FARMACIA, 2017, Vol.

65, 1
ORIGINAL ARTICLE

ANTICANCER ACTIVITY OF EUONYMUS EUROPAEUS FRUITS

EXTRACT ON HUMAN MELANOMA CELLS

BOGDAN SEVASTRE1, ORSOLYA SÁRPATAKI1, ROXANA LIANA STAN2*, MARIAN

TAULESCU1, ALEXANDRA CRISTINA SEVASTRE-BERGHIAN2, NELI KINGA OLAH3,4,
FLAVIA FURTUNA4, DANIELA HANGANU2, ADRIANA CORINA HANGAN2, MIHAI
CENARIU1, IOANA BÂLDEA2
1
University of Agricultural Science and Veterinary Medicine, 3-5 Calea Mănăștur Street, 400372, Cluj-Napoca, Romania
2
“Iuliu Hațieganu” University of Medicine and Pharmacy, 8 Victor Babeș Street, 400012, Cluj-Napoca, Romania
3
“Vasile Goldiș” West University, 94 Revoluției Street, 310025, Arad, Romania
4
PlantExtrakt Ltd, Rădaia Baciu, Cluj, Romania

*corresponding author: roxanaluc@yahoo.com

Manuscript received: November 2015

Abstract
Euonymus europaeus L. (Celastraceae) (EE), known as spindle tree, is a deciduous shrub commonly found all over European
continent, used in traditional medicine for the treatment of ectoparasites. The present study investigates the in vitro anticancer
effects of EE hydro-alcoholic extract prepared from fresh fruits. Qualitative and quantitative analysis of the EE extract
content where made by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Cell
Proliferation Assay and Anexin V-fluorescein isothiocyanate apoptosis detection where performed on both human melanoma
cell line and normal human fibroblasts, in serial doses ranging from 0.05 µg/mL up to 50 µg/mL, at 6 and 24 hours exposure.
Evodiamine, a potent anticancer compound was found in significant amounts. In vitro melanoma cells were inhibited in a
concentration-dependent manner (p < 0.001), (IC50 = 6.76 µg/mL), and they were almost ten-fold more sensitive than
fibroblasts (IC50 = 53.08 µg/mL). EE had a significant and selective antitumor activity.

Rezumat
Euonymus europaeus L. (Celastraceae) (EE), este un arbust răspândit pe întregul continent european, folosit în medicina
tradițională în tratamentul antiparazitar. Studiul de față investighează activitatea antitumorală a extractului hidroalcoolic de
EE in vitro. Analiza cantitativă și calitativă a extractului de EE a fost realizat prin HPLC și spectrometrie de masă (HPLC-
MS). Testele de proliferare celulară și de apoptoză (Anexin V - fluorescein isothiocyanate) au fost realizate pe culturi
fibroblastice dermice umane normale și pe o linia celulară de melanom uman, în doze seriate de la 0,05 µg/mL până la 50
µg/mL la 6 și 24 de ore de expunere. A fost identificată evodiamina, un compus cu activitate puternică antitumorală. In vitro,
proliferarea liniei de melanom a fost inhibată în manieră doză dependentă (p < 0,001), (IC50 = 6,76 pg/mL), și a fost aproape
de zece ori mai sensibilă decât linia de fibroblaste (IC50 = 53,08 pg/mL). Activitatea antitumorală a EE a fost exercitată
selectiv aupra celulelor tumorale.

Keywords: antitumor, Euonymus europaeus, evodiamine, cell viability

Introduction Eastern Asia, traditionally used for cancer treatment.

There are several pathways responsible for its
Despite some progress, cancer remains a major
remarkable anticancer properties, but two of them
research challenge. Research for new anticancer drugs
are of main importance. First, it exhibits antitumor
is a very active domain; natural products represent
properties by inducing apoptosis via mitochondrial
an important source of new drugs. Actually, more
pathway [10, 19]. Second, EA inhibits tumour
than 60% of the new therapeutic compounds are
invasion, mainly by suppression of the matrix
either isolated from natural products or incorporate
metalloprotease-9 (MMP-9) activity [4, 9], effect
synthetic compounds based on the chemical
provided by dihydroxycinnamic acid (caffeic acid)
structure of natural products [1, 5, 18].
[13]. The inhibition of MMP-2 and MMP-9 is mediated
Plants from the Celastraceae family have been used
by NK-kappaB pathway [6].
for centuries in the field of traditional medicine, in
Euonymus europaeus L., (Celastraceae) (EE) also
South America, China, and Africa, for the treatment of
known as the spindle tree, is the only European
various diseases, including cancer [7, 21]. One of the
representative of the Celastraceae family. The
most investigated was Euonymus alatus (Thunb)
dried fruits and seeds are used against external
Celastraceae (EA), a native species from South-
parasites as scabies, ticks and lice, while the seeds
56
 FARMACIA, 2017, Vol. 65, 1
are emetic and purgative [2]. Relatively few phyto-
chemical analysis on EE were performed, but they
revealed active compounds including sesquiterpene
polyesters belonging to the alatol, 3-deoxymatol,
3,4-dideoxymaytol families, evonimate alkaloids,
dihydro-β-agarofuran polyesters [7], and lectins
[17]. Evodiamine is a quinazolinecarboline alkaloid
initially isolated from Chinese herb Evodia rutaecarpa,
represent a promising chemotherapeutic agent in the
multiple-drug resistant cancer cells [12]. Evodiamine
has been shown to exhibit anticancer properties by
various mechanisms as cell cycle arrest, induction
of apoptosis [19].
In the present study, we investigated the anticancer
activity of EE. To differentiate the general cyto-
toxicity from a selective antitumor effect, we tested
the inhibitory potential on both normal fibroblastic
line and on melanoma cell line and then we
investigated apoptosis in EE treated cultures.
Materials and Methods
Plant materials and extraction. Fresh fruits of EE,
harvested in September 2011, from a forest near
Cluj-Napoca, Romania, were used to prepare the
hydro alcoholic extract. The harvest of fruits was
performed according to the Good Agricultural and
Collection Practices rules, from an unpolluted area,
at a minimum of 3 km distance from any circulated
road. The fruits were botanically identified by
Quality Control Laboratory of PlantExtrakt TC Ltd,
Rădaia, Cluj County, Romania. A voucher specimen
was filed and kept in the company’s archives (B.
no. 063811, CoA 4817/20.09.2011).
The sampling of the vegetable material was performed
according to the European Pharmacopoeia, and the
botanic identification, according to the German
Homeopathic Pharmacopoeia. The moisture of the
fresh vegetal material was 70%.
The freshly cut fruit was mixed with 90% vol.
ethanol. The extraction ratio was 1 part fruit to 0.7
parts solvent. The extraction was performed at
room temperature, by maceration and periodical
mixing (minimum 10 min x 2 times a day x 10
days). After 10 days, the relative density and the
dry residue were evaluated. The mixture of plant
and solvent was subsequently pressed, left at
maximum 30°C for 5 days and then filtered. The
resulting extract was the tincture used for the
further experiments. All preparation steps were
performed on an API-GMP certified production
flow at SC PlantExtrakt SRL in Rădaia, Cluj County,
Romania, according to method 2a from the German
Homoeopathic Pharmacopoeia [20].
Before use, the alcoholic solution was processed in
a rotary evaporator at 40°C, until 3/4 of the content
evaporated, then refilled with sterile saline solution
to initial volume for use on cell culture.
57
Standardization of the tincture. The aspect of the
tincture was determined by observation of countenance,
colour and smell. The relative density was measured
using an Anton Paar 35 DMA digital densitometer. The
result is the average of 3 independent determinations.
The dry residue was determined by drying at
110ºC, for 3 hours. The result is the average of 3
independent determinations. Firstly, the ethanol
content was determined by distillation, and secondly
by the relative density of the distillate solution
correlated with the values from the alcoholometric
tables.
Determination of evodiamine content. The evodiamine
content was determined by HPLC, using a method
provided by Phytolab and adapted. The determination
was performed using a Varian ProStar HPLC system.
We used a Phenomenex Luna C18 silicagel-C18
column, 150 mm x 4.6 µm with a pre-column of
5 mm x 4.6 µm, both having particles of 5 µm. As a
mobile phase, a solvent gradient with phosphoric acid
pH = 2.5, water and acetonitrile, with a 1 mL/min
flow rate were used. The detection was performed
with a DAD detector at 228 nm. The UV-Vis
spectra were recorded from 200 nm to 600 nm [22].
100 µL of EE extract from each concentration of
standard evodiamine solution were injected.
Evodiamine was used as the standard. For the
quantitative determination, a calibration curve with
concentration between 50 and 500 µg/mL was built.
The calibration curve’s equation is:
A = 60110 x C – 478977
and the correlation factor is: 0.9807. The
identification was made based on the comparison
between the retention time and UV-VIS spectra of
compounds separated from EE extract and the
standard evodiamine. The maximum absorption of
evodiamine is at 228 nm.
Cell culture. The assessment of antiproliferative
effect was performed both on normal human dermal
fibroblasts (HDFa - Invitrogen, Willow Creek,
USA) and on a human radial growth phase (RGP)
melanoma cell line (WM35). Fibroblasts were
cultivated in a Dulbecco's modified Eagle's medium
(DMEM) supplemented with 5% fetal calf serum,
50 µg/mL gentamicin and 5 ng/mL amphotericin
(Biochrom AG, Berlin, Germany). Melanoma cells
(obtained from M. Herlyn, the Wistar Institute,
Philadelphia, PA, USA) [8] were maintained in
RPMI medium supplemented with 5% fetal calf
serum, 50 µg/mL gentamicin and 5 ng/mL
amphotericin (Biochrom AG, Berlin, Germany).
Both cultures were incubated in a humid atmosphere
at 37°C and 5% CO2 [16].
Cell Proliferation Assay. The cell cytotoxicity assay
was performed using the CellTiter 96® Aqueous
Non-Radioactive Cell Proliferation Assay (Promega
Corporation Madison, USA) as specified by the
manufacturer. The cells were seeded at a density of
 FARMACIA, 2017, Vol. 65, 1
4
10 /well in ELISA 96-well micro titration flat
bottom plates and allowed to accommodate for 24 h
in normal growth conditions. The cultures were
then exposed to EE extract (prepared as described
above) in increasing concentrations, ranging from
0.05 to 50 µg/mL for 6 and 24 hours. Each experiment
was carried out in triplicate. Cell cultures treated
only with medium where used as controls.
After each individual time point, the E. europaeus
treated cells and their counterpart controls were
incubated for 2 hours with 20 µL of 3-(4,5-di-
methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulphenyl)-2H-tetrazolium MTS/phenazine
methosulfate (PMS) mixture in 100 µL culture
medium [3]. The absorbance of each sample was
read at 490 nm using an ELISA plate reader (Tecan,
Männedorf, Switzerland). The viability was evaluated
based on the comparison with control cells. The
IC50 values representing the extract concentration
required to inhibit 50% of cell proliferation were
calculated from the calibration curve by linear
regression using Microsoft Excel.
Evaluation of apoptosis. The Annexin V-fluorescein
isothiocyanate (FITC) apoptosis detection kit was
used in conjunction with the vital dye propidium
iodide (PI) (BD Biosciences, San Jose, CA, USA).
The induction of apoptosis was investigated by
assessing positive cells (%) for Annexin V-FITC
and/or PI. The Annexin V-FTC positive cells were
detected by green fluorescence, while the presence
of red fluorescence was the indicator for PI. The
viable cells (showing no apoptosis) were identified
as Annexin V (-)/PI (-); the apoptotic cells were
identified as Annexin V (+)/PI (-) (early apoptosis),
and Annexin V (+)/PI (+) (late apoptosis). The
differentiation among these three-cell-type populations
was made by flow cytometric detection.
WM35 and fibroblast cells were seeded on Petri
glass dishes (Nalgene, Nunc, USA) at a density of
5 x 104/cm2 for 24 hours in medium, then exposed
Figure 1.
HPLC chromatograms at 150 nm of standard evodiamine (A) and the compounds isolated from the ethanolic
extract of Euonymus europaeus (B)
58
to Euonymus europaeus extract in concentrations of
0.5 and 1 µg/mL for 6 and 24 hours, followed by
FITC Annexin V and PI staining, according to the
manufacturer’s instructions.
The flow cytometric analyses were performed at
room temperature with a BD FACSCanto II flow
cytometer (Becton Dickinson & Company, Franklin
Lakes, NJ, USA) equipped with two lasers as excitation
sources: blue (488 nm, air cooled, 20 mW solid
state) and red (633 nm, 17 mW HeNe). The cytometer
setup was performed using the BD Cytometer Setup &
Tracking beads (CS&T beads), while the compensation
was done using compensation beads (BD Comp-
Bead). The data was analysed using the BD FACSDiva
Software (Becton Dickinson). A total number of
10,000 events were recorded for each sample. The
fluorescence spectrum of Annexin V and PI were
detected using a 530/30 nm and a 575/26nm band-
pass (BP) filter, respectively.
Statistical analysis. All data are reported as the
mean ± SEM. The Gaussian distribution was
checked using the Shapiro-Wilk normality test.
Pearson’s correlation was used in order to assess the
correlation between normally distributed variables;
this interpretation was made according to the Colton
scale. Statistical values and figures were obtained
using GraphPad Prism version 5.0 for Windows,
GraphPad Software, San Diego California, USA.
Results and Discussion
Standardization of the hydro alcoholic extract. The
obtained hydro alcoholic extract was a clear,
brown-orange liquid. The quality parameters of the
extract were within the admissibility range of
German Homeopathic Pharmacopoeia, as follows:
relative density 0.953 (0.935 - 0.955), dry residue
5.76% (Min. 4%), ethanol content 45% vol. (40 -
50% vol.), digitoxin content of 0.0028% (Max.
0.01%). Evodiamine content was 0.404 g/mL [20].
 FARMACIA, 2017, Vol. 65, 1
Evodiamine presence in the EE was shown by UV-VIS spectra proved the presence of evodiamine
HPLC chromatogram (Figure 1), while the recorded in the extract (Figure 2).

Figure 2.
UV-Vis spectra of standard evodiamine (A) and the compound isolated from the ethanolic extract of Euonymus
europaeus (B)

In vitro cytotoxicity. Human melanoma cell line
(WM35) was compared to normal human dermal
fibroblasts for sensitivity to EE. The differences of
absorption percentage among different concentrations
were statistically covered for both fibroblasts and
VM35 cells (p < 0.001).
The dose-response curves showed that cancer cells
were, by far, more sensitive to EE treatment than
normal fibroblasts, the IC50 at 6h was almost ten-
fold higher. The IC50 values for VM35 cells were
6.76 ± 1.08 µg/mL after 6 h and 5.89 ± 0.93 µg/mL
after 24 h, while IC50 of the fibroblasts were 53.08
± 5.47 µg/mL and respectively 25.04 ± 3.73 µg/mL
(Figure 3).

Figure 3.
Comparative cytotoxicity of Euonymus europaeus extract on fibroblasts and VM35 cultures after 6 hours (A)
and 24 hours (B) of exposure at a different dose versus untreated cells (mean ± SEM) (n = 3)

The fibroblasts inhibition showed a very different visible after 6 h, and enhanced after 24 h, a dose
pattern, suffering a gradual dose-dependent and dependent reduction of cell concentration was
time-dependent inhibition of viability, already clearly visible.

59
 FARMACIA, 2017, Vol. 65, 1

Figure 4.
Cell density at 0.5 µg/mL and 5 µg/mL

Apoptosis/necrosis assessment. To determine whether

the growth inhibition is associated with cell death,
Annexin V Propidium Iodide double staining of
VM35 cells and fibroblasts followed by flow
cytometric analysis were performed. As shown in
Figure 4 in the upper left quadrant, the fluorescein
isothiocyanate conjugate of Annexin V (FITC-A)
staining in propidium iodide (PE-A) negative cells -
early apoptotic cells - represented less than 0.5% of
cells in all treatment conditions. This suggests that
apoptosis was insignificant, therefore all cells
positive for PE-A, positive or negative for FITC-A,
confined in the right side of the panel, were
considered as necrotic cells.
In tumour cells cultures, the percentage of necrotic
cells increased from 3.6% in control cells up to
18.6% at 1.0 µg/mL in 24 h. Fibroblasts were even
more sensitive. The control cells showed 8.3%
percentage of necrosis and 24 h later, the necrosis
in the fibroblast subjected to a dose of 1.0 µg/mL,
increased up to 72.1%.
Apoptosis was measured using flow cytometric
analysis. Figure 5 represents scatter plots of Annexin
V-fluorescein isothiocyanate (FITC-A) (y - axis)
versus vital dye propidium iodide (PE-A) labelling Figure 5.
(x - axes). Lower left quadrants (absence of both Scatter plots of Annexin V-fluorescein
markers) indicate viable cells; upper left quadrants isothiocyanate versus vital dye propidium iodide
(FITC-A positive, PE-A negative) indicate the labelling
apoptotic cells (early stage apoptosis). In the
present study, the necrotic cells are represented by The present study investigates the underlying
the right side of the panel (PE-A staining alone or mechanisms of tumoricidal properties of EE
together with FITC-A). Fibroblasts and VM35 cells extract, already observed in the preliminary study
were treated with Euonymus europaeus extract in [15]. The in vitro investigations were performed
doses of 0.5 µg/mL and 1.0 µg/mL, after 6 hours using a human melanoma cell line VM35 and, in
and 24 hours while untreated cells were used as order to check the specificity of inhibition against
controls. tumour cells, we conducted parallel studies on a
human fibroblasts cell line. As far as we know, no
other studies focusing on tumouricidal properties of
EE were ever published by other research groups.
Despite the fact that no studies about EE are
available, other Euonymus species, for example

60
 FARMACIA, 2017, Vol. 65, 1
Euonymus alatus, already proved to have antitumor
properties. Euonymus alatus exhibits antitumour
properties by induction of apoptosis via mitochondrial
pathway [10] and inhibits tumour invasion by
inducing the inhibition of matrix metalloprotease-9
(MMP-9) activity [9]. Comparative studies on
MMP-9 inhibition showed that Euonymus alatus
was among the most effective extracts providing an
inhibitory activity of up to 90% [14]. Another
antiproliferative mechanism was discovered for
mammary and genital tumours, respectively the
inhibition of aromatase activity. Aromatase is an
enzyme responsible for oestrogen synthesis. A large
proportion of breast cancers express their own
aromatase. Euonymus alatus was highly effective in
inhibiting the intracellular aromatase in myometrial and
leiomyomal cells, in a dose-dependent manner [11].
This is the first study showing the presence of
evodiamine in EE. Evodiamine was initially
isolated from a Chinese herb named Evodia
rutaecarpa and represents a promising chemo-
therapeutic agent in the multiple-drug resistant
cancer cells [12]. Therefore, in the present study,
the phytochemical investigation was focused on
revealing the potent and selective anticancer effect
of EE plant extract. Our in vitro studies proved that
EE has a strong dose-dependent inhibitory effect on
human melanoma cell line VM35. The effect was
present in relatively small concentrations of plant
extract, respectively 2.5 µg/mL. Remarkable, when
a similar concentration was added in normal human
fibroblasts, it had very low effect on cell viability.
In very high doses, the EE extract was able to
induce necrosis, but the apoptosis was insignificant.
This finding suggests that the apoptotic or necrotic
mechanism is not the main mechanism involved in EE
tumouricidal activity, but it might have other targets
e.g. cell cycle control, down regulation of protein
synthesis, enhancement of oxidative stress etc.
Commonly, the phytotherapeutic extracts responsible
for tumour inhibition contain more than one anti-
tumour compound. Therefore, all mechanisms
described above should be considered in further
studies in order to establish their applicative value
in cancer therapy.
Conclusions
The present study proves that the EE alcoholic
extract reduces the proliferation of VM35 melanoma
cell line. The induction of apoptosis is unlikely, but
other mechanisms, such as cell cycle control, are
more probably involved. Therefore, we consider
that further studies need to be carried out in order to
establish the applicative value of EE extract in
cancer therapy. Our future directions include to
performing in vivo antiproliferative studies and
survival analysis on a classic transplantable tumour
61
model - Ehrlich ascites carcinoma inoculated in
mice. This transplantable tumour model will give
us also the possibility to investigate the side effect
of tumour growth and the interaction between
tumour cells and the immune system.
Acknowledgement
This study was supported by an internal research
grant of the University of Agricultural Science and
Veterinary Medicine Cluj Napoca, Romania.
Declaration of interests
The authors report no declaration of interest.
References
1. Alam M.N., Bristi N.J., Rafiquzzaman M., Review
on in vivo and in vitro methods evaluation of
antioxidant activity. Saudi Pharmaceutical Journal,
2013; 21: 143-152.
2. Aronson J.K., Meyler’s Side Effects of Herbal
Medicines. Amsterdam, The Netherlands: Elsevier,
2009; 94-95.
3. Bălăceanu A., Mateescu D., Diaconu C., Sărsan A.,
Primary Malignant Fibrous Histiocytoma of the
Right Ventricle: a Case Report and Review of the
Literature. Journal of Ultrasound in Medicine
(Journal of American Society of Ultrasonography),
2010; 29(4): 655-658.
4. Cha B.Y., Park C.J., Lee D.G., Inhibitory effect of
methanol extract from Euonymus alatus on matrix
metalloproteinase-9. J. Ethnopharmacol., 2003; 85:
163-167.
5. Chabner B.A., Roberts T.G.Jr., Chemotherapy and
the war on cancer. Nat. Rev. Cancer, 2005; 5: 65-72.
6. Chung T.W., Moon S.K., Chang Y.C., Novel and
therapeutic effect of caffeic acid and caffeic acid
phenyl ester on hepatocarcinoma cells: Complete
regression of hepatoma growth and metastasis by
dual mechanism. FASEB J., 2004; 18: 1670-1681.
7. Descoins C.Jr., Bazzocchi I.L., Ravelo A.G., New
sesquiterpenes from Euonymus europaeus
(Celastraceae). Chem. Pharm. Bull. (Tokyo), 2002;
50: 199-202.
8. Herlyn D., Adachi K., Koprowski H., Herlyn M.,
Experimental model of human melanoma
metastasis. Cancer Treat. Res., 1991; 54: 105-118.
9. Jin U.H., Lee J.Y., Kang S.K., L phenolic compound,
5-caffeoylquinic acid (chlorogenic acid), is a new
type and strong matrix metalloproteinase-9 inhibitor:
isolation and identification from methanol extract of
Euonymus alatus. Life Sci., 2005; 77: 2760-2769.
10. Kim C.H., Kim D.I., Kwon C.N., Euonymus alatus
(Thunb.) Sieb induces apoptosis via mitochondrial
pathway as prooxidant in human uterine
leiomyomal smooth muscle cells. Int. J. Gynecol.
Cancer, 2006; 16: 843-848.
11. Lee T.K., Kim D.I., Han J.Y., Inhibitory effects of
Scutellaria barbata D. Don. and Euonymus alatus
Sieb. on aromatase activity of human leiomyomal
cells. Immunopharmacol. Immunotoxicol., 2004;
26: 315-327.
 FARMACIA, 2017, Vol. 65, 1
12. Liao C.H., Pan S.L., Guh J.H., Antitumor mechanism
of evodiamine, a constituent from Chinese herb
Evodiae fructus, in human multiple-drug resistant
breast cancer NCI/ADR-RES cells in vitro and in
vivo. Carcinogenesis, 2005; 26(5): 968-975.
13. Park W.H., Kim S.H., Kim C.H., A new matrix
metalloproteinase-9 inhibitor 3,4-dihydroxycinnamic
acid (caffeic acid) from methanol extract of
Euonymus alatus: isolation and structure determination.
Toxicology, 2005; 207(3): 383-390.
14. Seo U.K., Lee Y.J., Kim J.K., Large-scale and
effective screening of Korean medicinal plants for
inhibitory activity on matrix metalloproteinase-9.
Ethnopharmacol., 2005; 97: 101-106.
15. Sevastre B., Sarpataki O., Olah N.K., Stan R.L.,
Taulescu M., Marcus I., Cătoi C., Antitumor effect
of Euonymus europaeus on Ehrlich tumor cells in
vivo. Farmacia, 2014; 62(5): 907-917.
16. Stan R.L., Hangan A.C., Dican L., Sevastre B.,
Hanganu D., Cătoi C., Sarpataki O., Comparative
study concerning mistletoe viscotoxins antitumor
62
activity. Acta Biologica Hungarica, 2013; 64(3):
279-288.
17. Teneberg S., Alsen B., Angstrom J., Studies on
Gala3-binding proteins: comparison of the glycol-
sphingolipid binding specificities of Marasmius
oreades lectin and Euonymus europaeus lectin.
Glycobiology, 2003; 13: 479-486.
18. Vicaș L., Teuștea A., Vicaș S., Marian E., Jurca T.,
Mureșan M., Gligor F., Assessment of antioxidant
capacity of some extracts for further use in therapy.
Farmacia, 2015; 63(2): 267-274.
19. Yu H., Jin H., Gong W., Pharmacological actions
of multi-target-directed evodiamine. Molecules,
2013; 18: 1826-1843.
20. ***German Homeopathic Pharmacopoeia, ed.
2013, Deutsche Apotheke Verlag, Stuttgart, 2013.
21. ***Homoopathisches Arzneibuch, ed. 2010,
Amtliche Ausgabe, Deutscher Apotheker Verlag,
Govi-Verlag - Pharmazeutischer Verlag.
22. ***Phytolab HPLC method for determination of
Evodiamine, 2014.