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Enhanced pharmacokinetic behavior and hepatoprotective function of ginger extract-loaded supersaturable self-emulsifying drug delivery systems
Journal of Functional Foods 40 (2018) 156–163
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Enhanced pharmacokinetic behavior and hepatoprotective function of ginger extract-loaded supersaturable self-emulsifying drug delivery systems Mizuki Ogino a, Keisuke Yakushiji a, Hiroki Suzuki a, Kenichi Shiokawa b, Hiroshi Kikuchi b, Yoshiki Seto a, Hideyuki Sato a, Satomi Onoue a,⇑ a b
Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Japan Preventive Medical Laboratory Co., Ltd., 3-6-36 Toyoda, Suruga-ku, Shizuoka 422-8027, Japan
a r t i c l e
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Article history: Received 10 January 2017 Received in revised form 22 June 2017 Accepted 20 August 2017
Keywords: Ginger extract Hepatoprotective function Oral absorption Supersaturable self-emulsifying drug delivery systems
a b s t r a c t The aim of present study was to enhance the nutraceutical properties of ginger extract (GE) by employing supersaturable self-emulsifying drug delivery systems (S-SEDDS). SEDDS of GE (SEDDS/GE), consisting of medium-chain triglyceride, lysolecithin and glycerin, was produced. To prepare S-SEDDS of GE (S-SEDDS/GE), hydroxypropyl methylcellulose was added to the SEDDS/GE as a precipitation inhibitor. Physicochemical, pharmacokinetic, and hepatoprotective properties of GE formulations were characterized. Both formulations improved the dissolution behavior of GE due to the formation of fine micelles with a median diameter of ca. 110 nm. After oral administration of GE samples in rats, the relative bioavailabilities of 6-gingerol and 8-gingerol in the S-SEDDS/GE-treated group were ca. 3-fold higher than those of GE-treated group, respectively. Repeated oral administration of GE/S-SEDDS (100 mg-GE/kg) provided a hepatoprotective effect in a rat model of carbon tetrachloride-induced hepatotoxicity. From these observations, the S-SEDDS approach might be efficacious for enhancing the nutraceutical properties of GE. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Ginger (Zingiber officinale) has been widely used as a dietary condiment and medical plant (Baliga et al., 2011). Phytochemicals in ginger, such as 6-gingerol (6G), 8-gingerol (8G), 10-gingerol, and 6-shogaol (6S), have been identified as the major bioactive components (Semwal, Semwal, Combrinck, & Viljoen, 2015) showing various nutraceutical activities, including a gastro protective effect (Al-Yahya et al., 1989), thermoregulatory effect (Eldershaw, Colquhoun, Dora, Peng, & Clark, 1992), anti-inflammatory effect (Dugasani et al., 2010) and anti-oxidant activity (Dugasani et al., 2010; Lu et al., 2014). In the previous report, gingerols and shogaols could also have potent hepatoprotective effect due to the Abbreviations: ALT, alanine aminotransferase; ANOVA, analysis of variance; CCl4, carbon tetrachloride; COX-2, cyclooxygenase-2; DLS, dynamic light scattering; ESI, electrospray ionization; GI, gastrointestinal; 6G, 6-gingerol; 8G, 8-gingerol; GE, ginger extract; HPMC, hydroxypropyl methyl cellulose; MCT, medium-chain triglyceride; SEDDS, self-emulsifying drug delivery systems; 6S, 6-shogaol; SCF, supercritical fluid; S-SEDDS, supersaturable self-emulsifying drug delivery systems; TEM, transmission microscopy; UPLC, ultra-performance liquid chromatography. ⇑ Corresponding author. E-mail address: [email protected] (S. Onoue). https://doi.org/10.1016/j.jff.2017.08.035 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
inhibition of inflammatory and oxidative events (Atta et al., 2010; Yemitan & Izegbu, 2006). The mechanism on these functions of active chemicals are suppression of reactive oxygen species production by scavenging superoxide and inhibiting activity of xanthine oxidase (Dugasani et al., 2010), and reduction of prostaglandin E2 release by inhibiting activity of cyclooxygenase-2 (COX-2) enzyme and altering COX-2 mRNA levels (Lantz et al., 2007). A number of active compounds have been isolated from ginger (Sekiwa, Kubota, & Kobayashi, 2000; Sekiwa et al., 1999), and gingerols and shogaols likely possess essential properties for hepatoprotective function of lipophilic ginger extract (GE) (Dugasani et al., 2010). There has been growing interest in the therapeutic value of GE supplementation for hepatic injury. In spite of these attractive effects, lipophilic GE components exhibit poor solubility in water and low oral bioavailability, possibly resulting in the limited nutraceutical value. Recently, considerable attention has focused on lipid-based formulations like self-emulsifying drug delivery systems (SEDDS) as the solubilization technology for the application of poorly watersoluble materials to oral and injectable formulation. SEDDS are defined as isotropic mixtures of lipids, surfactants, co-surfactants, and substances that spontaneously form stable fine emulsions
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even under variable conditions in the gastrointestinal (GI) tract after oral administration (Kollipara & Gandhi, 2014). When exposed to aqueous media, such as the GI fluids, these systems form oil-in-water emulsions and present fine emulsion droplets. The emulsification can improve dissolution and oral absorption of compounds by providing a large interfacial area for substance release. In addition, supersaturable SEDDS (S-SEDDS) were designed to enhance the oral absorption of poorly water-soluble compounds compared with conventional SEDDS (Gao & Morozowich, 2006). In general, the S-SEDDS formulations contain a water-soluble polymeric precipitation inhibitor, such as polyvinylpyrrolidone, hypromellose acetate succinate or hydroxypropyl methylcellulose (HPMC), to maintain supersaturation state by steric and/or specific interactions with lipophilic chemicals, possibly leading to decreasing the precipitation and recrystallization. Although studies on several drugs demonstrated that the SSEDDS formulations could result in higher oral bioavailability compared with that of the conventional SEDDS formulations (Gao et al., 2003, 2004; Lee et al., 2015), far less is known about its feasibility to offer improved biopharmaceutical properties of GE. These findings prompted us to evaluate the applicability of S-SEDDS technology as a viable formulation option for GE. This study is the first attempt to design and develop a S-SEDDS formulation of GE. A GE-loaded conventional SEDDS formulation was designed for comparison. The physicochemical properties of SEDDS/GE and S-SEDDS/GE were characterized in terms of the particle distribution and dissolution property. Pharmacokinetic studies on GE, SEDDS/GE and S-SEDDS/GE were conducted to clarify the possible improvement in oral absorption of active ingredients. After repeated oral administration of GE and S-SEDDS/GE, hepatoprotective function was evaluated in a rat model of acute hepatotoxicity induced by carbon tetrachloride (CCl4)-treatment. 2. Materials and methods 2.1. Materials GE was supplied by Japan Preventive Medical Laboratory Co., Ltd. (Shizuoka, Japan), and coconard MTÒ, MCT, was supplied by Kao (Tokyo, Japan). SLP-paste lyso, lysolecithin, was purchased from Tsuji Seiyu Co., Ltd. (Mie, Japan) and glycerin was purchased from Wako Pure Chemical Indurstries, Ltd. (Osaka, Japan). HPMC (6 mPas) was supplied by Shin-Etsu Chemical (Tokyo, Japan). All other chemicals and solvents were of reagent or HPLC grade. 2.2. UPLC/ESI-MS analysis of active ingredients in GE The contents of active ingredients in GE were quantified using a Waters Acquity UPLC system (Waters, Milford, MA) equipped with a single quadrupole detector. A KINETEX C18 (particle size: 2.6 lm, column size: 2.1 mm 50 mm; Phenomenex) was used, and the column temperature was maintained at 60 °C during analysis. The gradient elution system was used to separate the components with a gradient mobile phase consisting of Milli-Q containing 5 mM ammonium acetate (A) and methanol (B) with a flow rate of 0.25 mL/min (0–0.5 min, 40% A; 0.5–3.0 min, 40–5% A; 3.0– 3.5 min, 5% A; and 3.5–4.0 min, 5%–40% A). Analysis was carried out using selected ion recording for specific m/z 317, 345, 294 and 372 for 6-gingerol [M+Na]+, 8-gingerol [M+Na]+, 6-shogaol [M+Na]+ and tamoxifen [M+H]+, respectively. 2.3. Preparation of GE formulations SEDDS formulations with GE were prepared by dissolving GE into a mixture of MCT, lysolecithin and glycerin at 50 °C and vor-
Table 1 Composition of GE formulations.
GE MCT Lysolecithin Glycerin HPMC
SEDDS/GE (wt%)
S-SEDDS/GE (wt%)
5 47.5 38 9.5 –
5 45 36 9 5
texing until all of the materials were completely dissolved. For the S-SEDDS formulation, HPMC was added into the SEDDS formulation, and then, the mixture was vortexed vigorously to obtain a uniform HPMC suspension. The compositions of the tested GE formulations are shown in Table 1. 2.4. Physicochemical properties of GE formulation 2.4.1. Transmission microscopy (TEM) An aliquot of SEDDS/GE or S-SEDDS/GE suspended in distilled water (1 mg-GE/mL) was placed on a Formvar 200 mesh Cu (Nisshin EM, Tokyo, Japan). The sample was allowed to stand for 15– 30 s, and then any excess solution was removed by blotting. The samples were visualized by negative staining with 1% (w/v) molybdenum solution and observed under an H-7600 transmission electron microscope (Hitachi, Tokyo, Japan). 2.4.2. Dynamic light scattering (DLS) Water suspended SEDDS/GE and S-SEDDS/GE (100 mg-GE/mL) were analyzed by a Zetasizer Nano ZS (MALVERN, Worcestershire, UK) to evaluate the micelle size distributions. For the physicochemical characterization of emulsion, transitions of the median particle size of emulsion and polydispersity index (PDI) value were measured. 2.4.3. Dissolution test Dissolution tests were conducted for 120 min using distilled water (50 mL, 37 °C) by a magnetic stirrer SST-66 (Shimadzu, Kyoto, Japan). GE, SEDDS/GE, and S-SEDDS/GE were weighed (25 mg-GE) in the dissolution vessel with a constant staring at 50 rpm, and then the samples were collected at the determined periods (0, 5, 10, 20, 30, 45, 60, 90 and 120 min). After centrifugation at 3000g for 5 min, the supernatants were collected and diluted with 50-fold volume of methanol. The released amounts of 6G, 8G, and 6S were assayed by Waters UPLC/ESI-MS system as described in Section 2.2. UPLC/ESI-MS analysis of active ingredients in GE. 2.5. Animals Male Sprague-Dawley rats (ca. 200 ± 50 g, 6–9 weeks of age; Japan SLC, Shizuoka, Japan) were housed by 2 rats in cage at 24 ± 1 °C and 55 ± 5% RH, and the room was maintained on a 12/12 h light/dark cycle. Rats can freely access to food and water. All animal experiments carried out in this study were approved by the Institutional Animal Care and Ethical Committee of the University of Shizuoka. 2.6. Pharmacokinetic behavior of GE samples Blood samples (300 mL) were collected from the tail veins of unanesthetized rats at the indicated times (0.18, 0.33, 0.5, 1, 2, 4, 8 and 12 h) after oral administration of GE (300 mg/kg) dissolved in 1 mL medium-chain triglyceride and SEDDS/GE or S-SEDDS/GE (100 mg-GE/kg) suspended in 2.5 mL of distilled water. Plasma samples were obtained after centrifugation of each blood sample
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at 10,000g. Concentrations of 6G and 8G in the plasma samples were assayed by UPLC/ESI-MS system. In brief, acetonitrile (150 lL) and internal standard (tamoxifen) (150 lL) were added to the plasma sample (50 lL) and centrifuged at 10,000g for 10 min. The supernatant was analyzed by Waters Acquity UPLC system (Waters, Milford, MA) as described in Section 2.2. 2.7. Hepatoprotective function of GE samples 2.7.1. Rat model of acute hepatic injury For the preparation of hepatic injury model rats, rats were treated by oral administration of CCl4 (0.7 mL/kg) with a same amount of corn oil. Corn oil (1.4 mL/kg) was administered orally as a control group. GE (100 mg/kg) dissolved in 1 mL MCT or S-SEDDS/GE (100 mg-GE/kg) dispersed in 2.5 mL of distilled water, was orally administered at 0.5 h and 12 h before and 12 h after CCl4treatment. At 24 h after CCl4 administration, blood samples (100 mL) were obtained from the tail vein of rats for biomarker analysis. After centrifugation of blood sample at 10,000g for 10 min, plasma samples were collected and kept frozen below 80 °C until analysis. 2.7.2. Histological examination For the histological examination, liver tissues were removed 24 h after CCl4-administration and fixed by 10% formalin neutral buffer. Fixed small pieces of liver were washed with phosphate buffered saline (pH7.4) three times and immersed in 30% sucrose containing 0.1% sodium azide at 4 °C for 24 h. Tissues were embedded in O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan), and frozen by liquid nitrogen. Embedded liver tissues were cut into 10 mm-thick sections on a cryostat. Prepared liver sections were stained by hematoxylin and eosin (H&E) staining for visualization of liver tissues. 2.7.3. Measurement of alanine aminotransferase (ALT) Plasma ALT activity was analyzed in accordance with a previously reported procedure (Bergmeyer, Scheibe, & Wahlefeld, 1978). Briefly, assay mixture (100 lL) added to plasma sample (10 lL). Mixtures were pre-incubated for 10 min at 30 °C, and then 15 mM 2-oxoglutarate (10 lL) was added to each sample. The absorbance at 340 nm was measured continuously every 30 s up to 5 min by a plate reader, Safire (Tecan, Männedorf, Switzerland). ALT activity was assessed by the following equation: DA340, change in absorbance at 340 nm for 1 min; e, molar decadic absorbance coefficient (b-NADH: 6220 M1 cm1); and L, the length of light path.
ALT ¼ ðDA340 12Þ=ðe LÞ 2.8. Statistical analysis Statistical analysis was carried out using one-way analysis of variance with pairwise comparison by Fisher’s least significant difference procedure. A P value of 0.05 or less was considered statistically significant. 3. Results 3.1. Physicochemical characterizations of GE formulations GE was extracted from ginger as an oily material by supercritical fluid (SCF) method with carbon dioxide, and the contents of 6G, 8G, and 6S in GE were quantified as 22%, 5%, and 3% w/v, respectively. Both SEDDS/GE and S-SEDDS/GE, self-emulsifying oily formulations, could disperse rapidly when introduced into aqueous
Fig. 1. Transmission microscopic images of (A) SEDDS/GE and (B) S-SEDDS/GE dispersed in distilled water. Each bar represents 300 nm.
media. After dispersion of the GE formulations in distilled water, the morphology was characterized by TEM observation (Fig. 1). They uniformly formed a number of spherical droplets, suggesting self-emulsification of GE formulations. The size distributions of SEDDS/GE and S-SEDDS/GE are shown in Fig. 2. Droplet size analysis shows the size and size distribution of formed emulsion droplets determining the dissolution rate of low water-soluble chemicals and extent of substance release as well as absorption. The median droplet size and PDI value, the parameters on emulsifying performance, of GE formulations were calculated (Table 2), indicating the narrow size distribution of the micronized droplets. This indicates on increase in surface area of GE formulations for faster release of active compounds compared with GE. DLS analysis of each formulation was also carried out at 3 h after suspension of these formulations in distilled water to clarify the stability of emulsion. As shown in Fig. 2, the particle size distribution of SEDDS/GE significantly changed at 3 h after suspension compared to those of S-SEDDS/GE, possibly indicating the collapse of nanoemulsion structure and precipitation of active component in GE. From these findings, S-SEDDS/GE has higher colloidal stability than SEDDS/GE to obtain better dissolution and oral absorption behavior. The dissolution profiles of active ingredients in GE and GE formulations were evaluated in distilled water up to 120 min (Fig. 3). GE exhibited limited dissolution of 6G, 8G, and 6S even after 120 min of testing. In contrast, compared with GE, there was marked enhancement in dissolution of 6G, 8G, and 6S in SSEDDS/GE with higher dissolution amounts of them at 120 min, by 4.2-, 13- and 25-fold, respectively. SEDDS/GE could also
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Fig. 2. Particle distributions of emulsion droplets of (A) SEDDS/GE and (B) S-SEDDS/ GE dispersed in distilled water after the indicated period. The solid line represents the particle size distribution at 0 min after dispersion; and the dotted line represents the particle size distribution at 3 h after dispersion.
Table 2 Particle size analysis of GE formulations suspended in water. Initial
SEDDS/GE S-SEDDS/GE
Stored for 3 h
d50 (nm)
PDI
d50 (nm)
PDI
108 114
0.507 0.455
200 134
0.502 0.418
d50, median diameter; and PDI, polydispersity index. Each formulation was suspended in distilled water with or without storage at room temperature for 3 h after suspension.
improve the dissolution properties of active components, while 6G, 8G, and 6S released from SEDDS/GE at 120 min were only 3.0-, 3.9and 13-fold that of GE, respectively. Formation of fine droplets could be a major reason for the increased dissolution rate of GE formulations compared with GE. On the basis of these physicochemical characterizations, S-SEDDS/GE would be an efficacious formulation of GE offering improved water-solubility of active components and thereby higher oral absorption. 3.2. Pharmacokinetic behavior of GE samples Several studies have clarified the pharmacokinetics of major active ingredients in GE, while their low bioavailability possibly
Fig. 3. Dissolution profiles of GE samples in water. d, GE; ., SEDDS/GE; and 4, SSEDDS/GE. Each bar represents mean ± S.E. of 3 experiments.
due to extensive first pass metabolism (Asami et al., 2010; Jiang, Wang, & Mi, 2008) and lipophilicity (Bakht et al., 2014) was reported. In order to evaluate the possible improvement in absorption of active ingredients in GE, the pharmacokinetic behaviors of GE samples were assessed in rats (Fig. 4). The blood concentration-time profiles of 6G and 8G were monitored in rats after oral administration of GE (300 mg-GE/kg) and GE formulations (100 mg-GE/kg). The 6S level in plasma of rats with any GE
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ingredients. The reasons for the data discrepancy may indicate: low release rate from emulsion droplets, poor membrane permeability, biliary excretion, rapid distribution, and metabolism of gingerols, although the exact reasons are still unclear. In contrast, in rats with oral S-SEDDS/GE (100 mg-GE/kg), oral absorption of 6G and 8G were found to be higher, with the relative oral bioavailabilities being 267% (6G) and 292% (8G) compared to the GE-group (Table 3). In the plasma concentration-time profiles of GE samples, there was prolonged systemic exposure of 6G and 8G in oral GE and S-SEDDS/GE compared with intravenously-administered GE (3 mg/kg, data not shown). This phenomenon could be observed with lymphatic absorption, possibly offering an extended half-life and long dosing interval for supplement use. These findings suggested that S-SEDDS technology would be an effective approach for improving the oral absorption of GE. However, the rapid elevation of component concentrations in plasma after oral administration of S-SEDDS/GE might lead to unexpected side effects, therefore a further study on dose-setting of S-SEDDS/GE for human use will be necessary. 3.3. Hepatoprotective function of GE and S-SEDDS/GE
Fig. 4. Plasma concentration-time profiles of 6G (A) and 8G (B) after oral administration of GE samples to rats. d, GE (300 mg/kg); s, GE (100 mg/kg); ., SEDDS/GE (100 mg-GE/kg); and 4, S-SEDDS/GE (100 mg-GE/kg). Data represent mean ± S.E. of 5–6 experiments.
treatment was below the detectable limit. After oral administration of GE (100 mg/kg), it was challenge to detect 6G and 8G in rat plasma, while oral administration of GE at a relatively high dose (300 mg/kg) resulted in their slight absorption. The limited oral bioavailability of active ingredients in GE was consistent with the poor dissolution behavior of GE in aqueous media. However, in spite of the improved dissolution behavior, the 6G and 8G levels in rat plasma were negligible after oral administration of SEDDS/ GE (100 mg-GE/kg), indicating poor absorption of these active
Some previous studies demonstrated the hepatoprotective function of GE, although repeated administration of GE in the long term was necessary for expression of therapeutic potential (Atta et al., 2010; Yemitan & Izegbu, 2006). The observations of enhanced systemic exposure in S-SEDDS/GE prompted us to assess the protective effects of S-SEDDS/GE against CCl4-induced hepatotoxicity in rats, since the improved systemic exposure could be key factor to obtain better pharmacological actions (0.7 mL/kg). CCl4 has been used as a hepatotoxin to prepare rat models of acute hepatic injury (Hayashi et al., 2008). CCl4 can be metabolized to reactive radicals which may bind to biomolecules such as proteins, lipids and nucleic acids, resulting in primary liver necrosis and hepatocyte apoptosis (Weber, Boll, & Stampfl, 2003). A previous study reported that plasma toxic biomarker levels gradually elevated after CCl4-treatment, indicating significant hepatocellular damage by metabolized CCl4 (Gewiese-Rabsch, Drucker, Malchow, Scheller, & Rose-John, 2010). Rat livers were sectioned at 24 h after CCl4-challenge and subjected to H&E staining to visualize acute hepatic injury (Fig. 5A). Oral administration of CCl4 caused a distorted tissue architecture with prominent necrosis and apoptosis in the liver (Fig. 5Aii); these symptoms can be found in acute liver disease, including viral hepatitis, alcoholic hepatitis and drug-induced hepatitis. Parenchymal injury was also evaluated by plasma ALT levels as a biomarker of hepatotoxicity (Fig. 5B). The significant elevation of plasma ALT level after CCl4challenge indicated hepatocellular damage by CCl4-treatment. After repeated treatment of GE (100 mg/kg) to model rats, slight hepatocyte injury was still found (Fig. 5Aiii) with a ca. 48%
Table 3 Pharmacokinetic parameters of 6G and 8G after administration of GE samples.
GE
Cmax (ng/mL)
AUC0–12 (hng/mL)
Relative BA (%)
(300 mg/kg, p.o.)
6G 8G
68.0 ± 26.9 15.8 ± 4.26
314 ± 67.0 68.0 ± 20.4
100 100
(100 mg/kg, p.o.)
6G 8G
N.D. N.D.
N.D. N.D.
N.D. N.D.
SEDDS/GE (100 mg-GE/kg, p.o.)
6G 8G
N.D. N.D.
N.D. N.D.
N.D. N.D.
S-SEDDS/GE (100 mg-GE/kg, p.o.)
6G 8G
41.9 ± 4.41 24.5 ± 9.70
280 ± 14.4 66.1 ± 22.8
267 292
GE
Cmax: maximum concentration; AUC0–12: area under the curve of plasma concentration vs. time from t = 0 to t = 12; BA: oral bioavailability; and N.D.: not detected. Data represent mean ± S.E. of 5–6 determinations.
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Fig. 5. (A) Histological features of liver sections at 24 h after CCl4-challenge with or without repeated oral administration of GE or S-SEDDS/GE (100 mg-GE/kg, 3 times). (i) Rats treated with corn oil, (ii) CCl4-treated rats, (iii) CCl4-treated rats with GE and (iv) CCl4-treated rats with S-SEDDS/GE. The insets highlight the histological changes of liver tissues. White and black bars represent 250 lm and 100 lm, respectively. (B) ALT level in rat plasma was also measured. Data represent mean ± S.E. of 5–6 experiments. * P < 0.05; **P < 0.01 with respect to control group and #P < 0.01 with respect to CCl4-treated rats with vehicle group.
reduction in ALT level compared to the CCl4-treated group (Fig. 5B). In contrast, repeated oral administration of S-SEDDS in the same dosing regimen (100 mg-GE/kg) attenuated hepatotoxicity with a prominent reduction of necrosis and apoptosis at 24 h after CCl4challenge (Fig. 5Aiv), followed by ca. 65% reduction in the ALT level (Fig. 5B). The hepatoprotective effect of the excipients, MCT and lecithin, on chemical-induced liver dysfunction was limited in previous report (Conti, Malandrino, & Magistretti, 1992; Kono et al., 2003), suggesting that these attenuations could be attributable to
the improved oral absorption of GE. No statistically significant difference was seen in the plasma ALT level between GE and S-SEDDS/ GE-treated groups (P value = 0.61), although the histological investigation and ALT data suggested that repeated treatment with SSEDDS/GE (100 mg-GE/kg) was more effective than repeated oral GE (100 mg/kg) in terms of hepatoprotection. The increased nutraceutical function in the liver of S-SEDDS against acute hepatotoxicity was in agreement with the improved physicochemical and pharmacokinetic properties compared to GE.
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4. Discussion The present investigation is the first to develop and characterize two GE formulations, SEDDS/GE and S-SEDDS/GE, and there was marked improvement of dissolution behavior of active components in both of the novel GE formulations. Although orallytaken SEDDS/GE failed to increase the oral absorption of active chemicals in GE, S-SEDDS could enhance the biopharmaceutical properties in GE, offering improved nutraceutical value of GE. S-SEDDS/GE exhibited two superior physicochemical properties in the current study compared with SEDDS/GE. First, S-SEDDS/GE exceeded SEDDS/GE in terms of colloidal stability, suggesting that co-existing HPMC, acting as a colloid stabilizer, prevented the aggregation of emulsion droplets. Theoretically, GI absorption of lipophilic chemicals can be enhanced by nano-emulsification due to the increased surface area available for direct contact with the intestinal mucosa (Gursoy & Benita, 2004); therefore, the high colloidal stability might be attributable to the improved bioavailability. Second, the S-SEDDS approach could achieve a higher amount of dissolved active components compared with SEDDS, possibly owing to the reduction in precipitation of free chemicals by HPMC. Precipitation inhibitors can prevent nucleation and/or crystal growth of free compounds by steric and/or specific interactions with lipophilic compounds, leading to the reduction in precipitation of free compounds. The chemicals can be released from emulsion droplets and absorbed in GI tract; thereby, the inhibition of precipitation of free chemicals can contribute to enhanced bioavailability. To decrease the compound’s precipitation, the SEDDS needs to contain a high amount of surfactant, although this might cause side effects in the GI tract and reduce the chemical release rate (Buyukozturk, Benneyan, & Carrier, 2010). In contrast, S-SEDDS contains a lower amount of surfactant than conventional SEDDS. Oral GE and S-SEDDS/GE resulted in prolonged systemic exposure of 6G and 8G compared with intravenously-administered GE. This phenomenon might be partly explained by lymphatic uptake of these lipophilic components. Generally, lipid-based formulations tended to be absorbed by the lymphatic pathway (Gursoy & Benita, 2004; Mu, Holm, & Mullertz, 2013), possibly leading to an augmentation of oral bioavailability by bypassing the liver and thereby reducing the first-pass metabolism. Dietary lipids have been thought to affect lipophilic drugs in the GI absorption and pharmacological effects due to their influence on GI physiological factors and thereby lymphatic absorption (Singh, 1999; Yasui-Furukori et al., 2003). A significant pharmacokinetic interaction between quazepam and food was observed (Yasui-Furukori et al., 2003), and administration of quazepam with food has been regulatorily contraindicated in Japan. However, in a clinical study, an emulsifying formulation with small-sized emulsion droplets diminished the influence of meals on the pharmacokinetic behavior of lipophilic chemicals in healthy volunteers (Fatouros, Karpf, Nielsen, & Mullertz, 2007). Therefore, the influence of food intake on pharmacokinetics of S-SEDDS/GE might be lower compared with GE, and further study should help to clarify the food effects on absorption of active ingredients in S-SEDDS/GE. The present study also demonstrated the hepatoprotective potential of GE and S-SEDDS/GE against CCl4-induced hepatotoxicity in rats. Gingerols and shogaols could protect membrane lipids from oxidation by reactive radicals since they have a phenolic OH moiety, acting as potent radical scavengers (Sabina, Pragasam, Kumar, & Rasool, 2011). Of all the components tested here, 6S has exhibited the most potent anti-oxidant and antiinflammatory properties (Dugasani et al., 2010; Lu et al., 2014), while 6S in plasma was negligible, possibly due to rapid metabolism and elimination (Asami et al., 2010). However, the first pass metabolites of shogaols, including paradols and zingerons, have
also been reported to be potent hepatoprotective agents (Cheong et al., 2016; Chung, Jung, Surh, Lee, & Park, 2001); herein, shogaols, as well as gingerols, might be essential constituents for hepatoprotection by ginger in spite of their low bioavailability. Some metabolites of gingerols and shogaols by first pass effect in liver are still effective, and this might be one possible reason for the data discrepancy between hepatoprotective outcomes and limited oral absorption of GE (Pfeiffer, Heuschmid, Kranz, & Metzler, 2006). Nevertheless, the metabolism of phenolic chemicals in ginger differs between species (Gauthier, Douat, Vachon, & Beaudry, 2011; Zick et al., 2008), and there might be different nutraceutical outcomes in humans compared with those in rats depending on the generation mass of metabolites with negligible anti-oxidative ability, including chemicals glucronized and sulfated at phenolic OH. The human efficacy of S-SEDDS/GE will need to be verified carefully. Enhancement in systemic exposure of ginger active ingredients was effective for increasing the hepatoprotection, suggesting that other formulations that improve water solubility might also be efficacious for increasing the nutraceutical value of GE. Previous studies demonstrated the enhancement in hepatoprotective function of lipophilic food active components, such as nobiletin and coenzyme Q10, with the use of a solid dispersion approach (Onoue et al., 2013, 2014). Lipid-based liquid formulations, including SEDDS and S-SEDDS, might suit the physical properties of GE, although liquid formulations generally show less storage stability than solid formulations (Chen et al., 2011; Onoue et al., 2012); therefore, careful consideration should be paid to its storage conditions. Careful selection of the most suitable formulation approach might be valuable for providing better GE products in terms of stability, manufacturability, manufacturing cost, and safety, as well as nutraceutical potential. S-SEDDS might be a promising dosage form because of easy manufacturing with low cost, low safety concerns upon selection of suitable excipients, and masked unpleasant taste by encapsulation of this liquid formulation into gelatin capsules (Pieroni & Torry, 2007). In conclusion, S-SEDDS technology could enhance the dissolution and pharmacokinetic properties of active ingredients in GE, offering potent hepatoprotective function and other nutraceutical effects. The S-SEDDS approach might be available for improving the nutraceutical value of other lipophilic active ingredients, as well as GE. Acknowledgements The authors are grateful to Japan Preventive Medical Laboratory Co., Ltd. (Shizuoka, Japan) for kindly providing GE prepared by SCF method. This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 15K18928; Y. Seto and 16K18950; H. Sato) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from the Takeda Science Foundation. We wish to thank Mr. Takumi Suzuki, University of Shizuoka, for the technical support on the animal studies. References Al-Yahya, M. A., Rafatullah, S., Mossa, J. S., Ageel, A. M., Parmar, N. S., & Tariq, M. (1989). Gastroprotective activity of ginger Zingiber officinale rosc., in albino rats. American Journal of Chinese Medicine, 17(1–2), 51–56. https://doi.org/10.1142/ s0192415x89000097. Asami, A., Shimada, T., Mizuhara, Y., Asano, T., Takeda, S., Aburada, T., ... Aburada, M. (2010). Pharmacokinetics of [6]-shogaol, a pungent ingredient of Zingiber officinale Roscoe (Part I). Journal of Natural Medicines, 64(3), 281–287. https:// doi.org/10.1007/s11418-010-0404-y. Atta, A. H., Elkoly, T. A., Mouneir, S. M., Kamel, G., Alwabel, N. A., & Zaher, S. (2010). Hepatoprotective effect of methanol extracts of Zingiber officinale and Cichorium intybus. Indian Journal of Pharmaceutical Sciences, 72(5), 564–570. https://doi. org/10.4103/0250-474x.78521.
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Enhanced pharmacokinetic behavior and hepatoprotective function of ginger extract-loaded supersaturable self-emulsifying drug delivery systems Mizuki Ogino a, Keisuke Yakushiji a, Hiroki Suzuki a, Kenichi Shiokawa b, Hiroshi Kikuchi b, Yoshiki Seto a, Hideyuki Sato a, Satomi Onoue a,⇑ a b
Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Japan Preventive Medical Laboratory Co., Ltd., 3-6-36 Toyoda, Suruga-ku, Shizuoka 422-8027, Japan
a r t i c l e
i n f o
Article history: Received 10 January 2017 Received in revised form 22 June 2017 Accepted 20 August 2017
Keywords: Ginger extract Hepatoprotective function Oral absorption Supersaturable self-emulsifying drug delivery systems
a b s t r a c t The aim of present study was to enhance the nutraceutical properties of ginger extract (GE) by employing supersaturable self-emulsifying drug delivery systems (S-SEDDS). SEDDS of GE (SEDDS/GE), consisting of medium-chain triglyceride, lysolecithin and glycerin, was produced. To prepare S-SEDDS of GE (S-SEDDS/GE), hydroxypropyl methylcellulose was added to the SEDDS/GE as a precipitation inhibitor. Physicochemical, pharmacokinetic, and hepatoprotective properties of GE formulations were characterized. Both formulations improved the dissolution behavior of GE due to the formation of fine micelles with a median diameter of ca. 110 nm. After oral administration of GE samples in rats, the relative bioavailabilities of 6-gingerol and 8-gingerol in the S-SEDDS/GE-treated group were ca. 3-fold higher than those of GE-treated group, respectively. Repeated oral administration of GE/S-SEDDS (100 mg-GE/kg) provided a hepatoprotective effect in a rat model of carbon tetrachloride-induced hepatotoxicity. From these observations, the S-SEDDS approach might be efficacious for enhancing the nutraceutical properties of GE. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Ginger (Zingiber officinale) has been widely used as a dietary condiment and medical plant (Baliga et al., 2011). Phytochemicals in ginger, such as 6-gingerol (6G), 8-gingerol (8G), 10-gingerol, and 6-shogaol (6S), have been identified as the major bioactive components (Semwal, Semwal, Combrinck, & Viljoen, 2015) showing various nutraceutical activities, including a gastro protective effect (Al-Yahya et al., 1989), thermoregulatory effect (Eldershaw, Colquhoun, Dora, Peng, & Clark, 1992), anti-inflammatory effect (Dugasani et al., 2010) and anti-oxidant activity (Dugasani et al., 2010; Lu et al., 2014). In the previous report, gingerols and shogaols could also have potent hepatoprotective effect due to the Abbreviations: ALT, alanine aminotransferase; ANOVA, analysis of variance; CCl4, carbon tetrachloride; COX-2, cyclooxygenase-2; DLS, dynamic light scattering; ESI, electrospray ionization; GI, gastrointestinal; 6G, 6-gingerol; 8G, 8-gingerol; GE, ginger extract; HPMC, hydroxypropyl methyl cellulose; MCT, medium-chain triglyceride; SEDDS, self-emulsifying drug delivery systems; 6S, 6-shogaol; SCF, supercritical fluid; S-SEDDS, supersaturable self-emulsifying drug delivery systems; TEM, transmission microscopy; UPLC, ultra-performance liquid chromatography. ⇑ Corresponding author. E-mail address: [email protected] (S. Onoue). https://doi.org/10.1016/j.jff.2017.08.035 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
inhibition of inflammatory and oxidative events (Atta et al., 2010; Yemitan & Izegbu, 2006). The mechanism on these functions of active chemicals are suppression of reactive oxygen species production by scavenging superoxide and inhibiting activity of xanthine oxidase (Dugasani et al., 2010), and reduction of prostaglandin E2 release by inhibiting activity of cyclooxygenase-2 (COX-2) enzyme and altering COX-2 mRNA levels (Lantz et al., 2007). A number of active compounds have been isolated from ginger (Sekiwa, Kubota, & Kobayashi, 2000; Sekiwa et al., 1999), and gingerols and shogaols likely possess essential properties for hepatoprotective function of lipophilic ginger extract (GE) (Dugasani et al., 2010). There has been growing interest in the therapeutic value of GE supplementation for hepatic injury. In spite of these attractive effects, lipophilic GE components exhibit poor solubility in water and low oral bioavailability, possibly resulting in the limited nutraceutical value. Recently, considerable attention has focused on lipid-based formulations like self-emulsifying drug delivery systems (SEDDS) as the solubilization technology for the application of poorly watersoluble materials to oral and injectable formulation. SEDDS are defined as isotropic mixtures of lipids, surfactants, co-surfactants, and substances that spontaneously form stable fine emulsions
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even under variable conditions in the gastrointestinal (GI) tract after oral administration (Kollipara & Gandhi, 2014). When exposed to aqueous media, such as the GI fluids, these systems form oil-in-water emulsions and present fine emulsion droplets. The emulsification can improve dissolution and oral absorption of compounds by providing a large interfacial area for substance release. In addition, supersaturable SEDDS (S-SEDDS) were designed to enhance the oral absorption of poorly water-soluble compounds compared with conventional SEDDS (Gao & Morozowich, 2006). In general, the S-SEDDS formulations contain a water-soluble polymeric precipitation inhibitor, such as polyvinylpyrrolidone, hypromellose acetate succinate or hydroxypropyl methylcellulose (HPMC), to maintain supersaturation state by steric and/or specific interactions with lipophilic chemicals, possibly leading to decreasing the precipitation and recrystallization. Although studies on several drugs demonstrated that the SSEDDS formulations could result in higher oral bioavailability compared with that of the conventional SEDDS formulations (Gao et al., 2003, 2004; Lee et al., 2015), far less is known about its feasibility to offer improved biopharmaceutical properties of GE. These findings prompted us to evaluate the applicability of S-SEDDS technology as a viable formulation option for GE. This study is the first attempt to design and develop a S-SEDDS formulation of GE. A GE-loaded conventional SEDDS formulation was designed for comparison. The physicochemical properties of SEDDS/GE and S-SEDDS/GE were characterized in terms of the particle distribution and dissolution property. Pharmacokinetic studies on GE, SEDDS/GE and S-SEDDS/GE were conducted to clarify the possible improvement in oral absorption of active ingredients. After repeated oral administration of GE and S-SEDDS/GE, hepatoprotective function was evaluated in a rat model of acute hepatotoxicity induced by carbon tetrachloride (CCl4)-treatment. 2. Materials and methods 2.1. Materials GE was supplied by Japan Preventive Medical Laboratory Co., Ltd. (Shizuoka, Japan), and coconard MTÒ, MCT, was supplied by Kao (Tokyo, Japan). SLP-paste lyso, lysolecithin, was purchased from Tsuji Seiyu Co., Ltd. (Mie, Japan) and glycerin was purchased from Wako Pure Chemical Indurstries, Ltd. (Osaka, Japan). HPMC (6 mPas) was supplied by Shin-Etsu Chemical (Tokyo, Japan). All other chemicals and solvents were of reagent or HPLC grade. 2.2. UPLC/ESI-MS analysis of active ingredients in GE The contents of active ingredients in GE were quantified using a Waters Acquity UPLC system (Waters, Milford, MA) equipped with a single quadrupole detector. A KINETEX C18 (particle size: 2.6 lm, column size: 2.1 mm 50 mm; Phenomenex) was used, and the column temperature was maintained at 60 °C during analysis. The gradient elution system was used to separate the components with a gradient mobile phase consisting of Milli-Q containing 5 mM ammonium acetate (A) and methanol (B) with a flow rate of 0.25 mL/min (0–0.5 min, 40% A; 0.5–3.0 min, 40–5% A; 3.0– 3.5 min, 5% A; and 3.5–4.0 min, 5%–40% A). Analysis was carried out using selected ion recording for specific m/z 317, 345, 294 and 372 for 6-gingerol [M+Na]+, 8-gingerol [M+Na]+, 6-shogaol [M+Na]+ and tamoxifen [M+H]+, respectively. 2.3. Preparation of GE formulations SEDDS formulations with GE were prepared by dissolving GE into a mixture of MCT, lysolecithin and glycerin at 50 °C and vor-
Table 1 Composition of GE formulations.
GE MCT Lysolecithin Glycerin HPMC
SEDDS/GE (wt%)
S-SEDDS/GE (wt%)
5 47.5 38 9.5 –
5 45 36 9 5
texing until all of the materials were completely dissolved. For the S-SEDDS formulation, HPMC was added into the SEDDS formulation, and then, the mixture was vortexed vigorously to obtain a uniform HPMC suspension. The compositions of the tested GE formulations are shown in Table 1. 2.4. Physicochemical properties of GE formulation 2.4.1. Transmission microscopy (TEM) An aliquot of SEDDS/GE or S-SEDDS/GE suspended in distilled water (1 mg-GE/mL) was placed on a Formvar 200 mesh Cu (Nisshin EM, Tokyo, Japan). The sample was allowed to stand for 15– 30 s, and then any excess solution was removed by blotting. The samples were visualized by negative staining with 1% (w/v) molybdenum solution and observed under an H-7600 transmission electron microscope (Hitachi, Tokyo, Japan). 2.4.2. Dynamic light scattering (DLS) Water suspended SEDDS/GE and S-SEDDS/GE (100 mg-GE/mL) were analyzed by a Zetasizer Nano ZS (MALVERN, Worcestershire, UK) to evaluate the micelle size distributions. For the physicochemical characterization of emulsion, transitions of the median particle size of emulsion and polydispersity index (PDI) value were measured. 2.4.3. Dissolution test Dissolution tests were conducted for 120 min using distilled water (50 mL, 37 °C) by a magnetic stirrer SST-66 (Shimadzu, Kyoto, Japan). GE, SEDDS/GE, and S-SEDDS/GE were weighed (25 mg-GE) in the dissolution vessel with a constant staring at 50 rpm, and then the samples were collected at the determined periods (0, 5, 10, 20, 30, 45, 60, 90 and 120 min). After centrifugation at 3000g for 5 min, the supernatants were collected and diluted with 50-fold volume of methanol. The released amounts of 6G, 8G, and 6S were assayed by Waters UPLC/ESI-MS system as described in Section 2.2. UPLC/ESI-MS analysis of active ingredients in GE. 2.5. Animals Male Sprague-Dawley rats (ca. 200 ± 50 g, 6–9 weeks of age; Japan SLC, Shizuoka, Japan) were housed by 2 rats in cage at 24 ± 1 °C and 55 ± 5% RH, and the room was maintained on a 12/12 h light/dark cycle. Rats can freely access to food and water. All animal experiments carried out in this study were approved by the Institutional Animal Care and Ethical Committee of the University of Shizuoka. 2.6. Pharmacokinetic behavior of GE samples Blood samples (300 mL) were collected from the tail veins of unanesthetized rats at the indicated times (0.18, 0.33, 0.5, 1, 2, 4, 8 and 12 h) after oral administration of GE (300 mg/kg) dissolved in 1 mL medium-chain triglyceride and SEDDS/GE or S-SEDDS/GE (100 mg-GE/kg) suspended in 2.5 mL of distilled water. Plasma samples were obtained after centrifugation of each blood sample
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at 10,000g. Concentrations of 6G and 8G in the plasma samples were assayed by UPLC/ESI-MS system. In brief, acetonitrile (150 lL) and internal standard (tamoxifen) (150 lL) were added to the plasma sample (50 lL) and centrifuged at 10,000g for 10 min. The supernatant was analyzed by Waters Acquity UPLC system (Waters, Milford, MA) as described in Section 2.2. 2.7. Hepatoprotective function of GE samples 2.7.1. Rat model of acute hepatic injury For the preparation of hepatic injury model rats, rats were treated by oral administration of CCl4 (0.7 mL/kg) with a same amount of corn oil. Corn oil (1.4 mL/kg) was administered orally as a control group. GE (100 mg/kg) dissolved in 1 mL MCT or S-SEDDS/GE (100 mg-GE/kg) dispersed in 2.5 mL of distilled water, was orally administered at 0.5 h and 12 h before and 12 h after CCl4treatment. At 24 h after CCl4 administration, blood samples (100 mL) were obtained from the tail vein of rats for biomarker analysis. After centrifugation of blood sample at 10,000g for 10 min, plasma samples were collected and kept frozen below 80 °C until analysis. 2.7.2. Histological examination For the histological examination, liver tissues were removed 24 h after CCl4-administration and fixed by 10% formalin neutral buffer. Fixed small pieces of liver were washed with phosphate buffered saline (pH7.4) three times and immersed in 30% sucrose containing 0.1% sodium azide at 4 °C for 24 h. Tissues were embedded in O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan), and frozen by liquid nitrogen. Embedded liver tissues were cut into 10 mm-thick sections on a cryostat. Prepared liver sections were stained by hematoxylin and eosin (H&E) staining for visualization of liver tissues. 2.7.3. Measurement of alanine aminotransferase (ALT) Plasma ALT activity was analyzed in accordance with a previously reported procedure (Bergmeyer, Scheibe, & Wahlefeld, 1978). Briefly, assay mixture (100 lL) added to plasma sample (10 lL). Mixtures were pre-incubated for 10 min at 30 °C, and then 15 mM 2-oxoglutarate (10 lL) was added to each sample. The absorbance at 340 nm was measured continuously every 30 s up to 5 min by a plate reader, Safire (Tecan, Männedorf, Switzerland). ALT activity was assessed by the following equation: DA340, change in absorbance at 340 nm for 1 min; e, molar decadic absorbance coefficient (b-NADH: 6220 M1 cm1); and L, the length of light path.
ALT ¼ ðDA340 12Þ=ðe LÞ 2.8. Statistical analysis Statistical analysis was carried out using one-way analysis of variance with pairwise comparison by Fisher’s least significant difference procedure. A P value of 0.05 or less was considered statistically significant. 3. Results 3.1. Physicochemical characterizations of GE formulations GE was extracted from ginger as an oily material by supercritical fluid (SCF) method with carbon dioxide, and the contents of 6G, 8G, and 6S in GE were quantified as 22%, 5%, and 3% w/v, respectively. Both SEDDS/GE and S-SEDDS/GE, self-emulsifying oily formulations, could disperse rapidly when introduced into aqueous
Fig. 1. Transmission microscopic images of (A) SEDDS/GE and (B) S-SEDDS/GE dispersed in distilled water. Each bar represents 300 nm.
media. After dispersion of the GE formulations in distilled water, the morphology was characterized by TEM observation (Fig. 1). They uniformly formed a number of spherical droplets, suggesting self-emulsification of GE formulations. The size distributions of SEDDS/GE and S-SEDDS/GE are shown in Fig. 2. Droplet size analysis shows the size and size distribution of formed emulsion droplets determining the dissolution rate of low water-soluble chemicals and extent of substance release as well as absorption. The median droplet size and PDI value, the parameters on emulsifying performance, of GE formulations were calculated (Table 2), indicating the narrow size distribution of the micronized droplets. This indicates on increase in surface area of GE formulations for faster release of active compounds compared with GE. DLS analysis of each formulation was also carried out at 3 h after suspension of these formulations in distilled water to clarify the stability of emulsion. As shown in Fig. 2, the particle size distribution of SEDDS/GE significantly changed at 3 h after suspension compared to those of S-SEDDS/GE, possibly indicating the collapse of nanoemulsion structure and precipitation of active component in GE. From these findings, S-SEDDS/GE has higher colloidal stability than SEDDS/GE to obtain better dissolution and oral absorption behavior. The dissolution profiles of active ingredients in GE and GE formulations were evaluated in distilled water up to 120 min (Fig. 3). GE exhibited limited dissolution of 6G, 8G, and 6S even after 120 min of testing. In contrast, compared with GE, there was marked enhancement in dissolution of 6G, 8G, and 6S in SSEDDS/GE with higher dissolution amounts of them at 120 min, by 4.2-, 13- and 25-fold, respectively. SEDDS/GE could also
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Fig. 2. Particle distributions of emulsion droplets of (A) SEDDS/GE and (B) S-SEDDS/ GE dispersed in distilled water after the indicated period. The solid line represents the particle size distribution at 0 min after dispersion; and the dotted line represents the particle size distribution at 3 h after dispersion.
Table 2 Particle size analysis of GE formulations suspended in water. Initial
SEDDS/GE S-SEDDS/GE
Stored for 3 h
d50 (nm)
PDI
d50 (nm)
PDI
108 114
0.507 0.455
200 134
0.502 0.418
d50, median diameter; and PDI, polydispersity index. Each formulation was suspended in distilled water with or without storage at room temperature for 3 h after suspension.
improve the dissolution properties of active components, while 6G, 8G, and 6S released from SEDDS/GE at 120 min were only 3.0-, 3.9and 13-fold that of GE, respectively. Formation of fine droplets could be a major reason for the increased dissolution rate of GE formulations compared with GE. On the basis of these physicochemical characterizations, S-SEDDS/GE would be an efficacious formulation of GE offering improved water-solubility of active components and thereby higher oral absorption. 3.2. Pharmacokinetic behavior of GE samples Several studies have clarified the pharmacokinetics of major active ingredients in GE, while their low bioavailability possibly
Fig. 3. Dissolution profiles of GE samples in water. d, GE; ., SEDDS/GE; and 4, SSEDDS/GE. Each bar represents mean ± S.E. of 3 experiments.
due to extensive first pass metabolism (Asami et al., 2010; Jiang, Wang, & Mi, 2008) and lipophilicity (Bakht et al., 2014) was reported. In order to evaluate the possible improvement in absorption of active ingredients in GE, the pharmacokinetic behaviors of GE samples were assessed in rats (Fig. 4). The blood concentration-time profiles of 6G and 8G were monitored in rats after oral administration of GE (300 mg-GE/kg) and GE formulations (100 mg-GE/kg). The 6S level in plasma of rats with any GE
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ingredients. The reasons for the data discrepancy may indicate: low release rate from emulsion droplets, poor membrane permeability, biliary excretion, rapid distribution, and metabolism of gingerols, although the exact reasons are still unclear. In contrast, in rats with oral S-SEDDS/GE (100 mg-GE/kg), oral absorption of 6G and 8G were found to be higher, with the relative oral bioavailabilities being 267% (6G) and 292% (8G) compared to the GE-group (Table 3). In the plasma concentration-time profiles of GE samples, there was prolonged systemic exposure of 6G and 8G in oral GE and S-SEDDS/GE compared with intravenously-administered GE (3 mg/kg, data not shown). This phenomenon could be observed with lymphatic absorption, possibly offering an extended half-life and long dosing interval for supplement use. These findings suggested that S-SEDDS technology would be an effective approach for improving the oral absorption of GE. However, the rapid elevation of component concentrations in plasma after oral administration of S-SEDDS/GE might lead to unexpected side effects, therefore a further study on dose-setting of S-SEDDS/GE for human use will be necessary. 3.3. Hepatoprotective function of GE and S-SEDDS/GE
Fig. 4. Plasma concentration-time profiles of 6G (A) and 8G (B) after oral administration of GE samples to rats. d, GE (300 mg/kg); s, GE (100 mg/kg); ., SEDDS/GE (100 mg-GE/kg); and 4, S-SEDDS/GE (100 mg-GE/kg). Data represent mean ± S.E. of 5–6 experiments.
treatment was below the detectable limit. After oral administration of GE (100 mg/kg), it was challenge to detect 6G and 8G in rat plasma, while oral administration of GE at a relatively high dose (300 mg/kg) resulted in their slight absorption. The limited oral bioavailability of active ingredients in GE was consistent with the poor dissolution behavior of GE in aqueous media. However, in spite of the improved dissolution behavior, the 6G and 8G levels in rat plasma were negligible after oral administration of SEDDS/ GE (100 mg-GE/kg), indicating poor absorption of these active
Some previous studies demonstrated the hepatoprotective function of GE, although repeated administration of GE in the long term was necessary for expression of therapeutic potential (Atta et al., 2010; Yemitan & Izegbu, 2006). The observations of enhanced systemic exposure in S-SEDDS/GE prompted us to assess the protective effects of S-SEDDS/GE against CCl4-induced hepatotoxicity in rats, since the improved systemic exposure could be key factor to obtain better pharmacological actions (0.7 mL/kg). CCl4 has been used as a hepatotoxin to prepare rat models of acute hepatic injury (Hayashi et al., 2008). CCl4 can be metabolized to reactive radicals which may bind to biomolecules such as proteins, lipids and nucleic acids, resulting in primary liver necrosis and hepatocyte apoptosis (Weber, Boll, & Stampfl, 2003). A previous study reported that plasma toxic biomarker levels gradually elevated after CCl4-treatment, indicating significant hepatocellular damage by metabolized CCl4 (Gewiese-Rabsch, Drucker, Malchow, Scheller, & Rose-John, 2010). Rat livers were sectioned at 24 h after CCl4-challenge and subjected to H&E staining to visualize acute hepatic injury (Fig. 5A). Oral administration of CCl4 caused a distorted tissue architecture with prominent necrosis and apoptosis in the liver (Fig. 5Aii); these symptoms can be found in acute liver disease, including viral hepatitis, alcoholic hepatitis and drug-induced hepatitis. Parenchymal injury was also evaluated by plasma ALT levels as a biomarker of hepatotoxicity (Fig. 5B). The significant elevation of plasma ALT level after CCl4challenge indicated hepatocellular damage by CCl4-treatment. After repeated treatment of GE (100 mg/kg) to model rats, slight hepatocyte injury was still found (Fig. 5Aiii) with a ca. 48%
Table 3 Pharmacokinetic parameters of 6G and 8G after administration of GE samples.
GE
Cmax (ng/mL)
AUC0–12 (hng/mL)
Relative BA (%)
(300 mg/kg, p.o.)
6G 8G
68.0 ± 26.9 15.8 ± 4.26
314 ± 67.0 68.0 ± 20.4
100 100
(100 mg/kg, p.o.)
6G 8G
N.D. N.D.
N.D. N.D.
N.D. N.D.
SEDDS/GE (100 mg-GE/kg, p.o.)
6G 8G
N.D. N.D.
N.D. N.D.
N.D. N.D.
S-SEDDS/GE (100 mg-GE/kg, p.o.)
6G 8G
41.9 ± 4.41 24.5 ± 9.70
280 ± 14.4 66.1 ± 22.8
267 292
GE
Cmax: maximum concentration; AUC0–12: area under the curve of plasma concentration vs. time from t = 0 to t = 12; BA: oral bioavailability; and N.D.: not detected. Data represent mean ± S.E. of 5–6 determinations.
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Fig. 5. (A) Histological features of liver sections at 24 h after CCl4-challenge with or without repeated oral administration of GE or S-SEDDS/GE (100 mg-GE/kg, 3 times). (i) Rats treated with corn oil, (ii) CCl4-treated rats, (iii) CCl4-treated rats with GE and (iv) CCl4-treated rats with S-SEDDS/GE. The insets highlight the histological changes of liver tissues. White and black bars represent 250 lm and 100 lm, respectively. (B) ALT level in rat plasma was also measured. Data represent mean ± S.E. of 5–6 experiments. * P < 0.05; **P < 0.01 with respect to control group and #P < 0.01 with respect to CCl4-treated rats with vehicle group.
reduction in ALT level compared to the CCl4-treated group (Fig. 5B). In contrast, repeated oral administration of S-SEDDS in the same dosing regimen (100 mg-GE/kg) attenuated hepatotoxicity with a prominent reduction of necrosis and apoptosis at 24 h after CCl4challenge (Fig. 5Aiv), followed by ca. 65% reduction in the ALT level (Fig. 5B). The hepatoprotective effect of the excipients, MCT and lecithin, on chemical-induced liver dysfunction was limited in previous report (Conti, Malandrino, & Magistretti, 1992; Kono et al., 2003), suggesting that these attenuations could be attributable to
the improved oral absorption of GE. No statistically significant difference was seen in the plasma ALT level between GE and S-SEDDS/ GE-treated groups (P value = 0.61), although the histological investigation and ALT data suggested that repeated treatment with SSEDDS/GE (100 mg-GE/kg) was more effective than repeated oral GE (100 mg/kg) in terms of hepatoprotection. The increased nutraceutical function in the liver of S-SEDDS against acute hepatotoxicity was in agreement with the improved physicochemical and pharmacokinetic properties compared to GE.
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4. Discussion The present investigation is the first to develop and characterize two GE formulations, SEDDS/GE and S-SEDDS/GE, and there was marked improvement of dissolution behavior of active components in both of the novel GE formulations. Although orallytaken SEDDS/GE failed to increase the oral absorption of active chemicals in GE, S-SEDDS could enhance the biopharmaceutical properties in GE, offering improved nutraceutical value of GE. S-SEDDS/GE exhibited two superior physicochemical properties in the current study compared with SEDDS/GE. First, S-SEDDS/GE exceeded SEDDS/GE in terms of colloidal stability, suggesting that co-existing HPMC, acting as a colloid stabilizer, prevented the aggregation of emulsion droplets. Theoretically, GI absorption of lipophilic chemicals can be enhanced by nano-emulsification due to the increased surface area available for direct contact with the intestinal mucosa (Gursoy & Benita, 2004); therefore, the high colloidal stability might be attributable to the improved bioavailability. Second, the S-SEDDS approach could achieve a higher amount of dissolved active components compared with SEDDS, possibly owing to the reduction in precipitation of free chemicals by HPMC. Precipitation inhibitors can prevent nucleation and/or crystal growth of free compounds by steric and/or specific interactions with lipophilic compounds, leading to the reduction in precipitation of free compounds. The chemicals can be released from emulsion droplets and absorbed in GI tract; thereby, the inhibition of precipitation of free chemicals can contribute to enhanced bioavailability. To decrease the compound’s precipitation, the SEDDS needs to contain a high amount of surfactant, although this might cause side effects in the GI tract and reduce the chemical release rate (Buyukozturk, Benneyan, & Carrier, 2010). In contrast, S-SEDDS contains a lower amount of surfactant than conventional SEDDS. Oral GE and S-SEDDS/GE resulted in prolonged systemic exposure of 6G and 8G compared with intravenously-administered GE. This phenomenon might be partly explained by lymphatic uptake of these lipophilic components. Generally, lipid-based formulations tended to be absorbed by the lymphatic pathway (Gursoy & Benita, 2004; Mu, Holm, & Mullertz, 2013), possibly leading to an augmentation of oral bioavailability by bypassing the liver and thereby reducing the first-pass metabolism. Dietary lipids have been thought to affect lipophilic drugs in the GI absorption and pharmacological effects due to their influence on GI physiological factors and thereby lymphatic absorption (Singh, 1999; Yasui-Furukori et al., 2003). A significant pharmacokinetic interaction between quazepam and food was observed (Yasui-Furukori et al., 2003), and administration of quazepam with food has been regulatorily contraindicated in Japan. However, in a clinical study, an emulsifying formulation with small-sized emulsion droplets diminished the influence of meals on the pharmacokinetic behavior of lipophilic chemicals in healthy volunteers (Fatouros, Karpf, Nielsen, & Mullertz, 2007). Therefore, the influence of food intake on pharmacokinetics of S-SEDDS/GE might be lower compared with GE, and further study should help to clarify the food effects on absorption of active ingredients in S-SEDDS/GE. The present study also demonstrated the hepatoprotective potential of GE and S-SEDDS/GE against CCl4-induced hepatotoxicity in rats. Gingerols and shogaols could protect membrane lipids from oxidation by reactive radicals since they have a phenolic OH moiety, acting as potent radical scavengers (Sabina, Pragasam, Kumar, & Rasool, 2011). Of all the components tested here, 6S has exhibited the most potent anti-oxidant and antiinflammatory properties (Dugasani et al., 2010; Lu et al., 2014), while 6S in plasma was negligible, possibly due to rapid metabolism and elimination (Asami et al., 2010). However, the first pass metabolites of shogaols, including paradols and zingerons, have
also been reported to be potent hepatoprotective agents (Cheong et al., 2016; Chung, Jung, Surh, Lee, & Park, 2001); herein, shogaols, as well as gingerols, might be essential constituents for hepatoprotection by ginger in spite of their low bioavailability. Some metabolites of gingerols and shogaols by first pass effect in liver are still effective, and this might be one possible reason for the data discrepancy between hepatoprotective outcomes and limited oral absorption of GE (Pfeiffer, Heuschmid, Kranz, & Metzler, 2006). Nevertheless, the metabolism of phenolic chemicals in ginger differs between species (Gauthier, Douat, Vachon, & Beaudry, 2011; Zick et al., 2008), and there might be different nutraceutical outcomes in humans compared with those in rats depending on the generation mass of metabolites with negligible anti-oxidative ability, including chemicals glucronized and sulfated at phenolic OH. The human efficacy of S-SEDDS/GE will need to be verified carefully. Enhancement in systemic exposure of ginger active ingredients was effective for increasing the hepatoprotection, suggesting that other formulations that improve water solubility might also be efficacious for increasing the nutraceutical value of GE. Previous studies demonstrated the enhancement in hepatoprotective function of lipophilic food active components, such as nobiletin and coenzyme Q10, with the use of a solid dispersion approach (Onoue et al., 2013, 2014). Lipid-based liquid formulations, including SEDDS and S-SEDDS, might suit the physical properties of GE, although liquid formulations generally show less storage stability than solid formulations (Chen et al., 2011; Onoue et al., 2012); therefore, careful consideration should be paid to its storage conditions. Careful selection of the most suitable formulation approach might be valuable for providing better GE products in terms of stability, manufacturability, manufacturing cost, and safety, as well as nutraceutical potential. S-SEDDS might be a promising dosage form because of easy manufacturing with low cost, low safety concerns upon selection of suitable excipients, and masked unpleasant taste by encapsulation of this liquid formulation into gelatin capsules (Pieroni & Torry, 2007). In conclusion, S-SEDDS technology could enhance the dissolution and pharmacokinetic properties of active ingredients in GE, offering potent hepatoprotective function and other nutraceutical effects. The S-SEDDS approach might be available for improving the nutraceutical value of other lipophilic active ingredients, as well as GE. Acknowledgements The authors are grateful to Japan Preventive Medical Laboratory Co., Ltd. (Shizuoka, Japan) for kindly providing GE prepared by SCF method. This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 15K18928; Y. Seto and 16K18950; H. Sato) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from the Takeda Science Foundation. We wish to thank Mr. Takumi Suzuki, University of Shizuoka, for the technical support on the animal studies. References Al-Yahya, M. A., Rafatullah, S., Mossa, J. S., Ageel, A. M., Parmar, N. S., & Tariq, M. (1989). Gastroprotective activity of ginger Zingiber officinale rosc., in albino rats. American Journal of Chinese Medicine, 17(1–2), 51–56. https://doi.org/10.1142/ s0192415x89000097. Asami, A., Shimada, T., Mizuhara, Y., Asano, T., Takeda, S., Aburada, T., ... Aburada, M. (2010). Pharmacokinetics of [6]-shogaol, a pungent ingredient of Zingiber officinale Roscoe (Part I). Journal of Natural Medicines, 64(3), 281–287. https:// doi.org/10.1007/s11418-010-0404-y. Atta, A. H., Elkoly, T. A., Mouneir, S. M., Kamel, G., Alwabel, N. A., & Zaher, S. (2010). Hepatoprotective effect of methanol extracts of Zingiber officinale and Cichorium intybus. Indian Journal of Pharmaceutical Sciences, 72(5), 564–570. https://doi. org/10.4103/0250-474x.78521.
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