Article Cite This: J. Agric. Food Chem. 2019, 67, 5746−5753
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Bioavailability Enhancement of Astilbin in Rats through Zein− Caseinate Nanoparticles Dan Zheng and Qing-Feng Zhang* Jiangxi Key Laboratory of Natural Product and Functional Food, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, People’s Republic of China
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S Supporting Information *
ABSTRACT: Astilbin-encapsulated zein−caseinate nanoparticles were fabricated through the antisolvent method. The encapsulation and loading efficiency of astilbin in the nanoparticles were determined by high-performance liquid chromatography. The nanoparticles were characterized by particle size, ζ potential, redispersibility, scanning electron microscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy, and differential scanning calorimetry (DSC). Under the optimal formulation of astilbin, zein, and sodium caseinate with a mass ratio of 1:1:2, the size and ζ potential of the nanoparticles were 152.9 nm and −40.43 mV, respectively, while the encapsulation and loading efficiency of astilbin were 80.1 and 21.8%, respectively. The nanoparticles had good redispersibility in water without a particle size change after freeze drying. The nanoparticles showed a spherical shape with a smooth surface. XRD and DSC analyses showed that astilbin existed in amorphous form in the nanoparticles. The interactions between astilbin and the protein were found, and astilbin was encapsulated in nanoparticles rather than adsorbed. The diffusion of astilbin from nanoparticles was significantly faster than that of astilbin suspensions in both simulated gastric and intestinal fluids. Astilbin was relatively stable in simulated intestinal fluids, and the encapsulation in the nanoparticles showed a slight stability improvement effect. A pharmacokinetic study showed that the absolute bioavailability of astilbin was improved from 0.32 to 4.40% in rats through nanoparticle fabrication. KEYWORDS: astilbin, zein, nanoparticles, pharmacokinetics, bioavailability
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INTRODUCTION
the oral bioavailability of astilbin solid dispersions was about 205% of that of astilbin.16 Zein is the main storage protein of corn, with more than 50% of hydrophobic amino acids. Hence, the protein is insoluble in water but has good solubility in 50−95% ethanol− aqueous solution.17 With the decrease of the ethanol concentration, zein will lose its solubility and is able to selfassemble into micro- or nanoparticles,17 fiber,18 and film.19 The specific structure is dependent upon the preparation method and conditions, such as pH, zein concentration, shearing rate, etc.17 The unique self-assembly ability of zein to form nanoparticles makes it a promising delivering carrier for nutraceuticals and drugs.20 The internal hydrophobic environment of zein nanoparticles is suitable for encapsulating hydrophobic compounds. Furthermore, zein is an edible protein with natural biocompatibility and biodegradability and is a generally recognized as safe (GRAS) biomaterial.17 The encapsulation in zein nanoparticles could significantly improve the stability, dispersibility, and bioavailability of guest compounds.17,21 Penalva et al. fabricated the resveratrol- and quercetin-loaded zein nanoparticles through a desolvation method. The size of nanoparticles was 307 nm, with resveratrol loading of 80 μg/mg. The oral bioavailability of resveratrol was enhanced by 19.2-fold in rats.22 Similar results were found in quercetin-loaded zein nanoparticles.23 Zou et al. designed
Astilbin is the rhamnoside of dihydroquercetin. The flavonoid is presented in many plant and plant-based foods with a variety of bioactivities.1−4 Particularly, the unique selective immunosuppressive activity of astilbin has attracted increasing attention.5−9 Pharmacological studies revealed that astilbin could significantly inhibit delayed hypersensitivity,5,6 collageninduced arthritis,7,8 and immune liver injury.9 It has reported that astilbin could selectively inhibit excess cellular immunity without affecting humoral immunity. Its strong immunomodulatory activity shows no obvious side effects. Other compounds with similar activity have yet to be found. Astilbin may be an attractive candidate for novel immunomodulator development.10 However, astilbin is a poorly soluble compound, with a solubility of 0.221 g/L in water at 298 K.11 It is also unstable in alkaline solution, and the isomerization and decomposition were found.12 As a result of the low solubility and permeability, the oral bioavailability of astilbin is very poor. Wang found that the apparent permeability coefficient of astilbin was less than 1 × 10−6 cm/s in Caco-2 cell, and its oral bioavailability was only 0.066% in rats.13 The study of Lei et al. showed that the oral bioavailability of astilbin was 2.01% in rats.14 Hence, improving the bioavailability of astilbin is critical for its practical application. By development of a self-microemulsifying drug delivery system, the bioavailability of astilbin was enhanced 5.56-fold in beagle dogs.15 He et al. prepared the solid dispersions of astilbin in PVP K30−Tween 80 combined carries. A pharmacokinetic study in beagle dogs showed that © 2019 American Chemical Society
Received: Revised: Accepted: Published: 5746
January 2, 2019 March 28, 2019 May 2, 2019 May 2, 2019 DOI: 10.1021/acs.jafc.9b00018 J. Agric. Food Chem. 2019, 67, 5746−5753
Article
Journal of Agricultural and Food Chemistry
Briefly, the nanoparticles were dissolved in 70% ethanol. After appropriate dilution, a 10 μL aliquot of sample was injected in an Agilent 1260 HPLC system (Agilent Technologies, Palo Alto, CA, U.S.A.). The HPLC conditions were the same as we described before.12 SEM Analysis. The freeze-dried nanoparticles (Ast−Zein1:1) and its redispersion solution were used for morphology characterization by SEM (FEI Quanta 250 FEG, FEI, Inc., Hillsboro, OR, U.S.A.). The redispersion solution was first dropped on a conductive glass and then dried naturally. Before imaging, the samples were coated with a thin layer of gold first. DSC Analysis. DSC analysis was carried on a NETZSCH DSC 214 Polyma instrument (Netzsch, Germany). About 5 mg of freezedried nanoparticles (Ast−Zein1:1) was added to a standard aluminum pan and hermetically sealed. The pan was placed in a glass desiccator for 24 h to exclude the influence of moisture. An empty sealed aluminum pan was used as a control. Samples were heated from 50 to 300 °C at a rate of 12 °C/min with a constant purging of dry nitrogen at a rate of 50 mL/min. The thermal characteristics of astilbin and physical mixture of zein and sodium caseinate (with a mass ratio of 1:2) were also analyzed. XRD Analysis. XRD patterns of Ast−Zein1:1 nanoparticles and astilbin were performed on a Bruker D8 ADVANCE diffractometer (Bruker, Germany). The instrument was equipped with a copper anode. The working accelerating voltage and tube current were 40 kV and 40 mA, respectively. The pattern was recorded over an angular range from 5° to 60° in continuous mode with a step size of 0.02° and scanning speed of 2°/min. FTIR Analysis. FTIR spectroscopy of Ast−Zein1:1 nanoparticles, astilbin, and physical mixture of zein and sodium caseinate (with a mass ratio of 1:2) was recorded on a Nicolet 5700 FTIR spectrometer (Nicolet, Madison, WI, U.S.A.) by the KBr method. In Vitro Diffusion of Astilbin from the Nanoparticle in SGF and SIF. SGF (pH 1.2) and SIF (pH 6.8) were prepared in accordance with USP XXII.19. Ast−Zein1:1 nanoparticles and astilbin were dispersed in 5 mL of SGF or SIF (with enzyme) with an astilbin concentration of 3 mg/mL. The solution was put into a dialysis bag (molecular cutoff of 7 kDa) and then placed in a beaker containing 500 mL of SGF or SIF (without enzyme) under magnetic stirring at 37 °C. At different time intervals, the astilbin concentration in the beaker was analyzed by HPLC. Stability of Free Astilbin and Ast−Zein1:1 Nanoparticles in SIF. Astilbin solution was prepared in 60% ethanol with a concentration of 1 mg/mL. Ast−Zein1:1 nanoparticles were dispersed in water with an astilbin concentration of 1 mg/mL. A 1.0 mL aliquot of the two solutions was mixed with 9 mL of SIF (without enzyme), respectively. The mixture was incubated at 37 °C in a water bath. At different time intervals, the remaining astilbin was determined by HPLC. Pharmacokinetic Studies in Rats. The animal studies were complied with the guidelines of Jiangxi Agricultural University on animal care. Sprague Dawley (SD) rats were obtained from Hunan Slack Jing Da Laboratory Animal Co., Ltd. (Changsha, Hunan, China). The rats were bred in controlled environmental conditions with a 12:12 h light/dark cycle and temperature of 23−25 °C. Before the pharmacokinetic study, rats fasted overnight with access to water. A total of 18 rats were randomly divided into three groups (n = 6) with a mean weight of 300 ± 15 g. For nanoparticle colloidal solution, freeze-dried Ast−Zein1:1 nanoparticles were dispersed in warm water (∼40 °C) with an astilbin concentration of 6 mg/mL. Similarly, astilbin suspensions were prepared by suspending astilbin powder (pass through 100 mesh, with average size of about 150 μm) in warm water (∼40 °C) directly with a concentration of 6 mg/mL. Because the solubility of astilbin is only about 0.2 mg/mL, it will precipitate soon. Thus, before oral (po) administration, the suspensions should be shocked intensely. Groups I and II were orally administered 1 mL of nanoparticle colloidal solution and astilbin suspensions with a dose of 20 mg of astilbin/kg of body weight, respectively. Group III received intravenous (iv) administration of 0.3 mL of astilbin solution (2 mg/mL in 10% ethanol) through the caudal vein with a dose of 2
TPGS-1000-emulsified zein nanoparticles to improve the oral bioavailability of daidzin. A pharmacokinetic study showed that Cmax of daidzein in mice plasma and the bioavailability were significantly increased.24 Sodium caseinate is the sodium salt of casein, which is the main protein of milk. Sodium caseinate has superb water solubility and is widely used for emulsifying and thickening functions in the food industry. It has been reported that sodium caseinate can adsorb on the surface of zein nanoparticles as a result of its amphiphilic and charged nature, which can prevent the aggregation of colloids.25 Thus, sodium caseinate can be used as a stabilizer for zein nanoparticles. In the present study, zein−caseinate nanoparticles were fabricated to improve the bioavailability of astilbin. The formulation was optimized through comparing the encapsulation and loading efficiency of astilbin. The nanoparticles were characterized by the particle size, ζ potential, redispersibility, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The diffusion profile of astilbin from the nanoparticles in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was also studied. Finally, a pharmacokinetic study was carried out to confirm the bioavailability enhancement of astilbin in rats.
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MATERIALS AND METHODS
Chemicals. Astilbin (>98%) were purified from Rhizoma Smilacis Glabrae in our laboratory and was identified by ultraviolet (UV), infrared (IR), mass spectrometry (MS), and nuclear magnetic resonance (NMR). High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were purchased from Anhui Tedia High Purity Solvents Co., Ltd. (Anqin, Anhui, China). Zein was obtained from Sigma Co., Ltd. (St. Louis, MO, U.S.A.). Sodium caseinate, pepsin, and pancreatin were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Milli-Q water was used throughout the study. All other reagents used were analytical-grade. Preparation of Astilbin-Encapsulated Zein−Caseinate Nanoparticles. The nanoparticles were prepared by the antisolvent method described elsewhere.17 The mass ratio of zein/astilbin in the nanoparticles was varied from 1:1 to 6:1, while the mass ratio of zein/ sodium caseinate was maintained at 1:2. Briefly, 100 mg of zein mixed with 100, 50, 25, or 16.7 mg of astilbin was dissolved in 10 mL of 70% ethanol solution. The mixture was dropped into 30 mL of water containing 200 mg of sodium caseinate using an injection syringe under quick magnetic stirring. The dropping rate was about 2 mL/ min. After stirring for another 60 min, the nanoparticles suspensions were concentrated to 25 mL by rotary evaporators at 50 °C. The suspensions were subsequently centrifuged at 2000g for 10 min to remove large particles and free astilbin. Then, a little part of the suspensions was used for encapsulated astilbin determination, particle size, and ζ potential analyses, and the rest was freeze-dried. Particle Size, Polydispersity Index (PDI), and ζ Potential Analyses. The particle size and ζ potential of nanoparticles were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Ltd., U.K.). The measurement was replicated 3 times for each sample. Encapsulation Efficiency (EE) and Loading Efficiency (LE). The encapsulated astilbin in nanoparticles was determined by the HPLC method. The EE and LE were calculated with the equations: EE =
astilbin encapsulated in nanoparticles (mg) × 100% total astilbin used (mg)
LE =
astilbin encapsulated in nanoparticles (mg) × 100% weight of dry nanoparticles (mg) 5747
DOI: 10.1021/acs.jafc.9b00018 J. Agric. Food Chem. 2019, 67, 5746−5753
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Journal of Agricultural and Food Chemistry Table 1. Particle Size, ζ Potential, EE, and LE of Nanoparticles Prepared with Different Formulations (n = 3)a mass ratio Ast−Zein1:1 Ast−Zein1:2 Ast−Zein1:4 Ast−Zein1:6 Ast−Zein1:1b Ast−Zein2:1b
particle size (nm) 152.9 142.4 141.4 132.0 153.9 145.8
± ± ± ± ± ±
1.2 0.9 0.9 1.7 1.4 0.7
a b b c a ab
ζ potential (mV)
PDI 0.15 0.12 0.17 0.15 0.21 0.23
± ± ± ± ± ±
0.02 0.02 0.02 0.04 0.01 0.00
−40.43 −44.87 −41.20 −41.40 −32.85 −36.80
cd d bc cd ab a
± ± ± ± ± ±
1.19 1.33 1.31 0.91 0.35 0.00
c d c c a b
EE (%) 80.18 75.68 76.57 75.88
± ± ± ±
LE (%)
4.34 a 10.27 a 7.35 a 3.46 a
21.86 9.87 4.77 3.89
± ± ± ±
1.74 0.59 0.34 0.18
a b c d
a
Different letters in columns mean significant difference (ANOVA; p < 0.05). bFreeze-dried sample redispersed in water.
mg of astilbin/kg of body weight. The iv solution was prepared by 5 times dilution of astilbin dissolved in 50% ethanol (10 mg/mL) with water. Blood samples (∼200 μL) were obtained from tail veins into K2−ethylenediaminetetraacetic acid (EDTA)-pretreated tubes at 0.167, 0.5, 1, 2, 3, 4, 6, and 8 h after administration. After centrifugation at 2000g for 10 min, the plasma samples were obtained and stored at −80 °C before analysis. An aliquot of 50 μL of plasma sample was mixed with 150 μL of methanol containing 350 ng/mL rutin [used as the internal standard (IS)]. Then, a 5 μL aliquot of acetic acid was added. The mixture was vortexed for 2 min to precipitate endogenous proteins. After centrifugation at 10000g for 10 min, the supernatant was transferred to a new 1.5 mL tube and concentrated to about 100 μL on a 75 °C water bath for 12 min. The solution was centrifuged at 10000g for 10 min again, and the supernatant was used for astilbin determination. The astilbin concentration was determined on an Agilent 1260 HPLC system equipped with an autosampler and a diode array detector (DAD). A symmetry C18 column (4.6 × 250 mm, 5.0 μm, Waters, Milford, MA, U.S.A.) was used. The mobile phase consisted of 24% acetonitrile and 76% water (with 0.1% acetic acid). The flow rate was 1.0 mL/min with a column temperature of 40 °C and detection wavelengths of 291 nm (for astilbin) and 355 nm (for rutin). The injection volume was 50 μL, and the analysis duration was 10 min. A calibration curve was obtained in triplicates by plotting the peak area ratio (astilbin/rutin) against the astilbin concentration (16, 32, 64, 128, 256, 515, 721, 1030, 5150, and 10 300 ng/mL in plasma). The regression equation is Y = 2.2604X, with linear coefficient of 0.9995, where Y is the peak area ratio and X is the concentration of astilbin in plasma (μg/mL). The pharmacokinetic data were simulated by Drug and Statistics (DAS) software (version 2.0) using a non-compartmental statistical model to calculate the pharmacokinetic parameters, including the maximal serum concentration (Cmax), the time in which Cmax is reached (tmax), the area under the concentration−time curve (AUC), the mean residence time (MRT), the clearance (CL), and the half-life in the terminal phase (t1/2). Furthermore, the absolute bioavailability (Fr, %) of astilbin was calculated by the equation Fr (%) =
The PDI of all formulations was less than 0.2, indicating the uniform distribution of the particle size. Luo et al. prepared the nanoparticles with different zein/caseinate ratios (from 1:0 to 1:2). The particle size was between 130 and 170 nm, and more caseinate resulted in a smaller particle size.26 The particle sizes of resveratrol- and quercetin-loaded zein nanoparticles were 307 and 358 nm, respectively.22,23 The typical size of zein nanoparticles is in the range of 100−400 nm, which was easily affected by the preparation conditions, such as formulation compositions, alcohol concentration, temperature, and even the adding speed.17,27 ζ potential is an important characteristic for the stability of colloidal suspensions, which can provide electrostatic repulsion and prevent the aggregation of nanoparticles. It is generally recognized that the colloids tend to coagulate when the absolute value of ζ potential is smaller than 10 mV.28 The higher the absolute value of ζ potential, the more stable the colloids found. In the present study, the ζ potential of all formulations ranged from −40.43 to −44.87 mV, indicating strong electrostatic stabilization of the nanoparticles. Although the zein nanoparticles are easy to fabricate, the major obstacle is its aggregation during practical applications. The drying process (lyophilization or spray drying) would cause the aggregation of zein nanoparticles and lead the loss of redispersibility.17 Besides, the isoelectric point of zein protein is around pH 6.2.25 Thus, when used as the oral drug delivery vehicle, the electrostatic repulsion between the colloidal particle in the intestine is very weak (as a result of the small ζ potential near the isoelectric point), which may result in aggregation. To improve the stability of zein nanoparticles, many stabilizers were tested in the literature, such as TPGS 1000,24 sodium caseinate,25 lecithin,29 etc. Among them, the use of sodium caseinate has attracted much attention. Sodium caseinate can adsorb on the surface of zein nanoparticles and shift the isoelectric point to around 5.0. After freeze drying, the zein−caseinate nanoparticles was redispersed in water without major changes in either the particle size or surface potential.25 Furthermore, the presence of sodium caseinate could significantly enhance cell uptake of zein nanoparticles in a concentration- and time-dependent manner in Caco-2 cells.26 Patel et al. has studied the effects of the mass ratio between sodium caseinate and zein in detail (from 0:1 to 2:1).25 It was found that, when the mass ratio between sodium caseinate and zein was less than 1.25:1, the freeze-dried nanoparticles showed incomplete redispersion. The more sodium caseinate assured better redispersibility of the dried colloidal particles. In the present study, the optimal mass ratio of 2:1 between sodium caseinate and zein was used. Results showed that the freeze-dried nanoparticles could be quickly redispersed in water. Using Ast−Zein1:1 nanoparticles as an example, the particle size was almost unchanged after freeze drying (Table 1). However, the PDI increased from 0.14 to 0.21, indicating
AUCoral × 100 AUCiv
Statistical Analysis. Data were expressed as the mean ± standard deviation of triplicates. Data analysis and plotting were performed with software of Origin 7.0 (Origin Lab Co., Northampton, MA, U.S.A.). One-way analysis of variance (ANOVA) was used for statistical analysis. Differences were considered significant with p < 0.05.
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RESULTS AND DISCUSSION Optimization of Nanoparticle Formulation. To optimize the formulation of nanoparticles, different mass ratios of astilbin/zein from 1:1 to 1:6 were studied. The particle size, PDI, ζ potential, EE, and LE of different nanoparticles were summarized in Table 1. The results showed that the mean size of nanoparticles ranged from 132.0 to 152.9 nm. Increasing the zein mass in the formulation slightly decreased the particle size. 5748
DOI: 10.1021/acs.jafc.9b00018 J. Agric. Food Chem. 2019, 67, 5746−5753
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Journal of Agricultural and Food Chemistry the slight decrease of size uniformity. Besides, the ζ potential also decreased, but the absolute value was still bigger than 30 mV. In Figure 1, a very small peak with a particle size of
characterization and bioavailability tests. The astilbin content in the freeze-dried nanoparticles was 21.86%. The practical LE was in accordance with the EE. Physicochemical Characterization of Ast−Zein1:1 Nanoparticles. Morphological Observation. In Figure 2A, the SEM image showed that, after freeze drying, although some individual particles were found, most of them were aggregated to each other. However, when redispersed in water, the particles became individuals immediately (Figure 2B). The results confirmed the good redispersibility of the freeze-dried product. It may be attributed to the sodium caseinate layer adsorbed on the particle surface, which has superb solubility in water. After redispersing in water, all particles showed a spherical shape with a smooth surface. The particle size was in accordance with the result of DLS analysis. XRD Analysis. The XRD patterns of astilbin and Ast− Zein1:1 nanoparticles are shown in Figure S1A of the Supporting Information. The major characteristic peaks of astilbin at diffraction angles of 7.24°, 9.37°, 13.68°, 16.30°, 21.85°, and 44.50° are present, indicating the highly crystalline nature of astilbin. However, all of these sharp peaks disappeared in the nanoparticles, and only a wide peak within the diffraction angle of 15−30° was found. The results revealed that astilbin lost its crystal structure in the nanoparticles. When forming nanoparticles, the molecular interaction between astilbin and the protein may suppress its crystallization. Similar results were found in many other studies. Both crystalline quercetin and curcumin existed in amorphous form in the zeinbased nanoparticles.29,30 Patel et al. concluded that crystalline compounds will convert to amorphous form when encapsulated in the colloidal particles.30 DSC Analysis. Figure S1B of the Supporting Information illustrated the DSC thermograms of astilbin, Ast−Zein1:1 nanoparticles, and the physical mixture of zein and sodium caseinate (2:1, wt/wt). The DSC curve of astilbin showed a sharp endothermic peak at 180.6 °C, which indicated the crystal melting of the compound. However, this sharp endothermic peak was unobserved in the astilbin-encapsulated nanoparticles, which indicated that astilbin dispersed in amorphous forms. The result was in accordance with XRD analysis. In the physical mixture of zein and sodium caseinate, a wider peak was found between 101.3 and 121.7 °C, which may be due to the denaturation of the protein. This wide peak was
Figure 1. Particle size distribution of Ast-Zein1:1 nanoparticles before and after freeze drying.
around 5 μm was found, which indicated that the aggregation of some nanoparticles (∼0.6%) also occurred during the drying process. Similar results were also found in other nanoparticle samples (Ast−Zein1:2 in Table 1). Besides the particle size and ζ potential, EE and LE are also very important characteristics of nanoparticles for drug or nutraceutical delivery. The EE values of nanoparticles with different astilbin/zein ratios were around 80%, and no significant difference was found between the formulations. Astilbin is poorly soluble in water. When zein self-assembles into the nanoparticles, its hydrophobic amino acid residues will form many inner hydrophobic environments. Hence, astilbin could be well-encapsulated in the nanoparticles through hydrophobic interactions. Because EE showed no difference between the formulations, it is clear that the more zein used in the formulation, the smaller the LE of the nanoparticles. Thus, the formulation of astilbin, zein, and sodium caseinate with a mass ratio of 1:1:2 (Ast−Zein1:1) was used to prepare the nanoparticles for future
Figure 2. SEM images of (A) freeze-dried Ast−Zein1:1 nanoparticles and (B) after redispersion in water. 5749
DOI: 10.1021/acs.jafc.9b00018 J. Agric. Food Chem. 2019, 67, 5746−5753
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Journal of Agricultural and Food Chemistry also found in the astilbin-encapsulated nanoparticles. However, the peak shape was changed. These results showed that some interactions between astilbin and the protein occurred, and astilbin was encapsulated in nanoparticles rather than adsorbed. FTIR Analysis. The FTIR spectra of the protein mixture, Ast−Zein1:1 nanoparticles, and astilbin are shown in Figure S1C of the Supporting Information. The protein mixture exhibited characteristic peaks at 3405 cm−1 (stretching vibration of O−H), 2961 cm−1 (vibration adsorption of C− H), 1633 cm−1 (amide I), and 1384 cm−1 (stretching vibration of −CH3). The amide I peak is caused by the stretching vibration of CO, mainly as a result of the α-helical component of the secondary structure of zein.31 The amide I peak also appeared in the nanoparticles at the same position without intensity reducing, indicating that the α-helical structure of zein in the nanoparticles was preserved. The strong peak at 1641 cm−1 in the spectra of astilbin was due to the CO stretching vibrations. Besides, many characteristic peaks were found between 800 and 1500 cm−1 as a result of the aromatic bending and stretching and −OH phenolic bending. However, many characteristic peaks of astilbin disappeared in the spectra of Ast−Zein1:1 nanoparticles, for instance, 1473, 1295, 1180, 976, 818 cm−1, etc. The results implied that astilbin might interact with zein through noncovalent binding, such as hydrogen-bonding and hydrophobic interactions. Wang et al. showed that astilbin could interact with human serum albumin with a bind constant of about 2.4 × 104 L mol−1. The hydrophobic interaction is the major force for the interaction, and a hydrogen-bonding interaction is also found.32 The molecular interaction may suppress the crystallization of astilbin, which causes it to exist in amorphous form in the nanoparticles, as reflected by XRD and DSC analyses. Diffusion of Astilbin from the Nanoparticles in SGF and SIF. The stability of plain zein nanoparticles was sensitive to electrolytes. The colloidal dispersion was aggregated instantly with an ionic strength higher than 30 mM NaCl. The addition of caseinate can stabilize the zein colloidal dispersion and prevent the aggregation, even up to 1.5 M NaCl.25 In the present study, the Ast−Zein1:1 nanoparticles were almost immediately dispersed in both SGF and SIF and no obvious aggregation was found. Figure 3 showed the diffusion profiles of astilbin suspensions and Ast−Zein1:1 nanoparticles in SGF and SIF at 37 °C. As shown, the diffusion of astilbin from nanoparticles was significantly faster than that of astilbin suspensions. At 30 min, the release of astilbin from nanoparticles was 35.4% in SGF, while this value was only 8.6% for astilbin suspensions. No significant release difference was found between SGF and SIF. Because the solubility of astilbin was very poor, the low concentration gradient across the membrane caused the slow diffusion rate. However, when encapsulated in the zein−caseinate nanoparticles with amorphous form, the excellent dispersion of nanoparticles in water significantly improves its solubility. The higher dissolution pressure caused the faster diffusion rate. For drugs with low solubility, improving its release profile is a crucial step to enhance the oral bioavailability.33 The transepithelial transport rates of astilbin across Caco-2 cell monolayers was positively linearly correlated with its concentration.34 Stability of Free Astilbin and Ast−Zein1:1 Nanoparticles in SIF. Besides the diffusion profile, the stability of
Figure 3. Diffusion profiles of astilbin suspensions and Ast−Zein1:1 nanoparticles in SGF and SIF at 37 °C (n = 3).
astilbin in the nanoparticles may also be concerned. The aqueous stability of astilbin was previously studied in detail with the effects of pH, temperature, and solvent.12 The results showed that astilbin was relatively stable in acidic solution. However, its isomerization and decomposition were found in neutral and alkaline solution. In the present study, the stability of astilbin in free form and zein nanoparticles was compared in SIF (without enzyme) with incubation of 4 h at 37 °C. The results showed that astilbin was relatively stable in SIF, and the stability improvement effect of encapsulation in the nanoparticles was slight. About 87.9 and 77.1% astilbin remained after 2 and 4 h of incubation in free astilbin solution, respectively, while these values were 90.5 and 79.9% for astilbin-encapsulated zein−caseinate nanoparticles, respectively (Figure 4). As we reported previously,12 most disappeared astilbin was isomerized into neoisoastilbin and the decomposed part was rare (
