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Co-delivery of curcumin-loaded nanoemulsion and Phaleria macrocarpa extract to NIH 3T3 cell for antifibrosis
Journal of Drug Delivery Science and Technology 39 (2017) 123e130
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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Co-delivery of curcumin-loaded nanoemulsion and Phaleria macrocarpa extract to NIH 3T3 cell for antifibrosis Heni Rachmawati a, b, *, Miranti A. Novel a, Risya M. Nisa a, Guntur Berlian c, Olivia M. Tandrasasmita c, Annisa Rahma a, Catur Riani a, Raymond R. Tjandrawinata c a b c
School of Pharmacy, Bandung Institute of Technology, Ganesha 10, Bandung 40132, Indonesia Research Center for Nanosciences and Nanotechnology, Bandung Institute of Technology, Ganesha 10, Bandung 40132, Indonesia Dexa Laboratories of Biomolecular Sciences, Cikarang - Jababeka, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2016 Received in revised form 14 February 2017 Accepted 19 March 2017 Available online 20 March 2017
Antifibrotic effect of curcumin-loaded nanoemulsion and its combination with Phaleria macrocarpa extract (PM) was determined through evaluation on collagen expression in fibroblast NIH/3T3 cells. Prior to activity study, cytotoxic effect of blank nanoemulsion in vitro was determined on different cell types using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The genes expression of collagens were determined with quantitative real time PCR. The amount of collagen protein was determined using Sirius Red/Fast Green Collagen Staining Kit. Stability of curcumin-loaded nanoemulsion towards hepatic metabolism was evaluated as well. Cells viability was preserved after treatment with curcumin- and curcumin-PM nanoemulsion. Both preparations decreased procollagen a1(1) and collagen IV genes and collagen protein. Nanoemulsion demonstrated protective effect against hepatic metabolism of curcumin. Curcumin nanoemulsion and curcumin-PM nanoemulsion showed a promising antifibrotic agent. © 2017 Elsevier B.V. All rights reserved.
Keywords: Liver fibrosis Collagen Procollagen a1(1) Curcumin Nanoemulsion Phaleria macrocarpa
1. Introduction Liver fibrosis is a reversible wound healing response characterized by the accumulation of extracellular matrix (ECM) following liver injury. When fibrosis is not controlled, it can further progress into cirrhosis. Cirrhosis is the end-stage consequence of fibrosis of the hepatic parenchyma, resulting in nodule formation that may alter hepatic function and blood flow [7]. Liver fibrosis is strongly associated with oxidative stress, increased the level of transforming growth factor beta, hepatocyte death, and chronic inflammation [10]. Collagen forms a small component of the total protein of normal liver. As the liver progresses into fibrosis, the total content of collagens and non-collagenous protein increases, accompanied by a shift in the type of ECM from the normal low density basement membrane-like matrix to high density interstitial type matrix. Type I collagen is present in the highest level in almost all organs and it is present in scar tissue, while type IV collagen is the main component
* Corresponding author. School of Pharmacy, Bandung Institute of Technology, Ganesha 10, Bandung 40132, Indonesia. E-mail addresses: [email protected], [email protected] (H. Rachmawati). http://dx.doi.org/10.1016/j.jddst.2017.03.015 1773-2247/© 2017 Elsevier B.V. All rights reserved.
of basement membrane [10,8]. Oxidative stress is thought to play an important role in stellate cell activation and the stimulation of extracellular matrix production. Meanwhile, liver injury is typically associated with inflammation process that drives the fibrogenic cascade [28]. Thus, a wide variety of antioxidants and antiinflammatory agents have received attention as potential antifibrotic compounds. Curcumin, 1,7-bis (4-hydroxy-3-methoxyphenol)-1,6heptadiene-3, 5-dione, is the primary active substance isolated from Curcuma domestica rhizome [38]. Curcumin has been shown to exhibit antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial activities [32]. It is also efficient in the treatment of liver disease such as liver fibrosis [1,36,6,15,23]. Curcumin attenuated Advanced Glycation End products (AGEs) mediated induction of Receptor for AGE (RAGE) gene expression, which is crucial in the activation of Hepatic Stellate Cell (HSC) [40]. Curcumin also inhibits TGF-b signaling pathway to activate HSC [3]. The pharmacological safety and efficacy of curcumin make it a potential compound for treating a wide variety of human diseases [15,18,13,30,23]. Unfortunately, several studies have shown that limited activity of curcumin in oral distribution due to its poor bioavailability, limited tissue distribution, rapid metabolism, and short half-life. Lipophilic
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drugs such as curcumin present a major challenge in oral distribution due to the low solubility and bioavailability [4,21,11,3,31]. Phaleria macrocarpa (PM), a tropical plant of Indonesian origin and is known to contain various bioactive compounds. The major component of PM is flavonoids but it also contains alkaloids, tannins, and saponins [5]. This plant has recently been used as a traditional herbal drug. Several parts of PM have been commonly used for medicinal treatments, including stems, leaves, and fruits [2]. This plant has been reported to have a hepatoprotective effect in rats induced by carbon tetrachloride (CCl4). This effect relates to its antioxidant activity, particularly by increasing superoxide dismutase levels in the liver [35]. Fruit extract of PM is used traditionally to treat a wide variety of diseases such as diabetes, rheumatoid arthritis, inflammation, and cancer [19]. It has demonstrated the ability to decrease blood glucose level in diabetic animal as a-glucosidase inhibitor. Other studies reported PM to be cytotoxic against the leukemia cell line L1210, myeloma cell, and human cancer cell EC9706 [16,14,37,39]. Considering the potential use of curcumin and Phaleria macrocarpa extract on preventing the progression of liver disease and the clinical issue due to low bioavailability, we describe the development of nanotechnology-based carrier system to improve the value of particularly curcumin. A spontaneous nanoemulsion aimed for oral administration was established to load reasonable amount of curcumin for a therapeutic purpose. Standard physical characterizations for nanocarrier systems were carried out to obtain an ideal property of the nanoemulsion. Combination of curcumin-loaded nanoemulsion and Phaleria macrocarpa extraxt was tested in vitro using fibroblast NIH 3T3 cell, a model of collagen producing cell. The gene expression of procollagen a1 and collagen IV, and the protein level of collagen were determined. 2. Materials and methods 2.1. Materials Curcumin was purchased from PT. Phytochemindo Lestari, Indonesia. Phaleria macrocarpa (PM) aqueous extract was kindly provided by Dexa Laboratory of Biomolecular Sciences, Indonesia. Glyceryl monooleate (GMO) 40 was purchased from PT. Tritunggal Arthamakmur, Indonesia. Tween 20 and 2-propanol-bioreagent were purchased from Sigma Aldrich. DMSO and ethanol were purchased from Merck, Germany. Polyethylene glycol (PEG) 400 was purchased from PT. Brataco, Indonesia. Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimum Essential Medium (EMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin-streptomycin, bovine serum, fetal bovine serum, and trypsin-EDTA were purchased from Gibco, USA. GoScript Reverse Transcription System, and 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent were purchased from Promega, Madison, USA. EvaGreen Super Mix was purchased from Bio-Rad, USA. DNAse/RNAse Free Water and Chlorofom were purchased from MP Biomedicals. TRIzol® reagent was purchased from Ambion. Sirius Red/Fast Green Staining Kit was purchased from Chondrex, UK. Gel Red Staining was purchased from Biotium, USA. Double distilled water was purchased form IPHA Laboratories, Indonesia.
cell line) were purchased from ATCC, Rockville, MD, USA. 2.3. Animals Male healthy Wistar rats with the weight of 250 g obtained from animal breeding laboratory, School of Pharmacy, Bandung Institute of Technology, Indonesia, were used for evaluation of curcumin nanoemulsion hepatic enzymatic metabolism ex vivo. The animal was treated under standard handling both for diet and environmental conditions. The animal experimentation was approved by the Local Committee for Care and Use of Laboratory Animals (01/ KEPHP-ITB/06e2015) and was performed according to the guidelines on animal experimentation. 2.4. Methods 2.4.1. Preparation of nanoemulsion Curcumin nanoemulsion was prepared by dissolving curcumin in GMO 40, followed by mixing with magnetic stirrer for 15 min at 100 rpm. Tween 20 was then added and the mixture was stirred for 15 min. PEG 400 was added, followed by constant stirring for 2 h. The resulting oil phase was sonicated using an ultrasonicator bath for 1 h. Distilled water was added to the oil phase with oil-to-water ratio of 1:5. Mild stirring was then conducted for 15 min until a transparent and homogenous nanoemulsion was formed. Curcumin-PM nanoemulsion was prepared using the same procedure except the extract of PM was dissolved in water prior to mixing with the oil phase. All procedures to prepare nanoemulsion were performed at room temperature. 2.4.2. Characterization of nanoemulsion Globule size and polydispersity index of the nanoemulsion were measured by Delsa Nano™ particle size analyzer (DelsaTM Nano, Beckman Coulter, Brea, CA). The morphology of nanoemulsion was observed using a transmission electron microscope (TEM; JEM 1400, JEOL, Tokyo, Japan). About 10 mL of sample was dropped in the specimen place and covered with a 400 mesh grid. After 1 min, 10 mL of uranyl acetate was dropped on top of the grid, and this sample was allowed to dry for 30 min before observation under the electron microscope. This procedure was used to confirm the particle size in the nanoemulsion as measured using the particle size analyzer. 2.4.3. Liver homogenate preparation Homogenate of the liver was prepared according to Olson et al. [22] with small modification. Male healthy Wistar rats were sacrificed under ether anesthesia, and the livers were isolated. The livers were rinsed with physiological sodium chloride solution, finely chopped, weighed then added with 0.42 M of icy-cold phosphate buffer solution (PBS) pH 7.4, followed by gentle mixing. Five milliliters of buffer was sufficient for 1 g of liver. Then, the mixture was put into Dounce tube and homogenized with a homogenizer at 200 rpm for 2 min. Subsequently, the mixture was centrifuged at 9000 rpm at temperature of 4 C for 10 min. The supernatant was collected and further centrifugation was applied. The supernatant from the second centrifugation so called liver S9 fraction, was transferred into a microcentrifuge tube and stored in a 20 C freezer for further analysis.
2.2. Cell lines NIH/3T3 (mouse fibroblast cell line), 3T3-SA (Swiss albino mouse fibroblast cell line), RSC-96 (rat neuron Schwann cell line), RAW 264.7 (mouse macrophage cell line), RBL-2H3 (rat basophilic leukemia cell line), CHO-K1 (Chinese hamster ovary cell line), HepG2 (human hepatoma cell line) and NCI-H292 (human tumor
2.4.4. Determination of curcumin stability in liver S9 fraction Curcumin nanoemulsion and pure curcumin in DMSO were treated with liver S9 fraction in a ratio of 1:1. Each preparation was incubated for 30 min, 1 h, and 2 h at 37 C. Immediately after incubation, the mixture was mixed by using vortex mixer for 5 min and 200 mL of the sample was added with 80 mL of double-distilled
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water and remixed by using vortex mixer for 30 s. Curcumin was extracted by adding 3.5 mL of ethyl acetate and 20 mL of methanol, and the extraction process was performed using a roller mixer for 30 min. The mixture was centrifuged at 5000 rpm for 5 min and the organic phase was collected and steam-dried at 30 C. The residue was reconstituted with 100 mL of acetonitrile:water 1:1 and mixed by using vortex mixer for 1 min. Curcumin content was further analyzed by High Performance Liquid Chromatography (HPLC). Samples were injected into a C18 Column (150 3.9 mm), and curcumin was detected using a UV detector at 430 nm. Gradient elution system was used with a constant flow rate at 1 mL/min. The mobile phase was a mixture of acetonitrile and 0.01 M of ammonium acetate buffer (pH 4.5). In the first 30 min, the composition of acetonitrile was raised gradually from 5 to 45%. In the next 20 min the amount of acetonitrile was increased, reaching a final ratio of acetonitrile and ammonium acetate buffer at 95:5. 2.4.5. Cell culture Mouse NIH/3T3 fibroblast cell was cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 C. The atmosphere was maintained under 5% of CO2. After reaching their confluence, the cells were subcultured with trypsin-EDTA. 2.4.6. Cytotoxic assay/MTS assay Cytotoxic effect of curcumin, PM extract, curcumin nanoemulsion, and curcumin-PM nanoemulsion on NIH/3T3 cells were determined using MTS assay according to the manufacturer's protocol (Promega, Madison, USA). Briefly, cells were trypsinized and plated into 96-well plate at density of 104 cells/well. Cells were cultured overnight at 37 C with 5% CO2. Cells were exposed to different concentration of curcumin (2, 4, 6, 8, 16, and 32 mg/mL), PM extract (21, 42, 84, 126, and 168 mg/mL), curcumin nanoemulsion (1, 2, 3, 4, 5, and 6 mg/mL), curcumin-PM extract (1e20 mg/ mL), and curcumin-PM nanoemulsion (1e20 mg/mL). Cells were incubated overnight and then added with 20 mL of MTS to each well. The cells were reincubated for 2 h at 37 C with 5% CO2. The absorbance was measured at 490 nm using ELISA microplate reader. The result was represented as percentage of cell viability, compared to untreated controls. The IC50 valued was calculated using BioStat Software. Further, a similar protocol for in vitro cytotoxic study on blank nanoemulsion was determined by MTS assay using several cell lines including NIH/3T3 (mouse fibroblast cell line), 3T3-SA (Swiss albino mouse fibroblast cell line), RSC-96 (rat neuron Schwann cell line), RAW 264.7 (mouse macrophage cell line), RBL-2H3 (rat basophilic leukemia cell line), CHO-K1 (Chinese hamster ovary cell line), HepG2 cell (human hepatoma cell line), and NCI-H292 (human tumor cell line). 2.4.7. Antifibrotic assay The antifibrotic assay of the preparation was performed by determining the reduction effect on collagen expression both at gene and protein levels [26,25]. NIH/3T3 cells were placed into 6-well plate at density of 105 cell/ mL. Cells were cultured overnight at 37 C with 5% CO2. Different concentrations of curcumin (2, 4, 6, and 8 mg/mL), PM extract (21, 42, and 168 mg/mL), curcumin nanoemulsion (0.5, 1, 1.5, and 2 mg/ mL), curcumin-PM extract (1e20 mg/mL), and curcumin-PM nanoemulsion (1e20 mg/mL) subsequently were added into cultured cells. Treated cells were incubated for 24 hs at 37 C with 5% CO2. 2.4.8. RNA extraction and gene expression analysis Total RNA was extracted with TRIzol® reagent according to a standard protocol. cDNA was prepared with GoScript kit and RTPCR method according to the manufacturer's method. Gene
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expression of procollagen a1 and collagen IV was analyzed with quantitative real time PCR. The PCR mixtures (20 mL) contained 2 mL of cDNA, primers (1 mM concentrations, Table 1), and EvaGreen master mix. The replication process involved initial denaturing step at 95 C for 30 s, followed by 35 cycle of denaturation step at 95 C for 5 s, 56 C for 5 s, and 65 C for 5 s. Each measurement was performed in three replicates. Data was analyzed using Bio-Rad CFX manager software and calculation of gene expression levels were normalized to the signal of the house keeping gene BAM. 2.4.9. Effects of nanoemulsion on collagen synthesis The amount of collagen protein was measured with Sirius Red/ Fast Green Staining Kit. NIH/3T3 mouse fibroblast cell were plated into 24-well plate at density of 3.104 cell/well. Cells were cultured overnight at 37 C with 5% CO2. Different concentrations of curcumin, PM extract, curcumin nanoemulsion, curcumin-PM extract, and curcumin-PM nanoemulsion were added to the cells. Then, the cells were incubated for 24 h at 37 C with 5% CO2. Further, culture medium was removed and the cells were washed with PBS. Kahle fixative reagent was added into cells and the cells were incubated for 10 min at room temperature. Fixative reagent was removed, followed by washing 0.2 mL of PBS. Dye solution was added to the cells and subsequently the cells were incubated at room temperature for 30 min. Dye solution was removed carefully and the cells were rinsed with 0.5 mL of distilled water repeatedly until the color faded. Stained cells were observed under a light microscope. Further, the cells were added with 1 mL of Dye Extraction Buffer and mixed gently until the color was eluted. The absorbance was measured with spectrophotometer at 540 nm and 605 nm. The amount of collagen was calculated using the following formula:
A eðA605 0:291Þ collagen mg=well ¼ 540 0:0378 non collagen protein
mg=well ¼
A605 0:00204
(1)
(2)
2.4.10. Statistical analysis Data were presented as mean ± SD. All data were subjected to an unpaired, two-tailed distribution student t-test. Differences were considered significant at p < 0.05. 3. Results 3.1. Characterization of nanoemulsion Table 2 shows physical characteristics of curcumin nanoemulsion. As presented, the mean globule diameter was around 30 nm. Polydispersity index was relatively low, being less than 0.5. Analysis of globule morphology by TEM confirmed the encapsulation of curcumin in the oil phase which is expected to prevent degradation of curcumin in GI tract (Fig. 1).
Table 1 PCR primers sequence. Gene
Nucleotide sequences
Procollagen a1
Upstream: 50 -AGCCTGAGCCAGCAGATTGA-30 Downstream: 50 -CCAGGTTGCAGCCTTGGTTA-30 Upstream: 50 -AACAACGTCTGCAACTTCGC-30 Downstream: 50 -CTTCACAAACCGCACACCTG-30 Upstream: 50 -ACCCACACTGTGCCCATCTA-30 Downstream: 50 -CGGAACCGCTCATTGCC-30
Collagen IV BAM
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H. Rachmawati et al. / Journal of Drug Delivery Science and Technology 39 (2017) 123e130 Table 2 Physical characteristics (mean ± SD, n ¼ 3).
3.3. In vitro cytotoxicity of
curcumin-loaded
nanoemulsion
Characteristics
Result
Globule diameter (nm) Polydispersity Index
27.3 ± 6.03 0.393 ± 0.13
3.2. Physical stability in the presence of liver S9 fraction Curcumin is a subject to metabolism by the liver, resulting in reduction inactive products after oral administration: curcumin glucuronide and curcumin sulfate conjugates [29]. This evidence was indicated by the decrease in curcumin content after exposure to liver S9 fraction. It was found that curcumin in nanoemulsion system exhibited lower metabolism rate as compared to pure curcumin (Fig. 2). The decrease in curcumin concentration after 2 h s incubation was 37.71% for pure curcumin, and 15.69% for curcuminloaded nanoemulsion.
The effects of pure curcumin, PM extract, curcumin nanoemulsion, and curcumin-PM nanoemulsion on cell viability are shown in Fig. 3. Pure curcumin decreased the viability of NIH/3T3 cells (Fig. 3a). The lowest cell viability was observed in preparation with the highest curcumin concentration. In fact, curcumin exhibited cytotoxic effect at a concentration of 16 mg/mL and above. However, cell viability was well preserved at concentration range of 2e8 mg/mL. The calculated IC50 of curcumin on NIH/3T3 cells was 17.8 mg/mL. Meanwhile, PM extract was able to maintain cell viability at concentration up to 168 mg/mL, despite slight decrease in cell viability (Fig. 3b). The predicted IC50 of PM extract on NIH/ 3T3 cells was 383.3 mg/mL. Formulation of curcumin into nanoemulsion resulted in greater cytotoxicity, limiting its safe concentration at 2 mg/mL (Fig. 3c). However, combination of curcumin and PM extract with respective concentration of 4 mg/mL and 20 mg/mL did not show any cytotoxic effect in NIH/3T3 cell (Fig. 3d). The blank nanoemulsion did not demonstrate any cytotoxic effect on NIH/ 3T3, 3T3-SA, RSC-96, RAW 264.7, RBL-2H3, CHO-K1, HepG2, and NCI-H292 cell lines (Fig. 3e).
Fig. 1. Morphological analysis of nanoemulsion with Transmission Electron Microscopy (TEM). Image was captured with 20000 magnification (a) and 10000 magnification (b).
Fig. 2. The stability profile of curcumin in the presence of liver S9 fraction.
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Fig. 3. In vitro study on cells viability of different treatments. Curcumin (a), PM extract (b), curcumin nanoemulsion (c), curcumin-PM extract and curcumin-PM nanoemulsion (d), unloaded nanoemulsion (e).
3.4. Procollagen a1(1) and collagen IV genes expression The effect of pure curcumin, PM extract, curcumin nanoemulsion, and curcumin-PM nanoemulsion on gene expression of procollagen a1(1) and collagen IV are illustrated in Fig. 4. Overall, tested preparations demonstrated the ability to suppress the expression of both genes significantly (p < 0.05). Both curcumin and PM extract showed inhibitory effect in dose-dependent manner (Fig. 4a and b). Gene suppression exhibited by curcumin in nanoemulsion form was significantly greater. The ratio of collagen gene to b-actin was around 0.4 in pure curcumin at concentration of 4 mg/mL. After the treatment with curcumin nanoemulsion, the gene:b-actin ratio was twice lower (approximately 0.2) at curcumin concentration of 2 mg/mL (Fig. 4c). Combination of curcumin (4 mg/mL) and PM extract (20 mg/mL) showed even greater suppression of procollagen a1 and collagen IV at gene level as compared to individual component at similar concentration (Fig. 4d). 3.5. Histological examination and quantification of collagen protein The result of histological examination of NIH/3T3 cells is shown in Fig. 5. The red color on NIH/3T3 cell indicates the presence of collagen protein. Fig. 6 shows the quantification of the protein after treatment. It is clear that the amount of collagen detected on the cells after treatment with curcumin-PM extract and nanoemulsion of curcumin-PM extract was significantly lower (p < 0.05).
However, there was no significant difference between curcuminPM extract and curcumin-PM nanoemulsion in terms of reduction of collagen production. Similarly, collagen reduction resulted from curcumin-PM extract combination did not differ significantly compared to single treatment of each compound. Therefore, it is suggested that no synergistic effect is demonstrated by a combination of curcumin and PM on the modulation of collagen synthesis. 4. Discussion Well documented problem of curcumin leading to inefficient therapeutic outcome is its low bioavailability, not only due to low solubility but also sensitive to in vitro and in vivo conditions [24,21,11,3,31]. The effect of curcumin to treat liver fibrosis is also reported by many investigators [1,36,6,15,23]. Liver fibrosis (scarring) occurs in advanced liver disease, where normal hepatic tissue is replaced with collagen-rich extracellular matrix and, if left untreated, results in cirrhosis. Curcumin inhibits hepatic cirrhosis in an animal model and exhibits multiple biological actions in hepatic stellate cells (HSCs), which play an important role in the pathogenesis of liver fibrosis. A nanoemulsion with a mean globule diameter around 30 nm has been successfully developed. The curcumin loaded in the nanoemulsion is expected to have an improved bioavailability since nano-sized droplets have higher surface area available for absorption [20,33,12]. Moreover, enhanced bioavailability of curcumin is
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Fig. 4. Effect of curcumin (a), PM extract (b), curcumin nanoemulsion (c), and curcumin-PM nanoemulsion (d) on procollagen a1 and collagen IV gene expression in NIH/3T3 mouse fibroblast cells after 24 h incubation. *p < 0.05.
Fig. 5. The collagen expression by Sirrius/fast green staining on mouse fibroblast NIH 3T3 cells before treatment (a) and after treatment with curcumin-PM nanoemulsion (b).
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Fig. 6. Inhibitory effect of curcumin on collagen protein expression in mouse Fibroblast NIH 3T3 cells. A: untreated cell, B: PM extract (20 mg/mL), C: curcumin (4 mg/mL), D: curcumin (2 mg/mL) - PM (10 mg/mL), E: curcumin (4 mg/mL) -PM (20 mg/mL), F: curcumin nanoemulsion (1 mg/mL), G: curcumin nanoemulsion (2 mg/mL), H: curcumin (1 mg/mL) PM (5 mg/mL) nanoemulsion, I: curcumin (2 mg/mL) - PM (10 mg/mL) nanoemulsion. *p < 0.05.
also obtained as the stability against liver metabolism was increased in the nanoemulsion form, as demonstrated in this report. Curcumin is a subject to metabolism by the liver, resulting in reduction products depending on the route of administration. After oral administration of curcumin, the drug is extensively metabolized by the liver to curcumin glucoronide and curcumin sulfate which has no bioactivity [29]. This event was indicated by the decrease in curcumin content after exposure to liver S9 fraction, already detected 0.5 h after incubation and continuing to decrease up to for 2 h observation. Lower metabolism rate of curcumin in nanoemulsion indicates a greater stability due to physical protection provided by the nanoemulsion system which controlled and sustained the release of curcumin from the oil globule to liver homogenate. Formulation into nanoemulsion requires careful consideration regarding toxicity. This is due to the high amount of surfactant applied in the formulation [12]. In this in vitro study, no cytotoxic effect was generated from the excipients used to form nanoemulsion. Secondly, it was found that formulation of curcumin and PM extract into nanoemulsion resulted in lower cell viability, in comparison with that of pure curcumin and pure PM. This indicates the beneficial formulation in lowering dose of drugs while retaining the efficacy. However, this also means that dose adjustment is required in formulation of curcumin-PM extract nanoemulsion to avoid any toxicity. Findings in this study have proven that curcumin- and curcumin-PM extract nanoemulsions significantly suppressed the expression of procollagen a1(1) and collagen IV at the gene level, and collagen at the protein level. This has clinical significance, considering that inhibition of collagen synthesis is one of the strategies of liver fibrosis therapy [17,34]. Particularly on the reduction of collagen expression at the protein level, tissue staining using Sirius red provides evidence upon histological visualization of this protein. The potential effect of both curcumin and PM extract are widely reported [9]. described the important evidences of Proliverenol, a bioactive compound in PM extract we used for this study, for liver disease. Proliverenol decreased the expression of transaminase enzymes, such as ALT. Proliverenol appears to suppress both the gene expression of ALT and the protein leakage from cytoplasm into the culture medium. The mechanism resulting the
hepatoprotective effect of proliverenol is via up-regulating APEX1 that represses DNA fragmentation and down-regulates the NF-kB, TNFa and caspase-8. The activity of collagen synthesis reduction demonstrated by curcumin nanoemulsion and curcumin-PM nanoemulsion in this report might be due to their antioxidant and anti-inflammatory activities. Oxidative stress is known to play a crucial role in hepatic fibrogenesis by stimulating HSC activation and increasing collagen synthesis [40]. In another study, curcumin has been reported to increase the activity of matrix metalloproteinase (MMP-1 and MMP-2), which are responsible for degradation of collagen, particularly type I and IV [27]. The ability of curcumin and PM extract in inhibiting collagen synthesis at the gene and protein levels suggest that these compounds are quite promising for novel antifibrotic therapy.
5. Conclusion Nanoemulsion provides numerous advantages including improved stability and oral absorption. Thus, it is expected that the application of nanoemulsion in oral delivery system will continue to increase, particularly for drugs with poor solubility, poor absorption, or high susceptibility to metabolic instability. Curcumin nanoemulsion has demonstrated its ability in suppressing gene expression of procollagen a1 and collagen IV, as well as the collagen protein, the main fibrotic markers. This effect is strengthen when co-delivered with PM extract. The nanoemulsion system offered protection against metabolic degradation of curcumin due to the enzymes in the liver, bringing its promising use as an oral liver antifibrotic therapy. Our findings showed that only a smaller dose of curcumin and PM extract was required in the nanoemulsion system to achieve the desired biological activity in vitro. This indicates that incorporation of drugs into nanoemulsion potentially results in better efficacy. Nevertheless, we suggest in vivo study regarding toxicity, pharmacokinetic profiles, and bioavailability in normal subjects and those with liver fibrosis for future work.
Conflict of interest The authors declare no conflict of interest.
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Acknowledgement The authors would like to thank Dexa Laboratory Biomolecular Sciences (DLBS) for provision of research facilities. Great appreciation is also addressed to Bandung Institute of Technology for financing this project under ‘ITB Innovative Research 2015’ research grant. References [1] R. Afrin, S. Arumugam, A. Rahman, M.I. Wahed, V. Karuppagounder, M. Harima, H. Suzuki, S. Miyashita, K. Suzuki, H. Yoneyama, K. Ueno, K. Watanabe, Curcumin ameliorates liver damage and progression of NASH in NASH-HCC mouse model possibly by modulating HMGB1-NF-kB translocation, Int. Immunopharmacol. 44 (2007) 174e182. [2] R. Altaf, M. Asmawi, A. Dewa, A. Sadikun, M. Umar, Phytochemistry and medicinal properties of Phaleria macrocarpa (Scheff.) Boerl. extracts, Pharmacogn. Rev. 7 (2013) 73e80. [3] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807e818. [4] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807e818. [5] S. Andrean, A. Prasetyo, T. Kristijarti, Hudaya, The extraction and activity test of bioactive compounds in Phaleria macrocarpa as antioxidants, Procedia Chem. 9 (2014) 94e101. l, E. Mura r, M. Cebov nova , The effect of [6] P. Janega Barta, P. Baba a, O. Pecha curcumin on liver fibrosis in the rat model of microsurgical cholestasis, Food Funct. 6 (2015) 2187e2193. [7] R. Bataller, D. Brenner, Liver fibrosis, J. Clin. Invest. 115 (2005) 209e218. [8] D. Behrens, M. Villone, G. Koch, L. Brunner, H. Sorokin, Robenek, et al., The epidermal basement membrane is a composite of separate laminin- or collagen iv-containing networks connected by aggregated perlecan, but not by nidogens, J. Biol. Chem. 287 (2012) 18700e18709. [9] Berlian, O.M. Tandrasasmita, R.R. Tjandrawinata, Standardized bioactive fraction of Phaleria macrocarpa(Proliverenol) prevents ethanol-induced hepatotoxicity via down-regulation of NF-kB-TNFa-caspase-8 pathway, As.Pac. J. Trop. Biom 6 (2016) 686e691. [10] T. Boyer, T. Wright, M. Manns, D. Zakim, Zakim and Boyer's Hepatology, first ed., Saunders/Elsevier, Philadelphia, 2006. [11] J. Cuomo, G. Appendino, A.S. Dern, E. Schneider, T.P. McKinnon, M.J. Brown, et al., Comparative absorption of a standardized curcuminoid mixture and its lecithin formulation, J. Nat. Prod. 74 (2011) 664e669. [12] N. Date, R. Desai, M. Dixit, Nagarsenker, Self-nanoemulsifying drug delivery systems: formulation insights, applications and advances, Nanomedicine 5 (2010) 1595e1616. [13] B.B. Dhillon, R.A. Aggarwal, R.A. Newman, A.B. Wolff, J.L. Kunnumakkara, Abbruzzese, et al., Phase II trial of curcumin in patients with advanced pancreatic cancer, Clin. Cancer Res. 14 (2008) 4491e4499. [14] M. Easmin, M. Sarker, S. Ferdosh, S. Shamsudin, K. Yunus, M. Uddin, et al., Bioactive compounds and advanced processing technology:Phaleria macrocarpa(sheff.) Boerl, a review, J. Chem. Technol. Biotechnol. 90 (2014) 981e991. [15] S.C. Gupta, S. Patchva, W. Koh, B.B. Aggarwal, Discovery of curcumin, a component of golden spice, and its miraculous biological activities, Clin. Exp. Pharmacol. Physiol. 39 (2012) 283e299. [16] W. Hendig, K.W. Ermin, Benzophenone glucoside isolated from the ethyl acetate extract of the bark of the mahkota dewa (Phaleria macrocarpa (Scheff.) Boerl.) and its inhibitory activity leukeumin L1210 cell line, Indones. J. Chem. 9 (2009) 142e145. [17] M.H. Ismail, M. Pinzani, Reversal of liver fibrosis, Saudi J. Gastroenterol. 15 (2009) 72e79. [18] M. Kanai, K. Yoshimura, M. Asada, A. Imaizumi, C. Suzuki, S. Matsumoto, et al., A phase I/II study of gemcitabine-based chemotherapy plus curcumin for patients with gemcitabineresistant pancreatic cancer, Cancer Chemother. Pharmacol. 68 (2011) 157e164.
[19] S. Muhamad, P. Indriati, Nanoencapsulation of the flavonoids isolated from Phaleria macrocarpa leaf by casein micelle, Int. J. Pharm. Bio. Sci. 3 (2012) 471e478. [20] E. Nazarzadeh, T. Anthonypillai, S. Sajjadi, On the growth mechanisms of nanoemulsions, J. Colloid Interface Sci. 397 (2013) 154e162. [21] M.J. Nirmala, M. Amitava, N. Chandrasekaran, A nanoemulsion drug delivery system for an aqueous insoluble drug, Int J. Chem Tech Res. 6 (2012) 2020e2022. [22] O.E. Olson, E.E. Fager, et al., Folic acid activity in homogenates of rat liver, J. Biol. Chem. 174 (1948) 319e326. [23] A. Oppenheimer, Turmeric (curcumin) in biliary diseases, Lancet 229 (1937) 619e621. [24] G.S. Patchva, B.B. Aggarwal, Therapeutic roles of curcumin: lessons learned from clinical trials Subash c, AAPS J. 15 (2013). [25] H. Rachmawati, L. Beljaars, C. R-Smit, H.I. Bakker, D.K.F. Meijer, K. Poelstra, IL10 modified with mannose 6-phosphate yields a liver-selective cytokine, Drug Metab. Dispos. 35 (2007) 814e821. [26] H. Rachmawati, A. Mattos-Pinto, C. Reker-Smit, K. Poelstra, L. Beljaars L, Modification of Interleukin-10 with Mannose 6-Phosphate Groups Yields a Liver-specific Cytokine with Antifibrotic Effects in Bile-duct Ligated Rats in the Book “Pharmacology”, publisher Intech, Croatia, March 2012. ISBN 979-953307-482-4. [27] H.W. Ryu, S.P. Kim, K.S. Lee, J.W. Cho, Curcumin induced decreased expression of type I collagen in human skin fibroblast through down-regulation of Smad2/3 expressions, Korean J. Dermatol 50 (2012) 1e7. [28] D.M. Samuel, M. Marco, S. Stefania, R. Chiara, A. Laura, T. Luciano, et al., Cellular and molecular mechanism of hepatic fibrosis leading to liver cancer, Transl. Gastrointest. Cancer 1 (2012) 88e94. [29] K. Shaiju, M. Kakarala, T.R. Mack, A.C. James A, P.N. Daniel, D. Zora, D.E. Brenner, Pharmacokinetics of curcumin conjugate metabolites in healthy human subjects, Cancer Epidemiol. Biomarkers Prev. 17 (2008) 1411e1417. [30] R.A. Sharma, H.R. McLelland, K.A. Hill, C.R. Ireson, S.A. Euden, M.M. Manson, et al., Pharmacodynamic and pharmacokinetic study of oral curcuma extract in patients with colorectal cancer, Clin. Cancer Res. 7 (2001) 1894e1900. [31] G. Shoba, D. Joy, T. Joseph, M. Majeed, R. Rajendran, P. Srinivas, Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers, Planta Med. 64 (1998) 353e356. [32] Stefanska, Curcumin ameliorates hepatic fibrosis in type 2 diabetes mellitus insights into its mechanisms of action, Br. J. Pharmacol. 166 (2012) 2209e2211. [33] D.Q. Sun, X.B. Wei, X. Xue, Z.I. Fang, M.R. Ren, H.Y. Lou, X.M. Zhang, Enhanced oral absorption and therapeutic effect of acetylpuerarin based on D-a-tocopheryl polyethylene glycol 1000 succinate nanoemulsions, Int. J. Nanomedicine 9 (2014) 3413e3423. [34] S. Swarnakar, K. Ganguly, P. Kundu, A. Banerjee, P. Maity, A. Sharma, Curcumin regulates expression and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer, J. Biol. Chem. 280 (2004) 9409e9415. [35] H.J. Triastuti, J. Park, Choi, Phaleria macrocarpa suppresses oxidative stress in Alloxan-induced diabetic rats by enhancing hepatic antioxidant enzyme activity, Nat. Product. Sci. 15 (2009) 37e43. [36] P. Wu, R. Huang, Y.L. Xiong, C. Wu C, Protective effects of curcumin against liver fibrosis through modulating DNA methylation, Chin. J. Nat. Med. 14 (2016) 255e264. [37] Y. Zhang, P. Zhang, G. Dai, H. Liu, Study on the synthesis and bioactivity of novel mahkoside A derivatives, Synth. Commun. 37 (2007) 3319e3328. €mme, antifibrotic effects [38] C. Zhang, C. Huang, R. Yang, J. Liu, J. Wang, D. Niu, Bro of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts, Respir. Res. 12 (2011) 1e12. [39] S. Zhang, Z. Gong, W. Li, X. Wang, T. Ling, Antifibrotic effect of curcumin in TGF-b1-induced myofibroblasts from human oral mucosa. Asian Pac, J. Cancer P 13 (2012) 289e294. [40] Z. Zhang, L. Zhang, D. Chen, X. Kong, C. Lu Zhang, et al., Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells, J. Cell. Mol. Med. 18 (2014) 1392e1406.
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Co-delivery of curcumin-loaded nanoemulsion and Phaleria macrocarpa extract to NIH 3T3 cell for antifibrosis Heni Rachmawati a, b, *, Miranti A. Novel a, Risya M. Nisa a, Guntur Berlian c, Olivia M. Tandrasasmita c, Annisa Rahma a, Catur Riani a, Raymond R. Tjandrawinata c a b c
School of Pharmacy, Bandung Institute of Technology, Ganesha 10, Bandung 40132, Indonesia Research Center for Nanosciences and Nanotechnology, Bandung Institute of Technology, Ganesha 10, Bandung 40132, Indonesia Dexa Laboratories of Biomolecular Sciences, Cikarang - Jababeka, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2016 Received in revised form 14 February 2017 Accepted 19 March 2017 Available online 20 March 2017
Antifibrotic effect of curcumin-loaded nanoemulsion and its combination with Phaleria macrocarpa extract (PM) was determined through evaluation on collagen expression in fibroblast NIH/3T3 cells. Prior to activity study, cytotoxic effect of blank nanoemulsion in vitro was determined on different cell types using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The genes expression of collagens were determined with quantitative real time PCR. The amount of collagen protein was determined using Sirius Red/Fast Green Collagen Staining Kit. Stability of curcumin-loaded nanoemulsion towards hepatic metabolism was evaluated as well. Cells viability was preserved after treatment with curcumin- and curcumin-PM nanoemulsion. Both preparations decreased procollagen a1(1) and collagen IV genes and collagen protein. Nanoemulsion demonstrated protective effect against hepatic metabolism of curcumin. Curcumin nanoemulsion and curcumin-PM nanoemulsion showed a promising antifibrotic agent. © 2017 Elsevier B.V. All rights reserved.
Keywords: Liver fibrosis Collagen Procollagen a1(1) Curcumin Nanoemulsion Phaleria macrocarpa
1. Introduction Liver fibrosis is a reversible wound healing response characterized by the accumulation of extracellular matrix (ECM) following liver injury. When fibrosis is not controlled, it can further progress into cirrhosis. Cirrhosis is the end-stage consequence of fibrosis of the hepatic parenchyma, resulting in nodule formation that may alter hepatic function and blood flow [7]. Liver fibrosis is strongly associated with oxidative stress, increased the level of transforming growth factor beta, hepatocyte death, and chronic inflammation [10]. Collagen forms a small component of the total protein of normal liver. As the liver progresses into fibrosis, the total content of collagens and non-collagenous protein increases, accompanied by a shift in the type of ECM from the normal low density basement membrane-like matrix to high density interstitial type matrix. Type I collagen is present in the highest level in almost all organs and it is present in scar tissue, while type IV collagen is the main component
* Corresponding author. School of Pharmacy, Bandung Institute of Technology, Ganesha 10, Bandung 40132, Indonesia. E-mail addresses: [email protected], [email protected] (H. Rachmawati). http://dx.doi.org/10.1016/j.jddst.2017.03.015 1773-2247/© 2017 Elsevier B.V. All rights reserved.
of basement membrane [10,8]. Oxidative stress is thought to play an important role in stellate cell activation and the stimulation of extracellular matrix production. Meanwhile, liver injury is typically associated with inflammation process that drives the fibrogenic cascade [28]. Thus, a wide variety of antioxidants and antiinflammatory agents have received attention as potential antifibrotic compounds. Curcumin, 1,7-bis (4-hydroxy-3-methoxyphenol)-1,6heptadiene-3, 5-dione, is the primary active substance isolated from Curcuma domestica rhizome [38]. Curcumin has been shown to exhibit antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial activities [32]. It is also efficient in the treatment of liver disease such as liver fibrosis [1,36,6,15,23]. Curcumin attenuated Advanced Glycation End products (AGEs) mediated induction of Receptor for AGE (RAGE) gene expression, which is crucial in the activation of Hepatic Stellate Cell (HSC) [40]. Curcumin also inhibits TGF-b signaling pathway to activate HSC [3]. The pharmacological safety and efficacy of curcumin make it a potential compound for treating a wide variety of human diseases [15,18,13,30,23]. Unfortunately, several studies have shown that limited activity of curcumin in oral distribution due to its poor bioavailability, limited tissue distribution, rapid metabolism, and short half-life. Lipophilic
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drugs such as curcumin present a major challenge in oral distribution due to the low solubility and bioavailability [4,21,11,3,31]. Phaleria macrocarpa (PM), a tropical plant of Indonesian origin and is known to contain various bioactive compounds. The major component of PM is flavonoids but it also contains alkaloids, tannins, and saponins [5]. This plant has recently been used as a traditional herbal drug. Several parts of PM have been commonly used for medicinal treatments, including stems, leaves, and fruits [2]. This plant has been reported to have a hepatoprotective effect in rats induced by carbon tetrachloride (CCl4). This effect relates to its antioxidant activity, particularly by increasing superoxide dismutase levels in the liver [35]. Fruit extract of PM is used traditionally to treat a wide variety of diseases such as diabetes, rheumatoid arthritis, inflammation, and cancer [19]. It has demonstrated the ability to decrease blood glucose level in diabetic animal as a-glucosidase inhibitor. Other studies reported PM to be cytotoxic against the leukemia cell line L1210, myeloma cell, and human cancer cell EC9706 [16,14,37,39]. Considering the potential use of curcumin and Phaleria macrocarpa extract on preventing the progression of liver disease and the clinical issue due to low bioavailability, we describe the development of nanotechnology-based carrier system to improve the value of particularly curcumin. A spontaneous nanoemulsion aimed for oral administration was established to load reasonable amount of curcumin for a therapeutic purpose. Standard physical characterizations for nanocarrier systems were carried out to obtain an ideal property of the nanoemulsion. Combination of curcumin-loaded nanoemulsion and Phaleria macrocarpa extraxt was tested in vitro using fibroblast NIH 3T3 cell, a model of collagen producing cell. The gene expression of procollagen a1 and collagen IV, and the protein level of collagen were determined. 2. Materials and methods 2.1. Materials Curcumin was purchased from PT. Phytochemindo Lestari, Indonesia. Phaleria macrocarpa (PM) aqueous extract was kindly provided by Dexa Laboratory of Biomolecular Sciences, Indonesia. Glyceryl monooleate (GMO) 40 was purchased from PT. Tritunggal Arthamakmur, Indonesia. Tween 20 and 2-propanol-bioreagent were purchased from Sigma Aldrich. DMSO and ethanol were purchased from Merck, Germany. Polyethylene glycol (PEG) 400 was purchased from PT. Brataco, Indonesia. Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimum Essential Medium (EMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin-streptomycin, bovine serum, fetal bovine serum, and trypsin-EDTA were purchased from Gibco, USA. GoScript Reverse Transcription System, and 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent were purchased from Promega, Madison, USA. EvaGreen Super Mix was purchased from Bio-Rad, USA. DNAse/RNAse Free Water and Chlorofom were purchased from MP Biomedicals. TRIzol® reagent was purchased from Ambion. Sirius Red/Fast Green Staining Kit was purchased from Chondrex, UK. Gel Red Staining was purchased from Biotium, USA. Double distilled water was purchased form IPHA Laboratories, Indonesia.
cell line) were purchased from ATCC, Rockville, MD, USA. 2.3. Animals Male healthy Wistar rats with the weight of 250 g obtained from animal breeding laboratory, School of Pharmacy, Bandung Institute of Technology, Indonesia, were used for evaluation of curcumin nanoemulsion hepatic enzymatic metabolism ex vivo. The animal was treated under standard handling both for diet and environmental conditions. The animal experimentation was approved by the Local Committee for Care and Use of Laboratory Animals (01/ KEPHP-ITB/06e2015) and was performed according to the guidelines on animal experimentation. 2.4. Methods 2.4.1. Preparation of nanoemulsion Curcumin nanoemulsion was prepared by dissolving curcumin in GMO 40, followed by mixing with magnetic stirrer for 15 min at 100 rpm. Tween 20 was then added and the mixture was stirred for 15 min. PEG 400 was added, followed by constant stirring for 2 h. The resulting oil phase was sonicated using an ultrasonicator bath for 1 h. Distilled water was added to the oil phase with oil-to-water ratio of 1:5. Mild stirring was then conducted for 15 min until a transparent and homogenous nanoemulsion was formed. Curcumin-PM nanoemulsion was prepared using the same procedure except the extract of PM was dissolved in water prior to mixing with the oil phase. All procedures to prepare nanoemulsion were performed at room temperature. 2.4.2. Characterization of nanoemulsion Globule size and polydispersity index of the nanoemulsion were measured by Delsa Nano™ particle size analyzer (DelsaTM Nano, Beckman Coulter, Brea, CA). The morphology of nanoemulsion was observed using a transmission electron microscope (TEM; JEM 1400, JEOL, Tokyo, Japan). About 10 mL of sample was dropped in the specimen place and covered with a 400 mesh grid. After 1 min, 10 mL of uranyl acetate was dropped on top of the grid, and this sample was allowed to dry for 30 min before observation under the electron microscope. This procedure was used to confirm the particle size in the nanoemulsion as measured using the particle size analyzer. 2.4.3. Liver homogenate preparation Homogenate of the liver was prepared according to Olson et al. [22] with small modification. Male healthy Wistar rats were sacrificed under ether anesthesia, and the livers were isolated. The livers were rinsed with physiological sodium chloride solution, finely chopped, weighed then added with 0.42 M of icy-cold phosphate buffer solution (PBS) pH 7.4, followed by gentle mixing. Five milliliters of buffer was sufficient for 1 g of liver. Then, the mixture was put into Dounce tube and homogenized with a homogenizer at 200 rpm for 2 min. Subsequently, the mixture was centrifuged at 9000 rpm at temperature of 4 C for 10 min. The supernatant was collected and further centrifugation was applied. The supernatant from the second centrifugation so called liver S9 fraction, was transferred into a microcentrifuge tube and stored in a 20 C freezer for further analysis.
2.2. Cell lines NIH/3T3 (mouse fibroblast cell line), 3T3-SA (Swiss albino mouse fibroblast cell line), RSC-96 (rat neuron Schwann cell line), RAW 264.7 (mouse macrophage cell line), RBL-2H3 (rat basophilic leukemia cell line), CHO-K1 (Chinese hamster ovary cell line), HepG2 (human hepatoma cell line) and NCI-H292 (human tumor
2.4.4. Determination of curcumin stability in liver S9 fraction Curcumin nanoemulsion and pure curcumin in DMSO were treated with liver S9 fraction in a ratio of 1:1. Each preparation was incubated for 30 min, 1 h, and 2 h at 37 C. Immediately after incubation, the mixture was mixed by using vortex mixer for 5 min and 200 mL of the sample was added with 80 mL of double-distilled
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water and remixed by using vortex mixer for 30 s. Curcumin was extracted by adding 3.5 mL of ethyl acetate and 20 mL of methanol, and the extraction process was performed using a roller mixer for 30 min. The mixture was centrifuged at 5000 rpm for 5 min and the organic phase was collected and steam-dried at 30 C. The residue was reconstituted with 100 mL of acetonitrile:water 1:1 and mixed by using vortex mixer for 1 min. Curcumin content was further analyzed by High Performance Liquid Chromatography (HPLC). Samples were injected into a C18 Column (150 3.9 mm), and curcumin was detected using a UV detector at 430 nm. Gradient elution system was used with a constant flow rate at 1 mL/min. The mobile phase was a mixture of acetonitrile and 0.01 M of ammonium acetate buffer (pH 4.5). In the first 30 min, the composition of acetonitrile was raised gradually from 5 to 45%. In the next 20 min the amount of acetonitrile was increased, reaching a final ratio of acetonitrile and ammonium acetate buffer at 95:5. 2.4.5. Cell culture Mouse NIH/3T3 fibroblast cell was cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 C. The atmosphere was maintained under 5% of CO2. After reaching their confluence, the cells were subcultured with trypsin-EDTA. 2.4.6. Cytotoxic assay/MTS assay Cytotoxic effect of curcumin, PM extract, curcumin nanoemulsion, and curcumin-PM nanoemulsion on NIH/3T3 cells were determined using MTS assay according to the manufacturer's protocol (Promega, Madison, USA). Briefly, cells were trypsinized and plated into 96-well plate at density of 104 cells/well. Cells were cultured overnight at 37 C with 5% CO2. Cells were exposed to different concentration of curcumin (2, 4, 6, 8, 16, and 32 mg/mL), PM extract (21, 42, 84, 126, and 168 mg/mL), curcumin nanoemulsion (1, 2, 3, 4, 5, and 6 mg/mL), curcumin-PM extract (1e20 mg/ mL), and curcumin-PM nanoemulsion (1e20 mg/mL). Cells were incubated overnight and then added with 20 mL of MTS to each well. The cells were reincubated for 2 h at 37 C with 5% CO2. The absorbance was measured at 490 nm using ELISA microplate reader. The result was represented as percentage of cell viability, compared to untreated controls. The IC50 valued was calculated using BioStat Software. Further, a similar protocol for in vitro cytotoxic study on blank nanoemulsion was determined by MTS assay using several cell lines including NIH/3T3 (mouse fibroblast cell line), 3T3-SA (Swiss albino mouse fibroblast cell line), RSC-96 (rat neuron Schwann cell line), RAW 264.7 (mouse macrophage cell line), RBL-2H3 (rat basophilic leukemia cell line), CHO-K1 (Chinese hamster ovary cell line), HepG2 cell (human hepatoma cell line), and NCI-H292 (human tumor cell line). 2.4.7. Antifibrotic assay The antifibrotic assay of the preparation was performed by determining the reduction effect on collagen expression both at gene and protein levels [26,25]. NIH/3T3 cells were placed into 6-well plate at density of 105 cell/ mL. Cells were cultured overnight at 37 C with 5% CO2. Different concentrations of curcumin (2, 4, 6, and 8 mg/mL), PM extract (21, 42, and 168 mg/mL), curcumin nanoemulsion (0.5, 1, 1.5, and 2 mg/ mL), curcumin-PM extract (1e20 mg/mL), and curcumin-PM nanoemulsion (1e20 mg/mL) subsequently were added into cultured cells. Treated cells were incubated for 24 hs at 37 C with 5% CO2. 2.4.8. RNA extraction and gene expression analysis Total RNA was extracted with TRIzol® reagent according to a standard protocol. cDNA was prepared with GoScript kit and RTPCR method according to the manufacturer's method. Gene
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expression of procollagen a1 and collagen IV was analyzed with quantitative real time PCR. The PCR mixtures (20 mL) contained 2 mL of cDNA, primers (1 mM concentrations, Table 1), and EvaGreen master mix. The replication process involved initial denaturing step at 95 C for 30 s, followed by 35 cycle of denaturation step at 95 C for 5 s, 56 C for 5 s, and 65 C for 5 s. Each measurement was performed in three replicates. Data was analyzed using Bio-Rad CFX manager software and calculation of gene expression levels were normalized to the signal of the house keeping gene BAM. 2.4.9. Effects of nanoemulsion on collagen synthesis The amount of collagen protein was measured with Sirius Red/ Fast Green Staining Kit. NIH/3T3 mouse fibroblast cell were plated into 24-well plate at density of 3.104 cell/well. Cells were cultured overnight at 37 C with 5% CO2. Different concentrations of curcumin, PM extract, curcumin nanoemulsion, curcumin-PM extract, and curcumin-PM nanoemulsion were added to the cells. Then, the cells were incubated for 24 h at 37 C with 5% CO2. Further, culture medium was removed and the cells were washed with PBS. Kahle fixative reagent was added into cells and the cells were incubated for 10 min at room temperature. Fixative reagent was removed, followed by washing 0.2 mL of PBS. Dye solution was added to the cells and subsequently the cells were incubated at room temperature for 30 min. Dye solution was removed carefully and the cells were rinsed with 0.5 mL of distilled water repeatedly until the color faded. Stained cells were observed under a light microscope. Further, the cells were added with 1 mL of Dye Extraction Buffer and mixed gently until the color was eluted. The absorbance was measured with spectrophotometer at 540 nm and 605 nm. The amount of collagen was calculated using the following formula:
A eðA605 0:291Þ collagen mg=well ¼ 540 0:0378 non collagen protein
mg=well ¼
A605 0:00204
(1)
(2)
2.4.10. Statistical analysis Data were presented as mean ± SD. All data were subjected to an unpaired, two-tailed distribution student t-test. Differences were considered significant at p < 0.05. 3. Results 3.1. Characterization of nanoemulsion Table 2 shows physical characteristics of curcumin nanoemulsion. As presented, the mean globule diameter was around 30 nm. Polydispersity index was relatively low, being less than 0.5. Analysis of globule morphology by TEM confirmed the encapsulation of curcumin in the oil phase which is expected to prevent degradation of curcumin in GI tract (Fig. 1).
Table 1 PCR primers sequence. Gene
Nucleotide sequences
Procollagen a1
Upstream: 50 -AGCCTGAGCCAGCAGATTGA-30 Downstream: 50 -CCAGGTTGCAGCCTTGGTTA-30 Upstream: 50 -AACAACGTCTGCAACTTCGC-30 Downstream: 50 -CTTCACAAACCGCACACCTG-30 Upstream: 50 -ACCCACACTGTGCCCATCTA-30 Downstream: 50 -CGGAACCGCTCATTGCC-30
Collagen IV BAM
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H. Rachmawati et al. / Journal of Drug Delivery Science and Technology 39 (2017) 123e130 Table 2 Physical characteristics (mean ± SD, n ¼ 3).
3.3. In vitro cytotoxicity of
curcumin-loaded
nanoemulsion
Characteristics
Result
Globule diameter (nm) Polydispersity Index
27.3 ± 6.03 0.393 ± 0.13
3.2. Physical stability in the presence of liver S9 fraction Curcumin is a subject to metabolism by the liver, resulting in reduction inactive products after oral administration: curcumin glucuronide and curcumin sulfate conjugates [29]. This evidence was indicated by the decrease in curcumin content after exposure to liver S9 fraction. It was found that curcumin in nanoemulsion system exhibited lower metabolism rate as compared to pure curcumin (Fig. 2). The decrease in curcumin concentration after 2 h s incubation was 37.71% for pure curcumin, and 15.69% for curcuminloaded nanoemulsion.
The effects of pure curcumin, PM extract, curcumin nanoemulsion, and curcumin-PM nanoemulsion on cell viability are shown in Fig. 3. Pure curcumin decreased the viability of NIH/3T3 cells (Fig. 3a). The lowest cell viability was observed in preparation with the highest curcumin concentration. In fact, curcumin exhibited cytotoxic effect at a concentration of 16 mg/mL and above. However, cell viability was well preserved at concentration range of 2e8 mg/mL. The calculated IC50 of curcumin on NIH/3T3 cells was 17.8 mg/mL. Meanwhile, PM extract was able to maintain cell viability at concentration up to 168 mg/mL, despite slight decrease in cell viability (Fig. 3b). The predicted IC50 of PM extract on NIH/ 3T3 cells was 383.3 mg/mL. Formulation of curcumin into nanoemulsion resulted in greater cytotoxicity, limiting its safe concentration at 2 mg/mL (Fig. 3c). However, combination of curcumin and PM extract with respective concentration of 4 mg/mL and 20 mg/mL did not show any cytotoxic effect in NIH/3T3 cell (Fig. 3d). The blank nanoemulsion did not demonstrate any cytotoxic effect on NIH/ 3T3, 3T3-SA, RSC-96, RAW 264.7, RBL-2H3, CHO-K1, HepG2, and NCI-H292 cell lines (Fig. 3e).
Fig. 1. Morphological analysis of nanoemulsion with Transmission Electron Microscopy (TEM). Image was captured with 20000 magnification (a) and 10000 magnification (b).
Fig. 2. The stability profile of curcumin in the presence of liver S9 fraction.
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Fig. 3. In vitro study on cells viability of different treatments. Curcumin (a), PM extract (b), curcumin nanoemulsion (c), curcumin-PM extract and curcumin-PM nanoemulsion (d), unloaded nanoemulsion (e).
3.4. Procollagen a1(1) and collagen IV genes expression The effect of pure curcumin, PM extract, curcumin nanoemulsion, and curcumin-PM nanoemulsion on gene expression of procollagen a1(1) and collagen IV are illustrated in Fig. 4. Overall, tested preparations demonstrated the ability to suppress the expression of both genes significantly (p < 0.05). Both curcumin and PM extract showed inhibitory effect in dose-dependent manner (Fig. 4a and b). Gene suppression exhibited by curcumin in nanoemulsion form was significantly greater. The ratio of collagen gene to b-actin was around 0.4 in pure curcumin at concentration of 4 mg/mL. After the treatment with curcumin nanoemulsion, the gene:b-actin ratio was twice lower (approximately 0.2) at curcumin concentration of 2 mg/mL (Fig. 4c). Combination of curcumin (4 mg/mL) and PM extract (20 mg/mL) showed even greater suppression of procollagen a1 and collagen IV at gene level as compared to individual component at similar concentration (Fig. 4d). 3.5. Histological examination and quantification of collagen protein The result of histological examination of NIH/3T3 cells is shown in Fig. 5. The red color on NIH/3T3 cell indicates the presence of collagen protein. Fig. 6 shows the quantification of the protein after treatment. It is clear that the amount of collagen detected on the cells after treatment with curcumin-PM extract and nanoemulsion of curcumin-PM extract was significantly lower (p < 0.05).
However, there was no significant difference between curcuminPM extract and curcumin-PM nanoemulsion in terms of reduction of collagen production. Similarly, collagen reduction resulted from curcumin-PM extract combination did not differ significantly compared to single treatment of each compound. Therefore, it is suggested that no synergistic effect is demonstrated by a combination of curcumin and PM on the modulation of collagen synthesis. 4. Discussion Well documented problem of curcumin leading to inefficient therapeutic outcome is its low bioavailability, not only due to low solubility but also sensitive to in vitro and in vivo conditions [24,21,11,3,31]. The effect of curcumin to treat liver fibrosis is also reported by many investigators [1,36,6,15,23]. Liver fibrosis (scarring) occurs in advanced liver disease, where normal hepatic tissue is replaced with collagen-rich extracellular matrix and, if left untreated, results in cirrhosis. Curcumin inhibits hepatic cirrhosis in an animal model and exhibits multiple biological actions in hepatic stellate cells (HSCs), which play an important role in the pathogenesis of liver fibrosis. A nanoemulsion with a mean globule diameter around 30 nm has been successfully developed. The curcumin loaded in the nanoemulsion is expected to have an improved bioavailability since nano-sized droplets have higher surface area available for absorption [20,33,12]. Moreover, enhanced bioavailability of curcumin is
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Fig. 4. Effect of curcumin (a), PM extract (b), curcumin nanoemulsion (c), and curcumin-PM nanoemulsion (d) on procollagen a1 and collagen IV gene expression in NIH/3T3 mouse fibroblast cells after 24 h incubation. *p < 0.05.
Fig. 5. The collagen expression by Sirrius/fast green staining on mouse fibroblast NIH 3T3 cells before treatment (a) and after treatment with curcumin-PM nanoemulsion (b).
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Fig. 6. Inhibitory effect of curcumin on collagen protein expression in mouse Fibroblast NIH 3T3 cells. A: untreated cell, B: PM extract (20 mg/mL), C: curcumin (4 mg/mL), D: curcumin (2 mg/mL) - PM (10 mg/mL), E: curcumin (4 mg/mL) -PM (20 mg/mL), F: curcumin nanoemulsion (1 mg/mL), G: curcumin nanoemulsion (2 mg/mL), H: curcumin (1 mg/mL) PM (5 mg/mL) nanoemulsion, I: curcumin (2 mg/mL) - PM (10 mg/mL) nanoemulsion. *p < 0.05.
also obtained as the stability against liver metabolism was increased in the nanoemulsion form, as demonstrated in this report. Curcumin is a subject to metabolism by the liver, resulting in reduction products depending on the route of administration. After oral administration of curcumin, the drug is extensively metabolized by the liver to curcumin glucoronide and curcumin sulfate which has no bioactivity [29]. This event was indicated by the decrease in curcumin content after exposure to liver S9 fraction, already detected 0.5 h after incubation and continuing to decrease up to for 2 h observation. Lower metabolism rate of curcumin in nanoemulsion indicates a greater stability due to physical protection provided by the nanoemulsion system which controlled and sustained the release of curcumin from the oil globule to liver homogenate. Formulation into nanoemulsion requires careful consideration regarding toxicity. This is due to the high amount of surfactant applied in the formulation [12]. In this in vitro study, no cytotoxic effect was generated from the excipients used to form nanoemulsion. Secondly, it was found that formulation of curcumin and PM extract into nanoemulsion resulted in lower cell viability, in comparison with that of pure curcumin and pure PM. This indicates the beneficial formulation in lowering dose of drugs while retaining the efficacy. However, this also means that dose adjustment is required in formulation of curcumin-PM extract nanoemulsion to avoid any toxicity. Findings in this study have proven that curcumin- and curcumin-PM extract nanoemulsions significantly suppressed the expression of procollagen a1(1) and collagen IV at the gene level, and collagen at the protein level. This has clinical significance, considering that inhibition of collagen synthesis is one of the strategies of liver fibrosis therapy [17,34]. Particularly on the reduction of collagen expression at the protein level, tissue staining using Sirius red provides evidence upon histological visualization of this protein. The potential effect of both curcumin and PM extract are widely reported [9]. described the important evidences of Proliverenol, a bioactive compound in PM extract we used for this study, for liver disease. Proliverenol decreased the expression of transaminase enzymes, such as ALT. Proliverenol appears to suppress both the gene expression of ALT and the protein leakage from cytoplasm into the culture medium. The mechanism resulting the
hepatoprotective effect of proliverenol is via up-regulating APEX1 that represses DNA fragmentation and down-regulates the NF-kB, TNFa and caspase-8. The activity of collagen synthesis reduction demonstrated by curcumin nanoemulsion and curcumin-PM nanoemulsion in this report might be due to their antioxidant and anti-inflammatory activities. Oxidative stress is known to play a crucial role in hepatic fibrogenesis by stimulating HSC activation and increasing collagen synthesis [40]. In another study, curcumin has been reported to increase the activity of matrix metalloproteinase (MMP-1 and MMP-2), which are responsible for degradation of collagen, particularly type I and IV [27]. The ability of curcumin and PM extract in inhibiting collagen synthesis at the gene and protein levels suggest that these compounds are quite promising for novel antifibrotic therapy.
5. Conclusion Nanoemulsion provides numerous advantages including improved stability and oral absorption. Thus, it is expected that the application of nanoemulsion in oral delivery system will continue to increase, particularly for drugs with poor solubility, poor absorption, or high susceptibility to metabolic instability. Curcumin nanoemulsion has demonstrated its ability in suppressing gene expression of procollagen a1 and collagen IV, as well as the collagen protein, the main fibrotic markers. This effect is strengthen when co-delivered with PM extract. The nanoemulsion system offered protection against metabolic degradation of curcumin due to the enzymes in the liver, bringing its promising use as an oral liver antifibrotic therapy. Our findings showed that only a smaller dose of curcumin and PM extract was required in the nanoemulsion system to achieve the desired biological activity in vitro. This indicates that incorporation of drugs into nanoemulsion potentially results in better efficacy. Nevertheless, we suggest in vivo study regarding toxicity, pharmacokinetic profiles, and bioavailability in normal subjects and those with liver fibrosis for future work.
Conflict of interest The authors declare no conflict of interest.
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Acknowledgement The authors would like to thank Dexa Laboratory Biomolecular Sciences (DLBS) for provision of research facilities. Great appreciation is also addressed to Bandung Institute of Technology for financing this project under ‘ITB Innovative Research 2015’ research grant. References [1] R. Afrin, S. Arumugam, A. Rahman, M.I. Wahed, V. Karuppagounder, M. Harima, H. Suzuki, S. Miyashita, K. Suzuki, H. Yoneyama, K. Ueno, K. Watanabe, Curcumin ameliorates liver damage and progression of NASH in NASH-HCC mouse model possibly by modulating HMGB1-NF-kB translocation, Int. Immunopharmacol. 44 (2007) 174e182. [2] R. Altaf, M. Asmawi, A. Dewa, A. Sadikun, M. Umar, Phytochemistry and medicinal properties of Phaleria macrocarpa (Scheff.) Boerl. extracts, Pharmacogn. Rev. 7 (2013) 73e80. [3] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807e818. [4] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807e818. [5] S. Andrean, A. Prasetyo, T. Kristijarti, Hudaya, The extraction and activity test of bioactive compounds in Phaleria macrocarpa as antioxidants, Procedia Chem. 9 (2014) 94e101. l, E. Mura r, M. Cebov nova , The effect of [6] P. Janega Barta, P. Baba a, O. Pecha curcumin on liver fibrosis in the rat model of microsurgical cholestasis, Food Funct. 6 (2015) 2187e2193. [7] R. Bataller, D. Brenner, Liver fibrosis, J. Clin. Invest. 115 (2005) 209e218. [8] D. Behrens, M. Villone, G. Koch, L. Brunner, H. Sorokin, Robenek, et al., The epidermal basement membrane is a composite of separate laminin- or collagen iv-containing networks connected by aggregated perlecan, but not by nidogens, J. Biol. Chem. 287 (2012) 18700e18709. [9] Berlian, O.M. Tandrasasmita, R.R. Tjandrawinata, Standardized bioactive fraction of Phaleria macrocarpa(Proliverenol) prevents ethanol-induced hepatotoxicity via down-regulation of NF-kB-TNFa-caspase-8 pathway, As.Pac. J. Trop. Biom 6 (2016) 686e691. [10] T. Boyer, T. Wright, M. Manns, D. Zakim, Zakim and Boyer's Hepatology, first ed., Saunders/Elsevier, Philadelphia, 2006. [11] J. Cuomo, G. Appendino, A.S. Dern, E. Schneider, T.P. McKinnon, M.J. Brown, et al., Comparative absorption of a standardized curcuminoid mixture and its lecithin formulation, J. Nat. Prod. 74 (2011) 664e669. [12] N. Date, R. Desai, M. Dixit, Nagarsenker, Self-nanoemulsifying drug delivery systems: formulation insights, applications and advances, Nanomedicine 5 (2010) 1595e1616. [13] B.B. Dhillon, R.A. Aggarwal, R.A. Newman, A.B. Wolff, J.L. Kunnumakkara, Abbruzzese, et al., Phase II trial of curcumin in patients with advanced pancreatic cancer, Clin. Cancer Res. 14 (2008) 4491e4499. [14] M. Easmin, M. Sarker, S. Ferdosh, S. Shamsudin, K. Yunus, M. Uddin, et al., Bioactive compounds and advanced processing technology:Phaleria macrocarpa(sheff.) Boerl, a review, J. Chem. Technol. Biotechnol. 90 (2014) 981e991. [15] S.C. Gupta, S. Patchva, W. Koh, B.B. Aggarwal, Discovery of curcumin, a component of golden spice, and its miraculous biological activities, Clin. Exp. Pharmacol. Physiol. 39 (2012) 283e299. [16] W. Hendig, K.W. Ermin, Benzophenone glucoside isolated from the ethyl acetate extract of the bark of the mahkota dewa (Phaleria macrocarpa (Scheff.) Boerl.) and its inhibitory activity leukeumin L1210 cell line, Indones. J. Chem. 9 (2009) 142e145. [17] M.H. Ismail, M. Pinzani, Reversal of liver fibrosis, Saudi J. Gastroenterol. 15 (2009) 72e79. [18] M. Kanai, K. Yoshimura, M. Asada, A. Imaizumi, C. Suzuki, S. Matsumoto, et al., A phase I/II study of gemcitabine-based chemotherapy plus curcumin for patients with gemcitabineresistant pancreatic cancer, Cancer Chemother. Pharmacol. 68 (2011) 157e164.
[19] S. Muhamad, P. Indriati, Nanoencapsulation of the flavonoids isolated from Phaleria macrocarpa leaf by casein micelle, Int. J. Pharm. Bio. Sci. 3 (2012) 471e478. [20] E. Nazarzadeh, T. Anthonypillai, S. Sajjadi, On the growth mechanisms of nanoemulsions, J. Colloid Interface Sci. 397 (2013) 154e162. [21] M.J. Nirmala, M. Amitava, N. Chandrasekaran, A nanoemulsion drug delivery system for an aqueous insoluble drug, Int J. Chem Tech Res. 6 (2012) 2020e2022. [22] O.E. Olson, E.E. Fager, et al., Folic acid activity in homogenates of rat liver, J. Biol. Chem. 174 (1948) 319e326. [23] A. Oppenheimer, Turmeric (curcumin) in biliary diseases, Lancet 229 (1937) 619e621. [24] G.S. Patchva, B.B. Aggarwal, Therapeutic roles of curcumin: lessons learned from clinical trials Subash c, AAPS J. 15 (2013). [25] H. Rachmawati, L. Beljaars, C. R-Smit, H.I. Bakker, D.K.F. Meijer, K. Poelstra, IL10 modified with mannose 6-phosphate yields a liver-selective cytokine, Drug Metab. Dispos. 35 (2007) 814e821. [26] H. Rachmawati, A. Mattos-Pinto, C. Reker-Smit, K. Poelstra, L. Beljaars L, Modification of Interleukin-10 with Mannose 6-Phosphate Groups Yields a Liver-specific Cytokine with Antifibrotic Effects in Bile-duct Ligated Rats in the Book “Pharmacology”, publisher Intech, Croatia, March 2012. ISBN 979-953307-482-4. [27] H.W. Ryu, S.P. Kim, K.S. Lee, J.W. Cho, Curcumin induced decreased expression of type I collagen in human skin fibroblast through down-regulation of Smad2/3 expressions, Korean J. Dermatol 50 (2012) 1e7. [28] D.M. Samuel, M. Marco, S. Stefania, R. Chiara, A. Laura, T. Luciano, et al., Cellular and molecular mechanism of hepatic fibrosis leading to liver cancer, Transl. Gastrointest. Cancer 1 (2012) 88e94. [29] K. Shaiju, M. Kakarala, T.R. Mack, A.C. James A, P.N. Daniel, D. Zora, D.E. Brenner, Pharmacokinetics of curcumin conjugate metabolites in healthy human subjects, Cancer Epidemiol. Biomarkers Prev. 17 (2008) 1411e1417. [30] R.A. Sharma, H.R. McLelland, K.A. Hill, C.R. Ireson, S.A. Euden, M.M. Manson, et al., Pharmacodynamic and pharmacokinetic study of oral curcuma extract in patients with colorectal cancer, Clin. Cancer Res. 7 (2001) 1894e1900. [31] G. Shoba, D. Joy, T. Joseph, M. Majeed, R. Rajendran, P. Srinivas, Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers, Planta Med. 64 (1998) 353e356. [32] Stefanska, Curcumin ameliorates hepatic fibrosis in type 2 diabetes mellitus insights into its mechanisms of action, Br. J. Pharmacol. 166 (2012) 2209e2211. [33] D.Q. Sun, X.B. Wei, X. Xue, Z.I. Fang, M.R. Ren, H.Y. Lou, X.M. Zhang, Enhanced oral absorption and therapeutic effect of acetylpuerarin based on D-a-tocopheryl polyethylene glycol 1000 succinate nanoemulsions, Int. J. Nanomedicine 9 (2014) 3413e3423. [34] S. Swarnakar, K. Ganguly, P. Kundu, A. Banerjee, P. Maity, A. Sharma, Curcumin regulates expression and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer, J. Biol. Chem. 280 (2004) 9409e9415. [35] H.J. Triastuti, J. Park, Choi, Phaleria macrocarpa suppresses oxidative stress in Alloxan-induced diabetic rats by enhancing hepatic antioxidant enzyme activity, Nat. Product. Sci. 15 (2009) 37e43. [36] P. Wu, R. Huang, Y.L. Xiong, C. Wu C, Protective effects of curcumin against liver fibrosis through modulating DNA methylation, Chin. J. Nat. Med. 14 (2016) 255e264. [37] Y. Zhang, P. Zhang, G. Dai, H. Liu, Study on the synthesis and bioactivity of novel mahkoside A derivatives, Synth. Commun. 37 (2007) 3319e3328. €mme, antifibrotic effects [38] C. Zhang, C. Huang, R. Yang, J. Liu, J. Wang, D. Niu, Bro of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts, Respir. Res. 12 (2011) 1e12. [39] S. Zhang, Z. Gong, W. Li, X. Wang, T. Ling, Antifibrotic effect of curcumin in TGF-b1-induced myofibroblasts from human oral mucosa. Asian Pac, J. Cancer P 13 (2012) 289e294. [40] Z. Zhang, L. Zhang, D. Chen, X. Kong, C. Lu Zhang, et al., Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells, J. Cell. Mol. Med. 18 (2014) 1392e1406.