Iranian Red Crescent Medical Journal. 2013 November; 15(11): e8871. DOI: 10.5812/ircmj.8871
Published online 2013 November 5. Research Article
Cytotoxic Properties of Some Medicinal Plant Extracts from Mazandaran,
Iran
Farkhondeh Nemati 1,*, Abbas Ali Dehpouri 1, Bahman Eslami 1, Vahid Mahdavi 1, Sepideh
Mirzanejad 1
1Department of Biology, Qaemshahr Branch, Islamic Azad University, Qaemshahr, IR Iran
*Corresponding Author: Farkhondeh Nemati, Qaemshahr Branch, Islamic Azad University, Qaemshahr, IR Iran. Tel: +98-9113140559, Fax: +98-1232253326. Email:, E-mail: farkhondehne-
mati@gmail.com.
Received: October 31, 2012; Revised: April 1, 2013; Accepted: May 21, 2013
Background: It was shown that plants derived agents are being used for treatment of cancer. In this study, crude ethanolic extract of
Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L., Orobanche orientalis G. Beck were screened in vitro for cytotoxic activity on Hela
(Human cervical carcinoma) cell line.
Objectives: We performed the present study to evaluate the in vitro cytotoxic activity of four plant extracts that we gathered from north
of Iran, Mazandaran
Materials and Methods: Hela cells were treated with various concentrations of individual samples (0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2.5, 5,
7.5 and 10 mg/ml) for 72 hours. Cell proliferation measured by MTT assay.
Results: Result from the performed assay showed that ethanolic extract of Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L. has
more significant cytotoxicity effect on Hela cell line than Orobanche orientalis G. Beck.
Conclusions: Extracts of the Consolida orientalis L., Ferula assa-foetida L., Coronilla varia L. could be considered as potential sources of
anticancer compounds but further studies are necessary for isolation and identification of biologically active substances.
Keywords: Cytotoxins; Medicinal Plant; Iran
1. Background
Plants involve bioactive secondary metabolites and
3. Materials and Methods
because of their complex structure, researches in this
domain are notably being remarked by scientists (1, 2).
3.1. Plant Materials
Scientists collect different parts of many plants, prepare
extracts, and test the extract for finding new and novel Samples of Consolida orientalis L., Ferula assa-foetida L.,
chemotherapeutics to treat cancer, as well as viral and Coronilla varia L., Orobanche orientalis G. Beck were col-
microbial infection. Cytotoxic screening of plants is the lected from different parts of Mazandaran, Iran. Voucher
preliminary methods to identify active compounds of specimens are deposited with the faculty of biology her-
plants (3, 4). barium (as NO 720-722, 720-456, 720-036 and 720-807).
2. Objectives 3.2. Preparation of Plant Extracts
In the course of our screening studies for the anticancer The plant materials were air dried at room tempera-
compounds from plants, we performed the present study ture for about 10 days and grounded into powder. Dry
to evaluate the in vitro cytotoxic activity of four plant ex- powder was extracted with ethanol for about 7 days at
tracts that we gathered from north of Iran, Mazandaran, room temperature. Dry ethanolic extracts were obtained
by using human cervix carcinoma cell line, HeLa. after removing the solvent by evaporation. Dry ethano-
Implication for health policy/practice/research/medical education:
Cancer is a major public health burden in both developed and developing countries. Plant derived agents are being used for the treatment of cancer. A
number of promising agents such as flavopiridol, roscovitine, combretastatin A-4, betulinic acid and silvestrol are in clinical or preclinical development.
So, it is anticipated that plants can provide potential anticancer proprties.
Copyright © 2013, Kowsar Corp.; Published by Kowsar Corp. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nemati F et al.
lic extracts were then dissolved in dimethyl sulphoxide
4. Results
(DMSO) to obtain appropriate solutions of the extracts.
Cytotoxicity activity of four plants extracts were carried
out against HeLa cell line at different concentrations to
3.3. Cell Line and Culture Medium
determine the IC50 (50% growth inhibition) by MTT assay.
HeLa (human cervical carcinoma) cell line obtained Results of different concentrations of Consolida orienta-
from Pasteur, Tehran, Iran, was used in this study. Cells lis L. including 0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2.5, 5, 7.5
were cultured in liquid medium (RPMI1640) supplement- and 10 mg/ml are tabulated in Table 1, and graphgically
ed 10% Fetal Bovine Serum (FBS), 100 u/ml penicillin and represented in Figure 1. MTT assay of Consolida orientalis
100 µg/ml streptomycin, and maintained under an atmo- L. shows significant effect on HeLa cell in concentration
sphere of 5% CO2 and 95% air at 37oC (5).
range between 10 mg/ml to 1 mg/ml compared with con-
trol. The highest cytotoxicity of this extract against HeLa
3.4. In Vitro Assay for Cytotoxic Activity cell was found in 5 and 2.5 mg/ml concentration with
82.45 and 80.49 percent of cell growth inhibition. It was
For testing, cells were washed by phosphate buffer sa-
found that the percentage of growth inhibition to be
line (PBS) and harvested by tripsinization and were plat-
increasing with increasing concentration of test com-
ed in 96 well plates (one cells/well) and incubated under
pounds, and IC50 value of this assay was 1.6 mg/ml.
5% CO2 and 95% air at 37oC for 24 hours. The cells were
treated with different concentrations of plants extracts
Table 1. Cytotoxicity Activity of Consolida orientalis L. Extracts
including 0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2.5, 5, 7.5 and
Against HeLa Cell Line at Different Concentrations by MTT Assay
10 mg/ml. Dilution of stock solutions was made in cul-
ture medium yielding final extracts concentrations with Concentrations of C. Absorbance, Inhibition, IC50,
orientalis L., mg/ml Mean Ä… SEM % mg/ml
a final DMSO concentration of 0.1%. This concentration
of DMSO did not affect cell viability. Control cells were
0.03125 0.649 Ä… 0.03 -17.48 -
incubated in culture medium only. All concentrations of
0.0625 0.578 Ä… 0.01 -3.94 -
plants extracts were in triplicates on the same cell batch.
0. 125 0.619 Ä… 0.04 -10.12 -
0.25 0.633 Ä… 0.09 -10.43 -
3.5. MTT Assay
0.5 0.540 Ä… 0.06 7.59 -
Growth of tumoral cells quantitated by the ability of
1 0.433 Ä… 0.07 29.24 1.6
living cells to reduce the yellow dye 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide (MTT) 2.5 0.168 Ä… 0.06 80.49 -
to a blue formazan product (6). At the end of 72 hours in-
5 0.155 Ä… 0.05 82.45 -
cubation, the medium in each well was replaced by MTT
7.5 0.209 Ä… 0.06 71.78 -
solution (20 cell/well, 5 mg/ml in phosphate-buffered
10 0.215 Ä… 0.06 71.07 -
saline), the plates were incubated for 4 hours under 5%
Control 0.583 Ä… 0.08 - -
CO2 and 95% air at 37ºC. MTT reagent was removed and
the formazan crystals produced by viable cells were dis-
solved in 100 DMSO and gently shaken. The absorbance
% Cell inhibition Vs Concentation
was then determined by ELISA reader at 492 nm.
100
The percentage growth inhibition was calculated using
80
following formula,
60
% cell inhibition = 100- [(At-Ab)/(Ac-Ab)] × 100
Where, At = absorbance value of test compound, Ab =
40
Absorbance value of blank and Ac = Absorbance value of
20
control.
0
The effects of extracts were expressed by IC50 values
0.5 1 2.5 5 7.5 10
-20
(the drug concentration reducing the absorbance of
treated cells by 50% with respect to untreated cells). -40 Concentrations [mg/ml]
Figure 1. Growth Inhibition of Consolida orientalis L. Extracts Against HeLa
3.6. Statiscal Analysis
Cell Line by MTT Assay
Experimental results are expressed as mean Ä… SEM. All
measurements were replicated three times. The data
Ethanolic extract of Ferula assa-foetida L. has signifi-
were analyzed by an analysis of variance (P < 0.05). The
cant cytotoxicity effect on Hela cell line in concentration
IC50 values were calculated from linear regression anal- range between 10 mg/ml to 2.5 mg/ml by using MTT assay
ysis.
compared with control. This extract also exerts the high
2 Iran Red Cres Med J. 2013;15(11):e8871
% Cell inhibitio
Nemati F et al.
cytotoxicity against HeLa cell in 2.5 mg/ml concentration
% Cell inhibition Vs Concentation
with 89.65 and 80.49 percent of cell growth inhibition.
100
IC50 value of Ferula assa-foetida L. on Hela cell was 0.61
80
mg/ml by MTT assay (Table 2, Figure 2).
60
Table 2. Cytotoxicity Activity of Ferula assa-foetida L. Extracts
40
Against HeLa Cell Line at Different Concentrations by MTT Assay
20
Concentrations of F. Absorbance, Inhibition, IC50,
0
0.03125 0.0625 0.125 0.25 0.5 1 2.5 5 7.5 10
assa-foetida L., mg/ml Mean Ä… SEM % mg/ml
-20
0.03125 0.825 Ä… 0.04 -50.33 -
-40
0.0625 0.706 Ä… 0.03 -24.33 -
-60 Concentrations
0. 125 0.677 Ä… 0.02 -18.28 -
0.25 0.676 Ä… 0.09 -18.55 -
Figure 2. Growth Inhibition of Ferula assa-foetida L. Extracts Against HeLa
Cell Line by MTT Assay.
0.5 0.709 Ä… 0.008 -25.30 -
1 0.482 Ä… 0.02 22.65 0.61
Ethanolic extract of Coronilla varia L. has significant
2.5 0.155 Ä… 0.02 89.65 -
cytotoxicity effect on Hela cell line in concentration
5 0.226 Ä… 0.03 77.76 -
range between 10 mg/ml to 2.5 mg/ml by using MTT
7.5 0.213 Ä… 0.03 80.39 -
assay. The highest cytotoxicity of this extract against
10 0.335 Ä… 0.03 54.19 -
HeLa cell was found in 5 mg/ml concentration with
94.18% of cell growth inhibition. IC50 value of Coronilla
Control 0.592 Ä… 0.02 - -
varia L. on HeLa cell was 0.5 mg/ml by MTT assay (Table
3, Figure 3).
Table 3. Cytotoxicity Activity of Coronilla varia L. Extracts Against HeLa Cell Line at Different Concentrations by MTT Assay
Concentrations of C. varia L., mg/ml Absorbance, Mean Ä… SEM Inhibition, % IC50, mg/ml
0.03125 0.563 Ä… 0.01 23.63 -
0.0625 0.492 Ä… 0.007 33.70 -
0. 125 0.452 Ä… 0.02 41.95 -
0.25 0.451 Ä… 0.01 42.06 -
0.5 0.379 Ä… 0.02 54.42 -
1 0.326 Ä… 0.01 63.89 0.5
2.5 0.191 Ä… 0.04 86.59 -
5 0.146Ä… 0.002 94.18 -
7.5 0.155 Ä… 0.006 92.48 -
10 0.161 Ä… 0.06 90.98 -
Control 0.715 Ä… 0.1 - -
5. Discussion
% Cell Inhibition Vs Concentration
It was found that ethanolic extract of Orobanche orienta-
120
lis G. Beck showed no significant cytotoxic ativity against
100 HeLa cell line except in 0.5 mg/ml concentration with
42.66% of cell growth inhibition (Table 4, Figure 4).
80
The comparison between IC50 of these extracts shows
60
that the ethanolic extract of Coronilla varia L. has lower
40
IC50 value than the others and could be considered as po-
20
tential source of anticancer compounds (Table 5).
0
Overall, this study evaluate that ethanolic extract of
0.03125 0.0625 0.125 0.25 0.5 1 2.5 5 7.5 10
Consolida orientalis L., Ferula assa-foetida L., Coronilla varia
Concentrations
L. has potential cytotoxic activity on Hela cell, indicating
the presence of cytotoxic compounds in these extracts.
Figure 3. Growth Inhibition of Coronilla varia L. Extracts Against HeLa Cell
Line by MTT Assay
This study provides only basic data, further studies are
Iran Red Cres Med J. 2013;15(11):e8871 3
% Cell inhibitio
% Cell Inhibition
Nemati F et al.
necessary for isolation and identification of biologically active substances from these extracts.
Table 4. Cytotoxicity Activity of Orobanche orientalis G. Beck Extracts Against HeLa Cell Line at Different Concentrations by MTT Assay
Concentrations of O. orientalis G. Beck, mg/ml Absorbance, Mean Ä… SEM Inhibition, % IC50, mg/ml
0.03125 1.374 Ä… 0.14 -2.6 -
0.0625 1.297 Ä… 0.14 12 -
0. 125 1.263 Ä… 0.11 15.33 -
0.25 1.210 Ä… 0.08 23 -
0.5 1.093 Ä… 0.05 42.66 -
1 1.149 Ä… 0.05 32.33 -
2.5 1.271 Ä… 0.05 10 -
5 1.529 Ä… 0.1 -33.33 -
7.5 1.782 Ä… 0.2 -71 -
10 1.899 Ä… 0.1 -97 -
Control 1.341 Ä… 0.1 - -
Authors Contribution
% Cell Inhibition Vs Concentration
None Declared.
60
40
Financial Disclosure
20
0
0.03125 0.0625 0.125 0.25 0.5 1 2.5 5 7.5 10 Authors declare there is no conflict of interests.
20-
40-
60-
Funding/Support
80-
100-
This Research Project was fully sponsored by Islamic
120-
Azad University, Qaemshahr Branch, with grant number
Concentrations
51073900607013.
Figure 4. Growth Inhibition of Orobanche orientalis G. Beck Extracts
Against HeLa Cell Line by MTT Assay
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1. Kinghorn A. Plant Secondary Metabolites as Potential Anticancer
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Table 5. The Comparison Between IC50 of Four Extracts
2. Wink Michael, Alfermann AWilhelm, Franke Rochus, Wetterauer
Bernhard, Distl Melanie, Windhovel J, et al. Sustainable biopro-
IC50 a, mg/ml Hela a
duction of phytochemicals by plant in vitro cultures: anticancer
agents. Plant Genetic Resources. 2005;3(2):90 100.
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3. Hashemi SA, Abediankenari S, Ghasemi M, Azadbakht M, Youse-
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degh, Elaheh Davari, Hoong CheahYew, Abdullah NoorRain. Cy-
a
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5. Freshney RIan. Culture of animal cells: a manual of basic
technique.New York: Wiley; 1994.
Acknowledgements
6. Mosmann Tim. Rapid colorimetric assay for cellular growth and
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This study was supported by grants from Islamic Azad
Immunol Meth. 1983;65(1 2):55 63.
University of Qaemshahr.
4 Iran Red Cres Med J. 2013;15(11):e8871
% Cell Inhibition
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