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The anticancer activity of five species of Artemisia on Hep2 and HepG2 cell lines

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It has been reported that several Artemisia species (Astraceae) possess cytotoxic activity against different human cell lines. In this study, the toxicity of the A. kulbadica, A. sieberi, A. turanica, A. santolina and A. diffusa against human Caucasian hepatocyte carcinoma (HepG-2) and human Caucasian larynx carcinoma (Hep-2) cell lines have been investigated. These plants were collected from Khorasan province, northeast of Iran. Different concentrations (200, 400, 600, 800, 1000, 1200, 1600 and 3200 μ g/mL) of ethanol extract of each sample were prepared. The cytotoxic effects of these concentrations against two human tumor cell lines Hep2 and HepG2 were determined by quantitative MTT assay. Results showed concentration- and time-dependent toxicity. In all extracts, toxic effects were significantly higher on HepG2 cells compared with Hep2 cells. HepG2 cells are rich in phase I and phase II metabolic enzymes and it is probable that metabolic activation of some active ingredients of the extracts were converted to more toxic metabolites and caused more toxicity in these cells.
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Pharmacologyonline 3: 327-339 (2009) Emami et al.
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The Anticancer Activity of Five Species of Artemisia on
Hep2 and HepG2 Cell Lines
Seyed Ahmad Emami, Nasser Vahdati-Mashhadian*, Remisa Vosough and Mohammad
Bagher Oghazian
Drug Research Centre, School of Pharmacy, Mashhad University of Medical Sciences
(MUMS), Mashhad, I R Iran
Summary
It has been reported that several Artemisia species (Astraceae) possess cytotoxic activity
against different human cell lines. In this study, the toxicity of the A. kulbadica, A. sieberi,
A. turanica, A. santolina and A. diffusa against human Caucasian hepatocyte carcinoma
(HepG-2) and human Caucasian larynx carcinoma (Hep-2) cell lines have been investigated.
These plants were collected from Khorasan province, northeast of Iran. Different
concentrations (200, 400, 600, 800, 1000, 1200, 1600 and 3200 µg/mL) of ethanol extract of
each sample were prepared. The cytotoxic effects of these concentrations against two human
tumor cell lines Hep2 and HepG2 were determined by quantitative MTT assay.
Results showed concentration- and time-dependent toxicity. In all extracts, toxic effects were
significantly higher on HepG2 cells compared with Hep2 cells. HepG2 cells are rich in phase I
and phase II metabolic enzymes and it is probable that metabolic activation of some active
ingredients of the extracts were converted to more toxic metabolites and caused more toxicity in
these cells.
Keywords: Hep2, HepG2, Artemisia spp., Hepatotoxicity, MTT assay.
* Author of correspondence:
Nasser Vahdati-Mashhadian
Drug research center
School of Pharmacy
Mashhad University of Medical Sciences
Mashhad, 91775-1365, Iran
E-mail: vahdatin@mums.ac.ir
Fax No. +98-511-8823251
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328
Introduction
The genus Artemisia is one of the largest and most widely distributed of the nearly 100 genera
in the tribe Anthemideae of the Asterceae (Compositae). This is a large and heterogeneous
genus, numbering over 400 species distributed mainly in the temperate zone of Europe, Asia and
North America. These species are Perennial, biennial and annual herbs or small shrubs,
frequently aromatic. Leaves are alternate, capitula small, usually pendent, racemose, paniculate
or capitate inflorescences, rarely solitary. Involucral bracts stand in few rows, receptacle flat to
hemispherical, without scales, sometimes hirsute. Florets are all tubular, Achenes obvoid,
subterete or compressed, smooth, finely striate or 2-ribbed; pappus absent or sometimes a small
scarious ring (1-3).
The genus in Iran has about 34 species which two of them are endemic to the country (3-
6)..These plants contain monoterpenes, sesquiterpenes, sesquiterpene lactones, flovonoides,
coumarins, sterols, polyacetylenes etc. (1).
Artemisia species have been shown to have wide range of pharmacological and toxicological
effects, including antimalarial (7, 8), cytotoxic (9), antifungal (10-12) and antioxidant (12-14)
activities.
In this study, the anticancer activity of five species of the genus against two human cancer cell
lines (Hep2 and HepG2) was investigated.
Materials and Methods
Plant material
Five species of Artemisia were collected from different parts of Iran (Table 1). Dr. V.
Mozaffarian, Research Institute of Forest and Rangelands, Ministry of Jahad-E-Agriculture Iran,
confirmed the identity of the plants. Voucher specimens of the species have been deposited in the
Herbarium of National Botanical Garden of Iran (TARI).
Table 1- Characteristics of collected Artemisia species
Artemisia species Location Collection
time
A. diffusa Krasch.
ex Poljak. Mazdavand, Khorasan Razavi province
(height 900 m) Aug. 19, 2007
A. kulbadica
Boiss. & Buhse Islamabad, Shahrabad road, Northern
Khorasan province (height 907 m) Aug. 5, 2007
A. santolina
Schrenk Daghe Akbar, Southern Khorasan province
(height 1460 m) Sep. 20, 2007
A. sieberi Besser Samie Abad, Khorasan Razavi province
(height 909 m) Sep. 15, 2007
A. turanica Krash Samie Abad, Khorasan Razavi province
(height 808 m) Sep. 15, 2007
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Cell cultures and treatments
HepG-2 and Hep-2 cells were purchased from Pasteur Institute Collection of Cell Cultures,
Tehran, Iran and were cultured in DMEM (Sigma) supplemented with 10% FBS (Gibco, USA),
L-glutamine (2 mM, Jaber ibn Hayan, Iran), penicillin (100 IU/mL, Jaber ibn Hayan, Iran) and
streptomycin (100 µg/mL, Jaber ibn Hayan, Iran) under standard conditions and was sterilized by
0.22 µm microbiological filters (Millipore, Irland) after preparation and kept at 4°C before using.
The cells were subcultured in the ratio 1:3 twice per week. Passages 1–15 were used for
experiments. Cells were seeded at a density of 5000 cells/well in 96-well plates (Nunc,
Denmark). Incubations with various concentrations of the extracts were started 24 h after
seeding and continued for 24 and 48 hours.
Extraction Procedure
The shade dried aerial parts of each species (100 g) were chopped in small pieces and then
crushed into powder by a blinder. Each sample was macerated in ethanol 70% (v/v) for 48 hours
and then extracted by a percolator. The extracted solutions were concentrated at 50°C under
reduced pressure to dryness.
Cytotoxicity assay
The cytotoxic effect of obtained extracts against previously mentioned human tumor cell lines
was determined by a rapid colorimetric assay, using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide, Sigma, Germany] and compared with untreated controls (15).
This assay is based on the metabolic reduction of soluble MTT by mitochondrial enzyme activity
of viable tumor cells, into an insoluble colored formazan product, which can be measured
spectrophotometrically after dissolving in dimethyl sulfoxide (DMSO, Significancema,
Germany) (16). Briefly, 200 µL of HepG-2 cells (2.5
×
104 cells per ml mL of media) were
seeded in ten columns of 96-well microplates and incubated for 24 h (37°C, 5% CO2 air
humidified). Then, for 8 columns, 20 µL of prepared concentrations (200, 400, 600, 800, 1000,
1200, 1600 and 3200 µg/mL) of each extract was added to each column and incubated for
another 24 and 48 h in the same condition. Two columns of each microplate were specified to
blank (containing only DMEM) and control (containing 5% ethanol 96%), respectively. To
evaluate cell survival, 20 µL of MTT solution (5 mg/mL in phosphate buffer solution) was added
to each well and incubated for 3-4 h. Then, almost all old medium containing MTT was gently
replaced by 200 µL of DMSO and 20 µL of glycine buffer (0.1 M, Biogen, Iran) and then
pipetted to dissolve any formed formazan crystals. The first column contained blank (20 µL
glycine, 200 µL DMSO) and last column contained culture without cells. Finally, the
microplates were incubated at room temperature for 30 min. The same procedure was carried
out for Hep-2 cells as well.
The absorbance of each well was measured by an ELISA reader (STAT FAX 303, USA) at a
wavelength of 570 nm. Determination of percent of growth inhibition was carried out using the
following equilibrium: Cell survival (%) = T/C × 100
Where C is the mean absorbance of control group and T is the mean absorbance of test group.
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330
Statistical analysis
The data were expressed as standard error of mean (SEM) of 8 independent experiments.
Wherever appropriate, the data were subjected to statistical analysis by one-way analysis of
variance (one-way ANOVA) followed by Tukey-Kramer test and Paired-Sample T Test for
multiple comparisons. A value of p<0.05 was considered significant. SPSS 11.5 software was
used for the statistical analysis. Curves were plotted by Graphpad Prism 4 Demo.
Results
24-hour exposure
Results of the MTT cytotoxicity assay for different concentrations of ethanol extracts of A.
kulbadica, A. diffusa, A. sieberi, A. santolina and A. turanica against HepG-2 and Hep-2 cell
lines are presented in Figures 1 through 5, respectively. Results showed a concentration-
dependent toxicity for all the extracts and the overall toxicity on HepG-2 cells is more than that
on Hep-2 cells. The calculated IC50 values for the above mentioned Artemisia species against
Hep2 cells were 951.11, 1054.75, 231.59, 1093.82 and 975.54 and against HepG2 cells were
<200, 520.70, <200, 514.82 and 259.49, respectively. Evaluation of IC50 indicated that the
extract of A. kulbadica was most toxic against HepG2 and A. sieberi was the most toxic against
Hep2 cells.
48-hour exposure
Results have shown a similar pattern to 24-hour exposure experiments with higher toxicity,
possibly due to longer exposure (results not shown).
The order of survival cells are as follows:
HepG2 (After 24 h):
A. diffusa > A. santolina > A. turanica > A. sieberi > A. kulbadica
Hep2 (After 24 h):
A. santolina > A. kulbadica = A. turanica = A. diffusa > A. sieberi
According to graphs, Minimum toxic dose of extracts of Artemisia as follows:
A. kulbadica=400 µg/ml A. sieberi=800 µg/ml
A. turanica=1000 µg/ml A. santolina=1600 µg/ ml A. diffusa=1600µg/ml
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**
*** *** *** *** *** *** ***
Discussion
In our investigation, the in vitro toxicity of the ethanol extracts of some Iranian Artemisia
species were shown against two cancer cell lines: Hep-2 and HepG-2. This is a concentration-
dependent effect in the range of 200 through 3200 µg/mL. Collectively, this toxicity is stronger
against HepG-2 compared with Hep-2 cell lines.
The toxicity of Artemisia species on cancer cells has been shown in vitro (17-19) and in vivo (7,
20). Also, we previously have shown a similar effect for some other Iranian Artemisia species
(21). Artemisinin, the active ingredient of Artemisia annua, showed cell toxicity against human
lymphoid leukemia (Molt-4) cells (22). Cesquiterpenlactones, terpenoids and flavonoids are
other antitumor substances extracted from Artemisia species (23, 24). Artesunate, a
semisynthetic derivative of artemisinin, showed both in vitro and in vivo anticancer effects (25).
It also may induce antiangiogenic effects that may contribute to its anticancer effects. The
predominant effect of the extracts or the active ingredients of Artemisia species is apoptosis.
They induce apoptosis in various cell lines via activation of caspases, depolarization of the
mitochondrial membrane potential and down-regulation of Bcl-2 expression (18) or cell cycle
arrest (26, 27). Hu, et al. observed morphological changes typical of apoptosis, including
condensed chromatin and a reduction in volume with exposure of another human hepatoma cell
line to components of Artemisia capillaries thunberg (26). Kim et al. (2007) suggested the use
of Artemisia fukudo as a preventive measure against cancer (27). Toxicity of Artemisia species
on HepG2 observed through inhibition of transeaminase (23).
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
Concentration(µ /ml)
%Absorption
Concentration (µg/ml)
0(alcohol)
Toxicity of A. Kulbadica a
g
ainst
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Figure 1- Results of the MTT assay of different concentrations of Artemisia kulbadica on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و **: 0.001 < p value < 0.01و(*: 0.01 < p value < 0.05
0
0(alc)
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400
600
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1000
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1600
3200
0
25
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%Abs
Concentration (µg/ml)
0(alcohol)
%Absorption
Toxicit
y
of A.
ieberi a
g
ainst He
p
2
Concentration (µg/ml)
%Absorption
**
**
**
*** ***
***
***
***
Toxicit
y
of A.
ieberi a
g
ainst He
p
-G2
0(alcohol)
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0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
Figure 2- Results of the MTT assay of different concentrations of Artemisia sieberi on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و (**: 0.001 < p value < 0.01
**
**
** ** ** **
***
**
%Absorption
Concentration (µg/ml)
0(alcohol)
Pharmacologyonline 3: 327-339 (2009) Emami et al.
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0
0(alc)
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3200
0
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conc(µ /ml)
%Abs
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
Figure 3- Results of the MTT assay of different concentrations of Artemisia turanica on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و **: 0.001 < p value < 0.01و(*: 0.01 < p value < 0.05
***
*
**
**
**
***
***
***
0(alcohol)
Concentration (µg/ml)
%Absorption
Toxicit
y
of A. turanica a
g
ainst
*
*
*
**
**
**
***
0
(
alcohol
)
Concentration
(/l)
%Absor
p
tion
*
Toxicit
y
of A. turanica a
g
ainst
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0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
Figure 4- Results of the MTT assay of different concentrations of Artemisia santolina on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و **: 0.001 < p value < 0.01و(*: 0.01 < p value < 0.05
***
***
*
*
**
**
***
***
0
(
alcohol
)
Concentration
(/l)
%Absor
p
tion
Toxicit
y
of A. santolina a
g
ainst
*
*
*
*
**
**
**
***
0
(
alcohol
)
Concentration
(/l)
%Absor
p
tion
Toxicit
y
of A. santolina a
g
ainst
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0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
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75
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conc(µ /ml)
%Abs
0
0(alc)
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1200
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3200
0
10
20
30
40
50
60
70
80
90
100
110
conc(µ /ml)
%Abs
Figure 5- Results of the MTT assay of different concentrations of Artemisia diffusa on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و **: 0.001 < p value < 0.01و(*: 0.01 < p value < 0.05
***
*
*
** **
**
***
***
0
(
alcohol
)
Concentration (µg/ml)
%Absor
p
tion
Toxicit
y
of A. diffusa a
g
ainst He
p
-
*
*
*
**
**
**
**
***
0
(
alcohol
)
Concentration
(/l)
%Absor
p
tion
Toxicit
y
of A. diffusa a
g
ainst He
p
2
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Artemisinin itself produced rapid apoptosis rather than necrosis against human lymphoid
leukemia (Molt-4) cells (22). Based on these previous studies, the mechanism of toxicity of
Artemisia species is predominantly apoptosis, although, necrosis is also another possibility,
especially in higher concentrations.
In our experiments, the extracts showed more potent activity against HepG2 cells than Hep2
cells. HepG2 cells are of liver origin and express a wide range of phase I and phase II enzymes
such as cytochrome P450 enzymes, hydrolase, nitroreductase, catalase, peroxidase,
NAD(P)H:cytochrome c reductase, cytochrome P450 reductase, epoxide hydrolase,
sulfotransferase, glutathione S-transferase (GST), and N-acetyl transferase (28). Some of these
enzymes present in higher concentrations in growing than in confluent cells (29). Our
experiments were carried out in the growing phase of HepG2 and Hep2 cells, thus a high
metabolic activity could be estimated. Eupatilin, a pharmacologically active flavone derived
from Artemisia species, is extensively metabolized by cytochrome P450 (CYP) and UDP-
glucuronosyltransferase (UGT) enzymes in human liver microsomes. Activation reactions with
rifampin have been shown in HepG2 cells, a drug that undergo metabolic activation in the liver
and causes hepatotoxicity (30). In our previous study, similar results were observed in these two
cell lines (31). Because of high metabolic capacity of HepG2 cells for activation and
deactivation of xenobiotics, part of the higher toxicity of the plants extracts against HepG-2
compared with Hep2 cells may be due to the activation reactions of ingredients of the extracts.
Complementary studies will elucidate the uncovered aspects of anticancer toxicity of these
plants.
Acknowledgement
The authors would like to thank the authorities in research council of Mashhad University of
Medical Sciences and School of Pharmacy (MUMS) for their supports.
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... Overall, both the methanol and ethyl acetate extracts of A. rutifolia displayed higher inhibitory effects against P. aeruginosa as compared to B. subtilis at lower concentrations. Values are the average of at least three readings (±SD) antioxidant, [40][41][42] antimicrobial, 40,41,[43][44][45][46][47] antiviral, [48][49][50][51][52][53] antimalarial, [54][55][56][57][58] anticancerous, [59][60][61][62] antidiabetic/hypoglycemic, [63][64][65][66][67][68] anti-inflammatory, 61,69,70 and anthelmintic activities [71][72][73] . ...
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Recently, most researches have focused on the biological activities of the extracts obtained from different Artemisia species due to the presence of essential compounds with strong activity against some gram-negative and gram-positive bacteria. In this study, five extracts of Artemisia rutifolia Stephan ex Spreng. from the northeastern Gilgit-Baltistan region of Pakistan were analyzed for total flavonoid and total phenolic contents and their antibacterial activities against some clinical and phyto-pathogenic bacterial strains were assessed with agar disk diffusion method. Results indicated that the methanol, ethanol, chloroform, ethyl acetate and n-hexane extracts of A. rutifolia are rich in flavonoids and phenols and all the tested extracts showed the broad spectrum growth inhibition of the tested gram positive (Bacillus subtilis and Staphylococcus aureus) and gram negative bacterial strains (Escherichia coli and Pseudomonas aeruginosa). Overall, methanol and ethyl acetate extracts showed better activities even at lower concentrations (5 mg/ 248 Acta Pharmaceutica Sciencia. Vol. 60 No. 3, 2022 ml) where B. subtilis and P. aeruginosa were the most susceptible strains. Hence, the MICs of these two effective extracts (methanol and ethyl acetate extracts) were tested against most susceptible bacterial strains (B. subtilis and P. aeruginosa) at 1-4 mg/ml conc. Results of MICs showed that both the methanol and ethyle acetat extracts were effective against B. subtilis and P. aeruginosa at 3 and 4 mg/ml concentrations where ethyl acetate extract exhibited higher inhibitory effect than the methanol extract. Therefore, extracts of A. rutifolia could be used as operational sources against pathogenic bacterial diseases.
... Ahamad et al. (2019) reported that the different parts and extracts of the A. absinthium have wide pharmacological and clinical activities. So, its essential oil shows antimicrobial activity (Dulger et al., 1999), the water extract of the leaves -some nematicidal activity (Vijayalakshmi et al., 1976), the methanolic extract of aerial part -anti-inflammatory activity and analgesic activity (Ahamad et al., 2019), aqueous and methanolic extracts -hepatoprotective action (Gilani and Janbaz, 1995), methanolic extract -in-vitro anticancer activity (Emami et al., 2009), different extracts -antiulcer activity (Shafi et al., 2004), the crude ethanolic extract and crude aqueous extract of aerial parts -anthelmintic effects (Tariq et al., 2009). In our study, A. absinthium L. was revealed to be used to treat the diarrhea (colibacillosis), internal and external parasites, wounds, scabies, ringworm, paronychia contagiosa, necrobacillosis and sheepox. ...
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... [45][46][47][48][49][50] Different species of Artemisia are very important in terms of cytotoxic and anti-proliferative activities and several reports have been published about their anti-cancer effects. [51][52][53][54] A. scoparia is a species of Artemisia with anti-nociceptive, anti-inflammatory, and antipyretic effects. [55] Moreover, this species can be considered as a strong source of natural cytotoxic compounds to inhibit cancer cell growth. ...
... For example the herb Artemisia absinthium (Wormwood) shows anti-angiogenic effects in tumor cell lines [3]. In another study Artemisia significantly inhibited cell proliferation and promoted apoptosis in two human breast cancer cell lines [4]. ...
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... Various therapeutic bioactivities including leishmanicidal effects, antioxidant, antifungal, antitumor, antimalarial, anti-inflammation and neuroprotective have been reported for different species of Artemisia [15,[26][27][28][29][30]. It has been shown that the extracts of different species of Artemisia [31][32][33] and green synthesized nanoparticles from different species of Artemisia have cytotoxic and anticancer properties [29,34]. Artemisia ciniformis is one of the species of Artemisia which is widely grown in Iran [35]. ...
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... The large increase in displaced persons, refugees, and migrants seen in 2014 Courses and Facilities that provide training in refugees and migrant health can be found in many countries, including, Australia (Emami et al., 2009), Canada (Jock, 2015) and the USA (Mental & Certificate, 2012). In Europe, in addition to training and education available at the national level, at the regional level, there are training modules provided under the EC CARE program. ...
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A general introduction to the genus Artemisia
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