Biotechnology for Sustainability by Subhash Bhore, K. Marimuthu and M. Ravichandran [E - HTML preview

G0/G1 phase. PDEE increased the apoptotic cell population from 11.4% in case of control to

49.6% at 100 μg/ml. Further, PDEE induces loss of mitochondrial membrane potential (∆Ψm)

to 99.5% at 100 μg/ml from 24.4% in control cells. These primary results depict the free radical

scavenging potential and anticancer activity of P. denticulata extracts. These findings may

serve as foundation to develop an anticancer drug from medically important P. denticulata.

Keywords: Apoptosis; DNA damage; DPPH; MiaPaCa-2 cells; Primula denticulata

1. Introduction

cases and 13.5 million deaths by 2030.

Pancreatic cancer is the fourth most

Cancer after cardiovascular dis-

common cause of cancer-related deaths

eases is the second leading mortality

across the world with incidence equalling

cause and is rapidly becoming a global

mortality and continues to pose an enor-

pandemic. The worldwide incidence and

mous challenge to clinicians and cancer

mortality of cancer in 2008 were 12.66

scientists (Hariharan et al., 2008). Among

and 7.56 million cases respectively. Ac-

all pancreatic cancers, pancreatic ductal

cording to (World Health Organization,

adenocarcinoma (PDAC) is the most

2010) report, the global cancer burden is

common epithelial, exocrine pancreatic

expected to nearly double to 21.4 million

malignancy, representing more than 80%

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Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al.

of the malignant neoplasms of the pancre-

tine,

podophyllotoxin,

camptothecin,

as (Alexakis et al., 2004).

combretastatins, flavopiridol, bruceatin

Consumption of fruits and vegeta-

etc, with diverse chemical structures have

bles is known to impart reduction in the

been isolated from plants. Several biolog-

incidence of ischemic heart disease and

ically active analogues such as taxotere,

some types of cancer, particularly stom-

isotaxel (taxol analogues) topotecan, iri-

ach, oesophagus, lung, oral cavity and

notecan, rubitecan, lurtotecan, 9-Amino

pharynx, endometrial, pancreas and colon

CPT (camptothecin analogues), etoposide,

cancers (Mathew et al., 2004). Similarly

teniposide (podophyllotoxin analogues),

natural antioxidant supplements (ascorbic

vinorelbine, hydravin (Vinca alkaloid de-

acid, tocopherols, anthocyanin, β-carotene

rivatives) have been synthesised from the-

and other polyphenols have been associ-

se front line anticancer lead molecules

ated with lower incidences of cancers and

(Vandana et al., 2005). As a result, em-

cancer related diseases (Fleischauer et al.,

phasis has now been shifted towards the

2003). During some pathophysiological

screening of apoptotic inducers from nat-

conditions, excess amount of reactive ox-

ural sources particularly from plants in

ygen species (ROS) is being generated by

the form of extracts or as isolated com-

certain external agents such as UV-

pounds that specifically increase apoptot-

radiations, drugs, pollution, other xenobi-

ic cell death in cancerous cells.

otics and as well as by endogenous chem-

Primula denticulata Sm. (Primu-

icals, especially stress hormones (adrena-

laceae) is an important member of genus

lin and noradrenalin). The superoxide

primula, which represent more than 400

dismutase (SOD) and other defence

species (Richards, 1993). P. denticulata is

mechanisms in living organisms are una-

commonly known as drumstick primula

ble to scavenge excess of ROS complete-

or tooth-leaved primula. P. denticulata is

ly, which causes damage to cellular mole-

20-30 cm tall perennial rarely annual, de-

cules such as DNA, RNA, enzymes, lipids

ciduous, clump-forming plant with com-

etc. that results in fluidity of bio-

pact heads of many flowers. The plant is

membranes (Dean and David, 1993) and

widely distributed from eastern Afghani-

development of degenerative diseases in-

stan and northern Pakistan, across the

cluding caners, cardiovascular, neuro-

Himalaya to Yunnan, Sichuan and Gui-

degenerative, Alzheimer’s and inflamma-

zhou in China. In Kashmir Himalaya, the

tory diseases (Shahidi et al., 1992; Gerber

species is widely distributed (Map-1). The

et al., 2002; Di Matteo and Esposito,

species thrives best in moist, shady

2003; Sreejayan and Rao, 1996). Hence

slopes, mostly near melting glaciers and

there is growing interest in natural poly-

moist meadows, ranging in altitude from

phenolic compounds, present in medicinal

2100 – 4050 meters.

and dietary plants that might help attenu-

ate oxidative damage (Silva et al., 2005).

2. Materials and methods

The increased incidence of differ-

ent types of cancers during the last few

2.1. Chemicals

decades and the modern techniques for

Sulphorhodamine-B

(SRB),

separation, structure elucidation, screen-

RPMI-1640 medium, fetal bovine serum

ing and combitorinial synthesis have led

(FBS), streptomycin, sodium bicarbonate,

to the development of new anticancer

5-Fluorouracil, paclitaxel, gentamycin

drugs, drug combinations and chemother-

sulphate,

trypsin,

1,1-diphenyl-2-

apy strategies by exploration of enormous

picrylhydrazyl (DPPH), folin–Ciocalteu

pool of biological, synthetic and natural

reagent, catechin, gallic acid were pro-

products (Mukherjee et al., 2001). So far

cured from Sigma-Aldrich. Trichloroace-

several potential anticancer lead mole-

tic acid (TCA), butylated hydroxytoluene

cules such as taxol, vincristine, vinblas-

(BHT), thiobarbituric acid (TBA), hydro-

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Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al.

N

Map 1: Distribution of Primulla denticulata Sm. in Kashmir Himalaya, J&K- India.

gen peroxide (H2O2), ferric chloride, di-

tude of 2650 m. The healthy plant species

methyl sulfoxide (DMSO), potassium fer-

were randomly collected by hand-picking

ricyanide were purchased from Merck.

and later identified by Dr. Anzar A.

The other reagents used were all of ana-

Khuroo at department of Botany, Univer-

lytical grade.

sity of Kashmir. A specimen under

voucher number KASH-1743 was pre-

2.2. Collection and identification of plant

served for future reference.

Primula denticulata Sm. at flow-

ering stage was collected from Gulmarg

2.3. Extract preparation

region of Kashmir Himalaya (latitude

Fresh and healthy leaves of P.

34°3'27" N; longitude 74°23'9" E) at alti-

denticulata (1Kg) were cleaned with dou-

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Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al.

ble distilled water, dried under shade (25±

pable of donating hydrogen or electron.

2°C) for 5-6 days. The dried plant materi-

100 μl of different concentrations (100-

al was ground to powder form. The plant

600 μg/ml) of plant extract or standard

powder was extracted in soxhelt apparatus

antioxidant was added to 1 ml DPPH so-

using ethanol as solvent at desirable tem-

lution (0.5 mM). The solution was slight-

perature. The filtered extract was concen-

ly shaken and kept stand for 30 min at

trated using Buchi rotavapour and stored

room temperature under dark conditions.

in glass vials at 4oC until used.

The yellow colour solution was read at

517 nm against ethanol (Brand-Williams

2.4. Estimation of total phenolics

et al., 1995). The free radical inhibition

The total phenolic content in leaf

was calculated as:

extract of P. denticulata was determined

Percentage inhibition = [(Ac-As)/Ac] x

by Folin–Ciocalteu method as adopted by

100

Slinkard and Singleton (1977) with slight

Where, Ac and As are the absorbance of

modifications. To 0.2 ml of plant extract

control and sample respectively

(1mg/ml) was added to 2.5 ml of 10% di-

Butylated hydroxytoluene and α- tocoph-

luted Folin–Ciocalteu reagent and 2 ml of

erol were used as positive control.

2.5% aqueous Na2CO3. The reaction mix-

●

ture was incubated at room temperature

2.7. Hydroxyl radical (HO ) scavenging

with intermittent shaking. The blue colour

assay

solution was read at 765 nm on UV–

Deoxyribose assay was used to

visible spectrophotometer. The absorb-

evaluate the hydroxyl radical scavenging

ance of solution was compared against

potential of P. denticulata leaf extract

●

standard Gallic acid (50 mg %) calibra-

(Halliwell et al., 1987). The HO generat-

tion curve.

ed in Fenton reaction attack deoxyribose

to form products that upon heating with

2.5. Estimation of total flavonoids

thiobarbituric acid at low pH yield a pink

The aluminium chloride colori-

chromogen (TBARS). A reaction mixture

metric method as described by Mcdonald

containing deoxyribose (25 mM), FeCl3

et al., (2001) was used to determine the

(10 mM), ascorbic acid (100 mM), H2O2

total flavonoid content of leaf extract. The

(2.8 mM) in 10 mM KH2PO4 (pH 7.4)

principle of this method is based on fla-

with or without plant extract at various

vonoid–aluminium complex formation,

concentrations (20-120 µg/ml) and incu-

which shows absorbance maximum at 430

bated at 37 0C for 1h. Then 1 ml of TBA

nm. Briefly 0.5 ml (1mg/ml) of extract

(1% w/v) and 1 ml of TCA (3% w/v)

was mixed with 1.5 ml of ethanol, 0.1 ml

were added and heated at 100 0C for 20

of 10% AlCl3, 0.1 ml of 1M potassium

min. Absorbance of TBARS was read at

acetate and 2.8 ml of distilled water. After

532 nm. Deoxyribose oxidation inhibition

5 min of incubation, the absorbance was

was calculated as:

read at 430 nm. Flavonoid concentration

Percentage inhibition = [(A-B)/A] ×100

was expressed as milligrams of catechin

Where, A is malonaldehyde produced

equivalents per gram dry weight.

when treated with extract and B is malo-

naldehyde produced without extract. Bu-

2.6. DPPH assay

tylated hydroxytoluene and α- tocopherol

DPPH assay is one of the most ex-

were taken as the positive control.

tensively used method for determining the

antioxidant potential of any biological

2.8. DNA damage assay

sample. DPPH is a purple stable free radi-

The Prevention of oxidative DNA

cal which is reduced to yellow colour

damage by PDEE was determined by

complex 1,1-diphenyl-2-picrylhydrazine

method as previously described by Ghan-

(DPPH-H) by compounds which are ca-

ta et al., (2007). Calf thymus DNA

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Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al.

(0.37μg) with and without plant extract

at room temperature. The optical density

(10, 30, 50, 80 and 100 μg) was incubated

was read at 570 nm using ELISA reader.

with 20 mM ferric nitrate 30 mM H2O2 in

The experiments were done in triplicates.

20.0 mM phosphate buffer (pH 7.4) in a

Percentage cell growth was calculated as:

final reaction mixture volume of 20 μl for

Percentage cell viability = [At/Ac] ×100

1h at 37 oC. Oxidative DNA damage was

Percentage cell growth inhibition = (100-

induced by hydroxyl radicals generated in

percentage cell viability)

Fenton reaction (Ani et al., 2006). Bro-

Where At and Ac are absorbance of treat-

mophenol blue (0.25%) and glycerol

ed and control cells, respectively.

(30%) were added to terminate reaction

mixture, followed by gel electrophoresis

2.11. DNA content and cell cycle phase

in 0.7% agarose. The gel was then visual-

distribution

ized and photographed on gel doc.

Human pancreatic (MiaPaca-2)

cells were seeded in 6-well culture plates

2.9. Cells culture

5

with cell density 2x10 cells/ml/well and

Pancreatic cell line (MiaPaca-2),

incubated for 24 hrs. After incubation, the

Lung cell line (A-549), Prostate cell line

cells were treated with PDEE (0, 30, 50

(PC-3), Leukaemia cell line (THP-1), Co-

and 100 mg/ml) and again incubated for

lon cell line (HCT-116) and Lung cell line

48 hrs. After 48 hrs treatment cells were

(HOP-62) were purchased from National

collected by 5 min centrifugation at 1000

Cancer Institute, U.S.A and European

rpm. The harvested cells were washed

collection of cell culture, UK. Cells were

twice with phosphate buffer solution and

cultured in RPMI-1640 and MEM medi-

fixed with 70 % ethanol at -20 ºC for 1h.

um supplemented with nutrients and anti-

The cells were then stained with DNA

biotics. Cells were grown at 37 oC and 5%

staining solution containing propidium

CO2 level with relative humidity of 98%.

iodide (20 mg/ml) and triton X-100 (1%)

in PBS for 30 min in dark. FACScan was

2.10. Anticancer activity

used to measure DNA content. For each

The anticancer activity of PDEE

data file, data was collected from 10,000

against different human cancer cell lines

cells. Cell Quest (Becton, USA) was used

was evaluated by SRB assay as described

for analysis of histograms.

by Monks et al., 1991. Briefly 100µl of

cell suspension (1x105 cells/well) were

2.12. Loss of Mitochondrial Membrane

cultured in 96-well plates and incubated

Potential (ΛΨm)

overnight at 37 0C and 5% CO2 level. 20

Flow cytometry was used to meas-

µl test material at various final concentra-

ure the mitochondrial membrane potential

tions (10-100 µg/ml) was added. Paclitax-

loss (ΛΨm). Human pancreatic (MiaPaca-

el (1 µM) and 5-fluorouracil (20 µM)

2) cells were plated in 6-well cultural

were used as standard anticancer drugs.

6

plates with cell density of 1x10

Cell growth was stopped after 48 hrs of

cells/ml/well and incubated for 24 hrs at 5

incubation by adding 50 μl of 50% TCA

% CO2 level. The cells were then treated

in each well and incubated further for 1h

with PDEE (0, 30, 50 and 100 mg/ml) and

at 4 0C. The plates were then washed, air

again incubated for 48 hrs. Rhodamine-

dried and stained with 50 μl of 0.4% SRB

123, a cell permeable cationic dye was

dye in 1% acetic acid, followed by incu-

added one hour before termination of ex-

bation for 30 min at room temperature.

periment and again incubated for 30 min.

The unbound dye was then removed by

The cells were washed with PBS and pel-

washing with 1% acetic acid and kept

lets were collected by centrifugation. The

overnight for drying. In each well, 100 μl

collected pellets were re-suspended in 300

of 10 mM tris-base was added to solubil-

ml of PBS. Florescence of Rh-123 in cells

ise the dye followed by stirring for 5 min

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Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al.

were analysed at 485 nm by flow cytome-

them to act as strong reducing agents, hy-

ter (Jung et al., 2006).

drogen donor’s metal chelaters and singlet

oxygen quenchers (Miguel, 2010). The

2.13. Statistical analysis

total flavonoid content in DPEE was

All of the experiments were done in

found to be 7.06 ± 1.31 (mg catechin/g

triplicate. The data were recorded as

dry extract). Flavonoids are known exhib-

means ± standard deviations and were

it many biological activities like antioxi-

analysed with SPSS software.

dant, anticancer, antimicrobial and anti-

inflammatory properties (Hodek et al.,

3. Results and discussion

2002).

3.1. Total phenolic and flavonoid content

3.2. DPPH free radical scavenging activi-

Polyphenolic compounds are very

ty

important plant bioactive constituents be-

DPPH is a purple stable free radi-

cause of their scavenging potential due to

cal at room temperature with characteris-

the presence of hydroxyl groups (Hatano

tic absorbance at 517 nm. The nitrogen

et al., 1989). Folin-Ciocalteu method is

free radical of DPPH is easily quenched

most widely used to measure the poly-

by an antioxidant to yellow coloured

phenol contents, with the basic mecha-

complex

(1,1-diphenyl-2-picrylhydra-

nism of electron transfer and reducing

zine). The decolourization of purple col-

ability (Prior and Schaich, 2005). Using

our is stoichiometric depending on the

this quantitative assay, we found that the

number of electrons gained (Soares et al.,

total phenolic content (TPC) of ethanolic

1997; Mokbel et al., 2006; Singh et al.,

leaf extract of P. denticulate was found

2002). DPPH radical scavenging potential

to be 12.24 ± 2.11 (mg GAE/g dry ex-

of PDEE at different concentrations in-

tract) as depicted in Figure 1. Considera-

vestigated in the present study was deter-

ble attention has been received by poly-

mined together with standard antioxidants

phenols for their physiological role as an-

(BHT and α-tocopherol) at the same con-

tioxidant and anticancer agents (Othman

centrations (Figure 2). PDEE showed sig-

et al., 2007). Polyphenolic compounds h-

nificant scavenging effect on DPPH free

Figure 1: Represents the total phenolic

and flavonoid content of PDEE. Each

value represents the mean ± SD (n = 3).