Arch. Pharm. Res. DOI 10.1007/s12272-014-0395-4
RESEARCH ARTICLE
Preparation and characterization of mucoadhesive enteric-coating ginsenoside-loaded microparticles Jong-Suep Baek • Won-Gi Yeon • Cho-A Lee Sung-Joo Hwang • Jeong-Sook Park • Dong-Chool Kim • Cheong-Weon Cho
•
Received: 1 November 2013 / Accepted: 7 April 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Ginsenoside saponins are phytochemically extracted from red ginseng and have been regarded as the principal components manifesting the pharmacologic activities. Saponins are very soluble in water but poorly absorbed when orally administrated. Moreover, they have some disadvantages including the decomposition in acid medium. The aim of this study was to develop oral formulation of ginsenosides composed of enteric-coating polymer and mucoadhesive polymer considering the low stability in acid medium and the low permeability of saponins. Ginsenoside-loaded microparticles were prepared by spray dryer. The influences of various parameters such as the ratio of saponin to polymer, feed concentration, feed rate, inlet/outlet temperature and additional excipients during spray-drying were investigated. In vitro release profile of ginsenoside-loaded microparticles using additional excipients, ginsenoside saponin Rg1 or Rb1 showed an 18 or 13 % release in pH 1.2 when ethyl cellulose was added. Also, ginsenoside-loaded microparticles exhibited mucoadhesive properties in the presence of chitosan. The application of these polymers is being considered as the Jong-Suep Baek and Won-Gi Yeon have equally contributed to this work. J.-S. Baek W.-G. Yeon C.-A. Lee J.-S. Park D.-C. Kim (&) C.-W. Cho (&) College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, 220 Gung-dong, Yuseong-gu, Taejon 305-764, South Korea e-mail: [email protected] C.-W. Cho e-mail: [email protected] S.-J. Hwang College of Pharmacy, Yonsei University, 162-1 Songdo-dong, Yeonsu-gu, Inchon 406-840, South Korea
potential strategy for improvement of bioavailability in saponin delivery, orally. Keywords Ginsenoside Eudragit L100-55 Microparticle Spray-drying Mucoadhesive Oral delivery
Introduction Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), which has been used as a tonic in traditional medicine for over 2,000 years, is one of the most widely used herbal medicines worldwide (Tyler 1995). Ginseng cultivated in Korea is classified into three types, depending on how it is processed such as fresh ginseng (less than 4 years old), white ginseng (4–6 years old and dried after peeling), and red ginseng (harvested when 6 years old, steamed and dried). Red ginseng is not skinned before it is steamed or otherwise heated and subsequently dried. In the course of the steaming process, a ginseng starch is gelatinized, causing an increase in saponin content. Traditionally, a red ginseng has been used to restore and enhance normal wellbeing, and is often referred to as an adaptogenic (Coon and Ernst 2002). Ginsenoside saponins extracted from red ginseng have been regarded as the principal components manifesting the pharmacological activities. Ginsenosides have a four-ring, steroid-like structure with sugar moieties attached, and, thus far, more than 40 different ginsenosides have been identified and isolated from the root of P. ginseng (Nah and McCleskey 1994). Based on their chemical structures, ginsenosides are generally divided into two groups based on their chemical structures: panaxadiols and panaxatriols; the former includes Rb1, Rb2, Rc, Rd, Rg3, Rh2, and Rh3,
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acidic environment and enhance localization in the intestinal membranes.
Materials and methods Materials and apparatus
Fig. 1 Chemical structure of ginsenoside Rg1 and Rb1
and the latter includes Re, Rf, Rg1, Rg2, and Rh1 (Seo et al. 2005). Ginsenoside Rg1 (Fig. 1), one of the main triol saponins, processes the properties of exciting the central nervous system, anti-fatigue, and hydrolysis. Ginsenoside Rb1 (Fig. 1), which belongs to the diol saponins, showing an effective anti-inflammatory action, an obvious vasodilator effect, and a tranquilizing function to the central nervous system (Benishin et al. 1991). Red ginseng saponins are very soluble in water but poorly absorbed when administrated orally. It was reported that the amount of absorbed ginsenoside Rg1 via oral administration was within the range 1.9–20.0 % of the dose (Odani et al. 1983) and little ginsenoside Rb1 was absorbed from gastro-intestine by oral administration to rats (Takino et al. 1982). The low oral bioavailability of ginsenosides was caused by hydrolysis in the stomach acid environment (Hasegawa et al. 1996; Kanaoks et al. 1994), metabolism in the intestine, and elimination in the liver. The bioavailability of ginsenoside Rg1 and Rb1 after administration via portal vein was 50.56 and 59.49 %, respectively (Han and Fang 2006; Han et al. 2006). Currently, there are some attempts of commercial formulation of saponin including tablet and injection. However, the oral bioavailability of saponin is not satisfying and it is reported that some adverse reactions such as epistasis, allergy and even anaphylactic shock might be caused by applying saponin injection (Chen et al. 2010). In order to avoid such serious adverse reactions as well as improving the oral bioavailability of saponin, it is necessary to develop a more appropriate formulation is needed. Therefore, we formulated to ginsenoside-loaded microparticles composed of enteric and mucoadhesive polymers for improvement of oral absorption. Applying Eudragit L100-55 as enteric polymer and chitosan as mucoadhesive polymer, we expect that ginsenosides will avoid decomposition in the
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Crude saponin was provided by Korea Ginseng Co. (Daejeon, Korea). Eudragit L100-55 for enteric polymer was gifted from Evonic (Darmstadt, Germany) and ethyl cellulose from Colorcon (USA). Mannitol was purchased from Sigma-Aldrich Korea (Yong-In, Korea). Chitosan (200–500 mPa s, 0.5 % in 0.5 % acetic acid at 20 °C) was purchased from Tokyo Kasei Kogyo Co. LTD. (Tokyo, Japan). Chito-oligosaccharide was purchased from Kitto Life Co. (Kyongki-Do, Korea). All other chemicals were commercial products of analytical or reagent grade and were used without further purification. Measurement of ginsenoside Rg1 and Rb1 contents Ginsenoside Rg1 and Rb1 contents of crude saponin were analyzed by HPLC analysis, an Agilent 1100 series HPLC apparatus (Agilent, USA). Phenomenex Luna C18 column (250 9 4.6 mm, 5 lm) was used at 35 °C. A binary gradient elution system consisted of acetonitrile (A) and water (B). Separation was achieved using the following gradient program: 0–20 min, 20 % of A, 20–25 min, 25 % of A, 25–45 min, 35 % of A, 45–50 min, 50 % of A. The flow rate was at 0.8 mL/min and sample injection volume was 10 lL. The effluent was monitored at 203 nm. pH stability of Rg1 and Rb1 The stability of Rg1 and Rb1 according to pH was studied. A certain amount of saponin was dissolved into pH 1.2, 4.0 or 6.8 buffers, respectively. The concentration of Rg1 was 58.07 lg/mL and that of Rb1 was 340.2 lg/mL. The samples were placed in water bath at 37 °C, protected against gas and light. One mL of the solution were withdrawn at 1, 2 and 4 h after placing, and filtered through a 0.45 lm filter. The filtrates were analyzed for content of Rg1 and Rb1 by HPLC. Preparation of microparticles using spray-drying The ginsenoside-loaded microparticles were prepared by spray-dryer (EYELA, Japan). To prepare feed solution, the crude saponin was previously dissolved in water and Eudragit L100-55 was added. After stirring of the mixture during 15 min, ethanol was added to the mixture. The ratio of water and ethanol was 3:1 to prepare the homogenous
Mucoadhesive enteric-coating ginsenoside-loaded microparticles
solution. The previously prepared feed solution was introduced into the heated evaporator chamber with compressed hot air through the inner capillary the two flow spray nozzle, in order to produce liquid droplets. The evaporator chamber is received the flow of heated air in order to evaporate solvent from droplet and to solute in form of small particles. The formed particles were moved to the cyclone. The particles were collected at particles recovery apparatus and the gaseous stream passed through the filter and aspirated by an aspiration pump. Determination of encapsulation efficiency and particle size A certain amount of spray-dried particles equivalent to 25 mg of saponin was dissolved in 10 mL sodium phosphate buffer:methanol (90:10, v/v), sonicated for 10 min and centrifuged at 3,000 rpm for 10 min. One mL of supernatant was filtered through a membrane filter with a pore size of 0.45 lm and analyzed using HPLC. The encapsulation efficiency was calculated as below. Encapsulation efficiency ð%Þ Weight of the drug in particles ¼ 100: Weight of the feeding drugs Ten mg of spray-dried particles was dispersed in 1 mL distilled water and measured using particle size-zeta potential analyzer (ELS-8000 Particle size analyzer, Otsuka Electronics, Japan). Surface morphology of ginsenoside-loaded microparticles The morphology and surface characteristics of ginsenosideloaded microparticles were examined by scanning electron microscopy, SEM (JEOL JSM 7500 Field Emission Scanning Electron Microscope, Thermo, USA). The sample was mounted onto metal stubs using double sided adhesive tape onto which the samples were applied. The stubs were sputter-coated with gold particles in a sputter coater for 2 min. In vitro release profiles of ginsenoside from microparticles Microparticles equivalent to 75 mg of saponin were accurately weighted and placed in a vessel containing 225 mL of 0.1 N HCl. After 120 min, 75 mL of 0.2 M tri-sodium phosphate pre-equilibrated to 37 ± 0.5 °C was added to each vessel and the pH of the solution adjusted to pH 6.8 ± 0.05. The paddle method using UDT-804 (Logan
Table 1 Decomposition constant of Rg1 and Rb1 in different pH Medium
Decomposition constant Rg1
Rb1
pH 1.2
62.12
57.18
pH 4.0
16.96
16.25
pH 6.8
1.87
1.81
Instruments Corporation) was employed to assess the dissolution behavior of the microparticles. The speed of the paddle was set at 100 rpm and the temperature of the solution was 37 ± 0.5 °C. Each dissolution test was carried out in triplicate. Samples were withdrawn at 30, 120, 180, 240 and 360 min, respectively, and filtered through a 0.45 lm filter. The filtrates were analyzed for ginsenoside by HPLC. Mucoadhesion test of ginsenoside-loaded microparticles The mucoadhesion of ginsenoside-loaded microparticles was evaluated with commercially available porcine mucin particles (Takeuchi et al. 2005). It was expected that the surface property of the mucin particles (Sigma, Steinheim, Switzerland) might be changed by the adhesion of the ginsenoside-loaded microparticles if the ginsenoside-loaded microparticles has a mucoadhesive property. Mucin particle was briefly suspended in distilled water with a concentration of 1 % w/v and then mixed with an appropriate amount of ginsenoside-loaded microparticles. The occurrence of such change was detected by measuring the zeta potential (ELS-8000, Otsuka Electronics, Japan).
Results and discussion Characterization of crude saponin Ginsenoside Rg1 and Rb1 are the principal components of red ginseng saponin. To quantify ginsenoside Rg1 and Rb1 in crude saponin, stock solution of ginsenoside Rg1, Rb1 and saponin solution was analyzed by HPLC. Retention times of ginsenoside Rg1 and Rb1 were 22.83 and 28.14 (data not shown), showing a good linearity (R2 = 0.9985 and 1), respectively. The concentration range of Rg1 was 1–330 lg/mL and that of Rb1 was 0.9–720 lg/mL. Crude saponin contains 5.74 % of ginsenoside Rg1 and 6.12 % of ginsenoside Rb1. Ginsenoside Rg1 and Rb1 had degraded considerably within 4 h in pH 1.2. The decomposition constant of ginsenoside Rg1 was 62.12, 16.96 or 1.87 in pH 1.2, 4.0 or 6.8, respectively and that of Rb1 was 57.18, 16.25 or 1.81 in pH 1.2, 4.0 or 6.8, respectively (Table 1)
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J.-S. Baek et al. Table 2 Formulation of ginsenoside-loaded microparticle with different parameters of process conditions (solvent composition; ethanol: DW = 3:1 mixture)
Formulations
Saponin (mg)
Eudragit L100-55 (mg)
Concentration (mg/mL)
Outlet temperature (°C)
Inlet temperature (°C)
Flow rate (mL/min)
F1
600
1,800
25
55
110
5
F2
600
1,800
50
55
110
5
F3
600
1,800
12.5
55
105
5
F4
600
1,800
25
55
120
10
F5
600
1,800
25
55
100
F6
600
1,800
25
45
85
5
F7 F8
600 1,200
1,800 1,200
25 25
65 55
130 105
5 5
F9
250
2,250
25
55
105
5
2.5
Table 3 Formulation of ginsenoside-loaded microparticles using additional excipients (solvent composition; ethanol: DW = 3:1 mixture, feed concentration; 25 mg/mL, outlet temperature; 55 °C, flow rate; 5 mL/min) Formulations
Saponin (mg)
Eudragit L100-55 (mg)
Ethyl cellulose (mg)
F26
600
1,800
300
F27 F28
600 600
1,800 1,800
600 600
F29
600
1,800
600
suggesting that ginsenoside Rg1 was more sensitive to acid environment than ginsenoside Rb1. Therefore, saponin needed to be protected by enteric-coating to prevent the degradation in the stomach of Rg1 and Rb1 in this study. Also, it was reported that the enteric coated pellets of Panax notoginseng saponins have good acid resistance to avoid P. notoginseng saponins from degrading in gastric acid (Lai et al. 2009). Preparation and characterization of ginsenoside-loaded microparticles Ginsenoside-loaded microcapsules were prepared by the spray-drying process. First of all, the optimal condition for the preparation of ginsenoside-loaded microparticles was examined. To prepare the feed solution, the crude saponin was previously dissolved in water and then enteric-coating polymer, Eudragit L100-55 was added. After stirring of the mixture for 15 min, ethanol was added to the mixture. The ratio of water and ethanol was 3:1 to prepare the homogenous solution. The prepared feed solution was introduced into the heated evaporator chamber with compressed hot air coming through the inner capillary of the two flow spray nozzle, in order to produce liquid droplets. The evaporator chamber is received the flow of heated air in order to evaporate solvent from droplet to solute in the form of small particles. The formed particles were moved to the cyclone. The particles were collected via the particles
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Chito-oligosaccharide (mg)
Chitosan (mg)
300 300
recovery apparatus and the gaseous stream passed through the filter and aspirated by an aspiration pump. The preparation variables such as the concentration of feed solution, the ratio of saponin to polymer, flow rate, inlet temperature and outlet temperature were examined as F1–9 (Table 2). After optimizing the spray-drying process, the effect of additional excipients on ginsenoside-loaded microcapsules by spray-drying was investigated as F26–29 (Table 3). Raw material of ginsenoside seems to have crystalline (Fig. 2a) and ginsenoside after spray-drying (Fig. 2b) shows to be condensed due to high inlet temperature of spray-drying and be slightly expended during spray-drying. Eudragit L100-55 after spray-drying appears a hollow circle with various sizes. F1–3 seems to be much corrugated with very small size. It was observed that the feed concentration didn’t influence particle morphology but inlet temperature and flow rate influenced particle morphology. Also, it was suggested that the greater difference between inlet temperature and outlet temperature caused particles to be expended because a sudden decrease of temperature induced quick evaporation of solvent in particle (F4–7). The more amount of Eudragit L100-55 made wrinkle of particle thick (F8, 9). The particle size and encapsulation efficiency of ginsenoside-loaded microparticles were shown in Table 4. The particle size varied from 698.0 to 1,273.1 nm for F1–9. The particle size was decreased in order of the ratio of saponin to polymer from the lowest to the highest. However, other
Mucoadhesive enteric-coating ginsenoside-loaded microparticles
Fig. 2 SEM images of ginsenoside-loaded microparticles. a Raw saponin (9200), b saponin after spray-drying, c Eudragit L100-55 after spraydrying, d F1, e F2, f F3, g F4, h F5, i F6, j F7, k F8, and l F9 (X5K)
Table 4 Particle size and encapsulation efficiency of ginsenosideloaded microparticles Formulations
Particle size (nm)
Encapsulation efficiency (%) Rg1
Rb1
F1
860.1 ± 96.2
93.5
95.6
F2
1,245.4 ± 80.2
92.7
96.4
F3 F4
958.9 ± 35.8 786.1 ± 63.7
91.4 96.3
92.4 96.2
F5
869.2 ± 48.6
90.2
97.8
F6
894.1 ± 45.7
93.3
92.4
F7
860.8 ± 40.4
91.1
90.7
F8
1,273.1 ± 130.2
98.8
98.2
F9
698.0 ± 50.8
93.1
94.1
F26
1,350.0 ± 50.2
87.78
92.79
F27
1,420.4 ± 28.4
88.98
91.19
F28
1,781.8 ± 108.5
90.26
89.41
F29
1,801.8 ± 85.4
95.20
93.20
parameters such as the feed concentration, flow rate, inlet temperature and outlet temperature didn’t show the significant effects on the particle size. The encapsulation efficiency was in the 90.07–98.20 % range but other
variables for spray-drying did not have any significant effects on the encapsulation efficiency. The additional polymer-type excipients showed generally increased particle size from 1,350.0 ± 50.2 to 1,801.8 ± 85.4 nm compared to ginsenoside-loaded microparticles without additional excipients. Also, the encapsulation efficiency was lower in the 89.41–93.20 % range compared to ginsenoside-loaded microparticles without additional excipients. In vitro release profiles of ginsenoside-loaded microparticles An enteric dosage form should not allow significant release of drug in the stomach, but provide rapid dissolution of drug in the intestine. Hydroxypropyl methyl cellulose phthalate, polyvinyl acetate phthalate and methacrylic acid/ methyl methacrylate copolymers are commonly used for enteric coating. These polymers are weak acids, possessing carboxyl groups in a substantial proportion of their monomeric units. Rapid dissolution of these polymers requires pH values that are much higher than those normally present in the stomach (Bozdag et al. 1999). In this study, ginsenoside-loaded microparticles were spray-dried
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Fig. 3 In vitro release of Rg1 and Rb1 in pH 1.2 from ginsenosideloaded microparticles by spray-drying with different process conditions
with Eudragit L100-55 soluble above pH 5.5. When a ratio of Eudragit L100-55 to saponin (F8) was 1:1, the released % of Rg1 or Rb1 in pH 1.2 was 43.0 or 45.0 %. When a ratio of Eudragit L100-55 to saponin (F1) was increased to 1:3, the released % of Rg1 or Rb1 in pH 1.2 was 28.2 or 28.5 %. When increased to 1:9 (F9), the released % of Rg1 or Rb1 in pH 1.2 was 25.2 or 24.6 %. As the ratio of Eudragit L100-55 to saponin was increased, the released % of Rg1 and Rb1 in pH 1.2 was decreased. On the other hand, when the feed concentration was 12.5 mg/mL (F3), the released % of Rg1 or Rb1 in pH 1.2 was 25.7 or 24.1 %. When the feed concentration was increased to 25 mg/mL (F1), the released % of Rg1 or Rb1 in pH 1.2 was 28.2 or 28.5 %. When increased to 50 mg/mL (F2), the released % of Rg1 or Rb1 in pH 1.2 was 31.9 or 35.7 %. As the feed concentration was increased, the released % of Rg1 and Rb1 in pH 1.2 was increased implying no encapsulating the saponin with enteric-coating polymer, Eudragit L100-55. However, outlet temperature or flow rate didn’t have significantly effect on the release of Rg1 or Rb1 in pH 1.2 (Fig. 3). On the other hand, to decrease the dissolution of Rg1 and Rb1 from gastric pH and increase the bioavailability of Rg1 and Rb1 by mucoadhesion, the additional polymertype excipients such as ethyl cellulose, chito-oligosaccharide and chitosan were used (Fig. 4). Ethyl cellulose was hydrophobic polymer to prepare sustained release products including film coated tablet (Rowe 1992), microsphere (Akbuga 1991; Eldrige et al. 1990) and matrix tablet for both soluble and poorly soluble drugs (Shaikh et al. 1987). For drugs with high water solubility, hydrophobic polymer is suitable along with a hydrophilic matrix for developing sustained release dosage forms (Enayatifard et al. 2009). When 300 mg of ethyl cellulose was added (F26), the released % of Rg1 or Rb1 was 21.89 or 26.14 % in pH 1.2 and 79 or 84.96 % at 4 h in pH 6.8. When the amount of ethyl cellulose was increased to 600 mg (F27), the released
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Fig. 4 In vitro release of Rg1 and Rb1 from ginsenoside-loaded microparticles using additional excipients in pH 1.2 and 6.8. a Rg1, and b Rb1
% of Rg1 or Rb1 was 16.18 or 19.93 % in pH 1.2 and 80.08 or 82.07 % at 4 h in pH 6.8. This may be attributed to decreased penetration of the solvent molecules in the presence of the hydrophobic polymer, leading to reduced diffusion of the drug from the matrix. According to penetration theory, when a matrix is composed of a water-soluble drug and a water-insoluble polymer, release of drug occur by dissolution of the active ingredient through capillaries composed of linking drug particle clusters and the pore network (Holman and Leuenberger 1988; Leuenberger et al. 1987). On the other hand, chitosan is a hydrophilic, biodegradable and biocompatible positively charged polysaccharide of low toxicity, which in recent years has found applications in cosmetic, biotechnology and drug delivery systems. This polymer has mucoadhesive properties due to its positive charges at neutral pH that enable an electrostatic interaction with mucous or a negatively charged mucosal surface (Lim et al. 2012). When chito-oligosaccharide was additionally added (F28), the released % of Rg1 or Rb1 was 21.3 or 30.4 % in pH 1.2 and 81.23 or 80.47 % at 4 h in pH 6.8. When additional chitosan was added (F29), the released % of Rg1 or Rb1 was 20.63 or 28.47 % in pH 1.2 and 86.2 or 84 % at 4 h in pH 6.8. The increased release of Rg1 or Rb1 in pH
Mucoadhesive enteric-coating ginsenoside-loaded microparticles
concentration were the critical factors to dissolution. When ethyl cellulose was additionally added, the release of Rg1 or Rb1 in pH 1.2 was decreased. When chito-oligosaccharide or chitosan was additionally added, the zetapotential with mucin particles was increased suggesting ginsenoside-loaded microparticles have a mucoadhesive property. In future, these ginsenoside-loaded microparticles will be tested to identify whether the oral absorption of ginsenoside Rg1 and Rb1 could be increased by in vivo test. Fig. 5 Zeta-potential of ginsenoside-loaded microparticles incubated with mucin particles
1.2 and 6.8 was due to hydrophilic property of chito-oligosaccharide or chitosan. Mucoadhesion test of ginsenoside loaded microparticles When the mucin particles suspensions were mixed with the ginsenoside-loaded microparticles, the zeta-potential of the mucin particles was changed as shown in Fig. 5. The zeta potential of 1 % mucin particle suspensions alone (pH 4.1) or saponin solution was -13.2 ± 3 or -10.5 ± 2 mV, respectively and that of 1 % mucin particle suspensions when simply incubated with 10 mg of chitosan was -1.3 ± 2 mV, suggesting the value of zeta potential was increased due to adhesion of chitosan into mucin particle. The zeta-potential of F26 and F27 mixed with mucin particles containing ethyl cellulose showed the lower values than F1 without ethyl cellulose, it might be contributed to characteristics of ethyl cellulose possessing carboxyl groups. The zeta potential values of each F28 or F29 formulation alone was 3.05 ± 1.2 or 7.12 ± 2.9, respectively. After interaction between mucin particles with F28 or F29, the zeta potential values was 0.34 ± 2.8 or 5.12 ± 3.4, respectively. The addition of chito-oligosaccharide or chitosan (F28 or F29) resulted in change to positive charge suggesting that this polymer has mucoadhesive properties due to its positive charges at neutral pH and that enable an electrostatic interaction with mucous or a negatively charged mucosal surface (Takeuchi et al. 2005).
Conclusion The ginsenoside-loaded microparticles could be successfully prepared by spray-drying process using enteric-coating polymer and mucoadhesive polymer. When the preparation variables during spray-drying were examined, both the ratio of saponin to Eudragit L100-55 and feed
Acknowledgments This work was supported by the Priority Research Centers Program (2009-0093815) through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology. Also, we thank Ms. Jiyoon Song for proofreading this manuscript.
References Akbuga, J. 1991. Furosemide-loaded ethyl cellulose microspheres prepared by spherical crystallization technique. International Journal of Pharmaceutics 76: 193–198. Benishin, C.G., R. Lee, L.C. Wang, and H.J. Liu. 1991. Effects of ginsenoside Rb1 on central cholinergic metabolism. Pharmacology 42: 223–229. Bozdag, S., S. Calis, and M. Sumnu. 1999. Formulation and stability evaluation of enteric-coated omeprazole formulations. STP Pharma Sciences 9: 321–327. Chen, Y.M., X.W. Li, and G.H. Zhu. 2010. Analysis of 103 cases with adverse drug reactions induced by Panax notoginseng saponins injection. Chinese Herbal Medicine: Materia Medica 35: 237–239. Coon, J.T., and E. Ernst. 2002. Panax ginseng: A systematic review of adverse effects and drug interactions. Drug Safety 25: 323–344. Eldrige, J.H., C.J. Hommond, J.A. Meulbroek, J.K. Staas, R.M. Gilley, and T.R. Tice. 1990. Controlled vaccine release in the gut-associated lymphoid tissues. Part 1. Orally administered biodegradable microspheres target the Peter’s patches. Journal of Controlled Release 11: 205–214. Enayatifard, R., M. Saeedi, J. Akbari, and Y.H. Tabatabaee. 2009. Effect of hydroxypropyl methylcellulose and ethyl cellulose content on release profile and kinetics of diltiazem HCl from matrices. Tropical Journal of Pharmaceutical Research 8: 425–432. Han, M., and X.L. Fang. 2006. Difference in oral absorption of ginsenoside Rg1 between in vitro and in vivo models. Acta Pharmacologica Sinica 27: 499–505. Han, M., X. Sha, Y. Wu, and X. Fang. 2006. Oral absorption of ginsenoside Rb1 using in vitro and in vivo models. Planta Medica 72: 398–404. Hasegawa, H., J.H. Sung, S. Matsumiya, and M. Uchiyama. 1996. Main ginseng saponin metabolites formed by intestinal bacteria. Planta Medica 62: 453–457. Holman, L.E., and H. Leuenberger. 1988. The relationship between solid fraction and mechanical properties of compacts—The percolation theory model approach. International Journal of Pharmaceutics 46: 35–44. Kanaoks, M., T. Akao, and K. Kobashi. 1994. Metabolism of ginseng saponins, ginsenoside, by human intestinal flora. Journal of Traditional Medicine 11: 241–246.
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J.-S. Baek et al. Lai, C., Q. Zheng, M. Yang, P. Hu, J. Pan, and C. Peng. 2009. Preparation and prescription of enteric coated pellets of Panax notoginseng saponins pellets. Zhongguo Zhong Yao Za Zhi 34: 1359–1363. Leuenberger, H., B.D. Rohera, and C. Haas. 1987. Percolation theory—A novel approach to solid dosage form design. International Journal of Pharmaceutics 38: 109–115. Lim, J.H., S.K. You, J.S. Baek, C.J. Hwang, Y.G. Na, S.C. Shin, and C.W. Cho. 2012. Surface-modified gemcitabine with mucoadhesive polymer for oral delivery. Journal of Microencapsulation 29: 487–496. Nah, S.Y., and E.W. McCleskey. 1994. Ginseng root extract inhibits calcium channels in rat sensory neurons through a similar path, but different receptor, as mu-type opioids. Journal of Ethnopharmacology 42: 45–51. Odani, T., H. Tanizawa, and Y. Takino. 1983. Studies on the absorption, distribution excretion and metabolism of ginseng saponins II. Chemical and Pharmaceutical Bulletin 31: 292–298. Rowe, R.C. 1992. Molecular weight dependence of the properties of ethyl cellulose and hydroxypropyl methyl cellulose films. International Journal of Pharmaceutics 88: 405–408.
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Seo, J.Y., J.H. Lee, N.W. Kim, E. Her, S.H. Chang, N.Y. Ko, Y.H. Yoo, J.W. Kim, D.W. Seo, J.W. Han, Y.M. Kim, and W.S. Choi. 2005. Effect of a fermented ginseng extract, BST204, on the expression of cyclooxygenase-2 in murine macrophages. International Immunopharmacology 5: 929–936. Shaikh, N.A., S.E. Abidi, and L.H. Block. 1987. Evaluation of ethyl cellulose as a matrix for prolonged release formulations Part 1. Water soluble drugs-acetaminophen theophylline. Drug Development and Industrial Pharmacy 14: 1345–1369. Takeuchi, H., J. Thongborisute, and Y. Matsui. 2005. Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systems. Advanced Drug Delivery Reviews 57: 1583–1594. Takino, Y., T. Odani, H. Tanizawa, and T. Hayashi. 1982. Studies on the absorption, distribution, excretion and metabolism of ginseng saponin I. Chemical and Pharmaceutical Bulletin 30: 2196–2201. Tyler, V.E. 1995. Herbal remedies. The Journal of Pharmacy Technology 11: 214–220.
RESEARCH ARTICLE
Preparation and characterization of mucoadhesive enteric-coating ginsenoside-loaded microparticles Jong-Suep Baek • Won-Gi Yeon • Cho-A Lee Sung-Joo Hwang • Jeong-Sook Park • Dong-Chool Kim • Cheong-Weon Cho
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Received: 1 November 2013 / Accepted: 7 April 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Ginsenoside saponins are phytochemically extracted from red ginseng and have been regarded as the principal components manifesting the pharmacologic activities. Saponins are very soluble in water but poorly absorbed when orally administrated. Moreover, they have some disadvantages including the decomposition in acid medium. The aim of this study was to develop oral formulation of ginsenosides composed of enteric-coating polymer and mucoadhesive polymer considering the low stability in acid medium and the low permeability of saponins. Ginsenoside-loaded microparticles were prepared by spray dryer. The influences of various parameters such as the ratio of saponin to polymer, feed concentration, feed rate, inlet/outlet temperature and additional excipients during spray-drying were investigated. In vitro release profile of ginsenoside-loaded microparticles using additional excipients, ginsenoside saponin Rg1 or Rb1 showed an 18 or 13 % release in pH 1.2 when ethyl cellulose was added. Also, ginsenoside-loaded microparticles exhibited mucoadhesive properties in the presence of chitosan. The application of these polymers is being considered as the Jong-Suep Baek and Won-Gi Yeon have equally contributed to this work. J.-S. Baek W.-G. Yeon C.-A. Lee J.-S. Park D.-C. Kim (&) C.-W. Cho (&) College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, 220 Gung-dong, Yuseong-gu, Taejon 305-764, South Korea e-mail: [email protected] C.-W. Cho e-mail: [email protected] S.-J. Hwang College of Pharmacy, Yonsei University, 162-1 Songdo-dong, Yeonsu-gu, Inchon 406-840, South Korea
potential strategy for improvement of bioavailability in saponin delivery, orally. Keywords Ginsenoside Eudragit L100-55 Microparticle Spray-drying Mucoadhesive Oral delivery
Introduction Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), which has been used as a tonic in traditional medicine for over 2,000 years, is one of the most widely used herbal medicines worldwide (Tyler 1995). Ginseng cultivated in Korea is classified into three types, depending on how it is processed such as fresh ginseng (less than 4 years old), white ginseng (4–6 years old and dried after peeling), and red ginseng (harvested when 6 years old, steamed and dried). Red ginseng is not skinned before it is steamed or otherwise heated and subsequently dried. In the course of the steaming process, a ginseng starch is gelatinized, causing an increase in saponin content. Traditionally, a red ginseng has been used to restore and enhance normal wellbeing, and is often referred to as an adaptogenic (Coon and Ernst 2002). Ginsenoside saponins extracted from red ginseng have been regarded as the principal components manifesting the pharmacological activities. Ginsenosides have a four-ring, steroid-like structure with sugar moieties attached, and, thus far, more than 40 different ginsenosides have been identified and isolated from the root of P. ginseng (Nah and McCleskey 1994). Based on their chemical structures, ginsenosides are generally divided into two groups based on their chemical structures: panaxadiols and panaxatriols; the former includes Rb1, Rb2, Rc, Rd, Rg3, Rh2, and Rh3,
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acidic environment and enhance localization in the intestinal membranes.
Materials and methods Materials and apparatus
Fig. 1 Chemical structure of ginsenoside Rg1 and Rb1
and the latter includes Re, Rf, Rg1, Rg2, and Rh1 (Seo et al. 2005). Ginsenoside Rg1 (Fig. 1), one of the main triol saponins, processes the properties of exciting the central nervous system, anti-fatigue, and hydrolysis. Ginsenoside Rb1 (Fig. 1), which belongs to the diol saponins, showing an effective anti-inflammatory action, an obvious vasodilator effect, and a tranquilizing function to the central nervous system (Benishin et al. 1991). Red ginseng saponins are very soluble in water but poorly absorbed when administrated orally. It was reported that the amount of absorbed ginsenoside Rg1 via oral administration was within the range 1.9–20.0 % of the dose (Odani et al. 1983) and little ginsenoside Rb1 was absorbed from gastro-intestine by oral administration to rats (Takino et al. 1982). The low oral bioavailability of ginsenosides was caused by hydrolysis in the stomach acid environment (Hasegawa et al. 1996; Kanaoks et al. 1994), metabolism in the intestine, and elimination in the liver. The bioavailability of ginsenoside Rg1 and Rb1 after administration via portal vein was 50.56 and 59.49 %, respectively (Han and Fang 2006; Han et al. 2006). Currently, there are some attempts of commercial formulation of saponin including tablet and injection. However, the oral bioavailability of saponin is not satisfying and it is reported that some adverse reactions such as epistasis, allergy and even anaphylactic shock might be caused by applying saponin injection (Chen et al. 2010). In order to avoid such serious adverse reactions as well as improving the oral bioavailability of saponin, it is necessary to develop a more appropriate formulation is needed. Therefore, we formulated to ginsenoside-loaded microparticles composed of enteric and mucoadhesive polymers for improvement of oral absorption. Applying Eudragit L100-55 as enteric polymer and chitosan as mucoadhesive polymer, we expect that ginsenosides will avoid decomposition in the
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Crude saponin was provided by Korea Ginseng Co. (Daejeon, Korea). Eudragit L100-55 for enteric polymer was gifted from Evonic (Darmstadt, Germany) and ethyl cellulose from Colorcon (USA). Mannitol was purchased from Sigma-Aldrich Korea (Yong-In, Korea). Chitosan (200–500 mPa s, 0.5 % in 0.5 % acetic acid at 20 °C) was purchased from Tokyo Kasei Kogyo Co. LTD. (Tokyo, Japan). Chito-oligosaccharide was purchased from Kitto Life Co. (Kyongki-Do, Korea). All other chemicals were commercial products of analytical or reagent grade and were used without further purification. Measurement of ginsenoside Rg1 and Rb1 contents Ginsenoside Rg1 and Rb1 contents of crude saponin were analyzed by HPLC analysis, an Agilent 1100 series HPLC apparatus (Agilent, USA). Phenomenex Luna C18 column (250 9 4.6 mm, 5 lm) was used at 35 °C. A binary gradient elution system consisted of acetonitrile (A) and water (B). Separation was achieved using the following gradient program: 0–20 min, 20 % of A, 20–25 min, 25 % of A, 25–45 min, 35 % of A, 45–50 min, 50 % of A. The flow rate was at 0.8 mL/min and sample injection volume was 10 lL. The effluent was monitored at 203 nm. pH stability of Rg1 and Rb1 The stability of Rg1 and Rb1 according to pH was studied. A certain amount of saponin was dissolved into pH 1.2, 4.0 or 6.8 buffers, respectively. The concentration of Rg1 was 58.07 lg/mL and that of Rb1 was 340.2 lg/mL. The samples were placed in water bath at 37 °C, protected against gas and light. One mL of the solution were withdrawn at 1, 2 and 4 h after placing, and filtered through a 0.45 lm filter. The filtrates were analyzed for content of Rg1 and Rb1 by HPLC. Preparation of microparticles using spray-drying The ginsenoside-loaded microparticles were prepared by spray-dryer (EYELA, Japan). To prepare feed solution, the crude saponin was previously dissolved in water and Eudragit L100-55 was added. After stirring of the mixture during 15 min, ethanol was added to the mixture. The ratio of water and ethanol was 3:1 to prepare the homogenous
Mucoadhesive enteric-coating ginsenoside-loaded microparticles
solution. The previously prepared feed solution was introduced into the heated evaporator chamber with compressed hot air through the inner capillary the two flow spray nozzle, in order to produce liquid droplets. The evaporator chamber is received the flow of heated air in order to evaporate solvent from droplet and to solute in form of small particles. The formed particles were moved to the cyclone. The particles were collected at particles recovery apparatus and the gaseous stream passed through the filter and aspirated by an aspiration pump. Determination of encapsulation efficiency and particle size A certain amount of spray-dried particles equivalent to 25 mg of saponin was dissolved in 10 mL sodium phosphate buffer:methanol (90:10, v/v), sonicated for 10 min and centrifuged at 3,000 rpm for 10 min. One mL of supernatant was filtered through a membrane filter with a pore size of 0.45 lm and analyzed using HPLC. The encapsulation efficiency was calculated as below. Encapsulation efficiency ð%Þ Weight of the drug in particles ¼ 100: Weight of the feeding drugs Ten mg of spray-dried particles was dispersed in 1 mL distilled water and measured using particle size-zeta potential analyzer (ELS-8000 Particle size analyzer, Otsuka Electronics, Japan). Surface morphology of ginsenoside-loaded microparticles The morphology and surface characteristics of ginsenosideloaded microparticles were examined by scanning electron microscopy, SEM (JEOL JSM 7500 Field Emission Scanning Electron Microscope, Thermo, USA). The sample was mounted onto metal stubs using double sided adhesive tape onto which the samples were applied. The stubs were sputter-coated with gold particles in a sputter coater for 2 min. In vitro release profiles of ginsenoside from microparticles Microparticles equivalent to 75 mg of saponin were accurately weighted and placed in a vessel containing 225 mL of 0.1 N HCl. After 120 min, 75 mL of 0.2 M tri-sodium phosphate pre-equilibrated to 37 ± 0.5 °C was added to each vessel and the pH of the solution adjusted to pH 6.8 ± 0.05. The paddle method using UDT-804 (Logan
Table 1 Decomposition constant of Rg1 and Rb1 in different pH Medium
Decomposition constant Rg1
Rb1
pH 1.2
62.12
57.18
pH 4.0
16.96
16.25
pH 6.8
1.87
1.81
Instruments Corporation) was employed to assess the dissolution behavior of the microparticles. The speed of the paddle was set at 100 rpm and the temperature of the solution was 37 ± 0.5 °C. Each dissolution test was carried out in triplicate. Samples were withdrawn at 30, 120, 180, 240 and 360 min, respectively, and filtered through a 0.45 lm filter. The filtrates were analyzed for ginsenoside by HPLC. Mucoadhesion test of ginsenoside-loaded microparticles The mucoadhesion of ginsenoside-loaded microparticles was evaluated with commercially available porcine mucin particles (Takeuchi et al. 2005). It was expected that the surface property of the mucin particles (Sigma, Steinheim, Switzerland) might be changed by the adhesion of the ginsenoside-loaded microparticles if the ginsenoside-loaded microparticles has a mucoadhesive property. Mucin particle was briefly suspended in distilled water with a concentration of 1 % w/v and then mixed with an appropriate amount of ginsenoside-loaded microparticles. The occurrence of such change was detected by measuring the zeta potential (ELS-8000, Otsuka Electronics, Japan).
Results and discussion Characterization of crude saponin Ginsenoside Rg1 and Rb1 are the principal components of red ginseng saponin. To quantify ginsenoside Rg1 and Rb1 in crude saponin, stock solution of ginsenoside Rg1, Rb1 and saponin solution was analyzed by HPLC. Retention times of ginsenoside Rg1 and Rb1 were 22.83 and 28.14 (data not shown), showing a good linearity (R2 = 0.9985 and 1), respectively. The concentration range of Rg1 was 1–330 lg/mL and that of Rb1 was 0.9–720 lg/mL. Crude saponin contains 5.74 % of ginsenoside Rg1 and 6.12 % of ginsenoside Rb1. Ginsenoside Rg1 and Rb1 had degraded considerably within 4 h in pH 1.2. The decomposition constant of ginsenoside Rg1 was 62.12, 16.96 or 1.87 in pH 1.2, 4.0 or 6.8, respectively and that of Rb1 was 57.18, 16.25 or 1.81 in pH 1.2, 4.0 or 6.8, respectively (Table 1)
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J.-S. Baek et al. Table 2 Formulation of ginsenoside-loaded microparticle with different parameters of process conditions (solvent composition; ethanol: DW = 3:1 mixture)
Formulations
Saponin (mg)
Eudragit L100-55 (mg)
Concentration (mg/mL)
Outlet temperature (°C)
Inlet temperature (°C)
Flow rate (mL/min)
F1
600
1,800
25
55
110
5
F2
600
1,800
50
55
110
5
F3
600
1,800
12.5
55
105
5
F4
600
1,800
25
55
120
10
F5
600
1,800
25
55
100
F6
600
1,800
25
45
85
5
F7 F8
600 1,200
1,800 1,200
25 25
65 55
130 105
5 5
F9
250
2,250
25
55
105
5
2.5
Table 3 Formulation of ginsenoside-loaded microparticles using additional excipients (solvent composition; ethanol: DW = 3:1 mixture, feed concentration; 25 mg/mL, outlet temperature; 55 °C, flow rate; 5 mL/min) Formulations
Saponin (mg)
Eudragit L100-55 (mg)
Ethyl cellulose (mg)
F26
600
1,800
300
F27 F28
600 600
1,800 1,800
600 600
F29
600
1,800
600
suggesting that ginsenoside Rg1 was more sensitive to acid environment than ginsenoside Rb1. Therefore, saponin needed to be protected by enteric-coating to prevent the degradation in the stomach of Rg1 and Rb1 in this study. Also, it was reported that the enteric coated pellets of Panax notoginseng saponins have good acid resistance to avoid P. notoginseng saponins from degrading in gastric acid (Lai et al. 2009). Preparation and characterization of ginsenoside-loaded microparticles Ginsenoside-loaded microcapsules were prepared by the spray-drying process. First of all, the optimal condition for the preparation of ginsenoside-loaded microparticles was examined. To prepare the feed solution, the crude saponin was previously dissolved in water and then enteric-coating polymer, Eudragit L100-55 was added. After stirring of the mixture for 15 min, ethanol was added to the mixture. The ratio of water and ethanol was 3:1 to prepare the homogenous solution. The prepared feed solution was introduced into the heated evaporator chamber with compressed hot air coming through the inner capillary of the two flow spray nozzle, in order to produce liquid droplets. The evaporator chamber is received the flow of heated air in order to evaporate solvent from droplet to solute in the form of small particles. The formed particles were moved to the cyclone. The particles were collected via the particles
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Chito-oligosaccharide (mg)
Chitosan (mg)
300 300
recovery apparatus and the gaseous stream passed through the filter and aspirated by an aspiration pump. The preparation variables such as the concentration of feed solution, the ratio of saponin to polymer, flow rate, inlet temperature and outlet temperature were examined as F1–9 (Table 2). After optimizing the spray-drying process, the effect of additional excipients on ginsenoside-loaded microcapsules by spray-drying was investigated as F26–29 (Table 3). Raw material of ginsenoside seems to have crystalline (Fig. 2a) and ginsenoside after spray-drying (Fig. 2b) shows to be condensed due to high inlet temperature of spray-drying and be slightly expended during spray-drying. Eudragit L100-55 after spray-drying appears a hollow circle with various sizes. F1–3 seems to be much corrugated with very small size. It was observed that the feed concentration didn’t influence particle morphology but inlet temperature and flow rate influenced particle morphology. Also, it was suggested that the greater difference between inlet temperature and outlet temperature caused particles to be expended because a sudden decrease of temperature induced quick evaporation of solvent in particle (F4–7). The more amount of Eudragit L100-55 made wrinkle of particle thick (F8, 9). The particle size and encapsulation efficiency of ginsenoside-loaded microparticles were shown in Table 4. The particle size varied from 698.0 to 1,273.1 nm for F1–9. The particle size was decreased in order of the ratio of saponin to polymer from the lowest to the highest. However, other
Mucoadhesive enteric-coating ginsenoside-loaded microparticles
Fig. 2 SEM images of ginsenoside-loaded microparticles. a Raw saponin (9200), b saponin after spray-drying, c Eudragit L100-55 after spraydrying, d F1, e F2, f F3, g F4, h F5, i F6, j F7, k F8, and l F9 (X5K)
Table 4 Particle size and encapsulation efficiency of ginsenosideloaded microparticles Formulations
Particle size (nm)
Encapsulation efficiency (%) Rg1
Rb1
F1
860.1 ± 96.2
93.5
95.6
F2
1,245.4 ± 80.2
92.7
96.4
F3 F4
958.9 ± 35.8 786.1 ± 63.7
91.4 96.3
92.4 96.2
F5
869.2 ± 48.6
90.2
97.8
F6
894.1 ± 45.7
93.3
92.4
F7
860.8 ± 40.4
91.1
90.7
F8
1,273.1 ± 130.2
98.8
98.2
F9
698.0 ± 50.8
93.1
94.1
F26
1,350.0 ± 50.2
87.78
92.79
F27
1,420.4 ± 28.4
88.98
91.19
F28
1,781.8 ± 108.5
90.26
89.41
F29
1,801.8 ± 85.4
95.20
93.20
parameters such as the feed concentration, flow rate, inlet temperature and outlet temperature didn’t show the significant effects on the particle size. The encapsulation efficiency was in the 90.07–98.20 % range but other
variables for spray-drying did not have any significant effects on the encapsulation efficiency. The additional polymer-type excipients showed generally increased particle size from 1,350.0 ± 50.2 to 1,801.8 ± 85.4 nm compared to ginsenoside-loaded microparticles without additional excipients. Also, the encapsulation efficiency was lower in the 89.41–93.20 % range compared to ginsenoside-loaded microparticles without additional excipients. In vitro release profiles of ginsenoside-loaded microparticles An enteric dosage form should not allow significant release of drug in the stomach, but provide rapid dissolution of drug in the intestine. Hydroxypropyl methyl cellulose phthalate, polyvinyl acetate phthalate and methacrylic acid/ methyl methacrylate copolymers are commonly used for enteric coating. These polymers are weak acids, possessing carboxyl groups in a substantial proportion of their monomeric units. Rapid dissolution of these polymers requires pH values that are much higher than those normally present in the stomach (Bozdag et al. 1999). In this study, ginsenoside-loaded microparticles were spray-dried
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Fig. 3 In vitro release of Rg1 and Rb1 in pH 1.2 from ginsenosideloaded microparticles by spray-drying with different process conditions
with Eudragit L100-55 soluble above pH 5.5. When a ratio of Eudragit L100-55 to saponin (F8) was 1:1, the released % of Rg1 or Rb1 in pH 1.2 was 43.0 or 45.0 %. When a ratio of Eudragit L100-55 to saponin (F1) was increased to 1:3, the released % of Rg1 or Rb1 in pH 1.2 was 28.2 or 28.5 %. When increased to 1:9 (F9), the released % of Rg1 or Rb1 in pH 1.2 was 25.2 or 24.6 %. As the ratio of Eudragit L100-55 to saponin was increased, the released % of Rg1 and Rb1 in pH 1.2 was decreased. On the other hand, when the feed concentration was 12.5 mg/mL (F3), the released % of Rg1 or Rb1 in pH 1.2 was 25.7 or 24.1 %. When the feed concentration was increased to 25 mg/mL (F1), the released % of Rg1 or Rb1 in pH 1.2 was 28.2 or 28.5 %. When increased to 50 mg/mL (F2), the released % of Rg1 or Rb1 in pH 1.2 was 31.9 or 35.7 %. As the feed concentration was increased, the released % of Rg1 and Rb1 in pH 1.2 was increased implying no encapsulating the saponin with enteric-coating polymer, Eudragit L100-55. However, outlet temperature or flow rate didn’t have significantly effect on the release of Rg1 or Rb1 in pH 1.2 (Fig. 3). On the other hand, to decrease the dissolution of Rg1 and Rb1 from gastric pH and increase the bioavailability of Rg1 and Rb1 by mucoadhesion, the additional polymertype excipients such as ethyl cellulose, chito-oligosaccharide and chitosan were used (Fig. 4). Ethyl cellulose was hydrophobic polymer to prepare sustained release products including film coated tablet (Rowe 1992), microsphere (Akbuga 1991; Eldrige et al. 1990) and matrix tablet for both soluble and poorly soluble drugs (Shaikh et al. 1987). For drugs with high water solubility, hydrophobic polymer is suitable along with a hydrophilic matrix for developing sustained release dosage forms (Enayatifard et al. 2009). When 300 mg of ethyl cellulose was added (F26), the released % of Rg1 or Rb1 was 21.89 or 26.14 % in pH 1.2 and 79 or 84.96 % at 4 h in pH 6.8. When the amount of ethyl cellulose was increased to 600 mg (F27), the released
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Fig. 4 In vitro release of Rg1 and Rb1 from ginsenoside-loaded microparticles using additional excipients in pH 1.2 and 6.8. a Rg1, and b Rb1
% of Rg1 or Rb1 was 16.18 or 19.93 % in pH 1.2 and 80.08 or 82.07 % at 4 h in pH 6.8. This may be attributed to decreased penetration of the solvent molecules in the presence of the hydrophobic polymer, leading to reduced diffusion of the drug from the matrix. According to penetration theory, when a matrix is composed of a water-soluble drug and a water-insoluble polymer, release of drug occur by dissolution of the active ingredient through capillaries composed of linking drug particle clusters and the pore network (Holman and Leuenberger 1988; Leuenberger et al. 1987). On the other hand, chitosan is a hydrophilic, biodegradable and biocompatible positively charged polysaccharide of low toxicity, which in recent years has found applications in cosmetic, biotechnology and drug delivery systems. This polymer has mucoadhesive properties due to its positive charges at neutral pH that enable an electrostatic interaction with mucous or a negatively charged mucosal surface (Lim et al. 2012). When chito-oligosaccharide was additionally added (F28), the released % of Rg1 or Rb1 was 21.3 or 30.4 % in pH 1.2 and 81.23 or 80.47 % at 4 h in pH 6.8. When additional chitosan was added (F29), the released % of Rg1 or Rb1 was 20.63 or 28.47 % in pH 1.2 and 86.2 or 84 % at 4 h in pH 6.8. The increased release of Rg1 or Rb1 in pH
Mucoadhesive enteric-coating ginsenoside-loaded microparticles
concentration were the critical factors to dissolution. When ethyl cellulose was additionally added, the release of Rg1 or Rb1 in pH 1.2 was decreased. When chito-oligosaccharide or chitosan was additionally added, the zetapotential with mucin particles was increased suggesting ginsenoside-loaded microparticles have a mucoadhesive property. In future, these ginsenoside-loaded microparticles will be tested to identify whether the oral absorption of ginsenoside Rg1 and Rb1 could be increased by in vivo test. Fig. 5 Zeta-potential of ginsenoside-loaded microparticles incubated with mucin particles
1.2 and 6.8 was due to hydrophilic property of chito-oligosaccharide or chitosan. Mucoadhesion test of ginsenoside loaded microparticles When the mucin particles suspensions were mixed with the ginsenoside-loaded microparticles, the zeta-potential of the mucin particles was changed as shown in Fig. 5. The zeta potential of 1 % mucin particle suspensions alone (pH 4.1) or saponin solution was -13.2 ± 3 or -10.5 ± 2 mV, respectively and that of 1 % mucin particle suspensions when simply incubated with 10 mg of chitosan was -1.3 ± 2 mV, suggesting the value of zeta potential was increased due to adhesion of chitosan into mucin particle. The zeta-potential of F26 and F27 mixed with mucin particles containing ethyl cellulose showed the lower values than F1 without ethyl cellulose, it might be contributed to characteristics of ethyl cellulose possessing carboxyl groups. The zeta potential values of each F28 or F29 formulation alone was 3.05 ± 1.2 or 7.12 ± 2.9, respectively. After interaction between mucin particles with F28 or F29, the zeta potential values was 0.34 ± 2.8 or 5.12 ± 3.4, respectively. The addition of chito-oligosaccharide or chitosan (F28 or F29) resulted in change to positive charge suggesting that this polymer has mucoadhesive properties due to its positive charges at neutral pH and that enable an electrostatic interaction with mucous or a negatively charged mucosal surface (Takeuchi et al. 2005).
Conclusion The ginsenoside-loaded microparticles could be successfully prepared by spray-drying process using enteric-coating polymer and mucoadhesive polymer. When the preparation variables during spray-drying were examined, both the ratio of saponin to Eudragit L100-55 and feed
Acknowledgments This work was supported by the Priority Research Centers Program (2009-0093815) through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology. Also, we thank Ms. Jiyoon Song for proofreading this manuscript.
References Akbuga, J. 1991. Furosemide-loaded ethyl cellulose microspheres prepared by spherical crystallization technique. International Journal of Pharmaceutics 76: 193–198. Benishin, C.G., R. Lee, L.C. Wang, and H.J. Liu. 1991. Effects of ginsenoside Rb1 on central cholinergic metabolism. Pharmacology 42: 223–229. Bozdag, S., S. Calis, and M. Sumnu. 1999. Formulation and stability evaluation of enteric-coated omeprazole formulations. STP Pharma Sciences 9: 321–327. Chen, Y.M., X.W. Li, and G.H. Zhu. 2010. Analysis of 103 cases with adverse drug reactions induced by Panax notoginseng saponins injection. Chinese Herbal Medicine: Materia Medica 35: 237–239. Coon, J.T., and E. Ernst. 2002. Panax ginseng: A systematic review of adverse effects and drug interactions. Drug Safety 25: 323–344. Eldrige, J.H., C.J. Hommond, J.A. Meulbroek, J.K. Staas, R.M. Gilley, and T.R. Tice. 1990. Controlled vaccine release in the gut-associated lymphoid tissues. Part 1. Orally administered biodegradable microspheres target the Peter’s patches. Journal of Controlled Release 11: 205–214. Enayatifard, R., M. Saeedi, J. Akbari, and Y.H. Tabatabaee. 2009. Effect of hydroxypropyl methylcellulose and ethyl cellulose content on release profile and kinetics of diltiazem HCl from matrices. Tropical Journal of Pharmaceutical Research 8: 425–432. Han, M., and X.L. Fang. 2006. Difference in oral absorption of ginsenoside Rg1 between in vitro and in vivo models. Acta Pharmacologica Sinica 27: 499–505. Han, M., X. Sha, Y. Wu, and X. Fang. 2006. Oral absorption of ginsenoside Rb1 using in vitro and in vivo models. Planta Medica 72: 398–404. Hasegawa, H., J.H. Sung, S. Matsumiya, and M. Uchiyama. 1996. Main ginseng saponin metabolites formed by intestinal bacteria. Planta Medica 62: 453–457. Holman, L.E., and H. Leuenberger. 1988. The relationship between solid fraction and mechanical properties of compacts—The percolation theory model approach. International Journal of Pharmaceutics 46: 35–44. Kanaoks, M., T. Akao, and K. Kobashi. 1994. Metabolism of ginseng saponins, ginsenoside, by human intestinal flora. Journal of Traditional Medicine 11: 241–246.
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J.-S. Baek et al. Lai, C., Q. Zheng, M. Yang, P. Hu, J. Pan, and C. Peng. 2009. Preparation and prescription of enteric coated pellets of Panax notoginseng saponins pellets. Zhongguo Zhong Yao Za Zhi 34: 1359–1363. Leuenberger, H., B.D. Rohera, and C. Haas. 1987. Percolation theory—A novel approach to solid dosage form design. International Journal of Pharmaceutics 38: 109–115. Lim, J.H., S.K. You, J.S. Baek, C.J. Hwang, Y.G. Na, S.C. Shin, and C.W. Cho. 2012. Surface-modified gemcitabine with mucoadhesive polymer for oral delivery. Journal of Microencapsulation 29: 487–496. Nah, S.Y., and E.W. McCleskey. 1994. Ginseng root extract inhibits calcium channels in rat sensory neurons through a similar path, but different receptor, as mu-type opioids. Journal of Ethnopharmacology 42: 45–51. Odani, T., H. Tanizawa, and Y. Takino. 1983. Studies on the absorption, distribution excretion and metabolism of ginseng saponins II. Chemical and Pharmaceutical Bulletin 31: 292–298. Rowe, R.C. 1992. Molecular weight dependence of the properties of ethyl cellulose and hydroxypropyl methyl cellulose films. International Journal of Pharmaceutics 88: 405–408.
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Seo, J.Y., J.H. Lee, N.W. Kim, E. Her, S.H. Chang, N.Y. Ko, Y.H. Yoo, J.W. Kim, D.W. Seo, J.W. Han, Y.M. Kim, and W.S. Choi. 2005. Effect of a fermented ginseng extract, BST204, on the expression of cyclooxygenase-2 in murine macrophages. International Immunopharmacology 5: 929–936. Shaikh, N.A., S.E. Abidi, and L.H. Block. 1987. Evaluation of ethyl cellulose as a matrix for prolonged release formulations Part 1. Water soluble drugs-acetaminophen theophylline. Drug Development and Industrial Pharmacy 14: 1345–1369. Takeuchi, H., J. Thongborisute, and Y. Matsui. 2005. Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systems. Advanced Drug Delivery Reviews 57: 1583–1594. Takino, Y., T. Odani, H. Tanizawa, and T. Hayashi. 1982. Studies on the absorption, distribution, excretion and metabolism of ginseng saponin I. Chemical and Pharmaceutical Bulletin 30: 2196–2201. Tyler, V.E. 1995. Herbal remedies. The Journal of Pharmacy Technology 11: 214–220.
