Research and Development on Nanotechnology in Indonesia,Vol.2, No.1, 2015, pp. 37- 48 ISSN : 2356-3303

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Self- Nanoemulsion Containing Combination of Curcumin and Silymarin : Formulation and Characterization

Utami Wulandari Rachmadi1), Dahlia Permatasari1), Annisa Rahma1),

Heni Rachmawati1) 1)

Jl. Ganesha, Bandung 40132, Indonesia Sekolah Farmasi, InstitutTeknologi Bandung

* e-mail: utamiwr@gmail.com

Received : 15 January 2015 Accepted : 18 February 2015

ABSTRACT

A nanoemulsion containing curcumin and sylimarin has been developed in order to increase the bioavailability of both drugs. The formulation was prepared by using self-nanoemulsifying method. Oil phase in nanoemulsion consisted of glycerylmonooleate as oil phase, Tween 20 as surfactant, and polyethylene glycol 400 as co-surfactant in a ratio of 1:8:1. Nanoemulsion was formed spontaneously by the addition of deionized water into oil phase under mild stirring. Optimization were performed to obtain maximum loading capacity of curcumin and silymarin in nanoemulsion. Characterization of nanoemulsion included visual appearance, droplet size and morphology, polydispersity index, entrapment efficiency, loading capacity, physical stability, and stability under normal storage condition for 2 weeks. Findings show that nanoemulsion which contained 15 mg of curcumin and 25 mg of silymarin resulted in the most desirable characteristics, including transparent visual appearance; spherical droplet shape; droplet sizeof 24.967 ± 1.026 nm; polydispersity index of 0.340 ± 0.054; entrapment efficiency of curcumin and silymarin of 99.796 ± 0.287% and 99.349 ± 2.297%, respectively. This nanoemulsion remained stable following centrifugation at 12,000 rpm for 15 minutes and after four cycles of freeze-thaw treatment. The results of stability study did not show significant difference of entrapment efficiency, drug content, and loading capacity for both curcumin and silymarin in nanoemulsion after being stored for 2 weeks. The stability of silymarin and curcumin in this nanoemulsion were higher compared to conventional emulsion. Self-nanoemulsion containing combination of curcumin and silymarin with sufficient characteristics has been successfully developed as a candidate of efficient drug delivery system.

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Keywords: Curcumin, silymarin, nanoemulsion, self- nanoemulsification, bioavaibility INTRODUCTION Curcumin and silymarin are two compounds which have shown a number of potent pharmacological activities. Curcumin is a polyphenolic compound found in Turmeric (Curcuma longa) [1] that has been found to exhibit a wide range of activities including antioxidant, anti-inflammatory, hepatoprotector, and anti-cancer [2, 3, 4, 5]. Whereas, silymarin from Silybum marianum has been widely known as a hepatoprotective agent [6]. In some studies, administration of these two compounds simultaneously can produce a synergistic pharmacological effects in terms of hepatoprotective activity [7], anticancer [8], and antioxidants [9]. However, both compounds has a very low bioavailability when administered orally. Curcumin is poorly soluble in water, which leads to poor absorption. In addition, curcumin undergoes rapid metabolism in various tissues, especially the liver and instestinal,converted into several derivatives product and conjugation metabolites such as glucuronide and sulfate conjugates [2]. Silymarin also has a low bioavailability due to its poor absorption, rapid metabolism and excretion. The absorption of silymarin is impeded by its low solubility in water (0.04 mg/mL) and low permeability to the intestinal wall [10]. Nanoemulsion is one of drug delivery systems that has been widely developed as a hydrophobic drug carrier. Nanoemulsion can increase both solubility and dispersibility of hydrophobic drugs in aqueous environment. The small droplet size in nanoemulsion allows them to be readily transferred through the intestinal cell membrane, and thus significantly improves the absorption and bioavailability of hydrophobic drugs [13]. In addition, the presence of surfactant and cosurfactant that form a continuous film on the droplet surface will protect the active compound from chemical or enzymatic degradation that can impair the effectiveness of the drug [13]. In recent years, nanoemulsion has been studied as a carrier system for silymarin or curcumin and the results showed significant increases in their bioavailability [11, 12]. Moreover, several studies reported increased pharmacological activity when nanoemulsion was used as a carrier system. For instance, anti-inflammatory effects of curcumin [2] and hepatoprotective effect of silymarin were improved in nanoemulsion preparation [12]. In this study, nanoemulsion containing combination of curcumin and silymarin were prepared with SNE method (self- nanoemulsification) or spontaneous nanoemulsification. SNE is defined as an isotropic mixture of oil, surfactant, and cosurfactant which will spontaneously form oil-in-water nanoemulsion when mixed together in aqueous environment under mild stirring. In SNE, interfacial

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tension was set to be very low so that the interfacial energy becomes equal or lower than the entropy of the system. As a consequence, zero or negative value of free energy in the system will be achieved, leading to a higher thermodynamic stability of SNE [14]. MATERIAL AND METHOD Material Curcumin was obtained from PT. Phytochemindo Lestari (Indonesia), Silymarin was obtained from PT. Javaplant (Indonesia), Glyceryl monooleate 40 (GMO 40) was purchased from PT. Tritunggal Arthamakmur, (Indonesia), Polysorbate 20 (Tween 20) was purchased from Sigma Aldrich; Polyethylene glycol 400 (PEG 400) was purchased from PT. Brataco (Bandung, Indonesia), Dimethylsulfoxide (DMSO) was purchased from Merck (Germany), acetonitrile was purchased from J.T. Baker (United States), orthophosporic acid was purchased from Merck (Germany), sodium hydroxide was purchased from Sekolah Farmasi, Institut Teknologi Bandung, (Indonesia), double distilled water was purchased from IPHA Laboratories (Bandung, Indonesia), and deionized water was purchased from Laboratorium Kimia, Institut Teknologi Bandung (Indonesia). All reagents used were analytical grade. Preparation and Optimization of Nanoemulsion Containing Curcumin and Silymarin Various amount of each curcumin and silymarin (5, 10, 15, 20, 25, 30, and 35 mg) was added into 1 gram of the oil phase composed of glycerin monooleate, Tween 20, and PEG 400 in the ratio of 1:8:1. The mixture were stirred for 2 hours at 100 rpm and sonicated in a bath-type sonicator (Nagoya S Ultrasonic Cleaner GB-928) for 1 hour. Deionized water as the water phase was added to the oil phase at the ratio of 5:1, under mild stirring. Nanoemulsion was then formed spontaneously. Optimization of curcumin and silymarin in nanoemulsion was performed by considering the following parameters: loading capacity, droplet size, droplet size distribution, and entrapment efficiency. Droplet Size and Droplet Size Distribution Droplet size and droplet size distribution in the nanoemulsion was analyzed by Photon Correlation Spectroscopy (DelsaTM

C Nano Particle Analyzer, Beckman Coulter). To perform the measurement, the sample was introduced into the disposable celland the both vesicle size and vesicle size distribution were read in light intensity of 3,000 to 30,000.

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Entrapment Efficiency Curcumin and silymarin content assay was performed using high performance liquid chromatography (HPLC, Hewlett Packard Agilent Series 1100) by a direct method. Prior to analysis by HPLC, nanoemulsion was centrifuged at 14000 rpm for 20 minutes. Supernatant obtained from centrifugation was then diluted using the mobile phase and filtered using 0.22 µm membrane. HPLC separation was performed using a C18 column (reversed phase, 150 mm × 4.6 mm with particle size of 5 µm, Inertsil ODS-3). Elution was performed with a gradient method using a mobile phase consisted of acetonitrile and 0.1% orthophosphoric acid at the ratio of 35:65 (v/v) during the first 5 minutes, 55:45 (v/v) at 6-10 minutes, and 35:65 (v/v) at 11-12 minutes. The detection wavelength used was 288 nm at 0-7 minutes and 430 nm at 8-12 minutes. The flow rate and elution time was 1 mL/min and 12 minutes, respectively. A total of 50 mL of sample was injected into the HPLC system. A calibration curve was prepared using a standard solution of curcumin and silymarin in DMSO. Dilution was done using the mobile phase to produce a series of concentration ranging from 10 to 35 ppm. The percentage of entrapment efficiency was obtained using the following equation:

% EE Curcumin = amount of curcumin encapsulated in droplet (mg)

amount of curcumin added into nanoemulsion (mg) × 100%

% EE Silymarin = amount of silymarin encapsulated in droplet (mg)

amount of curcumin added into nanoemulsion (mg) × 100%

Loading Capacity Sample preparation for the determination of loading capacity was similar to determination of entrapment efficiency but the loading capacity was determined by the equation below:

LC Curcumin = amount of curcumin encapsulated in droplet (mg)

amount of oil phase used in nanoemulsion (g)

LC Silymarin = amount of silymarin encapsulated in droplet (mg)

amount of oil phase used in nanoemulsion (g)

Morphology of Nanoemulsion Droplets Transmission Electron Microscopy (TEM; JEM 1400, JEOL, Japan) was used to observe the morphology of nanoemulsion droplets. A drop of

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nanoemulsion containing curcumin and silymarin (10 uL) was dripped in a specimen glass and covered with a grid of 400 mesh. After 1 minute, 10 mL of uranyl acetate was dripped above the grid. The grid was dried for 30 minutes and then observed with TEM. Stability Study of Nanoemulsion Thermodynamic stability of nanoemulsion was studied by performing centrifugation and freeze-thaw treatment. Centrifugation was performed at 12,000 rpm for 15 minutes. Freeze thaw treatment was performed for 4 cycles, wheresamples were stored for 2 days at 40 °C and 2 days at 4°C in each cycle. Physical and chemical stability of nanoemulsion at room temperature was observed for 14 days. Changes in the droplet size, polydispersity index, entrapment efficiency, as well as content of curcumin and silymarin in nanoemulsion were observed at day 0, 3, 6, 9, and 12. Curcumin and silymarin content during the storage was determined and compared against conventional emulsion to evaluate the chemical stability of nanoemulsion Data Analysis All experiments were done in triplicate (n = 3). All data is presented as mean value ± standard deviation. Statistical data analysis was performed using Student's t test unpaired and one-way ANOVA. P- Value < 0.05 was considered as the minimal level of significance. RESULTS AND DISCUSSION A proper ratio of oil to surfactant to cosurfactant is required in order to prepare a nanoemulsion using SNE method. An appropriate composition is able to produce sufficiently low interfacial tension to form nanoemulsion spontaneously [14]. Nanoemulsion formulas used in this study was based on optimization in our previous works [15] which used glyceryl monooleate (GMO) as lipid, Tween 20 as surfactant, and polyethylene glycol 400 (PEG 400) as cosurfactant with optimum ratio of 1:8:1. Tween 20 has a high hydrophilic-lipophilic balance (HLB) at 16.7 [16], which tends to form oil-in-water nanoemulsion. Surfactants with high HLB can reduce interfacial tension significantly, leading to spontaneous formation of nanoemulsion. PEG 400 as cosurfactant also decreases the surface tension and along with Tween 20, play an important role to form steric stability in nanoemulsion [17, 18]. Nanoemulsion preparation in this study used ultrasonic vibration at a frequency of 48 kHz to induce breaking down of the droplets into smaller size. The commonly used frequency is 20 kHz or more in order to produce sufficient shear force to break down the droplets [19].

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16,00 16,13 16,97 23,6535,60

50,80

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Silymarin Curcumin

Nanoemulsion has physical characteristics that can distinguish them from macroemulsion. Nanoemulsion droplets size are much smaller than the wavelength of visible light, resulting in weak light scattering and made the visual appearance of nanoemulsion become clear or translucent. In contrast, macroemulsion has a large droplet size, leading to a strong light scattering and thus the appearance is opaque [20]. As shown in Figure 1, all nanoemulsion had a clear yellow and transparent appearance, despite increasing curcumin and silymarin conte Smaller droplet size can improve dissolution rate and oral bioavaibility. In one study, it was shown that a substance with particle size less than 100 nm have significant increase in dissolution rate and oral bioavaibility. Therefore, in this study it was aimed to achieve droplet size below 100 nm to improve dissolution rate and bioavailability of curcumin and silymarin [23]

Figure 1 : Visual appearance of nanoemulsions with increasing amount of added curcumin and silymarin. From left to right: 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, and 35 mg of each curcumin and silymarin.

Figure 2 : Influence of curcumin and silymarin concentration on the droplet size of nanoemulsion.

Figure 3 : Influence of curcumin and silymarin concentration on the curcumin and silymarin entrapment efficiency in nanoemulsion.

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Amount of Curcumin : Silymarin (w/w) per 1 g oil phase

Parameter

Droplets size (nm)

Polydispersity Index

Entrapment Efficiency of

Curcumin (%)

Entrapment Efficiency of

Silymarin (%)

5 : 5 mg 16,00 ± 2,54 0,311 ± 0,06 99,785 ± 1,153 99,075 ± 2,718

10 : 10 mg 16,13 ± 2,14 0,310 ± 0,15 99,598 ± 1,824 99,764 ± 1,685

15 : 15 mg 16,97 ± 1,61 0,314 ± 0,14 99,249 ± 1,089 99,552 ± 1,734

20 : 20 mg 23,65 ± 1,89 0,411 ± 0,05 94,098 ± 0,846 97,344 ± 1,787

25 : 25 mg 35,60 ± 4,64 0,370 ± 0,03 87,616 ± 1,563 98,438 ± 0,975

30 : 30 mg 50, 80 ± 0,28 0,230 ± 0,26 77,808 ± 2,538 92,350 ± 2,572

35 : 35 mg 173,03 ± 11,76 0,353 ± 0,01 72,148 ± 0,791 83,687 ± 1,532

Data is shown as mean ± standard deviation, n = 3

Table 1 : Correlation between curcumin and silymarin concentration with nanoemulsion characteristics. Droplet size and droplet size distribution of each nanoemulsion then tested with PCS. From Figure 2, it can be seen that nanoemulsion with each curcumin and silymarin content up to 30 mg has a droplet size less than 100 nm. However, the addition of 35 mg of each curcumin and silymarin, resulted in increased average size up to the 173.03 nm. The deviation at each measurement was also quite large, which suggests that there might be a presence of free particles of curcumin / silymarin that were not dissolve completely in the oil phase. As the result, these free particles interfered the analysis. Therefore, it is necessary to analyze the entrapment efficiency on each nanoemulsion. Results shown in Figure 3, indicated that the increase in curcumin and silymarin resulted in decreased entrapment efficiency at some point, which indicates that the nanoemulsion system has a limited loading capacity.The decrease of curcumin entrapment efficiency occurred when the amount of curcumin was 20 mg or higher, while a decline in silymarin entrapment efficiency was seen in the addition of 30 mg of silymarin. This difference might be related to the solubility of curcumin and silymarin in the oil phase. The solubility of curcumin in the GMO, Tween 20, and PEG 400 was 0.154 mg/mL, 60 mg/mL, and 0.512 mg/mL, respectively [21, 22]. In contrast, silymarin has a higher solubility in GMO and Tween 20, being 33.2 mg/mL and 131.3 mg/mL, respectively [10]. It is suggested that higher solubility of a substance in the inner phase/oil would result in higher entrapment efficiency. Findings show that solubility of active compound also influenced the size of droplet. When the oil phase is no longer capable to dissolve the particles of the substance, the free particles (unencapsulated in droplets) results in an overall larger droplet

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size. Polydispersity index of all nanoemulsion were below 0.5, indicating uniform size of the droplet. The lower the polydispersity index value,the more uniform the droplet size [12]. Considering the results of droplet size, droplets size distribution, and entrapment efficiency analysis,the content of curcumin and silymarin which would be encapsulated in nanoemulsion needs to be adjusted to achieve maximum entrapment efficiency for both active compounds. Therefore, an optimized nanoemulsion were prepared with modification of curcumin and silymarin content at 15 mg and 35 mg, respectively. The resulting droplet size, droplet size distribution, and entrapment efficiency (shown in Table 2) indicate that nanoemulsion had desirable properties.

Data is shown as mean ± standard deviation, n = 3

Table 2 : Characteristics of optimized nanoemulsion containing 15 mg curcumin and 25 mg silymarin.

Evaluation of droplets morphology using TEM was conducted to determine the particle size and its distribution, as well as the shape of the nanoemulsion droplets. From Figure 4, it can be seen that the droplets have a spherical shape with diameter of 50 nm and fairly uniform size distribution.

Amount of Curcumin : Silymarin

(w/w) per 1 g oil

phase

Parameter

Droplet size (nm)

Polydispersity Index

Entrapment Efficiency of

Curcumin (%)

Entrapment Efficiency of

Silymarin (%)

Loading Capacity of Curcumin (mg/ 1 g oil

phase)

Loading Capacity of Silymarin (mg/ 1 g oil

phase) 15 : 25 24,967 ± 1,026 0,340 ± 0,054 99,796 ± 0,287 99,349 ± 2,297 14,956 ± 0,237 24,992 ± 0,465

Figure 4 : Transmission Electron Microscopy (TEM) image of optimized nanoemulsion containing 15 mg curcumin and 25 mg silymarin

Figure 5 : Visual appearance of (A) optimized nanoemulsion, and (B) conventional emulsion after centrifugation.

A B

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Physical stability study of optimized nanoemulsion using centrifugation showed no phase separation. In contrast, phase separation occurred in the conventional emulsion, indicated by the appearance of sediment at the bottom of centrifuge tube following centrifugation (Figure 5). The optimized nanoemulsion also shows satisfying stability after 4 cycles freeze thaw treatment. The appearance of nanoemulsion remained transparent and did not show phase separation. The results of centrifugation and freeze thaw test showed that the optimized nanoemulsion was thermodynamically stable, and thus would not undergo instabilites that are common in macroemulsion such as creaming, flocculation, and sedimentation [24]. Evaluation of physical and chemical stability of nanoemulsion was also conducted at room temperature for 14 days. Parameters which were evaluated are droplet size, droplet size distribution, entrapment efficiency, and the content of curcumin and silymarin.The results showed that the droplet size increased significantly (P <0.05) after being stored for 2 weeks, but the size was below 100 nm. Polydispersity index values were relatively stable and there was no significant difference (P ˃ 0.05) on entrapment efficiency and loading capacity of curcumin and silymarin during storage for 2 weeks. The visual appearance of nanoemulsion remained unchanged. The content of curcumin and silymarin in nanoemulsion were also analyzed and compared to conventional emulsion. There were significant differences (P <0.05) of curcumin and silymarin content between nanoemulsion and conventional emulsion after storage for 2 weeks.

An oil in water type emulsion can be used as a way to stabilize antioxidants which are susceptible to oxidation. Curcumin and silymarin as an antioxidant can be protected inside the droplet.The water as external phase will inhibit the diffusion of oxygen to prevent oxidative degradation of

Figure 6 : Comparison of Curcumin (A) and Silymarin (B) content in nanoemulsion and emulsion, at room temperature for 14 days.

B A

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curcumin and silymarin. Stabilization may also occure from hydrogen bond between oxyethylene group in Tween 20 and phenol group in silymarin and curcumin [25, 26]. In this study, nanoemulsion had denser interfacial layer compared to conventional emulsion due to high concentration of surfactant and addition of co-surfactant.These agents provided protection to the antioxidants inside the droplet in order to avoid chemical degradation of silymarin and curcumin. CONCLUSION Nanoemulsion containing combination of curcumin and silymarin has been succesfully developed. The characteristics of the preparation are summarized as the following: a clear yellow appearance; spherical globule shape; droplet size of 24.967 ± 1.026 nm; polydispersity index of 0.340 ± 0.054; entrapment efficiency of curcumin and silymarin of 99.796 ± 0.287% and 99.349 ± 2.297%, respectively; loading capacity of curcumin and silymarin of 14.956 ± 0.237 mg and 24.992 ± 0.465 mg in 1 gram of the oil phase, respectively. The physical and chemical stability of nanoemulsion was superior to conventional emulsion. Therefore, this formulation has a potential application as an ideal carrier system for curcumin and silymarin. REFERENCES [1] Akram, M., Shahabuddin., Ahmed, A., Usmanghani, A., Hannan, A., Mohiuddin, E., Asif, M. Curcuma longa and Curcumin : A Review Article. Plant Biology. 2010; 55(2): 65-70. [2] Wang, X., Jiang, Y., Wang, Y., Huang, M., Ho, C., Huang, Q. Enhancing Anti-Inflammation Activity of Curcumin through O/W Nanoemulsions. Food Chem. 2007;108 : 419-424. [3] Bhullar, K., Jha, A., Youssef, D., Rupasinghe, H. Curcumin and Its Carbocyclic Analogs : Structure- Activity in Relation to Antioxidant and Selected Biological Properties. Molecules. 2013; 18 : 5389-5404. [4] Campbell, F., Collett, G. Chemoprotective Properties of Curcumin. Future Oncol. 2005 ; 1(3) : 405-414. [5] Kiso, Y., Suzuki, Y., Watanabe, N., Oshima, Y., Hikino, H. Antihepatotooxic principles of Curcuma longa rhizomes. Planta Med. 1983 ; 49(3) : 185-187. [6] Dixit, N., Baboota, S., Kohli, K., Ahmed, S., Ali, J. A Review of Pharmacological Aspects and BioavaibilityEnhancemet Approach. Indian J Pharmacol. 2007; 39 :172-179. [7] Mekala, P., Hariharan, P., Punniamurthy, N. Inhibition of Aflatoxin Induced Liver Damage in Broilers by Curcumin and Silymarin. Toxicology International. 2007; 14 (2) : 147-151.

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