in vitro cytotoxicity and anticancer activity of four...

Rangasamy Dhanabalan et al. / Drug Invention Today 2014,6(2),88-95

Drug Invention Today Vol.6.Issue.2.July-December 2014 88-95

Research Article Available online through

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ABSTRACTFour plant species Solanum trilobatum, Spathodea campanulata, Syzygium jambos and Tylophora indica leaf extracts extracted withaqueous, methanol, ethanol and chloroform were screened for their anticancer activity in MCF-7 cells. Importantly, among the 16 extractstested significantly reliable (p<0.01) antiproliferation was exhibited by SJCLE (92.79%) and TICLE (79.97%) with IC

50 of 84.3 µg/mLand

90 µg/mLrespectively. The SJELE and STCLE displayed antiproliferative effect of 74.8% and 71% with IC50

concentrations 132.53 µg/mL and203.23 µg/mL respectively. A total of eight extracts such as SCMLE, SJALE, SCELE, STMLE, STELE, TIELE, SCCLE and SJMLE unveiled41.56-69.28% cytotoxicity against MCF-7 cell line at the highest concentration 300 μg/mL. Four extracts namely TIMLE, STALE, SCALE andTIALE showed least cytotoxicity 26.14%, 15.6%, 14.3% and 7.81% respectively at 300 μg/mL.

Key words: Anticancer, apoptosis, cytotoxicity, membrane blebbing

*Corresponding author.R.Dhanabalan,Department of Microbiology,Rathnavel Subramaniam College of Arts and Science,Coimbatore-641402, Tamil Nadu, India

In vitro cytotoxicity and anticancer activity of four folklore medicinal plantsused among tribal communities of Western Ghats, Coimbatore, Tamil Nadu

Rangasamy Dhanabalan1,*, Muthusamy Palaniswamy2, Joseph Devakumar1

1*Department of Microbiology, Rathnavel Subramaniam College of Arts and Science, Coimbatore-641402, Tamil Nadu, India 2Department of Microbiology, School of Life Sciences, Karpagam University, Coimbatore-641021, Tamil Nadu,India

Received on: 24-05-2014; Revised on: 28-06- 2014; Accepted on:12-07-2014

ISSN: 0975-7619

1. INTRODUCTION

Worldwide among leading causes of death in 2012, cancer accounts

for 8.2 million demises and figures as second foremost reason of death

in the United States.1 Aggressive existences of lung, colorectal, stom-

ach, liver and breast cancers are the chief cancerous deaths occurring

annually. Every year more than 60% of the world’s new cancer cases

occur in Africa, Asia and South and Central America accounting for

70% of the world’s cancer expiries. It is anticipated that in the next

two decades the world’s annual cancer cases will rise from 14 million

to 22 million cases.2

In the development of formularies and pharmacopoeias, data’s on

medicinal plants drive towards a new lane in the discovery of antican-

cer therapeutic agents. Herbal remedy has been approved worldwide

where the plants and plant metabolites are used in the treatment of

cancer. Records on plant dependent anticancer drugs from published

and unpublished sources were first circulated in December 1967 by

Hartwell with monumental information of 3000 species of plants with

anticancer property.3 The National Cancer Institute ratified 114,000

anticancer plant extracts from 35,000 plant species from 20 countries.4

The isolation of the vinca alkaloids, vinblastine5 and vincristine6 from

the Catharanthus roseus hosted a novel era in the use of plant con-

stituents as anticancer agents with a paramount significance applied

in the treatment of cancer at clinical level.7 The discovery has paved

a track to phytotherapy in cancer regimens with an advent of antican-

cer camptothecin derivatives, topotecan, irinotecan and etoposide

considerably examined in cancer rehabilitation.8-9 With a basic con-

cept from the literature reviews cited above the present investigation

was carried out to rule out the cytotoxic and anticancer efficacy of

four medicinal plants S.trilobatum, S.campanulata, S.jambos and

T. indica prevalent in the Western Ghats of Tamil Nadu.

2. MATERIALS AND METHODS

2.1. Collection of plant material

The plant samples were collected from different areas around West-

ern Ghats, Coimbatore, Tamil Nadu and authenticated by Dr. G.V.S.

Murthy, Scientist ‘F’ and Head, Botanical Survey of India, Southern

Regional Centre, Coimbatore. The voucher specimens Solanum

trilobatum L. (No.1269), Spathodea campanulata P. Beauv. (No.1371),

Syzygium jambos L. Alston (No.1408) and Tylophora indica (Burm.f.)

Merr (No.1194) were deposited in the Department of Microbiology,

RVS College of Arts and Science, Sulur for future references. The

Drug Invention Today Vol.6.Issue.2.July-December 2014


88-95

plant leaves were collected based on the information’s obtained from

tribal communities such as Malasars, Irulas and Konars living around

the Western Ghats.

2.2. Preparation of leaf extracts

The fresh disease free shade dried powdered leaves were defatted

with petroleum ether and extracted with aqueous and organic sol-

vents viz. methanol, ethanol and chloroform by cold maceration

method. After a week of socking, filtration was conducted with

whatmann filter paper no.1, and concentrated via. rotary vacuum

evaporator. The concentrated crude extracts were stored at 4oC for

further analysis.

2.3. In vitro cytotoxicity assay

2.3.1. Maintenance of cell lines

Human breast adenocarcinoma cells (MFC-7) were obtained from

National Centre for Cell Sciences (NCCS), Pune and preserved in

Eagles Minimum Essential Medium (EMEM) incubating under 370C,

5% CO2,

95% air and 100% relative humidity in a CO2 incubator. The

cell lines were maintained by weekly passage by changing the culture

medium twice a week.

2.3.2. Preparation of cell suspension

The monolayer cells were detached with trypsin-ethylenediamine

tetraacetic acid (EDTA) to make a single cell suspension. The viable

cells were counted using a hemocytometer and diluted with medium

containing 5% FBS to give final density of 1x105 cells/mL. About

100 µL of cell suspension was seeded into 96-well plates at a plating

density 1x104 cells/well and incubated at 370C provided with 5% CO2,

95% air and 100% relative humidity allowing for the attachment of

cells. After 24 hrs of incubation the cells in the plates were treated

with different concentration of plant extracts.

2.3.3. Cell treatment with test plant extracts

About 6 mg of plant extract was dissolved in 2 mL of serum free sterile

DMSO to obtain a stock concentration 6000 µg/2 mL. One mL of the

stock plant extract was kept undispensed and another 1 mL was serial

two fold diluted in sterile tubes containing 1mL of DMSO, so as to

obtain a drug concentration ranging from 3000, 1500, 750, 375 and

187.5 µg/mL. About 100 µL of diluted plant extract was dispensed to

the appropriate wells containing 100 µL of cell suspension followed

by an additional incubation of the plates for 48 hrs at 370C, 5% CO2,

95% air and 100% relative humidity. The cell suspension with DMSO

and Cytoxan (CTX) (10 µg/mL) served as negative and positive con-

trols respectively, and triplicates were maintained for all concentra-

tions.

2.3.4. MTT assay10

The 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide

(MTT) assay is a colorimetric assay for measuring the activity of

cellular mitochondrial enzymes (succinate dehydrogenases) reduces

(cleaves the tetrazolium ring) the yellow water soluble tetrazolium salt

dye MTT into insoluble purple colored formazan. Therefore, the quan-

tity of formazan produced remains directly proportional to the num-

ber of viable cells or the intensity of purple colored insoluble formazan

formed remains directly proportionate to the live cells/existence of

mitochondrial enzymes in the live cells. After 48 hours of incubation,

15µL of MTT (5mg/mL) in phosphate buffered saline (PBS) was added

to each well and incubated at 370C for 4 hrs until purple precipitates

were obviously visible under microscope. The medium together with

MTT was then flicked, formed formazan crystals were solubilized in

100µL of DMSO and absorbance recorded at 570 nm using a micro

plate reader. The percentage antiproliferation (cytotoxicity) was de-

termined by the following formula:

Percentageantiproliferation =

Average Abs of control wells - Average Absof plant extract wells

Average Abs of control wells

x100

2.4. Statistical analysis

Nonlinear regression graph was plotted using drug concentration

against percentage proliferation; a linear regression with trend line

was used to predict the IC50

concentration of extracts that inhibits

50% proliferation of MCF-7 cells. Statistical significance was deter-

mined by one-way analysis of variance (ANOVA) by Dunnett’s mul-

tiple comparison tests with control (DMSO) using Graph Pad Prism

software.

2.5. Morphological observation of MCF-7 cells

The cellular morphological observation was performed using Phase-

Contrast Inverted Microscope as described.11-12 In brief, the MCF-7

cells were plated at a density of 1×106 cells/mL into 6-well plates and

incubated for 24 hrs to adhere followed by the treatment with plant

extracts of determined IC50

concentrations and incubated at 370C for

24 hrs. The cells were then centrifuged (300×g for 10 min) and washed

twice with phosphate buffered saline (PBS) by discarding the super-

natant and remaining media. About 10μL of fluorescent dye acridine

orange (AO) (10μg/mL) was added into the cellular pellet at equal

volume, kept undisturbed for 2 hrs and a drop of stained cell suspen-

sion was observed under fluorescent (UV) microscope within an hour.

The live viable, early and late apoptotic and secondary necrotic cells

were determined based on the morphology under staining. The AO is

an intercalating nucleic acid-specific fluorochrome crosses the cell

membrane of live and early apoptotic cells with an emission of green

fluorescence, when bound to DNA. The criteria for cellular morpho-

logical identification are: viable cell nucleus will appear green with

unbroken structure, early apoptosis with bright-green condensed



chromatin in nucleus, late apoptosis have dense orange areas of

chromatin condensation and orange intact nucleus depicts second-

ary necrosis.

RESULTS AND DISCUSSION

2.3. In vitro cytotoxicity assay

A total of 16 extracts from 4 plant species were screened for their

cytotoxic activity (antiproliferation) against human mammary cancer

cells (MCF-7) and the cytotoxic assay results with significant IC50

values are depicted in Table 1. A complete growth arrest was noticed

in positive control treated with Cytoxan (Cyclophosphamide) and it

was worth noting that IC50

values ranged between 84- 275µg/mL,

though fairly high, still point subtly towards selective activity. A

dose dependent antiproliferation of plant extracts were represented

in Figure 1-4 and the percentage of antiproliferation were further

examined by analysis of variation (ANOVA) with Dunnett’s multiple

comparison test for the statistical significance.

Table 1. Percentage antiproliferation and IC50

concentration of plantextracts on MCF 7 cells

Plant extract Plant extract concentration (µg/mL)/

/ Drug Percentage antiproliferation IC50

18.75 37.5 7 5 150 300 (µg/mL)

STALE 0 1.93 3.02 6.88 15.66 NDSTMLE 4.245 14.82 27.1 44.32 53.12 242.3

STELE 1.809 11.2 24.35 35.36 57.48 247.48

STCLE 4.245 13.44 25.88 41.89 71.05 203.23SCALE 0 0.48 1.32 9.74 14.3 ND

SCMLE 2.227 9.25 17.93 30.82 41.56 >300

SCELE 5.428 9.67 13.22 29.5 51.63 275SCCLE 8.42 15.57 32.15 55.81 67.98 130.49

SJALE 0.765 2.38 1.79 14.26 41.93 >300

SJMLE 0.765 7.86 21.38 38.48 69.28 210.96SJELE 0.696 6.68 28.18 56.34 74.8 132.53

SJCLE 0.139 25.12 43.21 90.88 92.79** 84.3**

TIALE 0.128 0.19 4.88 5.53 7.81 NDTIMLE 0.45 2.96 7.21 14.74 26.14 >300

TIELE 6.76 15.84 25.75 57.69 65.03 131.94

TICLE 28.07 36.31 46.03 65.87 79.97** 90**

Cytoxan 97***

DMSO 0

Values in the parenthesis represents the percentage antiproliferation(cytotoxicity), ND-Not detected, IC

50- 50% inhibitory concentration, ***

and ** represents the statistical significance of treatment groups at the levelof p<0.001 and p<0.01 respectively compared with the negative control byDunnet's multiple comparison test.

STALE,STMLE, STELE & STCLE-S.trilobatum aqueous, methanol,ethanol and chloroform leaf extracts respectivelySCALE,SCMLE,SCELE & SCCLE-S.campanulata aqueous, methanol,ethanol and chloroform leaf extracts respectivelySJALE,SJMLE,SJELE & SJCLE-S.jambos aqueous, methanol, ethanoland chloroform leaf extracts respectively TIALE,TIMLE,TIELE & TICLE-T.indica aqueous, methanol, ethanol andchloroform leaf extracts respectively

Fig. 1Dose dependent antiproliferation of Solanum trilobatum leafextracts on MCF 7 cells

Fig. 2.Dose dependent antiproliferation of Spathodea campanulataleaf extracts on MCF 7 cells

Fig. 3. Dose dependent antiproliferation of Syzygium jambos leafextracts on MCF 7 cells

% a

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an

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fera

tion

% a

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Concentration g/mL

Concentration g/mL

Concentration g/mL



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Among four extracts of S.trilobatum the STCLE exhibited 71%

antiproliferation, STELE and STMLE displayed 57.48% and 53.12%

antiproliferation on MCF-7 cells at 300 µg/mL. Our result concurs

with the antiproliferative property of S.tilobatum ethanolic leaf ex-

tracts on Ehrlich Ascites Carcinoma (EAC) cells13 and inhibition of

mice melanoma and metastasis,14 which could be due to the presence

of flavonoid quercetin in the plant.15 The antiproliferative property of

S.trilobatum was substantiated by the American cancer society which

booms flavonol ‘Quercetin’ and its significance in anticancer therapy.16

Epidemiological studies17-18 recommends dietary flavonoids and its

role in reducing the risk of tumors in colon, breast, prostate, lung and

pancreas. A study submits the saponin fractions from S.trilobatum

(SFST) has a dose-dependent suppressive effect in cell prolifera-

tion.19 A partially purified Sobatum from S. trilobatum proved cyto-

toxic in Dalton’s Lymphoma ascites, Ehrlich ascites and tissue culture

cells (L929 and Vero).20

The chloroform leaf extracts from S.campanulata (SCCLE) exposed

67.98% antiproliferation, whereas the SCELE and SCMLE were effec-

tive at 51.63% and 41.56% on MCF-7 cells at 300 µg/mL. Six common

human dietary sources of anticancer flavonols from methanolic leaf

extracts of S.campanulata such as 1-O-caffeoyl- -D-glucopyranoside

kaempferol 3-O-(2-O- -D-xylopyranosyl)- -D-galactopyranoside ,

kaempferol 3-O-(6-O- -L-rhamnopyranosyl)- -D-galactopyranoside,

acteoside, kaempferol 3-O-(6-O--L-rhamnopyranosyl)- -D-

glucopyranoside and quercetin 3-O-(2-O- -D-xylopyranosyl)- -

D-galactopyranoside has been reported.21The dietary sources of fla-

vonols in S.campanulata might attribute to the antiproliferation in

synergistic or distinctive action.

The chloroform leaf extract of S.jambos (SJCLE) revealed highest

cytotoxicity 92.79% (p<0.01) against MCF-7 cells with an IC50

84.30

µg/mL. Besides the SJELE and SJMLE unveiled 74.8% and 69.28%

cytotoxicity with IC50

of 132.53 and 210.96 µg/mL respectively. Two

acylated flavonol glycosides myricitin and myricetin22 in addition to

quercetin23 were reported from the leaves of S.jambos. Reports rec-

ommend the inhibitory property of flavonol quercetin against mice

melanoma and invasive metastasis.14,16

At 300 µg/mL chloroform leaf extract of T.indica (TICLE) disclosed

79.97% (p<0.01) and TIELE revealed 65% antiproliferation on MCF-7

cells with IC50

values90 µg/mL and 131.94 µg/mL respectively. Effec-

tive anticancer activity of T.indica acetone and ether extracts on

BHK-21 cell line was reported.24 Unique class of antitumor tylophorine

analogs differing in mode of action from known antitumor drugs has

been reported.25 Polar phenanthrene-based tylophorine derivatives

(PBTs) N(2,3-methylenedioxy-6-methoxy-phenanthr-9-ylmethyl)-5-

aminopentanol and N-(2,3-methylenedioxy-6-methoxy-phenanthr-9-

ylmethyl)-l-2-piperidinemethanol has been proved to be potentially

cytotoxic against A-549 human cancer cells.26 Natural agents from

plant sources inducing apoptosis by impeding the proliferation of

malignant cells can be used in cancer chemotherapy and

chemoprevention. Therapeutic plants have posed a great attention in

the treatment of various types of cancer considering as safer and

effective anticancer agents.27 The consumption of flavonoid rich fruits

and vegetables remained to reduce the risk of cancer.28-30 Due to addi-

tive or synergistic and rival or confounding activity of plant extracts

on tumor cell lines provide an vital model for the analysis of biologi-

cal mechanisms produced in clinical effects.31-33 Similar kind of anti-

cancer studies has aided in the identification of plant metabolites

with effective anticancer activity on cancerous cells deprived of pro-

voking effects on normal cells.34

2.4. Morphological observation of MCF-7 cells

With the aid of microscopic technique the original morphological

criteria of apoptosis can be perceived and appraised determining the

structural alterations in cells . The microscopic examination remained

the gold standard for the precise detection of apoptosis with compre-

hensive information about cell lines. Normal inverted microscope was

applied to observe the morphological changes of MCF-7 cells in the

control and test plates after 72 hrs post treatment under 400X magni-

fication. The present study demonstrates that the highest

antiproliferative property was exhibited by SJCLE (92.79%) straggled

by TICLE (79.97%)>SJELE (74.8%)>STCLE (71%)>SJMLE (69.28)

>SCCLE (67.98%)>TICLE (65%)>STELE (57.48%)>STMLE (53.12%),

SCELE (51.63%)>SJALE (41.93%) and SCMLE (41.56%) via induction

of membrane blebbing on MCF-7 cells inducing apoptosis. The mi-

crographs of cells treated with higher concentration of extracts ex-

posed distinctive morphological membrane blebbing (Fig. 5a-f & 6a-

Fig. 4. Dose dependent antiproliferation of Tylophora indica leafextracts on MCF 7 cells

% a

nti

pro

life

rati

on

Concentration g/mL



f), a process associated with apoptosis such as cell shrinkage, chro-

matin condensation, DNA fragmentation and apoptotic body forma-

tion. The apoptotic micrographs endorse the apoptosis is a conse-

quence of chromatin margination and cytoplasm condensation with

typical compaction and segregation of the nuclear chromatin.

Fig. 5a-f. Micrographs of MCF 7 cells treated with chloroform leafextracts of S.jambos examined after 48 hrs

Fig. 5a. Control (DMSO) with confluent growth of cells

Fig. 5b-f.Progression of proliferation inhibition with increase in con-centration of plant extract AP: Apoptosis, MB: Membrane blebbing

Fig. 5b

Fig. 5c

Fig. 5d

Fig. 5e

Fig. 5f

Fig. 6a-f. Micrographs of MCF 7 cells treated with chloroform leafextracts of T.indica examined after 48 hrs

Fig. 6a. Control (DMSO) with confluent growth of cells



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Fig. 6b-f.Progression of proliferation inhibition with increase in con-centration of plant extract AP: Apoptosis, MB: Membrane blebbing

Fig. 6b

Fig. 6c

Fig. 6e

Fig. 6f

Fig. 6d

The morphological observation of treated cells exposed extremely

impaired extra and intracellular structures by reduction in number of

viable cells, in accordance with the cytotoxic property exhibited by

the plant fractions; however the controls remained with confluent

growth throughout the incubation period. Clear signs of apoptosis in

our investigation concur with the study35-36 determining the progres-

sion of cells internal environment condensation under treatment with

plant extracts. The condensation remains accompanied by nuclear

and cell outline convolutions resulting in a breach of nucleus into

detached ruins to produce membrane bounded apoptotic bodies by

budding of the cell. The antiproliferative property of extracts can be

evaluated by counting viable cancer cells, while the apoptogenic

property shall be determined by observing typical morphological

changes on apoptosis specifically membrane blebbing. Apoptosis

was well evident in the micrographs of cell lines treated in a dose

dependent manner of all the four plant extracts.



In the process of development and homeostasis in multicellular or-

ganisms, apoptosis remains exceedingly controlled and organized

cell death process regulating variety of physiological and pathologi-

cal conditions. The cytotoxicity micrographs in the present study

exposed the morphological characteristics of apoptotic cells contain-

ing chromatin condensation, fragmented nuclei and DNA concurring

with the previous report37 determining the apoptotic cellular morpho-

logical changes are due to condensation of chromatin and

oligonucleosomal DNA cleavage. The present study outcome indi-

cates clearly that the selected plants S.trilobatum, S.campanulata,

S.jambos and T.indica used as a folklore healthcare in traditional

medicine acts via programmed cell death. Out of four plants selected

the Syzygium jambos and Tylophora indica was found to be potent

in antiproliferative activity on MCF-7 cell lines. Additional studies

are crucial to define the molecular mechanisms and its pathways to

evaluate potential in vivo anticancer activity of the selected plant

fractions along with active components.

3. CONCLUSION

The impact of drug efficacy in therapy always remains instigated

from in vitro level before its execution to in vivo models. Plant fla-

vonoids play a major role in disease prevention and therapy, hence-

forth the flavonoid rich plants are explored with great implication in

the field of rehabilitation. Since the phytochemical analysis of

S.trilobatum, S.campanulata, S.jambos and T. indica leaf extracts

revealed the existence of flavonoids; the present cytotoxic study was

executed in accordance with a concept “flavonoids as anticancer

agents”. The MTT assay was implemented in the present study to

rule out the cytotoxic efficacy of selected plant extracts, as the method

provides an accurate and reliable quantification both in cancer and

noncancerous cell lines in terms of cell viability. The chloroform leaf

extracts of S.jambos was found to be potent antiproliferative but not

up to the level of Cytoxan. In the present study the IC50

concentration

of all the active extracts ranged from 84-275 µg/mLwith exception of

STALE, SCALE, SCMLE, SJALE, TIALE and TIMLE. The “high” IC50

values are likely may be due to very low concentration of compounds

of interest, which could considerably enrich the bioactivity in the

cytotoxic assay. Our study could further aim for the anticancer com-

pound identification with an execution on in vivo models.

CONFLICTS OF INTEREST

We declare that, we all authors have no conflict of interest.

ACKNOWLEDGEMENTThe authors are grateful to University Grants Commission (UGC) forthe financial support given to the present study under the MajorResearch Project [Sanction No. F. No. 40-125/2011/ 2010 (SR) dated:

24.07.2011].

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Source of support: UGC,India, Conflict of interest: None Declared

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