Curcumin-piperine mixtures in self-microemulsifying drug delivery system for ulcerative colitis therapy.

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Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Curcumin–piperine mixtures in self-microemulsifying drug delivery system for ulcerative colitis therapy

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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: Received 5 December 2014 Received in revised form 20 April 2015 Accepted 4 May 2015 Available online xxx

Curcumin (CUR) is a poorly water-soluble drug and its absorption is very low. In this study, CUR and piperine (PIP) were co-encapsulated into the nanoformulation called self-microemulsifying drug delivery system (SMEDDS) to improve the stability and water-solubility of CUR and enhance its anticolitis activity. The formulation of CUR–PIP-SMEDDS was prepared to encapsulate two hydrophobic 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 and transparent, and the microemulsion droplets appeared spherical without aggregation. The mean size 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)%, respectively. The vitro stability investigation of CUR–PIP-SMEDDS in colon tissue suggested that using SMEDDS as a delivery vehicle and co-encapsulated with PIP, CUR was more stable than drug solution in colons site. Meanwhile, the anti-inflammatory activity of CUR–PIP-SMEDDS was evaluated on DSSinduced colitis model. The results showed that CUR–PIP-SMEDDS exhibited definite anti-colitis activity by directing CUR–PIP-SMEDDS to inflammatory colon tissue through retention enema administration. Our study illustrated that the developed CUR–PIP-SMEDDS formulation was a potential carrier for developing colon-specific drug delivery system of CUR for ulcerative colitis treatment. ã 2015 Published by Elsevier B.V.

Chemical compounds studied in this article: Curcumin (PubChem CID: 969516) Piperine (PubChem CID638024)

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Keywords: Curcumin Piperine SMEDDS Preparation and characterization Stability Anti-colitis activity

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1. Introduction

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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

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* 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: [email protected] (J. Dai). 1 These authors contributed equally to this work and should be considered cofirst authors.

conventional medical treatment of UC relies on the use of aminosalicylates (5-aminosalicylic acid-based agents), corticosteroids, immunosuppressive drugs (e.g., azathioprine, 6-mercaptopurine, 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 Published by Elsevier B.V.

Please cite this article in press as: Li, Q., et al., Curcumin–piperine mixtures in self-microemulsifying drug delivery system for ulcerative colitis therapy. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.008

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2003). Therefore, there is a pressing need for developing better anti-inflammatory agents with increased efficacy and improved Q3 safety profiles. Recently, considerable attention has been devoted to identifying 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), antidepressive (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.,

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 microemulsion 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 potential 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

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Fig. 1. The chemical structure of curcumin (A) and piperine (B).


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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.

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2. Materials and methods

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2.1. Chemical and reagents

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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.

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2.2. Animals

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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.

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2.3. Preparation of CUR–PIP-SMEDDS formulation

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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 formulation was prepared by mixing the oil with surfactant and cosurfactant 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 cosurfactant 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.

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2.4. Characterization of CUR–PIP-SMEDDS formulation

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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) (JEM1400 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 formvarcoated 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.

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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., Worcestershire, 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.

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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).

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EEð%Þ ¼

Ce 100% Ct

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(1)

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).

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2.5. Anti-colitis activity of CUR–PIP-SMEDDS

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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; Q4 dissolved in fresh tap water) ad libitum for 7 consecutive days. The normal group was given water only. In the first group, designated

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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

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Diarrhea

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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 carboxymethycellulose 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–PIPSMEDDS (retention enema administration) once daily for 5 consecutive 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.

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.

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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.

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2.6. Statistical analysis

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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.

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3. Results

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3.1. Characterization of CUR–PIP-SMEDDS formulation

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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 microemulsion was clear and transparent solution with visible orangeyellow. 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 mg g1, which was significantly higher than that in the water. It is reported that the solubility of CUR in the aqueous medium was about 11 ng g1 (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 microemulsion 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.

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2.5.4. Determination of myeloperoxidase (MPO) activity MPO activity, an index of leukocyte recruitment, was determined according to the method described by Manktelow and Meyer (1986). In brief, colon tissue homogenate was extracted with hexadecyltrimethyl ammonium bromide. Then dimethoxybenzidine (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

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.


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3.1.2. Droplet size, zeta potential and encapsulation efficiency CUR–PIP-SMEDDS microemulsion was monodisperse with a mean particle size of 15.87 0.76 nm with PDI of 0.092 0.008 (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). Meanwhile, the mean EECUR and EEPIP values were (94.34 2.18)% and (90.78 2.56)%, respectively. 3.1.3. Co-encapsulated of CUR and PIP into SMEDDS can increase the stability of CUR CUR is relatively unstable, and this is one of the major barriers for clinical use of CUR to treat inflammation-related diseases. To determine whether CUR–PIP-SMDDES is more stable, free CUR, CUR–PIP mixture and CUR–PIP-SMEDDS were incubated at 37 C and sampled periodically to determine the concentration of CUR by HPLC. After incubation for 24 h at 37 C, we found free CUR, CUR– PIP mixture and CUR–PIP-SMEDDS degraded by 19.93%, 12.67% and 10.50% respectively in the artificial colon fluid (Fig. 4A). The degradation of CUR followed apparent first-order kinetics equation and the half-life were 19.67 h, 57.87 h and 75.23 h respectively in the artificial colon fluid. After incubation for 8 h at 37 C, we found free CUR, CUR–PIP mixture and CUR–PIP-SMEDDS degraded by 19.96%, 14.49% and 4.63% respectively in the mice colon tissue homogenate (Fig. 4B). The results show that CUR–PIP-SMEDDS could protect CUR from degration and metabolism of colon enzyme system to significantly improve the stability of CUR (P < 0.01–0.05).

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3.2. Anti-colitis activity of CUR–PIP-SMEDDS

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3.2.1. Effects on clinical symptoms We assessed the therapeutic effects of CUR–PIP-SMEDDS by using the acute DSS-induced UC model. The DSS-induced murine UC model used in this study is a well-established general prototype of intestinal tissue damage that accurately resembles the histological aspects of the tissue damage observed in patients suffering from inflammatory bowel disease (Goyal et al., 2014). In this study, we found that BALB/c mice subjected to the oral 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, gross blood adhering to the anus was noted. The DAI, an indicator of the severity of intestinal inflammation, was used to analyze the therapeutic benefit of CUR–PIP-SMEDDS treatment. In this experimental acute ulcerative colitis, treatment with CUR–PIPSMEDDS, mice showed remarkable improvements in body weight loss, intestinal bleeding and diarrhea, resulting in significant amelioration of UC as assessed by DAI. Fig. 5 shows the time course

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Time (h) Fig. 4. Degradation of free CUR, CUR–PIP mixture and CUR–PIP-SMEDDS in the artificial colon fluid (A) and the mice colon tissue homogenate (B).*P < 0.05, ** P < 0.01 vs. the free CUR. Data are represented as the mean SD; n = 3.

of the mice body weights (A) and DAI scores (B) after treatment with DSS. DAI scores of mice were increased in a time-dependent fashion. As shown in Fig. 5B, compared with the normal control group, the DAI scores were markedly increased in saline-treated mice with DSS-induced colitis (P < 0.01–0.05). By contrast, treatment with CUR–PIP-SMEDDS significantly reduced the DAI scores in mice induced by DSS (P < 0.01–0.05). We also found that retention enema administration group of CUR–PIP-SMEDDS exhibited the similar improvements to positive medicine group of 5-ASA (P > 0.05).

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3.2.2. Effects on the length of colon As shown in Fig. 6, the colonic length of the DSS-induced mice was significantly shorter compared with those of the normal group. However, the length of the shortened colon was significantly increased after the treatment of agents in mice with DSSinduced colitis (P < 0.01–0.001). The enema administration groups of CUR–PIP-SMEDDS and 5-ASA displayed the strongest efficacies to increase the length of the shortened colon induced by DSS, and the therapeutic effects were similar (P > 0.05).

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3.2.3. Effects on histological examination We characterized the histologic features of UC in BALB/c mice subjected to 5% DSS. In normal mice, no signs or only a very low level of polymorphonuclear leukocyte infiltration in the colon was observed (Fig. 7A). The severity of UC-like lesions was most marked in the colon of DSS-induced mice. Compared with normal mice, the distal colon of UC mice exhibited marked mucosal inflammation in all layers of the bowel wall, including a marked

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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 (retention 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 administration groups of CUR-PIP-SMEDDS exhibited the similar inhibitory activities to the positive medicine group of 5-ASA (P > 0.05).

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3.2.6. Effects on TNF-a and IL-6 levels DSS-induced UC was accompanied by the release of proinflammatory cytokines including TNF-a and IL-6. The levels of TNF-a and IL-6 in the supernatant of colon tissue homogenate from each group were measured to determine whether CUR–PIPSMEDDS inhibits the production of pro-inflammatory cytokines, therefore protecting the colon mucosa. As shown in Fig. 8C and D, the increase in the amount of TNF-a and IL-6 expression was significantly reduced after the treatment of agents in mice with DSS-induced colitis (P < 0.05–0.001). We could find that the administration groups of CUR–PIP-SMEDDS and 5-ASA displayed strong efficacies to inhibit the production of TNF-a and IL6 induced by DSS, and the therapeutic effects were similar (P > 0.05).

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Local treatment of UC has been the objective of countless 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 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 accumulate in the target inflamed tissues, and reside at the site of attachment for a prolonged period of time, owing to their large interfacial surface area and minute dimension (Constantinides, 1995). It is well documented that intestinal permeability is increased during inflammation, allowing permeation of the particles to the desired colon lesion site in a preferable manner

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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 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, effects on reducing the colonic MDA level compared to animals that received DSS alone.

##

CUR-PIP suspensions 6

Fig. 6. Effect of CUR–PIP-SMEDDS administration on the colon length. **P < 0.01, *** P < 0.001 vs. the DSS model; ###P < 0.001 vs. normal group. Data are represented as the mean SD; n = 8.

4

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Days 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 model; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. normal group. Data are represented as the mean SD; n = 8.


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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. 461 462 Q5 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497

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 DSSinduced 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–PIPSMEDDS 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 treatment 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 IL6 (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


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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. 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562

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 DSSinduced colitis model. Based on these results, the reduced TNFa 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 IL6 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 IL6 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

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.

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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 inflammatory mediators such as MPO activity, MDA content, as well as TNFa 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–PIPSMEDDS (retention enema administration) could directly act on the inflamed epithelium of the mice colon and released the drug

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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 anticolitis 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.

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Acknowledgments

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This work was supported by the National Natural Science Foundation of China (No. 30801549), Innovation Team Development Program of Beijing University of Chinese Medicine (No. 2011CXTD-13) and the Independent Project of Beijing University of Chinese Medicine (No. 2014-JYBZZ-XS-087).

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Drug therapy of ulcerative colitis.

5-ASA-loaded SiO2 nanoparticles-a novel drug delivery system targeting therapy on ulcerative colitis in mice.

Biological therapy for ulcerative colitis.

[Therapy of ulcerative colitis].

Drug management of ulcerative colitis.

Drug management of ulcerative colitis.

[Medical therapy of ulcerative colitis].

Microparticles as controlled drug delivery carrier for the treatment of ulcerative colitis: A brief review.

Patient preferences for surgical versus medical therapy for ulcerative colitis.

5-Aminosalicylic acid, a specific drug for ulcerative colitis.

Where do we stand on drug treatment for ulcerative colitis?

Microsponges based novel drug delivery system for augmented arthritis therapy.

Sustained drug-delivery system: a promising therapy for denture stomatitis?

Budesonide multi-matrix system formulation for treating ulcerative colitis.

Naltrexone therapy for Crohn's disease and ulcerative colitis.

Abdominal pain and the neurotrophic system in ulcerative colitis.

The Role of Cyclosporine Therapy in Ulcerative Colitis Treatment.

Dose-intensified granulocyte-monocyte apheresis in therapy refractory ulcerative colitis.

Oral 4-aminosalicylic acid therapy in ulcerative colitis.

Mesalamine in ulcerative colitis.

Mortality in ulcerative colitis.

Polyposis in ulcerative colitis.

EEG in ulcerative colitis.

Sulphasalazine in ulcerative colitis.