Abstract Background and objectives: Iran owns a rich and prestigious heritage of medicinal herbs but the majority of these plants have not yet undergone chemical, pharmacological and toxicological studies. In the present study some species form northern parts of Iran were evaluated for cytotoxicity. Methods: Sixteen medicinal plants were extracted with methanol and screened for their cytotoxic activities. The inhibition of cell growth for these extracts was evaluated against MCF-7, WEHI-164, HepG-2 and MDBK cell lines. Their 50% inhibitions of growth (IC 50 ) were determined by MTT assay. Moreover, cytotoxic evaluation of different fractions of the most potent species was performed. Results: Among examined samples, the IC 50 values of methanol extract of Centaurea bruguierana (DC.) Hand.-Mzt. on mentioned cell lines were found 47.30-87.40 µg/mL. In addition, the chloroform fraction of the species was cytotoxic with IC 50 values 17.00-23.03 µg/mL. Conclusion: It was concluded that the chloroform fraction of C. bruguierana was the best candidate for identification and isolation of active principles with cytotoxic effects. These results recommend further studies about this species

F Naghibi

M Mosaddegh

S Esmaeili

S Ghafari

S Sahranavard

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Available at: http://rjpharmacognosy.ir                                    Copy right
©
 2014 by the Iranian Society of Pharmacognosy 
*
Corresponding author: mmosaddegh@itmrc.org, Tel/Fax: +9821-88776027 
  
 
 
Research Journal of Pharmacognosy (RJP) 2(2), 2015: 47-51 
Received: Dec 2014 
Accepted: Feb 2015 
 
Original article 
 
 
Investigating the cytotoxic effect of some medicinal plants from northern 
parts of Iran 
 
S. Ghafari
1
, F. Naghibi
1,2
, S. Esmaeili
1,3
, S. Sahranavard
1,3
,
 
M. Mosaddegh
1,3*
 
 
1
Traditional Medicine & Materia Medica Research Center (TMRC), Shahid Beheshti University of Medical 
Sciences, Tehran, Iran. 
2
Department of Pharmacognosy, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, 
Iran. 
3
Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical 
Sciences, Tehran, Iran. 
 
Abstract 
Background and objectives: Iran owns a rich and prestigious heritage of medicinal herbs but the 
majority of these plants have not yet undergone chemical, pharmacological and toxicological studies. 
In the present study some species form northern parts of Iran were evaluated for cytotoxicity. 
Methods: Sixteen medicinal plants were extracted with methanol and screened for their cytotoxic 
activities. The inhibition of cell growth for these extracts was evaluated against MCF-7, WEHI-164, 
HepG-2 and MDBK cell lines. Their 50% inhibitions of growth (IC50) were determined by MTT 
assay. Moreover, cytotoxic evaluation of different fractions of the most potent species was performed. 
Results: Among examined samples, the IC50 values of methanol extract of Centaurea bruguierana 
(DC.) Hand.-Mzt. on mentioned cell lines were found 47.30-87.40 µg/mL. In addition, the chloroform 
fraction of the species was cytotoxic with IC50 values 17.00-23.03 µg/mL. Conclusion: It was 
concluded that the chloroform fraction of C. bruguierana was the best candidate for identification and 
isolation of active principles with cytotoxic effects. These results recommend further studies about 
this species. 
 
Keywords: Centaurea bruguierana, cytotoxic effect, medicinal plants, MTT, Iranian Traditional 
Medicine 
Introduction 
Natural products and related drugs are used to 
treat 87% of all categorized human diseases, 
including bacterial infections, cancers and 
immunological disorders [1]. So far, it has been 
estimated that only 5000 plant species have been 
properly studied for possible medical applications 
[2]. Furthermore, approximately 25% of 
prescribed drugs in the world originate from 
plants and over 3000 species of plants have been 
reported to have anticancer properties [3,4]. 
Plants continue to be a rich source of therapeutic 
agents. The active principles of many drugs are 
Ghafari
 
S. et al. 
 
48 RJP 2(2), 2015: 47-51 
 
found in plants and are produced as secondary 
metabolites [5]. Considering that there are 
250,000 to 300,000 plant species on the planet, 
the majority of this treasure awaits retrieval from 
plants [2]. Additionally, isolation and 
identification of some potent anti-tumour 
compounds from medicinal plants, such as 
colchicine, paclitaxle and podophyllotoxin, has 
encouraged scientists to screen different plant 
species against cancer cell lines [6].  
The screening of plants to investigate the 
cytotoxic effect and extraction with solvents of 
different polarity can improve the isolation of 
active compounds with the cytotoxic effect.  
To investigate their cytotoxic effect, we carried 
out a screening of some medicinal plants with 
reported antimicrobial effects [7,8]. These plants 
were from northern parts of Iran. 
 
Experimental 
Plant material    
Aerial parts of the selected plants (table 1) were 
collected from northern parts of Iran and 
identified by a qualified botanist at Traditional 
Medicine and Materia Medica Research Center 
(TMRC), Shahid Beheshti University of Medical 
Sciences, Tehran, Iran. The voucher specimens 
were deposited at the Herbarium of TMRC. 
 
Extraction and fractionation 
The aerial parts of the selected plants were 
crushed. Whilst being constantly shaken, 50g of 
each powdered dried material was macerated in 
500 mL methanol for 24 hours. The filtrates were 
evaporated to dryness. To investigate for 
cytotoxic effects, all methanol extracts of the 
plants were screened [9].  
The petroleum ether and chloroform fractions of 
the selected plants which showed IC50 less than 
100µg/mL were prepared successively with 
maceration method [9].  
 
Cell lines 
The cell lines used in this study were MCF-7 
(human    breast   adenocarcinoma),   WEHI-164  
(mouse fibro sarcoma), HepG-2 (human 
hepatocellular liver carcinoma) and MDBK 
(Madin-Darby bovine kidney). The cell lines 
were purchased from the Pasteur Institute, 
Tehran, Iran.  
 
In vitro cytotoxicity assay 
The cells were seeded in 96-well micro plates at 
8 ×10
3
 cells for MCF-7 and 10
4 
cells for WEHI-
164, HepG-2 and MDBK cells in triplicate. The 
cells were then incubated at 37 °C under 5% CO2 
 
Table 1. Scientific name, family, location and voucher number of medicinal plants 
No Scientific Name Family Location Voucher Number 
1 Astrodaucus orientalis (L.) Drude Apiaceae Tehran TMRC 1276 
2 Capparis spinosa L. Capparidacea Golestan TMRC 1295 
3 Centaurea bruguierana (DC.) Hand. – Mzt. Asteraceae North Khorasan TMRC 1291 
4 Cephalanthera caucasica kränzl Orchidaceae Mazandaran TMRC 1288 
5 Colutea persica Boiss. Fabaceae Mazandaran TMRC 1280 
6 Erodium oxyrrhynchum M.B.  Geraniaceae Tehran TMRC 1277 
7 Ficus carica L. var. genuina Boiss. Moraceae Tehran TMRC 1212 
8 Heracleum persicum Desf. Ex. Fischer Apiaceae Tehran TMRC   692 
9 Linaria pyramidata Lam. Scrophulariaceae North Khorasan TMRC 1293 
10 Marrubium vulgare L. Lamiaceae Mazandaran TMRC 1286 
11 Minuartia lineata Bornm. Caryophyllaceae Mazandaran TMRC 1279 
12 Paliurus spina-christi Mill. Rhamnaceae Mazandaran TMRC 1282 
13 Papaver bracteatum Lindl. Papaveraceae Ardabil TMRC 1297 
14 Pteropyrum aucheri Jaub. & Spach Polygonaceae Tehran TMRC 1285 
15 Roemeria refracta DC. Papaveraceae North Khorasan TMRC 1292 
16 Tamarix aralensis Bge. Tamaricaceae Gilan TMRC 1284 
Cytotoxicity of some medicinal plants from northern parts of Iran 
49 
 
atmosphere. After 24 h, the medium was replaced 
with a fresh one, containing different 
concentrations of samples to be tested.  
The initial concentration of samples was 100 
µg/mL in dimetylsulfoxide (DMSO), which was 
serially prepared with two fold dilutions to make 
six concentrations. 
The cells were exposed to each sample for 72 h at 
37 °C. The cytotoxicity of the plant extracts were 
determined by a rapid colorimetric method (MTT 
assay). They were then compared to the untreated 
controls [10]. The absorbance was recorded at 
570 nm by an ELISA plate reader. To calculate 
IC50, viability (%) versus log concentrations was 
graphed by Microsoft Excel program. Tamoxifen 
was used as the positive control. 
 
Results and Discussion 
In our study, the cytotoxic effect of 16 medicinal 
plant methanol extracts against four cell lines was 
determined using the MTT assay. The cytotoxic 
results are shown in table 2.  
Previous studies have revealed the in vitro 
cytotoxic effect of fruit and leaf extracts, as well 
as the latex of Ficus carica against HeLa cell line 
determined by MTT assay. The results have 
indicated that the latex and different extracts of 
Ficus carica could reduce the viability of the 
HeLa cells at concentrations as low as 2µg/mL in 
dose dependent manner [11]. The potential of 
anticancer activity of Astrodaucus orientalis 
extracts determined with MTT assay has been 
reported in human breast carcinoma cell line, T-
47D [12]. In other studies, cytotoxic activity of 
the essential oils of Heracleum persicum has 
been investigated showing cytotoxic effects in 
brine shrimp lethality assay [13]. 
According to table 2, except for Centaurea 
bruguierana, other methanol extracts did not 
show cytotoxicity against the cell lines.  
It could be argued that the above mentioned 
plants might have shown selective in vitro 
cytotoxicity against some cell lines and they were 
not toxic to the cell lines of the present 
study.There is also another possibility that these 
speciesmight have presented their cytotoxicity by 
a mechanism that could not be detected by MTT 
assay. Thus, other methods of evaluation could 
be suggested for further studies.  
As mentioned before, IC50 of Centaurea 
bruguierana methanol extract was less than 100 
µg/mL gainst MCF-7, WEHI-164, HepG-2 and 
MDBK cells. Centaurea is the largest genus of 
the family Asteraceae and represents 
approximately 47 species in Iran [14]. 
 
Table 2. IC50 of medicinal plants against MCF-7, WEHI-164, HepG-2 and MDBK cell lines 
Scientific name 
(IC50 µg/mL) 
MCF-7 WEHI-164 HepG-2 MDBK 
Astrodaucus orientalis (L.) Drude > 100 > 100 > 100 > 100 
Capparis spinosa L. > 100 > 100 > 100 > 100 
Centaurea bruguierana (DC.) Hand. – Mzt. 47.30 50.66 84.70 53.27 
Cephalanthera caucasica kränzl > 100 > 100 > 100 > 100 
Colutea persica Boiss. > 100 > 100 > 100 > 100 
Erodium oxyrrhnchum M.B. > 100 > 100 > 100 > 100 
Ficus carica L. var. genuina Boiss. > 100 > 100 > 100 > 100 
Heracleum persicum Desf. Ex. Fischer > 100 > 100 > 100 > 100 
Linaria pyramidata Lam. > 100 > 100 > 100 > 100 
Marrubium vulgare L. > 100 > 100 > 100 > 100 
Minuartia lineata Bornm. > 100 > 100 > 100 > 100 
Paliurus spina-christi Mill. > 100 > 100 > 100 > 100 
Papaver bracteatum Lindl > 100 > 100 > 100 > 100 
Pteropyrum aucheri Jaub. & Spach > 100 > 100 > 100 > 100 
Roemeria refracta DC. > 100 > 100 > 100 > 100 
Tamarix aralensis Bge. > 100 > 100 > 100 > 100 
Tamoxifen 3.69 19.1 4.38 4.39 
Ghafari
 
S. et al. 
 
50 RJP 2(2), 2015: 47-51 
 
Many of Centaurea species were used as anti-
diabetic, anti-rheumatic and anti-inflammatory 
agents, based on Iranian Traditional Medicine 
manuscripts [15] and ethnobotanical knowledge 
[16]. They were additionally used for their anti-
bacterial and cytotoxic effects [17].  
These species have been the subject of many 
investigations which has led to the isolation of 
various types of compounds. For example, 
sesquiterpene lactones, flavonoids and lignans 
[17]. There has been a report about investigation 
of C. bruguierana cytotoxicity by MTT assay. 
The data were collected from southern parts of 
Iran, against HT-29 (colon carcinoma), Caco-2 
(colon adenocarcinoma), T-47D (breast ductal 
carcinoma) and NIH-3T3 (Swiss embryo 
fibroblast) cell lines. The results had 
demonstrated cytotoxic activity against Caco-2 
cell line with an IC50 value of 10 µg/mL [18].  
In our study C. bruguierana was collected from 
northern parts of Iran and its methanol extract 
demonstrated  IC50 from 47.30 to 84.70µg/mL.  
The aim of our study was to acquire the most 
cytotoxic fraction of the selected plant; thus, C. 
bruguierana was our candidate for further 
fractionation. The cytotoxic results of its 
fractions are shown in table 3. 
 
Table 3. IC50 of fractions of Centaurea  bruguierana against 
cell lines 
 Extract (IC50 µg/mL) 
MCF-7 WEHI-164 HepG-2 MDBK 
Petroleum ether > 100 > 100 > 100 > 100 
Chloroform 23.03 14.44 21.10 17.00 
 
These results are in accordance with previous 
studies [18].  
According to table 3, the chloroform fraction of 
C. bruguierana showed the lowest IC50 against 
selected cell lines, in comparison with other 
fractions (IC50 ranging from 14.44 to 23.03 
µg/mL). Therefore the chloroform fraction of C. 
bruguierana is the best candidate for isolation 
and identification of active principles with 
cytotoxic effects as well as investigating their 
mechanism of inhibition.  
Acknowledgements 
This research was supported by a Grant (No. 111, 
145) from Traditional Medicine and Materia 
Medica Research Center, Shahid Beheshti 
University of Medical Sciences, Tehran, Iran. We 
are grateful to Dr. Elahe Davari, Dr. Hamid 
Moazzeni and Ms. Parisa Rabeei for their 
assistance in this research. 
 
Declaration of interest 
The authors declare that there is no conflict of 
interest. The authors alone are responsible for the 
content of the paper. 
 
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Investigating the cytotoxic effect of some medicinal plants from northern parts of Iran

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Investigating the cytotoxic effect of some medicinal plants from northern parts of Iran

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