Research Article  Open Access
Ultrafine Resveratrol Particles: Supercritical Antisolvent Preparation and Evaluation In Vitro and In Vivo
Abstract
Ultrafine resveratrol (uRes) particles were prepared through the SAS process. The orthogonal method was used to optimize the factors of the SAS process. The size of uRes reached 0.68 μm under the optimum conditions. The characterization of the uRes particles was tested by many analysis methods. The chemical structure of Res was unaffected by the SAS process. The degree of crystallinity of the uRes particles greatly reduced. The purity of the uRes particles increased from 98.5% to 99.2% during the SAS process. The uRes particles had greater saturation solubility and dissolution rate than the rawRes (rRes) particles. The radical scavenging activity and bioavailability of the uRes in vivo were 1.9 times of the rRes.
1. Introduction
Resveratrol (Res) (Figure 1) is a natural polyphenol [1] found in grapes [2]. Res in wine grapes reduces the risk of cardiovascular disease [3]. Res is found in 21 families, 31 genera, and 72 species of plants [4], such as peanuts [5], cocoa, and Polygonum cuspidatum [6]. Res is a natural health product that has potential use for treatment of several diseases because of its advantages such as the antitumor effect [7], antioxidant and free radical scavenging effect [8], antithrombotic effect [9], antibacterial effect [10], antiinflammatory effect [11], prevention and treatment of neurological diseases [12], and enhancement of body immunity [13]. However, the poor water solubility and low bioavailability of Res greatly reduce its effect and application value [14].
In recent years, considerable efforts have been made to reduce the size of Res particles and encase water soluble materials on the surface of the Res particles through an antisolvent precipitation technology. The antisolvent precipitation technology increases the solubility of Res. Kim et al. were able to make the saturation solubility of Res reach 66.8 and 56.2 µg/mL in pure water and PBS solution (pH 7.4), respectively, which is much higher than the 31.8 and 29.6 µg/mL of rRes in the same condition. And they make Res have a dissolution rate of 20% within 5 min and complete dissolution within 50 min [15]. However, this technology introduced other substances into Res; thus, high purity water soluble Res still remains unattainable.
The supercritical antisolvent (SAS) has been developed recently as a new nanopreparation technology [16, 17]. The SAS process has several advantages over the other methods: the size of the product particle is micro, the biological activity of samples (especially thermal sensitive materials) is kept, it uses minimal residual solvent, and it is environment friendly [18, 19]. The principle of the SAS process is that the CO_{2} gas becomes a supercritical fluid under high temperature and high pressure [20]. The sample organic solvent solution is then sprayed into the supercritical fluid vessel through the capillary nozzle by solution pump. When the sample organic solvent mixes with the supercritical fluid, the sample is recrystallized to precipitation. The process of recrystallization precipitation is so fast that mixed solutions have great supersaturation in only a short period. The grain size of recrystallization precipitation reaches the micro grade [21].
Because of the above advantages of SAS, the innovation of this paper aims to prepare uRes particles to solve the poor water solubility problem of Res using SAS process. And using the method of SAS in preparing uRes particles is rarely reported. Then, the orthogonal method was used to optimize the factors of the SAS process to prepare uRes particles under the best conditions. Moreover, the influences of characterization in uRes particles were inspected by SEM, FTIR, LCMS, XRD, TG, DSC, HPLC analyses, saturation solubility, dissolution rate evaluation in vitro, and bioavailability in vivo. And the uRes particles are compared with the results of previous researchers to prove the superiority.
2. Materials and Methods
2.1. Materials
Res (mass purity ≥ 98.5%) was purchased from Riotto Botanical Co., Ltd., China. Res standard substance (mass purity ≥ 99.9%) was purchased from Selleck Biological Technology Co. Ltd., USA. CO_{2} (mass purity ≥ 99.9%) was purchased from Harbin Liming Gas Co., Ltd., China. Ethanol (mass purity ≥ 99.7%) was supplied by Tianjin Tianli Chemistry Co., Ltd., China. Methanol (mass purity ≥ 99.9%) and acetonitrile (mass purity ≥ 99. 9%) were supplied by Beijing J&KCHEMICA Co., Ltd., China. Hydrochloric acid (mass purity ≥ 38%) was supplied by Tianjin Fuyu Fine Chemistry Co., Ltd., China. All materials were directly used without further purification.
2.2. Preparation of the uRes Particle
Figure 2 illustrates the operating procedure of the SAS process apparatus. CO_{2} gas was cooled by a cooler (3) through a valve (2). The cooled CO_{2} gas was then compressed by CO_{2} pump (4) and continuously introduced into a heat exchanger (5), which cooled the CO_{2} pump at the same time. The CO_{2} gas was preheated in the heat exchanger (5) and then entered into a precipitation chamber (8). The CO_{2} was liquefied in the precipitation chamber (8) and completely filled a stainless steel frit vessel (7) through a 200 nm pore. Upon achieving a steady state, Res ethanol solution from a solution supply (13) was pumped using a solution pump (14) and sprayed into the stainless steel frit vessel (7) through a valve (15) and a capillary nozzle (6). When the Res ethanol solution mixed with the CO_{2} supercritical fluid, Res was quickly recrystallized to micron precipitation. Simultaneously, the SCCO_{2}/ethanol mixture passes through the 200 nm pore of the stainless steel frit vessel (7), and then entered into a gasliquid separation vessel (10) through the valve (9); only the uRes precipitation was left. In the asliquid separation vessel (10), the CO_{2} supercritical fluid was gasified to enter the circulation through a valve (12), and the ethanol solution was recycled using a valve (11). Moreover, the CO_{2} pump (4) was stopped for 30 min when all of the Res ethanol solution was sprayed into the precipitation chamber (8); the steady state of the precipitation chamber (8) was maintained. The CO_{2} pump was then rebooted to keep the CO_{2} flowing for 30 min and remove the residual ethanol in uRes precipitation. Finally, the uRes particles were collected for further characterization analysis.
2.3. Optimization of the SAS Process
An orthogonal design L16 (4∧5) was selected to optimize the SAS process of the uRes particles (Table 1). The factors of the SAS process were pressure (MPa), temperature (°C), Res ethanol solution concentration (mg/mL), and sample solution flow rate (mL/min). Each factor level gained range through preliminary experiments. The particle size of the uRes particles was used as dependent variable. Data were analyzed using the orthogonal software assistant for optimum condition. Then the uRes particles under this condition were obtained for characterization by various analytical methods.

2.4. Particle Characterization
2.4.1. Scanning Electron Microscopy (SEM)
The surface morphologies of the Res samples were observed using scanning electronic microscopy (SEM) equipment (Quanta 200, FEI, USA). First, the rRes and uRes samples were glued on metal button by conducting resin and sprayed with a thin layer of gold on the surface. These metal buttons were then measured at a high voltage (12.5 KV). Based on the SEM pictures, the last particle size of the Res samples was calculated using the ImagePro Plus 6.0 software, providing data to support the further optimization of the SAS process.
2.4.2. Fourier Transform Infrared Spectroscopy (FTIR)
Spectra of rRes and uRes particles were collected for contrast using Fourier transform infrared (FTIR) equipment (IRAffinity1, Shimadzu, Japan). 2 mg of rRes and uRes samples was mixed with 200 mg KBr and made to flake. Flaking was then measured in the wavenumber range from 500 cm^{−1} to 4000 cm^{−1}.
2.4.3. Liquid ChromatographyMass Spectrometry (LCMS)
Liquid chromatographymass spectrometry (LCMS) equipment (API300, MDS Sciex, USA) was used to obtain the spectra of the rRes and uRes particles. The detection condition of LCMS was 100–1000 , the mobile phase was 50% (v/v) methanol, the flow rate was 0.2 mL/min, and the sample solution concentration was 2.5 mg/mL [22]. Other conditions were in accordance with the 2.5 detection method.
2.4.4. XRay Diffraction (XRD)
The crystallinity of the Res particles was analyzed using Xray diffraction (XRD) equipment (XPert Pro MPD, Philips, Netherlands). The detection conditions were at room temperature, Cuka radiation generated at 30 mA and 50 kV, scanned 3° to 60° (0.02° per minute). Then, the uRes particles were placed 4 months under low temperature and less light conditions and analyzed again.
2.4.5. Differential Scanning Calorimetry (DSC)
Differential scanning calorimetry (DSC) equipment (Q2000, TA, USA) was used to obtain the thermal behavior of the rRes and uRes particles. The 3.0 mg weight samples were placed in airtight aluminum pans and heated from 45°C to 300°C at a heating rate of 10°C/min.
2.4.6. Thermogravimetric Analysis (TGA)
Thermogravimetric analysis of the Res samples was performed using a Perkin Elmer Pyris TGA (PerkinElmer Co., USA). The experiments were carried out with a heating rate of 1°C/min under nitrogen flow of 50 mL/min. 5.0 mg of samples was placed in open aluminum pans, and a percentage weight loss from 32.5°C to 712.5°C was monitored.
2.5. Purity Detection
Res standard substance was prepared in different concentrations of aqueous solutions of the Res. These solutions were then detected using high performance liquid chromatography (HPLC) equipment (1525–2489, Watershed, USA) for linear regression equations.
The rRes and uRes were, respectively, weighed to 2.0 mg and dissolved under ultrasound in 3 mL of 95% ethanol for 30 min. The solutions were then centrifuged at 10000 r/min for 5 min. Finally, the supernatant was joined with the HPLC. The detection conditions were HPLC column (C18, 25 cm); detection wavelength (305 nm); mobile phase, acetonitrile and 0.2% glacial acetic acid water solution, 30 : 70 (V/V); injection volume (10 μL), and flow rate (1 min/mL) [23].
2.6. Saturation Solubility
To determine the saturation solubility of the uRes and rRes particles, 1 g of uRes and rRes was added to 5 mL of pure water and pH 7.4 phosphate buffer solution (PBS) to ensure excess samples. Each suspension liquid was then sealed and mixed at a speed of 75 rpm in a 25°C water bath kettle for 48 h. The solution was injected into HPLC through 0.22 µm filter membrane for testing according to the 2.5 detection method.
2.7. Dissolution Test
Approximately 5 mg of uRes and 5 mg of rRes particles were each placed in 500 mL of 0.2 mol/L hydrochloric acid solution, with 0.5% (w/w) Tween80 at 37°C and a rotor speed of 75 rpm [24]. Afterward, 1 mL of sample solution was transferred and taken through 0.22 µm filter membrane into the HPLC at 1, 2, 3, 4, 5, 10, 15, and 20 min. Finally, each sample solution was tested and repeated three times according to the 2.5 detection method.
2.8. DPPH Radical Scavenging Activity
The DPPH (1,1diphenyl2picrylhydrazyl radical 2,2diphenyl1(2,4,6trinitrophenyl) hydrazyl) researched radical scavenging activity of the uRes and rRes was used. First, different concentrations of uRes and rRes suspension water solutions (3.125 µg/mL to 500 µg/mL) were prepared. The solutions were then filtrated through the 0.45 µm membrane and 1.5 mL solution of water was added to 1.5 mL 0.1 mmol/L DPPH 95% ethanol solution, mixed, ultrasonically treated for 5 min, and placed for 30 min at room temperature in the dark. Finally, the absorbencies of the solutions were measured at 517 nm by UVdetector. The computation formula of the DPPH () is as follows: , where is the absorbency of the blank sample and is the sample absorbency. Using the IC_{50} analysis software, of the sample of the different suspension concentrations of the water solutions was calculated for IC_{50} values. IC_{50} was the suspension concentration when sample scavenged half of the free radical.
2.9. Bioavailability Studies
Six female SpragueDawley rats (weight between 200 and 250 g, age between 60 and 70 d) were prepared and divided into two groups, each group of 3 rats. Prior to all experiments, animals were fasted overnight and freely drunk water. Two groups of rats were, respectively, given rRes and uRes by gavage at the doses of 50 mg/kg (according to Res). After feeding, blood samples from the two groups were obtained at 0.25, 0.5, 1, 2, 3, 4, 6, and 8 h. The blood samples were then centrifuged at 10000 rpm for 10 min. The supernatants of the blood samples were mixed with methanol 1 : 3 (v/v) and centrifuged at 10000 rpm for 10 min. The supernatants of the mixed solutions were measured according to the 2.5 detection method. Experimental procedures and animal use and care protocols were approved by the Committee on Ethical Use of Animals of Harbin Medical University.
3. Results and Discussion
3.1. Optimization Studies
The SEM images for uRes particle size data, which the orthogonal assistant analyzed in Table 2, were measured using ImagePro Plus 6.0. Table 2 shows that the minimum particle size of the uRes is 0.7 ± 0.02 µm and the maximum particle size is 2.18 ± 0.24 µm. According to the value from Table 2, is the most important factor; the influencing factors of particle size is as follows: . In Figure 3, the trend of the particle size is erratic, rising then declining and then rising again under the conditions , , and , with , , and as the lowest points. The trend of particle size initially increased and then decreased under condition , and is the lowest point. Moreover, the fluctuation intensity is (i.e., pressure factor > temperature factor > concentration factor > flow rate factor); pressure is the most important factor. The optimum condition of the SAS process of the uRes particles is (precipitation temperature at 50°C, concentration at 9 mg/mL, precipitation pressure at 20 MPa, and flow rate at 1 mL/min) according to the value from Table 2 and Figure 3. Then the uRes particles was obtained under the optimum condition of and the particle size of the confirmatory test is 0.68 ± 0.03 µm. Then, the uRes particles under this condition were characterized by various analytical methods.
 
= (mean particle size at )/4, the mean values of mean particle size for a certain factor at each level with standard deviation. . 
3.2. Morphology
Compared with the crystal column form of rRes in Figure 4(a), the lamellar form of the uRes particles is easily distinguishable in Figure 4(b). The uRes particle size is smaller, generally, less than 2 µm, its crystal degree is lower, and its specific surface area is larger. According to the NoyesWhitney equation, the water solubility of the uRes particles is higher.
(a)
(b)
3.3. Purity Study
Figure S_{1} (see Figure S_{1} in the Supplementary Material available online at http://dx.doi.org/10.1155/2015/838513) shows the HPLC chromatograms of the rRes and uRes particles in vitro. A standard curve of Res is drawn using HPLC, and the regressive equation is with . The peak values of the rRes and micro particles are placed into regressive equation for the contents. The contents of the rRes and micro particles are at 98.5% ± 0.5 and 99.2% ± 0.5 (mean ± SD), confirming that the Res is purified in the SAS process. The reason is that similar Res molecules crystallize to each other and reject other molecules in SAS process. Impurity is separated from Res. So, the purity of uRes after recrystallization by SAS process is higher than rRes.
3.4. Chemical Structure Studies
3.4.1. FTIR Spectroscopy
The molecular structures of the rRes and uRes particles were examined using FTIR equipment. Figure 5 shows no significant differences between the two particles in 3331, 3014, 1888, 1593, 1512, 1328, 1149, 964, 833, 675, and 518 cm^{−1}. Therefore, the functional groups of the rRes and micro particles did not exhibit particular changes in the SAS process.
3.4.2. Liquid ChromatographyMass Spectrometry (LCMS)
The molecular weights of the rRes and uRes particles were 226.8 and 227.0, as shown in Figure 6. Results of the LCMS clearly showed that the two particles have the same molecular weight, and the molecular weight is the same as Res at 226.8 [24], indicating that the Res is accurate. Res did not exhibit any change in the SAS process.
(a)
(b)
3.5. Physical Structure Studies
3.5.1. XRay Diffraction Studies
Figure 7 shows that the XRD pattern of the rRes has a highintensity diffraction peak at = 6.6°, 16.3°, and 19.1°. Although the uRes particles have diffraction peaks at the same position as the XRD pattern, the intensity of the diffraction peaks of micro particles is far below the level of the raw particles. Results suggest that the crystallization degree of Res is significantly reduced in the SAS process; thus, improving the dissolution of Res in water can be beneficial. Figure 7 also shows that the XRD pattern of uRes particles which was placed 4 months under low temperature has no change. This proves that the uRes is stable.
3.5.2. Differential Scanning Calorimetry
Compared with the DSC patterns of the rRes and uRes particles in Figure 8, two similar sharp endothermic peaks were found at 267°C and 265°C. The peak of the uRes particles is far below the rRes. This illustrated that the crystallization degree of uRes is significantly reduced and is beneficial to improve the solubility and the bioavailability [25].
3.5.3. Thermogravimetric Analysis
Figure 9 shows that the TG curves of the rRes and uRes particles both have a slow weight drop below 260°C and a sudden weight drop between 260 and 712°C, indicating that both rRes and uRes have small water and volatile constituents to lose below 260°C. And the melting point of Res is 253–255°C. So the Res begins to decompose with temperature increasing at 260°C [26]. Figure 9 also shows the total weight loss of rRes and uRes is 50% and 53.5%, respectively, which indicates that uRes has more weight loss than rRes between 260 and 712°C. The reason is that the smaller the particle size is, the bigger the specific surface area is, the more the atoms are on the surface of particles, the more its surface energy will increase quickly, the higher the specific surface energy is, the worse the stability of the particles is, and the more the weight loss is at high temperatures [27]. So the uRes particles are smaller in size and lead to a high specific surface energy, which explains why the uRes has more weight loss than rRes.
3.6. Saturation Solubility and Dissolution Rate
Figure 10 shows that saturation solubility of uRes (75.1 µg/mL) in pure water is much higher than that of rRes (31.8 µg/mL). The saturation solubility of uRes (66.2 µg/mL) in PBS solution is much higher than the rRes (29.6 µg/mL) (pH 7.4). Thus, the saturation solubility of the uRes is 2.4 times and 2.3 times of the rRes in pure water and in PBS solution (pH 7.4). The solubility of rRes and uRes in pure water is slightly higher than in PBS solution (pH 7.4). Combined with the study of the physical structure of Res, this indicates that the saturation solubility of Res increases with the decrease of the particle size. The OstwaldFreundlich equation gives the principle of the above conclusion. The OstwaldFreundlich equation is defined as follows:where and are the particle radius; and are the solubility when the particle radius was and ; is the density of solid drugs; is the interfacial tension between solid drugs and liquid solvents; is molecular weight of the drug; is molar gas constant; and is thermodynamic temperature [28]. When the of uRes is smaller than the of rRes, the of uRes is greater than the of rRes. So the saturation solubility of Res increases with the decrease of the particle size. Kim et al. were able to make the saturation solubility of Res reach 66.8 and 56.2 µg/mL in pure water and PBS solution (pH 7.4), respectively [15]. So, compared with the results obtained by Kim et al., the uRes prepared using the SAS process is shown to have more saturation solubility and does not contain any subsidiary material.
Figure 11 shows that the uRes particles have a dissolution rate of 89.5% within 5 min and complete dissolution within 15 min, whereas the rRes particles have a dissolution rate of only 72.6% at 5 min, 77.3% at 15 min, and without completing dissolution 77.8% at 30 min. The results clearly indicate that uRes particles have a much faster dissolution rate than rRes. According to the NoyesWhitney equation, the main factors that affect the sample dissolution rate are the dissolution interface area, sample saturation concentration, and sample solution concentration constant [29]. The NoyesWhitney equations are defined as follows:where is dissolution rate; is dissolution interface area; is sample saturation concentration; is sample solution concentration; is diffusion coefficients; is dissolution medium quality; is boundary layer thickness. The sample dissolution rate is directly proportional to the interface area with dissolution medium [30]; as the interface area increases, the dissolution rate also increases. Decreased particle size leads to an increase in the effective surface area in the diffusion layer, thereby increasing the sample dissolution rate [31]. When the sample particle size decreases to less than 5 µm, the boundary layer thickness of the Prandtl boundary layer equation is reduced [32]. The constant will then be increased by the equation , substituted in the equation of ; the sample dissolution rate increases.
As shown in Figure 4, the uRes particles constitute tiny lamellar particles, whereas the rRes particles constituted large crystal columns. So the uRes particles are smaller than the rRes particles. The uRes particles have a much larger interface area with dissolution medium and faster dissolution rate than the rRes particles. According to Figure 12, the dissolution rate of the uRes is 1.3 times that of the rRes. Kim et al. make Res have a dissolution rate of 20% within 5 min and complete dissolution within 50 min [15]. Compared with the results of Kim et al., the uRes prepared by the SAS process has higher dissolution rate without any subsidiary material.
3.7. Radical Scavenging Activities
Six suspension concentration water solutions of rRes and uRes particles were tested through PDDH. As shown in Figure 12, the free radical scavenging rate of the uRes particles is much higher than the rRes particles. Using the IC_{50} calculation software, the IC_{50} value of rRes (14.55 µg/mL) was much greater than the uRes particles (7.75 µg/mL), indicating that the uRes particles only needed half of the rRes concentration to reach the same effect. The radical scavenging activity of the uRes is 1.9 times that of rRes. The dissolution rate of the uRes particles was larger than the rRes according to Figure 12. Therefore, the uRes particles greatly improved the free radical scavenging activity as a natural product and have potential in the health care products market.
3.8. Bioavailability Analysis
As shown in Figure 13, the result of bioavailability is the drug concentration of the uRes is higher than the rRes in rat plasma. The drug concentration of the uRes reached the maximum of 2.35 µg/mL at 15 min. Then the drug concentration of the uRes particles decreased slowly to 0.01 µg/mL in 8 h. The drug concentration of the rRes also reached the maximum of 1.95 µg/mL at 15 min. Then, the drug concentration of the rRes decreased to 0.01 µg/mL in 8 h. Through the OriginLab 8.5 software, comparing the AUC of the two groups, the uRes is 1.9 times of the rRes. The reason of the result is that the uRes has smaller particle size than rRes, which leads to the uRes having larger interface area and higher saturation solubility. So, the SAS method improves the bioavailability of the Res.
4. Conclusions
This paper was conducted to make Res reach micron level through the SAS process and to optimize the factors of the SAS process by orthogonal method. Using SEM, the microstructure of Res was observed to have fundamentally changed from the large crystal column of rRes to the tiny lamellar particle of uRes under the SAS process. The average particle size was also reduced from hundreds of microns of rRes to 0.68 µm of uRes. uRes has the same chemical structure and physical structure as rRes according to the FTIR, XRD, DSC, and TG data, indicating that the crystal degree of Res was greatly reduced and the structure did not change during the SAS process. Moreover, the SAS process purified the HPLCdetected Res from 98.5% to 99.2%. Finally, a comparison between the rRes and uRes particles through saturation solubility studies, dissolution rate test in vitro, radical scavenging activity, and bioavailability test in vivo showed that the saturation solubility of the uRes was 2.4 times and 2.3 times of the rRes in pure water and PBS solution (pH 7.4). Moreover, Kim et al. reported that the dissolution rate of the uRes was much faster than the Res. The radical scavenging activity and bioavailability of the uRes in vivo were 1.9 times of the rRes. Therefore, uRes particles made by the SAS process greatly increased the application value of Res particles as a new material.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgments
The authors would like to acknowledge the financial support from the Special Fund for Forestry Scientific Research in the Public Interest (20140470102) and the National Natural Science Foundation of China (no. 21203018).
Supplementary Materials
Fig. S1 shows the HPLC chromatograms of rRes (a) and uRes (b) particles. The contents of the rRes and uRes particles are at 98.5%± 0.5 and 99.2% ± 0.5 (mean ± SD), confirming that the Res is purified in the SAS process.
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Copyright © 2015 Kunlun Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.