pharmaceutics

Article Highly Water-Soluble Solid Dispersions of Honokiol: Preparation, Solubility, and Studies and Anti-Tumor Activity Evaluation

Li Wang 1,2, Weiwei Wu 1,2, Lingling Wang 1,2, Lu Wang 1,2 and Xiuhua Zhao 1,2,*

1 College of Chemistry, Chemical Engineering and Resource Utilization, Northeast Forestry University, Harbin 150040, China; [email protected] (L.W.); [email protected] (W.W.); [email protected] (L.W.); [email protected] (L.W.) 2 Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin 150040, China * Correspondence: [email protected]; Tel.: +86-451-82191517; Fax: +86-451-82102082

 Received: 18 September 2019; Accepted: 24 October 2019; Published: 1 November 2019 

Abstract: Honokiol (HK), a well-tolerated natural product, has many multiple pharmacological activities. However, its poor water solubility and low bioavailability limit its clinical application and development. The aim of this research was to prepare the solid dispersion (SD) formulation of honokiol (HK) with poloxamer-188 (PLX) as the carrier, thereby improving its solubility and oral bioavailability. Firstly, by investigating the relationship between the addition amount of the PLX and the solubility of HK, and the effects of solid dispersions with different ratios of HK–PLX on the solubility of HK, we determined that the optimum ratio of PLX to HK was (1:4). Then, the HK–PLX (1:4) SD of HK was prepared using the solvent evaporation method. The morphology of the obtained HK–PLX (1:4) SD was different from that of free HK. The HK in the HK–PLX (1:4) SD existed in amorphous form and formed intermolecular hydrogen bonds with PLX. Additionally, the solubility values of the HK–PLX (1:4) SD were about 32.43 0.36 mg/mL and 34.41 0.38 mg/mL ± ± in artificial gastric juice (AGJ) and in artificial intestinal juice (AIJ), respectively. Compared with free HK, the release rate and the bioavailability was also substantially improved for HK in its SD form. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay indicated that the HK–PLX (1:4) SD showed higher inhibition of HepG2 cells than free HK. Taken together, the present study suggests that the HK–PLX (1:4) SD could become a new oral drug formulation with high bioavailability and could produce a better response for clinical applications of HK.

Keywords: honokiol; solid dispersion; solubility; bioavailability; antitumor activity

1. Introduction Today, in the process of drug development, low water solubility is still a difficult problem that needs to be solved for drug candidates. Many insoluble drugs are lipid-soluble drugs with low water solubility and high permeability [1]. So far, various methods have been explored to enhance the dissolution properties of these insoluble drugs, such as using salt formation [2], cyclodextrins [3], self-emulsifying drug delivery systems (SEDDS) [4], liposomes [5], micelles [6], co-crystals [7], micro- or nanoparticles [8], and solid dispersion [9]. Among them, solid dispersion is a relatively more sophisticated technology that overcome the limitations of insoluble drugs in FDA available products. The solid dispersion technology recently reported in the literature and used in approved products is also becoming more popular [1]. Solid dispersion formulations with hydrophilic excipients have many merits, such as enhanced wettability, higher porosity, and having an amorphous state [10].

Pharmaceutics 2019, 11, 573; doi:10.3390/pharmaceutics11110573 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2019, 11, 573 2 of 15

Pharmaceutics 2019, 11, 573 2 of 15 Generally, the formation of intramolecular hydrogen bonds in the drug molecules reduces the polarityGenerally, of the molecules,the formation thus of reducingintramolecular the solubility hydrogen in bonds water. in The the formationdrug molecules of intermolecular reduces the polarityhydrogen of bonds the molecules, increases thethus solubility reducing of the the solubility drugs, probably in water. because The formation the formation of intermolecular of hydrogen hydrogenbonds between bonds drug increases molecules the solubility and water of molecules the drugs, accelerates probably the because dissolution the formation of the drugs. of hydrogen Honokiol bonds(HK) is between a bioactive drug natural molecules compound and extracted water molecule from the stem,s accelerates bark, and the roots dissolution of of o ffithecinalis drugs.[11]. Honokiol (HK) is a bioactive natural compound extracted from the stem, bark, and roots of Magnolia It is a small biphenolic (Figure1) with the molecular formula C 18H18O2 [12]. HK exists in officinalis [11]. It is a small biphenolic lignan (Figure 1) with the molecular formula C18H18O2 [12]. HK nature as a white crystalline powder, and its melting point is about 85 ◦C[13]. Moreover, HK has many existsdiverse in pharmacological nature as a white and crystalline biological powder, properties, and including its melting anti-anxiety point is about effects, 85 anti-oxidant °C [13]. Moreover, actions, HKanti-inflammatory has many diverse effects, pharmacological and anti-cancer and effects biological [14–16 ].properties, HK has been including extensively anti-anxiety researched effects, and anti-oxidantdeveloped because actions, of itsanti-inflammatory numerous applications effects, andand good anti-cancer safety. However,effects [14–16]. the phenolic HK has hydroxyl been extensivelygroups of HK researched are prone toand forming developed intramolecular because hydrogenof its numerous bonds, which applications lead to decreased and good solubility safety. However,in water, thus the limitingphenolic its hydroxyl application groups for various of HK diseasesare prone [17 ,to18 ].forming If a carrier-containing intramolecular oxygenhydrogen or bonds,hydroxyl which group lead forms to decreased intermolecular solubility hydrogen in water, bonds thus with limiting HK, then its theapplication hydrogen for bonds various between diseases the [17,18].drugs and If a the carrier-containing carriers hinder the oxygen formation or hydr of intermolecularoxyl group forms interaction intermolecular between hydrogen drug molecules, bonds withand asHK, such then the the solubility hydrogen of HK bonds is improved. between the drugs and the carriers hinder the formation of intermolecular interaction between drug molecules, and as such the solubility of HK is improved.

Figure 1. Chemical structure of honokiol. Figure 1. Chemical structure of honokiol. Hydrophilic poloxamer-188 (PLX) is a polyoxyethylene–polyoxypropylene copolymer. Due to its Hydrophilic poloxamer-188 (PLX) is a polyoxyethylene–polyoxypropylene copolymer. Due to low melting point, high drug loading, good hydrophilicity, and good safety, PLX is not only widely its low melting point, high drug loading, good hydrophilicity, and good safety, PLX is not only used in some traditional formulations, such as suppositories, tablets, and emulsions, but also can form widely used in some traditional formulations, such as suppositories, tablets, and emulsions, but also micelles to increase the solubility of insoluble drugs, increase the stability of drugs, and promote the can form micelles to increase the solubility of insoluble drugs, increase the stability of drugs, and absorption of drugs by the body. Additionally, it is a surfactant approved by the FDA [19]. Moreover, promote the absorption of drugs by the body. Additionally, it is a surfactant approved by the FDA PLX is also a commonly used carrier material for poorly water-soluble PLX is also a commonly used [19]. Moreover, PLX is also a commonly used carrier material for poorly water-soluble PLX is also a carrier material for poorly water-soluble drugs, which can ensure the high dispersibility of drugs commonly used carrier material for poorly water-soluble drugs, which can ensure the high in water and prevent drug aggregation [20,21]. Compared with polyvinylpyrrolidone (PVP) K30, dispersibility of drugs in water and prevent drug aggregation [20,21]. Compared with polyethylene glycol (PEG) 6000/4000, and Soluplus, the viscosity of PLX is lower, which means the polyvinylpyrrolidone (PVP) K30, polyethylene glycol (PEG) 6000/4000, and Soluplus, the viscosity solution is easily desiccated. Moreover, for some poorly water-soluble drugs, the solid dispersion of of PLX is lower, which means the solution is easily desiccated. Moreover, for some poorly PLX as a carrier has a higher solubility than other polymers, such as PEG 4000, PEG 6000, and PVP water-soluble drugs, the solid dispersion of PLX as a carrier has a higher solubility than other K30 [10,22]. Considering these properties, PLX can be recommended as a potential hydrophilic carrier polymers, such as PEG 4000, PEG 6000, and PVP K30 [10,22]. Considering these properties, PLX can for solid dispersion. be recommended as a potential hydrophilic carrier for solid dispersion. In addition, some technologies have been used to enhance the solubility and oral bioavailability In addition, some technologies have been used to enhance the solubility and oral bioavailability of free HK. For example, Xu and coworkers prepared an inclusion complex of HK with sulfobutyl of free HK. For example, Xu and coworkers prepared an inclusion complex of HK with sulfobutyl ether-β-cyclodextrin (SB-β-CD), and in vivo results showed that the AUC(0–t) and Cmax of the inclusion ether-β-cyclodextrin (SB-β-CD), and in vivo results showed that the AUC(0–t) and Cmax of the complex increased by approximately 158% and 123%, respectively, compared with free HK [14]. inclusion complex increased by approximately 158% and 123%, respectively, compared with free Han and coworkers prepared honokiol nanosuspensions using a solvent precipitation–ultrasonication HK [14]. Han and coworkers prepared honokiol nanosuspensions using a solvent method, and the nanosuspensions improved the oral bioavailability of honokiol in in vivo studies precipitation–ultrasonication method, and the nanosuspensions improved the oral bioavailability of in rats, showing a 3.94-fold Cmax increase and a 2.2-fold AUC (0–t) increase [15]. Godugu and honokiol in in vivo studies in rats, showing a 3.94-fold Cmax increase and a 2.2-fold AUC (0–t) coworkers prepared honokiol-loaded nanomicelles (HNK-NM) using amphiphilic polymer Vitamin E increase [15]. Godugu and coworkers prepared honokiol-loaded nanomicelles (HNK-NM) using Polyethylene Glycol Succinate. The nanomicellar formulations resulted in a significant increase in the amphiphilic polymer Vitamin E Polyethylene Glycol Succinate. The nanomicellar formulations oral bioavailability. Cmax increased by 4.06- and 3.60-fold and AUC by 6.26- and 5.83-fold in comparison resulted in a significant increase in the oral bioavailability. Cmax increased by 4.06- and 3.60-fold and AUC by 6.26- and 5.83-fold in comparison to 40 and 80 mg/kg oral application of free HK, respectively [23]. In addition, Zhang and coworkers prepared the pectin nanoparticles loaded with

Pharmaceutics 2019, 11, 573 3 of 15 to 40 and 80 mg/kg oral application of free HK, respectively [23]. In addition, Zhang and coworkers prepared the pectin nanoparticles loaded with honokiol and the in vitro release results indicated that the drug-loaded nanoparticles exhibited a higher drug release rate than free honokiol, showing effective sustained release [24]. Nevertheless, following these strategies, there is still a need for continuous exploration in terms of dissolution and solubility enhancement, and further studies are required for new formulations and to explore the development process. Solid dispersion (SD) technology is an effective and economical method to improve the solubility and release properties of drugs, and its preparation process is easy, simple, and reproducible. Hence, it is feasible to develop a new HK SD system with good water solubility, low toxicity, and which is suitable for clinical application and mass production. Moreover, the preparation of an oral SD formulation of HK using PLX as the carrier and the effects of this formulation on the physicochemical properties, solubility, and oral bioavailability of the HK have not been studied so far. In recent years, various technologies, such as melting method [25], spray-drying method [26], supercritical fluid method [27], hot melt extrusion technologies [28], solvent evaporation method [29,30], and freeze-drying method [31,32], have been extensively used in the preparation of SDs. However, some of these approaches have many disadvantages. For example, the melting method, spray-drying method, and hot melt extrusion method require high preparation temperature, which can cause degradation of the drugs. Further, the entire amount of drug used in the preparation cannot be completely melted into the carrier materials. In addition, the freeze-drying method and the supercritical fluid method require large equipment and high cost investment. Compared with other methods, the solvent evaporation approach has the advantages of low cost and convenient operation and regeneration, and has the potential to realize industrial production. In the present investigation, a HK–PLX solid dispersion was prepared using solvent evaporation method, and its physicochemical properties were characterized by SEM, FTIR, XRD, and DSC. Furthermore, the dissolution in vitro, the solubility and the bioavailability in vivo, and the MTT assay were also investigated and evaluated.

2. Materials and Methods

2.1. Materials Poloxamer-188 (PLX), hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG) 4000, PEG 6000, and polyvinylpyrrolidone (PVP) K30 were purchased from Aladdin (Shanghai, China). Honokiol (purity = 98.5%) was obtained from Baoji Haoxiang Biotechnology Co., Ltd (Baoji, China). Ethanol, acetonitrile, and were obtained from Shandong Yuwang Industrial Co., Ltd (Shandong, China).

2.2. Preparation of the HK SD Formulation Initially, the HK and the PLX were dissolved in ethanol for 5 min to reach the clear organic phase, and then the solution was stirred for 30 min under ambient conditions. After 30 min, the ethanol was evaporated using a rotary evaporator (SENCO, Shanghai, China) at 35 ◦C to obtain the SD formulation of HK with PLX (HK–PLX SD), and the obtained HK–PLX SD was then dried in a vacuum oven to remove the residual solvent. The removed ethanol could be recycled and reused. Finally, the SDs with different mass ratios of HK/PLX were obtained through the same process. The short names for as-synthesized HK SDs are as follows: HK–PLX 2:1, HK–PLX 1:1, HK–PLX 1:2, HK–PLX 1:3, HK–PLX 1:4, HK–PLX 1:5, HK–PLX 1:6, and HK–PLX 1:7. In addition, the SD formulation of the HK and other carriers (the mass ratio of HK to carriers was 1:5), including HPMC, PEG 4000, PEG 6000, and PVP K30, were also prepared via solvent evaporation method. Pharmaceutics 2019, 11, 573 4 of 15

2.3. Characterization of the HK SD Formulations

2.3.1. Scanning Electron Microscopy (SEM) Apparent morphology of the HK–PLX (1:4) SD and the free HK was examined by a SEM (FEI, Eindhoven, Netherlands). Dry samples were fixed on the sample tables with a conductive paste and made to be electrically conductive by sputter-coating with gold using the ion sputtering coating machine.

2.3.2. Fourier Transform Infrared Spectroscopy (FTIR) The structure of the HK–PLX (1:4) SD, the physical mixture (PM), and the free HK were examined 1 in the scanning range of 400–4000 cm− by FTIR spectrophotometer (SHIMADZU, Kyoto, Japan). Each sample (2 mg) was accurately weighed and mixed with KBr (200 mg) and then pellets were made to perform the measurements.

2.3.3. X-Ray Diffractometry (XRD) The crystal forms of the HK–PLX (1:4) SD, the PM, and the free HK were tested and evaluated by an X-ray diffractometer (Rigaku Corporation, Tokyo, Japan). Each sample was measured in the range of 5–60◦. Appropriate process parameters were: scanning speed 5◦/min; generator current 30 mA; generator tension (voltage) 40 kV.

2.3.4. Differential Scanning Calorimetry (DSC). Thermal characteristics of the HK–PLX (1:4) SD, the PM, and the free HK were detected by DSC (TA instruments, New Castle DE, USA). Samples of 4–6 mg were placed in a sealed aluminum pan and then heated from 40 ◦C to 400 ◦C in atmosphere at a heating rate of 10 ◦C/min.

2.3.5. Gas Chromatography (GC) Measurement The solvent residue of ethanol in the HK SD was inspected by an Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA). Firstly, 100 mg of HK SD powder was weighed and dissolved in (1 mL), and then centrifuged at 10,000 rpm for 10 min to obtain the supernatant. Then, 5 µL of the supernatant was injected into a GC system for analysis, with a split ratio of 20:1. The detection conditions were as follows: initial temperature was kept at 40 ◦C for 5 min, then the temperature was raised to 100 ◦C for 2 min at 10 ◦C/min, then 240 ◦C at 40 ◦C/min, and then maintained for 2 min. In addition, both the inlet and detector temperatures were set to 250 ◦C. The air flow rate was 400 mL/min, the nitrogen flow rate was 25 mL/min, and the hydrogen flow rate was 30 mL/min.

2.4. Solubility Tests of the HK SD Formulations

2.4.1. High Performance Liquid Chromatograph (HPLC) Method for HK HK was quantified by a Waters 1525-2489 high performance liquid chromatograph (Waters Corporation, Milford, MA, USA) consisting of a pump (Waters 1525 binary) and UV detector (Waters 2489 Tunable Absorbance Detector), which was equipped with a Diamonsil C18 reverse-phase column (4.6 250 mm, 5 µm, DIKMA, Beijing, China). A methanol (55%), acetonitrile (20%), and water (25%) × mixture was used as mobile phase. Other parameters of HPLC detection were as follows: A wavelength was 294 nm, the flow rate of the mobile phase was 1 mL/min, and the injection volume was 10 µL. A standard solution of HK was prepared as follows: HK (10 mg) was accurately weighed and dissolved in 10 mL methanol solution, and then the solution was diluted appropriately to obtain solutions of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.007813, and 0.003906 mg/mL. Using the concentration of HK standard solution as the abscissa and the absorbency as the y-coordinate, the linear chart was constructed, and the regression equation was Y = 11326308.72X 8679 (R2 = 0.9998). − The retention time of the HK was about 10–11 min. Pharmaceutics 2019, 11, 573 5 of 15

2.4.2. Solubility Tests Solid dispersions of HK with different polymers (the mass ratio of HK to polymer was fixed at 1:5) were also prepared by solvent evaporation method. Each solid dispersion (the content of HK was excessive) was weighed and placed in small beakers with 2 mL of deionized water, and then the beakers were sealed and placed in a water bath at 37 1.0 C for 4 h with constant stirring (100 rpm). ± ◦ After 4 h, the samples were taken out and centrifuged at 12,000 rpm for 10 min, and then filtered through a 0.22 µm membrane filter. The supernatant obtained was used for HPLC to determine HK concentration. The solubility of the SD of HK with PLX and the corresponding PMs in water were determined by HPLC detection. Firstly, the excess amounts of SD and PM samples (equivalent to 150 mg of free HK) were placed in 25 mL beakers, and then 10 mL distilled water was added to each beaker. Then, the beakers were sealed and placed in a 37 1.0 C water bath for 4 h with constant stirring (100 rpm). ± ◦ After 4 h, the samples were taken out and centrifuged at 12,000 rpm for 10 min, and then filtered through 0.22 µm membrane filter to remove the possible residual drugs. The final supernatant was assayed for HK by the HPLC system, as described above.

2.5. Dissolution Tests The equilibrium solubility studies of HK–PLX (1:4) SD, free HK, and the PM in artificial gastric juice (AGJ) (pH 1.2) were carried out and investigated with 0.4% Tween-80 and artificial intestinal juice (AIJ) (pH 6.8). Briefly, excess samples were weighed and placed in small vials containing 2 mL dissolution media. Then, each vial was placed in a 37 ◦C water bath and stirred for 48 h at 100 rpm. After 48 h, the samples were centrifuged at 12,000 rpm for 10 min and then filtered through 0.22 µm membrane filters. The final supernatant was diluted with methanol, and then the concentration of HK in the samples was determined by HPLC detection. The in vitro release of the HK–PLX (1:4) SD, the PM, and the free HK was analyzed in the two media mentioned above. Dissolution experiments were consistent with the sink conditions and were carried out at 37.0 0.5 C at a rotation speed of 100 rpm. Samples equivalent to 200 mg of HK were ± ◦ weighed and dispersed in a 250 mL beaker containing 200 mL of dissolution media. One milliliter of sample was taken out at predetermined times of 5, 10, 15, 20, 30, 45, 60, 90, 120, 240, 480, and 720 min, and the corresponding volume of dissolution medium was supplemented. The samples obtained were centrifuged at 12,000 rpm for 10 min and then filtered through 0.22 µm membrane filters to separate the excess drug. The final supernatant was diluted with methanol, and then the concentration of HK in the samples was determined by the HPLC detection.

2.6. In Vivo Pharmacokinetic Study

2.6.1. Animal Dosing Eighteen Sprague–Dawley rats (200–220 g) were maintained at 25 2 C and 50–60% relative ± ◦ humidity (RH) under natural light/dark conditions for one week before the experiment. In the experiment, the rats were randomized into three different groups, administered orally with free HK, the HK–PLX (1:4) SD, or the PM (at the dose of 50 mg/kg). The experimental protocols were approved by the Institutional Animal Care and Use Committee of Harbin Medical University (approval No. HMUIRB-2008-06) on June 23, 2006. Blood samples were collected from the retro-orbital plexus at different time intervals of 0.08, 0.17, 0.25, 0.33, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h. Samples were collected in 1.5 mL centrifuge tubes containing heparin, and were then centrifuged at 3000 rpm for 10 min. The upper plasma was taken and stored in a refrigerator at 40 C until analysis. − ◦ Pharmaceutics 2019, 11, 573 6 of 15

2.6.2. Determination of HK Content in Plasma Samples Plasma samples (200 µL) were placed in a clean 1.5 mL centrifugal tube. Then, 400 µL acetonitrile was added and vortexed for 30 s to precipitate plasma proteins, which was then centrifuged at 12,000 rpm for 10 min to separate the supernatant. Then, 50 mg sodium chloride was placed in a new centrifugal tube containing the supernatant. All the samples were placed in a 25 ◦C water bath for 10 min and then centrifuged at 12,000 rpm for 5 min. Finally, 10 µL of supernatant was analyzed for the amount of HK by HPLC method.

2.7. MTT Assay Inhibition rate of the HK–PLX (1:4) SD on HepG2 cell lines was evaluated by conventional methyl thiazole tetrazolium (MTT) cell survival assay. The assay was based on the reduction of MTT by the mitochon-drial dehydrogenase of viable cells into purple formazan crystals, which were dissolved in dimethyl sulfoxide (DMSO), and absorbance was then detected by the mark analyzer instrument. Firstly, HepG2 cell lines were seeded at a density of 1 104 cells per well in 96-well plates × and incubated for 24 h. Subsequently, the old medium was removed and the samples containing free HK and the HK–PLX (1:4) SD at different HK concentrations (HK: 900.0, 450.0, 225.0, 112.5, 56.25, 28.13, 14.06, and 7.03 µg/mL) were added to the plates (the samples containing free HK and the HK–PLX (1:4) SD with different HK concentration were dissolved with culture medium, and the obtained samples were filtered through a 0.45 µm pore diameter membrane, and then added in the plates). In addition, the corresponding PLX at different concentrations (PLX: 3600.0, 1800.0, 900.0, 450.0, 225.0, 112.52, 56.24, and 28.12 µg/mL) was also added to the plates. After 48 h incubation, the cells were washed and the fresh medium containing MTT was added into each plate, followed by incubation for another 4 h. Thereafter, the medium was removed and the formazan crystals were solubilized with DMSO (150 µL). After mild shaking for 10 min, the absorbance value (OD) was detected by the enzyme mark analyzer instrument (detection wavelength of 490 nm and reference wavelength of 630 nm) and compared with the blank control group.

2.8. Statistical Analysis The data were presented as mean standard deviation. The statistical analysis was analyzed ± by using one-sample t-test in the Origin software. A value of * p < 0.05 was accepted as statistical significance. For in vitro studies, 3–4 replicates were used each time. Three individual experiments were performed and results were averaged. For in vivo experiments, n = 6 was used.

3. Results and Discussion

3.1. Solubility Enhancement

3.1.1. Solubility of Different Solid Dispersions of HK In the experiments, we investigated the effects of different polymers, namely HPMC, PEG 4000, PEG 6000, PVP K30, and PLX, as carriers on the solubility of HK, and the measured solubility of the samples was shown in Figure2a. As seen in the figure, the solubility values of the free HK and the different solid dispersions of HK were about 0.014 0.002 mg/mL (free HK), 0.021 0.002 mg/mL ± ± (HK–HPMC 1:5), 0.101 0.003 mg/mL (HK–PEG 4000 1:5), 0.103 0.002 mg/mL (HK–PEG 6000 1:5), ± ± 0.023 0.002 mg/mL (HK–PVP K30 1:5), and 18.072 0.067 mg/mL (HK–PLX 1:5). As you can see from ± ± these results, the solubility of solid dispersion with PLX as the carrier was significantly higher than that of other solid dispersions and free HK, and was 1290.86 times that of the free HK. The solubility values of other solid dispersions were a little higher than that of free HK (1.5–7.35 times higher than that of free HK). Pharmaceutics 2019, 11, 573 7 of 15 Pharmaceutics 2019, 11, 573 7 of 15

FigureFigure 2. (a(a) )The The solubility solubility of of the the different different solid solid dispersions of honokiol (HK) (* pP << 0.05). ( (bb)) PhotographsPhotographs ofof di differentfferent solid solid dispersions dispersions in water. in water. (c) Drug (c solubility) Drug assolubility a function as of a poloxamer-188 function of poloxamer-188(PLX) concentration (PLX) in concentration HK–PLX physical in HK–PLX mixture. physical (d) The solubility mixture. of(d HK) The SD solubility with different of HK proportions SD with differentof PLX (* proportionsp < 0.05). of PLX (* P < 0.05). In addition, the appearance of different solid dispersions (their quality was identical) in water is In addition, the appearance of different solid dispersions (their quality was identical) in water is illustrated in Figure2b. In the figure, the HK–PLX 1:5 was clearer and more transparent than other illustrated in Figure 2b. In the figure, the HK–PLX 1:5 was clearer and more transparent than other solid dispersions. Therefore, in this paper, the PLX was chosen as the carrier. solid dispersions. Therefore, in this paper, the PLX was chosen as the carrier. 3.1.2. Solubility of PMs and SD of HK 3.1.2. Solubility of PMs and SD of HK The measured solubility of free HK, PMs of HK with PLX, and HK SD are shown in Figure2c,d. The measured solubility of free HK, PMs of HK with PLX, and HK SD are shown in Figure 2c As seen in Figure2d, free HK aqueous solubility was about 0.014 0.002 mg/mL after 4 h at 37 ◦C. and 2d. As seen in Figure 2d, free HK aqueous solubility was about± 0.014 ± 0.002 mg/mL4 after 4 h at In addition, the critical micelle concentration (CMC) of the PLX was about 4.8 10− M[33]. As might 37 °C. In addition, the critical micelle concentration (CMC) of the PLX was about× 4.8 × 10−4 M [33]. As be expected, the solubility of HK was linearly related to the concentration of surfactants above the CMC, might be expected, the solubility of HK was linearly related to the concentration of surfactants above which was in accordance with the micelle solubility equation ST = S0 + k (PT CMC)[34], as shown the CMC, which was in accordance with the micelle solubility equation ST = S−0 + k (PT-CMC) [34], as in Figure2c. When the content of PLX was 6% w/v, the solubility of HK was about 11.56 0.23 mg/mL, shown in Figure 2c. When the content of PLX was 6% w/v, the solubility of HK was about± 11.56 ± 0.23 which was about 825.7 times that of free HK. It was expected that the solubility of HK would mg/mL, which was about 825.7 times that of free HK. It was expected that the solubility of HK increase linearly with the further increase of PLX content, but that was not the case. When the would increase linearly with the further increase of PLX content, but that was not the case. When the content of PLX was 7.5%, 9%, and 10.5% w/v, the solubility of HK was about 12.4 0.15, 11.9 0.24, content of PLX was 7.5%, 9%, and 10.5% w/v, the solubility of HK was about 12.4 ±± 0.15, 11.9 ±± 0.24, and 12.9 0.12 mg/mL, respectively, and the magnitude of solubility enhancement was inconspicuous. and 12.9± ± 0.12 mg/mL, respectively, and the magnitude of solubility enhancement was Additionally, considering that the presence of large doses of surfactants in pharmaceutical products inconspicuous. Additionally, considering that the presence of large doses of surfactants in might cause unpredictable side effects in vivo, the amount of PLX did not need to be added too much. pharmaceutical products might cause unpredictable side effects in vivo, the amount of PLX did not Conversely, as expected, SD could enhance the solubility of HK more effectively. It was noteworthy need to be added too much. that the solubility of the SDs in Figure2d showed a significant increase; the di fference between the Conversely, as expected, SD could enhance the solubility of HK more effectively. It was different SDs was statistically significant (p < 0.05), and was obviously higher than that of the same noteworthy that the solubility of the SDs in Figure 2d showed a significant increase; the difference proportion of PMs. As could be seen, the drugs existed in the SD in amorphous form, which was between the different SDs was statistically significant (P < 0.05), and was obviously higher than that the critical factor for solubility enhancement. Additionally, when the mass ratio of HK to PLX was of the same proportion of PMs. As could be seen, the drugs existed in the SD in amorphous form, which was the critical factor for solubility enhancement. Additionally, when the mass ratio of HK to PLX was 1:4, the solubility of the SD was about 27.59 ± 0.33 mg/mL, while when the mass of PLX

Pharmaceutics 2019, 11, 573 8 of 15

Pharmaceutics 2019, 11, 573 8 of 15 1:4, the solubility of the SD was about 27.59 0.33 mg/mL, while when the mass of PLX increased increased further, the solubility of the corresponding± SD changed inconspicuously. Consequently, in further, the solubility of the corresponding SD changed inconspicuously. Consequently, in this paper, this paper, the HK–PLX (1:4) SD was selected and used for the following detections. the HK–PLX (1:4) SD was selected and used for the following detections.

3.2. Characterization of HK Formulations

3.2.1. SEM Results The free HKHK andand thethe HK–PLXHK–PLX (1:4)(1:4) SDSD were detecteddetected by SEM to observe their morphology, as shown inin FigureFigure3 .3. TheThe SEMSEM imageimage ofof freefree HKHK showedshowed irregularirregular blockblock particlesparticles ofof variousvarious sizessizes (Figure(Figure3 3a).a). However, However, the the HK–PLX HK–PLX (1:4) (1:4) SD SD showed showed block block particles particles with with porous porous structure structure (Figure (Figure3b), and3b), theand morphologythe morphology was diwasfferent different from from that ofthat free of HK. free Moreover, HK. Moreover, it could it could be seen be from seen the from figure the thatfigure no that HK no crystals HK crystals were observedwere observed in the in solid the solid dispersion, dispersion, which which suggested suggested that that the the HK HK might might be completelybe completely dispersed dispersed in thein the carrier carrier material. material.

Figure 3. Scanning electron microscopy (SEM) images of the samples: (a) SEM image of free HK; (Figureb) SEM 3. image Scanning of the electron HK–PLX microscopy (1:4) SD. (SEM) images of the samples: (a) SEM image of free HK; (b) SEM image of the HK–PLX (1:4) SD. 3.2.2. Physicochemical Properties Characterization 3.2.2.The Physicochemical possible interaction Properties between Characterization HK and PLX was determined by FTIR. The FTIR spectra of free 1 HK, theThe HK–PLX possible (1:4)interaction SD, PM between of HK with HK PLX, and andPLX PLX was from determined 4000 to 400 by cmFTIR.− are The shown FTIR inspectra Figure of4. 1 1 Thefree freeHK, HK the (curveHK–PLX a) exhibited (1:4) SD, characteristicPM of HK with peaks PLX, at and 3304 PLX cm− from(–OH 4000 vibration), to 400 cm 1638−1 are cm −shown(alkene in 1 1 1 CFigure=C vibration), 4. The free 1498 HK cm (curve− (C= Ca) aromatic exhibited stretching), characteristic 1217 peaks and 908 at cm 3304− (C–O), cm−1 (–OH and 989 vibration), and 825 cm1638− 1 (C–C).cm−1 (alkene The FTIR C=C spectra vibration), of PLX 1498 (curve cm d)−1 (C=C were characterizedaromatic stretching), by principal 1217 absorption and 908 cm peaks−1 (C–O), at 2885 and cm 989− 1 1 (C–Hand 825 stretch cm−1 aliphatic), (C–C). The 1338 FTIR cm spectra− (in-plane of PLX O–H (curve bend), d) and were 1106 characterized cm− (C–O stretching).by principal As absorption for PM of HKpeaks with at PLX2885 (curvecm−1 (C–H c), the stretch spectrum aliphatic), basically 1338 consisted cm−1 (in-plane of the overlapping O–H bend), peaks and 1106 of HK cm and−1 (C–O PLX. 1 Somestretching). characteristic As for absorption PM of HK peaks with of HKPLX at 3303,(curve 1638, c), 1497,the spectrum 906, and 825basically cm− were consisted easy to findof the in PM,overlapping suggesting peaks there of wasHK noand interaction PLX. Some between characteristic HK and absorption PLX. Meanwhile, peaks of these HK characteristicat 3303, 1638, peaks1497, of906, HK and almost 825 cm disappear−1 were easy in the to spectrum find in PM, of SD suggesting (curve b), there which was could no interaction be attributable between to interaction HK and betweenPLX. Meanwhile, HK and PLX.these Moreover,characteristic considering peaks of theHK chemicalalmost disappear structures in of the PLX spectrum and HK, of theSD hydrogen(curve b), bondswhich maycould be formedbe attributable between to PLX interaction and HK during between the formulationHK and PLX. of the Moreover, HK–PLX (1:4)considering SD, and thisthe interactionchemical structures played a vitalof PLX role and in solubilizing HK, the hydrogen the drug. bonds may be formed between PLX and HK during the formulation of the HK–PLX (1:4) SD, and this interaction played a vital role in solubilizing the drug.

Pharmaceutics 2019, 11, 573 9 of 15 PharmaceuticsPharmaceutics 20192019,, 1111,, 573 573 9 9of of 15 15

FigureFigure 4. 4.FTIRFTIR results results (wavenumbers: (wavenumbers: 400–4000 400–4000 cm cm−1) of1) ofthe the samples: samples: (a) ( afree) free HK; HK; (b ()b HK–PLX) HK–PLX (1:4) (1:4) Figure 4. FTIR results (wavenumbers: 400–4000 cm−−1) of the samples: (a) free HK; (b) HK–PLX (1:4) solidsolid dispersion dispersion (SD); (SD); (c ()c physical) physical mixture mixture (PM) (PM) of of HK HK and and PLX; PLX; (d ()d PLX.) PLX. solid dispersion (SD); (c) physical mixture (PM) of HK and PLX; (d) PLX. TheThe XRD XRD and and DSC DSC results results of of the the samples, samples, including including free free HK, HK, HK–PLX HK–PLX (1:4) (1:4) SD, SD, PM PM of of HK HK with with The XRD and DSC results of the samples, including free HK, HK–PLX (1:4) SD, PM of HK with PLX,PLX, and and PLX, PLX, are are shown shown in inFigure Figures 5 and5 and Figure6, respectively. 6, respectively. It can It be can seen be seen in Figure in Figure5a that 5a freethat HKfree PLX, and PLX, are shown in Figure 5 and Figure 6, respectively. It can be seen in Figure 5a that free HKhad had many many obvious obvious di ffdiffractionraction peaks peaks between between 5◦ and5° and 40 ◦40°,, and and its its DSC DSC curve curve (Figure (Figure6a) 6a) displayed displayed one HK had many obvious diffraction peaks between 5° and 40°, and its DSC curve (Figure 6a) displayed oneendothermic endothermic peak peak for for the the melting melting point point at at 86.7 86.7◦ °C,C, illustratingillustrating that the the drug drug existed existed in in nature nature as as a a one endothermic peak for the melting point at 86.7 °C, illustrating that the drug existed in nature as a crystal.crystal. Additionally, Additionally, the the diffraction diffraction patterns patterns of of PLX PLX (Figure 55d)d) hadhad two two distinct distinct di diffractionffraction peakspeaks at crystal. Additionally, the diffraction patterns of PLX (Figure 5d) had two distinct diffraction peaks at2 2θθ= =19.23 19.23°◦ and and 23.4 23.4°,◦, and and as as seen seen in in the the DSC DSC curves curves of of PLX PLX in in Figure Figure6d, 6d, the the melting melting point point peak peak of PLX of at 2θ = 19.23° and 23.4°, and as seen in the DSC curves of PLX in Figure 6d, the melting point peak of PLXwas was about about 53.8 53.8◦C, °C, indicating indicating that that PLX PLX existed existed as as a crystal. a crystal. The The XRD XRD spectrum spectrum of of the the PM PM (Figure (Figure5c) PLX was about 53.8 °C, indicating that PLX existed as a crystal. The XRD spectrum of the PM (Figure 5c)was was basically basically superimposed superimposed with with those those of HK of HK and and PLX, PLX, and someand some diffraction diffraction peaks peaks of HK of still HK existed still 5c) was basically superimposed with those of HK and PLX, and some diffraction peaks of HK still existed(considering (considering the PLX the dilution PLX dilution effect, theeffect, drug the peaks drug almost peaks disappeared almost disappeared due to the due high to percentagethe high existed (considering the PLX dilution effect, the drug peaks almost disappeared due to the high percentageof PLX). Moreover, of PLX). theMoreover, DSC curve the of DSC the PMcurve (Figure of 6thec) existedPM (Figure as two 6c) sharp existed endothermic as two peaksharp at percentage of PLX). Moreover, the DSC curve of the PM (Figure 6c) existed as two sharp endothermic53.8 and 86.9 peak◦C, at corresponding 53.8 and 86.9 °C, to the corresponding melting peaks to the of PLX melting and peaks HK, indicating of PLX and that HK, HK indicating existed in endothermic peak at 53.8 and 86.9 °C, corresponding to the melting peaks of PLX and HK, indicating thatPM HK in theexisted form in of PM crystal. in the The form endothermic of crystal. peakThe endothermic of HK was relatively peak of small,HK was which relatively was probably small, that HK existed in PM in the form of crystal. The endothermic peak of HK was relatively small, whichbecause was the probably PLX first because melted the at 53.8PLX◦ firstC during melted the at heating 53.8 °C process during ofthe DSC. heating Some process of the of HK DSC. might Some have which was probably because the PLX first melted at 53.8 °C during the heating process of DSC. Some ofdissolved the HK might in the PLX,have sodissolved the endothermic in the PLX, peak so of the HK endothermic in the PM was peak not obvious.of HK in As the seen PM in was Figure not5b, of the HK might have dissolved in the PLX, so the endothermic peak of HK in the PM was not obvious.the endothermic As seen in peak Figure of HK 5b, almostthe endothermic completely pe disappeared,ak of HK almost and thecompletely endothermic disappeared, peak shown and in the the obvious. As seen in Figure 5b, the endothermic peak of HK almost completely disappeared, and the endothermicspectrum of peak HK–PLX shown (1:4) in SDthe wasspectrum similar of to HK–PLX that of PLX,(1:4) indicatingSD was similar thatHK to that might of PLX, exist inindicating the SD in endothermic peak shown in the spectrum of HK–PLX (1:4) SD was similar to that of PLX, indicating thatan HK amorphous might exist form, in whichthe SDwas in an also amorphous consistent fo withrm, which XRD analysis. was also consistent with XRD analysis. that HK might exist in the SD in an amorphous form, which was also consistent with XRD analysis.

FigureFigure 5. 5.XRDXRD results results (2 (2θ θ= =5–405–40 °) ◦of) ofthe the samples: samples: (a) ( afree) free HK; HK; (b ()b HK–PLX) HK–PLX (1:4) (1:4) SD; SD; (c ()c physical) physical Figure 5. XRD results (2θ = 5–40 °) of the samples: (a) free HK; (b) HK–PLX (1:4) SD; (c) physical mixturemixture of of HK HK and and PLX; PLX; (d ()d PLX.) PLX. mixture of HK and PLX; (d) PLX.

Pharmaceutics 2019, 11, 573 10 of 15 Pharmaceutics 2019, 11, 573 10 of 15

Pharmaceutics 2019, 11, 573 10 of 15

FigureFigure 6. 6.DSCDSC results results (temperature (temperature range: range: 30–250 30–250 °C)◦C) of the samples: ((aa)) freefree HK;HK; ( b(b)) HK–PLX HK–PLX (1:4) (1:4) SD; SD;(c )(c physical) physical mixture mixture of of HK HK and and PLX; PLX; (d )(d PLX.) PLX.

3.2.3. ResidualIn addition, Solvent the Determination crystal structures of the SDs of HK prepared in different batches were also investigated, as shown in the supplementary materials (Figure S1). The results obtained showed In this work, the ethanol with low toxicity was applied to prepare the HK–PLX (1:4) SD. Figure that theFigure HK 6. could DSC results maintain (temperature its amorphous range: 30–250 form in°C) SD of forthe samples: a long time, (a) free which HK; ( illustratedb) HK–PLX that(1:4) the 7a,b display the results of the determination of residual ethanol solvents in the HK–PLX (1:4) SD by HK-PLX(1:4)-SDSD; (c) physical had mixture goodstability. of HK and PLX; (d) PLX. GC detection. In addition, a linear regression equation of peak area (Y) versus ethanol concentration (x3.2.3.) 3.2.3.was ResidualobtainedResidual SolventasSolvent Y = 1980.6 Determination Determination x + 221.13 (R2 = 0.999) over the range of 0.015625–1 mg/mL. According to the regression equation, the residual ethanol content in the HK–PLX (1:4) SD was 1733 ppm, In this work, the ethanol with low toxicity was applied to prepare the HK–PLX (1:4) SD. Figure7a,b which metIn this the work, minimum the ethanol standard with low(5000 toxicity ppm) was requ appliedired by to preparethe International the HK–PLX Conference (1:4) SD. Figureon display the results of the determination of residual ethanol solvents in the HK–PLX (1:4) SD by GC Harmonization7a,b display the(ICH) results and ofwas the suitable determination for pharmaceutical of residual ethanoluse. solvents in the HK–PLX (1:4) SD by detection.GC detection. In addition, In addition, a linear a linear regression regression equation equation of peak of peak area area (Y) versus(Y) versus ethanol ethanol concentration concentration (x) 2 was(x) was obtained obtained as Y as= 1980.6Y = 1980.6 x + 221.13x + 221.13 (R (R= 0.999)2 = 0.999) over over the the range range of 0.015625–1of 0.015625–1 mg /mg/mL.mL. According According to theto regressionthe regression equation, equation, the residual the residual ethanol ethanol content content in the HK–PLX in the HK–PLX (1:4) SD was (1:4) 1733 SD ppm, was which1733 ppm, met thewhich minimum met the standard minimum (5000 ppm)standard required (5000 by ppm) the International required by Conference the International on Harmonization Conference (ICH) on andHarmonization was suitable (ICH) for pharmaceutical and was suitable use. for pharmaceutical use.

Figure 7. (a) Gas chromatograms of ethanol standard solution; (b) The gas phase of the 100 mg/mL acetone solution of HK–PLX (1:4) SD.

3.3. Dissolution Study TheFigure drug 7. (solubilitya) Gas chromatograms and dissolution of ethanol are standarddisplaye solution;d in Figure (b) The 8. gasAs phaseseen ofin the Figure 100 mg 8A,/mL the Figure 7. (a) Gas chromatograms of ethanol standard solution; (b) The gas phase of the 100 mg/mL equilibriumacetone solubility solution of values HK–PLX of (1:4)the SD.free HK, the PM of HK, and PLX in AGJ and AIJ were about acetone solution of HK–PLX (1:4) SD. 0.063.3. ± Dissolution0.022, 12.14 Study ± 0.54 mg/mL, 0.04 ± 0.015, and 12.87 ± 0.45 mg/mL, respectively. This illustrates that3.3. the Dissolution PLX was Studybeneficial in increasing the solubility of HK, which might be because PLX, as a surfactant,The drugcould solubility increase and the dissolution solubility areof the displayed drugs inby Figure forming8. As micelles. seen in FigureHowever,8A, the the equilibrium HK–PLX solubilityThe valuesdrug solubility of the free and HK, dissolution the PM of are HK, displaye and PLXd inin AGJFigure and 8. AIJ As were seen about in Figure 0.06 8A,0.022, the (1:4) SD values assessed by the HPLC detection of about 32.43 ± 0.36 mg/mL and 34.41 ± 0.38 mg/mL± 12.14equilibrium0.54 mgsolubility/mL, 0.04 values0.015, of the and free 12.87 HK, th0.45e PM mg of/mL, HK, respectively. and PLX in AGJ This and illustrates AIJ were that about the in AGJ and± AIJ, respectively,± were obviously higher± than the others, and the difference between the groupsPLX0.06 was ±was 0.022, beneficial statistically 12.14 ± in 0.54 increasingsignificant mg/mL, the (0.04P < solubility 0.05),± 0.015, indicating ofand HK, 12.87 which that ± 0.45 the might mg/mL,HK be existed because respectively. as PLX,an amorphism as This a surfactant, illustrates in HK–PLXcouldthat the increase (1:4) PLX SD was the and solubility beneficial formed of intermolecularin the increasing drugs by the forminghydr soogenlubility micelles. bonds of HK, with However, which PLX. themightSo, HK–PLXthe be HK–PLX because (1:4) SD(1:4) PLX, values SD as a assessedsurfactant, by could the HPLC increase detection the solubility of about of 32.43 the drugs0.36 mgby /formingmL and 34.41micelles.0.38 However, mg/mL the in AGJHK–PLX and had better dissolution and bioavailability. ± ± AIJ,(1:4) respectively, SD values assessed were obviously by the HPLC higher detection than the of others, aboutand 32.43 the ± 0.36 difference mg/mL between and 34.41 the ± groups 0.38 mg/mL was in AGJ and AIJ, respectively, were obviously higher than the others, and the difference between the groups was statistically significant (P < 0.05), indicating that the HK existed as an amorphism in HK–PLX (1:4) SD and formed intermolecular hydrogen bonds with PLX. So, the HK–PLX (1:4) SD had better dissolution and bioavailability.

Pharmaceutics 2019, 11, 573 11 of 15 statistically significant (p < 0.05), indicating that the HK existed as an amorphism in HK–PLX (1:4) SD and formed intermolecular hydrogen bonds with PLX. So, the HK–PLX (1:4) SD had better dissolution and bioavailability.

Figure 8. Equilibrium solubility (* p < 0.05) (A) and the release profiles in artificial gastric juice (B) and

artificial intestinal juice (C) for free HK, the physical mixture, and the HK–PLX (1:4) SD.

Furthermore, the SD formulation of HK significantly improved drug dissolution compared to the drug powder. The release profiles of HK from the SD formulation compared to free HK are shown in Figure8B,C. It was found that the release percentage of HK was extremely low in AGJ (pH 1.2) and AIJ (pH 6.8) and the maximal percentages of the drug released at 12 h were 3.57% 0.36% and ± 5.48% 0.25%, respectively. The PM of HK and PLX was dissolved 88.96% 0.28% and 82.41% 0.43% ± ± ± in AGJ and AIJ at 12 h, and the dissolution rate of the PM was faster than that of free HK, indicating that the PLX could increase the dissolution rate of HK by forming micelles. The HK–PLX (1:4) SD exhibited much better dissolution behavior than free HK, and the percentages of the HK–PLX (1:4) SD release at 5 min were about 33.67% 0.19% in the AGJ and 46.18% 0.26% in the AIJ. Its release ± ± percentage reached a maximum of 100% at 45 min in the AGJ and at 30 min in the AIJ. As can be seen from the results, the HK–PLX (1:4) SD was obviously faster than both the PM and the free HK in AGJ and AIJ. The increased dissolution rate of the HK–PLX (1:4) SD was mainly attributed to the fact that the HK existed in the SD in an amorphous state and formed intermolecular hydrogen bonds with PLX. Therefore, the HK–PLX (1:4) SD preparation could be rapidly absorbed after oral administration of HK formulations and produced a better response for its clinical application.

1

Pharmaceutics 2019, 11, 573 12 of 15

3.4. Pharmacokinetic Behavior of HK Formulations

PharmaceuticsPlasma 2019 concentration–time, 11, 573 profiles and corresponding pharmacokinetic parameters of HK,12 PM of 15 of HK with PLX, and the HK–PLX (1:4) SD in rats are displayed in Figure9 and Table1, respectively. HK with content PLX in showed the plasma a slightly was determined higher plasma by the co HPLCncentration method. than As the shown free in HK, Figure while9, the the PM HK–PLX of HK (1:4)with SD PLX displayed showed asignificantly slightly higher higher plasma plasma concentration concentrations than of the HK free compared HK, while to the the HK–PLX PM and (1:4)the freeSD displayedHK. The difference significantly between higher the plasma groups concentrations was statistically of HK significant compared (P to < the0.05). PM Additionally, and the free HK.the CThemax divaluesfference of the between free HK, the the groups PM, was and statisticallythe HK–PLX significant (1:4) SD (werep < 0.05). 159.02 Additionally, ± 5.65 ng/mL, the 443.81Cmax values± 9.14 ng/mL,of the free and HK, 1109.87 the PM,± 7.24 and ng/mL, the HK–PLX respectively. (1:4) SDThe were AUC 159.02 values 5.65of the ng free/mL, HK, 443.81 the PM,9.14 and ng/ mL,the ± ± HK–PLXand 1109.87 (1:4) 7.24SD were ng/mL, 580.45 respectively. ± 11.15 ng/mL·h, The AUC 848.34 values ± of12.24 the freeng/mL·h, HK, the and PM, 2558.22 and the ± 8.15 HK–PLX ng/mL·h, (1:4) ± respectively.SD were 580.45 The results11.15 ngillustrated/mL h, 848.34 that the12.24 AUC ngand/mL theh, C andmax values 2558.22 of the8.15 PM ng were/mL slightlyh, respectively. higher ± · ± · ± · thanThe resultsthose of illustrated the free HK, that while the AUC the HK–PLX and the C (1:4)max values SD gave of significantly the PM were higher slightly AUC higher and than Cmax those values of comparedthe free HK, to whilethe PM the and HK–PLX the free (1:4) HK. SD Moreover, gave significantly the value higher of C AUCmax for and theC maxHK–PLXvalues (1:4) compared SD was to aboutthe PM 6.98-fold and the greater free HK. than Moreover, that of free the valueHK, and of Cthemax AUC(0for the→ HK–PLXt) of the HK–PLX (1:4) SD was(1:4) about SD was 6.98-fold about 4.41-foldgreater than greater that than of free that HK, of andfree theHK. AUC(0 Furthermore,t) of the the HK–PLX half-life (1:4)(t1/2) of SD the was HK–PLX about 4.41-fold (1:4) SD greater(0.37 ± → 0.05than h) that was of shorter free HK. than Furthermore, that of the the PM half-life (0.51 ± (0.12t ) ofh) theand HK–PLX the free HK (1:4) (0.74 SD (0.37 ± 0.15 0.05h). The h) was Tmax shorter of the 1/2 ± HK–PLXthan that (1:4) of the SD PM (0.5 (0.51 ± 0.080.12 h) was h) andalso theshorter free HKthan (0.74 that of0.15 the h).PM The(0.75T max± 0.09of h) the and HK–PLX the free (1:4) HK SD (2 ± ± ±(0.5 0.15 0.08h). Thus, h) was we also concluded shorter than that that the of HK–PLX the PM (0.75 (1:4) SD0.09 had h) andgood the oral free bioavailability HK (2 0.15 h). in Thus, rats ± ± ± comparedwe concluded to the that PM the of HK–PLXHK with (1:4)PLX SDand had the goodfree HK. oral bioavailability in rats compared to the PM of HK with PLX and the free HK.

Figure 9. The bioavailability result of the free HK, the physical mixture and the HK–PLX (1:4) SD Data Figureare presented 9. The asbioavailability the mean standard result of deviationthe free HK, (n = th6).e *physicalp < 0.05 mixture vs. free HK.and the HK–PLX (1:4) SD Data are presented as the mean± ± standard deviation (n = 6). *P < 0.05 vs. free HK. Table1. Pharmacokinetic parameters for HK in rats after oral administration of free HK, physical mixture of HK with PLX, and HK–PLX (1:4) SD. Data are presented as mean standard deviation (n = 6). * Table 1. Pharmacokinetic parameters for HK in rats after oral administration± of free HK, physical p < 0.05 (vs. free HK). mixture of HK with PLX, and HK–PLX (1:4) SD. Data are presented as mean ± standard deviation (n = 6).Pharmacokinetic * P < 0.05 (vs. free HK). Physical Mixture of Free HK HK–PLX (1:4) SD Parameters HK and PLX Pharmacokinetic Parameters Free HK Physical Mixture of HK and PLX HK–PLX (1:4) SD C maxC max(ng (ng/mL)/mL) 159.02 159.02 ± 5.65 443.81 443.81 ±9.14 9.14* * 1109.87 1109.877.24 ± 7.24* * ± ± ± Tmax (h) 2 0.15 0.75 0.09 * 0.5 0.08 * T max (h) 2 ±± 0.15 0.75± ± 0.09* ± 0.5 ± 0.08* t1/2 (h) 0.74 0.15 0.51 0.12 0.37 0.05 * t1/2 (h) 0.74 ±± 0.15 0.51± ± 0.12 0.37± ± 0.05* MRT(0–t) (h) 3.93 1.25 6.94 2.22 * 5.70 0.76 * MRT(0–t) (h) 3.93 ±± 1.25 6.94± ± 2.22* 5.70± ± 0.76* MRT(0– ) (h) 5.11 2.13 12.15 3.15 * 6.44 1.15 MRT(0–∞ ∞) (h) 5.11 ±± 2.13 12.15± ± 3.15* 6.44± ± 1.15 AUC(0–t) (ng/mL h) 580.45 11.15 848.34 12.24 * 2558.22 8.15 * AUC(0–t) (ng/mL·h)· 580.45 ±± 11.15 848.34± ± 12.24* 2558.22± ± 8.15* AUC(0– ) (ng/mL h) 637.91 13.42 1004.11 15.38 * 2588.70 10.54 * AUC(0–∞∞) (ng/mL·h)· 637.91 ±± 13.42 1004.11± ± 15.38* 2588.70± ± 10.54* Maximum plasma concentration(Cmax); Area under the curve (AUC(0–t), AUC(0– )); Half time of life (t1/2); MaximumTime to plasma peak (Tmax); concentration(Cmax); Mean residence time (MRT(0–t), Area under MRT(0– the)). curve (AUC(0–t),∞ AUC(0–∞)); Half time of ∞ life (t1/2); Time to peak (Tmax); Mean residence time (MRT(0–t), MRT(0–∞)). Furthermore, it had been reported that the honokiol nanosuspensions were prepared by the precipitation–ultrasonicationFurthermore, it had been method,reported andthat thethe honokiolhonokiol nanosuspensions existed as an amorphous were prepared form by in the precipitation–ultrasonication method, and the honokiol existed as an amorphous form in the honokiol nanosuspensions [27]. The pharmacokinetic test results in rats showed that the honokiol nanosuspension had a higher AUC(0→t) value in rats, which was about 2.2 times that of HK–CMCS. However, in this study, the in vivo in rats demonstrated that the AUC(0→t) value of the HK–PLX (1:4) SD preparation was approximately 4.41-fold greater than that of free HK,

Pharmaceutics 2019, 11, 573 13 of 15 honokiol nanosuspensions [27]. The pharmacokinetic test results in rats showed that the honokiol nanosuspension had a higher AUC(0 t) value in rats, which was about 2.2 times that of HK–CMCS. → However, in this study, the in vivo pharmacokinetics in rats demonstrated that the AUC(0 t) value → of the HK–PLX (1:4) SD preparation was approximately 4.41-fold greater than that of free HK, which indicated that the HK–PLX (1:4) SD should be the potential oral delivery system for clinical applications and could be better absorbed by the body.

3.5. MTT Study In this study, the inhibitory effect of the HK–PLX (1:4) SD on HepG2 cell lines was studied and compared with free HK. Cell viabilities of HepG2 cells after 48 h incubation with different concentrations of the HK–PLX (1:4) SD, free HK, and PLX are shown in Figure 10. It was found that the PLX had little inhibitory effect on the growth of the cell. However, both the HK–PLX (1:4) SD and the free HK have certain inhibitory effects on HepG2 cell growth, and with the increase of drug concentration, the ability to inhibit HepG2 cell growth was significantly improved. When the concentration exceeded 56.25 µg/mL, the inhibitory effect of the free HK on cells did not change significantly, which might be due to the low solubility of free HK. The HK–PLX (1:4) SD showed more effective inhibition than free HK and the difference between the groups was statistically significant (p < 0.05), which could be explained by the increased cellular uptake of the HK SD formulation.

Figure 10. Inhibitory rate of the free HK, the HK–PLX (1:4) SD, and the corresponding PLX (* p < 0.05).

4. Conclusions The SD formulation of HK with PLX ((1:4)) was prepared by the solvent evaporation method, and characterized by SEM, FTIR, XRD, and DSC. The SEM analysis illustrated that the SD of HK exhibited different morphology than free HK, and no HK crystals were observed in the SD. The XRD 1 and DSC results illustrated that the HK existed in the SD in amorphous form. The FTIR spectra indicated that there might be intermolecular hydrogen bonds between HK and PLX. Moreover, the solubility and dissolution experiments in AGJ and AIJ clearly indicated that the HK–PLX (1:4) SD had the higher solubility, with values of 32.43 0.36 mg/mL and 34.41 0.38 mg/mL, respectively. ± ± The dissolution rate of the HK–PLX (1:4) SD was also apparently higher than free HK in AGJ and AIJ. Moreover, the Cmax value and the AUC(0 t) value of the HK–PLX (1:4) SD (1109.87 7.24 ng/mL → ± and 2558.22 8.15 ng/mL h, respectively) were about 6.98- and 4.41-fold higher than those of free HK ± · (159.02 5.65 ng/mL and 580.45 11.15 ng/mL h, respectively); hence, the HK–PLX (1:4) SD had better ± ± · bioavailability than free HK. MTT study showed that the HK–PLX (1:4) SD generated higher inhibition of HepG2 cells than free HK. In addition, the solvent residual of the HK–PLX (1:4) SD was suitable for pharmaceutical use. Thus, it can be concluded that the HK–PLX (1:4) SD prepared in this study significantly improved the absorption of HK in vivo, and has potential clinical application value as a new oral drug formulation. Pharmaceutics 2019, 11, 573 14 of 15

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/11/11/573/s1, Figure S1: XRD diagram of the SDs of the HK prepared at different time, (a) a week ago, and (b) 16 months ago. Author Contributions: Conceptualization, X.Z. and W.W.; methodology, X.Z.; software, L.W. (Lingling Wang); validation, X.Z., L.W. (Li Wang) and W.W.; formal analysis, L.W. (Li Wang), L.W. (Lu Wang) and W.W.; investigation, L.W. (Li Wang); resources, X.Z. and L.W. (Li Wang); writing—original draft preparation, L.W. (Li Wang); writing—review and editing, L.W. (Li Wang) and X.Z.; visualization, W.W.; project administration, X.Z.; funding acquisition, X.Z. Funding: This research was funded by the Special Fund for Forestry Scientific Research in the Public Interest (201504701) and Heilongjiang Touyan Innovation Team Program. Conflicts of Interest: The authors declare no conflict of interest.

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