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Research Article
Volume 29 Issue 1 (2018) 1
Indonesian J. Pharm. Vol. 29 No. 1 : 1 – 9
ISSN-p : 2338-9427
DOI: 10.14499/indonesianjpharm29iss1pp1
In vitro evaluation of anti-inflammatory and anti-
diabetic effects of Euphorbia tithymaloides ethanol
extract
Theresia Galuh Wandita1, Najuma Joshi1, Joseph dela Cruz2, Seong Gu Hwang1*
1. Laboratory of Applied
Biochemistry, Department of
Animal Life and
Environmental Science,
Hankyong National
University, South Korea, 327
Jungang-ro, Anseong-si,
Gyeonggi-do, 456-749, South
Korea
2. Department of Basic
Veterinary Sciences, College
of Veterinary Medicine,
University of the Philippines
Los Banos, Philippines
Submitted: 11-11-2017
Revised: 25-12-2017
Accepted: 06-01-2017
*Corresponding author
Seong Gu Hwang
Email:
sghwang@hknu.ac.kr
ABSTRACT
Euphorbia tithymaloides L., a native plant of tropical and
subtropical areas in Asian countries which has been known as
traditional medicine with a wide range of healing effects, such as
anti-hemorrhagic, anti-diabetic, anti-bacterial, anti-inflammatory,
and anti-tumor activity. The present study was orchestrated to
evaluate the potential anti-inflammatory and anti-diabetic effects
of Euphorbia tithymaloides ethanol extract (ETE). The anti-
inflammatory and anti-diabetic activities were studied through
the treatment of RAW 264.7 murine macrophages cells and 3T3-
L1 adipocytes with various concentrations of ETE (50, 100, 200,
and 400µg/mL). The results showed that ETE below 400µg/mL
has no negative effect on RAW 264.7 cell proliferation. ETE
decreased nitric oxide production in macrophages RAW 264.7 cell
line and reduced the protein expression of cyclooxygenase 2,
interlukin-6, inducible nitric oxide synthase, tumor necrosis
factor-α and nuclear factor-kB in a dose-dependent manner. In
3T3-L1 preadipocytes, the increase of ETE concentration did not
affect cell viability, but significantly enhanced adipogenesis
through increase in differentiation and the expression of
peroxisome proliferator-activated receptor gamma, CEBPα,
glucose transporter type 4 and insulin receptor substrate 1. ETE
also stimulated 2-NBDG uptake through activation of the insulin
signaling pathway through insulin-sensitizing PPARγ agonists in a
dose-dependent manner. In conclusion, our findings that ETE has
immunomodulatory activity and anti-diabetic effects, which can
be used for treating inflammation and type II diabetes.
Keywords: Anti-diabetic, Euphorbia tithymaloides, Glucose uptake,
Immunomodulation, Nitric oxide
INTRODUCTION
Medicinal plants have been used since
ancient times. Owing to the side effects of
some modern medicines, The World Health
Organization Expert Committee has
recommended the use of traditional medicinal
plants for the treatment of some diseases.
Ethno-botanical knowledge suggests that
approximately about 800 plants have
immunomodulatory and anti-diabetic potential
(Singh, 2011). Medicinal plants may provide a
good alternative for treating diabetes mellitus
and inflammation, and counter the high cost,
side effects, and poor availability of the
currently available drugs in developing
countries such as the Philippines.
Inflammation is the most common type
of response employed by the body as a defense
mechanism against harmful stimuli originating
from the surrounding environment. The
inflammatory response is characterized by the
coordinated activation of various signaling
pathways that regulate the expression of both
pro- and anti-inflammatory mediators in
resident tissue cells and leukocytes recruited
from the blood. Nuclear factor kappa B (NF-
kB) plays an important role in the activation of
genes involved in the immune responses. This
transcription factor increases many cytokines
gene expression, such as interleukin (IL-1β, IL-
2, IL-6, IL-8, and IL-12), cyclooxygenase
(COX)-2, and tumor necrosis factor (TNF)-α.
Potential effects of Euphorbia tithymaloides
2 Volume 29 Issue 1 (2018)
They possess NF-kB binding sites and have
been shown to be transcriptionally regulated by
NF-kB. Moreover, NF-kB transcriptionally
regulates the gene for inducible nitric oxide
synthase (iNOS) (Ghosh et al., 2014).
Diabetes mellitus (type II diabetes) is
caused by defective insulin production or
insulin resistance. The prevalence of diabetes
has increased at an alarming rate in Asian
countries including the Philippines. In 2016, the
death of, 33.2 million people in the Philippines
was attributable to diabetes. Adipogenesis is
regulated by a complex gene expression such as
peroxisome proliferator-activated receptor
gamma and CCAAT/enhancer binding protein
(CEBPα). It is considered as a nuclear receptor
in adipocytes (Winrow et al., 1996; Rosen et al.,
2000; Vergara et al., 2015). Adipocytes are
turning up as a potential medicine for
treating diabetes mellitus (Hassan et al., 2007).
The mechanism involves insulin-triggered
relocalization of a glucose transporter (GLUT4),
which is highly expressed, and insulin-stimulated
glucose uptake in adipocytes by the rapid
translocation of GLUT4 from intracellular
stores to the plasma membrane (Choi et al.,
2009; Manaharan et al., 2013).
Euphorbia tithymaloides belongs to the
family Euphorbiaceae. It is a perennial succulent
spurge that has been used to treat inflammation
and cancer in Asian countries, especially the
Philippines. The root of this plant has been
shown to be effective against intestinal worms
and to reduce inflammation when ingested (Jain
and Srivastava, 2005; Heinrich et al., 1992; Adzu
et al., 2008). The extracts of the leaves have
been used to treat conditions such as asthma,
laryngitis,and persistent coughing. In an earlier
study, various bio-active compounds were
found in this plant such as octacosanol, beta-
sitosterol, cycloartenone, and oxime. One of
these, octacosanol, significantly decreased the
pro-inflammatory cytokines genes expression
in in-vitro and in-vivo studies (Oliveira et al., 2012;
Castaldo and Capasso, 2002; Njamen et al.,
2003). Galactose-specific lectin, also contained
in this plant might be useful for the treatment
of diabetes mellitus as mentioned by Joseph
and Priya (2011). Furthermore, there have been
no reports on the anti-inflammatory and anti-
diabetic activities of this plant in macrophages
and 3T3-L1 cell. Thus, our present study aimed
to evaluate the potential effects of E.
tithymaloides ethanol extract as an
immunomodulatory and anti-diabetic herbal
medicine.
MATERIALS AND METHODS
Reagents
RAW 264.7 macrophages cell line and
3T3-L1 mouse preadipocyte cells were obtained
from ATCC (Manassas, VA, USA). Dulbecco’s
modified Eagle’s medium (DMEM), bovine
calf serum (BCS), phosphate buffer saline
(PBS), fetal bovine serum (FBS), and 2-(N-(7-
nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-
deoxyglucose (2-NBDG) were purchased from
GIBCO/Invitrogen (Carlsbad, CA, USA).
Lipopolysaccharide (LPS), bovine serum
albumin (BSA), isobutyl-3-methyl-xanthine
(IBMX), insulin (INS), and dexamethasone
(DEX), were acquired from Sigma-Aldrich (St.
Louis, MO, USA). Primers used in these
experiments were purchased from Macrogen
(South Korea). The antibodies used in these
experiments were obtained from Cell Signaling
Technology (Danver, Massachusetts, USA).
Plant material
The dried whole plant of E. tithymaloides
was powdered and successively extracted in 80%
of ethanol for 24h. The ethanol extract of this
plant (ETE) was filtered, and evaporated under
vacuum. The freeze-dried sample was
powdered and stored at -80ºC until use.
Hereafter, the extract was mixed with the
medium.
Cell culture and cell viability assay
RAW 264.7 macrophages cell line and
3T3-L1 preadipocytes cells were grown in
DMEM with 10% FBS and DMEM with 10%
BCS, respectively. To activate macrophages,
10µL/mL of lipopolysaccharide was added to
the culture medium. To induce the
differentiation of 3T3-L1, the cells were
cultured with fresh induction of 1µM
dexamethasone, 0.5mM isobutyl-3-methyl-
xanthine, and 10µL/mL insulin.
Cell viability was determined by using
cell counting kit (CCK)-8 assay (Dojindo
Laboratories, Japan). The cells, RAW 264.7
macrophages cells (1x105 cell/well) and 3T3-L1
preadipocytes cells (1x104 cell/well) were
Theresia Galuh Wandita
Volume 29 Issue 1 (2018) 3
seeded and treated with various concentrations
of ETE (50, 100, 200, 400µg/mL) for 24h.
CCK solution (1µg/mL) was then added to
each well. The absorbance was quantified at
450nm by using an ELISA microplate reader.
Six replicates were performed for each assay.
Anti-inflammatory activity
Anti-inflammatory activity was
determined from the nitrite oxide production
and the expression of pro-inflammatory
cytokines. Nitric oxide production was
determined as the nitrite concentration in
culture medium by using a colorimetric assay.
The cells were seeded in a 96-well plate and
treated with different concentration of ETE.
Negative control (NC) and positive control
(PC) were designed to identify the effect of
ETE in RAW 264.7 macrophages cells.
Lipopolysaccharides were used as positive
control which helps to activate the cells. After
1h incubation, the cells were stimulated with
1µg/mL LPS for 24h. The supernatant was
mixed with Griess reagent, and the absorbance
at 540 nm was measured by using an ELISA
microplate reader (Tecan Infinite F50). The
concentration of NO was calculated by
reference to a linear standard curve generated
from serial dilutions of sodium nitrite in
working medium.
The protein and genes expression of
pro-inflammatory cytokines (iNOS, COX2, IL-
6, NF-kB and TNFα) in the macrophages cell
line RAW 264.7 were measured by using
reverse-transcription polymerase chain reaction
(RT-PCR) and western blotting, respectively.
Anti-diabetic activity
Anti-diabetic activity was determined by
a glucose uptake assay, Oil -Red -O staining,
triglyceride assay, and by analyzing adipogenic
markers expression. To determine the glucose
uptake in 3T3-L1 preadipocytes, a modified
method by Manaharan et al. (2013) was used in
the present study. 3T3-L1 cells were incubated
with different concentration of ETE (50, 100,
200, and 400µg/ml) and 10µL/mL insulin for
24h at 37ºC in 5% CO2 atmosphere. Thereafter,
after the addition of 250µmol/L 2-NBDG (N-
(7-Nitrobemz-2-oxa-1,3-diazol-4-yl)Amino)-2-
deoxyglucose), the cells were incubated for a
further 30 min. To remove the excess
fluorescent material in the wells, the cells were
washed twice with PBS after incubation. The
fluorescence retained in the cell monolayers at
540nm using an ELISA microplate reader.
Negative control (NC) and positive control (PC)
were designed to identify the effect of ETE in
3T3-L1 preadipocytes cells. The addition of
insulin was considered as positive control
which helps to enhance glucose uptake to the
cells.
To measure the accumulation of
triglycerides, adipocyte differentiation was
induced at the end of the treatment period,
which is a common measurement of PPARγ
agonist activity. The differentiated adipocytes
cells were fixed with formaldehyde and washed
with distilled water. Oil-Red-O solution (0.5%
in isopropanol) was used to stain the cells and
incubated for 1h. After washing, the fat
droplets in 3T3-L1 adipocytes were monitored
under microscope. The amount of triglyceride
was determined by isopropanol dissolution and
quantified by spectrophotometric analysis at
490 nm.
The adipogenic markers expression such
as PPARγ, C/EBPα, GLUT4, and IRS1 was
determined by western blotting.
Statistical analysis
All data were presented as the means ±
standard deviation (SD). SPSS was used for the
statistical data analysis. Between groups
comparison were made by ANOVA analysis. It
was followed by Duncan’s Multiple Range Test
(DMRT) where P-values less than 0.05 were
considered significantly different.
RESULTS AND DISCUSSIONS
In vitro anti-inflammatory activity
Inflammation is an early response to
tissue injury and foreign pathogens, and then
the normal tissue structure and function are
recovered. A normal inflammatory response
regulates expression of pro-inflammatory and
anti-inflammatory proteins. During these
inflammation responses, macrophages are
essential cells that bridged innate and adaptive
immunity (Park et al., 2016). Based on these
observations, it has been hypothesized that
inhibiting high-output NO, IL-6 and TNFα
production in macrophages, by blocking iNOS
and COX2 expressions or their activities, can
Potential effects of Euphorbia tithymaloides
4 Volume 29 Issue 1 (2018)
serve as the basis for the potential development
of anti-inflammatory drugs. Furthermore,
nuclear factor-κB (NF-kB)-dependent gene
expression plays an important role in
inflammatory responses and increases the
expression of genes encoding cytokines and
receptors involved in pro-inflammatory enzyme
pathways such as iNOS and COX2. Recently,
many studies have demonstrated the role of
phytochemicals in anti-inflammatory activity is
through downregulation of the NF-kB pathway
(Kundu and Surh, 2005).
The toxic concentration of ETE in LPS-
stimulated RAW 264.7 cells was assessed via
CCK-8 assay. Significant (P<0.05) differences
were observed in viability of macrophages
RAW 264.7 cells treated with different
concentration up to 400µg/ml ETE compared
with the control (Figure 1A). As the
concentration of ETE increased up to
200µg/mL, the cell viability increased to greater
than that of the control, and then started to
decline.
Figure 1. Effects of Euphorbia tithymaloides
ethanol extract on the proliferation of RAW
246.7 machrophages stimulated with LPS (A)
and 3T3-L1 preadipocytes (B) for 24h. The
data presented as the means ±SD (n=6). Means
with different superscript letters are significantly
different (P<0.05).
The nitrite detection method used in this
study was a spectrophotometric assay based on
the Griess reagent. In Figure 2A, the incubation
of LPS-stimulated RAW 264.7 cells with
ETE for 24h resulted in significant NO
production (14.4µM/L). However, increases
in the concentration of ETE up to 400µg/mL
significantly decreased NO production in RAW
264.7 macrophages (from 14.4µM/L to 8.9µM/L).
In addition, ETE inhibited expression of iNOS
in a dose dependent manner (Figure 2B).
Figure 2. Effects of Euphorbia tithymaloides
ethanol extract on NO production (A) and
iNOS expression (B) in RAW 264.7
machrophages.The data are presented as the
means ± SD (n=5). Means with different
superscript letters are significantly different
(P<0.05).
ETE significantly inhibited NF-kB
activation (Figure 3). The genes and protein
expression of inflammatory cytokines
decreased as the concentration of ETE
was increased to 400µg/mL. As calculated from
the images (Figure 3), ETE significantly
inhibited LPS-stimulated NF-kB activation
by 98% (P<0.05) in dose-dependent manner.
Theresia Galuh Wandita
Volume 29 Issue 1 (2018) 5
ETE also decreased the expression of
TNFα, IL-6, and COX2, which leads to the
amplification of inflammation. These results
indicated that ETE applied an anti-inflammatory
activity in LPS-stimulated RAW 264.7
macrophages by blocking NF-kB activation.
Figure 3. Effects of Euphorbia tithymaloides
ethanol extract on genes (A) and protein (B)
expression of pro-inflammatory cytokines in
RAW 264.7 macrophages
The result of this study revealed the
significant inhibitory activity of ETE in RAW
264.7 cells stimulated with LPS at doses up to
400µg/mL, which occurred through blockade
of the NF-kB signaling pathways. NF-kB is one
of the major expressions in the immune system
and, regulates many pro-inflammatory cytokines
expression that define responses to pathogen.
The NF-kB transcription factor was activated
by lipopolysaccharide in macrophages (Hayden
and Ghosh, 2008). The activation of NF-kB
leads to the induction of many pro-
inflammatory cytokines expression (Baeuerle,
1991). After the cells are activated, NF-kB
separates from inhibitory protein (IkBα) which
releases the p50/p65 heterodimer. Thereafter, it
translocates owing to the nuclear localization
signal and thereby induces the expression
of pro-inflammatory genes and proteins. TNFα
and interleukin function synergistically to
induce the expression of several
major pro-inflammatory mediators including
prostaglandins, leukotrienes, and NO.
NO was involved in the regulation of
diverse physiological and pathophysiological
mechanisms in immunological systems. It acts
as first line of defense against infection in the
immune system. However, in inflammatory
disorders, NO also acts as cytotoxic agent in
some pathological activity because of its free
oxygen radical (Alderton et al., 2001; Bogdan,
2001; Dawn and Bolli, 2002). LPS, which is
isolated from bacteria, stimulates the
macrophages production of NO by iNOS.
COX-2 is related with NO production and its
overproduction may cause inflammation and
carcinogenesis (Park et al., 2004). In this
study, NO production and COX-2 expression
decreased by ETE in dose-dependent manner.
In vitro anti-diabetic activity
Diabetes is associated with abnormal
metabolism of glucose which caused by various
factors. One of the requirements of ideal anti-
diabetic agents is enhanced glucose uptake in
most insulin target tissue (Navarro and Mora,
2006). Type II diabetes, formerly known as
non-insulin dependent DM, is the most
common form of DM characterized by
hyperglycemia, insulin resistance, and relative
insulin deficiency. This leads to a decrease in
glucose transport into the liver, muscle cells,
and fat cells. In skeletal muscle, insulin
resistance can result from high levels of
circulating fatty acid that disrupt insulin
signaling pathways (Saraswathy et al., 2017).
Insulin signaling, after binding to its specific
receptor in adipocytes, regulates the storage of
free fatty acids in the form of triglycerides;
however, massive adipocytes produce a number
of adipocytokines that consequently modulate
insulin signaling with excess free fatty acids
(Hassan et al., 2007). This present study discovered
that ETE increased lipid accumulation, and
significantly increased glucose uptake and
GLUT4 expression in 3T3-L1 adipocytes.
As shown in figure 1B, ETE was not
cytotoxic to cells below 400µg/ml. The cells
were expressed as a percentage of cell viability
compared with the untreated groups, which
were considered to be 100% viable. No
significant change in 3T3-L1 preadipocytes cell
Potential effects of Euphorbia tithymaloides
6 Volume 29 Issue 1 (2018)
proliferation was observed even when the
concentration of ETE was increased to
400µg/mL (P>0.05). Therefore, these concen-
trations of ETE were selected for further
experiments. ETE was found to significantly
(P<0.05) enhance adipogenesis in a dose-
dependent manner in 3T3-L1 preadipocytes
(Figure 4A). The lipid accumulation by ETE in
fully differentiated 3T3-L1 cells significantly
increased as the concentration was increased to
400µg/mL (Figure 4B).
Figure 4. Effects of Euphorbia tithymaloides
ethanol extract on triglyceride content (A) and
adipogenesis (B) in fully differenteated 3T3-L1
preadipocytes. The data are presented as the
means ± SD (n=6). Means with different
superscript letters are significantly different
(P<0.05).
2-NBDG assay was used to examine the
effects of ETE on glucose uptake. The results
indicated that ETE significantly increased
glucose uptake in a dose-dependent manner
(P<0.05). As shown in figure 5A, 400µg/mL
ETE caused a two fold increase in glucose
uptake in insulin-induced 3T3-L1 cells. Similarly,
GLUT4 protein expression was also increased
along with increasing the concentration of
ETE (Figure 5B). In contrast, 1µg/mL insulin
increased GLUT4 expression of positive
control and 400µg/mL ETE by 8 and 13- fold,
respectively.
Figure 5. Effects of Euphorbia tithymaloides
ethanol extract treatment for 24 h on glucose
uptake (A) and GLUT4 expression (B) in fully
differentiated 3T3-L1 preadipocytes. The data
are presented as the means ± SD (n=5). Means
with different superscript letters are significantly
different (P<0.05)
Figure 6. Effects of Euphorbia tithymaloides
ethanol extract on protein expression of
adipogenic markers in differentiated 3T3-L1
cells.
The expression of adipogenic markers,
such as PPARγ, C/EBPα, and IRS-1 also
significantly increased as the concentration of
ETE increased (Figure 6). The upregulation of
those expressions is very important in
adipogenesis. Similarly, ETE also played an
important role in the enhancement of insulin
sensitivity, as shown by the observed increase
in IRS-1 protein expression. These results
Theresia Galuh Wandita
Volume 29 Issue 1 (2018) 7
suggested that the excellent anti-diabetic action
of ETE occurred through the activation of
insulin signaling pathways by insulin-sensitizing
PPARγ agonists.
Adipogenesis is the process by which the
undifferentiated precursor cells differentiate
into fat cells. This is supported by the increase
in the adipocyte genes expression such as
PPARγ and C/EBPα. The activation of PPARγ
is a key process in adipocyte differentiation.
PPARγ regulates the genes expression related
with insulin signaling pathways, as well as
glucose and lipid metabolism in mature
adipocyte (Bouaboula et al., 2005). In the
present study, ETE activated PPARγ, which
induced the differentiation of 3T3-L1
preadipocyte. The results showed that ETE
enhances adipogenesis in the presence of
insulin in a dose-dependent manner; therefore,
it can be considered to have insulin-like activity.
Our results suggest that ETE may have the
ability to increase insulin sensitivity through the
activation of PPARγ. Insulin stimulation in
adipocytes leads to the translocation of
GLUT4 to the cell surface by binding to insulin
receptor proteins within the cell. Our results
admitted the insulin-like and insulin sensitive’s
properties of ETE in experiments through
the stimulation of 2-NBDG uptake into
adipocytes.
The present study provided evidence
that ETE enhances insulin sensitivity, as shown
by the increased expression of IRS-1 proteins.
Furthermore, it also significantly induced
GLUT4 expression, which serves as an
important role in insulin mediated glucose
transport. In summary, E. tithymaloides inhibited
NF-kB signaling pathways by blocking NF-kB
activation. Furthermore, it also enhanced
insulin sensitivity, which leads to enhanced
uptake of glucose into adipocyte cells. These
findings suggested that ETE is herbal
component with potential use in the
development of new immunomodulatory and
anti-diabetic agents for treating inflammation
and diabetes mellitus, respectively.
CONCLUSIONS
In our study, ETE administration
increased NO production, inhibited the
NF-kB pathway, and suppressed other
pro-inflammatory cytokines expression in LPS-
stimulated RAW 246.7 cells in a dose-
dependent manner. Furthermore, it also
enhanced adipogenesis, stimulated 2-NBDG
uptake, and increased insulin sensitivity through
PPARγ upregulation. ETE demonstrated a clear
potential to act as an immunomodulatory and
anti-diabetic herbal medicine for treating
inflammation and type II diabetes. However,
further in vivo studies are necessary to
verify the effectiveness of ETE in the
treatment of these conditions. Furthermore,
the identification of the active compounds in
ETE is required to elucidate the mechanisms
of action.
ACKNOWLEDGEMENT
The author’s contributions are as follows:
all authors collaborated in the design of the
experiment, Seong Gu Hwang supervised for
the whole duration of study; Joseph dela Cruz
and Najuma Joshi are responsible for the
writing and editing of the manuscript; Theresia
Galuh Wandita conducted the experiment and
also assisted in writing the manuscript. The
authors declare no conflicts of interest to this
work.
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