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Journal of Applied Pharmaceutical Science Vol. 6 (11), pp. 087-093, November, 2016
Available online at http://www.japsonline.com
Extracts of Codiaeum variegatum (L.) A. Juss is Cytotoxic on Human
Leukemic, Breast and Prostate Cancer Cell Lines
Mathias Tawiah Anim1, Christopher Larbie1*, Regina Appiah-Opong2, Isaac Tuffour2, Kofi Baffour-Awuah Owusu2,
1Department of Biochemistry and Biotechnology, KNUST, Kumasi, Ghana.
2Department of Clinical Pathology, NMIMR, University of Ghana, Legon, Ghana.
Received on: 26/05/2016
Revised on: 14/07/2016
Accepted on: 12/08/2016
Available online: 29/11/2016
The high cost of treatment of cancer coupled with the emergence of drug resistance makes it imperative for new
drug interventions to curb its occurrence. Hence, the objective of this research was to determine the
phytochemical, total phenolic content, antioxidant and antiproliferative effect of Codiaeum variegatum crude
extracts and fractions. The MTT cell viability and DPPH assays among others were used to determine the
selected properties of the plants. The presence of general glycosides, tannins, alkaloids, flavonoids and sterols
was observed in its stem bark and leaf. Triterpenoids were present in the leaf only while saponins were observed
in the stem bark only. Strong antioxidant activities were observed in both stem bark and leaf with EC50 values of
0.053±0.004 mg/mL and 1.396±0.073 mg/mL respectively. Both crude extracts showed antiproliferative activity
towards all cancer cell lines with the stem bark exhibiting the strongest cytotoxicity. However, both showed
strong cytotoxicity towards normal cells as well. The mechanism of cell death was determined to be apoptosis.
Further testing of fractions from the stem bark crude extract revealed an increase in cytotoxicity of its
chloroform fraction against Jurkat cells with an IC50 of 44.71±0.44 µg/mL. These results establish the
antiproliferative nature of this plant.
Cancer is a term used to describe a large group of
diseases characterized by the uncontrolled proliferation and
spread of abnormal cells (Hayflick, 1997). Its incidence and
mortality has risen tremendously over the past decade causing the
need for effective control measures (Ferlay et al., 2013). It is
estimated that the number of new cases is expected to rise by
about 70% over the next two decades (World Cancer Report,
2014). Recent statistics by the International Agency for Research
on Cancer (IARC) indicates a changing trend in recorded
incidence and mortality of which less developed countries now
record the highest number of cases (Ferlay et al., 2013). More
than 60% of the world’s total annual new cases occur in Africa,
* Corresponding Author
Email: e.kowlarbie @ gmail.com
Asia and Central and South America, with these regions
accounting for 70% of the world’s cancer deaths (World Cancer
Report, 2014). Though there are several treatment options
available, each comes with a high price tag often coupled with
adverse side effects. The recently evolving paradigm of drug
resistance to chemotherapeutic agents is also posing a great barrier
to reducing the incidence and mortality of cancer (Shervington and
Lu, 2008). Hence there is the need to exploit other remedies with
possibly less known adverse effects and from readily accessible
sources like plants. Plants could serve as a major source of
bioactive compounds with potential efficacy against cancers
(Talalay and Fahey, 2001). Codiaeum variegatum L. belongs to the
family Euphorbiaceae and it’s native to India, Philippines, Sri
Lanka, Thailand, Indonesia, Malaysia and some other Pacific
Islands (Stamps and Osborne, 2003). It is a common perennial
plant with over 200 varieties worldwide; each different from the
other with respect to the pattern and shades of colour as well as the
size and shape of it leaf (Figure 1).
Anim et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 087-093
Aside from its main purpose in serving as an ornamental,
the root decoction of C. variegatum is used to treat gastric ulcers.
The leaves have antibacterial and antiamoebic properties and can
be crushed and drunk to cure diarrhoea (Moundipa et al., 2005). In
indigenous Malaysian medicine, the plant is used as an anti-
infective and an anti-cancer agent (Ali et al., 1996). Research
conducted by Hassan et al. (2013) exposed the cytotoxicity effect
of C. variegatum cv. petra leaves on human caucasian breast
adenocarcinoma (MCF7), hepatocellular carcinoma (HepG2),
colon cell line (HCT116) and lung carcinoma cell line (A549) with
activities ranging from 17.3% to 98%.
Fig. 1: Codiaeum variegatum cv Gold Dust.
The purpose of this research was to evaluate the
cytotoxicity of 50% hydroethanolic extracts of C. variegatum CV
gold dust on leukemia (Jurkat), breast (MCF 7) and prostate (PC 3)
cancer cells. C. variegatum crude stem bark and leaf extracts were
also analyzed for their phytochemical constituents, antioxidant
activity, total phenolic content (TPC) and mode of cytotoxicity
MATERIAL AND METHODS
Cell lines and reagents
The cell lines used (Jurkat, MCF 7, PC 3, WRL 68,
HepG2) were obtained from RIKEN BioResource Centre Cell
Bank (Japan). Culture media (RPMI and α-MEM), 96 well plates,
(MTT) dye, isopropanol, HCl, trypan blue solution, absolute
ethanol, foetal bovine serum (FBS), antibiotics (penicillin and
streptomycin), 2, 2-diphenyl-1-picryl hydrazyl (DPPH), and
phosphate buffer saline were obtained from Sigma-Aldrich
Company (St. Louis, MO, USA).
Plant material and extraction process
Codiaeum variegatum cv. gold dust samples were
handpicked from the environs of the New Times Corporation
(5°34'10.5"N 0°13'20.5"W), North Industrial Area, Accra in April,
2014 before 9.00 am. Specimen of the plant was sent to the
Department of Herbal Medicine, Faculty of Pharmacy and
Pharmaceutical Sciences, KNUST, Kumasi for authentication by a
taxonomist and a voucher specimen was deposited at the
Herbarium for reference purpose (KNUST/HMI/2014/L094). The
plant was sorted into stem bark and leaves. The stem bark
component was chopped into pieces and both parts were washed
separately with water three times and air dried at room temperature
for three weeks. The dried samples were separately pulverized and
packaged in zip-locks for further use. Preparation of 50%
hydroethanolic extraction of the plant leaves and stems were
carried out separately, by suspending 50 g of the powder of each
part in 500 mL of 50% ethanol (50:50 v/v). The extraction was
done by cold maceration for 24 hrs at room temperature on a
shaker. The extracts were filtered through cotton wool,
concentrated using a rotary evaporator and freeze-dried to obtain
the C. variegatum hydroethanolic leaf and stem bark crude
Fractionation of C. variegatum stem bark hydroethanolic
extract Fractionation of the hydroethanolic stem bark extract of
C. variegatum was carried out in a separating funnel using
solvents of increasing polarity, petroleum ether, chloroform and
ethyl acetate. A mass of 1.5 g was dissolved in 15 mL of 50%
ethanolic solution and was successively partitioned with petroleum
ether, then chloroform and finally with ethyl acetate, each having a
volume of 30 mL, to obtain petroleum ether, chloroform and ethyl
acetate fractions. This was done for two to three times as polarity
increased. The remaining portion was designated as
The phytochemicals tested for were general glycosides,
anthracene glycosides, saponins, tannins, alkaloids, flavonoids,
sterols and triterpenoids. The presence of these phytochemicals in
the crude extracts was analyzed using standard methods (Trease
and Evans, 1989; Sofowora, 1993; Harborne, 1998).
Determination of total phenols
Total phenolic content (TPC) of leaf and stem bark was
determined using the Folin–Ciocalteau assay with slight
modification (Marinova et al., 2005). To a volume of 10 μL of
sample, 790 μL of distilled water was added. The concentration of
the leaf and stem bark extracts tested was 5 mg/mL. A volume of
50 μL of Folin–Ciocalteau reagent was added to the diluted
samples and thoroughly mixed. The mixtures were incubated in
the dark for 8 mins. Subsequently, 150 μL of 7% Na2CO3 was
added before incubation of the mixture for 2 hrs in the dark at
room temperature. Triplicate experiments were performed. The
absorbance was read at a wavelength of 750 nm using a microplate
reader (Tecan Infinite M200, Austria). Gallic acid (GA) was used
as the standard phenolic compound. A GA calibration curve was
plotted and used to determine the total phenolic content. The
results were expressed in milligrams of GA equivalents per gram
dry mass (mg GAE/g DM).
Anim et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 087-093 089
Determination of antioxidant activity
The antioxidant activity of C. variegatum leaf and stem
bark extracts was determined using the free radical scavenging
activity by DPPH method with some modification (Blois, 1958).
Methanolic solution of DPPH (0.5 mM) was added to equal
volumes of various concentrations of each extract (concentration
range 0-5 mg/mL). After 20 mins incubation at room temperature,
the absorbance was read at a wavelength of 517 nm (Tecan Infinite
M200 Pro plate reader, Austria). The inhibition concentration at
50% (IC50) value of each extract was calculated from the following
formula: % Antioxidant activity = [(A0−A1)/A0 × 100]
Where A0 is the absorbance of negative control (methanol), and A1
is the absorbance of test sample with DPPH. Butylated
hydroxytoluene (BHT) was used as standard control. Triplicate
experiments were performed. The half maximal effective
concentration (EC50) value, which is the concentration of the
extracts that can cause 50% free radical scavenging activity, was
L-RPMI and α-MEM culture media, respectively,
supplemented with 10% foetal bovine serum (FBS), containing
penicillin, streptomycin, and L-glutamine were maintained in
culture at 37°C in a humidified 5% CO2 atmosphere. The
tetrazolium-based colorimetric assay (MTT) was used to
determine the cytotoxicity of C. variegatum on the cancer and
normal cell lines (Ayisi et al., 2011). Triplicate experiments were
performed. Cells were seeded into the 96-well plates at the
concentration of 1×104 cells/well, treated with varying
concentrations of the plant extracts (0-1000 μg/mL) and incubated
as indicated above for 72 hrs. A color control plate was also setup
for each extract including the positive control, curcumin. MTT
solution (0.5 mg/mL) was added to each well on the plate, and
incubation continued for further 4 hrs. The reaction was stopped
with acidified isopropanol solution, and the plate incubated in the
darkness overnight at room temperature before reading the
absorbance at 570 nm using a microplate reader (Tecan Infinite
M200 Pro, Austria). The percentage cell viability was determined
The IC50 values were determined from the plot of percent cell
viability on the y-axis against extract concentrations on the x-axis.
Nuclear morphology examination (Hoechst staining)
MCF-7 cells were seeded at 1×106 cells /mL in a total
volume of 6 mL in sterile petri dishes and incubated for 24 hrs at
37 ºC in 5% CO2 to allow the cells to adhere to the dishes. The
cells were then treated with two different concentrations (20 and
40 µg/mL) of the most active crude extract (C. variegatum stem
bark) and standard urosolic acid (5.7 µg/mL) and then re-incubated
for 24 hrs at 37 ºC in 5% CO2. The cells were scraped from the
petri dishes with a cell lifter and transferred into 15 mL centrifuge
tubes. Centrifugation was done at 1000 rpm for 5 min and the
supernatant was discarded. The remaining cell pellets were re-
suspended in 1 mL of phosphate buffered saline (PBS). The cells
were then transferred into 1.5 mL eppendorf tubes and centrifuged
at 1000 rpm for 5 min. The supernatant was discarded and the cell
pellets were treated with 200 µL of 1% glutaraldehyde and then
incubated at room temperature for 30 min. The centrifugation was
subsequently repeated (as above) and the supernatant was
removed. A volume of 50 µL of PBS and 8 µL of Hoechst
solutions was finally added and mixed gently but uniformly. The
samples were applied on microscope slides, covered with cover
slips, mounted and examined on a fluorescent microscope
MCF-7 cells were seeded at 2×105 cells /mL in a total
volume of 3 mL in sterile petri dishes and incubated for 24 hrs at
37 ºC in 5% CO2. The cells were then treated with different
concentrations (20 and 40 µg/mL) of the most active crude extract
(C. variegatum stem bark) and standard curcumin and then re-
incubated for 24 hrs at 37 ºC in 5% CO2. The cells were scraped
from the petri dishes with a cell lifter and stirred gently and
uniformly. A volume of 100 µL was aliquoted into wells in a 96
well plate. An equal volume of the Guava Nexin Reagent was
aliquoted into each well, mixed thoroughly and incubated for 20
mins. The plates were read in a flow cytometer (Guava Easycyte,
Germany) (Vermes et al., 1995).
Data were analyzed by one-way analysis of variance and
the means assessed by Tukey’s test at 5% level of significance (p
< 0.05) using Graph pad Prism version 5.0. The results were
expressed as mean ± SD.
RESULTS AND DISCUSSION
Medicinal plants are of great importance to the general
health of individuals and communities. It is also an immense
source of medicines in pharmacognosy. Research by Ogunwenmo
et al. (2007) proved a difference in the phytochemical constituents
among various C. variegatum varieties. The presence of alkaloids,
saponins, and tannins were reported by Ogunwenmo et al. (2007)
to be varying in levels among varieties of this plant. With respect
to the C. variegatum cv gold dust tested in this research, both its
stem bark and leaf tested positive for general glycosides, tannins,
alkaloids, flavonoids and sterols as shown in table 1. However,
saponins were absent from the crude extract of C. variegatum
leaves, (probably due to the difference in variety as compared to
that used by Ogunwenmo et al., 2007) while its stem bark showed
no traces of triterpenoids. The presence of alkaloids, saponins,
tannins in such high concentrations could render this plant,
Anim et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 087-093
antibacterial and antiamoebic and this could be related to its use in
the treatment of diarrhoea (Moundipa et al., 2005).
Table 1: The phytochemical constituents present in hydroethanolic extracts of
The data show the intensities of observed colours or froths as compared to
+++ present at high concentration; ++ present in moderate concentration; +
present in low concentration; - absent
Total phenol content
The total phenolic content was extrapolated from the
standard calibration curve (
obtained from GA. Figure 2 shows the levels of TPC in the leaf
and stem bark extracts. The TPC of the stem bark extract was
significantly higher (p= 0.0005) as compared to the leaf extract as
shown in Figure 2.
Fig. 2: Total phenolic content of hydroethanolic extracts of C. variegatum stem
bark and leaf expressed as mean±standard deviation (p = 0.0005).
Figure 3 shows the antioxidant activity of C. variegatum
stem bark and leaf extracts. Both extracts and standard BHT
exhibited strong antioxidant activity in a dose dependent manner.
All the samples analysed showed an increase in antioxidant
activity with increasing concentrations, thus exhibiting a
concentration dependent pattern of free radical scavenging ability.
The stem bark extract of C. variegatum recorded the strongest
antioxidant activity. The stem bark of the C. variegatum appeared
to have stronger antioxidant activity as compared with the standard
(BHT) (p<0.0001). Members of the Euphorbiaceae plant family
possess strong antioxidant activities which are greatly associated
with the presence of phenolic compounds (Shahwar et al., 2010).
For instance, analysis of the leaf extract of C. variegatum cv spiral
and royal-like by HPLC-DAD showed that ellagic acid, a phenolic
compound may be responsible for its antioxidant activity (Saffoon
et al., 2014). Findings from this research proved that the stem bark
of the C. variegatum recorded the highest concentration of total
phenolics for this plant, suggesting that the observed antioxidant
activity could be partly attributed to the high levels of total
phenolics present in the sample. (Figure 3)
Fig. 3: Antioxidant activity of (A) BHT, (B) C. variegatum stem bark and C.
variegatum leaf. Each point represents three determinations.
Antiproliferative activity of extracts and curcumin
Table 2 shows the antiproliferative effect of the extracts
and standard (curcumin) on Jurkat cells, MCF 7, PC 3 and WRL
68 cell lines. The stem bark extract showed stronger inhibitory
effect on all the cell lines compared to the leaf extract. All the
extracts and curcumin inhibited the growth of the normal cell
WRL 68, indicating a poor selectivity (Table 2).
A previous study on C. variegatum cv petra established
the cytotoxicity of the leaves of this plant on human caucasian
breast adenocarcinoma (MCF7), hepatocellular carcinoma
(HepG2), colon cell line (HCT116) and lung carcinoma cell line
(A549) with activities ranging from 17.3% to 98% (Hassan et al.,
2013). This was confirmed in this research since both parts of the
plant showed various levels of cytotoxicity against MCF 7 cells,
with the stem bark of C. variegatum being the most cytotoxic part
with an IC50 of 35.55 ± 1.50 µg/mL. A similar trend was observed
with respect to leukaemia (Jurkat) and prostate (PC 3) cancer cell
lines where the stem bark recorded an IC50 of 59.71 ± 12.20
TPC (mg GAE/g)
A EC50 = 0.454 mg/mL
Anim et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 087-093 091
µg/mL and 52.54 ± 1.88 µg/mL as compared to 62.03 ± 8.49
µg/mL and 211.20 ± 77.09 µg/mL recorded by its leaf. This
suggests that the stem bark of C. variegatum could possibly be a
better source of bioactive compounds for chemotherapy than its
leaf. In elucidating the cytotoxic effect on the normal human liver
cell line (WRL 68), it was observed that the stem bark of C.
variegatum was the most toxic part. None of the extracts showed
good selectivity against the cancer cell lines with respect to the
normal human liver cells. This suggests that even the most active
part of the plant could in one way or the other cause damage to the
liver and probably other organs of the body when this medicine is
administered. However, further studies will be needed to eliminate
the toxic components of the extracts and isolate the active
Table 2: Cytotoxic activities of C. variegatum crude extracts.
IC50 values (µg/mL)
62.03 ± 8.49
1.84 ± 0.16
35.55 ± 1.50
84.44 ± 1.53
3.65 ± 0.08
52.54 ± 1.88
211.20 ± 77.09
8.10 ± 0.82
49.37 ± 2.7
74.55 ± 4.8
8.35 ± 0.40
Tabulated values represent mean ± standard deviation of three replicates.
p-values compare the statistical difference between the calculated means using
Tukey’s test. For each plant extract tested n=3
Nuclear morphology examination (Hoechst staining)
Apoptosis is often characterised by nuclear condensation,
chromosomal DNA fragmentation and the formation of cell
fragments called apoptotic bodies (Bruce et al., 2008). Cells
that are not undergoing cell death often possess a nucleus with the
normal, roughly spherical morphology. The most active crude
extract among all the extracts tested, C. variegatum stem bark, was
analysed to elucidate its molecular mechanism of action. The
effect of this selected crude extract on the nuclear morphology of
breast cancer cells (MCF 7) and its specific mode of action was
analysed using the Hoechst staining. The presence of fragmented
or shrunk nuclei was observed in C. variegatum stem bark treated
cells as well as standard urosolic acid. Figure 4 shows the effect of
C. variegatum stem bark extract on MCF 7 cells.
Hoechst staining showed that there were significant
morphological changes in nuclear chromatin similar to the changes
observed in the apoptotic mechanism of action by other members
of the Euphorbiaceae family on MCF 7 cells (Aslanturk and Celik,
Flow cytometry analysis
Figure 5 shows the mechanism of action of C.
variegatum stem bark on MCF 7 cells. The extract induced
cytotoxicity in a dose dependent manner and the mode of
cytotoxicity was confirmed to be apoptosis. (Figure 5)
Data generated from the flow cytometry assay indicated
and confirmed a dose-dependent and apoptotic mode of
cytotoxicity for C. variegatum stem bark at 20 and 40 µg/mL.
Emerging evidence has demonstrated that, the anticancer activities
of certain chemotherapeutic agents involved in the induction of
apoptosis have no side effects on normal tissues, and are thus
regarded as the preferred method of treating cancer (Xiao, 2007).
Hence, the apoptotic nature of C. variegatum renders it a good
candidate for chemoptherapy.
Fig. 4: Nuclear morphology of MCF 7 cells after 24 hrs of incubation without any treatment, control (A), treatment with 20 µg/mL (B), 40 µg/mL of C.
variegatum stem bark extract (C) and urosolic acid (standard) (D). Arrows point to nuclei that are fragmented/shrunk
Anim et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 087-093
Antiproliferative activity of stem bark fractions and curcumin
The antiproliferative activity of petroleum ether,
chloroform, ethyl acetate and hydroethanolic fractions against
Jurkat, MCF 7 and HepG2 was assessed. (Table 3).
Fractionation of the stem bark extract was performed to
verify if the fractions could elicit an increase in cytotoxic activity.
The fractions were tested against MCF 7, Jurkat and HepG2 cell
lines. With the exception of the chloroform extract of C.
variegatum stem bark which recorded an increase in cytotoxicity
towards Jurkat cells, with an IC50 of 44.71 ±0.44 µg/mL compared
to the extract; all the other fractions were relatively less toxic
against the cancer cells as compared to the crude extract.
This could be attributed to the fact that the active molecules in the
extract worked in a synergistic manner (or the activity of the active
compound was complemented by another compound) and
individually was not that effective. On the other hand, it is possible
that the solvents used for fractionation were unsuitable for the
This study provides information on the phytochemical
constituents, antioxidant and antiproliferative effect of C.
variegatum. From the study the stem bark of C. variegatum
exhibited a stronger free radical scavenging activity than its leaf,
though both had good antioxidant properties. However, further
Fig. 5: Flow cytometry evaluation of apoptotic effects on MCF 7 cells after 24 hrs of incubation without any treatment, control (A) and treatment with 20 µg/mL
(B), 40 µg/mL (C) of C. variegatum stem bark extract and curcumin (standard) (D). Inserted values represent percentage of cells of two replicates., Upper left
quadrant represents nuclear debris, upper right quadrant represents cells in late apoptotic stage, lower left quadrant represents non apoptotic cells and lower right
quadrant represents cells in early stage apoptosis.
Table 3: Cytotoxic activities of fractions of C. variegatum stem bark .
IC50 values (µg/mL)
Ethyl acetate fr.
560.27 ± 22.16
Tabulated values represent mean ± standard deviation of three replicates p-values compare the statistical difference between the calculated means u sing Tukey’s
test. For each fraction n=3
Anim et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 087-093 093
study on the stem bark of C. variegatum is needed to reveal its
Findings from this study indicates that C. variegatum has
antiproliferative effect on leukaemia (Jurkat), breast (MCF 7) and
prostate (PC 3) cancer cell lines and possibly contain bioactive
compounds with anticancer properties.
However, though the stem bark and leaf of C. variegatum
showed interesting anticancer activities both were very toxic to the
normal human liver cells (WRL 68), suggesting a possible health
risk to individuals who take the preparations of this extract for the
purpose of alleviating illnesses especially on chronic users.
However, it is imperative that this research is repeated in animal
models such as mice. This will help serve as an index of its
potential toxicity in humans.
Also, further fractionation of the stem bark revealed an
increase in cytotoxicity of its chloroform fraction towards Jurkat
relative to the crude extract, indicating the presence of some
bioactive compound(s) in this fraction.
In addition to the anticancer evaluation, the molecular
studies conducted on the C. variegatum stem bark crude extract
using the Hoechst staining and flow cytometry revealed an
apoptotic mechanism of action. This renders it a great candidate
with promising leads to medicines against cancer.
The Molecular Biology Laboratory of the Department of
Biochemistry and Biotechnology, Department of Pharmacognosy,
Kwame Nkrumah University of Science and Technology and the
Clinical Pathology Department of Noguchi Memorial Institute for
Medical Research, University of Ghana supported this research.
Conflict of Interests: There are no conflicts of interest.
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How to cite this article:
Anim MT, Larbie C, Opong RA, Tuffour I, Owusu KBA, Aning A.
Extracts of Codiaeum variegatum (L.) A. Juss Is Cytotoxic on
Human Leukemic, Breast and Prostate Cancer Cell Lines. J App
Pharm Sci, 2016; 6 (11): 087-093.