International Journal of Pharmaceutics 471 (2014) 366–376
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Enhancement of solubility, antioxidant ability and bioavailability of taxifolin nanoparticles by liquid antisolvent precipitation technique Yuangang Zu, Weiwei Wu, Xiuhua Zhao * , Yong Li, Weiguo Wang, Chen Zhong, Yin Zhang, Xue Zhao Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin, Heilongjiang 150040, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 April 2014 Received in revised form 18 May 2014 Accepted 28 May 2014 Available online 2 June 2014
Taxifolin is a kind of flavanonol, whose antioxidant ability is superior to that of ordinary flavonoids compounds owing to its special structure. However, its low bioavailability is a major obstacle for biomedical applications, so the experiment is designed to prepare taxifolin nanoparticles by liquid antisolvent precipitation (LAP) to improve its bioavailability. We selected ethanol as solvent, deionized water as antisolvent, and investigated primarily the type of surfactant and adding amount, drug concentration, volume ratio of antisolvent to solvent, precipitation temperature, dropping speed, stirring speed, stirring time factors affecting drug particles size. Results showed that the poloxamer 188 was selected as the surfactant and the particle size of taxifolin obviously reduced with the increase of the poloxamer 188 concentration, the drug concentration and the dropping speed from 0.08% to 0.45%, from 0.04 g/ml to 0.12 g/ml, from 1 ml/min to 5 ml/min, respectively, when the volume ratio of antisolvent to solvent increased from 2.5 to 20, the particle size of taxifolin first increased and then decreased, the influence of precipitation temperature, stirring speed, stirring time on particle size were not obvious, but along with the increase of mixing time, the drug solution would separate out crystallization. The optimum conditions were: the poloxamer 188 concentration was 0.25%, the drug concentration was 0.08 g/ml, the volume ratio of antisolvent to solvent was 10, the precipitation temperature was 25 C, the dropping speed was 4 ml/min, the stirring speed was 800 r/min, the stirring time was 5 min. Taxifolin nanosuspension with a MPS of 24.6 nm was obtained under the optimum conditions. For getting taxifolin nanoparticles, the lyophilization method was chosen and correspondingly g-cyclodextrin was selected as cryoprotectant from g-cyclodextrin, mannitol, lactose, glucose. Then the properties of raw taxifolin and taxifolin nanoparticles were characterized by scanning electron microscopy (SEM), fourier-transform infrared spectroscopy (FTIR), high performance liquid chromatography–mass spectrometry (LC–MS), Xray diffraction (XRD), differential scanning calorimetry (DSC), and thermo gravimetric (TG), and the conclusion was drawn that taxifolin nanoparticles can be converted into an amorphous form but its chemical construction cannot been changed. Furthermore, dissolving capability test, 2,2-diphenyl-1picrylhydrazyl (DPPH) radical-scavenging activity and reducing power assay, solvent residue test were also carried out. The experimental data showed that the solubility and the dissolution rate of taxifolin nanoparticles were about 1.72 times and 3 times of raw taxifolin, the bioavailability of taxifolin nanoparticles increased 7 times compared with raw taxifolin, and the antioxidant capacity of taxifolin nanoparticles was also superior to raw taxifolin. Furthermore, the residual ethanol of the taxifolin nanoparticles was less than the ICH limit for class 3 solvents of 5000 ppm or 0.5% for solvents and could be used for pharmaceutical. These results suggested that taxifolin nanoparticles might have potential value to become a new oral taxifolin formulation with high bioavailability. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Taxifolin Nanoparticles LAP Antioxidant ability Dissolution rate Bioavailability
1. Introduction
* Corresponding author. Tel.: +86 451 82191517; fax: +86 451 82102082. E-mail address: [email protected] (X. Zhao). http://dx.doi.org/10.1016/j.ijpharm.2014.05.049 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
Taxifolin (3,5,7,3,4-pentahydroxy flavanone or dihydroquercetin, Fig. 1) is a kind of flavanonol (Ma et al., 2012), which was widely distributed in barks of the genus Pinus or Larix and in the seeds of the genus Silybum (Zu et al., 2012). Recent years, taxifolin was also
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Fig. 1. The chemical structure of taxifolin.
found in fruits, especially grapes, oranges and grapefruit (AbadGarcia et al., 2009). It played a special role in maintaining normal functions of circulatory system because of its unique antioxidant activity and biological activity (Lee et al., 2012; Liang et al., 2013; Rogovskii et al., 2010), which effectively eliminate excess free radicals in the human body (Teselkin et al., 2000; Vladimirov et al., 2009), improve immune function, and reduce the formation of cancer cells (Weidmann, 2012; Zhang et al., 2013), prevent cardiovascular disease (Wang et al., 2006). At present, because the taxifolin possessed some medicinal properties, such as antitumor, anti-virus, antioxidant, it has been widely used in medicine, health care products, food industries (Yang et al., 2011). However, on account of the slight solubility of taxifolin, it was difficult to be absorbed and metabolized by the body, which greatly limited its bioavailability and efficacy (Shikov et al., 2009; Zinchenko et al., 2011; Zu et al., 2012). Solubility of drugs was associated with the specific surface area of materials, along with particle size of drugs reduced, the effective area contacting with media increased, so that the solubility and dissolution rate of drugs will improve (Rasenack and Muller, 2004). Therefore, in order to improve the solubility and bioavailability of taxifolin, it is important to prepare small and uniform amorphous taxifolin nanoparticles by micronization technology. In general, the micronization technology is mechanical grinding (Rogers et al., 2003), but the method has disadvantages of large energy consumption, low efficiency, wide distribution of particle size of products, etc. In recent years, with the continuous development of nanometer material technology, some micronization preparation methods and technologies of drugs with potential application value such as the liquid antisolvent precipitation (Azad et al., 2013; Dong et al., 2010; Meer et al., 2011; Zu et al., 2013), the supercritical fluid technology (Byrappa et al., 2008; Li et al., 2008; Park et al., 2013), the spray drying method (Hu et al., 2011; Tao et al., 2013; Tshweu et al., 2013), and so on were widely applied in improving the solubility of low solubility drugs. Currently, our group had a related document reported that micronized taxifolin were prepared by the supercritical antisolvent (SAS) technology and its granule morphology was almost needle-like with particle size distributing between 2 mm and 11 mm (Zu et al., 2012), but the technology existed the questions of large equipment investment and lower productivity. This experiment applied the LAP method, according to the dissolved properties of taxifolin, selected ethanol as the solvent and selected water as the antisolvent and engendered precipitations, then prepared taxifolin nanoparticles through lyophilization. The method compared with other micronization technologies, had some advantages including its process is simple, easy to operate, easy to industrialization and lower cost, was expected to realize industrial production (Chen et al., 2004). The experiment prepared small and uniform amorphous taxifolin nanoparticles by the LAP without changing taxifolin molecule structure. Through the single factor design, the type of surfactant and adding amount, drug concentration, volume ratio of
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antisolvent to solvent, precipitation temperature, dropping speed, stirring speed, stirring time factors affecting particles size of drugs were mainly investigated, and the appropriate conditions of micronization were obtained. The taxifolin nanoparticles were obtained by lyophilization, and its properties had been characterized by scanning electron microscopy (SEM), fourier-transform infrared spectroscopy (FTIR), high performance liquid chromatography–mass spectrometry (LC–MS), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermo gravimetric (TG). Finally solvent residue test, antioxidant test and dissolution test were also carried out to investigate if there was residual ethanol in the process of experiment, and the biological activity and the bioavailability of taxifolin nanoparticles. 2. Materials and methods 2.1. Materials Taxifolin (FW = 304, purity 98%) was purchased from Nanjing Zelang Medical Technological Co., Ltd. (Jiangsu, PR China); ethanol, acetonitrile, methanol, glacial acetic acid, hydrochloric acid, 2,2diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide, trichloroacetic acid, FeCl3, Na2HPO412H2O, NaH2PO42H2O, poloxamer 188, Tween-80 and g-cyclodextrin were obtained from Sigma, deionized water was prepared with Hitech-K flow water purification system (Hitech Instruments Co., Ltd., Shanghai, China). 2.2. Preparation of taxifolin nanoparticles The taxifolin nanoparticles were prepared by the LAP. In short, a certain amount of raw taxifolin were weighed and completely dissolved in a certain volume of ethanol, then the solution was poured into a certain volume of deionized water with a certain amount of surfactants by a peristaltic pump at a certain precipitation temperature under stirring with a certain speed intensity. After a period of time, the ethanol in the solution was removed from the solution by a spin steaming instrument, and then a certain amount of cryoprotectants were added into the solution and stirred evenly through a magnetic stirrer, finally the taxifolin nanoparticles were obtained by the lyophilizer at 50 C for 64 h. Under the same conditions, taxifolin nanoparticles without cryoprotectants were also prepared. Each experiment was repeated at least 3 times. 2.3. Optimization of the LAP of taxifolin nanopaticles Single factor method was used to investigate the optimization of operating conditions for amorphous taxifolin nanoparticles by the LAP process. Through preliminary experiments, several main factors influencing the particle size of taxifolin nanoparticles included the type of surfactant and adding amount, drug concentration, volume ratio of antisolvent to solvent, precipitation temperature, dropping speed, stirring speed, stirring time factors, every factor was respectively investigated in order to get the optimal condition, as follows: surfactants were poloxamer 188, carbomer, PEG-6000 and the surfactant concentration was studied at 0.08%–0.45%. The drug concentration was studied at 0.04– 0.12 mg/ml. The volume ratio of antisolvent to solvent was studied at 2.5–20. The precipitation temperature was investigated at 5– 45 C. The dropping speed was studied at 1–5 ml/min. The stirring speed was studied at 400–2000 r/min. The stirring time was studied at 1–90 min. Finally, the optimum condition of every factor was obtained based on the smallest particle size of every factor, respectively. The obtained nanoparticles were redispersed in deionized water containing a certain amount of g-cyclodextrins, prefrozen at 40 C for 2 h, and subsequently lyophilized at 50 C
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for 64 h to obtain the taxifolin nanoparticles. Taxifolin nanoparticles without g-cyclodextrins were lyophilized under the same freeze drying conditions. 2.4. The condition of HPLC HPLC method was implemented by a Waters chromatographic instrument. The chromatographic column: C18 reverse-phase column (250 mm 4.6 mm, 5 mm, China); The mobile phase: 30:70:0.1% (v/v/v) mixtures of acetonitrile, water and glacial acetic acid; the column temperature: indoor temperature; the flow rate: 1.0 ml/min; the detection wavelength: 289 nm; the injection volume: 10 ml. 2.5. Characterization of taxifolin nanoparticles 2.5.1. Scanning electron microscope (SEM) The morphology of raw taxifolin, taxifolin nanoparticles and taxifolin nanoparticles without g-cyclodextrins was analyzed by SEM. Dry samples were glued on the observation desks with double-sided conductive adhesive and plated gold by the ion sputtering coating machine, uniform and thin metal coating could be obtained, then the morphology and size of the samples were observed. 2.5.2. FTIR and high performance liquid chromatography–mass spectrometry (LC–MS) The molecular structures of raw taxifolin, taxifolin nanoparticles, taxifolin nanoparticles without g-cyclodextrins and g-cyclodextrin were researched using FTIR in the wave number range of 400–4000 cm1 at a resolution of 2 cm1. The KBr pellet method was adopted. Each sample was accurately weighed 2 mg, then mixed with KBr of 200 mg respectively, and grinded into fine powders, then pressed into transparent slices for analysis. The mass spectrums of raw taxifolin and taxifolin nanoparticles were obtained by analyst 1.4 of API 3000 (AB, USA). The raw taxifolin and the taxifolin nanoparticles were dissolved in methanol of chromatographic grade, and paired the solution of 1 mg/ml, respectively. According to the testing conditions, the solution of 1 mg/ml could be diluted. The mass spectrometer was operated in positive ion mode. 2.5.3. XRD The crystal form of raw taxifolin, taxifolin nanoparticles, taxifolin nanoparticles without g-cyclodextrins and g-cyclodextrin was detected and analyzed by an X-ray diffractometer with Cu Ka1 radiation at 30 mA and 40 kV. The four samples were scanned at a step length of 0.02 within 5 60 of 2u, and the scanning speed rate was 0.02 min1.
The specific process as follows: excessive and identical taxifolin amount of each sample were weighed and separately added into four vials containing 3 ml artificial gastric juice (pH 1.2), then all the samples were put in the water bath of 25 C, 100 r/min, after 48 h, 100 ml of solution was separately taken out and put into 2 ml centrifuge tube, and 900 ml of methanol was also added in every centrifuge tube, respectively (the volume ratio of solution to methanol was 1:9), after mixed evenly, the mixture was ultrasonically treated for 30 min, then centrifuged at 10,000 r/ min for 10 min by a centrifugal, the supernate of 10 ml was taken out and injected into the HPLC system. The analysis conditions were described in Section 2.4. 2.5.6. The dissolution rate test The dissolution rate of raw taxifolin, taxifolin nanoparticles and physical mixture of taxifolin with auxiliary materials was studied on the premise of maintaining the sink condition and detected by HPLC. The environmental conditions of the process including the speed and the dissolution temperature were set as 100 r/min, 37.0 0.5 C, respectively. The artificial gastric juice (pH 1.2) containing 0.4% Tween-80 was served as the dissolution medium. 84 mg of raw taxifolin, 194 mg of taxifolin nanoparticles equating with 64 mg of taxifolin (taxifolin content of taxifolin nanoparticles is 35.02% by HPLC detecting) and physical mixture of raw taxifolin of 84 mg with auxiliary materials were completely added into three beakers containing 200 ml of dissolution medium. 1 ml of dissolution medium was taken out each time at 3 min, 8 min, 13 min, 18 min, 23 min, 28 min, 38 min, 48 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, then 1 ml of dissolution medium was added to ensure that the volume of dissolution medium remained constant. 1 ml of dissolution medium was centrifuged at 10,000 r/ min for 10 min by the centrifugal, 100 ml of solution was taken out and put into 2 ml centrifuge tube, and 900 ml methanol was also added in the centrifuge tube, after mixed evenly, the mixture was ultrasonically treated for 30 min, then centrifuged for 10 min at 10,000 r/min, the supernate of 10 ml was taken out and injected into the HPLC system. The analysis conditions were described in Section 2.4. 2.5.7. Measurement of DPPH radical-scavenging activity and reducing powers 2.67 mg raw taxifolin and taxifolin nanoparticles (containing 2.67 mg taxifolin) were weighed and separately added into two vials containing 3 ml deionized water, then the mixture was put in the water bath of 25 C, 100 r/min for 30 min. The suspensions of each sample were centrifuged for 10 min with 10,000 r/min. The
2.5.4. DSC and TG The heating stability of raw taxifolin, taxifolin nanoparticles, taxifolin nanoparticles without g-cyclodextrins and g-cyclodextrin was studied by DSC (TA instruments, model, DSC 204), the four samples were operated at a heating rate of 10 C min1 from 30 C to 300 C. The thermal gravimetric of raw taxifolin, taxifolin nanoparticles, taxifolin nanoparticles without g-cyclodextrins and g-cyclodextrin was researched using TGA at a heating rate of 10 C min1 in a temperature range of 30600 C, and the whole process was carried out under dynamic nitrogen atmosphere. 2.5.5. Equilibrium solubility test The equilibrium solubility of raw taxifolin, taxifolin nanoparticles without g-cyclodextrins, taxifolin-g-cyclodextrins (8:6, 8:7, 8:8, 8:9, 8:10) nanoparticles were detected by HPLC method.
Fig. 2. The effect of the type of surfactant on the MPS taxifolin nanoparticles.
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supernate was obtained and diluted into different taxifolin concentrations (0.89–4.45 mg/ml). In addition, the antioxidant activities of g-cyclodextrin with corresponding concentration of taxifolin nanoparticles solution acted as the control and were measured. DPPH radical-scavenging activity measurement: 2 ml of each sample at different taxifolin concentrations (0.89–4.45 mg/ml) was added to 2 ml of ethanol DPPH solution (25 mg/l), the mixture was shaken energetically and placed at room temperature in the dark. After 30 min, the absorbance of the mixture was measured at 517 nm. The experiment repeated 3 times. The DPPH scavenging
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capacity (SC) of the samples was calculated using the following equation: ðAc Ai Þ 100% SCð%Þ ¼ Ac where Ac is the absorbance of the control, and Ai is the absorbance of the sample. Reducing power measurement: 2 ml of each sample at different taxifolin concentrations (0.89–4.45 mg/ml) were mixed with 2 ml of 0.2 mol/l phosphate buffer (pH 6.6) and 2 ml of 1% potassium
Fig. 3. The effect of each factor on the MPS taxifolin nanoparticles. (a) The amount of surfactant; (b) concentration of taxifolin solution; (c) the volume ratio of antisolvent to solvent; (d) precipitation temperature; (e) stirring speed; (f) stirring time; (g) dropping speed.
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ferricyanide, and then incubated at 50 C for 20 min. In order to stop the reaction, 10% trichloroacetic acid of 2 ml was added into the mixture, after mixed evenly, the mixture was centrifuged at 3000 r/min for 10 min. The supernatant was mixed with distilled water and 0.1% FeCl3 according to volume ratio of 1:1:0.2 (v/v/v), after 10 min, the absorbance of the samples was measured at 700 nm. The experiment repeated 3 times. 2.5.8. Bioavailability 6 female Sprague-Dawley rats (weight between 200 and 250 g) were randomly divided into two groups, each group of 3 rats. They were fasted and freely drunk water in the 12 h before doses. Two groups of rats were respectively given raw taxifolin and taxifolin nanoparticles by gavage at the doses of 50 mg/kg (according to taxifolin calculation), after oral administration, the raw taxifolin group and the taxifolin nanoparticles group were taken blood from rats eyeball at 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24 and 48 h and at 0.15, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h, respectively. Blood samples were put in the centrifuge tube containing heparin, and centrifuged at 3000 r/min for 10 min, the supernatant plasma were stored in the refrigerator of 4 C, and processed on the same day. The processing of blood samples: 0.2 ml of methanol and 0.08 ml of hydrochloric acid solution (10 mol l1) were put in 2 ml centrifuge tube, after mix evenly, the plasma of 0.08 ml was added in the centrifuge tube and vibrated for 30 s by a vortex mixer for blending, then centrifuged for 10 min at 8000 r/min, the supernate of 10 ml was taken out and injected into the HPLC system. The analysis conditions were described in Section 2.4. 2.5.9. GC measurement A gas chromatograph (GC) (Agilent 7890A, Palo Alto, CA, USA) was used to detect whether there was residual ethanol in taxifolin nanoparticles, which equipped with a G1540N-210 flame ionization detector and a HP-5 5% phenyl methyl siloxane capillary column (30.0 m 320 mm 0.25 mm, nominal). 10 mg taxifolin nanoparticles were weighed and dissolved into 1 ml deionized water, then centrifuged for 10 min at 10,000 r/min. The supernate of 2 ml was injected into the GC. The conditions of GC analysis of ethanol were as follows: the initial temperature was 40 C, maintained for 6 min raised to 240 C at the heating rate of 30 C min1 and maintained for 4 min. The make-up gas rate was 30.0 ml min1, the rate of H2 was 40.0 ml min1, and the rate of air was 400.0 ml min1. The sample size was 2 ml, and the split ratio was 20:1.
Fig. 4. (a) SEM images of raw taxifolin; (b) (c) SEM images of taxifolin nanoparticles without g-cyclodextrins; (d) (e) SEM images of taxifolin nanoparticles.
(Hecq et al., 2005; Terayama et al., 2004). In addition, on the premise of smaller particle size, the amount of pharmaceutical excipients added should be little as far as possible, so the poloxamer 188 of 0.25% was selected as the best condition. 3.1.2. Drug concentration In the process of the LAP, the drug concentration has a great influence on particle size. The relationship between drug
3. Result and analysis 3.1. Optimization Study 3.1.1. The effects of surfactant The effects of surfactant on the average particle size of taxifolin are shown in Figs. 2 and 3. From Fig. 2, the average particle size of taxifolin without surfactant and taxifolin containning poloxamer 188, carbomer, PEG-6000 of identical concentration were about 2088 nm, 43 nm, 1820.3 nm, 188.2 nm, respectively. Therefore, the experiment finally selected the poloxamer 188 as the surfactant from poloxamer 188, carbomer, PEG-6000. With the poloxamer 188 concentration ranging from 0.08% to 0.45%, the changes of particle size were examined. From Fig. 3(a), when the poloxamer 188 concentration changing from 0.08% to 0.25%, the average particle size was obviously reduced from 286 nm to 39 nm, but the poloxamer 188 concentration increased from 0.25% to 0.45%, the change of the average particle size was unconspicuous. The experimental results explained that the poloxamer 188 could effectively coat on the surface of drugs, inhibit the growth of particles and be beneficial to decrease the particle size of taxifolin
Fig. 5. FTIR spectra of each sample. (a) Raw taxifolin; (b) taxifolin nanoparticles; (c) taxifolin nanoparticles without g-cyclodextrins; (d) g-cyclodextrin.
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concentration and average particle size is shown in Fig. 3(b). With increasing the drug concentration from 0.04 g/ml to 0.08 g/ml, the average particle size decreased from 426 nm to 30 nm. When the drug concentration was greater than 0.08 g/ml, the average particle size scarcely changed. Therefore, the increase of drug concentration led to the average particle size decreasing significantly. This was because the advance of the drug concentration can increase the super saturation of system, resulting in the crystal nucleation rate was greater than the growth rate, which was favorable to generate tiny particles. However, when the drug concentration was excessive, the number of drug particles increased sharply on the interface of solid phase and liquid phase diffusion, which caused particles adhesion, reunion, and larger particles generation (Zhang et al., 2009; Zu et al., 2014). By comprehensive consideration, the drug concentration of 0.08 g/ml was selected the optimum condition. 3.1.3. Volume ratio of antisolvent to solvent The effects of volume ratio of antisolvent to solvent (2.5, 5, 10, 15, 20) on particle size were studied, as shown in Fig. 3(c). Firstly, with the volume ratio of antisolvent to solvent increasing from 2.5 to 10, the average particle size decreased from 40 nm to 20 nm. When the volume ratio was 10, the average particle size was the smallest at about 20 nm. But when the volume ratio was more than
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10, the average particle size began to increase and when the volume ratio was 20, the average particle size increased at about 74 nm. The results illustrated that the increase of the solvent ratio was beneficial to decrease the amount of ultrafine particles generated in the per unit volume of liquid, and reduce collision and agglomeration between particles, was conducive for small particles formation (Zhang et al., 2011). However, as the supersaturation increased further, the nucleation rate had a maximum. So the optimum ratio of antisolvent to solvent was 10. 3.1.4. Precipitation temperature, stirring speed, stirring time The influences of different temperature, stirring speed and stirring time on the particle size were studied. As shown in Fig. 3(d and e), the temperature (5, 15, 25, 35 and 45 C) and stirring speed (400, 800, 1200, 1600 and 2000 r/min) did not present a significant influence on the particle size. Therefore, for easy operation, 25 C was selected as the optimal temperature; the experiment can be performed at room temperature. When stirring speed was higher or lower than 800 r/min, the particle size became slightly bigger. So, the stirring speed of 800 r/min was selected as the optimal condition. From Fig. 3(f), as the growth of stirring time from 1 min to 60 min, the change of particle size was not obvious. After 60 min, crystallization and precipitation began to appear. The results showed that when the stirring time was short, the existence of the
Fig. 6. LC–MS/MS spectra of taxifolin nanoparticles and raw taxifolin.
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Fig. 7. XRD results of each sample. (a) Raw taxifolin; (b) taxifolin nanoparticles; (c) taxifolin nanoparticles without g-cyclodextrins; (d) g-cyclodextrin.
poloxamer 188 stabilizer could effectively control particle size and morphology of taxifolin particles, and limit the agglomeration of particles. When the stirring time was longer, too much energy was input into the system, and destroyed the relative balance of the system, led to agglomeration and growth of particles. Therefore, 5 min was selected as the optimal stirring time. 3.1.5. Dropping speed The influences of dropping speed (1, 2, 3, 4 and 5 ml/min) on the average particle size were examined. Under the condition of poloxamer 188 concentration of 0.25%, drug concentration of 0.08 g/ml, volume ratio of antisolvent to solvent of 10, stirring speed of 800 r/min, From Fig. 3(g), as dropping speed increased from 1 ml/min to 4 ml/min, the average particle size reduced from 30 nm to 17 nm, when dropping speed was more than 4 ml/min, the change of average particle size was no longer apparent. Therefore, the dropping speed of 4 ml/min was the optimal condition. Through the above analysis, the optimal conditions were: the poloxamer 188 was selected as surfactant, poloxamer 188 concentration was 0.25%, drug concentration was 0.08 g/ml, volume ratio of antisolvent to solvent was 10, precipitation
Fig. 8. DSC results of each sample. (a) Raw taxifolin; (b) taxifolin nanoparticles;(c) taxifolin nanoparticles without g-cyclodextrins; (d) g-cyclodextrin.
Fig. 9. TG curves of each sample. (a) Raw taxifolin; (b) taxifolin nanoparticles; (c) taxifolin nanoparticles without g-cyclodextrins; (d) g-cyclodextrin.
temperature was 25 C, dropping speed was 4 ml/min, stirring speed was 800 r/min and stirring time was 5 min. Taxifolin nanosuspension with a MPS of 24.6 nm were obtained under the optimum conditions. In addition, through preliminary experiments, g-cyclodextrin were selected as cryoprotectant from g-cyclodextrin, mannitol, lactose, glucose. In the end, taxifolin nanoparticles were obtained by lyophilization and used for the following detections. 3.2. Characterization of taxifolin nanoparticles 3.2.1. SEM analysis SEM images of raw taxifolin, taxifolin nanoparticles and taxifolin nanoparticles without g-cyclodextrins are shown in Fig 4. From Fig. 4(a), the granule morphology of the raw taxifolin was various block with particle size distributing between 0.54 mm and 16.6 mm, in Fig. 4(b and c), the granule morphology of the taxifolin nanoparticles without g-cyclodextrins was fibrous or irregular block and its particle size was about 10.1–19.3 mm. However, from Fig. 4(e and f), the taxifolin nanoparticles were almost spherical with particle size distributing between 148 nm and 167.2 nm, the reason for this result was that g-cyclodextrin had a cladding effect for the taxifolin by comparing with the granule morphology of the
Fig. 10. The equilibrium solubility of taxifolin nanoparticles.
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the FTIR and the MS results showed that the molecular structure of the taxifolin nanoparticles cannot be changed. 3.2.3. XRD analysis The XRD curves of raw taxifolin, taxifolin nanoparticles and taxifolin nanoparticles without g-cyclodextrins, g-cyclodextrin are shown in Fig. 7. From Fig. 7(a and c), The vibration intensity of the diffraction peaks of the taxifolin nanoparticles without g-cyclodextrins decreased compared with raw taxifolin, and the size of the vibration intensity reflects the strength of the material crystallization, this illustrated the crystallinity of taxifolin nanoparticles without g-cyclodextrins was lower than that of the raw taxifolin. In Fig. 7(b and d), the diffraction peaks of the taxifolin nanoparticles were less than that of the raw taxifolin, which indicated the particle size of the taxifolin nanoparticles decrease significantly, while the position of the diffraction peak of the taxifolin nanoparticles was different with that of the raw taxifolin, this might because the g-cyclodextrin induced a reaction of cladding for the taxifolin nanoparticles. Fig. 11. The dissolution profiles of raw taxifolin, taxifolin nanoparticles and physical mixture of taxifolin with auxiliary materials.
taxifolin nanoparticles without g-cyclodextrins. Therefore, the particle size of the taxifolin nanoparticles was much smaller than that of the raw taxifolin and the taxifolin nanoparticles without g-cyclodextrins, which was beneficial to improve the bioavailability of taxifolin. Previously, our group also prepared micronized taxifolin of needle-like with particle size distributing between 2 mm and 11 mm by SAS (Zu et al., 2012), but the granule morphology of taxifolin nanoparticles was different with micronized taxifolin of this paper. 3.2.2. FTIR and MS analysis The FTIR spectrograms of raw taxifolin, taxifolin nanoparticles and taxifolin nanoparticles without g-cyclodextrins, g-cyclodextrin are shown in Fig. 5. In Fig. 5(a and b), the molecular structures of the raw taxifolin and the taxifolin nanoparticles without g-cyclodextrins were almost coincident. From Fig. 5(c and d), the taxifolin nanoparticles and the raw taxifolin had some differences in 3200– 2700 cm1 and 1500–700 cm1, however, the peaks of g-cyclodextrinwere almost similar with that of the taxifolin nanoparticles in the two range, which explained the reasons of differences were taxifolin nanoparticles containing g-cyclodextrins. The mass spectrograms of raw taxifolin and taxifolin nanoparticles are shown in Fig. 6, in the diagram, the molecular weight of raw taxifolin and taxifolin nanoparticles were both 302.9. Thus,
3.2.4. DSC and TG analysis DSC analysis results are shown in the Fig. 8, from Fig. 8(a and c), the raw taxifolin had an obvious peak in the 241.89 C, while the taxifolin nanoparticles without g-cyclodextrins had a small peak at about 222.27 C, the solid melting point of the taxifolin nanoparticles without g-cyclodextrins changed, the reason for this phenomenon was that the crystallinity of the taxifolin nanoparticles without g-cyclodextrins reduced compared with the raw taxifolin. From Fig. 8(b and d), the taxifolin nanopaticles did not have an obvious melting process on account of g-cyclodextrins, this illustrated the taxifolin nanoparticles were amorphous form and was beneficial to improve the solubility and the bioavailability of taxifolin (Kim et al., 2008). TG analysis results are shown in Fig. 9, the Fig. 9 shows that the raw taxifolin and the taxifolin nanoparticles without g-cyclodextrins began to weightlessness from 240 C, the g-cyclodextrin began from 230 C to weightlessness, while the taxifolin nanoparticles lost weight fom 180 C. This might be because the particle size of the taxifolin nanoparticles was smaller than that of the raw taxifolin led to its higher specific surface area, so that the taxifolin nanoparticles more easily evaporated and rapidly decomposed. 3.2.5. Equilibrium solubility The equilibrium solubility of raw taxifolin, taxifolin nanoparticles without g-cyclodextrins, taxifolin-g-cyclodextrins (8:6, 8:7, 8:8, 8:9, 8:10) nanoparticles were 0.96, 1.24, 1.47, 1.65, 1.57, 1.67,
Fig. 12. The DPPH radical-scavenging activity and the reducing power of taxifolin nanoparticles and raw taxifolin.
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it meant that the auxiliary material was not the key factor in improving the dissolution rate of taxifolin. And the dissolution rate of the taxifolin nanoparticles was much faster than that of the raw taxifolin, in the 60 min, taxifolin nanoparticles achieving the maximum dissolution was about 93.1%, and in the 180 min, the dissolution of the raw taxifolin was about 79.03%, this was mainly because the particle size of the taxifolin nanoparticles was much smaller than that of the raw taxifolin. In addition, recently our group prepared micronized taxifolin by SAS, its solubility was about 0.02 mg/ml at 60 min (the dissolution medium was PBS) (Zu et al., 2012), this illustrated that the taxifolin nanoparticles prepared by the LAP process were amorphous and smaller so that the dissolution rate of the taxifolin nanoparticles increased. According to Noyes–Whitney equation, with the particle size of micronization products decreasing, the specific surface area of the drugs increased accordingly, thereby increased the contact area of the solid drugs and the dissolution medium, the dissolution of the drugs was also improved accordingly. Fig. 13. The plasma concentration of taxifolin nanoparticles and raw taxifolin.
and 1.46 mg/ml by HPLC detecting, respectively. From Fig. 10, the equilibrium solubility of taxifolin-g-cyclodextrins nanoparticles was better than that of taxifolin nanoparticles without g-cyclodextrins, so the g-cyclodextrin was conducive to improving the solubility of taxifolin, and the solubility of taxifolin-g-cyclodextrins (8:7) nanoparticles and taxifolin-g-cyclodextrins (8:9) nanoparticles were similar. However, the taxifolin would be made into oral drugs, the amount of pharmaceutical excipients should be less as far as possible. Consequently, the ratio of taxifolin to g-cyclodextrins of 8/7 was the optimal condition, and the solubility of taxifolin-g-cyclodextrins (8:7) nanoparticles were about 1.72 times of raw taxifolin. 3.2.6. The dissolution rate analysis The dissolution profiles of raw taxifolin, taxifolin nanoparticles and physical mixture of taxifolin with auxiliary materials are shown in Fig. 11. The dissolution rate of raw taxifolin was faster than that of physical mixture of taxifolin with auxiliary materials;
3.2.7. DPPH radical-scavenging activity and reducing power analysis Taxifolin was a natural antioxidant, but was not widely used because of its low bioavailability, the experiment prepared taxifolin nanoparticles by the LAP to improve the solubility of taxifolin, and applied the DPPH radical-scavenging activity and the reducing power test to compare the antioxidant ability of taxifolin nanoparticles and raw taxifolin. The experimental data obtained illustrated the antioxidant activity of g-cyclodextrin was very little and hardly influence the antioxidant activity of taxifolin nanoparticles. The experimental results of taxifolin nanoparticles and raw taxifolin are shown in Fig. 12, which revealed the radicalscavenging activities and the reducing powers of all the samples increased with the concentrations, but the radical-scavenging activities and the reducing powers of taxifolin nanoparticles were higher than that of raw taxifolin. In addition, from the halfinhibition concentrations (IC50) of the samples, the IC50 of the taxifolin nanoparticles and raw taxifolin was separately 1.654 mg/ ml, 3.093 mg/ml, it was also seen that the taxifolin nanoparticles had a higher DPPH radical-scavenging activity by lower IC50 than that of raw taxifolin.
Fig. 14. (a) The gas phase diagram of 10 mg/ml water solution of the taxifolin nanoparticles; (b) the gas phase diagram of 0.05 mg/ml ethanol solution.
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3.2.8. Bioavailability analysis Bioavailability experiment result is shown in Fig. 13, it could be seen from the figure, which the absorption of taxifolin nanoparticles group was significantly faster than that of raw taxifolin group in vivo, and the drug concentration in rat plasma of the taxifolin nanoparticles group was also higher than that of the raw drug group. The drug concentration of the taxifolin nanoparticles and the raw taxifolin in plasma of rats reached the maximum of 13.5 ng/ml and 1.3 ng/ml at 9 min and 2 h after gavage, respectively. By comparing the corresponding AUC values of the two groups, the result demonstrated that the AUC values of the taxifolin nanoparticles was 7 times higher than that of the raw taxifolin, this was because the amorphous taxifolin nanoparticles made the effective area of taxifolin contacting with media increase, which was beneficial to improve the bioavailability of taxifolin. So the oral bioavailability of the taxifolin nanoparticles was improved significantly compared with the raw taxifolin. 3.2.9. Solvent residue The detection of residual solvent is widely used for pharmaceutical. The experiment prepared taxifolin nanoparticles with ethanol as the solvent by the LAP, the detected results are shown in Fig. 14(a and b). Fig. 14(a) shows the gas phase diagram of 10 mg/ml water solution of the taxifolin nanoparticles. There was only one solvent peak, its retention time was 2.343 min. Fig. 14(b) shows the gas phase diagram of 0.05 mg/ml ethanol solution (10 mg/ml water solution of the taxifolin nanoparticles containing 0.5% ethanol). There were two solvent peak, after repeated testing, it was confirmed that a retention time of water was 2.304 min and a retention time of ethanol was 2.857 min. Therefore, the solvent peak was water peak in Fig. 14(a). Fig. 14(b) shows that the residual ethanol was less than the ICH limit for class 3 solvents of 5000 ppm or 0.5% for solvents, so the taxifolin nanoparticles conformed to ICH requirements and could be used for pharmaceutical. 4. Conclusions Taxifolin nanoparticles have been successfully prepared by the LAP technique using ethanol as solvent and deionized water as antisolvent. In this experiment, the poloxamer 188 and the g-cyclodextrin was selected as surfactant and cryoprotectant to inhibit the aggregation and particle growth, respectively. The optimal conditions as follows: poloxamer 188 concentration was 0.25%, drug concentration was 0.08 g/ml, volume ratio of antisolvent to solvent was 10, precipitation temperature was 25 C, dropping speed was 4 ml/min, stirring speed was 800 r/min and stirring time was 5 min. Taxifolin nanosuspension with a MPS of 24.6 nm was obtained under the optimum conditions, then the taxifolin nanoparticles with a MPS of 167.2 nm were achieved by mean of lyophilization. In addition, the FTIR and the MS results showed that the molecular structure of the taxifolin nanoparticles cannot be changed. The analysis results of XRD and DSC indicated that the prepared taxifolin nanoparticles were amorphous. The ethanol residual of taxifolin nanoparticles was less than the ICH limit for class 3 solvents of 5000 ppm or 0.5% for solvents by the GC analysis, and had a higher DPPH-scavenging activity and reducing powers than raw taxifolin. The dissolution test showed the taxifolin nanoparticles exhibited enhanced dissolution rate and solubility compared to the raw taxifolin, and the oral bioavailability of the taxifolin nanoparticles was improved significantly compared with the raw taxifolin in vivo. Therefore, the taxifolin nanoparticles may have great potential value to become a new oral txifolin formulation.
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Acknowledgments The authors would like to acknowledge the financial support from Ph.D. Research Startup Foundation of Heilongjiang Province (LBH-Q13005), and the National Natural Science Foundation of China (No. 21203018). References Abad-Garcia, B., Garmon-Lobato, S., Berrueta, L.A., Gallo, B., Vicente, F., 2009. A fragmentation study of dihydroquercetin using triple quadrupole mass spectrometry and its application for identification of dihydroflavonols in citrus juices. Rapid Commun. Mass Spectrom. 23, 2785–2792. Azad, M.A., Knieke, C., To, D., Dave, R., 2013. Preparation of concentrated stable fenofibrate suspensions via liquid antisolvent precipitation. Drug Dev. Ind. Pharm. . Byrappa, K., Ohara, S., Adschiri, T., 2008. Nanoparticles synthesis using supercritical fluid technology – towards biomedical applications. Adv. Drug Deliv. Rev. 60, 299–327. Chen, J.-F., Zhou, M.-Y., Shao, L., Wang, Y.-Y., Yun, J., Chew, N.Y., Chan, H.-K., 2004. 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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Enhancement of solubility, antioxidant ability and bioavailability of taxifolin nanoparticles by liquid antisolvent precipitation technique Yuangang Zu, Weiwei Wu, Xiuhua Zhao * , Yong Li, Weiguo Wang, Chen Zhong, Yin Zhang, Xue Zhao Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin, Heilongjiang 150040, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 April 2014 Received in revised form 18 May 2014 Accepted 28 May 2014 Available online 2 June 2014
Taxifolin is a kind of flavanonol, whose antioxidant ability is superior to that of ordinary flavonoids compounds owing to its special structure. However, its low bioavailability is a major obstacle for biomedical applications, so the experiment is designed to prepare taxifolin nanoparticles by liquid antisolvent precipitation (LAP) to improve its bioavailability. We selected ethanol as solvent, deionized water as antisolvent, and investigated primarily the type of surfactant and adding amount, drug concentration, volume ratio of antisolvent to solvent, precipitation temperature, dropping speed, stirring speed, stirring time factors affecting drug particles size. Results showed that the poloxamer 188 was selected as the surfactant and the particle size of taxifolin obviously reduced with the increase of the poloxamer 188 concentration, the drug concentration and the dropping speed from 0.08% to 0.45%, from 0.04 g/ml to 0.12 g/ml, from 1 ml/min to 5 ml/min, respectively, when the volume ratio of antisolvent to solvent increased from 2.5 to 20, the particle size of taxifolin first increased and then decreased, the influence of precipitation temperature, stirring speed, stirring time on particle size were not obvious, but along with the increase of mixing time, the drug solution would separate out crystallization. The optimum conditions were: the poloxamer 188 concentration was 0.25%, the drug concentration was 0.08 g/ml, the volume ratio of antisolvent to solvent was 10, the precipitation temperature was 25 C, the dropping speed was 4 ml/min, the stirring speed was 800 r/min, the stirring time was 5 min. Taxifolin nanosuspension with a MPS of 24.6 nm was obtained under the optimum conditions. For getting taxifolin nanoparticles, the lyophilization method was chosen and correspondingly g-cyclodextrin was selected as cryoprotectant from g-cyclodextrin, mannitol, lactose, glucose. Then the properties of raw taxifolin and taxifolin nanoparticles were characterized by scanning electron microscopy (SEM), fourier-transform infrared spectroscopy (FTIR), high performance liquid chromatography–mass spectrometry (LC–MS), Xray diffraction (XRD), differential scanning calorimetry (DSC), and thermo gravimetric (TG), and the conclusion was drawn that taxifolin nanoparticles can be converted into an amorphous form but its chemical construction cannot been changed. Furthermore, dissolving capability test, 2,2-diphenyl-1picrylhydrazyl (DPPH) radical-scavenging activity and reducing power assay, solvent residue test were also carried out. The experimental data showed that the solubility and the dissolution rate of taxifolin nanoparticles were about 1.72 times and 3 times of raw taxifolin, the bioavailability of taxifolin nanoparticles increased 7 times compared with raw taxifolin, and the antioxidant capacity of taxifolin nanoparticles was also superior to raw taxifolin. Furthermore, the residual ethanol of the taxifolin nanoparticles was less than the ICH limit for class 3 solvents of 5000 ppm or 0.5% for solvents and could be used for pharmaceutical. These results suggested that taxifolin nanoparticles might have potential value to become a new oral taxifolin formulation with high bioavailability. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Taxifolin Nanoparticles LAP Antioxidant ability Dissolution rate Bioavailability
1. Introduction
* Corresponding author. Tel.: +86 451 82191517; fax: +86 451 82102082. E-mail address: [email protected] (X. Zhao). http://dx.doi.org/10.1016/j.ijpharm.2014.05.049 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
Taxifolin (3,5,7,3,4-pentahydroxy flavanone or dihydroquercetin, Fig. 1) is a kind of flavanonol (Ma et al., 2012), which was widely distributed in barks of the genus Pinus or Larix and in the seeds of the genus Silybum (Zu et al., 2012). Recent years, taxifolin was also
Y. Zu et al. / International Journal of Pharmaceutics 471 (2014) 366–376
Fig. 1. The chemical structure of taxifolin.
found in fruits, especially grapes, oranges and grapefruit (AbadGarcia et al., 2009). It played a special role in maintaining normal functions of circulatory system because of its unique antioxidant activity and biological activity (Lee et al., 2012; Liang et al., 2013; Rogovskii et al., 2010), which effectively eliminate excess free radicals in the human body (Teselkin et al., 2000; Vladimirov et al., 2009), improve immune function, and reduce the formation of cancer cells (Weidmann, 2012; Zhang et al., 2013), prevent cardiovascular disease (Wang et al., 2006). At present, because the taxifolin possessed some medicinal properties, such as antitumor, anti-virus, antioxidant, it has been widely used in medicine, health care products, food industries (Yang et al., 2011). However, on account of the slight solubility of taxifolin, it was difficult to be absorbed and metabolized by the body, which greatly limited its bioavailability and efficacy (Shikov et al., 2009; Zinchenko et al., 2011; Zu et al., 2012). Solubility of drugs was associated with the specific surface area of materials, along with particle size of drugs reduced, the effective area contacting with media increased, so that the solubility and dissolution rate of drugs will improve (Rasenack and Muller, 2004). Therefore, in order to improve the solubility and bioavailability of taxifolin, it is important to prepare small and uniform amorphous taxifolin nanoparticles by micronization technology. In general, the micronization technology is mechanical grinding (Rogers et al., 2003), but the method has disadvantages of large energy consumption, low efficiency, wide distribution of particle size of products, etc. In recent years, with the continuous development of nanometer material technology, some micronization preparation methods and technologies of drugs with potential application value such as the liquid antisolvent precipitation (Azad et al., 2013; Dong et al., 2010; Meer et al., 2011; Zu et al., 2013), the supercritical fluid technology (Byrappa et al., 2008; Li et al., 2008; Park et al., 2013), the spray drying method (Hu et al., 2011; Tao et al., 2013; Tshweu et al., 2013), and so on were widely applied in improving the solubility of low solubility drugs. Currently, our group had a related document reported that micronized taxifolin were prepared by the supercritical antisolvent (SAS) technology and its granule morphology was almost needle-like with particle size distributing between 2 mm and 11 mm (Zu et al., 2012), but the technology existed the questions of large equipment investment and lower productivity. This experiment applied the LAP method, according to the dissolved properties of taxifolin, selected ethanol as the solvent and selected water as the antisolvent and engendered precipitations, then prepared taxifolin nanoparticles through lyophilization. The method compared with other micronization technologies, had some advantages including its process is simple, easy to operate, easy to industrialization and lower cost, was expected to realize industrial production (Chen et al., 2004). The experiment prepared small and uniform amorphous taxifolin nanoparticles by the LAP without changing taxifolin molecule structure. Through the single factor design, the type of surfactant and adding amount, drug concentration, volume ratio of
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antisolvent to solvent, precipitation temperature, dropping speed, stirring speed, stirring time factors affecting particles size of drugs were mainly investigated, and the appropriate conditions of micronization were obtained. The taxifolin nanoparticles were obtained by lyophilization, and its properties had been characterized by scanning electron microscopy (SEM), fourier-transform infrared spectroscopy (FTIR), high performance liquid chromatography–mass spectrometry (LC–MS), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermo gravimetric (TG). Finally solvent residue test, antioxidant test and dissolution test were also carried out to investigate if there was residual ethanol in the process of experiment, and the biological activity and the bioavailability of taxifolin nanoparticles. 2. Materials and methods 2.1. Materials Taxifolin (FW = 304, purity 98%) was purchased from Nanjing Zelang Medical Technological Co., Ltd. (Jiangsu, PR China); ethanol, acetonitrile, methanol, glacial acetic acid, hydrochloric acid, 2,2diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide, trichloroacetic acid, FeCl3, Na2HPO412H2O, NaH2PO42H2O, poloxamer 188, Tween-80 and g-cyclodextrin were obtained from Sigma, deionized water was prepared with Hitech-K flow water purification system (Hitech Instruments Co., Ltd., Shanghai, China). 2.2. Preparation of taxifolin nanoparticles The taxifolin nanoparticles were prepared by the LAP. In short, a certain amount of raw taxifolin were weighed and completely dissolved in a certain volume of ethanol, then the solution was poured into a certain volume of deionized water with a certain amount of surfactants by a peristaltic pump at a certain precipitation temperature under stirring with a certain speed intensity. After a period of time, the ethanol in the solution was removed from the solution by a spin steaming instrument, and then a certain amount of cryoprotectants were added into the solution and stirred evenly through a magnetic stirrer, finally the taxifolin nanoparticles were obtained by the lyophilizer at 50 C for 64 h. Under the same conditions, taxifolin nanoparticles without cryoprotectants were also prepared. Each experiment was repeated at least 3 times. 2.3. Optimization of the LAP of taxifolin nanopaticles Single factor method was used to investigate the optimization of operating conditions for amorphous taxifolin nanoparticles by the LAP process. Through preliminary experiments, several main factors influencing the particle size of taxifolin nanoparticles included the type of surfactant and adding amount, drug concentration, volume ratio of antisolvent to solvent, precipitation temperature, dropping speed, stirring speed, stirring time factors, every factor was respectively investigated in order to get the optimal condition, as follows: surfactants were poloxamer 188, carbomer, PEG-6000 and the surfactant concentration was studied at 0.08%–0.45%. The drug concentration was studied at 0.04– 0.12 mg/ml. The volume ratio of antisolvent to solvent was studied at 2.5–20. The precipitation temperature was investigated at 5– 45 C. The dropping speed was studied at 1–5 ml/min. The stirring speed was studied at 400–2000 r/min. The stirring time was studied at 1–90 min. Finally, the optimum condition of every factor was obtained based on the smallest particle size of every factor, respectively. The obtained nanoparticles were redispersed in deionized water containing a certain amount of g-cyclodextrins, prefrozen at 40 C for 2 h, and subsequently lyophilized at 50 C
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for 64 h to obtain the taxifolin nanoparticles. Taxifolin nanoparticles without g-cyclodextrins were lyophilized under the same freeze drying conditions. 2.4. The condition of HPLC HPLC method was implemented by a Waters chromatographic instrument. The chromatographic column: C18 reverse-phase column (250 mm 4.6 mm, 5 mm, China); The mobile phase: 30:70:0.1% (v/v/v) mixtures of acetonitrile, water and glacial acetic acid; the column temperature: indoor temperature; the flow rate: 1.0 ml/min; the detection wavelength: 289 nm; the injection volume: 10 ml. 2.5. Characterization of taxifolin nanoparticles 2.5.1. Scanning electron microscope (SEM) The morphology of raw taxifolin, taxifolin nanoparticles and taxifolin nanoparticles without g-cyclodextrins was analyzed by SEM. Dry samples were glued on the observation desks with double-sided conductive adhesive and plated gold by the ion sputtering coating machine, uniform and thin metal coating could be obtained, then the morphology and size of the samples were observed. 2.5.2. FTIR and high performance liquid chromatography–mass spectrometry (LC–MS) The molecular structures of raw taxifolin, taxifolin nanoparticles, taxifolin nanoparticles without g-cyclodextrins and g-cyclodextrin were researched using FTIR in the wave number range of 400–4000 cm1 at a resolution of 2 cm1. The KBr pellet method was adopted. Each sample was accurately weighed 2 mg, then mixed with KBr of 200 mg respectively, and grinded into fine powders, then pressed into transparent slices for analysis. The mass spectrums of raw taxifolin and taxifolin nanoparticles were obtained by analyst 1.4 of API 3000 (AB, USA). The raw taxifolin and the taxifolin nanoparticles were dissolved in methanol of chromatographic grade, and paired the solution of 1 mg/ml, respectively. According to the testing conditions, the solution of 1 mg/ml could be diluted. The mass spectrometer was operated in positive ion mode. 2.5.3. XRD The crystal form of raw taxifolin, taxifolin nanoparticles, taxifolin nanoparticles without g-cyclodextrins and g-cyclodextrin was detected and analyzed by an X-ray diffractometer with Cu Ka1 radiation at 30 mA and 40 kV. The four samples were scanned at a step length of 0.02 within 5 60 of 2u, and the scanning speed rate was 0.02 min1.
The specific process as follows: excessive and identical taxifolin amount of each sample were weighed and separately added into four vials containing 3 ml artificial gastric juice (pH 1.2), then all the samples were put in the water bath of 25 C, 100 r/min, after 48 h, 100 ml of solution was separately taken out and put into 2 ml centrifuge tube, and 900 ml of methanol was also added in every centrifuge tube, respectively (the volume ratio of solution to methanol was 1:9), after mixed evenly, the mixture was ultrasonically treated for 30 min, then centrifuged at 10,000 r/ min for 10 min by a centrifugal, the supernate of 10 ml was taken out and injected into the HPLC system. The analysis conditions were described in Section 2.4. 2.5.6. The dissolution rate test The dissolution rate of raw taxifolin, taxifolin nanoparticles and physical mixture of taxifolin with auxiliary materials was studied on the premise of maintaining the sink condition and detected by HPLC. The environmental conditions of the process including the speed and the dissolution temperature were set as 100 r/min, 37.0 0.5 C, respectively. The artificial gastric juice (pH 1.2) containing 0.4% Tween-80 was served as the dissolution medium. 84 mg of raw taxifolin, 194 mg of taxifolin nanoparticles equating with 64 mg of taxifolin (taxifolin content of taxifolin nanoparticles is 35.02% by HPLC detecting) and physical mixture of raw taxifolin of 84 mg with auxiliary materials were completely added into three beakers containing 200 ml of dissolution medium. 1 ml of dissolution medium was taken out each time at 3 min, 8 min, 13 min, 18 min, 23 min, 28 min, 38 min, 48 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, then 1 ml of dissolution medium was added to ensure that the volume of dissolution medium remained constant. 1 ml of dissolution medium was centrifuged at 10,000 r/ min for 10 min by the centrifugal, 100 ml of solution was taken out and put into 2 ml centrifuge tube, and 900 ml methanol was also added in the centrifuge tube, after mixed evenly, the mixture was ultrasonically treated for 30 min, then centrifuged for 10 min at 10,000 r/min, the supernate of 10 ml was taken out and injected into the HPLC system. The analysis conditions were described in Section 2.4. 2.5.7. Measurement of DPPH radical-scavenging activity and reducing powers 2.67 mg raw taxifolin and taxifolin nanoparticles (containing 2.67 mg taxifolin) were weighed and separately added into two vials containing 3 ml deionized water, then the mixture was put in the water bath of 25 C, 100 r/min for 30 min. The suspensions of each sample were centrifuged for 10 min with 10,000 r/min. The
2.5.4. DSC and TG The heating stability of raw taxifolin, taxifolin nanoparticles, taxifolin nanoparticles without g-cyclodextrins and g-cyclodextrin was studied by DSC (TA instruments, model, DSC 204), the four samples were operated at a heating rate of 10 C min1 from 30 C to 300 C. The thermal gravimetric of raw taxifolin, taxifolin nanoparticles, taxifolin nanoparticles without g-cyclodextrins and g-cyclodextrin was researched using TGA at a heating rate of 10 C min1 in a temperature range of 30600 C, and the whole process was carried out under dynamic nitrogen atmosphere. 2.5.5. Equilibrium solubility test The equilibrium solubility of raw taxifolin, taxifolin nanoparticles without g-cyclodextrins, taxifolin-g-cyclodextrins (8:6, 8:7, 8:8, 8:9, 8:10) nanoparticles were detected by HPLC method.
Fig. 2. The effect of the type of surfactant on the MPS taxifolin nanoparticles.
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supernate was obtained and diluted into different taxifolin concentrations (0.89–4.45 mg/ml). In addition, the antioxidant activities of g-cyclodextrin with corresponding concentration of taxifolin nanoparticles solution acted as the control and were measured. DPPH radical-scavenging activity measurement: 2 ml of each sample at different taxifolin concentrations (0.89–4.45 mg/ml) was added to 2 ml of ethanol DPPH solution (25 mg/l), the mixture was shaken energetically and placed at room temperature in the dark. After 30 min, the absorbance of the mixture was measured at 517 nm. The experiment repeated 3 times. The DPPH scavenging
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capacity (SC) of the samples was calculated using the following equation: ðAc Ai Þ 100% SCð%Þ ¼ Ac where Ac is the absorbance of the control, and Ai is the absorbance of the sample. Reducing power measurement: 2 ml of each sample at different taxifolin concentrations (0.89–4.45 mg/ml) were mixed with 2 ml of 0.2 mol/l phosphate buffer (pH 6.6) and 2 ml of 1% potassium
Fig. 3. The effect of each factor on the MPS taxifolin nanoparticles. (a) The amount of surfactant; (b) concentration of taxifolin solution; (c) the volume ratio of antisolvent to solvent; (d) precipitation temperature; (e) stirring speed; (f) stirring time; (g) dropping speed.
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ferricyanide, and then incubated at 50 C for 20 min. In order to stop the reaction, 10% trichloroacetic acid of 2 ml was added into the mixture, after mixed evenly, the mixture was centrifuged at 3000 r/min for 10 min. The supernatant was mixed with distilled water and 0.1% FeCl3 according to volume ratio of 1:1:0.2 (v/v/v), after 10 min, the absorbance of the samples was measured at 700 nm. The experiment repeated 3 times. 2.5.8. Bioavailability 6 female Sprague-Dawley rats (weight between 200 and 250 g) were randomly divided into two groups, each group of 3 rats. They were fasted and freely drunk water in the 12 h before doses. Two groups of rats were respectively given raw taxifolin and taxifolin nanoparticles by gavage at the doses of 50 mg/kg (according to taxifolin calculation), after oral administration, the raw taxifolin group and the taxifolin nanoparticles group were taken blood from rats eyeball at 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24 and 48 h and at 0.15, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h, respectively. Blood samples were put in the centrifuge tube containing heparin, and centrifuged at 3000 r/min for 10 min, the supernatant plasma were stored in the refrigerator of 4 C, and processed on the same day. The processing of blood samples: 0.2 ml of methanol and 0.08 ml of hydrochloric acid solution (10 mol l1) were put in 2 ml centrifuge tube, after mix evenly, the plasma of 0.08 ml was added in the centrifuge tube and vibrated for 30 s by a vortex mixer for blending, then centrifuged for 10 min at 8000 r/min, the supernate of 10 ml was taken out and injected into the HPLC system. The analysis conditions were described in Section 2.4. 2.5.9. GC measurement A gas chromatograph (GC) (Agilent 7890A, Palo Alto, CA, USA) was used to detect whether there was residual ethanol in taxifolin nanoparticles, which equipped with a G1540N-210 flame ionization detector and a HP-5 5% phenyl methyl siloxane capillary column (30.0 m 320 mm 0.25 mm, nominal). 10 mg taxifolin nanoparticles were weighed and dissolved into 1 ml deionized water, then centrifuged for 10 min at 10,000 r/min. The supernate of 2 ml was injected into the GC. The conditions of GC analysis of ethanol were as follows: the initial temperature was 40 C, maintained for 6 min raised to 240 C at the heating rate of 30 C min1 and maintained for 4 min. The make-up gas rate was 30.0 ml min1, the rate of H2 was 40.0 ml min1, and the rate of air was 400.0 ml min1. The sample size was 2 ml, and the split ratio was 20:1.
Fig. 4. (a) SEM images of raw taxifolin; (b) (c) SEM images of taxifolin nanoparticles without g-cyclodextrins; (d) (e) SEM images of taxifolin nanoparticles.
(Hecq et al., 2005; Terayama et al., 2004). In addition, on the premise of smaller particle size, the amount of pharmaceutical excipients added should be little as far as possible, so the poloxamer 188 of 0.25% was selected as the best condition. 3.1.2. Drug concentration In the process of the LAP, the drug concentration has a great influence on particle size. The relationship between drug
3. Result and analysis 3.1. Optimization Study 3.1.1. The effects of surfactant The effects of surfactant on the average particle size of taxifolin are shown in Figs. 2 and 3. From Fig. 2, the average particle size of taxifolin without surfactant and taxifolin containning poloxamer 188, carbomer, PEG-6000 of identical concentration were about 2088 nm, 43 nm, 1820.3 nm, 188.2 nm, respectively. Therefore, the experiment finally selected the poloxamer 188 as the surfactant from poloxamer 188, carbomer, PEG-6000. With the poloxamer 188 concentration ranging from 0.08% to 0.45%, the changes of particle size were examined. From Fig. 3(a), when the poloxamer 188 concentration changing from 0.08% to 0.25%, the average particle size was obviously reduced from 286 nm to 39 nm, but the poloxamer 188 concentration increased from 0.25% to 0.45%, the change of the average particle size was unconspicuous. The experimental results explained that the poloxamer 188 could effectively coat on the surface of drugs, inhibit the growth of particles and be beneficial to decrease the particle size of taxifolin
Fig. 5. FTIR spectra of each sample. (a) Raw taxifolin; (b) taxifolin nanoparticles; (c) taxifolin nanoparticles without g-cyclodextrins; (d) g-cyclodextrin.
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concentration and average particle size is shown in Fig. 3(b). With increasing the drug concentration from 0.04 g/ml to 0.08 g/ml, the average particle size decreased from 426 nm to 30 nm. When the drug concentration was greater than 0.08 g/ml, the average particle size scarcely changed. Therefore, the increase of drug concentration led to the average particle size decreasing significantly. This was because the advance of the drug concentration can increase the super saturation of system, resulting in the crystal nucleation rate was greater than the growth rate, which was favorable to generate tiny particles. However, when the drug concentration was excessive, the number of drug particles increased sharply on the interface of solid phase and liquid phase diffusion, which caused particles adhesion, reunion, and larger particles generation (Zhang et al., 2009; Zu et al., 2014). By comprehensive consideration, the drug concentration of 0.08 g/ml was selected the optimum condition. 3.1.3. Volume ratio of antisolvent to solvent The effects of volume ratio of antisolvent to solvent (2.5, 5, 10, 15, 20) on particle size were studied, as shown in Fig. 3(c). Firstly, with the volume ratio of antisolvent to solvent increasing from 2.5 to 10, the average particle size decreased from 40 nm to 20 nm. When the volume ratio was 10, the average particle size was the smallest at about 20 nm. But when the volume ratio was more than
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10, the average particle size began to increase and when the volume ratio was 20, the average particle size increased at about 74 nm. The results illustrated that the increase of the solvent ratio was beneficial to decrease the amount of ultrafine particles generated in the per unit volume of liquid, and reduce collision and agglomeration between particles, was conducive for small particles formation (Zhang et al., 2011). However, as the supersaturation increased further, the nucleation rate had a maximum. So the optimum ratio of antisolvent to solvent was 10. 3.1.4. Precipitation temperature, stirring speed, stirring time The influences of different temperature, stirring speed and stirring time on the particle size were studied. As shown in Fig. 3(d and e), the temperature (5, 15, 25, 35 and 45 C) and stirring speed (400, 800, 1200, 1600 and 2000 r/min) did not present a significant influence on the particle size. Therefore, for easy operation, 25 C was selected as the optimal temperature; the experiment can be performed at room temperature. When stirring speed was higher or lower than 800 r/min, the particle size became slightly bigger. So, the stirring speed of 800 r/min was selected as the optimal condition. From Fig. 3(f), as the growth of stirring time from 1 min to 60 min, the change of particle size was not obvious. After 60 min, crystallization and precipitation began to appear. The results showed that when the stirring time was short, the existence of the
Fig. 6. LC–MS/MS spectra of taxifolin nanoparticles and raw taxifolin.
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Fig. 7. XRD results of each sample. (a) Raw taxifolin; (b) taxifolin nanoparticles; (c) taxifolin nanoparticles without g-cyclodextrins; (d) g-cyclodextrin.
poloxamer 188 stabilizer could effectively control particle size and morphology of taxifolin particles, and limit the agglomeration of particles. When the stirring time was longer, too much energy was input into the system, and destroyed the relative balance of the system, led to agglomeration and growth of particles. Therefore, 5 min was selected as the optimal stirring time. 3.1.5. Dropping speed The influences of dropping speed (1, 2, 3, 4 and 5 ml/min) on the average particle size were examined. Under the condition of poloxamer 188 concentration of 0.25%, drug concentration of 0.08 g/ml, volume ratio of antisolvent to solvent of 10, stirring speed of 800 r/min, From Fig. 3(g), as dropping speed increased from 1 ml/min to 4 ml/min, the average particle size reduced from 30 nm to 17 nm, when dropping speed was more than 4 ml/min, the change of average particle size was no longer apparent. Therefore, the dropping speed of 4 ml/min was the optimal condition. Through the above analysis, the optimal conditions were: the poloxamer 188 was selected as surfactant, poloxamer 188 concentration was 0.25%, drug concentration was 0.08 g/ml, volume ratio of antisolvent to solvent was 10, precipitation
Fig. 8. DSC results of each sample. (a) Raw taxifolin; (b) taxifolin nanoparticles;(c) taxifolin nanoparticles without g-cyclodextrins; (d) g-cyclodextrin.
Fig. 9. TG curves of each sample. (a) Raw taxifolin; (b) taxifolin nanoparticles; (c) taxifolin nanoparticles without g-cyclodextrins; (d) g-cyclodextrin.
temperature was 25 C, dropping speed was 4 ml/min, stirring speed was 800 r/min and stirring time was 5 min. Taxifolin nanosuspension with a MPS of 24.6 nm were obtained under the optimum conditions. In addition, through preliminary experiments, g-cyclodextrin were selected as cryoprotectant from g-cyclodextrin, mannitol, lactose, glucose. In the end, taxifolin nanoparticles were obtained by lyophilization and used for the following detections. 3.2. Characterization of taxifolin nanoparticles 3.2.1. SEM analysis SEM images of raw taxifolin, taxifolin nanoparticles and taxifolin nanoparticles without g-cyclodextrins are shown in Fig 4. From Fig. 4(a), the granule morphology of the raw taxifolin was various block with particle size distributing between 0.54 mm and 16.6 mm, in Fig. 4(b and c), the granule morphology of the taxifolin nanoparticles without g-cyclodextrins was fibrous or irregular block and its particle size was about 10.1–19.3 mm. However, from Fig. 4(e and f), the taxifolin nanoparticles were almost spherical with particle size distributing between 148 nm and 167.2 nm, the reason for this result was that g-cyclodextrin had a cladding effect for the taxifolin by comparing with the granule morphology of the
Fig. 10. The equilibrium solubility of taxifolin nanoparticles.
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the FTIR and the MS results showed that the molecular structure of the taxifolin nanoparticles cannot be changed. 3.2.3. XRD analysis The XRD curves of raw taxifolin, taxifolin nanoparticles and taxifolin nanoparticles without g-cyclodextrins, g-cyclodextrin are shown in Fig. 7. From Fig. 7(a and c), The vibration intensity of the diffraction peaks of the taxifolin nanoparticles without g-cyclodextrins decreased compared with raw taxifolin, and the size of the vibration intensity reflects the strength of the material crystallization, this illustrated the crystallinity of taxifolin nanoparticles without g-cyclodextrins was lower than that of the raw taxifolin. In Fig. 7(b and d), the diffraction peaks of the taxifolin nanoparticles were less than that of the raw taxifolin, which indicated the particle size of the taxifolin nanoparticles decrease significantly, while the position of the diffraction peak of the taxifolin nanoparticles was different with that of the raw taxifolin, this might because the g-cyclodextrin induced a reaction of cladding for the taxifolin nanoparticles. Fig. 11. The dissolution profiles of raw taxifolin, taxifolin nanoparticles and physical mixture of taxifolin with auxiliary materials.
taxifolin nanoparticles without g-cyclodextrins. Therefore, the particle size of the taxifolin nanoparticles was much smaller than that of the raw taxifolin and the taxifolin nanoparticles without g-cyclodextrins, which was beneficial to improve the bioavailability of taxifolin. Previously, our group also prepared micronized taxifolin of needle-like with particle size distributing between 2 mm and 11 mm by SAS (Zu et al., 2012), but the granule morphology of taxifolin nanoparticles was different with micronized taxifolin of this paper. 3.2.2. FTIR and MS analysis The FTIR spectrograms of raw taxifolin, taxifolin nanoparticles and taxifolin nanoparticles without g-cyclodextrins, g-cyclodextrin are shown in Fig. 5. In Fig. 5(a and b), the molecular structures of the raw taxifolin and the taxifolin nanoparticles without g-cyclodextrins were almost coincident. From Fig. 5(c and d), the taxifolin nanoparticles and the raw taxifolin had some differences in 3200– 2700 cm1 and 1500–700 cm1, however, the peaks of g-cyclodextrinwere almost similar with that of the taxifolin nanoparticles in the two range, which explained the reasons of differences were taxifolin nanoparticles containing g-cyclodextrins. The mass spectrograms of raw taxifolin and taxifolin nanoparticles are shown in Fig. 6, in the diagram, the molecular weight of raw taxifolin and taxifolin nanoparticles were both 302.9. Thus,
3.2.4. DSC and TG analysis DSC analysis results are shown in the Fig. 8, from Fig. 8(a and c), the raw taxifolin had an obvious peak in the 241.89 C, while the taxifolin nanoparticles without g-cyclodextrins had a small peak at about 222.27 C, the solid melting point of the taxifolin nanoparticles without g-cyclodextrins changed, the reason for this phenomenon was that the crystallinity of the taxifolin nanoparticles without g-cyclodextrins reduced compared with the raw taxifolin. From Fig. 8(b and d), the taxifolin nanopaticles did not have an obvious melting process on account of g-cyclodextrins, this illustrated the taxifolin nanoparticles were amorphous form and was beneficial to improve the solubility and the bioavailability of taxifolin (Kim et al., 2008). TG analysis results are shown in Fig. 9, the Fig. 9 shows that the raw taxifolin and the taxifolin nanoparticles without g-cyclodextrins began to weightlessness from 240 C, the g-cyclodextrin began from 230 C to weightlessness, while the taxifolin nanoparticles lost weight fom 180 C. This might be because the particle size of the taxifolin nanoparticles was smaller than that of the raw taxifolin led to its higher specific surface area, so that the taxifolin nanoparticles more easily evaporated and rapidly decomposed. 3.2.5. Equilibrium solubility The equilibrium solubility of raw taxifolin, taxifolin nanoparticles without g-cyclodextrins, taxifolin-g-cyclodextrins (8:6, 8:7, 8:8, 8:9, 8:10) nanoparticles were 0.96, 1.24, 1.47, 1.65, 1.57, 1.67,
Fig. 12. The DPPH radical-scavenging activity and the reducing power of taxifolin nanoparticles and raw taxifolin.
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it meant that the auxiliary material was not the key factor in improving the dissolution rate of taxifolin. And the dissolution rate of the taxifolin nanoparticles was much faster than that of the raw taxifolin, in the 60 min, taxifolin nanoparticles achieving the maximum dissolution was about 93.1%, and in the 180 min, the dissolution of the raw taxifolin was about 79.03%, this was mainly because the particle size of the taxifolin nanoparticles was much smaller than that of the raw taxifolin. In addition, recently our group prepared micronized taxifolin by SAS, its solubility was about 0.02 mg/ml at 60 min (the dissolution medium was PBS) (Zu et al., 2012), this illustrated that the taxifolin nanoparticles prepared by the LAP process were amorphous and smaller so that the dissolution rate of the taxifolin nanoparticles increased. According to Noyes–Whitney equation, with the particle size of micronization products decreasing, the specific surface area of the drugs increased accordingly, thereby increased the contact area of the solid drugs and the dissolution medium, the dissolution of the drugs was also improved accordingly. Fig. 13. The plasma concentration of taxifolin nanoparticles and raw taxifolin.
and 1.46 mg/ml by HPLC detecting, respectively. From Fig. 10, the equilibrium solubility of taxifolin-g-cyclodextrins nanoparticles was better than that of taxifolin nanoparticles without g-cyclodextrins, so the g-cyclodextrin was conducive to improving the solubility of taxifolin, and the solubility of taxifolin-g-cyclodextrins (8:7) nanoparticles and taxifolin-g-cyclodextrins (8:9) nanoparticles were similar. However, the taxifolin would be made into oral drugs, the amount of pharmaceutical excipients should be less as far as possible. Consequently, the ratio of taxifolin to g-cyclodextrins of 8/7 was the optimal condition, and the solubility of taxifolin-g-cyclodextrins (8:7) nanoparticles were about 1.72 times of raw taxifolin. 3.2.6. The dissolution rate analysis The dissolution profiles of raw taxifolin, taxifolin nanoparticles and physical mixture of taxifolin with auxiliary materials are shown in Fig. 11. The dissolution rate of raw taxifolin was faster than that of physical mixture of taxifolin with auxiliary materials;
3.2.7. DPPH radical-scavenging activity and reducing power analysis Taxifolin was a natural antioxidant, but was not widely used because of its low bioavailability, the experiment prepared taxifolin nanoparticles by the LAP to improve the solubility of taxifolin, and applied the DPPH radical-scavenging activity and the reducing power test to compare the antioxidant ability of taxifolin nanoparticles and raw taxifolin. The experimental data obtained illustrated the antioxidant activity of g-cyclodextrin was very little and hardly influence the antioxidant activity of taxifolin nanoparticles. The experimental results of taxifolin nanoparticles and raw taxifolin are shown in Fig. 12, which revealed the radicalscavenging activities and the reducing powers of all the samples increased with the concentrations, but the radical-scavenging activities and the reducing powers of taxifolin nanoparticles were higher than that of raw taxifolin. In addition, from the halfinhibition concentrations (IC50) of the samples, the IC50 of the taxifolin nanoparticles and raw taxifolin was separately 1.654 mg/ ml, 3.093 mg/ml, it was also seen that the taxifolin nanoparticles had a higher DPPH radical-scavenging activity by lower IC50 than that of raw taxifolin.
Fig. 14. (a) The gas phase diagram of 10 mg/ml water solution of the taxifolin nanoparticles; (b) the gas phase diagram of 0.05 mg/ml ethanol solution.
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3.2.8. Bioavailability analysis Bioavailability experiment result is shown in Fig. 13, it could be seen from the figure, which the absorption of taxifolin nanoparticles group was significantly faster than that of raw taxifolin group in vivo, and the drug concentration in rat plasma of the taxifolin nanoparticles group was also higher than that of the raw drug group. The drug concentration of the taxifolin nanoparticles and the raw taxifolin in plasma of rats reached the maximum of 13.5 ng/ml and 1.3 ng/ml at 9 min and 2 h after gavage, respectively. By comparing the corresponding AUC values of the two groups, the result demonstrated that the AUC values of the taxifolin nanoparticles was 7 times higher than that of the raw taxifolin, this was because the amorphous taxifolin nanoparticles made the effective area of taxifolin contacting with media increase, which was beneficial to improve the bioavailability of taxifolin. So the oral bioavailability of the taxifolin nanoparticles was improved significantly compared with the raw taxifolin. 3.2.9. Solvent residue The detection of residual solvent is widely used for pharmaceutical. The experiment prepared taxifolin nanoparticles with ethanol as the solvent by the LAP, the detected results are shown in Fig. 14(a and b). Fig. 14(a) shows the gas phase diagram of 10 mg/ml water solution of the taxifolin nanoparticles. There was only one solvent peak, its retention time was 2.343 min. Fig. 14(b) shows the gas phase diagram of 0.05 mg/ml ethanol solution (10 mg/ml water solution of the taxifolin nanoparticles containing 0.5% ethanol). There were two solvent peak, after repeated testing, it was confirmed that a retention time of water was 2.304 min and a retention time of ethanol was 2.857 min. Therefore, the solvent peak was water peak in Fig. 14(a). Fig. 14(b) shows that the residual ethanol was less than the ICH limit for class 3 solvents of 5000 ppm or 0.5% for solvents, so the taxifolin nanoparticles conformed to ICH requirements and could be used for pharmaceutical. 4. Conclusions Taxifolin nanoparticles have been successfully prepared by the LAP technique using ethanol as solvent and deionized water as antisolvent. In this experiment, the poloxamer 188 and the g-cyclodextrin was selected as surfactant and cryoprotectant to inhibit the aggregation and particle growth, respectively. The optimal conditions as follows: poloxamer 188 concentration was 0.25%, drug concentration was 0.08 g/ml, volume ratio of antisolvent to solvent was 10, precipitation temperature was 25 C, dropping speed was 4 ml/min, stirring speed was 800 r/min and stirring time was 5 min. Taxifolin nanosuspension with a MPS of 24.6 nm was obtained under the optimum conditions, then the taxifolin nanoparticles with a MPS of 167.2 nm were achieved by mean of lyophilization. In addition, the FTIR and the MS results showed that the molecular structure of the taxifolin nanoparticles cannot be changed. The analysis results of XRD and DSC indicated that the prepared taxifolin nanoparticles were amorphous. The ethanol residual of taxifolin nanoparticles was less than the ICH limit for class 3 solvents of 5000 ppm or 0.5% for solvents by the GC analysis, and had a higher DPPH-scavenging activity and reducing powers than raw taxifolin. The dissolution test showed the taxifolin nanoparticles exhibited enhanced dissolution rate and solubility compared to the raw taxifolin, and the oral bioavailability of the taxifolin nanoparticles was improved significantly compared with the raw taxifolin in vivo. Therefore, the taxifolin nanoparticles may have great potential value to become a new oral txifolin formulation.
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