Preparation, characterization and in vitro evaluation of melatonin-loaded porous starch for enhanced bioavailability

Carbohydrate Polymers 202 (2018) 125–133

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Preparation, characterization and in vitro evaluation of melatonin-loaded porous starch for enhanced bioavailability

T

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Yuanyuan Lia, Xiuhua Zhaoa, , Lingling Wanga, Yanjie Liua, Weiwei Wua, Chen Zhongb, Qian Zhanga, Jianhang Yanga a b

Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin 150040, Heilongjiang, China College of Life Science, Northeast Forestry University, Harbin 150040, Heilongjiang, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Porous starch Melatonin (PubChem CID: 896) Release Cellular antioxidant activity Bioavailability

The present work aimed to apply porous starch (PS) as carrier to improve the oral bioavailability of poorly soluble drug melatonin (MLT). The drug loading and encapsulation efficiency of MLT-loaded porous starch (MPS) were optimized. The characteristics of MPS were analyzed using scanning electron microscopy, Brunauer–Emmett–Teller surface area, Fourier–transform infrared spectroscopy, x-ray diffraction, differential scanning calorimetry and thermo gravimetric analysis. Most MLT transformed into their amorphous form in the PS pores. MPS showed higher MLT solubility and cumulative release rate compared with raw MLT in SGF and SIF. MPS exhibited a higher inhibition to DCFH–DA-oxidized peroxyl radicals at a lower EC50 than that of the raw MLT. Furthermore, the plasma concentrations of MLT and MPS reached a Cmax of 134.26 and 291.77 ng/mL at 15 and 20 min, respectively. The AUC0–360min of the formulated MPS-treated group was approximately 2.34fold higher than that of raw MLT.

1. Introduction Porous starch (PS) is a sustained-release agentthat has attracted extensive attention for its biodegradability, biocompatibility, low cost, and no toxicity (Guo, Li, Liu, Meng, & Tang, 2013; Hemamalini & Dev, 2018; Jiang et al., 2017). Compared with other carrier materials, PS exhibits excellent adsorption property due to abundant pores or hollows that are formed from the surface to the center of the granules, resulting in stable pore structure (Zhang et al., 2012) and high pore volume and specific surface area (Benavent-Gil & Rosell, 2017; Wang et al., 2016). Therefore, PS can be widely used in food, pharmaceuticals, agriculture, cosmetics, and other industries (Belingheri, Giussani, RodriguezEstrada, Ferrillo, & Vittadini, 2015; Lei et al., 2018; Wang, Yuan, & Yue, 2015). PS has also been successfully used to prepare several chemicalbased drugs, such as lovastatin (Wu et al., 2011), carbamazepine (Ali, Fule, Sav, & Amin, 2013), probucol (Zhang et al., 2013) and ciprofloxacin (Gao, Li, Bi, Mao, & Adhikari, 2013). In recent years, studies on antioxidant and bioactivity of natural polymers such as biodegradable polypyrrole/dextrin (Zare, Lakouraj, & Mohseni, 2014) and polyaniline/dextrin (Zare & Lakouraj, 2014) conductive nanocomposite, multilayered electromagnetic bionanocomposite based on alginicacid (Zare, Lakouraj, Mohseni, & Motahari, 2015) have received extensive

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attention. As a natural glucose polymer, PS is used as a carrier to load MLT and its in vitro release, antioxidant activity and bioavailability have not been reported so far. Therefore, the current study aims to apply PS as a drug delivery carrier to improve the solubility, release profile, antioxidant activity and bioavailability of MLT. Melatonin (N-acetyl-5-methoxytryptamine; MLT) is a multifunctional molecule found in unicellular organisms, fungi, plants, and animals, as well as humans. Human MLT is mainly produced by the pineal gland and other nonendocrine organs (Gelaleti et al., 2017), which exhibit many physiological and behavioral functions, such as sleep–wake cycle (Hatonen et al., 1999; Zhdanova et al., 2002), hormonal secretion (Kaestner, Wiedemann, von Bueren, Hoffmann, & Ullrich, 2006), neuromodulation (Haridas, Kumar, & Manda, 2012; Tilden et al., 2003), and immune response regulation (Ciechanowska et al., 2015; Moore & Siopes, 2000). MLT acts as a scavenger of toxic oxygen derivatives (Reiter, Tan, Tamura, Cruz, & Fuentes-Broto, 2014; Reiter et al., 2003) and can reduce reactive oxygen species (Andreadou, Tsantili-Kakoulidou, Spyropoulou, & Siatra, 2003) and reactive nitrogen species (Tan et al., 2002). MLT also prevents oxidative and nitrosative damages to all macromolecules in cell compartments (Goodarzi et al., 2018). Plasma MLT levels are high at night and low during the day (Farhadi, Gharghani, & Farhadi, 2016), which decline

Corresponding author. E-mail address: [email protected] (X. Zhao).

https://doi.org/10.1016/j.carbpol.2018.08.127 Received 27 May 2018; Received in revised form 29 July 2018; Accepted 28 August 2018 Available online 30 August 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.

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suspension was centrifuged for 10 min at 8000 rpm. MLT concentrations in the supernatant were determined by high-performance liquid chromatography (HPLC), and the precipitate was dried at 50 °C for 6 h. Each experiment was repeated at least thrice. Through preliminary experiments, several main factors influencing DL and EE, including stirring time, MLT concentration and MLT mass ratio to PS were investigated. Stirring time, MLT concentration, and MLT to PS mass ratio was analyzed at 3–360 min, 20–120 mg/mL, and 1:1–1:5 to obtain the optimal conditions, respectively. The single factor method was used to investigate the effects of main factors on DL and EE. In this method, only a factor was changed while the other factors remained constant. When the stirring time was the factor to be tested, the other factors are fixed as follows: MLT concentration of 100 mg/mL, MLT to PS mass ratio of 1:3. The same method as described was used to investigate the other factors. Finally, the optimum condition was obtained based on the DL and EE. The experiment was conducted in triplicate. DL and EE were calculated using the following formulas:

with the advancement of age (Pandi-Perumal et al., 2013). Therefore, exogenous MLT may be useful, considering the natural decline of MLT and its potential therapeutic effects. However, MLT is poorly absorbed due to its low and variable bioavailability, short biological half-life, and extensive first-pass metabolism due to poor aqueous solubility by the human body (Hartter, Grozinger, Weigmann, Roschke, & Hiemke, 2000; Kikwai, Kanikkannan, Babu, & Singh, 2002; Topal, Altindal, & Gumusderelioglu, 2015; Vlachou, Eikosipentaki, & Xenogiorgis, 2006), severely limiting its clinical application and therapeutic efficiency. Changing the solubility of MLT can enhance its ability to dissociate from the lipid membrane into the water-based liquid, thus improving its absorption capacity. Nanotechnology-based drug delivery systems have been developed to improve the solubility, release profile, and bioavailability of MLT. Various biodegradable polymers, such as polyvinylpyrrolidone (Thakral, Wolf, Beilman, & Suryanarayanan, 2017), polylactic acid or polylactide (PLA) (Carbone et al., 2016; Pandey, Haldar, Vishwas, & Maiti, 2015), poly(lactic-co-glycolic acid) (Altindal & Gumusderelioglu, 2016), poly(ethylene glycol) (Chen et al., 2017), and cyclodextrins (Vlachou et al., 2017), have been extensively used as polymeric nanoparticles to deliver MLT for increased bioavailability and sustained release. Nanocapsule-containing MLT that is prepared by interfacial deposition (Schaffazick, Pohlmann, Mezzalira, & Guterres, 2006), MLT-loaded zein nanoparticles using supercritical CO2 antisolvent (Li & Zhao, 2017), MLT-cocrystal that is generated through melt crystallization (Yan, Chen, & Lu, 2015), and MLT-loaded silica that is coated with hydroxypropyl methylcellulose phthalate (Li et al., 2017) have also been applied to improve the dissolution rate of MLT. MLT nanoparticles have been successfully prepared using these methods, but these nanoformulation strategies may introduce problems of low drug loading (DL), encapsulation efficiency (EE), release rate. These techniques also require large equipment investment, high-energy input, low productivity, high cost and so on. Hence, discovering drug delivery systems that exhibit high water solubility, bioavailability, and stability is necessary. PS is obtained by enzymatic treatment of starch with low cost and high safety, it is suitable for oral MLT delivery systems. First, MLT was dissolved in acetone solution and then adsorbed into the PS to prepare MLT-loaded PS (MPS). The different experimental parameters on DL and EE were investigated. MLT-loaded PS was obtained under optimum conditions. The solid-state properties of the loaded MPS samples were characterized by scanning electron microscopy and energy dispersive spectrometer (SEM/EDS), Fourier-transform infrared (FTIR) spectroscopy, x-ray diffraction (XRD), thermal gravimetry (TG), and differential scanning calorimetry (DSC) to analyze the dispersal state of the loaded MLT compared with pure MLT. Additionally, the in vitro release rate, bioavailability in rats, and cellular antioxidant activity (CAA) were also analyzed.

DL (%) = (Mtotal−VSUP×CSUP)/WMPS × 100% EE (%) = (Mtotal−VSUP×CSUP)/Mtotal × 100% where Mtotal is the initial amount of MLT (mg), VSUP is the volume of the supernatant (mL), CSUP is the concentration of MLT in the supernatant (mg/mL) and WMPS is the total weight of precipitate (mg). MLT concentration was determined via HPLC. The chromatographic column is a C18 reverse-phase column (Diamonsil, 5 μm, 250 mm × 4.6 mm, Dikma Technologies). The mobile phase is a 50:50:0.2 (v/v/v) mixture of methanol, water and glacial acetic acid. The column temperature is indoor temperature, the flow rate, detection wavelength, injection volume of samples and standards are 1.0 mL/min, 224 nm and 10 μL, respectively. 2.3. Physicochemical characterization of melatonin-loaded porous starch 2.3.1. Morphology SEM/EDS (SEM, Quanta 200, FEI; the Netherlands) was used to observe the morphology and scan the elementary composition of the samples. Each sample was sputter coated with gold for 4 min before observation. All specimens were examined at an accelerating voltage of 10 kV. The total ray counting rate range, dead time, and count time were 2000–3000 cps, < 30%, and 100 s, respectively. 2.3.2. Brunauer–Emmett–Teller surface area Nitrogen adsorption/desorption experiments were examined using an ASAP2020 automated adsorption apparatus (Micrometric, Ltd, USA). All samples were degassed under a vacuum at 45 °C at least 3 h prior to measurement. The specific surface areas of starch were calculated according to the BET method.

2. Materials and methods 2.3.3. Fourier–transform infrared spectroscopy, x-ray diffraction, differential scanning calorimetry and thermo gravimetric analysis FTIR spectroscopy of samples were performed on an FTIR spectrophotometer (Shimadzu Corporation, Japan). Approximately 2 mg of samples and 200 mg of KBr were mixed to prepare a pellet. The blended KBr pellet was analyzed through spectrometry at 500–4000 cm−1 wave numbers and 2 cm−1 resolution. The x–ray diffractograms for MLT, PS, and MPS and physical mixture of MLT and PS (melatonin of 12.30% according to the drug loading of MPS detected by HPLC) were obtained using an x–ray diffractometer (Philips, Xper t-Pro; The Netherlands) with nickel and a filtered radiation of 35 kV and 40 mA. All samples were placed in a sample holder and with a scan range between 5° and 60° (2θ) at a rate of 5 deg/min. The samples were subjected to TG using a Thermo Gravimetrical Analyzer (Diamond TG/DTA Perkin–Elmer, Waltham, MA, USA). The samples were weighed in open aluminum pans and heated at 40 °C–500 °C under nitrogen purge at a heating rate of 10 °C/min.

2.1. Materials MLT (purity 98%) was purchased from Nanjing Zelang Medical Technological Co., Ltd. (Jiangsu, PR China). PS (corn starch treated with α-amylase and glucoamylase in weak acid) was obtained from Liaoning Lida Bio-technology Co., Ltd. Acetone was acquired from J&K Scientific Ltd. (Beijing, China). Circadin® is commercially available and was purchased from a local drug store. Deionized water was collected using a Hitech-K flow water purification system (Hitech Instruments Co., Ltd., Shanghai, China). 2.2. Preparation of melatonin-loaded porous starch MLT was loaded with PS using the following method. A certain amount of PS was added into a certain volume of MLT–acetone solution under stirring at room temperature. After a period of time, the 126

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as follows:

DSC (TA Instruments, Model DSC 204 and X-DSC7000) was conducted to analyze the thermal behavior of the samples. Approximately 5 mg of each sample was heated at 40 °C – 200 °C at a temperature heating rate of 10 °C/min under dry nitrogen atmosphere.

CAA unit = 100−(∫ SA / ∫ CA) × 100 where ∫ SA is the integrated area under the sample fluorescence versus time curve, and ∫ CA is the integrated area from the control curve. The Y value was defined here as the calculation according to the formula:

2.4. In vitro release and cellular antioxidant activity study 2.4.1. Saturation solubility measurement The saturation solubility of raw MLT and MPS was detected in a simulated gastric fluid (SGF) in HCl/NaCl buffer solution (pH 1.2) and simulated intestinal fluid (SIF) in KH2PO4/NaOH buffer (pH 7.4, containing pancreatic amylase of 1% and maltase of 1%), respectively. In brief, an excess amount of each sample was placed into a 5 mL dissolution medium in a capped vial and maintained at a temperature of 37 ± 0.5 °C with a paddle rotation speed of 100 rpm for 48 h. The suspension was centrifuged at 10,000 rpm for 10 min. The supernatant was diluted with sufficient methanol and subjected to HPLC. The test conditions are shown in Section 2.2. The drug concentration in the supernatant indicated the saturation solubility of MLT. The experiment was conducted in triplicate.

Y = CAA/ (100−CAA) The median effective concentration dose (EC50) was determined for the median effect plot of logY versus logC. 2.5. Bioavailability studies The bioavailability behaviors of MLT, commercially available MLT and MPS were investigated in rats after oral MLT and MPS administration. All animal studies were conducted according to NIH-approved protocols and in compliance with the Guide for the Care and Use of Laboratory Animals. Twelve healthy Sprague Dawley rats (250 ± 5 g) were randomly divided into three groups (n = 6) and fasted overnight with free access to water before administration. Groups A, B and C were administered with 2 mg/kg (according to MLT calculation) MLT, MPS and commercially available MLT dispersed in water, respectively. Blood samples were withdrawn from the retro-orbital plexus at 5, 10, 15, 20, 25, 30, 60, 120, 240, and 360 min after oral administration and collected into the centrifuge tube containing heparin. The samples obtained were centrifuged at 3000 rpm for 10 min to immediately separate the plasma. The plasma was stored at −20 °C until analysis. For the analysis, 1.2 mL of ethyl acetate was added into 200 μL of plasma and vortex-mixed for 2 min. The mixture was centrifuged at 1000 rpm for 10 min, and the supernatants were dried at 40 °C. The residue was dissolved in methanol and subjected to HPLC analysis as described above.

2.4.2. Release rate test Raw MLT (298 mg) and MPS (containing 298 mg of pure MLT) were dispersed in 5 mL of SGF (containing pancreatic amylase of 1% and maltase of 1%, pH 1.2) dissolution media and placed in Slide–A–Lyzer® dialysis bags (molecular weight cutoff of 3500; Thermo Fisher Scientific, Waltham, MA, USA), respectively. The dissolution media used in this study were 200 mL SGF (containing pancreatic amylase of 1% and maltase of 1%, pH 1.2) for the first 2 h, and 200 mL of SIF (containing pancreatic amylase of 1% and maltase of 1%, pH 7.4) maintained at a controlled temperature of 37 ± 0.5 °C and 100 rpm paddle rotation speed. Dissolution medium (0.5 mL) was collected at time intervals of 0.08, 0.17, 0.33, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36 and 48 h, then simultaneously supplemented with the same volume of the dissolution medium. The sample obtained was centrifuged at 10,000 rpm for 10 min. The drug concentrations in the supernatant were measured with the HPLC system, and the detection conditions were the same as above. The release profiles of MLT were plotted according to the cumulative release rate (%, w/w). Each experiment was conducted in triplicate.

2.6. Stability of melatonin in porous starch MPS was stored at room temperature (25 ± 2 °C/60 ± 5% humidity) for one year. The content of melatonin in PS determined after storage of 0, 1, 3, 6 and 12 Months detected by HPLC method, and mass spectrum of MLT in PS was detected after storage of 12 Months for stability test. In detail, a certain amount of MPS was added into excess methanol solution ultrasonic 30 min at room temperature, the suspension was centrifuged for 10 min at 8000 rpm, repeat this process three times, and combine the supernatant. MLT concentration of the supernatant was determined via HPLC. Mass spectrum structure of the supernatant and raw MLT dissolved in methanol detected by mass spectrometry (QTRAP® 5500 LC/MS/MS, America). The experiment was conducted in triplicate.

2.4.3. Cellular antioxidant activity test The MLT and MPS were subjected to CAA test with human hepatocellular carcinoma HepG2 cells as previously described (Phiphatwatcharaded et al., 2017; Wolfe & Liu, 2007; Zhao et al., 2015). The cells were grown in DMEM supplemented with 10% FBS, 10 mM Hepes, 2 mM L-glutamine, 5 μg/mL insulin, 0.05 μg/mL hydrocortisone, 50 units/mL penicillin, 50 μg/mL streptomycin and 100 μg/ mL gentamycin at 37 °C and 5% CO2. HepG2 cells were seeded at a density of 6 × 104/well on a 96-well microplate in 100 μL of growth medium/well. After 24 h of seeding, the growth medium was removed, and the wells were washed with PBS. Quintuplicate wells were treated with 100 μL treatment medium containing solvent control (blank control), control test, or the test groups (MLT and MPS) at proportions of 80, 120, 180, 300 and 480 μM of pure MLT. DCFH-DA (25 μM) was dissolved in the treatment medium for 1 h. The treatment medium was removed and the cells were washed twice with PBS. ABAP (600 μM) was then applied to the cells of control and test groups in 100 μL oxidant treatment medium. Fluorescence intensity was measured via fluorescence microplate readers at 37 °C, emission at 538 nm, and excitation at 485 nm every 5 min for 1 h.

3. Results and discussion 3.1. Effects of experimental parameters on melatonin-loaded porous starch The MPS preparation involved several experiments to select the optimal conditions and obtain the formulation composition and production conditions. The effect of stirring time, MLT concentration and mass ratio of MLT to PS were studied. As shown in Fig. 1A, DL increased slightly at 3–30 min of stirring time and tended to stabilize after 30 min. The amount of MLT and PS was fixed, whereas the adsorption process was rapid and was quickly stabilized. Fig. 1B shows that a variation in MLT concentration from 20 mg/mL to 120 mg/mL significantly affected the DL. The DL clearly increased with increasing MLT concentration. The result indicated that the opportunities for PS to contact MLT increased as the amount of MLT was elevated at a constant PS amount and stirring time. The effect of the mass ratio of MLT to PS on the DL

2.4.4. Cellular antioxidant activity quantification After blank subtraction of the initial fluorescence values, the area under the curve of fluorescence versus time was integrated to calculate the CAA value at each concentration of MLT and MPS and control test, 127

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Fig. 1. Influence of (A) stirring time, (B) MLT concentration, (C) mass ratio of MLT to MPS to drug loading, and (D) mass ratio of MLT to MPS to encapsulation efficiency on the drug loading and encapsulation efficiency of PS.

in PS without nitrogen. The nitrogen content in MPS occurred between that of the MLT and PS.

and EE at different stirring times and MLT concentrations is shown in Fig. 1C and D. The increased mass ratio of MLT to PS resulted in the reduction of DL and the increase of EE. The results can indicate that a certain concentration of MLT leads to a specified amount MLT. Furthermore, the quality of PS increased with the mass ratio of MLT to PS. The more PS content, the more pores, and the more MLT can get into the pore. Hence, the result is reasonable according to the calculation of DL and EE as discussed in Section 2.2. Therefore, 30 min stirring time, 120 mg/mL MLT concentration, and 1:5 mass ratio of MLT to PS were selected as the final optimal conditions regarding the formulation composition and production conditions. The DL and EE of 12.30% and 70.17% were obtained under the optimum conditions, respectively. However, the DL was higher than that of the 6.9% obtained in a study on MLT-loaded Zein nanoparticles using supercritical CO2 antisolvent (Li & Zhao, 2017), and the EE was approximately twice higher than that in our previous research on MLTloaded hydroxypropyl methylcellulose phthalate-coated silica (Li et al., 2017).

3.2.2. Brunauer–Emmett–Teller surface area assay The specific surface areas of PS and MPS were calculated using the BET method, as shown in Table 2. The surface area of the PS and the MPS were 3.567 and 2.146 m2/g, respectively. The surface area of the MPS was slightly lower than that of the PS. Therefore, most of the pores in PS were occupied by MLT at the end of the adsorption process, according to the results obtained from SEM, EDS and BET assay. 3.2.3. Fourier–transform infrared spectroscopy, x-ray diffraction, differential scanning calorimetry and thermo gravimetric analysis The FTIR spectra, XRD patterns, TG, and DSC of PS, MLT, and MPS are indicated in Fig. 3. The FTIR spectra of PS (Fig. 3A. a) was observed at approximately 3400 (−OH), 2929 (−CH2), 1642 (CeOeO, in a carbohydrate group), 1454 and 1368 (bending absorption of CeH in −CH2), 1415 (bending absorption of −CH2 associated with stretching vibration CeOeO), 1082 (CeOH), and 1016 cm−1 (CeO) (Jiang et al., 2017; Zhang et al., 2012). The characteristic absorption peaks of MLT (Fig. 3A. b) was shown at 3300 (NeH), 3080–2880 (CeH), 1624 (NeH bending vibration), 1555 (CeN), and 1213 cm−1 (CeOeC) (Pandey et al., 2015; Topal et al., 2015). When the characteristic absorption of MPS (Fig. 3A. c) was compared with the corresponding peak of MLT and PS, we observed that the peaks at 3300 (NeH) and 3080–2880 cm−1 (CeH stretching vibration from MLT) was very weak compared with those at the MLT and the peak at 1642 cm−1 (CeOeO, a carbohydrate group from PS) combined with the peak at 1624 cm−1 (NeH bending vibration from MLT). Furthermore, the characteristic NeH bending vibration was observed while the MPS exhibits the characteristic absorption peaks of MLT at 1555 and 1213 cm−1. The results indicated that most MLT was adsorbed in the pores of PS, and a small amount floated on the surface of the PS. Fig. 3B shows the XRD patterns of MLT, PS, MPS and physical

3.2. Melatonin-loaded porous starch characterization 3.2.1. Morphology studies The SEM analysis results of PS and MPS are shown in Fig. 2. The PS exhibited irregular polygonal or spherosome shapes with a smooth surface and broad particle size distribution ranging between 10 and 20 μm. The pore size was approximately 1.0–2.0 μm in diameter in the PS, and some macropores collapsed or merged with each other (Fig. 2A). The MPS (Fig. 2B) exhibited a similar appearance to PS, showing no evidence of rupture or breakage during the preparation process. The SEM photographs of MPS after mechanical crushing and grinding are shown in Fig. 2C. Some of the mircopores were breached and clear in texture. The elementary composition of MLT, PS and the broken MPS were investigated using EDS (Table 1). The weight percentage of carbon, nitrogen, and oxygen in MLT was 72.33%, 9.42%, and 18.26% in MPS and 56.71%, 7.25%, and 36.03% 128

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Fig. 2. SEM images of (A) PS, (B) MPS, and (C) mechanically crushed and ground MPS.

mixture of MLT and PS. Some sharp peaks of MLT at the diffraction angles of 2θ 16.26°, 18.94°, 24.14°, 24.88° and 26.02° (Fig. 3B, b) suggesting that MLT exhibits a crystalline structure. The XRD pattern of PS (Fig. 3B, a) showed three broad peaks at the range of 13–16, 16–20, and 20–25, confirming that the PS was amorphous (Jiang et al., 2017; Zhang et al., 2012). On the other hand, the characteristic peaks of the MPS (Fig. 3B, c) were less than those of the MLT and similar to those of the PS, which indicated that the crystallinity of MLT was significantly decreased. The characteristic peaks of physical mixture of MLT and PS (Fig. 3B, d) are basically consistent with MLT. The difference between the highest and lowest values of MLT and that of physical mixture of MLT at 2θ 16.26° was taken as IMLT and Imixture, respectively. According to Fig. 3B, b and Fig. 3B, d, the value of Imixture / IMLT×12.30% is about 99.6%, which indicates that the crystalline state of MLT in the physical mixture is basically the same as that of the raw MLT. According to the above results, we can preliminarily infer that the MLT in MPS is

Table 1 Energy dispersive spectrometer (EDS). Factor

1 2 3

Sample

Element (W t%)

PS MPS MLT

C

N

O

59.65 56.71 72.33

– 7.25 9.42

40.35 36.03 18.26

Table 2 Surface area. Factor

Sample

Surface area (m2/g)

1 2

PS MPS

3.567 2.146

Fig. 3. (A) FTIR spectra of (a) PS, (b) MLT, and (c) MPS; (B) XRD patterns of (a) PS, (b) MLT, and (c) MPS, (d) physical mixture of MLT and PS; (C) TG curves of (a) PS, (b) MLT, and (c) MPS; (D) DSC results of (a) PS, (b) MLT, and (c) MPS. 129

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Fig. 4. The preparation process of MPS.

of drying. The amorphous or low crystallinity of MLT may be due to hydrogen bonding and steric hindrance of interaction MLT and PS which is in good agreement with literature(Ali et al., 2013). The amorphous MLT has more free energy than that of its crystallization, which is conducive to the improvement of solubility.

basically amorphous. The TGA curves of MLT, PS, and MPS are shown in Fig. 3C. The retention rate of MLT (Fig. 3C, b), PS (Fig. 3C, a), and MPS (Fig. 3C, c) were 0.86%, 14.03%, and 13.33% at 600 °C, respectively. The content of MLT in MPS was 12.78% according to the retention rate, which was approximately equal to the analysis of HPLC. The DSC result of PS (Fig. 3D, a) showed no melting point peaks (Zhang et al., 2012). In the MLT thermogram (Fig. 3D, b), a sharp endothermic transition at 118 °C (Li & Zhao, 2017; Pandey et al., 2015) was observed, which is the melting point of MLT. For the MPS thermogram (Fig. 3D, c), the endothermic peak occurred at approximately 116 °C, and the endothermic amount (8.94 J/g) sharply decreased compared with that of the MLT (132.38 J/g). The endothermic peak of MPS was attributed to MLT. Thus, the calculated theoretical endothermic amount of MPS was 69.95 J/g according to the DL of 12.78%, as analyzed by TG. However, the actual result (8.94 J/g) was significantly lower than the theoretical value. The endothermic peak of MPS was attributed to MLT. Therefore, the results of DSC and XRD confirmed that MLT in MPS was mostly amorphous. The specific preparation process of the MPS is shown in Fig. 4. As PS was added to MLT–acetone solution, the pores in the PS were filled with MLT–acetone solution, and the molecular state of MLT was absorbed into the pores. The pores in the PS are small, meanwhile, hydrogen bonding interaction of MLT and PS may be form. These induces the molecules of MLT in which were not enough space and energy to fully convert to crystalline state, so the amorphous or low crystallinity gradually formed as the gradual evaporation of acetone during the process

3.3. In vitro release and Cellular antioxidant activity evaluation The saturation solubility of the MLT and MPS at 37 °C was 1.78 and 2.79 mg/mL in SGF (HCl/NaCl buffer solution, pH 1.2) and 1.85 and 2.96 mg/mL in SIF (KH2PO4/NaOH buffer, pH 7.4, pancreatic amylase of 1% and maltase of 1%), respectively. The equilibrium solubility of MPS was significantly higher than that of MLT in both SGF and SIF. The release profiles of MLT and MPS are illustrated in Fig. 5 C. The MPS showed a more rapid dissolution rate with a much higher cumulative amount of dissolved MLT in SGF within two hours, the cumulative release rate of MLT and MPS were 14.18% and 33.91% in 2 h, respectively. Two hours later, the dissolution rate of MPS still higher than that of MLT in SIF (KH2PO4/NaOH buffer, pH 7.4, pancreatic amylase of 1% and maltase of 1%), it was basically balanced and the cumulative release rate of MLT and MPS were 39.69% and 85.16%, respectively at 12th hour. The cumulative release rate of MPS at 12 h was higher than that of MLT-entrapped PLA nanoparticles (about 60%), as previously described (Pandey et al., 2015). In MPS, MLT exists in an amorphous form, and the solubility of amorphous MLT is higher than that of crystalline MLT. Compared with the crystalline MLT, the amorphous MLT is easy to form molecular state in the process of 130

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Fig. 5. Peroxyl radical-induced oxidation of DCFH-DA to DCF in HepG2 cells, and oxidation inhibition by (A) MLT and (B) MPS; (C) Cumulative release of melatonin in simulated gastric fluid and simulated intestinal fluid; (D) Median effect plots for the inhibition of peroxyl radical-induced DCFH-DA oxidation by MLT and MPS.

release, MLT exists in a molecular state after it release from MPS, which can not be characterized by x-ray diffraction, but the x-ray pattern of MPS show that the MLT became amorphous almost, the physical mixture of MLT and PS showed a pattern that was basically consistent with raw MLT. In CAA, the MLT and MPS at concentrations of 80, 120, 180, 300, and 480 μM according to MLT calculation generated peroxyl radicals from DCFH–DA-oxidized ABAP in a dose-dependent manner. The fluorescence intensity varied with time curves, as shown in Fig. 5A and B. The increase in fluorescence from DCFH–DA formation was inhibited by MLT in a dose-dependent manner. The fluorescein reaction of MLT (Fig. 5A) was different compared with that of MPS (Fig. 5B). The area under the curve of fluorescence versus time of MPS was less than that of MLT at the same concentration. The results can be explained that the MLT in the MPS exhibited a more rapid dissolution rate than raw MLT at the same concentration (Fig. 5C). The more dissolved is the state of MLT, the lower is the fluorescence intensity. We can calculate the free–radical-scavenging capacity (CAA units) according to the formula presented in Section 2.4.4. The Y value was defined according the CAA. The EC50 was determined for the median effect plot of logY versus logC (Fig. 5D). The EC50 is the concentration at which Y = 1 (CAA unit = 50). The EC50 value of MLT and MPS were 317.72 and 246.49 μM, according to the plot in Fig. 5 D. The results indicated that the amorphous MLT released from MPS was steady and rapid, and the penetration rate of the amorphous MLT was faster than that of the crystalline raw MLT. Thus, the MLT from MPS is efficient.

Fig. 6. Plasma drug concentration of commercially available MLT, MLT and MPS in rats.

The plasma concentration of MPS-treated rats was always higher than that of those treated with MLT and the market drug Circadin® after oral administration. The Cmax of commercially available MLT, MLT and MPS in rats were 120.11, 134.26 and 291.77 ng/mL, respectively. After oral administration, the plasma concentration of commercially available MLT and MLT reached a maximum concentration within 15 min, followed by a rapid fall. The plasma concentration of MPS reaching Cmax at 20 min maintained a significantly increased plasma drug concentration significantly for a prolonged period of time. These results may be due to the amorphous morphology of MLT and the twisty pores in the PS. Oral delivery of raw MLT presents a challenge because of its poor oral absorption. The main hurdle faced by raw MLT is its lack of solubility in the gastrointestinal tract. MLT is a small molecule with low water solubility and high permeability so that it is difficult to free from

3.4. Bioavailability study The plasma concentration–time curves of MLT and MPS following oral administration are shown in Fig. 6. 131

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and 174.2, respectively. The mass spectrogram of melatonin in the supernatant was identical with that of raw melatonin in Fig. 7B. It could be proved that MLT in the MPS were sufficiently stable when they are stored at room temperature for 12 months. 4. Conclusion The DL and EE of 12.30% and 70.17% of MPS were successfully prepared under optimum conditions, namely, 30 min stirring time, 120 mg/mL MLT concentration, and 1:5 mass ratio of MLT to PS. The physical and chemical properties of the MPS were investigated, and the SEM/EDS, BET and FTIR results revealed that MLT was mostly loaded in the pores of PS. The XRD, DSC and TG analysis for MPS showed that most of the MLT was transformed into an amorphous form in the pores of PS. The dissolving capability test results showed that the MLT solubility of MPS was obviously improved than that of raw MLT in artificial gastric juice and in artificial intestinal juice. The MPS showed higher MLT cumulative release rates compared with raw MLT in SGF and SIF. MPS exhibited a higher inhibition of peroxyl radicals in HepG2 cells by the lower EC50 than that of MLT. The bioavailability evaluation in rats displayed the Cmax of MLT and MPS of 134.26 and 291.77 ng/mL, which were reached at 15 and 20 min, respectively. The AUC0–360min of the group treated with formulation MPS was approximately 2.34-fold higher than that of raw MLT. Therefore, this study offers a new and simple approach to improve the solubility and bioavailability of MLT.

Fig. 7. Mass spectrogram of raw MLT and the supernatant from MPS that storage at room temperature for 12 months.

lipid membranes into aqueous liquids, resulting in low absorption rate. MLT is absorbed into the liver through the gastrointestinal tract, and the rich enzyme system in the liver has certain metabolic effects on it, which makes it suffer great losses before entering the systemic circulation. MLT-loaded PS can change MLT into an amorphous form, so the solubility of MLT is improved, the high solubility can increase the absorption of MLT, the pores of PS are twisty, it is possible that the release rate of MLT loaded by PS is slower than that of the amorphous type without carrier loading and the solubility of MLT in MPS is greater than that of crystalline MLT. MLT loaded with PS as carrier may improve its water solubility and make it continuously released from the pore of PS with a high concentration, the pores of PS are twisty may slower the release of MLT, thus reducing its metabolic rate in the liver and increasing its half-life. The process of MPS absorption in the gastrointestinal tract is shown in the Fig.S1 (see Fig. S1 in the supplementary material). The solubility of MLT in MPS is higher than that of raw MLT that results in a higher Cmax of MPS than raw MLT. Tmax of MPS greater than that of raw MLT were attributed to porosity of PS which resulted in MLT released from the carrier release gradually at a stable concentration in plasma compared with that of the crystalline raw MLT. The bioavailability is proportional to the AUC at the same dosage of oral administration. The findings were attributed to the amorphous MLT that was released from the carrier at a stable concentration and the elevated drug concentration in plasma compared with that of the crystalline raw MLT. The AUC0–360min of the group treated with formulation MPS was found to be 2.34-fold higher than that of the MLTtreated group. Although the AUC of MPS was slightly lower than that obtained in our previous study on MLT-loaded hydroxypropyl methylcellulose phthalate-coated silica (3.5 times higher than that of raw MLT) (Li et al., 2017), the safety of PS as a carrier was higher than that of silica. Furthermore, the preparation process of MPS was simpler than that of the MLT-loaded hydroxypropyl methylcellulose phthalatecoated silica. The significantly larger AUC0–360min and immediate plasma spike obtained from the MPS indicated that the MLT in the pores of PS facilitated the fast dissolution of the drug in the gastrointestinal fluid, which resulted in an enhanced oral bioavailability of the poorly soluble drug MLT.

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3.5. Stability study The content of MLT in PS determined after storage of 0, 1, 3, 6 and 12 Months were 12.30%, 12.31%, 12.29%, 12.31% and 12.28%, respectively. From the results, the content of melatonin in PS had no obvious change from 0 to 12 months. Mass spectrogram of raw MLT and the supernatant from MPS that storage at room temperature for 12 months were shown in Fig. 7. From Fig. 7A, the molecular ion peak of raw melatonin was [M+H] + (233.0), fragment ion (m/z) were 216.1 132

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