- Email: [email protected]
Enhancement of solubility and dissolution rate of baicalein, wogonin and oroxylin A extracted from Radix scutellariae
International Journal of Pharmaceutics 528 (2017) 602–610
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Enhancement of solubility and dissolution rate of baicalein, wogonin and oroxylin A extracted from Radix scutellariae Hang Yua , Jae-Sang Changa , Sun Young Kimb , Yoon G. Kimb , Hoo-Kyun Choia,* a b
College of Pharmacy, Chosun University, 309 Philmoondaero, Gwangju 61452, Korea College of Pharmacy, Dankook University, 119 Dandaero, Cheonan 31116, Korea
A R T I C L E I N F O
Article history: Received 14 March 2017 Received in revised form 30 May 2017 Accepted 18 June 2017 Available online 19 June 2017 Keywords: Solid dispersion Flavonoids Povidone K-30 Chemical stability Poor water solubility Pharmacokinetics
A B S T R A C T
Baicalein, wogonin, and oroxylin A are three major hydrophobic components in the extract of Radix scutellariae with wide spectrum of pharmacological applications. The purpose of this study was to enhance the solubility, dissolution rate and stability of baicalein, wogonin and oroxylin A by solid dispersion (SD) technique. SD of the extract with various polymers was prepared to select the best carrier. Solubility study, chemical stability study and dissolution study were performed to characterize the SD. The solubility of all three components, after forming solid dispersion with povidone K-30 (PVP K-30) was significantly increased in pH 6.8 medium at room temperature. Stability study conducted for 80 days elucidated that the SD in powder state was fairly stable without the aid of Vitamin C (VC). VC was required as antioxidant to impart stability to baicalein in aqueous medium. The dissolution test of the SD of three components, admixed with VC at the weight ratio of 1:6 (Radix scutellariae extract: VC, w/w) exhibited faster dissolution rate with 100% release of all components. Pharmacokinetic study of baicalein solid dispersion revealed that AUC and Cmax significantly increased by solid dispersion preparation. Thus, the current developed method, being simple, economical and effective, can be useful for the production of soluble dosage forms of the extract of Radix scutellariae in commercial scale. © 2017 Published by Elsevier B.V.
1. Introduction Radix scutellariae is an important herb in traditional Chinese medicine and is prepared from the roots of Scutellaria baicalensis Georgi (Labiatae family). Traditionally, it has been employed for detoxication and relief from fever. Nowadays, Radix scutellariae is widely used for the treatment of inflammation, fever, hepatitis, allergic diseases, hypertension (Wang et al., 1983). The chemical constituents of Radix scutellariae have been elucidated and are mostly consisted of flavonoids like baicalein, baicalin, wogonin and wogonoside. More than 60 flavonoids have been reported from Radix scutellariae, and most of them occur as glucuronides (Han et al., 2007). Among them, baicalin has been used as a phytochemical marker for the quality control of Radix scutellariae in Chinese pharmacopoeia (Qi et al., 1997; Wu et al., 2005). Pharmacological studies revealed that baicalin has anti-inflammatory, anti-allergic, anti-oxidant and hepatoprotective properties (Lu et al., 2007) whereas wogonin possessed anticancer activity (Lu et al., 2008). Even though the Radix scutellariae components
* Corresponding author. E-mail address: [email protected] (H.-K. Choi). http://dx.doi.org/10.1016/j.ijpharm.2017.06.068 0378-5173/© 2017 Published by Elsevier B.V.
showed many advantages, their low aqueous solubility, bioavailability, and sensitivity to light and temperature (Lei et al., 2006; Yao and Zhang, 2006) exerted problems in the drug delivery. Limited number of studies has been reported in the literature regarding the enhancement of solubility of the baicalein. One study utilized inclusion complex with hydroxypropyl-beta-cyclodextrin (HP-b-CD) and freeze drying process (Liu et al., 2006). Use of HPb-CD and freeze drying process can be costly and difficult to be commercialized. Hence, simple and convenient process that can sufficiently improve solubility and dissolution of major components of Radix scutellariae using cheaper excipients are more desirable. A solid dispersion is “the dispersion of one or more active ingredients in an inert carrier or matrix at solid state prepared by melting (fusion), solvent or melting-solvent method”(Chiou and Riegelman, 1971). It is widely used and is one of the most successful strategies to improve drug release and enhance oral bioavailability of poorly soluble drugs (Choi et al., 2016; Ha et al., 2015; Vasconcelos et al., 2007). The SD may exist as coarse suspension, fine suspension or solid solution depending upon the state of the drug in the carrier. To formulate successful SD, drug should be in finely reduced size, preferably in amorphous form. Dissolution of a drug with reduced particle size will be faster due to
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
increase in surface area and solubilization of amorphous state is much easier as no energy is required to break the crystal lattice. The state of the drug in the carrier is determined by drug-carrier interaction which in turn governs the release behavior of the drug from the SD. Hence, selection of carrier for SD is crucial and formation of successful SD is favored by the use of carrier having high affinity to the drug or high mixing capability. Hydrophilic polymers are generally used as carrier in the SD which enhances wettability and in turn solubility of the drug (Kang et al., 2010). In this study, solid dispersion formulation of Radix scutellariae was developed to enhance the solubility and dissolution rate of three major hydrophobic components. Various polymers were tested to select optimum carrier for the SD preparation of the extract of Radix scutellariae. The SD thus prepared was evaluated in terms of solubility, dissolution rate, stability, and pharmacokinetics. 2. Materials and methods 2.1. Materials Radix scutellariae extract was obtained from Wonkwang University (Iksan, Korea). Vitamin C was provided by Dong-A Pharm. Co. (Seoul, Korea). Poloxamer 407, poloxamer 188 and povidone K-30 (Kollidon1 30) were obtained from BASF (Ludwigshafen, Germany). Avicel1 101 (microcrystalline cellulose) was provided by Seoul Pharm. Co. (Seoul, Korea). Polyethylene glycol 3400 and 8000 (PEG 3400 and PEG 8000) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). AC-1709 was used as an internal standard for plasma sample analysis and was obtained from Professor Keon-Wook Kang at Seoul National University. All other chemicals were of reagent grade or above and were used without further purification. 2.2. HPLC analysis HPLC system (Shimadzu Scientific Instruments, Kyoto, Japan) equipped with a UV detector (SPD-10A), a pump (LC-10AD) and an automatic injector (SIL-10A) was used. The wavelength of UV detector was 254 nm and the flow rate was 1 mL/min. The mobile phase composed of 6.7% (v/v) acetic acid aqueous solution and acetonitrile at volume ratio of 1:1. The samples were analyzed through a reversed-phase column (Gemini 5 m C18, Phenomenex, USA) at the temperature of 30 C. 2.3. Component content analysis of Radix scutellariae extract 1 mg of Radix scutellariae extract was completely dissolved in 2 mL of methanol and suitably diluted. The concentration of three major components, viz. baicalein, wogonin and oroxylin A, in the sample solution was analyzed by HPLC and percentage of each of three components contained in the extract was calculated.
603
temperature for 12 h. The residue was ground in a mortar, passed through a 200 mm sieve and stored in a sealed vial for further investigation. Each solid dispersion thus prepared was subjected for solubility test at room temperature. Solid dispersion of baicalein for in vivo study was prepared by solvent method at the weight ratio of 1:5:1 (baicalein:PVP K-30: vitamin C) in a same way described above. 2.4.2. DSC study Thermal analysis was carried out using a differential scanning calorimeter (Pyris 6 DSC, Perkin Elmer, Waltham, MA, USA). Indium was used to calibrate temperature scale and enthalpic response. Samples were placed in aluminum pans and heated at a scanning rate of 10 C/min from 30 to 300 C. 2.5. Solubility study To determine the solubility of the components in SDs of Radix scutellariae extract with various polymers, excess amount of each of SD prepared with various polymers was added into 1 mL of distilled water. Effect of pH on the solubility of the components in the SD of the extract prepared with PVP K-30 was determined in each of 1 mL of pH 1.2, pH 4.0, pH 6.0 and pH 6.8 buffers. To investigate the effect of PVP K-30 concentration on the solubility of the components of Radix scutellariae in pH 6.8 buffer, PVP K-30 concentrations of 0.1 mg/mL, 1 mg/mL, 5 mg/mL and 10 mg/mL were used. In all cases, samples were stirred with magnetic bar at 750 rpm at room temperature for 12 h. The samples were then centrifuged at 12,000g for 10 min and the supernatants were analyzed by HPLC after appropriate dilution. 2.6. Stability study The chemical stability test of Radix scutellariae SD prepared with PVP K-30 in solid powder state was carried out for 80 days. The sample powder was stored at room temperature in tightly capped glass vial wrapped with aluminum foil to protect it from the light. At predetermined time intervals, sample powder was completely dissolved in methanol and the concentration of three components in Radix scutellariae SD was analyzed by HPLC. The chemical stability test of the SD in pH 6.8 buffer was carried out for 36 h in order to investigate the stability of three components of Radix scutellariae in intestinal environment. SD powder was completely dissolved in pH 6.8 buffer and the concentration of each component was determined by HPLC after 0 h, 3 h, 6 h, 12 h and 36 h. The effect of adding vitamin C (VC) on the chemical stability of the components in pH 6.8 buffer was evaluated using 1:1, 1:3, 1:6 and 1:12 w/w ratios of the extract of Radix scutellariae and VC. The samples were completely dissolved in pH 6.8 buffer and analyzed in the same manner as previous study. 2.7. Dissolution test
2.4. Preparation of solid dispersions 2.4.1. Method of preparation Solid dispersion of the Radix scutellariae extract with various polymers (PEG 3400, PEG 8000, PVP K-30, poloxamer 188 and poloxamer 407) at the weight ratio of 1:5 (Radix scutellariae extract: polymer) was prepared by solvent method. All three components of the extract of Radix scutellariae showed excellent solubility in methanol and hence used as solvent for the formation of solid dispersion. Briefly, the extract of Radix scutellariae and the polymer were separately dissolved in a minimum volume of methanol, and the two solutions were mixed together. The solvent was removed by evaporation under reduced pressure at room
Tablets of Radix scutellariae extract and its SD were prepared by rotary tablet press (PP-11D, Chamunda Pharma Machinery, India), and the formulations of the tablets are shown in Table 1. The dissolution test of the tablets was carried out using an USP dissolution apparatus II (DST 810, Labfine, Inc., Suwon, Korea). All of the tablets contained equivalent amount of the extract and each tablet was placed in dissolution vessel containing 300 mL of pH 6.8 buffer at 37 C. The paddle was rotated at 50 rpm/min. At predetermined time intervals, 2 mL of sample solution was withdrawn and equal volume of the medium was replenished. Sample solution was then centrifuged at 12,000g for 10 min and the supernatant was analyzed by HPLC.
604
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
Table 1 Formulations of various tablets used in dissolution test. Numbers represent weight in mg unless otherwise stated. Sample
Radix Scutellariae extract
PVP K-30
VC
Avicel
Ratio (extract:VC)
Control tablets SD tablets 1 SD tablets 2 SD tablets 3
17 17 17 17
0 85 85 85
102 0 51 102
68 238 187 136
1:6 1:0 1:3 1:6
2.8. Pharmacokinetics study 2.8.1. LC–MS/MS analysis For the analysis of baicalein concentration in rat plasma, a simple LC–MS/MS method was developed and used. HPLC system (UltiMate1 3000, Thermo Scientific, USA) equipped with a pump, autosampler, and column compartment was used and was connected to quadrupole tandem mass spectrometer (API 3200, SCIEX, USA) equipped with electrospray ionization (ESI–MS-MS). System control and data analysis were carried out with API MS software (Analyst 1.5.2). Chromatographic separation was achieved using a reversed-phase HPLC column (Luna C18, 50 20 mm i.d., 5 mm particle size, Phenomenex, Torrance, CA, USA) protected by a guard column (Phenomenex C18, 4 mm 2 mm, Phenomenex, Torrance, CA, USA). Column oven temperature was 40 C. The mobile phase of 0.1% formic acid in water/ acetonitrile (30: 70, v/v) was run at a flow rate of 300 mL/min. The electrospray ionization (ESI) mass spectrometer was operated in the positive ion mode. Multiple reaction monitoring (MRM) of the precursor-product ion transitions from m/z 271.120 to m/z 123.2 for baicalein and from m/z 310.102 to m/z 135.200 for internal standard (IS, AC-1709) was used for quantitation. Collision energy was 43 V for baicalein and IS. The optimized conditions were as follows; curtain gas (20 psi), nebulizer gas (50 psi), ionspray voltage (5500 V), turbo gas temperature (400 C). The run time was maintained at 3 min. 2.8.2. Preparation of stock solution and standard solution A stock solution of baicalein (1 mg/mL) was prepared in methanol and dilutions of stock solution were made with 0.1% formic acid in 50% methanol. Standard solutions of baicalein in rat plasma were prepared by spiking with an appropriate volume of the diluted stock solution, giving final concentrations of 1, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL. The IS (AC-1709) solution was prepared in methanol and diluted with 0.1% formic acid in 50% methanol to give a final concentration of 1 mg/mL. 2.8.3. Sample preparation Sample preparations involved protein precipitation with acetonitrile. An aliquot (50 mL) of plasma was spiked with 5 mL of IS (AC1709, 1 mg/mL) in a 1.5 mL polyethylene micro tube. Then, 150 mL of acetonitrile was added to the tube and vortexed for 30 s. Then, the mixture was centrifuged at 12,000 rpm for 5 min. A 150 mL of the supernatant samples was transferred into clean eppendorf tube and 5 mL of supernatant was directly injected onto HPLC column. 2.8.4. Calibration curve The calibration standards used were 1, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL of baicalein in rat plasma. These plasma samples were extracted as described above (sample preparation). Calibration curves of baicalein were constructed by the weighted regression method by plotting the peak area ratio of baicalein and IS (AC-1709) as a function of baicalein concentration in rat plasma. 2.8.5. Animal experiments All animal procedures were based on the guidelines issued by Dankook University’s Institutional Animal Care and Use
Committee, which adheres to the guidelines issued by the Institution of Laboratory of Animal Resources. Male SpragueDawley rats of 8 weeks of age were purchased from Charles River Laboratories (Samtaco Inc., Osan, South Korea). All rats were maintained in a light-controlled room kept at a temperature of 23 2 C with 12 h light and dark cycles and a relative humidity of 50 10%. The rats were housed in metabolic cages under filtered, pathogen-free air, with food (Samtaco Inc., Osan, South Korea) and water available ad libitum. The experiments were started after acclimation under these conditions for at least 1 week. The rats were fasted for at least 12 h prior to the start of the experiments and had free access to tap water. The rats were anaesthetized lightly with zoletil 50 and rompun (70: 30, v/v) and the carotid artery was cannulated using a polyethylene tube (PE 60, 0.76 mm I. d., 1.22 mm o.d., Becton Dickinson, Franklin Lakes, NJ, USA) for blood sampling. Then, each rat was housed individually in a rat metabolic cage and allowed to recover from the anesthesia for 3– 4 h before drug administration. 2.8.6. Oral administration in rat and sample collection Baicalein powder dissolved in PEG 400: DW mixture (20: 80, v/ v) and test formulation of baicalein (solid dispersion) dissolved in distilled water at doses of 15 mg/kg was administered orally using a gastric gavage tube in rat. Approximately 0.2 mL aliquot of blood was collected via the carotid artery at 0 (before administration), 5, 15, 30, 60, 90, 120, 180, 240, 360, 480, 600, 720 and 1440 min after the oral administration. Approximately 0.3 mL aliquot of heparinized normal saline (50 units/mL) was injected to flush the cannula immediately after each blood sampling to prevent blood clotting. Blood samples were immediately centrifuged and a aliquot of each plasma sample was stored at 80 C until used for the LC–MS/MS analysis. Urine samples were collected between 0 and 1440 min. At the end of 24 h, each metabolic cage was rinsed with 10 mL of distilled water and the rinsings were combined with the 24 h urine sample. After measuring the exact volume of the combined urine sample, aliquots of the combined urine sample were stored at 80 C freezer until used for the LC–MS/MS analysis. 2.8.7. Statistical analysis The Microsoft1 Excel was used for statistical analysis and a p value of less than 0.05 was considered to be statistically significant using unpaired t-test. 3. Results and discussions 3.1. Screening of carriers The solubility enhancement of the hydrophobic drug in solid dispersion was influenced by the properties of the polymers including molecular weight and surface activity (Leuner and Dressman, 2000). Hence, various carriers were evaluated to determine the optimal polymer for enhancing the solubility of all three components of Radix scutellariae extract when formulated as solid dispersion. As shown in Fig. 2, solid dispersion formed with PVP K-30 offered highest solubility of all three components among the screened carriers. The solubility of baicalein, wogonin and
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
Fig. 1. Chemical structure of baicalein, wogonin, and oroxylin A.
605
oroxylin A (Fig. 1) achieved through solid dispersion with PVP K-30 was 312.3 mg/mL, 21.9 mg/mL and 19.3 mg/mL, respectively in water. The enhancement in solubility of baicalein, wogonin and oroxylin A via the solid dispersion preparation was 35.1, 6.4 and 12.9 times higher as compared to the solubility of the components from the extract in water, respectively (Table 2). Solid dispersion with poloxamer 407 increased only the solubility of wogonin (12.6 mg/mL) and oroxylin A (9.1 mg/mL) significantly, however, relatively lower than that of solid dispersion prepared with PVP K30. Solid dispersions with all other polymers brought relatively insignificant improvement in solubility. It has been reported that materials with similar solubility parameters will have similar intermolecular interactions, which will favor miscibility during preparation of solid dispersion (Laitinen et al., 2009). Therefore, it can be assumed that among screened polymers, PVP K-30 exhibited more similarity in intermolecular interactions and better miscibility with the three hydrophobic components. Hence, PVP K30 was selected as the optimal carrier to form the solid dispersion of three components of the extract for further study. In order to confirm baicalein exists in amorphous form in solid dispersion prepared with PVP K-30, differential scanning calorimetry (DSC) was used. Only baicalein was used in this study, since it is the major component in the extract. The DSC thermograms of baicalein, PVP-K30, and baicalein solid dispersions are shown in Fig. 3. The DSC thermograms of pure baicalein exhibited endothermic peaks around 269 C, which corresponded to its intrinsic melting point. Although small melting peak of baicalein was observed when the ratio of baicalein to PVP K-30 was 1:1, no baicalein peak was observed when the ratio of baicalein to PVP K30 was 1:3 and 1:5, indicating that baicalein might have dispersed in molecular form in PVP K-30. 3.2. Component content of the extract and solubility Component content analysis of the extract showed that baicalein, wogonin and oroxylin A altogether constituted 72.9% (w/w) of the extract (Table 2). The major component of the extract was baicalein (61.2%, w/w) followed by wogonin (9.6%, w/w) and oroxylin A (2.1%, w/w).
Fig. 2. Solubility of the components of Radix Scutellariae extract after forming SD with various polymers in distilled water at room temperature (mean S.D., n = 3).
Table 2 Component content and solubility from the extract and SD with PVP K-30 in various pH medium at room temperature (mean S.D.a , n = 3). Components
Content (%) Solubility (mg/mL) Pure extract in water Pure extract in pH 6.8 SD in pH 1.2 SD in pH 4.0 SD in pH 6.0 SD in pH 6.8 SD + VC in pH 6.8 a
Standard deviation.
Baicalein
Wogonin
Oroxylin A
61.2 2.1
9.6 0.2
2.1 0.1
8.9 1.5 3.39 0.92 223.8 3.1 316.9 8.2 439.9 7.4 1091.3 135.2 2865.4 496.3
3.4 0.0 3.98 0.72 18.6 0.4 20.7 0.3 29.3 0.3 78.4 14.6 127.5 7.9
1.5 0.0 2.07 0.14 14.7 0.2 18.8 0.5 25.1 0.3 90.1 4.6 92.5 0.9 Fig. 3. Differential scanning calorimetry (DSC) thermograms of baicalein, PVP K-30, baicalein solid dispersions prepared at ratios of 1:1, 1:3, and 1:5 (baicalein:PVP K30).
606
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
As is shown in Table 2, solubility of three major components in the pure extract was extremely low in water. Upon formulating as solid dispersion with PVP K-30, all three components exhibited greatly enhanced solubility when measured in various pH buffers. It was found that solid dispersion technique was more successful in enhancing solubility of baicalein as compared to wogonin and oroxylin A. However, this difference in the extent of solubility enhancement of three components would have little impact on the dissolution of the extract owing to very low proportions of wogonin and oroxylin A in the extract as compared to baicalein. Moreover, solubility of three components from the extract showed pH dependency. As illustrated in Table 2, components’ solubility from solid dispersion with PVP K-30 displayed profound increase with increase in pH. Solubility of baicalein was 4.9 times higher at pH 6.8 as compared to the solubility at pH 1.2, whereas wogonin and oroxylin A exhibited 4.2 and 6.1 times higher solubility, respectively. However, it was found later that the components from the extract degraded in the aqueous medium (detail description is in section “stability test of the components in solid dispersion”). Baicalein is a well-known anti-oxidant and it is highly likely that the degradation is oxidative process. Therefore, vitamin C was selected as a model anti-oxidant to confirm degradation mechanism of baicalein and to find a way to prevent degradation during dissolution process. The solubility of the components was determined in pH 6.8 buffer in the presence of vitamin C (VC) (SD: VC = 1:6) as an antioxidant. In such case, concentration as high as 2865.4 mg/mL was obtained for baicalein, and for wogonin and oroxylin A, it was 127.5 mg/mL and 92.5 mg/mL, respectively (Table 2). The increased solubility of baicalein and wogonin in pH 6.8 buffer after incorporation of VC was thought to be antioxidative effect of VC. No significant difference was observed in case of oroxylin A. The solubility of a number of poorly soluble active drugs may be increased by simply mixing with PVP (Kibbe, 2000). Hence, the influence of the PVP K-30 on the solubility of three components from the extract in pH 6.8 medium was investigated to evaluate direct effect of PVP K-30, and the results are shown in Fig. 4. It shows that solubility of all three components increased as the concentration of PVP K-30 increased in pH 6.8 medium, however, the extent of increase was not directly proportional to that of PVP K-30. As the concentration of PVP K-30 increased from 0.1 mg/mL to 10 mg/mL in pH 6.8 buffer, the increase in solubility of baicalein, wogonin and oroxylin A was from 12.7 mg/mL to 18.3 mg/mL (1.4 times), 4.3 mg/mL to 11.8 mg/mL (2.7 times) and 2.4 mg/mL to 3.9 mg/mL (1.6 times), respectively. When compared with the solubility of pure extract in pH 6.8 buffer (Table 2), the increase in
Fig. 4. Solubility of three components from pure extract in various concentrations of PVP K-30 in pH 6.8 medium at room temperature (mean S.D., n = 3).
solubility of three components due to incorporation of PVP K-30 was relatively low. On the other hand, SD of PVP K-30 greatly enhanced the solubility of the three components in pH 6.8 buffer as compared with that from the solution of PVP K-30 in pH 6.8 buffer. At weight ratio of 1:5 of the extract and PVP K-30, solubility of the baicalein, wogonin and oroxylin A from the SD in pH 6.8 buffer was 1091.3 mg/mL, 78.4 mg/mL and 90.1 mg/mL, respectively, which were 59.8, 6.6 and 23.1 times higher than that of solubility in 10 mg/mL PVP K-30 solution in pH 6.8 buffer. Such enormous increase in solubility of the components may be attributable to reduction of crystal size of components and/or change of crystalline form of components into amorphous state (Karavas et al., 2007). 3.3. Stability test of the components in SD As mentioned earlier, Radix Scutellariae extract is known to have sensitivity towards external factors such as light and temperature8. The presence of numerous components in the herb extract increases the risk of interactions among its components and degradation under the influence of exterior environments such as humidity, air, light and heat. Therefore, content of the components in herb extract should be monitored periodically. Apart from its solubilizing utility, solid dispersion technique can also be used to impart chemical stability to the drug. It was assumed that polymeric matrix used in the solid dispersion would create sufficient physical or sterical hindrance for the entry of air, moisture and light, thereby preventing or reducing the rate of degradation of the drugs. Stability test of the three components in Radix Scutellariae SD powder was conducted for 80 days to ascertain the chemical stability. It was found that there was no significant change in concentration of all three components throughout the period of 80 days (data not provided). This confirmed chemical stability of the components in the SD throughout the experimental period. Like many of traditional herbs extract, Radix scutellariae extract showed stability problem in aqueous environment. It was found that among three components of the extract, baicalein was especially unstable at pH 6.8 buffer. It may due to the fact that the major component, baicalein, is a kind of flavonoids with potent antioxidative effect, indicating that it is prone to oxidation. To inhibit oxidation of baicalein, a stronger antioxidant is required. Vitamin C, a water soluble potent antioxidant, which had been previously used to prevent oxidation of polyphenolic compounds17, was our obvious choice. Hence, VC was mixed with SD at various weight proportions as an antioxidant. The stability test of each component in Radix Scutellariae SD with or without VC in pH 6.8 medium was carried out for 36 h and the results are shown in Fig. 5. It was observed that baicalein in the SD without VC showed relatively lower concentration as compared to the SDs with VC at 0 h (samples were analyzed immediately after complete dissolution). This might be due to instantaneous oxidation of the component during dissolution phase in the absence of VC. On prolonging the exposure time of the SD without VC in pH 6.8 buffer, further decrease in concentration of baicalein was observed. When VC was physically mixed with SD at the weight ratio of 1:1, the concentration of baicalein was maintained till 12 h and then decline in the concentration was observed. The decline in the concentration after 12 h may be due to insufficient amount of VC left in the buffer to prevent oxidation of baicalein. When weight ratio of the extract and VC in the SD was increased to 1:3 and beyond, baicalein showed complete stability in pH 6.8 buffer throughout the period of 36 h. Wogonin and oroxylin A showed relatively good stability in pH 6.8 buffer. The slightly lower initial concentration of both components was observed from the samples without VC, however, there was no decline in concentration of
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
607
shown in Table 1. In Fig. 6a, because of the low solubility of baicalein in pH 6.8 buffer, control group showed less than 10% release after 6 h. Baicalein in SD tablet 1 showed significantly higher release rate than control owing to high solubility. In the initial 30 min, the amount of baicalein released reached to about 70% and then gradually decreased. The release percentage declined to less than 50% at 6 h. The reason for the decline was due to
Fig. 5. Chemical stability test of Radix Scutellariae SD in pH 6.8 buffer at room temperature: (a) Radix Scutellariae SD without VC (b) SD and VC (Radix Scutellariae extract: VC = 1:1) (c) SD and VC (Radix Scutellariae extract: VC = 1:3) (d) SD and VC (Radix Scutellariae extract: VC = 1: 6) (e) SD and VC (Radix Scutellariae extract: VC = 1:12) (mean S.D., n = 3).
wogonin on prolonging the exposure time. A slight decrease in concentration of oroxylin A was observed after 12 h in case of SD tablet 1. On increasing the amount of VC in the SD at the weight ratio of 1:3 and beyond, no decrease in initial concentrations of both components was observed and no significant change in concentrations was observed over the period of 36 h. Hence, to prevent the oxidation of all three components in aqueous environment, the optimum weight ratio of the extract and VC in the physical mixture of SD and VC was found to be 1:3. 3.4. Dissolution test of the components from the extract Dissolution profiles of control and SD tablets were studied and the results are shown in Fig. 6. The compositions of the tablets are
Fig. 6. Dissolution test of the components from the extract of Radix scutellariae, formulated as tablets with varying composition: (a) baicalein (b) wogonin (c) oroxylin A (mean S.D., n = 3).
608
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
simultaneous oxidation of baicalein even though all the drug content was liberated into the aqueous medium. Baicalein in tablet 2 showed 100% release in the initial 30 min. The presence of VC in the tablet prevented initial oxidation, however, the amount of VC used was not sufficient enough to prevent oxidation for more than 2 h and hence the amount released declined after 2 h. In the SD tablet 3, baicalein showed 100% release in 6 h. The higher proportion of VC used in the tablets 3 adequately inhibited the oxidation of baicalein. The results indicated that by adjusting the amount of VC, the oxidation of baicalein can be controlled for a longer duration of time. Similarly in Fig. 6b, the amount of wogonin released from control tablets was very low, however, in all SD tablets, the amount of wogonin released was almost 100%.
Wogonin was relatively stable at pH 6.8 buffer when compared to baicalein. As shown in Fig. 6c, the dissolution of oroxylin A from control tablet was 22.8% at 6 h. SD tablet 1 showed approx. 80.3% release of oroxylin A whereas tablets 2 and 3 showed almost 100% release. Slightly lower dissolution of oroxylin A from tablet 1 may be due to insufficient amount of VC in the formulation for the prevention of oxidation of the component. This result is slightly deviated from the result of the chemical stability test. In chemical stability test, the sample containing same proportion of VC did not show significant decline of oroxylin A (‘B’ in Fig. 6c). This discrepancy can be explained by the fact that the volume of pH 6.8 buffer used in dissolution test was much larger than in chemical stability test which resulted in much lesser concentration
Fig. 7. LC–MS/MS chromatogram of baicalein and AC-1709 (IS): (a) double blank rat plasma samples (b) blank rat plasma samples spiked with IS (c) rat plasma samples spiked with baicalein at 1 ng/mL (LLOQ) (d) plasma obtained 90 min after oral administration of baicalein.
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
of VC in dissolution test. It also suggested that oroxylin A is relatively more prone to oxidation as compared with wogonin. The dissolution test illustrated that SD tablet 3 was the best formulation with 100% release of the components. Moreover, the amount of VC added in this composition completely inhibited the oxidation of the components in pH 6.8 buffer. 3.5. Pharmacokinetic study of baicalein In order to confirm whether increased dissolution rate of SD formulation prepared with PVP K-30 results in higher bioavailability, baicalein was selected for model compound for in vivo study. Vitamin C was also mixed with solid dispersion of baicalein to improve chemical stability. The ratio of components used in this study was 1:5:1 (baicalein:PVP K-30:vitamin C). 3.5.1. Specificity and linearity of analytical method Fig. 7 illustrates chromatograms of double blank rat plasma samples (Fig. 7a), blank rat plasma samples spiked with AC-1709 (IS) (Fig. 7b), rat plasma samples spiked with baicalein at 1 ng/mL (LLOQ) (Fig. 7c), plasma obtained 90 min after oral administration of baicalein (Fig. 7d). The retention times of baicalein and AC-1709 (IS) were 0.66 and 2.14 min, respectively. The overall run time was 3 min. No endogenous or extraneous peaks interfered with the analytes. The calibration curve of baicalein was constructed by plotting the ratio of the peak area of baicalein to that of AC-1709. There was an excellent linearity over the range of 1–2000 ng/mL with a mean correlation coefficient of 0.9995. The typical equation describing the calibration curve in rat plasma was y = 0.00264 x +-0.000397. 3.5.2. Pharmacokinetic analysis The mean arterial plasma concentration-time profiles of baicalein after oral administration of powder (n = 7) and the solid dispersion formulation (n = 9), 15 mg/kg, to rats are shown in Fig. 8, and the relevant pharmacokinetic parameters are also listed in Table 3. The plasma concentrations of baicalein were significantly higher after oral administration of the solid dispersion formulation than baicalein powder, and this resulted in significantly greater AUC24h (40.3% increase), AUCINF (44.1% increase) and Cmax (284.9% increase). The mean percentage of oral dose of baicalein excreted by urine at 24 h was also considerably increased after oral administration of the solid dispersion formulation than baicalein powder (23.0% vs 28.7%). The absorption of baicalein from rat gastrointestinal tract seemed to be rapid; the peak plasma
609
Table 3 Pharmacokinetic parameters of baicalein after oral administration of 15 mg/kg of baicalein in rat (mean S.D).
AUC24h (mg min/mL)* AUCINF (mg min/mL)* Cmax (ng/mL)* Tmax (h) MRT (h) T1/2 (h) Xu,24h (%) *
Control
Solid dispersion
12.9 2.3 16.1 5.8 45.8 20.4 0.08 0.0 11.6 3.4 7.6 3.2 23.0 5.3
18.1 3.8 23.2 6.4 176.3 66.0 0.2 0.2 11.0 2.9 10.0 2.7 28.7 16.2
Significantly different compared with control group (P < 0.05).
concentration of baicalein reached at 15 min after oral administration of baicalein powder, however, slightly delayed peak plasma concentration of baicalein was observed after oral administration of the solid dispersion formulation. The results clearly indicate that solid dispersion method can increase bioavailability of baicalein. 4. Conclusions The solubility of all three components was significantly increased after forming solid dispersion with hydrophilic polymer, PVP K-30, in pH 6.8 buffer. The three components viz. baicalein, wogonin and oroxylin A, exhibited chemical stability, over a monitored period of 80 days, when Radix scutellariae SD without addition of VC was stored as solid powder in air tight container protected from sunlight. In addition, the three components in the physical mixture of Radix scutellariae SD with VC at the ratio of 1:3 or beyond (Radix scutellariae extract: VC, w/w) showed stability in aqueous environment when checked for 36 h in pH 6.8 buffer. In dissolution test, all three components showed 100% release in pH 6.8 medium within 6 h from the physical mixture of SD with VC at the ratio 1:6 (Radix scutellariae extract: VC, w/w). Pharmacokinetic evaluation of baicalein showed that bioavailability could be significantly improved by solid dispersion owing to increased solubility and dissolution rate. Therefore, the present formulation, being efficient and cost effective, is viable for commercial utilization. Declaration of interest The authors declare that there is no conflict of interest. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01058691). References
Fig. 8. Mean plasma concentration-time profiles of control baicalein (*) and test baicalein (5) after oral administration of 15 mg/kg of baicalein in rat. Each point represents mean S.D (control n = 7, test n = 9).
Chiou, W.L., Riegelman, S., 1971. Pharmaceutical applications of solid dispersion systems. J. Pharm. Sci. 60, 1281–1302. Choi, Y.H., Chin, Y.-W., Yang, S.J., Pel, P., Kim, Y.-J., Kim, E.Y., Han, H.-K., 2016. Isolation of a lignan-enriched fraction from Schisandra chinensis and its effective solubilization via poloxamer 407-based solid dispersion formulation. J. Pharm. Invest. 46, 133–138. Ha, E.-S., Kim, J.-S., Baek I.-h. Hwang, S.-J., Kim, M.-S., 2015. Enhancement of dissolution and bioavailability of ezetimibe by amorphous solid dispersion nanoparticles fabricated using supercritical antisolvent process. J. Pharm. Invest. 45, 641–649. Han, J., Ye, M., Xu, M., Sun, J., Wang, B., Guo, D., 2007. Characterization of flavonoids in the traditional Chinese herbal medicine-Huangqin by liquid chromatography coupled with electrospray ionization mass spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 848, 355–362. Kang, J.-H., Yan, Y.-D., Kim, H.-C., Lee, S.-N., Yong, C.-S., Choi, H.-G., 2010. Enhanced dissolution of coenzyme Q10 using solid dispersions prepared by low temperature melting method. J. Pharm. Invest. 40, 277–283. Karavas, E., Georgarakis, E., Sigalas, M.P., Avgoustakis, K., Bikiaris, D., 2007. Investigation of the release mechanism of a sparingly water-soluble drug from
610
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
solid dispersions in hydrophilic carriers based on physical state of drug, particle size distribution and drug-polymer interactions. Eur. J. Pharm. Biopharm. 66, 334–347. Kibbe, A., 2000. Pharmaceutical Excipients, 3rd ed. American Pharmaceutical Association, Washington, DC. Laitinen, R., Suihko, E., Toukola, K., Björkqvist, M., Riikonen, J., Lehto, V.P., Järvinen, K., Ketolainen, J., 2009. Intraorally fast-dissolving particles of a poorly soluble drug: preparation and in vitro characterization. Eur. J. Pharm. Biopharm. 71, 271–281. Lei, W., Xueyan, W., Xueqin, Z., Zhihui, Z., Xinjian, Y., 2006. Study on the stability of baicalin in different solvents. China Pharmacist 2, 017. Leuner, C., Dressman, J., 2000. Improving drug solubility for oral delivery using solid dispersions. Eur. J. Pharm. Biopharm. 50, 47–60. Liu, J., Qiu, L., Gao, J., Jin, Y., 2006. Preparation, characterization and in vivo evaluation of formulation of baicalein with hydroxypropyl-bcyclodextrin. Int. J. Pharm. 312, 137–143. Lu, T., Song, J., Huang, F., Deng, Y., Xie, L., Wang, G., Liu, X., 2007. Comparative pharmacokinetics of baicalin after oral administration of pure baicalin, Radix
scutellariae extract and Huang-Lian-Jie-Du-Tang to rats. J. Ethnopharmacol. 110, 412–418. Lu, N., Gao, Y., Ling, Y., Chen, Y., Yang, Y., Gu, H.-Y., Qi, Q., Liu, W., Wang, X.-T., You, Q.D., 2008. Wogonin suppresses tumor growth in vivo and VEGF-induced angiogenesis through inhibiting tyrosine phosphorylation of VEGFR2. Life Sci. 82, 956–963. Qi, L., Zhou, R., Wang, Y., Zhu, Y., 1997. Study of major flavonoids in crude Scutellariae Radix by micellar electrokinetic capillary chromatography. J. Capill. Electrophor. 5, 181–184. Vasconcelos, T., Sarmento, B., Costa, P., 2007. Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. Drug Discov. Today 12, 1068–1075. Wang, Y.-S., Deng, W., Que, C., 1983. The Pharmacology and Application of Traditional Chinese Medicine. Beijing, People’s Medical Publishing House 78. Wu, S., Sun, A., Liu, R., 2005. Separation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi by high-speed counter-current chromatography. J. Chromatogr. A 1066, 243–247. Yao, Y., Zhang, L., 2006. Studies of stability of baicalein. Chin. J. Spectrosc. Lab. 23, 346–348.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Enhancement of solubility and dissolution rate of baicalein, wogonin and oroxylin A extracted from Radix scutellariae Hang Yua , Jae-Sang Changa , Sun Young Kimb , Yoon G. Kimb , Hoo-Kyun Choia,* a b
College of Pharmacy, Chosun University, 309 Philmoondaero, Gwangju 61452, Korea College of Pharmacy, Dankook University, 119 Dandaero, Cheonan 31116, Korea
A R T I C L E I N F O
Article history: Received 14 March 2017 Received in revised form 30 May 2017 Accepted 18 June 2017 Available online 19 June 2017 Keywords: Solid dispersion Flavonoids Povidone K-30 Chemical stability Poor water solubility Pharmacokinetics
A B S T R A C T
Baicalein, wogonin, and oroxylin A are three major hydrophobic components in the extract of Radix scutellariae with wide spectrum of pharmacological applications. The purpose of this study was to enhance the solubility, dissolution rate and stability of baicalein, wogonin and oroxylin A by solid dispersion (SD) technique. SD of the extract with various polymers was prepared to select the best carrier. Solubility study, chemical stability study and dissolution study were performed to characterize the SD. The solubility of all three components, after forming solid dispersion with povidone K-30 (PVP K-30) was significantly increased in pH 6.8 medium at room temperature. Stability study conducted for 80 days elucidated that the SD in powder state was fairly stable without the aid of Vitamin C (VC). VC was required as antioxidant to impart stability to baicalein in aqueous medium. The dissolution test of the SD of three components, admixed with VC at the weight ratio of 1:6 (Radix scutellariae extract: VC, w/w) exhibited faster dissolution rate with 100% release of all components. Pharmacokinetic study of baicalein solid dispersion revealed that AUC and Cmax significantly increased by solid dispersion preparation. Thus, the current developed method, being simple, economical and effective, can be useful for the production of soluble dosage forms of the extract of Radix scutellariae in commercial scale. © 2017 Published by Elsevier B.V.
1. Introduction Radix scutellariae is an important herb in traditional Chinese medicine and is prepared from the roots of Scutellaria baicalensis Georgi (Labiatae family). Traditionally, it has been employed for detoxication and relief from fever. Nowadays, Radix scutellariae is widely used for the treatment of inflammation, fever, hepatitis, allergic diseases, hypertension (Wang et al., 1983). The chemical constituents of Radix scutellariae have been elucidated and are mostly consisted of flavonoids like baicalein, baicalin, wogonin and wogonoside. More than 60 flavonoids have been reported from Radix scutellariae, and most of them occur as glucuronides (Han et al., 2007). Among them, baicalin has been used as a phytochemical marker for the quality control of Radix scutellariae in Chinese pharmacopoeia (Qi et al., 1997; Wu et al., 2005). Pharmacological studies revealed that baicalin has anti-inflammatory, anti-allergic, anti-oxidant and hepatoprotective properties (Lu et al., 2007) whereas wogonin possessed anticancer activity (Lu et al., 2008). Even though the Radix scutellariae components
* Corresponding author. E-mail address: [email protected] (H.-K. Choi). http://dx.doi.org/10.1016/j.ijpharm.2017.06.068 0378-5173/© 2017 Published by Elsevier B.V.
showed many advantages, their low aqueous solubility, bioavailability, and sensitivity to light and temperature (Lei et al., 2006; Yao and Zhang, 2006) exerted problems in the drug delivery. Limited number of studies has been reported in the literature regarding the enhancement of solubility of the baicalein. One study utilized inclusion complex with hydroxypropyl-beta-cyclodextrin (HP-b-CD) and freeze drying process (Liu et al., 2006). Use of HPb-CD and freeze drying process can be costly and difficult to be commercialized. Hence, simple and convenient process that can sufficiently improve solubility and dissolution of major components of Radix scutellariae using cheaper excipients are more desirable. A solid dispersion is “the dispersion of one or more active ingredients in an inert carrier or matrix at solid state prepared by melting (fusion), solvent or melting-solvent method”(Chiou and Riegelman, 1971). It is widely used and is one of the most successful strategies to improve drug release and enhance oral bioavailability of poorly soluble drugs (Choi et al., 2016; Ha et al., 2015; Vasconcelos et al., 2007). The SD may exist as coarse suspension, fine suspension or solid solution depending upon the state of the drug in the carrier. To formulate successful SD, drug should be in finely reduced size, preferably in amorphous form. Dissolution of a drug with reduced particle size will be faster due to
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
increase in surface area and solubilization of amorphous state is much easier as no energy is required to break the crystal lattice. The state of the drug in the carrier is determined by drug-carrier interaction which in turn governs the release behavior of the drug from the SD. Hence, selection of carrier for SD is crucial and formation of successful SD is favored by the use of carrier having high affinity to the drug or high mixing capability. Hydrophilic polymers are generally used as carrier in the SD which enhances wettability and in turn solubility of the drug (Kang et al., 2010). In this study, solid dispersion formulation of Radix scutellariae was developed to enhance the solubility and dissolution rate of three major hydrophobic components. Various polymers were tested to select optimum carrier for the SD preparation of the extract of Radix scutellariae. The SD thus prepared was evaluated in terms of solubility, dissolution rate, stability, and pharmacokinetics. 2. Materials and methods 2.1. Materials Radix scutellariae extract was obtained from Wonkwang University (Iksan, Korea). Vitamin C was provided by Dong-A Pharm. Co. (Seoul, Korea). Poloxamer 407, poloxamer 188 and povidone K-30 (Kollidon1 30) were obtained from BASF (Ludwigshafen, Germany). Avicel1 101 (microcrystalline cellulose) was provided by Seoul Pharm. Co. (Seoul, Korea). Polyethylene glycol 3400 and 8000 (PEG 3400 and PEG 8000) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). AC-1709 was used as an internal standard for plasma sample analysis and was obtained from Professor Keon-Wook Kang at Seoul National University. All other chemicals were of reagent grade or above and were used without further purification. 2.2. HPLC analysis HPLC system (Shimadzu Scientific Instruments, Kyoto, Japan) equipped with a UV detector (SPD-10A), a pump (LC-10AD) and an automatic injector (SIL-10A) was used. The wavelength of UV detector was 254 nm and the flow rate was 1 mL/min. The mobile phase composed of 6.7% (v/v) acetic acid aqueous solution and acetonitrile at volume ratio of 1:1. The samples were analyzed through a reversed-phase column (Gemini 5 m C18, Phenomenex, USA) at the temperature of 30 C. 2.3. Component content analysis of Radix scutellariae extract 1 mg of Radix scutellariae extract was completely dissolved in 2 mL of methanol and suitably diluted. The concentration of three major components, viz. baicalein, wogonin and oroxylin A, in the sample solution was analyzed by HPLC and percentage of each of three components contained in the extract was calculated.
603
temperature for 12 h. The residue was ground in a mortar, passed through a 200 mm sieve and stored in a sealed vial for further investigation. Each solid dispersion thus prepared was subjected for solubility test at room temperature. Solid dispersion of baicalein for in vivo study was prepared by solvent method at the weight ratio of 1:5:1 (baicalein:PVP K-30: vitamin C) in a same way described above. 2.4.2. DSC study Thermal analysis was carried out using a differential scanning calorimeter (Pyris 6 DSC, Perkin Elmer, Waltham, MA, USA). Indium was used to calibrate temperature scale and enthalpic response. Samples were placed in aluminum pans and heated at a scanning rate of 10 C/min from 30 to 300 C. 2.5. Solubility study To determine the solubility of the components in SDs of Radix scutellariae extract with various polymers, excess amount of each of SD prepared with various polymers was added into 1 mL of distilled water. Effect of pH on the solubility of the components in the SD of the extract prepared with PVP K-30 was determined in each of 1 mL of pH 1.2, pH 4.0, pH 6.0 and pH 6.8 buffers. To investigate the effect of PVP K-30 concentration on the solubility of the components of Radix scutellariae in pH 6.8 buffer, PVP K-30 concentrations of 0.1 mg/mL, 1 mg/mL, 5 mg/mL and 10 mg/mL were used. In all cases, samples were stirred with magnetic bar at 750 rpm at room temperature for 12 h. The samples were then centrifuged at 12,000g for 10 min and the supernatants were analyzed by HPLC after appropriate dilution. 2.6. Stability study The chemical stability test of Radix scutellariae SD prepared with PVP K-30 in solid powder state was carried out for 80 days. The sample powder was stored at room temperature in tightly capped glass vial wrapped with aluminum foil to protect it from the light. At predetermined time intervals, sample powder was completely dissolved in methanol and the concentration of three components in Radix scutellariae SD was analyzed by HPLC. The chemical stability test of the SD in pH 6.8 buffer was carried out for 36 h in order to investigate the stability of three components of Radix scutellariae in intestinal environment. SD powder was completely dissolved in pH 6.8 buffer and the concentration of each component was determined by HPLC after 0 h, 3 h, 6 h, 12 h and 36 h. The effect of adding vitamin C (VC) on the chemical stability of the components in pH 6.8 buffer was evaluated using 1:1, 1:3, 1:6 and 1:12 w/w ratios of the extract of Radix scutellariae and VC. The samples were completely dissolved in pH 6.8 buffer and analyzed in the same manner as previous study. 2.7. Dissolution test
2.4. Preparation of solid dispersions 2.4.1. Method of preparation Solid dispersion of the Radix scutellariae extract with various polymers (PEG 3400, PEG 8000, PVP K-30, poloxamer 188 and poloxamer 407) at the weight ratio of 1:5 (Radix scutellariae extract: polymer) was prepared by solvent method. All three components of the extract of Radix scutellariae showed excellent solubility in methanol and hence used as solvent for the formation of solid dispersion. Briefly, the extract of Radix scutellariae and the polymer were separately dissolved in a minimum volume of methanol, and the two solutions were mixed together. The solvent was removed by evaporation under reduced pressure at room
Tablets of Radix scutellariae extract and its SD were prepared by rotary tablet press (PP-11D, Chamunda Pharma Machinery, India), and the formulations of the tablets are shown in Table 1. The dissolution test of the tablets was carried out using an USP dissolution apparatus II (DST 810, Labfine, Inc., Suwon, Korea). All of the tablets contained equivalent amount of the extract and each tablet was placed in dissolution vessel containing 300 mL of pH 6.8 buffer at 37 C. The paddle was rotated at 50 rpm/min. At predetermined time intervals, 2 mL of sample solution was withdrawn and equal volume of the medium was replenished. Sample solution was then centrifuged at 12,000g for 10 min and the supernatant was analyzed by HPLC.
604
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
Table 1 Formulations of various tablets used in dissolution test. Numbers represent weight in mg unless otherwise stated. Sample
Radix Scutellariae extract
PVP K-30
VC
Avicel
Ratio (extract:VC)
Control tablets SD tablets 1 SD tablets 2 SD tablets 3
17 17 17 17
0 85 85 85
102 0 51 102
68 238 187 136
1:6 1:0 1:3 1:6
2.8. Pharmacokinetics study 2.8.1. LC–MS/MS analysis For the analysis of baicalein concentration in rat plasma, a simple LC–MS/MS method was developed and used. HPLC system (UltiMate1 3000, Thermo Scientific, USA) equipped with a pump, autosampler, and column compartment was used and was connected to quadrupole tandem mass spectrometer (API 3200, SCIEX, USA) equipped with electrospray ionization (ESI–MS-MS). System control and data analysis were carried out with API MS software (Analyst 1.5.2). Chromatographic separation was achieved using a reversed-phase HPLC column (Luna C18, 50 20 mm i.d., 5 mm particle size, Phenomenex, Torrance, CA, USA) protected by a guard column (Phenomenex C18, 4 mm 2 mm, Phenomenex, Torrance, CA, USA). Column oven temperature was 40 C. The mobile phase of 0.1% formic acid in water/ acetonitrile (30: 70, v/v) was run at a flow rate of 300 mL/min. The electrospray ionization (ESI) mass spectrometer was operated in the positive ion mode. Multiple reaction monitoring (MRM) of the precursor-product ion transitions from m/z 271.120 to m/z 123.2 for baicalein and from m/z 310.102 to m/z 135.200 for internal standard (IS, AC-1709) was used for quantitation. Collision energy was 43 V for baicalein and IS. The optimized conditions were as follows; curtain gas (20 psi), nebulizer gas (50 psi), ionspray voltage (5500 V), turbo gas temperature (400 C). The run time was maintained at 3 min. 2.8.2. Preparation of stock solution and standard solution A stock solution of baicalein (1 mg/mL) was prepared in methanol and dilutions of stock solution were made with 0.1% formic acid in 50% methanol. Standard solutions of baicalein in rat plasma were prepared by spiking with an appropriate volume of the diluted stock solution, giving final concentrations of 1, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL. The IS (AC-1709) solution was prepared in methanol and diluted with 0.1% formic acid in 50% methanol to give a final concentration of 1 mg/mL. 2.8.3. Sample preparation Sample preparations involved protein precipitation with acetonitrile. An aliquot (50 mL) of plasma was spiked with 5 mL of IS (AC1709, 1 mg/mL) in a 1.5 mL polyethylene micro tube. Then, 150 mL of acetonitrile was added to the tube and vortexed for 30 s. Then, the mixture was centrifuged at 12,000 rpm for 5 min. A 150 mL of the supernatant samples was transferred into clean eppendorf tube and 5 mL of supernatant was directly injected onto HPLC column. 2.8.4. Calibration curve The calibration standards used were 1, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL of baicalein in rat plasma. These plasma samples were extracted as described above (sample preparation). Calibration curves of baicalein were constructed by the weighted regression method by plotting the peak area ratio of baicalein and IS (AC-1709) as a function of baicalein concentration in rat plasma. 2.8.5. Animal experiments All animal procedures were based on the guidelines issued by Dankook University’s Institutional Animal Care and Use
Committee, which adheres to the guidelines issued by the Institution of Laboratory of Animal Resources. Male SpragueDawley rats of 8 weeks of age were purchased from Charles River Laboratories (Samtaco Inc., Osan, South Korea). All rats were maintained in a light-controlled room kept at a temperature of 23 2 C with 12 h light and dark cycles and a relative humidity of 50 10%. The rats were housed in metabolic cages under filtered, pathogen-free air, with food (Samtaco Inc., Osan, South Korea) and water available ad libitum. The experiments were started after acclimation under these conditions for at least 1 week. The rats were fasted for at least 12 h prior to the start of the experiments and had free access to tap water. The rats were anaesthetized lightly with zoletil 50 and rompun (70: 30, v/v) and the carotid artery was cannulated using a polyethylene tube (PE 60, 0.76 mm I. d., 1.22 mm o.d., Becton Dickinson, Franklin Lakes, NJ, USA) for blood sampling. Then, each rat was housed individually in a rat metabolic cage and allowed to recover from the anesthesia for 3– 4 h before drug administration. 2.8.6. Oral administration in rat and sample collection Baicalein powder dissolved in PEG 400: DW mixture (20: 80, v/ v) and test formulation of baicalein (solid dispersion) dissolved in distilled water at doses of 15 mg/kg was administered orally using a gastric gavage tube in rat. Approximately 0.2 mL aliquot of blood was collected via the carotid artery at 0 (before administration), 5, 15, 30, 60, 90, 120, 180, 240, 360, 480, 600, 720 and 1440 min after the oral administration. Approximately 0.3 mL aliquot of heparinized normal saline (50 units/mL) was injected to flush the cannula immediately after each blood sampling to prevent blood clotting. Blood samples were immediately centrifuged and a aliquot of each plasma sample was stored at 80 C until used for the LC–MS/MS analysis. Urine samples were collected between 0 and 1440 min. At the end of 24 h, each metabolic cage was rinsed with 10 mL of distilled water and the rinsings were combined with the 24 h urine sample. After measuring the exact volume of the combined urine sample, aliquots of the combined urine sample were stored at 80 C freezer until used for the LC–MS/MS analysis. 2.8.7. Statistical analysis The Microsoft1 Excel was used for statistical analysis and a p value of less than 0.05 was considered to be statistically significant using unpaired t-test. 3. Results and discussions 3.1. Screening of carriers The solubility enhancement of the hydrophobic drug in solid dispersion was influenced by the properties of the polymers including molecular weight and surface activity (Leuner and Dressman, 2000). Hence, various carriers were evaluated to determine the optimal polymer for enhancing the solubility of all three components of Radix scutellariae extract when formulated as solid dispersion. As shown in Fig. 2, solid dispersion formed with PVP K-30 offered highest solubility of all three components among the screened carriers. The solubility of baicalein, wogonin and
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
Fig. 1. Chemical structure of baicalein, wogonin, and oroxylin A.
605
oroxylin A (Fig. 1) achieved through solid dispersion with PVP K-30 was 312.3 mg/mL, 21.9 mg/mL and 19.3 mg/mL, respectively in water. The enhancement in solubility of baicalein, wogonin and oroxylin A via the solid dispersion preparation was 35.1, 6.4 and 12.9 times higher as compared to the solubility of the components from the extract in water, respectively (Table 2). Solid dispersion with poloxamer 407 increased only the solubility of wogonin (12.6 mg/mL) and oroxylin A (9.1 mg/mL) significantly, however, relatively lower than that of solid dispersion prepared with PVP K30. Solid dispersions with all other polymers brought relatively insignificant improvement in solubility. It has been reported that materials with similar solubility parameters will have similar intermolecular interactions, which will favor miscibility during preparation of solid dispersion (Laitinen et al., 2009). Therefore, it can be assumed that among screened polymers, PVP K-30 exhibited more similarity in intermolecular interactions and better miscibility with the three hydrophobic components. Hence, PVP K30 was selected as the optimal carrier to form the solid dispersion of three components of the extract for further study. In order to confirm baicalein exists in amorphous form in solid dispersion prepared with PVP K-30, differential scanning calorimetry (DSC) was used. Only baicalein was used in this study, since it is the major component in the extract. The DSC thermograms of baicalein, PVP-K30, and baicalein solid dispersions are shown in Fig. 3. The DSC thermograms of pure baicalein exhibited endothermic peaks around 269 C, which corresponded to its intrinsic melting point. Although small melting peak of baicalein was observed when the ratio of baicalein to PVP K-30 was 1:1, no baicalein peak was observed when the ratio of baicalein to PVP K30 was 1:3 and 1:5, indicating that baicalein might have dispersed in molecular form in PVP K-30. 3.2. Component content of the extract and solubility Component content analysis of the extract showed that baicalein, wogonin and oroxylin A altogether constituted 72.9% (w/w) of the extract (Table 2). The major component of the extract was baicalein (61.2%, w/w) followed by wogonin (9.6%, w/w) and oroxylin A (2.1%, w/w).
Fig. 2. Solubility of the components of Radix Scutellariae extract after forming SD with various polymers in distilled water at room temperature (mean S.D., n = 3).
Table 2 Component content and solubility from the extract and SD with PVP K-30 in various pH medium at room temperature (mean S.D.a , n = 3). Components
Content (%) Solubility (mg/mL) Pure extract in water Pure extract in pH 6.8 SD in pH 1.2 SD in pH 4.0 SD in pH 6.0 SD in pH 6.8 SD + VC in pH 6.8 a
Standard deviation.
Baicalein
Wogonin
Oroxylin A
61.2 2.1
9.6 0.2
2.1 0.1
8.9 1.5 3.39 0.92 223.8 3.1 316.9 8.2 439.9 7.4 1091.3 135.2 2865.4 496.3
3.4 0.0 3.98 0.72 18.6 0.4 20.7 0.3 29.3 0.3 78.4 14.6 127.5 7.9
1.5 0.0 2.07 0.14 14.7 0.2 18.8 0.5 25.1 0.3 90.1 4.6 92.5 0.9 Fig. 3. Differential scanning calorimetry (DSC) thermograms of baicalein, PVP K-30, baicalein solid dispersions prepared at ratios of 1:1, 1:3, and 1:5 (baicalein:PVP K30).
606
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
As is shown in Table 2, solubility of three major components in the pure extract was extremely low in water. Upon formulating as solid dispersion with PVP K-30, all three components exhibited greatly enhanced solubility when measured in various pH buffers. It was found that solid dispersion technique was more successful in enhancing solubility of baicalein as compared to wogonin and oroxylin A. However, this difference in the extent of solubility enhancement of three components would have little impact on the dissolution of the extract owing to very low proportions of wogonin and oroxylin A in the extract as compared to baicalein. Moreover, solubility of three components from the extract showed pH dependency. As illustrated in Table 2, components’ solubility from solid dispersion with PVP K-30 displayed profound increase with increase in pH. Solubility of baicalein was 4.9 times higher at pH 6.8 as compared to the solubility at pH 1.2, whereas wogonin and oroxylin A exhibited 4.2 and 6.1 times higher solubility, respectively. However, it was found later that the components from the extract degraded in the aqueous medium (detail description is in section “stability test of the components in solid dispersion”). Baicalein is a well-known anti-oxidant and it is highly likely that the degradation is oxidative process. Therefore, vitamin C was selected as a model anti-oxidant to confirm degradation mechanism of baicalein and to find a way to prevent degradation during dissolution process. The solubility of the components was determined in pH 6.8 buffer in the presence of vitamin C (VC) (SD: VC = 1:6) as an antioxidant. In such case, concentration as high as 2865.4 mg/mL was obtained for baicalein, and for wogonin and oroxylin A, it was 127.5 mg/mL and 92.5 mg/mL, respectively (Table 2). The increased solubility of baicalein and wogonin in pH 6.8 buffer after incorporation of VC was thought to be antioxidative effect of VC. No significant difference was observed in case of oroxylin A. The solubility of a number of poorly soluble active drugs may be increased by simply mixing with PVP (Kibbe, 2000). Hence, the influence of the PVP K-30 on the solubility of three components from the extract in pH 6.8 medium was investigated to evaluate direct effect of PVP K-30, and the results are shown in Fig. 4. It shows that solubility of all three components increased as the concentration of PVP K-30 increased in pH 6.8 medium, however, the extent of increase was not directly proportional to that of PVP K-30. As the concentration of PVP K-30 increased from 0.1 mg/mL to 10 mg/mL in pH 6.8 buffer, the increase in solubility of baicalein, wogonin and oroxylin A was from 12.7 mg/mL to 18.3 mg/mL (1.4 times), 4.3 mg/mL to 11.8 mg/mL (2.7 times) and 2.4 mg/mL to 3.9 mg/mL (1.6 times), respectively. When compared with the solubility of pure extract in pH 6.8 buffer (Table 2), the increase in
Fig. 4. Solubility of three components from pure extract in various concentrations of PVP K-30 in pH 6.8 medium at room temperature (mean S.D., n = 3).
solubility of three components due to incorporation of PVP K-30 was relatively low. On the other hand, SD of PVP K-30 greatly enhanced the solubility of the three components in pH 6.8 buffer as compared with that from the solution of PVP K-30 in pH 6.8 buffer. At weight ratio of 1:5 of the extract and PVP K-30, solubility of the baicalein, wogonin and oroxylin A from the SD in pH 6.8 buffer was 1091.3 mg/mL, 78.4 mg/mL and 90.1 mg/mL, respectively, which were 59.8, 6.6 and 23.1 times higher than that of solubility in 10 mg/mL PVP K-30 solution in pH 6.8 buffer. Such enormous increase in solubility of the components may be attributable to reduction of crystal size of components and/or change of crystalline form of components into amorphous state (Karavas et al., 2007). 3.3. Stability test of the components in SD As mentioned earlier, Radix Scutellariae extract is known to have sensitivity towards external factors such as light and temperature8. The presence of numerous components in the herb extract increases the risk of interactions among its components and degradation under the influence of exterior environments such as humidity, air, light and heat. Therefore, content of the components in herb extract should be monitored periodically. Apart from its solubilizing utility, solid dispersion technique can also be used to impart chemical stability to the drug. It was assumed that polymeric matrix used in the solid dispersion would create sufficient physical or sterical hindrance for the entry of air, moisture and light, thereby preventing or reducing the rate of degradation of the drugs. Stability test of the three components in Radix Scutellariae SD powder was conducted for 80 days to ascertain the chemical stability. It was found that there was no significant change in concentration of all three components throughout the period of 80 days (data not provided). This confirmed chemical stability of the components in the SD throughout the experimental period. Like many of traditional herbs extract, Radix scutellariae extract showed stability problem in aqueous environment. It was found that among three components of the extract, baicalein was especially unstable at pH 6.8 buffer. It may due to the fact that the major component, baicalein, is a kind of flavonoids with potent antioxidative effect, indicating that it is prone to oxidation. To inhibit oxidation of baicalein, a stronger antioxidant is required. Vitamin C, a water soluble potent antioxidant, which had been previously used to prevent oxidation of polyphenolic compounds17, was our obvious choice. Hence, VC was mixed with SD at various weight proportions as an antioxidant. The stability test of each component in Radix Scutellariae SD with or without VC in pH 6.8 medium was carried out for 36 h and the results are shown in Fig. 5. It was observed that baicalein in the SD without VC showed relatively lower concentration as compared to the SDs with VC at 0 h (samples were analyzed immediately after complete dissolution). This might be due to instantaneous oxidation of the component during dissolution phase in the absence of VC. On prolonging the exposure time of the SD without VC in pH 6.8 buffer, further decrease in concentration of baicalein was observed. When VC was physically mixed with SD at the weight ratio of 1:1, the concentration of baicalein was maintained till 12 h and then decline in the concentration was observed. The decline in the concentration after 12 h may be due to insufficient amount of VC left in the buffer to prevent oxidation of baicalein. When weight ratio of the extract and VC in the SD was increased to 1:3 and beyond, baicalein showed complete stability in pH 6.8 buffer throughout the period of 36 h. Wogonin and oroxylin A showed relatively good stability in pH 6.8 buffer. The slightly lower initial concentration of both components was observed from the samples without VC, however, there was no decline in concentration of
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
607
shown in Table 1. In Fig. 6a, because of the low solubility of baicalein in pH 6.8 buffer, control group showed less than 10% release after 6 h. Baicalein in SD tablet 1 showed significantly higher release rate than control owing to high solubility. In the initial 30 min, the amount of baicalein released reached to about 70% and then gradually decreased. The release percentage declined to less than 50% at 6 h. The reason for the decline was due to
Fig. 5. Chemical stability test of Radix Scutellariae SD in pH 6.8 buffer at room temperature: (a) Radix Scutellariae SD without VC (b) SD and VC (Radix Scutellariae extract: VC = 1:1) (c) SD and VC (Radix Scutellariae extract: VC = 1:3) (d) SD and VC (Radix Scutellariae extract: VC = 1: 6) (e) SD and VC (Radix Scutellariae extract: VC = 1:12) (mean S.D., n = 3).
wogonin on prolonging the exposure time. A slight decrease in concentration of oroxylin A was observed after 12 h in case of SD tablet 1. On increasing the amount of VC in the SD at the weight ratio of 1:3 and beyond, no decrease in initial concentrations of both components was observed and no significant change in concentrations was observed over the period of 36 h. Hence, to prevent the oxidation of all three components in aqueous environment, the optimum weight ratio of the extract and VC in the physical mixture of SD and VC was found to be 1:3. 3.4. Dissolution test of the components from the extract Dissolution profiles of control and SD tablets were studied and the results are shown in Fig. 6. The compositions of the tablets are
Fig. 6. Dissolution test of the components from the extract of Radix scutellariae, formulated as tablets with varying composition: (a) baicalein (b) wogonin (c) oroxylin A (mean S.D., n = 3).
608
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
simultaneous oxidation of baicalein even though all the drug content was liberated into the aqueous medium. Baicalein in tablet 2 showed 100% release in the initial 30 min. The presence of VC in the tablet prevented initial oxidation, however, the amount of VC used was not sufficient enough to prevent oxidation for more than 2 h and hence the amount released declined after 2 h. In the SD tablet 3, baicalein showed 100% release in 6 h. The higher proportion of VC used in the tablets 3 adequately inhibited the oxidation of baicalein. The results indicated that by adjusting the amount of VC, the oxidation of baicalein can be controlled for a longer duration of time. Similarly in Fig. 6b, the amount of wogonin released from control tablets was very low, however, in all SD tablets, the amount of wogonin released was almost 100%.
Wogonin was relatively stable at pH 6.8 buffer when compared to baicalein. As shown in Fig. 6c, the dissolution of oroxylin A from control tablet was 22.8% at 6 h. SD tablet 1 showed approx. 80.3% release of oroxylin A whereas tablets 2 and 3 showed almost 100% release. Slightly lower dissolution of oroxylin A from tablet 1 may be due to insufficient amount of VC in the formulation for the prevention of oxidation of the component. This result is slightly deviated from the result of the chemical stability test. In chemical stability test, the sample containing same proportion of VC did not show significant decline of oroxylin A (‘B’ in Fig. 6c). This discrepancy can be explained by the fact that the volume of pH 6.8 buffer used in dissolution test was much larger than in chemical stability test which resulted in much lesser concentration
Fig. 7. LC–MS/MS chromatogram of baicalein and AC-1709 (IS): (a) double blank rat plasma samples (b) blank rat plasma samples spiked with IS (c) rat plasma samples spiked with baicalein at 1 ng/mL (LLOQ) (d) plasma obtained 90 min after oral administration of baicalein.
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
of VC in dissolution test. It also suggested that oroxylin A is relatively more prone to oxidation as compared with wogonin. The dissolution test illustrated that SD tablet 3 was the best formulation with 100% release of the components. Moreover, the amount of VC added in this composition completely inhibited the oxidation of the components in pH 6.8 buffer. 3.5. Pharmacokinetic study of baicalein In order to confirm whether increased dissolution rate of SD formulation prepared with PVP K-30 results in higher bioavailability, baicalein was selected for model compound for in vivo study. Vitamin C was also mixed with solid dispersion of baicalein to improve chemical stability. The ratio of components used in this study was 1:5:1 (baicalein:PVP K-30:vitamin C). 3.5.1. Specificity and linearity of analytical method Fig. 7 illustrates chromatograms of double blank rat plasma samples (Fig. 7a), blank rat plasma samples spiked with AC-1709 (IS) (Fig. 7b), rat plasma samples spiked with baicalein at 1 ng/mL (LLOQ) (Fig. 7c), plasma obtained 90 min after oral administration of baicalein (Fig. 7d). The retention times of baicalein and AC-1709 (IS) were 0.66 and 2.14 min, respectively. The overall run time was 3 min. No endogenous or extraneous peaks interfered with the analytes. The calibration curve of baicalein was constructed by plotting the ratio of the peak area of baicalein to that of AC-1709. There was an excellent linearity over the range of 1–2000 ng/mL with a mean correlation coefficient of 0.9995. The typical equation describing the calibration curve in rat plasma was y = 0.00264 x +-0.000397. 3.5.2. Pharmacokinetic analysis The mean arterial plasma concentration-time profiles of baicalein after oral administration of powder (n = 7) and the solid dispersion formulation (n = 9), 15 mg/kg, to rats are shown in Fig. 8, and the relevant pharmacokinetic parameters are also listed in Table 3. The plasma concentrations of baicalein were significantly higher after oral administration of the solid dispersion formulation than baicalein powder, and this resulted in significantly greater AUC24h (40.3% increase), AUCINF (44.1% increase) and Cmax (284.9% increase). The mean percentage of oral dose of baicalein excreted by urine at 24 h was also considerably increased after oral administration of the solid dispersion formulation than baicalein powder (23.0% vs 28.7%). The absorption of baicalein from rat gastrointestinal tract seemed to be rapid; the peak plasma
609
Table 3 Pharmacokinetic parameters of baicalein after oral administration of 15 mg/kg of baicalein in rat (mean S.D).
AUC24h (mg min/mL)* AUCINF (mg min/mL)* Cmax (ng/mL)* Tmax (h) MRT (h) T1/2 (h) Xu,24h (%) *
Control
Solid dispersion
12.9 2.3 16.1 5.8 45.8 20.4 0.08 0.0 11.6 3.4 7.6 3.2 23.0 5.3
18.1 3.8 23.2 6.4 176.3 66.0 0.2 0.2 11.0 2.9 10.0 2.7 28.7 16.2
Significantly different compared with control group (P < 0.05).
concentration of baicalein reached at 15 min after oral administration of baicalein powder, however, slightly delayed peak plasma concentration of baicalein was observed after oral administration of the solid dispersion formulation. The results clearly indicate that solid dispersion method can increase bioavailability of baicalein. 4. Conclusions The solubility of all three components was significantly increased after forming solid dispersion with hydrophilic polymer, PVP K-30, in pH 6.8 buffer. The three components viz. baicalein, wogonin and oroxylin A, exhibited chemical stability, over a monitored period of 80 days, when Radix scutellariae SD without addition of VC was stored as solid powder in air tight container protected from sunlight. In addition, the three components in the physical mixture of Radix scutellariae SD with VC at the ratio of 1:3 or beyond (Radix scutellariae extract: VC, w/w) showed stability in aqueous environment when checked for 36 h in pH 6.8 buffer. In dissolution test, all three components showed 100% release in pH 6.8 medium within 6 h from the physical mixture of SD with VC at the ratio 1:6 (Radix scutellariae extract: VC, w/w). Pharmacokinetic evaluation of baicalein showed that bioavailability could be significantly improved by solid dispersion owing to increased solubility and dissolution rate. Therefore, the present formulation, being efficient and cost effective, is viable for commercial utilization. Declaration of interest The authors declare that there is no conflict of interest. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01058691). References
Fig. 8. Mean plasma concentration-time profiles of control baicalein (*) and test baicalein (5) after oral administration of 15 mg/kg of baicalein in rat. Each point represents mean S.D (control n = 7, test n = 9).
Chiou, W.L., Riegelman, S., 1971. Pharmaceutical applications of solid dispersion systems. J. Pharm. Sci. 60, 1281–1302. Choi, Y.H., Chin, Y.-W., Yang, S.J., Pel, P., Kim, Y.-J., Kim, E.Y., Han, H.-K., 2016. Isolation of a lignan-enriched fraction from Schisandra chinensis and its effective solubilization via poloxamer 407-based solid dispersion formulation. J. Pharm. Invest. 46, 133–138. Ha, E.-S., Kim, J.-S., Baek I.-h. Hwang, S.-J., Kim, M.-S., 2015. Enhancement of dissolution and bioavailability of ezetimibe by amorphous solid dispersion nanoparticles fabricated using supercritical antisolvent process. J. Pharm. Invest. 45, 641–649. Han, J., Ye, M., Xu, M., Sun, J., Wang, B., Guo, D., 2007. Characterization of flavonoids in the traditional Chinese herbal medicine-Huangqin by liquid chromatography coupled with electrospray ionization mass spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 848, 355–362. Kang, J.-H., Yan, Y.-D., Kim, H.-C., Lee, S.-N., Yong, C.-S., Choi, H.-G., 2010. Enhanced dissolution of coenzyme Q10 using solid dispersions prepared by low temperature melting method. J. Pharm. Invest. 40, 277–283. Karavas, E., Georgarakis, E., Sigalas, M.P., Avgoustakis, K., Bikiaris, D., 2007. Investigation of the release mechanism of a sparingly water-soluble drug from
610
H. Yu et al. / International Journal of Pharmaceutics 528 (2017) 602–610
solid dispersions in hydrophilic carriers based on physical state of drug, particle size distribution and drug-polymer interactions. Eur. J. Pharm. Biopharm. 66, 334–347. Kibbe, A., 2000. Pharmaceutical Excipients, 3rd ed. American Pharmaceutical Association, Washington, DC. Laitinen, R., Suihko, E., Toukola, K., Björkqvist, M., Riikonen, J., Lehto, V.P., Järvinen, K., Ketolainen, J., 2009. Intraorally fast-dissolving particles of a poorly soluble drug: preparation and in vitro characterization. Eur. J. Pharm. Biopharm. 71, 271–281. Lei, W., Xueyan, W., Xueqin, Z., Zhihui, Z., Xinjian, Y., 2006. Study on the stability of baicalin in different solvents. China Pharmacist 2, 017. Leuner, C., Dressman, J., 2000. Improving drug solubility for oral delivery using solid dispersions. Eur. J. Pharm. Biopharm. 50, 47–60. Liu, J., Qiu, L., Gao, J., Jin, Y., 2006. Preparation, characterization and in vivo evaluation of formulation of baicalein with hydroxypropyl-bcyclodextrin. Int. J. Pharm. 312, 137–143. Lu, T., Song, J., Huang, F., Deng, Y., Xie, L., Wang, G., Liu, X., 2007. Comparative pharmacokinetics of baicalin after oral administration of pure baicalin, Radix
scutellariae extract and Huang-Lian-Jie-Du-Tang to rats. J. Ethnopharmacol. 110, 412–418. Lu, N., Gao, Y., Ling, Y., Chen, Y., Yang, Y., Gu, H.-Y., Qi, Q., Liu, W., Wang, X.-T., You, Q.D., 2008. Wogonin suppresses tumor growth in vivo and VEGF-induced angiogenesis through inhibiting tyrosine phosphorylation of VEGFR2. Life Sci. 82, 956–963. Qi, L., Zhou, R., Wang, Y., Zhu, Y., 1997. Study of major flavonoids in crude Scutellariae Radix by micellar electrokinetic capillary chromatography. J. Capill. Electrophor. 5, 181–184. Vasconcelos, T., Sarmento, B., Costa, P., 2007. Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. Drug Discov. Today 12, 1068–1075. Wang, Y.-S., Deng, W., Que, C., 1983. The Pharmacology and Application of Traditional Chinese Medicine. Beijing, People’s Medical Publishing House 78. Wu, S., Sun, A., Liu, R., 2005. Separation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi by high-speed counter-current chromatography. J. Chromatogr. A 1066, 243–247. Yao, Y., Zhang, L., 2006. Studies of stability of baicalein. Chin. J. Spectrosc. Lab. 23, 346–348.