International Journal of Pharmaceutics 490 (2015) 22–31

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Curcumin–piperine mixtures in self-microemulsifying drug delivery

system for ulcerative colitis therapy
Qiuping Li 1, Wenwen Zhai 1, Qiaoli Jiang, Ruixue Huang, Lehuan Liu, Jundong Dai * ,
Weihong Gong, Shouying Du, Qing Wu
Department of Chinese Medicinal Pharmaceutics, School of Chinese Materia Medica, Eastern Campus, Beijing University of Chinese Medicine, Beijing 100102,
PR China

A R T I C L E I N F O A B S T R A C T

Article history: Curcumin (CUR) is a poorly water-soluble drug and its absorption is very low. In this study, CUR and
Received 5 December 2014 piperine (PIP) were co-encapsulated into the nanoformulation called self-microemulsifying drug
Received in revised form 20 April 2015 delivery system (SMEDDS) to improve the stability and water-solubility of CUR and enhance its anti-
Accepted 4 May 2015
colitis activity. The formulation of CUR-PIP-SMEDDS was prepared to encapsulate two hydrophobic
Available online 6 May 2015
components CUR and PIP, and then was characterized by assessing appearance, morphology, particle size,
zeta potential and drug encapsulation efficiency. The appearance of CUR-PIP-SMEDDS remained clarified
Chemical compounds studied in this article:
and transparent, and the microemulsion droplets appeared spherical without aggregation. The mean size
Curcumin (PubChem CID: 969516)
Piperine (PubChem CID638024)
of microemulsion droplet formed from CUR-PIP-SMEDDS was 15.87
0.76 nm, and the drug
encapsulation efficiency of SMEDDS for CUR and PIP were (94.34
2.18)% and (90.78
2.56)%,
Keywords: respectively. The vitro stability investigation of CUR-PIP-SMEDDS in colon tissue suggested that using
Curcumin SMEDDS as a delivery vehicle and co-encapsulated with PIP, CUR was more stable than drug solution in
Piperine colons site. Meanwhile, the anti-inflammatory activity of CUR-PIP-SMEDDS was evaluated on DSS-
SMEDDS induced colitis model. The results showed that CUR-PIP-SMEDDS exhibited definite anti-colitis activity
Preparation and characterization by directing CUR-PIP-SMEDDS to inflammatory colon tissue through retention enema administration.
Stability
Our study illustrated that the developed CUR-PIP-SMEDDS formulation was a potential carrier for
Anti-colitis activity
developing colon-specific drug delivery system of CUR for ulcerative colitis treatment.
ã 2015 Elsevier B.V. All rights reserved.

1. Introduction conventional medical treatment of UC relies on the use of

As a subcategory of inflammatory bowel diseases, ulcerative
colitis (UC) is an idiopathic and chronic disorder of unknown
etiology, which starts in the rectum and generally extends
proximally in a continuous manner through part of, or the entire,
colon (Ordás et al., 2012). The incidence of UC is increasingly
worldwide, and the disease remains incurable (Cosnes et al., 2011).
Furthermore, UC places a substantial economic burden because it
reduces the quality of life and the ability to work, and increases
disability (Cohen et al., 2010; Kappelman et al., 2008). The
* Corresponding author at: Department of Chinese Medicinal Pharmaceutics,
School of Chinese Materia Medica, Eastern Campus, Beijing University of Chinese
Medicine, No.6 Wangjing Zhonghuan South Road, Chaoyang District, Beijing
100102, PR China. Tel.: +86 10 84738616; fax: +86 10 84738611.
E-mail address: daijd@163.com (J. Dai).
1
These authors contributed equally to this work and should be considered co-
first authors.
aminosalicylates (5-aminosalicylic acid-based agents), corticoste-
roids, immunosuppressive drugs (e.g., azathioprine, 6-mercapto-
purine, cyclosporin), and antibiotics, with the goals of achieving
clinical or mucosal remission (Feuerstein and Cheifetz, 2014; Head
and Jurenka, 2003). However, these conventional therapies are in
many instances ineffective or cannot be tolerated by the patients.
Aminosalicylates are the most common protocols for maintaining
remission of UC and usually well tolerated by the patients, but
frequently induce side effects, such as hemolytic anemia, acute
pancreatitis, hepatitis, abdominal pain, nausea, infection, diarrhea,
renal failure and pericarditis (Hanauer, 2004). Corticosteroids are
the mainstay for acute episodes of UC, while dose-dependent
reaction is evident, and disease recurs on reducing the dose,
whereas high doses are accompanied by serious side effects, such
as osteoporosis, cataracts and myopathy (Head and Jurenka, 2003).
Other agents used for UC are immunomodulators and antibiotics;
however, these agents still have been largely ineffective or are
accompanied by severe adverse effects for use (Head and Jurenka,

http://dx.doi.org/10.1016/j.ijpharm.2015.05.008
0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
 Q. Li et al. / International Journal of Pharmaceutics 490 (2015) 22–31
2003). Therefore, there is a pressing need for developing better
anti-inflammatory agents with increased efficacy and improved
safety profiles.
Recently, considerable attention has been devoted to identify-
ing naturally occurring chemopreventive polyphenol substances,
particularly those present in dietary and medicinal plants.
Remarkably, further studies have demonstrated that CUR could
exert excellent anti-colitis effects with the virtues of high
acceptance by patients, good efficacy, relatively safety and low
cost (Baliga et al., 2012; Lahiff and Moss, 2011). CUR (Fig. 1A), a
highly lipophilic bioactive substance extracted from the rhizomes
of the herb Curcuma longa L., has a long history of use for centuries
in Asia, both in traditional medicine and in cooking as turmeric
which gives food an exotic natural yellow color. It has been found to
have a wide variety of biological and pharmacological effects,
including anti-inflammatory (Singla et al., 2014; Wang et al.,
2014b), antioxidant (Yu et al., 2014a; Zhao et al., 2014), anti-
depressive (Sanmukhani et al., 2014; Zhang et al., 2014), memory
improvement (Choudhary et al., 2013; Wang et al., 2014a),
antitumor (Lim et al., 2014; Yu et al., 2014b), and hepatoprotective
activities (Nabavi et al., 2014; Zheng et al., 2014). To date, a number
of studies in the animal models (Qureshi et al., 1992; Shankar et al.,
1980) and in patients (Cheng et al., 2001; Lao et al., 2006) have not
discovered any toxicity associated with the use of CUR even at quite
high doses. With the beneficial properties of low cost, reliable
sources and excellent anti-inflammatory effects, CUR can be used
as potential drug for the treatment of UC in patients. However, in
spite of its promising pharmacological effects and safety, CUR has
not yet been used as a clinical therapeutic agent because of poor
bioavailability, which appears to be due to its low aqueous
solubility, extensive hepatic and intestinal metabolism, and rapid
systemic elimination. Therefore, it is very important for clinical use
to increase its aqueous solubility and decrease its metabolic
clearance simultaneously in the further studies.
In order to increase the absorption of CUR in vivo, various
formulations have been prepared such as polymeric micelles (Gong
et al., 2013), oil-in-water emulsions (Hu et al., 2012), solid lipid
nanoparticles (Sun et al., 2013) and liposomes (Karewicz et al.,
2013). Amongst all these formulation technologies, an approach
named self-microemulsifying drug delivery system (SMEDDS),
isotropic and thermodynamically transparent stable solution
consisting of oil, surfactant, co-surfactant and drug mixtures,
has many properties that make it appealing as a universal vehicle
for insoluble lipophilic drugs delivery (Kohli et al., 2010; Qi et al.,
O
H
O
O
H
O
H H
N
O
OH
(A)
Fig. 1. The chemical structure of curcumin (A) and piperine (B).
23
2011). Previous studies have shown that SMEDDS can be utilized to
enhance the solubility, dissolution and oral absorption for
insoluble lipophilic drugs, such as curcumin (Cui et al., 2009;
Setthacheewakul et al., 2010), felodipine (Ansari et al., 2014),
docetaxel (Seo et al., 2013), berberine hydrochloride (Zhu et al.,
2013) and zedoary essential oil (Zhao et al., 2010). The system
could emulsify spontaneously to form fine oil-in-water micro-
emulsion with nanometric droplet size upon gentle agitation in
aqueous media such as gastrointestinal (GT) fluids. The potential
advantages of SMEDDS include not only increasing drug solubility
but also improving release and absorption properties through
enhancing permeation across the inflamed mucosal tissues and
droplet size reduction, providing a large interfacial surface area for
drug absorption in colon site (Constantinides, 1995). The oil used in
the formulation is probably to protect the drug from enzyme
degradation. An alternative approach suggested for improving the
delivery of CUR is co-administration with piperine (Fig. 1B).
Piperine (PIP), an important bioactive compound of black pepper,
could enhance CUR’s absorption and has the ability to inhibit
metabolizing enzymes, retard clearance of CUR via P glycoprotein
efflux pump as well as through the down-regulation of NF-kB
release and provide protection against oxidative damage (Kakarala
et al., 2010; Rinwa and Kumar, 2012; Sharma et al., 2010). It is
reported that CUR bioavailability was increased by 2000% at
45 minutes after co-administering CUR orally with PIP in humans
(Shoba et al., 1998). Intestinal absorption of CUR was also
evidenced relatively higher when administered concomitantly
with PIP, and it stayed significantly longer in the body tissues
(Suresh and Srinivasan, 2010). In view of these findings, PIP could
significantly improve the level of serum, extent of absorption and
the efficacy of CUR in both rats and humans with no adverse
effects. Therefore, CUR-PIP-SMEDDS could be a promising poten-
tial therapeutic formulation for the treament of UC. However, no
data exist on the anti-colitis activity of CUR-PIP-SMEDDS in
experimental colitis to date. We therefore chose to investigate the
efficacy of CUR-PIP-SMEDDS in experimental colitis in order to
demonstrate the improved anti-inflammatory effects of CUR.
In the present study, an attempt was made to improve the
stability, solubility and therapeutic effects of CUR for UC therapy by
formulating it as CUR-PIP-SMEDDS through retention enema
administration. Under conditions of colon fluid, CUR-PIP-SMEDDS
containing PIP could inhibit the metabolic transformation of CUR
and obtain higher extent absorption and therapeutic effect during
ulcerative colitis therapy. Therefore, we provided a novel strategy
O H H
O
H H
O
(B)
24 Q. Li et al. / International Journal of Pharmaceutics 490 (2015) 22–31
to localize potent drugs in close proximity possible to the inflamed
mucosal tissues to achieve maximal local drug concentrations in
the study. The primary objectives of this study were to characterize
a CUR-PIP-SMEDDS formulation and evaluate its anti-colitis
activity. The co-delivery system was characterized in terms of
appearance, morphology, particle size, zeta potential, drug
encapsulation efficiency and stability property in vitro. Meanwhile,
anti-colitis activity of CUR-PIP-SMEDDS was evaluated on a model
of DSS-induced UC in male BALB/c mice.
2. Materials and methods
2.1. Chemical and reagents
CUR (80% pure, with 15% of demethoxycurcumin and 4% of
bisdemethoxycurcumin as impurities) was purchased from Tianjin
Fu Chen Chemical Reagents Factory (Tianjin, China). PIP and
5-aminosalicylic acid (5-ASA) were purchased from Sigma-Aldrich
Corporation (St. Louis, USA). CUR and PIP standards were
purchased from the National Institutes for Food and Drug Control
(Beijing, China). Capryol 90 and Transcutol HP were purchased
from Gattefosse (Saint-Priest, France). Cremophor RH40 was
obtained from BASF (Ludwigshafen, Germany). Dextran sulfate
sodium (DSS) was purchased from MP Biomedicals Inc. (Santa Ana,
USA). Methanol and acetonitrile were of high HPLC grade
(Emerson, USA). Pure water was supplied by Wahaha Group Co.,
Ltd. (Hangzhou, China). All other reagents were of analytical
grade.
2.2. Animals
Forty-eight specific pathogen-free male BALB/c mice weighing
18–22 g were purchased from Vital River Laboratories (Beijing,
China). The animals were housed in standard cages with wood
shavings. Eight animals/cage were maintained in an animal room
with a carefully controlled ambient temperature (20–25  C) and
artificial illumination (12 h light/dark cycle) and were provided
with standard diet. Animal welfare and experimental procedures
were strictly in accordance with Principles of Laboratory Animal
Care for animal experiments and approved by Beijing University of
Chinese Medicine Committee on Animal Care and Use.
2.3. Preparation of CUR-PIP-SMEDDS formulation
The CUR-PIP-SMEDDS formulation was prepared in accordance
with our previous study (Li et al., 2014). Briefly, Capryol 90,
Cremophor RH40 and Transcutol HP were selected as oil,
surfactant and co-surfactant, separately. Blank SMEDDS formula-
tion was prepared by mixing the oil with surfactant and co-
surfactant at the ratio of 10:60:30 (w/w/w) with magnetic stirring
at 100 rpm for 10 min at 70  C. CUR and PIP were added to the blank
SMEDDS and mixed by gentle magnetic stirring (50 rpm) until a
transparent preparation was obtained, then the CUR-PIP-SMEDDS
was prepared (containing CUR 50 mg/g and PIP 1 mg/g). In the
previous study, ternary phase diagram and a simplex lattice
experiment design were adopted to optimize the composition of
CUR-PIP-SMEDDS. The concentrations of oil, surfactant and co-
surfactant were chosen as the independent variables and kept their
total concentration constant. The solubility of CUR and PIP in
SMEDDS and the mean particle size of formed microemulsion by
diluting CUR-PIP-SMEDDS with distilled water were taken as
responses, respectively. The Simplex-Lattice experiment design for
a three-component system was represented (Rispoli and Shah,
2009; Zhang et al., 2012) with the aid of Design Expert
8.06 software.
2.4. Characterization of CUR-PIP-SMEDDS formulation
2.4.1. Appearance and morphology
The appearance of CUR-PIP-SMEDDS was evaluated under
different conditions. The morphology of CUR-PIP-SMEDDS was
observed by transmission electron microscope (TEM) (JEM-1400
plus, JEOL, Tokyo, Japan). CUR-PIP-SMEDDS was diluted with
deionized water at 1:500 and mixed by slightly shaking. One drop
of dilute samples was carefully placed on 200-mech formvar-
coated copper TEM grid followed by staining with 2% aqueous
solution of phosphotungstic acid for 2 min. The excess solution on
the grid was removed using a piece of fine filter paper, and the
samples were allowed to air dry before observation under the TEM.
2.4.2. Droplet size and zeta potential
CUR-PIP-SMEDDS samples were diluted 100-fold with 37  C
deionized water and shaken up to form microemulsion samples
prior to measurement. The mean particle size distribution and zeta
potential of microemulsion samples were measured using a
Zetasizer Nano ZS analyser (Malvern Instruments Co., Worcester-
shire, UK). Measurements were performed with at least three
different batches to obtain an average value and standard deviation
for the particle diameter and zeta potential.
2.4.3. Determination of drug encapsulation efficiency
0.1 g CUR-PIP-SMEDDS was diluted with 10 ml of deionized
water under gentle stirring at 37  C and was centrifuged at
5000 rpm for 20 min to remove the undissolved drugs. A fixed
amount of supernatant was diluted to a suitable concentration
with methanol and the content of CUR and PIP existed in
supernatant was called the concentration of encapsulated drug
(Ce) (Lin et al., 2014). To determine the concentration of total drug
(Ct), 25 mg Cur-PIP-SMEDDS was dissolved by methanol and the
concentration of two drugs was measured using an established
high performance liquid chromatography (HPLC) method. The
chromatographic separation was performed on a C18 column
(5 mm, 250  4.6 mm; Merck Purospher STAR, China) with the
mobile phase composed of acetonitrile-4% acetic acid aqueous
(55:45, vol/vol) at a flow rate of 1.0 ml/min. The wavelength of
detection was 342 nm. The encapsulation efficiency (EE, %) was
calculated using Eq. (1).
Ce
EEð%Þ ¼  100% (1)
Ct
2.4.4. Stability tests in vitro
To determine whether CUR-PIP-SMEDDS is more stable in colon
site, free CUR, CUR-PIP mixture and CUR-PIP-SMEDDS were
incubated in the artificial colon fluid (PBS pH 7.8-8.0) and the
mice colon tissue homogenate in the dark at 37  C. At different time
points, 1 ml of each sample was taken to determine the
concentration of CUR. The concentrations of CUR, CUR-PIP mixture
and CUR-PIP-SMEDDS at the 0-min were considered as 1.00. The
fold reduction of the concentrations at each point was determined
by comparison to the 0-min. The experiments were repeated three
times for each time point (n = 3).
2.5. Anti-colitis activity of CUR-PIP-SMEDDS
2.5.1. Experimental design
A total of 48 mice were allotted in 6 groups, 8 mice each. Acute
ulcerative colitis was induced by oral administration of 5% (wt/vol)
dextran sulphate sodium (DSS) (molecular weight 36–50 kDa;
dissolved in fresh tap water) ad libitum for 7 consecutive days. The
normal group was given water only. In the first group, designated
 Q. Li et al. / International Journal of Pharmaceutics 490 (2015) 22–31
Table 1
Scoring system to calculate the disease activity index.
Scoring of disease activity index (DAI)
Score Weight loss Stool consistency Visible blood in feces
0 None Normal None
1 1–5% Loose Slight bleeding
2 6–10%
3 11–15%
4 >15% Diarrhea Gross bleeding
Note: DAI value is calculated as the sum of scores of weight loss, stool consistency,
and blood in feces.
as the normal group, colitis was not induced and normal saline was
given by retention enema on day 2 once daily for 5 consecutive
days. The second group, designated as the DSS colitis group,
received 5% DSS for 7 days and blank SMEDDS given by retention
enema on day 2 once daily for 5 consecutive days. The mice of other
4 groups received 5% DSS for 7 days and were treated respectively
with 5-ASA (5-ASA dispersed in 1% (wt/vol) sodium carboxyme-
thycellulose solution, retention enema administration), CUR-PIP
suspensions (CUR and PIP dispersed in 1% (wt/vol) sodium
carboxymethycellulose solution, retention enema administration),
CUR-PIP-SMEDDS (intragastric administration, ig) and CUR–PIP-
SMEDDS (retention enema administration) once daily for 5 conse-
cutive days. Administration of each agent was started on day 2 after
DSS-induced colitis. All CUR-PIP-SMEDDS formulations were
dosed in the oil phase, without prior addition of water. CUR and
5-ASA were dosed at 100 mg/kg of body weight, while PIP was
dosed at 2 mg/kg of body weight for all the studies. Meanwhile,
body weight, stool consistency and stool blood were recorded
daily. Disease activity index (DAI) was determined according to the
parameters outline in Table 1.
2.5.2. Morphological analysis
At the end of the experiment, animals were euthanized by
cervical dislocation and the entire colon was immediately
removed, gently flushed with saline, placed on an ice-cold plate,
cleaned of fat and mesentery, and patted dry with filter paper. The
colon length was measured between the ileo-cecal junction and
the proximal rectum.
2.5.3. Histopathological examination
Colon samples were kept in 10% formalin solution for 24 h and
then embedded in paraffin blocks. Serial sections 4-mm-thick were
prepared and stained by hematoxylin and eosin (H&E) for
histological evaluation.
2.5.4. Determination of myeloperoxidase (MPO) activity
MPO activity, an index of leukocyte recruitment, was deter-
mined according to the method described by Manktelow and
Meyer (1986). In brief, colon tissue homogenate was extracted
with hexadecyltrimethyl ammonium bromide. Then dimethox-
ybenzidine (DMB) was oxidized by MPO in presence of hydrogen
peroxide, and the optical density was measured at 460 nm at room
temperature (25  C). Optical density was a direct measure of
enzymatic activity. The final MPO activity was represented as U/mg
colonic tissue.
2.5.5. Determination of malondialdehyde (MDA) content
Lipid peroxidation was assessed as MDA content of the colon
according to the method described by Mihara and Uchiyama
(1978). In short, the colorimetric determination of MDA is based on
the reaction of one molecule of the reactive aldehyde with two
molecules of thiobarbituric acid at low pH (2–3) and at a
temperature of 95  C for 45 min. The resultant pink color was
extracted by n-butanol, and the absorbance was determined at
25
535 and 520 nm spectrophotometrically. The difference in optical
density between both wavelengths was used as a measure of
colonic MDA content. The final value of MDA was represented as
nmol/mg protein.
2.5.6. Determination of tumor necrosis factor-alpha (TNF-a) and
interleukin-6 (IL-6) levels
The levels of TNF-a and IL-6 in homogenized colonic tissue
were measured by quantitative enzyme-linked immunosorbent
assay (ELISA) kits according to the manufacturer’s instructions
(eBioscience, Inc., San Diego, CA, USA). The final values of TNF-a
and IL-6 were expressed as pg/mg protein.
2.6. Statistical analysis
The data were expressed as mean
standard deviation (SD).
The statistical significance of the difference in each parameter
among the groups was evaluated using one-way analysis of
variance (ANOVA) followed by the Fisher’s protected least
significant difference (PLSD) comparison tests for post hoc t-tests.
Criterion for statistically significant difference was chosen to be at
P < 0.05. Scores were analyzed by using the Wilcoxon’s test.
3. Results
3.1. Characterization of CUR-PIP-SMEDDS formulation
3.1.1. Appearance and morphology
CUR-PIP-SMEDDS could form O/W microemulsion immediately
when diluted with deionized water. As shown in the appearance of
Fig. 2, CUR-PIP-SMEDDS was clear and transparent liquid with
visible deep red at room temperature. CUR-PIP-SMEDDS micro-
emulsion was clear and transparent solution with visible orange-
yellow. When the same content of CUR and PIP were dispersed in
1% (wt/vol) sodium carboxymethycellulose solution (CUR-PIP
suspensions), it was yellow suspension. The solubility of CUR in
CUR-PIP-SMEDDS was 40.90
0.70 mgg1, which was significant-
ly higher than that in the water. It is reported that the solubility of
CUR in the aqueous medium was about 11 ngg1 (Kaminaga et al.,
2003; Yu and Huang, 2010). Therefore, it could be concluded that
CUR-PIP-SMEDDS has increased the solubility of CUR in the water
about 4  106-fold. The morphology of CUR -PIP-SMEDDS micro-
emulsion was observed by TEM and the observation results were
displayed in Fig. 3. The TEM image shows that most microemulsion
droplets appeared as perfect spherical shape with approximate
size and uniform dispersion.
Fig. 2. Appearance of CUR-PIP-SMEDDS under different conditions: (A) appearance
of CUR-PIP-SMEDDS at room temperature; (B) appearance of CUR-PIP-SMEDDS
diluted 100-fold with deionized water; (C) appearance of CUR-PIP suspensions.
26 Q. Li et al. / International Journal of Pharmaceutics 490 (2015) 22–31

(A)
100 ** **
CUR
**
** ** CUR-PIP
95 **
**

Fraction of CUR /%
** CUR-PIP-SMEDDS
** **
90 **
**

** **
85

80

75
0 5 10 15 20 25
Fig. 3. TEM micrograph of CUR–PIP-SMEDDS with the scale bar for the image
representing 100 nm. Time (h)
(B)
100 **
** **
3.1.2. Droplet size, zeta potential and encapsulation efficiency ** **
CUR-PIP-SMEDDS microemulsion was monodisperse with a * **
** **
95
mean particle size of 15.87
0.76 nm with PDI of 0.092
0.008 **

Fraction of CUR /%
(mean
SD; n = 6). The mean zeta potential of CUR-PIP-SMEDDS
**
was detected to be 0.799
0.081 mV (mean
SD; n = 6). Mean- 90
while, the mean EECUR and EEPIP values were (94.34
2.18)% and **
(90.78
2.56)%, respectively. **
85
3.1.3. Co-encapsulated of CUR and PIP into SMEDDS can increase the CUR
stability of CUR CUR-PIP
80
CUR is relatively unstable, and this is one of the major barriers
for clinical use of CUR to treat inflammation-related diseases. To CUR-PIP-SMEDDS
determine whether CUR-PIP-SMDDES is more stable, free CUR, 75
CUR-PIP mixture and CUR-PIP-SMEDDS were incubated at 37  C 0 2 4 6 8
Time (h)
and sampled periodically to determine the concentration of CUR by
HPLC. After incubation for 24 h at 37  C, we found free CUR, CUR- Fig. 4. Degradation of free CUR, CUR-PIP mixture and CUR-PIP-SMEDDS in the
PIP mixture and CUR-PIP-SMEDDS degraded by 19.93%, 12.67% and artificial colon fluid (A) and the mice colon tissue homogenate (B).*P < 0.05,
**
10.50% respectively in the artificial colon fluid (Fig. 4A). The P < 0.01 vs. the free CUR. Data are represented as the mean
SD; n = 3.

degradation of CUR followed apparent first-order kinetics equation

of the mice body weights (A) and DAI scores (B) after treatment
and the half-life were 19.67 h, 57.87 h and 75.23 h respectively in
with DSS. DAI scores of mice were increased in a time-dependent
the artificial colon fluid. After incubation for 8 h at 37  C, we found
fashion. As shown in Fig. 5B, compared with the normal control
free CUR, CUR-PIP mixture and CUR-PIP-SMEDDS degraded by
group, the DAI scores were markedly increased in saline-treated
19.96%, 14.49% and 4.63% respectively in the mice colon tissue
mice with DSS-induced colitis (P < 0.01–0.05). By contrast,
homogenate (Fig. 4B). The results show that CUR-PIP-SMEDDS
treatment with CUR-PIP-SMEDDS significantly reduced the DAI
could protect CUR from degration and metabolism of colon
scores in mice induced by DSS (P < 0.01–0.05). We also found that
enzyme system to significantly improve the stability of CUR
retention enema administration group of CUR-PIP-SMEDDS
(P < 0.01–0.05).
exhibited the similar improvements to positive medicine group
of 5-ASA (P > 0.05).
3.2. Anti-colitis activity of CUR-PIP-SMEDDS
3.2.2. Effects on the length of colon
3.2.1. Effects on clinical symptoms
As shown in Fig. 6, the colonic length of the DSS-induced mice
We assessed the therapeutic effects of CUR-PIP-SMEDDS by
was significantly shorter compared with those of the normal
using the acute DSS-induced UC model. The DSS-induced murine
group. However, the length of the shortened colon was signifi-
UC model used in this study is a well-established general prototype
cantly increased after the treatment of agents in mice with DSS-
of intestinal tissue damage that accurately resembles the
induced colitis (P<0.01–0.001). The enema administration groups
histological aspects of the tissue damage observed in patients
of CUR-PIP-SMEDDS and 5-ASA displayed the strongest efficacies
suffering from inflammatory bowel disease (Goyal et al., 2014). In
to increase the length of the shortened colon induced by DSS, and
this study, we found that BALB/c mice subjected to the oral
the therapeutic effects were similar (P > 0.05).
administration of 5% DSS regularly developed ulcerative colitis
with weight loss, severe diarrhea and rectal prolapse accompanied
by extensive wasting disease in untreated mice. In severe cases, 3.2.3. Effects on histological examination
gross blood adhering to the anus was noted. The DAI, an indicator We characterized the histologic features of UC in BALB/c mice
of the severity of intestinal inflammation, was used to analyze the subjected to 5% DSS. In normal mice, no signs or only a very low
therapeutic benefit of CUR-PIP-SMEDDS treatment. In this level of polymorphonuclear leukocyte infiltration in the colon was
experimental acute ulcerative colitis, treatment with CUR-PIP- observed (Fig. 7A). The severity of UC-like lesions was most
SMEDDS, mice showed remarkable improvements in body weight marked in the colon of DSS-induced mice. Compared with normal
loss, intestinal bleeding and diarrhea, resulting in significant mice, the distal colon of UC mice exhibited marked mucosal
amelioration of UC as assessed by DAI. Fig. 5 shows the time course inflammation in all layers of the bowel wall, including a marked
 Q. Li et al. / International Journal of Pharmaceutics 490 (2015) 22–31 27

increase in the thickness of muscle layer, severe submucosal

edema, depletion of goblet cells, inflammatory cells infiltration and
crypt abscesses (Fig. 7B). However, CUR-PIP-SMEDDS protected
against both the infiltration of inflammatory cells and the mucosal
damage, resulting in a significant reduction of histopathology
damage. Microscopically, the groups of CUR-PIP-SMEDDS (reten-
tion enema) and 5-ASA exhibited virtually the same normal
histology with no or only a very low level of inflammatory cells
infiltration, edema or crypt abscess (Fig. 7C and F). The group of
CUR-PIP suspensions produced a slight inflammatory cells
infiltration and crypt abscess in colonic mucosa with no
submucosal edema (Fig. 7D). The group of CUR-PIP-SMEDDS (ig)
produced a slight inflammatory cells infiltration and gland
abnormal growth in colonic mucosa with no submucosal edema
(Fig. 7E). The results showed that therapeutic effects were
correlated with formulation and administration route.

3.2.4. Effects on MPO activity

In the experiment, we found that MPO activity was correlated
with the development of colonic inflammation. As shown in
Fig. 8A, colitis induced by DSS significantly elevated MPO activity,
whereas administration of CUR-PIP-SMEDDS strongly inhibited
MPO activity in mice with DSS-induced colitis. We could find that
Fig. 6. Effect of CUR-PIP-SMEDDS administration on the colon length. **P < 0.01,
administration groups of CUR-PIP-SMEDDS exhibited the similar ***
P < 0.001 vs. the DSS model; ###P < 0.001 vs. normal group. Data are represented
inhibitory activities to the positive medicine group of 5-ASA as the mean
SD; n = 8.
(P > 0.05).

3.2.5. Effects on MDA content

As shown in Fig. 8B, CUR-PIP-SMEDDS (retention enema) and 5-
ASA substantially decreased the colonic content of MDA by about
(A) 22 35% and 29% respectively, compared to DSS model group, and there
was no significant difference between the two groups (P > 0.05).
Treatment with CUR-PIP-SMEDDS (ig) also exerted, to some extent,
20 *
* ***
effects on reducing the colonic MDA level compared to animals
*** that received DSS alone.
Body weight (g)

*** ***
***
#
*** 3.2.6. Effects on TNF-a and IL-6 levels
18 *** ***
Normal group DSS-induced UC was accompanied by the release of pro-
inflammatory cytokines including TNF-a and IL-6. The levels of
DSS colitis ### ***

5-ASA * TNF-a and IL-6 in the supernatant of colon tissue homogenate

16 CUR-PIP suspensions ### from each group were measured to determine whether CUR-PIP-
CUR-PIP-SMEDDS (ig) SMEDDS inhibits the production of pro-inflammatory cytokines,
CUR-PIP-SMEDDS ### therefore protecting the colon mucosa. As shown in Fig. 8C and D,
14 the increase in the amount of TNF-a and IL-6 expression was
0 2 4 6 8
significantly reduced after the treatment of agents in mice with
Days
DSS-induced colitis (P < 0.05–0.001). We could find that the
(B) ##
10 administration groups of CUR-PIP-SMEDDS and 5-ASA displayed
Normal group
## strong efficacies to inhibit the production of TNF-a and IL-
DSS colitis
* 6 induced by DSS, and the therapeutic effects were similar
Disease activity index (DAI)

8 5-ASA (P > 0.05).

##
CUR-PIP suspensions
**
** 4. Discussion
6 CUR-PIP-SMEDDS (ig)
**
**
CUR-PIP-SMEDDS
**
Local treatment of UC has been the objective of countless
4
**
** reports in recent years, where the activity of some drugs is
## **
** correlated with their concentration in the colonic mucosa (Frieri
#
et al., 2000). However, a major drawback of colonic delivery
*
2 * systems is their inability to target the mucosa after colon arrival.
* One approach to improve this situation is to use nanoparticle drug
carriers, which possess improved capability to adsorb and
0
0 2 4 6 8 accumulate in the target inflamed tissues, and reside at the site
Days of attachment for a prolonged period of time, owing to their large
interfacial surface area and minute dimension (Constantinides,
Fig. 5. Effect of CUR–PIP-SMEDDS administration on the time-course changes in
the mice body weights (A) and DAI (B). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the DSS
1995). It is well documented that intestinal permeability is
model; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. normal group. Data are represented as increased during inflammation, allowing permeation of the
the mean
SD; n = 8. particles to the desired colon lesion site in a preferable manner
28 Q. Li et al. / International Journal of Pharmaceutics 490 (2015) 22–31
Fig. 7. Histologic analysis of the colon section in BALB/c mice (H&E 40): (A) normal group; (B) DSS colitis group; (C) 5-ASA retention enema administration group; (D) CUR–
PIP suspensions retention enema administration group; (E) CUR–PIP-SMEDDS ig group; (F) CUR–PIP-SMEDDS retention enema administration group.
when compared to the normal intestine (Ekstrom and Andersson,
2000; McGuckin et al., 2009). Lamprecht et al. (2005) have already
shown that a significant deposition of nanoparticles retained in the
mucosa of DNBS induced rats after systemic administration,
probably due to the retention effect of the inflamed tissue. In our
study, CUR-PIP-SMEDDS formulation with negative charge was
prepared. Tirosh et al. (2009) have recently shown that targeting
the inflamed mucosa could be accomplished with negatively
charged dosage forms for the topical treatment of UC. Therefore,
CUR and PIP were delivered via anionic SMEDDS to the inflamed
mucosa of experimental colitis-induced mice were more effective
in ameliorating the induced inflammation compared with their
aqueous suspensions.
As a relatively non-toxic natural product combined with
excellent anti-inflammatory and antioxidant properties, CUR has
been used to attenuate inflammation in the animal models of
colitis and is effective in patients with UC (Arafa et al., 2009; Holt
et al., 2005). Unfortunately, dosing CUR alone, researchers would
face the dilemma of poor absorption, it is thus very vital for clinical
application to improve absorption of CUR in the further studies (Liu
et al., 2013). In the present study, the CUR-PIP-SMEDDS was
prepared to encapsulate two hydrophobic components CUR and
PIP, which could significantly improve the stability, solubility and
anti-colitis activity of CUR. This is, to our knowledge, the first
evaluation of the efficacy of CUR-PIP-SMEDDS in an experimental
colitis. In order to investigate the topical therapeutic effects of
CUR-PIP-SMEDDS on UC, we selected a model of colitis induced by
DSS in mice, treatment through retention enema administration.
The model exhibits many symptoms and signs similar to those seen
in human UC, such as diarrhea, bloody feces, body weight loss,
mucosal ulceration and shortening of colon length (Okayasu et al.,
1990; Sartor, 2006; Strober et al., 2002). In addition, the DSS-
induced UC animal model has a variety of advantages over others,
such as simple experimental methods, reproducibility of the time-
course of development as well as colitis severity among individual
mice, and relative uniformity of the induced lesions (Camuesco
et al., 2005; Dieleman et al., 1998). Therefore, this model is thought
to be reliable for testing drug formulations or phytochemicals for
UC treatment (Araki et al., 2006; Kitajima et al., 1999; Tang et al.,
2014).
In the present study, we measured the severity of clinical
symptoms by assessing the body weight loss, stool consistency and
stool blood, and finally, evaluated the therapeutic effects of CUR-
PIP-SMEDDS treatment. Our findings demonstrated that CUR-PIP-
SMEDDS treatment significantly suppressed DSS-induced colitis in
mice by improving their body weight and stool consistency as well
as decreasing intestinal bleeding. In addition, the DSS-induced
colitis exhibited mucosal inflammation with crypt abscesses and
regenerating epithelium in the mucosa. CUR-PIP-SMEDDS treat-
ment greatly reduced the infiltration of leukocytes and mucosal
damage, resulting in significant amelioration of histopathological
lesion and preserving colon length.
Studies have demonstrated that treatment with CUR on UC
could significantly lower MPO activity and MDA content (Arafa
et al., 2009; Salh et al., 2003). MPO is an enzyme found in
neutrophils and its activity in the colon is related linearly to
neutrophil infiltration (Eiserich et al., 1998). The assessment of
MPO activity is well established for quantifying intestinal
inflammation, such as UC (Krawisz et al., 1984). The activity of
MPO, which is a major enzyme in the formation of ROS leading to
tissue damage, increased in the colitis model. Lipid peroxidation, as
estimated by the MDA concentration, was elevated in the inflamed
UC mucosa. The amount of MDA was associated with epithelial
catalase expression and neutrophilic myeloperoxidase activity
(Arafa et al., 2009). The suppression of MPO activity and MDA
production by CUR–PIP-SMEDDS treatment in the DSS-induced
colitis model is effective for the inhibition of UC progression.
It is well known that there is an inflammatory cascade within
the gut tissues of UC that is characterized by the recruitment of
circulating leukocytes into the gut tissues and the aberrant
expression of pro-inflammatory cytokines such as TNF-a and IL-
6 (Pedersen et al., 2014; Takac et al., 2014). In the present study, we
have also shown the development of such a cascade of
inflammatory events in colitis induced by DSS. Analysis of
 Q. Li et al. / International Journal of Pharmaceutics 490 (2015) 22–31
Fig. 8. Effects of CUR–PIP-SMEDDS administration on MPO activity (A), MDA content (B), TNF-a (C) and IL-6 (D) levels in colonic tissues. *P < 0.05, **P < 0.01, ***P < 0.01 vs. the
DSS model. ###P < 0.001 vs. normal group. Data are represented as the mean
SD; n = 8.
inflammatory cytokine production in colon tissue homogenate
revealed a significant reduction in the levels of TNF-a and IL-6 in
mice treated with CUR-PIP-SMEDDS, compared to the DSS-induced
colitis model. Based on these results, the reduced TNF-a and IL-
6 production in the colonic tissues represents a possible means for
decreasing the severity of UC. Indeed, we found that the
administration of CUR-PIP-SMEDDS not only reduced DAI and
histopathological lesion, but also downregulated TNF-a and IL-
6 production, limited the inflammatory response, and thereby
significantly ameliorated the severity of DSS-induced colitis. In
fact, in agreement with our findings, numerous studies have also
demonstrated that CUR could simultaneously block the activation
of multiple transcription factors (Bharti et al., 2003a,b). Therefore,
as a strategy, we believe that CUR-PIP-SMEDDS may be an
efficacious and promising remedy in the treatment of UC because
its therapeutic targets are multiple transcription pathways.
The study has shown that the difference between the CUR-PIP
suspensions treatment and the CUR-PIP-SMEDDS treatment,
which can be seen from the DAI, colonic length, histological
analysis and MPO activity. However, the difference was not
statistically significant from the MDA content, TNF-a and IL-
6 levels. The reasons may be that MPO activity is related linearly to
neutrophil infiltration (Eiserich et al., 1998) and the assessment of
MPO activity is well established for quantifying intestinal
inflammation (Krawisz et al., 1984), while the other inflammatory
biomarkers, such as MDA content, TNF-a and IL-6 levels, do not
share such close relationship with the severity of inflammation
just like MPO activity. Furthermore, the above phenomenon
29
perhaps also may be ralated to the difference of sample points
between histological analysis samples and the samples of
inflammation biomarkers determination. Further research should
be carried out in light of this information.
5. Conclusion
In this study, CUR-PIP-SMEDDS formulation was prepared with
small particle size and high drug encapsulation efficiency which
could improve the stability and water-solubility of CUR. We
demonstrated that CUR-PIP-SMEDDS really could target the
injured epithelium of colon on DSS-induced colitis through
retention enema administration, as shown by the reduction in
DAI and histopathological lesion, and downregulating inflamma-
tory mediators such as MPO activity, MDA content, as well as TNF-
a and IL-6 levels. These findings suggest that CUR-PIP-SMEDDS
may be a useful therapeutic approach to the treatment of UC, with
high specificity and successful colon targeted.
Our experiment results have shown that treatment with CUR-
PIP-SMEDDS (retention enema administration) has an equivalent
effect to 5-ASA in maintaining remission of UC, while the
therapeutic effects of CUR-PIP-SMEDDS (ig) were relatively
weaker, and CUR-PIP suspensions exerted the weakest efficacy.
The results were due to that CUR-PIP-SMEDDS could significantly
increase the solubility and stability of CUR, compared to CUR-PIP
suspensions. In comparison to CUR-PIP-SMEDDS (ig), CUR-PIP-
SMEDDS (retention enema administration) could directly act on
the inflamed epithelium of the mice colon and released the drug
30 Q. Li et al. / International Journal of Pharmaceutics 490 (2015) 22–31
immediately to increase the local concentration of CUR in colonic
lesion site, which could provide sustained exposure of therapeutic
agents to sites of pathology, reduce side effects and finally improve
therapeutic effects. Taken together, we provide evidence that
SMEDDS can co-deliver CUR and PIP that in turn enhances the anti-
colitis activity of CUR through increasing the solubility and
stability of CUR, and improving the local concentration of drugs
in colonic lesion site through retention enema administration.
Therefore, it is plausible to develop CUR-PIP- SMEDDS for oral
colon-specific drug delivery system and it may be a potential
carrier for colon delivery of CUR.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 30801549), Innovation Team Develop-
ment Program of Beijing University of Chinese Medicine (No. 2011-
CXTD-13) and the Independent Project of Beijing University of
Chinese Medicine (No. 2014-JYBZZ-XS-087).
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