, 0.5 %

SDS) for 15 min. at 55°C. Lysed cells were then digested with proteinase-K (200 µg/ml)

(Sigma Chemical Co., USA) at 55°C for 1 h followed by incubation with100 �g/ml

DNase-free RNase (Sigma Chemical Co., USA) at 55°C for 90 min. The DNA was

extracted with 250 µl of phenol: chloroform: isoamylalcohol (25:24:1) for 1 min and

centrifuged at 12,000g for 5 min twice. The aqueous phase was further extracted with

chloroform: isoamylalcohol (24:1) and centrifuged. DNA precipitated from aqueous phase

with 0.1 volume of 2M NaCl and 2.5 volumes of chilled ethanol was kept at 20°C

overnight. The precipitate was centrifuged at 12,000g for 10 min. The DNA pellet was

washed in 80% ethanol, dried, dissolved in 100 µl Tris-EDTA buffer (pH 8.0) and

electrophoresed in 1.5 % agarose gel at 50 V for 1.5 h. The gel was photographed by using

Bio-Rad Gel documentation system.

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3.9 Statistical analysis

All in vitro experiments were done in triplicate, and each data point represents the

average of at least 3 independent experiments. The data are reported as the mean ± SD.

The comparisons were made between controls and treated cultures using unpaired

Student’s t-tests, and the difference was considered to be statistically significant if p <

0.05 (�), highly significant if p < 0.01 (��), and extremely significant if p < 0.001 (��).

Erythrina suberosa Roxb.

E. suberosa Tree

E. suberosa Stem Bark E. suberosa Stem and Leaves

Plate 1

Anagallis arvensis L.

A. arvensis Whole Plant

A. arvensis in its natural habitat A. arvensis Flowers

Plate 2

Plant material

95% Alcoholic extract 50% Alcoholic-aqueous extract Aqueous extract

Pet. Ether fraction

(PE1,2) EtOAc fraction

EA1,2,3

Ethanol fraction

ET1,2 (13.5%)

Eth:Water fraction

EW1,2

Flow Chart 1. Preparation of various extracts and fractions from Plant Material

Fig. 2A. Stages of cell lines. Images obtained from ATCC website

Fig. 2B. Stages of cell lines. Images obtained from ATCC website

Fig. 2C. Stages of cell lines. Images obtained from ATCC website

Table 3. List of tissues, cell lines, medium, cell density and positive controls used

Tissue Cell lines Medium No. of

cells/well Positive control

Breast MCF-7 MEM 8,000

Adriamycin T-47D RPMI-1640 10,000

CNS SF-295 RPMI-1640 10,000 Adriamycin

Colon

502713 RPMI-1640 8,000

5-Fluorouracil SW-620 RPMI-1640 10,000

COLO 205 RPMI-1640 15,000

HCT-15 RPMI-1640 10,000

Liver HEP-2 MEM 8,000 Mitomycin C

Lung HOP-62 RPMI-1640 10,000

Paclitaxel A-549 RPMI-1640 8,000

Ovary OVCAR-5 RPMI-1640 20,000

Paclitaxel IGR-OV-1 RPMI-1640 10,000

Neuroblastoma IMR-32 MEM 10,000 Adriamycin

Prostrate DU-145 RPMI-1640 10,000

Mitomycin C PC-3 RPMI-1640 8,000

Renal 786-0 RPMI-1640 10,000 5-Fluorouracil

Leukemia HL-60 RPMI-1640 40,000 Camptothecin

Normal Lung

Fibroblast

MRC-5 MEM 10,000 -

WI-38 MEM 10,000 -

Normal Monkey

Kidney CV-1 MEM 10,000 -

Table 4. Methods employed for detection of Apoptosis

Flow cytometric

assays

• Cell Cycle analysis

• Phosphatidylserine externalisation by Annexin V/PI

• Change in Mitochondrial Membrane Potential by

Rhodamine-123

Colorimetric

assays

• Bcl-2 level change

• Cytochrome c release

• Caspase activity

� Caspase 9

� Caspase 8

� Caspase 3

� Caspase 6

• Nitric Oxide estimation

Fluorimetric

assay • Reactive Oxygen Species (ROS) level by DCFH-DA

Assessment of

DNA

• BrdU incorporation assay

• DNA Ladder Assay by Gel Electrophoresis

Seeded human cell lines (100 µl/well) in 96-well culture plates

Incubated for 24 hr at 5% CO2, 95% RH & 37ºC

Added the test material (100 µl/well) in desired concentration

Incubated for desired time at 5% CO2, 95% RH & 37ºC

Reaction terminated by addition of chilled 50% TCA (50 µl/well)

Kept at 4ºC for 1 hr

Washed the plates 5 times with Distilled Water

Allowed the plates to dry

Added Sulforhodamine B into plates (100 µl/well)

30 mins at room temp.

Washed the plate 4 times with 1% acetic acid

Allowed the plates to dry

Added Tris-Hcl buffer (pH 10.5; 100 µl/well) to dissolve the dye

Read the plates on ELISA reader at 540 nM

Fig. 3. Sulforhodamine-B (SRB) assay

Seeded human cell lines (100 µl/well) in 96-well culture plates

Incubated for 24 hr at 5% CO2, 95% RH & 37ºC

Added the test material (100 µl/well) in desired concentration

Incubated for desired time at 5% CO2, 95% RH & 37ºC

Add MTT before 2-3 hours of completion of incubation

Observe the formation of formazan crystals under microscope

Centrifuge the plates and aspirate the supernatent

Dissolve the Formazan in DMSO & read plates at 540nm

Fig. 4. MTT colorimetric assay

Normal

Sarcoma-180 (Ascites) Sarcoma-180 (Solid)

Ehlrich Ascites Carcinoma (EAC) Lymphoid leukemia (L1210)

Murine Tumor Models

Plate 3

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shodhganga.inflibnet.ac.inshodhganga.inflibnet.ac.in/bitstream/10603/29717/10/10... · 2018-07-02 · Pangara (Marathi) or Mandara (Bengali, Sanskrit). It is grown on road side, residential