Inhibition of tumor cells growth and stimulation of lymphocytes by Euphorbia species

Abstract

Plants have been shown to possess a number of beneficial anticancer and immunomodulatory properties. In this study the possible in vitro antitumor activity and immunomodulatory effects of five species of Euphorbia, an important genus of Euphorbiaceae, including E. petiolata, E. hebecarpa, E. osyridea, E. microciadia, and E. heteradenia were investigated using cytotoxicity and cell proliferation assays. Among different tumor cell lines, the most sensitive cell line to methanolic extracts of the plants was determined as follows. Hela cervical cancer cells to E. hebecarpa and E. microciadia, K562 leukemia cells to E. petiolata and E. heteradenia, and Fen bladder cancer cells to E. osyridea. The methanolic extracts were then fractionated into hexane, n-butanol, ethyl acetate, and water and the effect of these fractions was tested for cytotoxic activity on the selected cell lines. The results indicated the significant stronger antiproliferatory effect of the hexane factions in all the plants when compared with other ones. The methanolic extracts of the plants were also studied for their effects on the activation of the lymphocytes. All of the extracts showed stimulatory effects on the proliferation of the lymphocytes at lower concentrations. After further fractionation of the extracts, the butanolic and hexane fractions showed the highest activity on the lymphocyte activation. In conclusion, all the plants studied had the capacity to inhibit proliferation of tumor cells with beneficial immunomodulatory effects on the lymphocytes. This dual effect of the plants indicates their value for further investigations as antitumor agents.

Full text

Introduction
Plants have been used in the treatment of cancer for
centuries. In recent years, various medicinal plants
have been evaluated for their antitumor activity.(1–5)
Eorts have been applied to identify the new potential
components as anticancer drugs. A variety of plant
extracts and isolated components have been investi-
gated for their cytotoxic potential on various leuke-
mia or solid tumor-derived cell lines.(6–10) Medicinal
plants belonging to Euphorbia genus are among those
that have been widely studied with promising eects.
Euphorbia is the largest genus of the plant family
Euphorbiaceae with over 1000 species ranging from
annuals to trees and a broad distribution in both tem-
perate and tropical regions.(11) e Euphorbia is named
after Greek surgeon Euphorbus who is supposed to
have used the species’ milky latex in his medicine in
Africa.(12) In Iran, over 70 species have been reported,
17 of which are endemic.
(13)
Several plants of this family
are known to have traditional medical uses against skin
infections, gonorrhea, migraines, intestinal parasites,
and warts.(14) ey have also been traditionally used for
treatment of cancers and viral diseases.(15) e antitu-
mor activity of several species of the genus Euphorbia,
against sarcoma 180 ascites, leukemia in mice, and
cytotoxic activity against certain cancer cell lines has
been reported.(16–18) In our previous study, the cyto-
toxic activity Euphorbia cheiradenia, which is wildly
grown in Iran, against Jurkat and K562 leukemia cell
lines was demonstrated.
(19)
Furthermore, we found that
E. cheiradenia has the capacity to enhance proliferation
Immunopharmacology and Immunotoxicology
Immunopharmacology and Immunotoxicology, 2011; 33(1): 34–42
2011
33
1
34
42
Address for Correspondence: Zahra Amirghofran, Immunology Department, Medical School, Shiraz University of Medical Science, 71345-1798 Shiraz, Iran.
E-mail: amirghz@sums.ac.ir
08 October 2009
06 January 2010
13 February 2010
0892-3973
1532-2513
© 2011 Informa Healthcare USA, Inc.
10.3109/08923971003699018
RESEARCH ARTICLE
Inhibition of tumor cells growth and stimulation of
lymphocytes by Euphorbia species
Zahra Amirghofran1,2, Saeed Malek-hosseini1, Hossein Gholmoghaddam1, and Fatemeh Kalalinia1
1Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran, and 2Medicinal and Natural Products
Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract
Plants have been shown to possess a number of beneficial anticancer and immunomodulatory properties. In
this study the possible in vitro antitumor activity and immunomodulatory effects of five species of Euphorbia,
an important genus of Euphorbiaceae, including E. petiolata, E. hebecarpa, E. osyridea, E. microciadia, and
E. heteradenia were investigated using cytotoxicity and cell proliferation assays. Among different tumor
cell lines, the most sensitive cell line to methanolic extracts of the plants was determined as follows. Hela
cervical cancer cells to E. hebecarpa and E. microciadia, K562 leukemia cells to E. petiolata and E. heteradenia,
and Fen bladder cancer cells to E. osyridea. The methanolic extracts were then fractionated into hexane,
n-butanol, ethyl acetate, and water and the effect of these fractions was tested for cytotoxic activity on
the selected cell lines. The results indicated the significant stronger antiproliferatory effect of the hexane
factions in all the plants when compared with other ones. The methanolic extracts of the plants were also
studied for their effects on the activation of the lymphocytes. All of the extracts showed stimulatory effects
on the proliferation of the lymphocytes at lower concentrations. After further fractionation of the extracts,
the butanolic and hexane fractions showed the highest activity on the lymphocyte activation. In conclusion,
all the plants studied had the capacity to inhibit proliferation of tumor cells with beneficial immunomodula-
tory effects on the lymphocytes. This dual effect of the plants indicates their value for further investigations
as antitumor agents.
Keywords: Euphorbia species; tumor cell line; lymphocytes
IPI
470423
(Received 08 October 2009; revised 06 January 2010; accepted 13 February 2010)
ISSN 0892-3973 print/ISSN 1532-2513 online © 2011 Informa Healthcare USA, Inc.
DOI: 10.3109/08923971003699018 http://www.informahealthcare.com/ipi
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Eect of Euphorbia species on tumor cell lines and lymphocytes proliferation 35
and activation of human lymphocytes.(20) Lymphocytes
are the major cells orchestrating host–defense against
cancer cells. An augmentation in the function of these
cells can help control or even eradicate malignant
tumors. erefore, medicinal herbs possessing immu-
nostimulatory eects in addition to antitumor activity
can be good candidates for defense against cancer
cells.
In the present study, the antitumoral activity and
immunomodulatory eects of ve other Euphorbia
species that are native to Iran are reported. ese spe-
cies included E. petiolata, E. hebecarpa Boiss, E. osyridea
Boiss, E. microciadia Boiss, and E. heteradenia Boiss.
To the best of our knowledge, the cytotoxic and immu-
nomodulatory activities of these plants have not been
previously reported.
Materials and methods
Plant materials
Aerial parts of E. petiolata, E. hebecarpa, E. osyridea,
E. microciadia, and E. heteradenia were collected
from Fars province and authenticated by Dr Khosravi
from Department of Biology, Shiraz University, Shiraz,
Iran. Voucher specimens were deposited in the Shiraz
University Herbarium. A methanol extract from each
plant was obtained by maceration of the plant in
3 × 1500 mL methanol at room temperature for 48 h. e
methanol extracts were ltered and concentrated under
reduced pressure. e yield (w/w) of extracts was 8.5%
for E. petiolata, 7.4% E. hebecarpa, 11% E. osyridea,
5.4% E. microciadia, and 11.1% E. heteradenia. e
methanol extract was then suspended in water (500 mL)
and successively re-extracted by 500 mL each (three
times) of hexane, ethyl acetate, and n-butanol. All frac-
tions including the nal remaining water fractions were
concentrated under reduced pressure using a rotary
evaporator and then freeze-dried. For the bioassay test,
samples were dissolved in dimethyl sulfoxide (DMSO)
and further diluted in culture medium (RPMI 1640).
Cell lines and cell culture
Tumor cell lines including Hela (cervix epitheloid carci-
noma), MCF-7 (Michigan Cancer Foundation-7; breast
carcinoma), Fen (bladder carcinoma), K562 (myelog-
enous leukemia), and Jurkat (T-cell leukemia) prepared
from Iranian cell bank were used in this study. All the
cell lines were kept in RPMI 1640 medium (Sigma, St
Louis, MO) supplemented with 10% fetal calf serum
(Gibco--BRL, Eggenstein, Germany) in culture asks
at 37°C in 5% humidied CO2 incubator. e cells were
fed until conuence (2 × 106) and were expanded by
trypsinization (for adherent cells) and subcultured at
lower numbers in new culture asks. e viability of
cells determined by Trypan blue was more than 95%.
Negative control cells were treated with DMSO and
RPMI. e nal concentration of DMSO in control wells
was as equal as tests (maximum 0.1% v/v).
In vitro cytotoxicity assay
A colorimetric assay using 3-(4,5-dimethylthiazoyl-2,5-
diphenyltetrazolium bromide (MTT) was performed.
Briey, appropriate concentrations of the tumor cells,
determined in a previous examination, were added onto
at-bottomed microculture plates in the absence or pres-
ence of the various concentrations of the extracts and/or
fractions (in triplicate). e cells were incubated at 37°C in
a 5% humidied CO
2
incubator for 48 h. en, 10 µL MTT
(5 mg/mL, Sigma) was added to each well and incubation
was continued for a further 4 h at 37°C. Solubilization
solution containing isopropanol (100 µL/well)
and 10% SDS in 0.01 M HCl was added into each well.
After complete solubilization of the dye, the plates were
read at 570 nm on an ELISA reader. e reference wave-
length was 690 nm. e mean optical density (OD) ± SD
for each group of replicates was calculated. Percentage
inhibition of cells exposed to various treatments was
obtained as follows. % Inhibition= 100 – [(Test OD/
negative control OD) × 100].
Lymphocyte proliferation assay
Peripheral blood lymphocytes (PBLs) from at least
ve healthy male individuals (25–35 years) with their
consent were separated by gradient centrifugation with
Ficoll-Hypaque. e viability of cells was more than 98%.
e eect of the extracts and the resultant fractions on
the mitogen-induced PBLs (1 × 105/well) was determined
using 5-bromo-2′-deoxy-uridine (BrdU) incorporation
assay (ELISA kit, Roche Diagnostics GmbH, Mannheim,
Germany). Phytohemagglutinin (PHA) at a suboptimal
concentration (1:3500 diluted, Gibco, Germany) was
added to the culture of PBLs as the mitogen. Cells were
cultured in at-bottom 96-well plates for 48 h in the
presence or absence of various concentrations of the
extracts or fractions. Controls were extract-untreated
cells exposed to PHA and DMSO at a concentration as
equal as the tests. After labeling with BrdU for the nal
18 h of the incubation period, DNA was denatured and
the cells were incubated with anti-BrdU monoclonal
antibody for detecting incorporated BrdU. e absorb-
ance related to the BrdU level was measured with a
microplate reader at 450 nm. e stimulation index (SI)
was calculated according to the following formula: (OD
of treated cells/OD of corresponding control) × 100. All
experiments were plated in triplicate wells and were
performed at least three times.
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36 Z. Amirghofran et al.
Statistical analysis
e data were presented as mean ± SD and the analysis
was performed using SPSS 11.5 Software. e Student’s
t-test determined whether the results had statistical sig-
nicance. e level of signicance was set at P < 0.05.
Results
Eects of the extracts on the growth of tumor cells
In this study, the methanolic extract of the plants were
prepared and their cytotoxic activity on ve dierent types
of tumor cells was investigated (Figure 1). Dierent frac-
tions from the methanolic extracts were also obtained and
tested on the most sensitive cell line to each methanolic
extract (Figure 2). Fractions were used at 0.01–200 μg/mL
and half maximal inhibitory concentration (IC50) was
then determined (Table 1). cis-Platin was used as a ref-
erence compound. e results obtained for each extract
was as follows.
E. hebecarpa
e highest cytotoxic activity of methanolic extract was
against Hela tumor cells (Table 1). At a concentration
of 84 μg/mL, 50% Hela cells growth was inhibited. After
Fen
−20
0
20
40
60
80
100
200100501010.10.01
%Inhibition
Concentration (µg/ml)
Mcf-7
0
20
40
60
80
100
200100501010.10.01
% Inhibition
Concentration (µg/ml)
K562
0
20
40
60
80
100
200100501010.10.01
% Inhibition
Concentration (µg/ml)
Jurkat
0
20
40
60
80
100
200100501010.10.01
% Inhibition
Concentration (µg/ml)
Hela
−20
0
20
40
60
80
100
200100501010.10.01
% Inhibition
E. petiolate
E. hebecarpa
E. osyridea
E. microciadia
E. heteradenia
Concentration (µg/ml)
Figure 1. Eect of methanolic extracts of the Euphorbia plants on dierent tumor cell lines determined by MTT colorimetic assay. Result was
expressed as the mean percentage of cell growth inhibition in three dierent experiments. Standard deviation was <2.1 in all concentrations.
Control, extract-untreated cells containing DMSO in the nal concentration equal to test wells. DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimeth-
ylthiazoyl-2,5-diphenyltetrazolium bromide.
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Eect of Euphorbia species on tumor cell lines and lymphocytes proliferation 37
Hela cells, Jurkat with IC
50
of 110 μg/mL and K562 with
IC
50
of 127 μg/mL were more sensitive than other cells.
e least inhibitory eects of this extract was observed
on MCF-7 cells (IC50 > 200 μg/mL). e eects of dif-
ferent fractions obtained from the methanolic extract
on the most sensitive cell line, Hela cells, showed that
hexane fraction with IC50 of 19 μg/mL was the most
eective fraction.
E. microciadia
At a concentration of 100 μg/mL, more than 50% growth
inhibition of Hela, K562, and Jurkat cells was observed.
is extract was mostly eective on Hela cells with IC50
of 70 μg/ml. e IC50 obtained for the eects of all the
fractions of this extract on Hela cells except hexane was
more than 200 μg/mL. 50% growth inhibition of Hela
cells was observed at 30.1 μg/mL of the hexane fraction
(P < 0.05).
E. petiolata
e order of sensitivity of dierent cell lines to this
extract was K562 > Fen > Hela > Jurkat > MCF-7. At 120
and 127 μg/mL of this extract, 50% of K562 and Fen
cells proliferation were inhibited. Among various frac-
tions, inhibitory eect of the hexane fraction on K562
cells (IC50 79.4 μg/mL) was the strongest (P < 0.05).
Other fractions did not show 50% inhibitory eects at
concentrations <200 μg/mL.
E. heteradenia / K562
0
20
40
60
80
100
0.01 0.1 11050 100 200
%Inhibition
Concentration (µg/ml)
E. microciadia / Hela
0
20
40
60
80
100
0.01 0.1 11050 100 200
%Inhibition
Concentration (µg/ml)
E. petiolata / K562
0
20
40
60
80
100
0.01 0.1 11050 100 200
% Inhibition
Concentration (µg/ml)
E. hebecarpa / Hela
0
20
40
60
80
100
0.01 0.1 11050 100 200
%Inhibition
Concentration (µg/ml)
E. osyridea / Fen
0
20
40
60
80
100
0.01 0.1 11050 100 200
% Inhibition
water
ethyl acetate
hexane
n-butanol
Concentration (µg/ml)
Figure 2. Eect of various extracts of the Euphorbia plants on selected tumor cell lines determined by MTT colorimetic assay. Result was expressed
as the mean percentage of cell growth inhibition in three dierent experiments. Standard deviation was <1.5 in all concentrations. Control,
extract-untreated cells containing DMSO in the nal concentration equal to test wells. DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazoyl-
2,5-diphenyltetrazolium bromide.
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38 Z. Amirghofran et al.
E. osyridea
e methanolic extract of this plant had inhibitory eects
on the growth of dierent cell lines but this eect did not
reach to 50%. In calculation of IC25, Fen cells and then
Hela and K562 were more sensitive than the others (IC25:
2.5, 14, and 70 μg/mL respectively). e eect of the
various fractions of this extract on Fen cells showed the
maximum activity for the hexane fraction with IC50 of
49 μg/mL (P < 0.05).
E. heteradenia
e cytotoxic activity of this extract on dierent cell lines
was almost weak. It was mostly active on K562 and Hela
cells with IC
25
of 68 and 79 μg/mL. e eects of various
fractions of this extract on K562 cells showed that both
hexane and butanol fractions were eective and caused
50% inhibition of these cells at concentrations of 20.4
and 28.1 μg/mL, respectively (P < 0.05). Ethyl acetate and
aqueous fractions did not show notable eects on this
cell line.
Eects of the extracts on the lymphocytes proliferation
e methanolic extract of the plants was investigated
for their possible immunomodulatory eects using
BrdU cell proliferation assay. PBLs were stimulated
with PHA and then exposed to various concentra-
tions of the extracts (0.01–100 μg/mL). All methanolic
extracts had the ability to increase lymphocyte prolif-
eration at lower concentrations and inhibit at higher
concentrations (Figure 3). Almost in all the extracts,
the maximum stimulation eect was at 10 µg/mL
Table 1. Eect of the methanolic extracts and dierent fractions of the Euphorbia plants on the growth of tumor cell lines using MTT colorimetric
assay. e methanolic extracts of the plants were fractionated into hexane, ethyl acetate, butanol and aqueous and then cells were exposed to
dierent concentrations of the fractions. IC50 or IC25 (µg/mL) obtained for each fraction is demonstrated.
E. petiolataaE. hebecarpaaE. osyrideabE. microciadiaaE. heteradeniab
Eect of methanolic extracts
Jurkat 165 110 121 96 >200
K562 120 127 70 86 68
Hela 144 84 14 70 79
Fen 127 140 2.5 135 142.5
Mcf-7 176 >200 >200 163 195
E. petiolatacE. hebecarpadE. osyrideaeE. microciadiadE. heteradeniac
Eect of dierent fractions
Water >200 >200 >200 >200 >200
Butanol >200 158 >200 >200 28.1
Hexane 79.4 19 49 30.1 20.4
Ethyl acetate >200 >200 >200 >200 >200
Cis-platin 19.4 23.4 3.1 19.4 23.4
IC50, half maximal inhibitory concentration; MTT, 3-(4,5-dimethylthiazoyl-2,5-diphenyltetrazolium bromide.
aValues are represented IC50. bValues represented IC25. cIC50 on K562 cells. dIC50 on Hela cells. eIC50 on Fen cells.
0
0.5
1
1.5
2
2.5
E. osyridea E. heteradenia E. petiolata E. microciadia E. hepecarpa
Extract concentration (µg/ml)
Stimulation Index
0.01
0.1
1
10
50
100
200
*
*
*
*
*
*
**
**
*
*
*
**
*
Figure 3. Eect of methanolic extracts of the Euphorbia plants on cell proliferation of the mitogen-induced PBLs determined by DNA (BrdU)
incorporation assay. Result was expressed as the mean of stimulation indices. Control; PHA-treated cells lacking the extract and containing DMSO
in the nal concentration equal to test wells. e error bar is the standard deviation of the means between three dierent experiments. Asterisks
represent statistically signicance stimulatory eect compared to control (P < 0.05). BrdU, 5-bromo-2′-deoxy-uridine; DMSO, dimethyl sulfoxide;
PBLs, peripheral blood lymphocytes; PHA, phytohemagglutinin.
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Eect of Euphorbia species on tumor cell lines and lymphocytes proliferation 39
(SI, 1.8–1.9). e results of the eect of various fractions
on the lymphocytes are demonstrated in Figure 4 and
were as follows.
E. hebecarpa
Two fractions of this plant showed the maximum stimula-
tory eect on the lymphocytes, butanol and ethyl acetate.
e butanol fraction showed stimulation indices of 1.49
and 1.28 at concentrations of 1 and 10µg/mL, respectively
(P < 0.05). e SI of ethyl acetate fraction was 1.33 and
1.45 at concentrations of 50 and 100 µg/mL (P < 0.05).
E. microciadia
Butanol fraction showed the strongest eect on the lym-
phocytes with SI of 1.6 at 10 µg/mL (P < 0.05). Hexane
fraction at 1 µg/mL and ethyl acetate at 50 µg/mL
showed a weaker stimulatory eect than butanol
extract.
E. petiolata
Hexane and butanol fractions of this plant were eective
on the proliferation of the lymphocytes at concentrations
of 1 and 10 µg/mL (maximum SI, 1.7) (P < 0.05). Other
Euphorbia hebecarpa
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0.01 0.1 11050 100
Stimulation Index
water
ethyl acetate
hexane
n-butanol
Concentration (µg/ml)
Euphorbia Osyridea
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0.01 0.1 11050 100
Stimulation Index
water
ethyl acetate
hexane
n-butanol
Concentration (µg/ml)
Concentration (µg/ml)
Euphorbia microciadia
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0.01 0.1 11050 100
Stimulation Index
water
ethyl acetate
hexane
n-butanol
Concentration (µg/ml)
Euphorbia heteradenia
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0.01 0.1 11050 100
Stimulation Index
water
ethyl acetate
hexane
n-butanol
Concentration (µg/ml)
Euphorbia petiolate
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0.01 0.1 11050 100
Stimulation Index
water
ethyl acetate
hexane
n-butanol
Figure 4. Eect of various extracts of the Euphorbia plants on cell proliferation of the mitogen-induced PBLs determined by DNA (BrdU) incorpora-
tion assay. Result was expressed as the mean of stimulation indicies in three dierent experiments. Standard deviation was <0.2 in all concentrations.
Control; PHA-treated cells lacking the extract and containing DMSO in the nal concentration equal to test wells. BrdU, 5-bromo-2′-deoxy-uridine;
DMSO, dimethyl sulfoxide; PBLs, peripheral blood lymphocytes; PHA, phytohemagglutinin.
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40 Z. Amirghofran et al.
extracts did not increase cell proliferation compared with
the control.
E. osyridea
e butanol as well as ethyl acetate fractions were more
eective in the incorporation of BrdU into DNA than the
other extracts. e maximum stimulatory activity of buta-
nol fraction was at 10 µg/mL (SI, 1.5) and that of ethyl
acetate was at 50 µg/mL (SI, 1.47 µg/mL) (P < 0.05).
E. heteradenia
Hexane extract at 1 µg/mL with SI of 1.45 was more
eective on the cell proliferation (P < 0.05). e maxi-
mum SI for butanol and ethyl acetate fractions was
equal to 1.26.
As appeared from the results mentioned above, the
hexane extract of E. heteradenia and E. petiolata was
the most stimulatory fraction. In terms of other plants,
the butanol extract was more eective (Figure 5).
It is noted that, since activation of lymphocytes could
enhance the reduction of MTT dye in the mitochondria
and interfere with the eects of the extracts, MTT assay
was only used for testing the proliferation of tumor cells
and this was a limitation of our study.
Discussion
In the present study, the antitumor activity of the extracts
of ve native species of the Euphorbia plants was inves-
tigated on tumor cell lines derived from leukemia
or solid tumor origins. e methanolic extract of the
plants were rst studied on the cell lines. Generally, all
the extracts more and less showed cytotoxic activity in
various concentrations. e maximum cytotoxic activ-
ity of the extracts was against Hela tumor cells (in terms
of E. hebecarpa and E. microciadia), K562 (in terms of
E. petiolata and E. heteradenia), and Fen cells (in terms
of E. osyridea). ese inhibitory eects indicate the dier-
ence between the sensitivity of tumor cell lines possibly
due to dissimilar origin of the cells. e cell lines were
then treated with various fractions of the methanolic
extracts including hexane, n-butanol, ethyl acetate, and
aqueous.
e hexane extract in all the plants exhibited the
maximum antiproliferatory eects on the related cancer
cells. A comparison of the IC50 values obtained for the
hexane extracts showed the following order of activity:
E. hebecarpa > E. heteradenia > E. microciadia >
E. osyridea > E. petiolata. In several previous studies
the antitumor activity of other species of the Euphorbia
plants has been demonstrated. Antileukemic activity
of the extract of E. esula against the P-388 lymphocytic
leukemia in mice has been shown.(21) E. chamaesyce L.
has demonstrated antitumor activity against a panel
of human cancer cell lines.(22) In our previous study,
E. cheiradenia showed cytotoxic activity against Jurkat
and K562 leukemia cell lines.(20) e ability of this plant
to inhibit leukemia cell lines was shown to be due to the
induction of apoptosis in the cells.
Another new nding of this study was that the treat-
ment of PBLs with the extracts induced proliferation of
the lymphocytes. In our previous study, E. cheiradenia
had promising immunostimulatory properties on cell-
mediated and humoral antibody-mediated activation of
T and B cells. e methanolic extract of this herb showed
a signicant dose-dependent stimulatory eect on the
proliferation of the lymphocytes and the secretion of
IL-2. is extract showed also a dose-related eect
on in vivo cellular immunity and humoral antibody
synthesis.(20) In this study, the methanolic extract of the
plants showed stimulatory eects on the proliferation
of the lymphocytes. is eect was observed at lower
concentrations and replaced with an inhibitory eect
as the concentration of the extract increased. Various
fractions prepared from the methanolic extracts were
also tested to nd the fractions with best activity on
the lymphocytes. Butanolic fractions from most of the
plants showed a signicant impact on the activation of
the mitogen-induced lymphocytes. e hexane fractions
also eectively stimulate the BrdU incorporation of lym-
phocytes. In case of E. heteradenia and E. petiolata, the
highest activity belonged to the hexane fraction which
may indicate the presence of a compound in this frac-
tion with both cytotoxic and immunostimulatory eects.
e hexane extract was also eective in other plants.
It is possible that the same components involve in the
Butanolic extract
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0.01 0.1 11050 100
Concentration (mg/ml)
Stimulation Index
E. osyridea
E. heteradenia
E. petiolate
E. microciadia
E. hebecarpa
Figure 5. Eect of butanolic extracts of Euphorbia plants on the pro-
liferation of the mitogen-induced PBLs determined by DNA (BrdU)
incorporation assay. Result was expressed as the mean of stimula-
tion indices in three dierent experiments. Control, PHA-treated
cells lacking the extract and containing DMSO in the nal concen-
tration equal to test wells. BrdU, 5-bromo-2′-deoxy-uridine; DMSO,
dimethyl sulfoxide; PHA, phytohemagglutinin; PBLs, peripheral blood
lymphocytes.
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Eect of Euphorbia species on tumor cell lines and lymphocytes proliferation 41
inhibition of tumor cell growth at high concentration but
stimulation of lymphocytes at low concentration. is
suggests the presence of compounds such as polysac-
charides, lectins, or novel phytochemicals in these
extracts. In some previous studies on other medicinal
plants, both immunostimulatory and antitumor activi-
ties have been observed. ese dual eects have mostly
been attributed to the dierent compounds particularly
certain polysaccharides presented in the plants that can
directly inhibit the proliferation of mammalian tumor
cells and stimulate the immune system by acting as a
mitogen.(23,24) Mitogen-activated lymphocytes have been
shown to kill several types of cancer cells.
(25)
More stud-
ies and in vivo data are necessary to prove the benets
of these eects together and the mechanism involved.
It is also likely that dierent ingredients are responsible
for the observed eects. Euphorbia plants are rich in
phenolics, aromatic esters, steroids, diterpenoids, tetra-
cyclic triterpenoids, pentacyclic triterpenoids, essential
oils, and several bioactive constituents.(12,26–28) In previ-
ous studies, searching for the presence of agents with
antitumoral eects has been lead to identify new com-
pounds in Euphorbia species. Euphoportlandols A and
B, tetracylic diterpene polyesters from E. portlandica,(29)
3-ingenyl angelate, a hydrophobic diterpene ester iso-
lated from the plant E. peplus,(18) euphol, a triterpene
alcohol from E. kansui,(30) bioactive diterpene esters
isolated from E. cornigera,(31) and jolknolide B from
E. scherina
(32)
have been introduced as new anticancer
agents. e majority of in vitro phytochemical antican-
cer studies on non-nutritive plant substances have
focused on plant compounds such as avonoids and
terpenoids.
(25)
Several avonoids, tannins, alkanes, ster-
ols, and cycloartane triterpenoids have been isolated
from E. petiolata.(12) Moreover, in a study performed on
the chemical constituents of some species of Euphorbia
grown in Iran, diterpenoids with myrsinane skeletons
was isolated from E. heteradenia and E. cheiradenia.
e diterpenoids were biologically active with diverse
tumor-promoting and anticancer activities.(12) A few
immunostimulatory compounds from Euphorbia spe-
cies have also been reported. Ingenols from E. kansai
has shown stimulatory eects on macrophages.(33)
e mitogenic activity on the murine spleen lym-
phocytes and/or human T-lymphocytes on the latex of
E. neriifolia(34) and E. marginata,(35) and E. characias L.(36)
due to lectins presented in the plant extracts have been
shown. On the contrary, immunosuppressive eects
of an ethyl acetate fraction from E. royleana has been
reported,(37) which indicated the presence of dierent
compounds with a variety of mode of actions on the
immune system in Euphorbia species.
In conclusion, results of this study showed that the
hexane fraction of the plants studied had cytotoxic
activity on dierent tumor cells which was in line with
the anticancer activity reported for some other species
of Euphorbia genus. On the other hand, the extracts
enhanced lymphocyte activation and proliferation indi-
cating the immunomodulatory activity of them. Whether
a single active or several compounds are responsible
for these eects requires further studies. e ability of
immunostimulation with antitumor activity by these
plants also suggests their value for in vivo studies to nd
how these eects can work together against cancer.
Declaration of interest
is work was supported by grant nos 3311 and 3937 from
Shiraz University of Medical Sciences. e authors report
no conicts of interest. e authors alone are responsible
for the content and writing of the paper.
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