Artificial Cells, Nanomedicine, and Biotechnology

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Development of enteric-coated microspheres

of embelin for their beneficial pharmacological
potential in ulcerative colitis

Nidhi, Ankita Dadwal, Supandeep Singh Hallan, Saurabh Sharma & Neeraj
Mishra

To cite this article: Nidhi, Ankita Dadwal, Supandeep Singh Hallan, Saurabh Sharma &
Neeraj Mishra (2016): Development of enteric-coated microspheres of embelin for their
beneficial pharmacological potential in ulcerative colitis, Artificial Cells, Nanomedicine, and
Biotechnology, DOI: 10.1080/21691401.2016.1202258

To link to this article: http://dx.doi.org/10.1080/21691401.2016.1202258

Published online: 07 Jul 2016.

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 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2016
http://dx.doi.org/10.1080/21691401.2016.1202258

ORIGINAL PAPER

Development of enteric-coated microspheres of embelin for their beneficial

pharmacological potential in ulcerative colitis
Nidhia, Ankita Dadwala, Supandeep Singh Hallana, Saurabh Sharmab and Neeraj Mishraa
a
Department of Pharmaceutics, I.S.F. College of Pharmacy, Moga, Punjab, India; bDepartment of Pharmacology, I.S.F. College of Pharmacy,
Moga, Punjab, India

ABSTRACT ARTICLE HISTORY

The aim of the present study is to develop embelin-loaded enteric-coated microspheres and investigate Received 7 March 2016
their pharmacological potential in acetic acid induced ulcerative colitis. The optimized formulation of Revised 9 April 2016
embelin-loaded microspheres has shown significant sustained release of embelin. Further this formula- Accepted 13 June 2016
tion significantly reduced the ulcer activity score and oxidative stress, and attenuated the inflammatory Published online 4 July 2016
changes. Thus it may be concluded that embelin-loaded enteric-coated microspheres have shown KEYWORDS
delayed release capacity than plain embelin and exerts colon ulcer protective effect in rats. Colon; Eudragit S 100;
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multiparticulate carrier; pH
dependent; time dependent

Introduction extensively documented to exert its pleotropic protective

The therapeutic strategy for treating inflammatory bowel dis-
ease is now focused on the use of anti-inflammatory agents
(Xu et al. 2004). Glucocorticoids and aminosalicylate have
been used for the treatment of inflammatory bowel disease,
but their side effects remain a major clinical problem. Plant
remedies play an important role in the therapy of many
inflammatory disease conditions including inflammatory
bowel disease (Mahgoub 2003, Medhi et al. 2008). A growing
body of literature suggests that inflammatory bowel disease
results from a deregulated immune response to normal bac-
terial antigens. This uncontrolled immune system activation
results in the sustained overproduction of reactive metabolites
of oxygen and nitrogen. Some of the intestinal and/or colonic
injury and dysfunction observed in inflammatory bowel dis-
ease is due to elaboration of these reactive species. In many
studies, it has been reported that antioxidants showed benefi-
cial effects on experimental colitis. Quinone derivatives from
plants are known to possess protective effects against colitis
induced by acetic acid (Thippeswamy et al. 2011). Targeted
drug delivery to colon via oral route is highly desirable for
ameliorative localization and controlled release of drug, for
the selective treatment of inflammatory bowel diseases such
as ulcerative colitis (UC), Crohn’s disease, colon cancer,
amoebiasis, etc. (Glavas-Dodov 2013).
The commonly used drugs for UC are sulfasalazine, mesal-
amine, olsalazide, balsalazide, budesonide, etc. But these
drugs show various side effects such as diarrhea, nausea,
vomiting, which result in maximum removal of drug from
body. Therefore, it necessitates using drugs from herbal origin,
which hardly shows any side effects. Recently, embelin is
effects. It is commonly known as false black pepper or
vidanga and is known for its anti-inflammatory (Chitra et al.
1994) and antioxidant activities (Joshi et al. 2007). Embelin
has also been reported to inhibit NF-jB activation by blocking
its signaling pathways, which further inhibits pro-inflammatory
gene expression in inflamed mucosa (Ahn et al. 2007).
Furthermore, Kumar et al. (2011) proved that embelin amelio-
rates dextran sodium sulfate-induced colitis by suppressing
the levels of pro-inflammatory cytokines, such as TNF-a, IL-1b,
IL-6 in the colonic tissues.
Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), a
major constituent of Embelia ribes Burm., is a naturally occur-
ring alkyl substituted hydroxyl benzoquinone. The plant is
indicated in traditional medicine for the treatment of various
diseases (Thippeswamy et al. 2011). The fruit is bitter in taste,
used to treat fever, inflammatory diseases and a variety of
gastrointestinal ailments for thousands of years (Gupta et al.
1976). Embelin is reported to possess anti-inflammatory, anal-
gesic (Chitra et al. 1994), antioxidant (Joshi et al. 2007) and
wound healing (Swamy et al. 2007) activities. It is also
reported to impair the inflammatory signaling through inhib-
ition of nuclear factor kappa B (NF-kappa B) activity (Ahn
et al. 2007). It is reported to possess potent antioxidant (Joshi
et al. 2007) and anti-inflammatory (Chitra et al. 1994) activ-
ities. Various animal models of experimental colitis to screen
drugs effective against inflammatory bowel disease have been
established and acetic acid-induced colitis is an animal model
that mimics some of the acute inflammatory responses seen
in UC (Gonzalez et al. 1999). Boreddy et al., has already
reported the protective effect of embelin against acetic acid

CONTACT Dr. Neeraj Mishra, Associate Professor neerajdops@rediffmail.com Department of Pharmaceutics, ISF College of Pharmacy, Moga 146001, Punjab,
India
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
 2 NIDHI ET AL.
induced UC in rats. It has also been reported that embelin
treatment significantly decreased clinical activity score, gross
lesion score, percent affected area, and wet colon weight
when compared to acetic induced colitis (Thippeswamy et al.
2011). Among these approaches, polymer-based microspheres
are found to be a promising approach to increase drug stabil-
ity by either encapsulating or dispersing drug in polymeric
matrix. The drug released from microspheres by different
mechanism depends on composition of polymer and its pre-
parative techniques (Faisant et al. 2002). Eudragit S 100, a co-
polymer of methacrylic acid and methyl methacrylate ester is
a commonly used pH sensitive polymer for targeting drugs to
intestine (Ibekwe et al. 2006). Although, it protects drug to
dissolve in upper intestinal tract, due to its pH sensitive prop-
erties, it starts dissolving at pH >6. This lack of site specificity
needs another hydrophobic polymer for sustained drug
release (Davis et al. 1986). Ethyl cellulose is one of the best
encapsulating hydrophobic delayed release polymers, which
controls the release behavior of drug. Thus, by combining
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both pH-dependent and time-controlled polymer, site-specific
drug delivery to colon can be easily achieved. It has also been
reported that optimal particle size of carrier for improved
localization in colonic region is the utmost importance and it
should be in the range of 4–15 lm, in order to achieve exces-
sive accumulation in inflamed region by internalization of
macrophages (Glavas-Dodov 2013). It is possible by using mul-
tiparticulate drug delivery system in which blend of combined
pH-dependent, i.e., Eudragit S 100 and delayed release, i.e.,
ethyl cellulose polymers has been used.
The aim of present study is to develop pH-dependent sus-
tained-release embelin-loaded microspheres prepared by
using combination of crosslinking technique and w/o emul-
sion solvent evaporation technique employing combined pH-
dependent, i.e., Eudragit S 100 and delayed release, i.e., ethyl
cellulose hydrophobic polymers for the treatment of UC.
Materials and methods
Materials
Embelin was purchased from Indo Fine Chemicals,
Hillsborough Township, NJ. Eudragit S 100 was procured as
free gift sample from Rohm Pharma, Darmstadt, Germany.
Span 80 was purchased from Himedia Laboratories (Mumbai,
India). Glutaraldehyde was purchased from CDH. Petroleum
ether, ethyl cellulose, methanol, acetone, diethyl ether and
liquid light paraffin were purchased from Loba Chemie,
Mumbai, India. Dianisidine was purchased from Sigma
(St. Louis, MO). Mesalamine was purchased from Sun Pharma
(Sikkim, India). All other chemicals were of analytical grade.
Methods
Preparation of embelin containing microparticles
As per the studies of Thippeswamy et al. (2011), we have
taken 50 mg/kg of embelin. It was loaded in microspheres
and prepared by using combined techniques of solvent evap-
oration and crosslinking method. In this method, polymer
solution was prepared by dissolving ethyl cellulose in 5 ml
methanol and Eudragit S 100 in 5 ml acetone. Embelin was
also dissolved in methanolic solution using bath sonicator
(Steryl Medi Equip System, 40050, Chennai). This polymeric
methanolic solution was mixed with above polymeric acetone
solution under magnetic stirring (Tarsons Product Pvt. Ltd.,
Kolkata, India). This drug polymer solution was taken in syr-
inge and dropped into 20 ml of liquid light paraffin containing
Span 80 (2% w/v) as an emulsifier. After complete dispersion
of polymer–drug solution into continuous oil phase, stirring
was continued for 15 min using mechanical stirrer at 1500 rpm
and thereafter, 0.2 ml of glutaraldehyde was added into con-
tinuous phase. Simultaneously, 0.2 ml of NaOH (0.1 M) was
added into the mixture. Stirring proceeded for 2 h until com-
plete removal of solvent. Microspheres get hardened and
were washed successively with petroleum ether to remove
adhering oil. For the removal of crosslinking agent, micro-
spheres were washed with buffer solution of pH 1.2.
Microspheres were allowed to dry at room temperature and
kept in desiccator until further use (Shahi et al. 2014). To
assess the impact of varying manufacturing parameters on
particle size, entrapment efficiency, and drug release, the
basic protocol was modulated step-by-step.
Surface morphology and particle size analyses
The particle size and surface morphology were analyzed by
using motic and scanning electron microscope. The micro-
spheres were dried and coated with gold palladium and
placed under the atmosphere of argon for at least 150 s for
getting thin 20 nm film. These coated microspheres were
examined in scanning electron microscope (JEOL JSM-840,
Tokyo, Japan) for surface morphology and particle size
analyses.
Drug entrapment efficiency
The amount of embelin entrapped in microspheres was
determined by centrifugating 12 ml of microparticle solution
at 6000 rpm for 30 min in ultracentrifuge (Sigma
Laborzentrifugen, SV Instruments Pvt. Ltd., Gurgaon, India).
After 30 min, supernatant was collected and analyzed using
UV-spectrophotometry (Perkin Elmer, 1900). Entrapment effi-
ciency was calculated by using the following formula:
total drug  free drug
% Entrapment efficiency ¼  100
total drug
The entrapment efficiency of embelin was determined in
triplicate for each formulation. The results were expressed as
mean ± standard deviation.
In vitro drug release study
In vitro release study of microspheres was carried out using
dialysis bag method. For this study, microspheres equivalent
to 5 mg of embelin were measured and added to dialysis
membrane-70 (Himedia, LA393-10MT). This dialysis membrane
was taken into 50 ml of simulated gastric fluid of pH 1.2 in a
conical flask for 2 h. After 2 h, this buffer was replaced with a
fresh simulated intestinal buffer of pH 6.8 for small intestine.

Thereafter, it was replaced with a buffer of pH 7.4 for colon.
This flask was taken in an incubator shaker and the speed of
shaker was maintained at 60 rpm at 37 ± 0.5  C. At specific
time intervals, samples (5 ml) were withdrawn and filtered.
Same volume (5 ml) of the phosphate buffer pH 7.4 was
replaced after each sampling. The drug content in the sample
was determined spectrophotometrically at 292.37 nm (Perkin
Elmer, Waltham, MA) (Rai et al. 2016) (Figure 2).
In vitro release kinetics
Different release kinetic models were applied on release
data in order to determine the drug release mechanism.
The R2 values determine the mechanism of drug release.
Greater the R2 values, more is the probability of determin-
ing release kinetics. Release kinetics was determined by
using BCP software.
In vivo study
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In vivo study was carried out as per the approved protocol by
Institutional Animal Ethical Committee (IAEC) formed as per
the norms of Committee for Prevention, Control and
Supervision of Experiments on Animals (CPCSEA). Healthy wis-
tar rats (180–200 g) were subjected to standard laboratory
conditions (i.e., room temperature, 25 ± 2  C; relative humidity,
55 ± 5%; 12/12 h light/dark cycles) with free access to com-
mercial rodent diet and water.
Induction of ulcerative colitis
A solution of 1.5 ml of acetic acid (3%, v/v) in 0.9% saline was
instilled into the lumen of the colon to a distance of 8 cm via
rectal route with help of a polypropylene tube of 2 mm diam-
eter and maintained in a supine trendelenburg position for
30 s to prevent the leakage of the intracolonic instillate. After
72 h of single dose administration of acetic acid (8th day),
stool consistency and change in body weight were measured
and the animals were anaesthetized with ether and sacrificed
by cervical dislocation. The colon was dissected out. It was
flushed gently with saline and weighed. It is used for macro-
scopic scoring, histopathological and biochemical estimations
(Thippeswamy et al. 2011).
Evaluation of ulcerative colitis
Clinical activity score The ulcer activity score was reported
by Thippeswamy et al. (2011). Colitis was quantified with a
clinical score assessing weight loss, stool consistency and
bleeding of the colon (measured by guaiac reaction, hemoc-
cult) as described previously. No weight loss was counted as
0 point, weight loss of 1–5% as 1 point, 5–10% as 2 points,
10–20% as 3 points, and 20% as 4 points. For stool consist-
ency, the stool was collected in a thick paper and observed, 0
points were given for well-formed pellets, 2 points for pasty
and semiformed stools that did not stick to the anus, and 4
points for liquid stools that did stick to the anus. Bleeding
was scored 0 points for no blood in hemoccult, 2 points for
positive hemoccult, and 4 points for gross bleeding. These
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 3
scores were added and divided by 3, forming a total clinical
score that ranged from 0.0 (healthy) to 4.0 (maximal activity
of colitis).
Macroscopic characters The severity of colitis was evaluated
by an independent observer who was blinded to the treat-
ment. For each animal, the distal 10 cm portion of the colon
was removed and cut longitudinally, slightly cleaned in
physiological saline to remove fecal residues and weighed.
Macroscopic inflammation scores were assigned based on
clinical features of the colon using the following scoring pat-
tern. No visible change was counted as 0 point, hyperemia at
sites as 1 point, lesions having diameter l mmor or less
counted as 2 points, lesions having diameter 2 mmor or less
(number b5, 5–10 and N10) as 3, 4, and 5 points, respectively
and lesions having diameter more than 2 mm (number b5,
5–10, N10) counted as 6, 7, and 8 points, respectively. Weight
of rats was taken on first and last day, and then change in
weight was calculated for all the groups according to the
given formula.
%Change in weight ¼ ðWeight on first day  Weight on last dayÞ=
Weight on first day  100
Biochemical studies Colon was homogenized in 10% (w/v) of
ice-cold potassium phosphate buffer (pH 7.4) using Elvenjan
homogenizer (Remi Motors Ltd., Mumbai, India) and the hom-
ogenate was used for the measurement of myeloperoxidase
activity (MPO), lipid peroxidation, and reduced glutathione
(GSH).
Colonic MPO activity Colonic MPO activity was reported by
Thippeswamy et al. (2011). It has been calculated by using
the following formula:
MPO activity ¼ X=weight of piece of tissue taken
where X ¼ 10  change in absorbance per min=
 volume of supernatant taken in the final concentration:
Colonic lipid peroxides concentration (LPO) Lipid peroxide
level was estimated according to the standard protocol
reported by Ohkawa et al. 1979. The amount of lipid peroxide
was determined by using the following formula:
ð3  OD of sampleÞ=ð0:156  mg prÞ
¼ n moles of MDA/mg pr (Ohkawa et al. 1979):
Colonic glutathione level (GSH) Glutathione level was esti-
mated according to the standard protocol reported by Moron
et al. (1979). The amount of GSH was calculated by the follow-
ing formula:
ð3  OD of sampleÞ=ð13:6  mg prÞ=0:25
¼ n moles of GSH/mg pr (Moron et al. 1979):
Histopathological study
Histopathological studies were done by the standard protocol
of Thippeswamy et al. (2011).
 4 NIDHI ET AL.

Experimental groups Effect of amount of crosslinking agent on particle size and

Animals were divided into five groups. Each group consists of
six animals. Group I normal control, Group II UC control rats
(1.5 ml of acetic acid (3%, v/v) in 0.9% saline), Group III UC
rats þ embelin (50 mg/kg., p.o), Group IV UC rats þ embelin
microspheres (50 mg/kg., p.o), Group V UC rats þ mesalamine
(100 mg/kg., p.o). Acetic acid was administered to Groups II,
III, IV, and V for induction of UC. On the 4th day of the treat-
ment, the animals were fasted overnight with access to water
and libitum. On the 5th day after 1 h of the aforementioned
treatments, the animals (Groups II, III, IV, and V) were sacri-
ficed and assessment of UC was done.
entrapment efficiency
Microspheres prepared by using different amounts of cross-
linking agent were found to be fragile. Crosslinking of poly-
mers with glutaraldehyde is very necessary for controlled
drug delivery to colon. In the present study, with varying
amounts of crosslinking agent from 0.1 to 0.3 ml, particle size
of microspheres was also increased from 17 lm to 24.3 lm
and entrapment efficiency also increases from 73.6 ± 4.4 to
81.2 ± 3.4% as given in Table 1. This is due to the formation
of rigid network, which prevents the drug from leaching out
during crosslinking of microspheres.

Statistical analyses Effect of Span 80 on particle size analysis

The values were expressed as mean ± SD. The statistical ana-
lysis was carried out by one-way ANOVA followed by compari-
son test of Bonferroni. P values <0.05 were considered as
significant (Thippeswamy et al. 2011).
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On increasing Span 80 concentration from 0.5 to 2%, particle
size of microspheres goes on decreasing, but to some extent.
After this, it again goes on increasing. Optimized microsphere
F11 formulation has shown size and entrapment efficiency

12.6 lm ± 2.1 size and 79.5 ± 2.3%, respectively, by using 2%

Results
Particle size analyses
Enteric-coated microsphere was prepared by using combined
technique of solvent evaporation and crosslinking method.
The microspheres were found to have spherical surface as
shown in Figure 1. The effect of formulation variables on
entrapment efficiency, particle size, and drug release of embe-
lin-loaded microspheres is shown in Table 1.
Effect of ethyl cellulose and Eudragit S 100 on particle size
and entrapment efficiency
Polymer concentration has substantial impact on average
diameter of prepared microspheres. With increase in polymer
concentration, entrapment efficiency goes on decreasing. This
is due to the fact that with increase in polymer concentration,
viscosity also increases which leads to large solvent droplet,
making it difficult for drug to diffuse inside droplet, which
ultimately decreases entrapment efficiency.
span, 1:1:0.3 EC:ES 100 drug ratio and 0.2 ml crosslinking
agent. This is due to the fact that low concentration of surfac-
tant is unable to cover the entire organic droplet. Therefore
on increasing Span 80 concentration, it covers the entire
droplet surface and forms a surface coat over entire droplets,
which help in the miscibility of continuous phase with dis-
persed phase and form a stable emulsion with relatively larger
droplets.
In vitro drug release
In vitro release study of microspheres was carried out using
dialysis bag method. As shown in Figure 2, it can be con-
cluded that with increase in the concentration of hydrophobic
polymer, % drug release was decreased after 24 h. F1 (EC:ES
100, 1:1 w/w) exhibited nearly 84.1% drug release within 24 h
of incubation in dissolution medium compared to only 66.5%
drug release in F2 (EC:ES 100, 2:1 w/w). There is a great
impact of concentration of both the polymers in drug release.
F3 also shows nearly same drug release as that of F1 within
24 h of incubation in dissolution medium. But during first 5 h,
F3 shows nearly 49.4% of drug release as compared to F1

Table 1. Effect of formulation parameters on particle size and entrapment

efficiency.
Entrapment
Formulation Parameters Values Particle size efficiency
F1 EC:ES 100:drug 1:1:0.3 13.5 lm ± 1.2 81.2 ± 3.4%
F2 2:1:0.3 21.3 lm ± 1.6 75.5 ± 1.2%
F3 1:2:0.3 17.5 lm ± 3.9 53.5 ± 3.9%
F4 0:1:0.3 8.7 lm ± 4.3 54.9 ± 4.34%
F5 Crosslinking agent 0.1 ml 5.08 lm ± 0.8 73.6 ± 4.4%
F6 0.2 ml 11.7 lm ± 1.7 79.5 ± 3.1%
F7 0.3 ml 26.1 lm ± 2.4 81.2 ± 3.4%
F8 Span 80 0.5% 28.5 lm ± 1.4 84.8 ± 1.5%
F9 1% 23.2 lm ± 1.5 83.5 ± 2.3%
F10 1.5% 19.9 lm ± 3.8 80.8 ± 1.8%
F11 2% 12.6 lm ± 2.1 79.5 ± 2.3%
F12 2.5% 17.4 lm ± 0.8 60.1 ± 2.8%
Bold values indicate the optimized values also have been considered as the
best values of size, concentration of crosslinking agent and span to avail max-
Figure 1. Scanning electron microscopic (SEM) image of polymeric microparticle. imum entrapment efficiency of formulation.

Figure 2. % Drug release with respect to time. Values expressed as mean ± SD
(n ¼ 3).
Table 2. Drug release kinetics of different formulations.
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Zero First Peppas
S.no. Formulation order (R2) order (R2) model (R2)
1 F1 (1:1) 0.9588 0.9961 0.7947
2 F2 (1:2) 0.7176 0.8253 0.5947
3 F3 (2:1) 0.7129 0.8817 0.6108
4 F4 (0:1) 0.5283 0.7957 0.4974
which shows only 19% of drug release. This is due to the fact
that with increase in concentration of ethylcellulose, i.e., hydro-
phobic polymer, viscosity goes on increasing, which leads to
large particle size and less surface area for drug to expose in
release medium, which decreases drug release in the first 5 h.
Thus, showed delayed release. Another reason might be
decreasing the wettability of microspheres in medium due to
increased concentration of hydrophobic polymer, i.e., ethyl cel-
lulose around the drug. Thus, dissolution rate of drug
decreases. While, with increase in concentration of Eudragit S
100, it creates pores around drug at pH 6.8 during first 5 h.
Thus, on exposing it in release medium, medium diffuses inside
particles and drug starts releasing faster from that pores. But
due to the presence of ethyl cellulose, only 50% of drug was
released during the first 5 h, and 82% of drug was released
after 24 h. F4 showed 72.8% of drug release during the first 5 h
in which no ethyl cellulose was incorporated. This can be
explained by the fact that at pH 6.8, eudragit starts dissolving
and whole drug was released from matrix due to absence of
any delayed release polymer, i.e., ethyl cellulose. Thus, 90% of
drug was released during 24 h. This type of formulation is suit-
able for targeting in small intestine.
In vitro release kinetics
The kinetics of embelin release from the prepared micro-
spheres was studied by fitting the release data to zero order,
first order, Peppas model, and Higuchi model. Table 2 shows
different drug release kinetic models.
It was found that F1 (1:1) shows both zero-order and first-
order kinetics, which indicates that R2 value of F1 in case of
zero order was found to be 0.9588 and in case of first order is
0.9961, which shows that it follows diffusion as well as
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 5
dissolution mechanism. Eudragit S 100 polymer was dissolved
at pH 6.8, where it shows first-order release and after dissol-
ution, drug starts diffusing out of ethyl cellulose and follows
the zero-order kinetics and provides sustained drug release.
In vivo study
Effect of embelin on ulcer activity scores
Acetic acid produces significant inflammation followed by
ulcers in the colon assessed in terms of increase in scores for
% weight change, stool consistency, lesion score, and macro-
scopic scores. Embelin-loaded microspheres (F11) has shown
% weight change, stool consistency, lesion score, and macro-
scopic scores, 2 ± 0.03, 1 ± 0.05, 2 ± 0.05, and 2 ± 0.03, respect-
ively, which are comparable with results obtained with
marketed formulation (Tables 3 and 4). It can be concluded
from above studies that embelin-loaded microspheres (F11)
has significantly (P < 0.05) reduced the ulcer activities.
Higuchi
model (R2)
0.8594 Effect of embelin on myeloperoxidase level in acetic acid
0.9427 induced colitis in rats
0.9279
0.8508 Intrarectal administration of acetic acid showed significant
increase in the concentration of MPO, i.e., 16 lmol/min/mg tis-
sue, while embelin-loaded microparticle (F11) shows signifi-
cant decrease in the concentration of MPO, i.e., 8.8 lmol/min/
mg as compared to disease control. Plain embelin (50 mg/kg)
significantly reduced biochemical parameter (11.3 lmol/min/
mg) as compared to disease control but less than embelin-
loaded microspheres (Figure 3).
Effect of embelin on lipid peroxidation in acetic acid
induced colitis in rats
Lipid peroxidase is an enzyme that occurs in colonic tissue.
Increased lipid peroxide generates more and more reactive
free radicals, which exhausts cellular antioxidants and ultim-
ately favors the consequent development of further inflamma-
tion and ulceration. It is assumed that embelin-loaded
microsphere treatment improves colonic oxidative balance in
colitis induced animals, because microspheres were able to
reduce the level of malondialdehyde, a good indicator of lipid
peroxidation.
Intrarectal administration of acetic acid showed significant
increase in the concentration of LPO, i.e., 166.3 lmol of MDA/
mg pr, while embelin-loaded microspheres show significantly
decreased concentration of LPO, i.e., 101.33 lmol of MDA/
mg pr as compared to disease control. Plain embelin (50 mg/
kg) significantly reduced biochemical parameter (126.3 lmol
of MDA/mg pr) as compared to disease control but less than
embelin-loaded microparticles (Figure 4).
Effect of embelin on reduced glutathione level in acetic
acid induced colitis in rats
Pre-treatment of embelin-loaded microspheres reversed the
depletion of GSH and restored the level towards normal.
 6 NIDHI ET AL.

Table 3. Macroscopic scoring for evaluation of disease in different treatment groups.

Treatment Scores (% weight loss) Stool consistency Lesion scores Macroscopic scores
Group I (normal control) 0.0 ± 0 0.0 ± 0 0.0 ± 0 0
Group II (colitis control) 3 ± 0.02a 4 ± 0.05a 4 ± 0.01a 4 ± 0.01a
Group III (acetic acid þ plain embelin (50 mg/kg) 2 ± 0.04b 2 ± 0.03b 3 ± 0.02b 3 ± 0.02b
Group IV (acetic acid þ embelin-loaded microparticles) 2 ± 0.03b 1 ± 0.05b 2 ± 0.05b 2 ± 0.03b
Group V (acetic acid þ mesalamine) 1 ± 0.05b 1 ± 0.05b 1 ± 0.01b 1 ± 0.03b
Values are given as mean ± SEM; values are statistically significant at ap < 0.05 as compared to normal and bp < 0.05 as compared to colitis
control rats.

Table 4: Scoring chart for the evaluation of disease.

For weight change Stool consistency Lesion score
0 score – 0% 0 score – well-formed pellets 0 score – no
1 score – 1–5% 1 score – hard 1 score – hyperemia at sites
2 score – 5–10% 2 score – semisolid 2 score – lesion having diameter less than 1 mm
3 score – 10–20% 3 score – pasty 3 score – lesion having diameter less than 2 mm
4 score – above 20% 4 score – liquid diameter 4 score – lesion having diameter less than 3
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Figure 3. Effect of embelin on MPO level of acetic acid induced colitis in female
wistar rats. Values are given as mean ± SEM; values are statistically significant at
a
p < 0.05 as compared to normal and bp < 0.05 as compared to colitis
control rats.
Figure 5. Effect of embelin on GSH level of acetic acid induced colitis in female
wistar rats. Values are given as mean ± SEM; values are statistically significant at
a
p < 0.05 as compared to normal and bp < 0.05 as compared to colitis
control rats.

MDA/mg pr as compared to disease control. Plain embelin

(50 mg/kg) significantly increased GSH (4 l mol of GSH/mg pr)
as compared to disease control but not as much potent as
embelin-loaded microparticles. Standard drug mesalamine sig-
nificantly increases GSH (6.3 lmol of GSH/mg pr) as compared
to colitis control. Figure 5 shows the effect of embelin on
GSH level in acetic acid induced colitis in rats.

Figure 4. Effect of embelin on LPO level of acetic acid induced colitis in female
wistar rats. Values are given as mean ± SEM; values are statistically significant at
a
p < 0.05 as compared to normal and bp < 0.05 as compared to colitis
control rats.
Intrarectal administration of acetic acid showed significant
decrease in the concentration of GSH, i.e., 2.66 lmol of
GSH/mg pr, while embelin-loaded microparticle shows signifi-
cant increase in the concentration of GSH, i.e., 5.7 lmol of
Histopathological study
Embelin-loaded microspheres showed minimal damage of
mucosa with slight submucosal edema and mild inflammatory
cell infiltration. Embelin-loaded microspheres and standard
drug, i.e., mesalamine showed remarkable recovery of colonic
mucosa from acetic acid induced colitis damage. Figure 6
shows the histological image of colonic tissue of normal con-
trol group received CMC as vehicle. Normal control group
shows papillary mucosa having a lining of columnar epithe-
lium. The nucleus was placed in the basal portion and the
cytoplasm fills the majority of the cell. The submucosa

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Figure 6. (A) Normal control. (B) Colitis control. (C) Plain embelin (50 mg/kg)þacetic acid. (D) Embelin-loaded microspheres þ acetic acid. (E) Standard drug, i.e., mesal-
amine (100 mg/kg)þacetic acid.
showed an infiltrate of lymphocytes while the deeper zone
showed muscular layer. The case of acetic acid induced colitis
group showed the massive destruction of epithelium, sub-
mucosal edema and inflammatory cell infiltration in sub-
mucosa and muscularis. Plain embelin at 50 mg/kg b.w.
showed minimum damage of mucosa with submucosal
edema and mild inflammatory cell infiltration. But embelin-
loaded microspheres showed almost complete recovery of
colonic mucosa from acetic acid induced colitis damage as
compared to other groups. Thus, it has been concluded that
embelin-loaded microspheres showed comparable results with
standard drug, i.e., mesalamine, which also showed a healthy
colonic epithelium as shown in Figure 6.
Discussion
Acetic acid induced colitis is a model wherein inflammatory
mediators such as reactive oxygen species, vasoactive amines,
and eicosanoids play a prominent role (Carty et al. 2000). The
underlying pathophysiological mechanisms involved include
colon structure and mucosa barrier destruction by chemical
stimulation, enhanced vessel permeability, increased
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 7
inflammatory mediators, promotion of fibrin hydrolysis, and
disturbance of cruor process.
Myeloperoxidase is an enzyme present in neutrophils
and in a much lower concentration in monocytes and macro-
phages. The level of MPO activity is directly proportional to
the neutrophil concentration in the inflamed tissue. Therefore,
a measurement of MPO activity has been considered a quanti-
tative and sensitive assay for acute intestinal inflammation. In
addition, increased MPO activity has been reported to be an
index of neutrophil infiltration and inflammation (Choudhary
et al. 2001). Embelin at both the doses exhibited a significant
decrease in the MPO levels when compared to acetic acid
induced colitis control. Increased lipid peroxides that occur in
colonic tissue can initiate a vicious cycle that generates more
and more reactive metabolites, which exhausts cellular antiox-
idants, vitamins C and E, and ultimately favors the consequent
development of further inflammation and ulceration. Embelin
at both the doses significantly inhibited the increase in the
lipid peroxides activity in the colonic tissue. It is therefore rea-
sonable to assume that the embelin treatment improves
colonic oxidative balance in colitis induced animals, because
embelin was able to reduce the level of malondialdehyde, a
 8 NIDHI ET AL.
good indicator of lipid peroxidation (Ohkawa et al. 1979). GSH
is involved in the synthesis and repair of DNA, assists the
recycling of vitamins C and E, blocks free radical damage,
enhances the antioxidant activity of vitamin C, facilitates the
transport of amino acids and plays a critical role in detoxifi-
cation (Chavan et al. 2005). Pre-treatment of embelin
reversed the depletion of GSH and restored the levels
towards the normal. LDH, a cytosolic enzyme is involved in
the biochemical regulation reaction of the body tissues and
fluids. An elevation of LDH in serum indicates a shift
towards anaerobiosis resulting in the enhanced production
of lactic acid (Manna et al. 2004). In the present study, the
embelin pre-treatment altered the serum LDH level induced
by acetic acid towards the normal. The clinical, macroscopic,
and biochemical evidence for the protective effect of embe-
lin on acetic acid induced colitis in rats was well correlated
by the histopathological studies. The histological science of
inflammation such as leukocyte infiltration, edema and tissue
injury was found to be low following the pre-treatment with
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embelin. The results obtained from embelin-treated acetic
acid induced colitis in the present study are in well correl-
ation with earlier results of its ability to inhibit carrageen, an
induced paw edema in rats, inhibition of NF-kappa B activa-
tion, which makes it a potentially effective suppressor of
tumor cell survival, proliferation, invasion, angiogenesis, and
inflammation (Ahn et al. 2007, Chitra et al. 1994, Gupta
et al. 1976). In addition, embelin is known to suppress the
NF-kappa B activation induced by TNF-a and various other
inflammatory and carcinogenic agents (Ahn et al. 2007). The
results from the present study are also in consistent with
the earlier study of its ability to scavenge physiologically
relevant oxidizing radicals (Joshi et al. 2007). In the present
study, combined pH-dependent and delayed release embe-
lin-loaded microspheres were examined in vitro and in vivo
to elucidate their gastrointestinal behavior. In order to
improve the local targeting, it is essential to use a suitable
polymeric carrier system, which is able to deliver the drugs
specifically to inflamed region and avoid the gastrointestinal
absorption as far as possible (Tozaki et al. 2002). With the
advancement in novel formulation technology, the complete
knowledge and understanding of cellular and molecular
pathogenesis of UC along with microparticulate system could
overcome the drawbacks of conventional therapy and pro-
vide efficacious delivery of newly developed drugs. At pre-
sent, various anti-inflammatory drugs are available in market
such as sulfasalazine, mesalamine, balsalazide, but their side
effects always remain a major clinical problem. Therefore,
present study emphasis on the use of embelin (2,5-dihy-
droxy-3-undecyl-1,4-benzoquinone) from herbal origin, which
is reported to possess anti-inflammatory and antioxidant
activities. Thus, embelin-loaded microspheres were developed
by combining the concept of pH dependent and delayed
release for targeting drugs specifically to inflamed local site
of colon. In addition, particle size ranging 4–15 lm is opti-
mum, in order to achieve large surface area and prolonged
residence time in colon. Embelin-loaded microspheres (F1)
showed average particle size of 13.5 lm ± 1.2, which was
considered to be suitable for macrophages to accumulate
particles in the inflamed region. Other important parameters
were also optimized based on particle size and entrapment effi-
ciency such as Span 80 and crosslinking agent. The surfactant
as well as crosslinking agent play a major role in the particle
size, entrapment efficiency, and release study. With increase in
Span 80 concentration, particle size, and entrapment efficiency
goes on decreasing. This was due to the reason that high con-
centration of surfactant forms a coating over entire organic
droplet and gets miscible with continuous phase and forms sta-
ble emulsion, which decreases the particle size. Thus, because
of small particle size, more micelle formation containing drug
takes place and these micelles easily miscible with continuous
phase causing increased drug diffusion, which ultimately
reduces entrapment efficiency. But, due to the presence of
ethyl cellulose, viscosity of medium increases, which increases
particle size. The data indicated that increasing ethyl cellulose
concentration resulted in decrease of entrapment efficiency.
This was due to the reason that at high polymer concentration,
larger droplets were formed resulting in the inability of drug to
diffuse inside the particles and consequently, entrapment effi-
ciency decreases. In addition, with increase in crosslinking
agent, particle size as well as entrapment efficiency increases.
This was due to the formation of rigid network in the medium,
which prevents the drug from leaching out of polymer. In vitro
study also concluded that F1 formulation showed both zero-
and first-order releases. Very less amount of drug was released
up to 2 h in the presence of simulated gastric fluid using pepsin
(pH 1.2). This is due to the presence of pH-sensitive and time-
controlled polymer, which prevents the drug from releasing
into a gastric environment, but with increase in time, drug
started releasing at pH 6 and followed first-order release but
with further increase in time, it started showing zero-order
release. Thus, controlled drug delivery was achieved, which is
considered to be a promising approach for UC. The in vivo
study concluded that pre-treatment of embelin prevents acetic
acid-induced colitis in rats, and this effect is due to its antioxi-
dant and anti-inflammatory actions.
Conclusion
This study has demonstrated that embelin-loaded micro-
spheres (F1) formulation consisted of 1:1:0.3 w/w/w
EC:ES100:embelin ratio using 2% Span 80 for stabilization
could deliver drug specifically to colon. The drug release is
pH- and time dependent. This approach produces time-
dependent and pH-dependent sustained release as compared
to other conventional dosage forms. The study examined the
application of microparticulate system in enhancing stability
and controlled release with brightening results concerning the
therapeutic efficacy using blend of both pH-dependent and
time controlled polymers.
Acknowledgements
The authors are thankful to Mr. Praveen Garg, Chairman, ISF College of
Pharmacy, Moga, Punjab, for his continuous support and encouragement.
Disclosure statement
The authors have declared no conflict of interest. The authors alone are
responsible for the content and writing of the paper.

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