pharmaceutics

Article
Manufacturing Different Types of Solid Dispersions
of BCS Class IV Polyphenol (Daidzein) by Spray
Drying: Formulation and Bioavailability
Gean Pier Panizzon 1 , Fernanda Giacomini Bueno 2 , Tânia Ueda-Nakamura 1 ,
Celso Vataru Nakamura 1 and Benedito Prado Dias Filho 1, *
1 Post-graduate Program in Pharmaceutical Sciences, Laboratory of Technological Innovation in the
Development of Drugs and Cosmetics, State University of Maringá, Maringá 87020-900, Paraná, Brazil;
gean_panizzon@yahoo.com.br (G.P.P.); tunakamura@uem.br (T.U.-N.); cvnakamura@uem.br (C.V.N.)
2 Medical and Pharmaceutical Sciences Center, Western Paraná State University, Cascavel 85819-110, Paraná,
Brazil; buenofgb@gmail.com
* Correspondence: bpdfilho@uem.br; Tel.: +55-044-3041-5049




Received: 16 August 2019; Accepted: 16 September 2019; Published: 25 September 2019


Abstract: Daidzein (DZ) is a polyphenolic compound belonging to Biopharmaceutical Classification

System class IV, which shows that it may have limited therapeutic effects due to its low solubility
and poor bioavailability. This study aimed to obtain high-purity DZ and prepare and characterize
different types of solid dispersions (SDs) in order to enhance aqueous solubility and bioavailability.
Excipients were investigated in order to manufacture different types of solid dispersions (SDs).
Second-generation solid dispersions (SG), third-generation solid dispersions (TG), and second- and
third-generation pH-modulated solid dispersions (SD and TG pHM-SD) were produced via spray
drying. The SDs were characterized and tested for in vitro DZ release and oral bioavailability. SDs
have shown increased aqueous solubility and in vitro release rate. Solid-state characterization showed
that DZ was in an amorphous state in most of the formulations. The enhanced aqueous solubility of
TG-pHM SD was reflected by an increase in oral bioavailability, which significantly increased the
maximum plasma concentration approximately 20-fold and decreased the time to reach the maximum
plasma concentration. The production of pHM SDs that contain DZ via spray drying is a simple
and effective approach for oral drug delivery, which has the potential to greatly reduce the dose and
enhance therapeutics effects.

Keywords: daidzein; solid dispersion; polyphenols; bioavailability enhancement; spray drying

1. Introduction
In recent years, there have been many studies on Daidzein (DZ), which is a soybean isoflavone
aglycone. Biological effects of DZ include the prevention of osteoporosis [1] and breast cancer [2].
It also has neuroprotective [3] and cardioprotective effect [4], in addition to reducing hyperglycemia [5].
Thus, numerous DZ-based dosage-form formulations and dietary supplements have been produced
worldwide, considering that most of them are polyphenols belonging to Biopharmaceutical
Classification System (BCS) class II or IV. DZ is a weak acid (pKa = 9.6 for the hydroxyl group
at position 40 and pKa = 7.50 at position 7) with logP 2.51 [6] and high crystallinity [7] (Figure 1).

Pharmaceutics 2019, 11, 492; doi:10.3390/pharmaceutics11100492 www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2019, 11, 492 2 of 17
Pharmaceutics 2019, 11, x FOR PEER REVIEW 2 of 17

Figure 1.
Figure Chemical structure
1. Chemical structure of
of Daidzein.
Daidzein.

Daidzein’s low
Daidzein’s low aqueous
aqueous solubility
solubility and
and permeability
permeability make make itit aa member
member of of BCS
BCS class
class IV
IV [8][8] and
and
limit its bioavailability and biological effects [9]. Drugs with these physicochemical
limit its bioavailability and biological effects [9]. Drugs with these physicochemical characteristics characteristics
have limited
have limitedapplications
applications in nutraceutical
in nutraceutical and dosage-form
and dosage-form formulations, and technological
formulations, strategies
and technological
are required to overcome oral delivery limitations. Solid dispersions (SDs)
strategies are required to overcome oral delivery limitations. Solid dispersions (SDs) are defined as are defined as dispersions
of a drug into
dispersions of aadrug
solid-state inert matrix.
into a solid-state inertThey haveThey
matrix. beenhave considered the most the
been considered successful strategy
most successful
for enabling drug solubility and bioavailability [10]. According to
strategy for enabling drug solubility and bioavailability [10]. According to their composition, SDs their composition, SDs cancanbe
classified as first generation, in which carriers and drugs are in a crystalline state
be classified as first generation, in which carriers and drugs are in a crystalline state [11]. This state is [11]. This state is more
thermodynamically
more thermodynamically stable than
stable amorphous
than amorphousSDs. Second-generation
SDs. Second-generation SDs (SG) employSDs (SG) amorphous
employ
polymers and the drug may be molecularly dissolved or dispersed.
amorphous polymers and the drug may be molecularly dissolved or dispersed. Third-generation Third-generation SDs (TG) have SDs
similar
(TG) havecharacteristics to SG, but atosurfactant
similar characteristics SG, but a is added toisthe
surfactant system
added to increase
to the system the drug dissolution
to increase the drug
and reduce and
dissolution problemsreduce ofproblems
precipitation and recrystallization
of precipitation [11].
and recrystallization [11].
Recently, agents that are able to generate
Recently, agents that are able to generate a modulated pH a modulated pH inin the
the microenvironment
microenvironment and and change
change
drug release
drug release from
fromsolidsolidoral
oraldosage
dosageformsforms have
have been
beenadded
added to SDs
to SDs[12].[12].
Since then,
Since pH-modulated
then, pH-modulated SDs
(pHM-SD) have been explored as an effective alternative to overcome
SDs (pHM-SD) have been explored as an effective alternative to overcome the solubility and the solubility and bioavailability
issues of weak-acid
bioavailability andofweak-base
issues weak-acid drugs
and[13]. Such pHM-SD
weak-base drugscan be broadly
[13]. Such pHM-SD employedcan because almost
be broadly
two-thirds because
employed of substances
almost with low solubility
two-thirds are weakwith
of substances acidslow or bases with are
solubility pH-dependent
weak acids solubility
or bases with [12].
The most suitable excipients for producing SDs are the ones that have
pH-dependent solubility [12]. The most suitable excipients for producing SDs are the ones that have better miscibility and drug
affinity.
better Consequently,
miscibility and drugsuchaffinity.
selection needs to be optimized
Consequently, such selection by screening
needs to procedures
be optimized [13].
by Food
screening and
pharmaceutical industries commonly use spray drying in various
procedures [13]. Food and pharmaceutical industries commonly use spray drying in various applications. Spray drying of poorly
water-solubleSpray
applications. drugsdrying
is mainly aimed
of poorly at generating
water-soluble amorphous
drugs is mainly materials
aimed atand is the technique
generating amorphous of
choice forand
materials particle
is thesize reduction
technique [10], which
of choice is suitable
for particle for the small-scale
size reduction [10], which manufacturing
is suitable formethods
the small- or
early development and also for the large-scale production of formulations
scale manufacturing methods or early development and also for the large-scale production of of SD dosage forms [14].
Some studiesofhave
formulations assessed
SD dosage formsthe [14].
enhancement
Some studies of DZhave solubility
assessedby theSDs [15]. Later,ofFeng
enhancement et.al [16]
DZ solubility
reported
by SDs [15].theLater,
enhancement
Feng et.alof[16]DZreported
bioavailability by the SDs of
the enhancement obtained with polyvinylpyrrolidone.
DZ bioavailability by the SDs obtained
Although these
with polyvinylpyrrolidone. studies have shown the feasibility of obtaining only SG with daidzein, none
of them evaluated
Although thesedifferent
studies have typesshown
of SDs. the Therefore,
feasibility ofinobtaining
this study, onlyweSG purified, produced,
with daidzein, noneand of
assessed four types of DZ SDs, including SG, TG, SG-pHM, and TG-pHM
them evaluated different types of SDs. Therefore, in this study, we purified, produced, and assessed SDs using the spray-drying
technique
four typesand of optimized excipients.SG,
DZ SDs, including We TG,
also assessed
SG-pHM,their andphysicochemical
TG-pHM SDs using characteristics through a
the spray-drying
scanning electron
technique microscopy
and optimized (SEM), differential
excipients. We also assessedscanning calorimetry
their (DSC), X-ray
physicochemical powder diffraction
characteristics through
(XRPD), and gel permeation chromatography (GPC).
a scanning electron microscopy (SEM), differential scanning calorimetry (DSC),We have also determined an in vitropowder
X-ray release
profile using a flow-through cell apparatus (USP apparatus IV).
diffraction (XRPD), and gel permeation chromatography (GPC). We have also determined an in Finally, the pharmacokinetic profile
vitro
of purified DZ and optimized SDs was compared in rats using a validated
release profile using a flow-through cell apparatus (USP apparatus IV). Finally, the pharmacokinetic high-performance liquid
chromatography–tandem
profile of purified DZ andmass spectrometry
optimized SDs was(HPLC-MS/MS)
compared in rats method.
using a validated high-performance
liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method.
2. Materials and Methods
2. Materials andstandard
Analytical Methodsof DZ, genistein and glycitein (IS; internal standard) were purchased from
Tecpar (Curitiba, Brazil; purity ≥98%). Soybean extract (~20% of DZ) was purchased from Galena®
Analytical standard of DZ, genistein and glycitein (IS; internal standard) were purchased from
(Campinas, Brazil). Polyvinylpyrrolidone K90 (PVP), benzalkonium chloride (BAK; Fluka® , ≥95%,
Tecpar (Curitiba, Brazil; purity ≥98%). Soybean extract (~20% of DZ) was purchased from Galena®
Milwaukee, WI, USA), sodium dodecyl sulfate (SDS), and Type HP-2 β-glucuronidase/sulfatase
(Campinas, Brazil). Polyvinylpyrrolidone K90 (PVP), benzalkonium chloride (BAK; Fluka®, ≥95%,
from Helix pomatia (197114 units/mL β-glucuronidase; 876 units/mL sulfatase) were purchased from
Milwaukee, WI, USA), sodium dodecyl sulfate (SDS), and Type HP-2 β-glucuronidase/sulfatase from
Helix pomatia (197114 units/mL β-glucuronidase; 876 units/mL sulfatase) were purchased from
Sigma-Aldrich® (St Louis, MO, USA). Purified water was supplied by a Milli-Q system (Millipore
Pharmaceutics 2019, 11, 492 3 of 17

Sigma-Aldrich® (St Louis, MO, USA). Purified water was supplied by a Milli-Q system (Millipore
Co., Bedford, MA, USA). We have also used acetonitrile, acetic acid (HPLC grade; J.T. Baker Chemical
Co., Phillipsburg, NJ, USA), dimethyl sulfoxide (Sigma-Aldrich® ), Rompun® (2%, Bayer), Ketamine
Agener (10%, Agener União, São Paulo, Brazil) and Na2 CO3 (Merck, Darmstadt, Germany). All other
used reagents and solvents were of analytical grade and were used as received.

2.1. Extraction and Purification of DZ

The purification of DZ from soybean extract was carried out in accordance with Zhang et al. [17]
with modifications. Soybean extract (50 g) was dispersed in 0.1% (v/v) acetic acid (1 L) and kept under
magnetic stirring (1 h, 1000 rotations per minute [rpm], C-Mag HS, Ika, Germany). The dispersion was
then centrifuged (4 × 30 min, 15,000 g, 4 ◦ C, Optima LK-90, Beckman Coulter, Krefeld, Germany), the
supernatant was discharged and the slurry was freeze-dried (Alpha-2, Martin Christ, Osterode am Harz,
Germany). Then, freeze-dried slurry was dispersed in 20% EtOH (v/v) and kept under magnetic stirring
(1 h, 1000 rpm). The supernatant was discharged, the EtOH excess was removed by rotavapor (r-200,
Buchi, Flawil, Switzerland), and the slurry was freeze-dried. We have carried out twice the dispersion,
rotavaporing and freeze-drying with EtOH. The freeze-dried material was added to 2 L of anhydrous
EtOH, kept under magnetic stirring for 4 h (1000 rpm, 80 ◦ C), and vacuum-filtered (0.45 µm). In the end,
anhydrous EtOH was removed under reduced pressure (MiVac Quattro, Genevac, Ipswich, UK).

2.2. Solubility of DZ and Screening of Excipients for SD Manufacturing

The drug solubility was evaluated in water, buffered solutions (pH 1.2–10; USP, 35), or a binary
system of EtOH and water (20–80% EtOH, v/v). The screening of excipients was evaluated with 10
hydrophilic polymers, six surfactants, and four alkalizers at three different concentrations (0.5%, 1%,
and 2%, w/v). Excess DZ (20 mg) was added to 1.5 mL of each solution. The samples were subjected
to constant shaking in a metabolic bath (48 h, 50 rpm, 37 ± 0.5 ◦ C, MA093, Marconi), centrifuged
(5000× g, 15 min, Hettich Mikro 220, Tuttlingen, Germany), filtered (0.22 µm), diluted, and analyzed
by HPLC (Waters, Alliance 2695 equipped with photodiode array detector Waters 2998, Milford,
MA, USA). Empower software (version 3) (Milford, MA, USA) was used for instrument control and
data acquisition.

2.3. Manufacturing of SDs by Spray Drying Technique

Table 1 shows the experimental design for manufacturing SG, TG, SG-pHM, and TG-pHM SDs.

Table 1. Experimental design for manufacturing solid dispersions (SDs).

Volume of Solvent
SDs DZ (g) PVP (g) SDS (%) Na2 CO3 (g) Solvent
(mL)
2nd generation (SG)
F1 2.5 5 - - 80% EtOH 1000
F2 2.5 10 - - 80% EtOH 1000
3rd generation (TG)
F3 2.5 10 5 - 80% EtOH 1000
F4 2.5 10 10 - 80% EtOH 1000
2nd generation + pH-modulated (SG-pHM)
F5 2.5 5 - 5 Water 1000
F6 2.5 10 - 5 Water 1000
3rd generation + pH modulated (TG-pHM)
F7 2.5 5 5 5 Water 1000
F8 2.5 10 5 5 Water 1000
F9 2.5 5 10 5 Water 1000
F10 2.5 10 10 5 Water 1000
Pharmaceutics 2019, 11, 492 4 of 17

In order to manufacture SG, we have dissolved PVP and DZ in 80% EtOH (v/v) using an ultrasonic
bath (40 ◦ C, 1 h, Unique, Brazil). To manufacture TG, SDS was also added. To manufacture SG-pHM
and TG-pHM SDs, we have initially dissolved Na2 CO3 and DZ in purified water so that PVP and (in
the case of TG-pHM) SDs were dissolved under moderate magnetic stirring (30 min, 1500 rpm). A pilot
spray dryer (model LM MSD 1.0, Labmaq, Brazil) was used. The following conditions were used: 0.7
mm pressure atomizer nozzle, feed solution flow rate of 6 mL/min, 40 L/h atomizing air flow, three-bar
air pressure, 110 ± 2 ◦ C inlet temperature, and 80 ± 2 ◦ C outlet temperature. The spray dryer process
parameters (i.e., inlet temperature, airflow rate, feed solution flow rate, and feed concentration) were
selected by assessing the yield, morphology, and glass transition temperature (Tg) of the polymer.

2.4. Saturation Solubility

Saturation solubility of purified DZ, physical mixtures (PMs), and spray-dried formulations were
determined by the shake flask method. Excess DZ (25 mg), corresponding to the weight of the SDs
and PMs containing the same proportions of each component, were added to 1.5 mL of purified water.
The samples were subjected to the same procedure as described in solubility assay and then diluted
and analyzed by HPLC (Waters, Alliance 2695) equipped with photodiode array detector Waters 2998.
Empower software (version 3) was used for instrument control and data acquisition

2.5. Characterization of Solid Dispersion

Loading efficiency (LE), SEM, and particle size measurement have been carried out in accordance
with Panizzon et al. [18] with modifications. The amount of DZ was determined by using HPLC.
Analyses were carried out on a Waters 2695 Alliance (Milford, MA, USA) equipped with PDA detector
(Waters 2998), and software Empower 3 (Milford, MA, USA). The used mobile phase was: Channel
A: 0.1% acetic acid in 18.2 MΩ/cm water (solution A) and Channel B: 0.1% acetic acid in acetonitrile
(solution B). The used gradient in Channel B was: 14% (8 min), 14% to 21% (4 min), 21% (3 min),
21% to 29% (5 min), 29% to 40% (3 min), 40% to 50% (2 min), 50% (5 min), 50% to 14% (5 min), 14%
(5 min). The injection volume was 20 µL and the UV–Vis detection system was monitored at 254 nm.
The flow rate was 1.0 mL/min and the temperature was 30 ◦ C. The separation was carried out on a
Gemini C18, 250 × 4.6 mm, 5-µm column (Phenomenex Inc., Torrance, CA, USA). We have placed
the samples in tubes containing acetonitrile (10 mL) and shaken them (500 rpm × 5 min). Distilled
water (10 mL) was added and shaken again for 60 min. The mixture was filtered through a 0.22 µm
membrane and injected.
Morphology and mean particle size were examined by SEM (Shimadzu SS-550, Kyoto, Japan).
The material was fixed on double-sided tape attached to an aluminum support, then coated with
gold/palladium under argon atmosphere, and examined under a scanning electron microscope.
The mean particle size was determined with the aid of an Image-Pro® Plus image analyzer (Silver
Spring, MD, USA), by measuring the Ferret’s diameter of at least one thousand particles present in
the photomicrographs. DZ content (DZC), loading efficiency and span were calculated by using the
following equations (1)–(3):

DZC (mg/g) = weight of DZ in formulation/weight of formulation (1)

Loading efficiency (%) = (weight of DZ/theoretical weight of DZ) × 100 (2)

Span = (d(0.9) − d(0.1))/d(0.5) (3)

2.6. X-ray Diffraction (XRPD)

X-ray Diffraction patterns have been obtained by using an X-ray diffractometer (Bruker-AXS,
Karlsruhe, Germany). The specifications were 2◦ /min, 2θ, scanning from 10◦ to 70◦ , Cu-Kα X-radiation
(λ = 1.5418 Å), 40 mA current, and 40 kV voltage.
Pharmaceutics 2019, 11, 492 5 of 17

2.7. Differential Scanning Calorimetry (DSC)

DSC analyses have been carried out by using a calorimeter (Q20, TA instruments, New Castle, DE,
USA) operating at the following conditions: heating rate of 10 ◦ C/min, nitrogen flow rate of 50 mL/min,
and temperature range from 25 to 350 ◦ C. Samples (6–9 mg) were placed on an aluminum DSC pan
and hermetically sealed with a lid (Tzero® , TA instruments, New Castle, DE, USA). An empty pan was
used as a reference. Indium standard was used to calibrate the DSC temperature and enthalpy scale.
Data acquisition and analysis were carried out by using Advantage software v.5.22 (TA Instruments,
New Castle, DE, USA).

2.8. Gel Permeation Chromatography (GPC)

The molecular weight profile changes of PVP were determined by GPC on a Waters Alliance 2695
(Waters) equipped with an evaporative light scattering detector Waters 2424. Data analyses were carried
out in Empower version 3 (Waters). Gel permeation chromatography columns: Ultrahydrogel-500,
Ultrahydrogel-250, and Ultrahydrogel-120 (Waters) were connected in order to decrease pore size and
maintained at 45 ◦ C. The detector nebulizer was tuned in cooling mode, the drift tube temperature was
kept at 80 ◦ C, and the nitrogen pressure was kept at 50 psi. The used mobile phase was acetonitrile:water
(20:80, v/v), with a flow rate of 0.8 mL/min and an injection volume of 10 µL. Molecular weight calibration
was carried out by using Pullulan Calibration Kit (Shodex standard, Showa Denko, Knagawa, Japan)
standards with a molecular weight range of 21.7–3050 kDa.

2.9. Dissolution Profile, Daidzein-Release Mechanism, and Kinetics

The in vitro dissolution profile of DZ and SDs was determined by using flow-through cell (USP
Apparatus 4) (EC 7smart, Sotax Co., Aesch, Switzerland) on open mode. Twenty milligrams of DZ,
calculated from the LE results, were added between the layers of glass beads (2 × 1 g; 1 mm) in the
equipment cells. A glass microfiber filter (GF/B, Whatman, Maidstone, England, 1 µm pores) was
coupled to the cells in order to prevent the passage of undissolved particles. Phosphate buffer (75 mM,
pH 6.8) (USP 35) was used as a dissolution medium (4 mL/min, 37 ± 0.5 ◦ C) and after 5, 15, 30, 60,
90, and 150 min, and aliquots of each sample were collected and analyzed by HPLC. The pH of
the dissolution medium was monitored immediately after every sampling time using the pH meter
(DM-22, Digimed, São Paulo, Brazil). The samples were filtered (0.22 µm), diluted, and analyzed by
HPLC. Results have been reported as cumulative percentage of DZ dissolved in the medium over time.
The DZ-release mechanism and kinetics SDs have been carried out in accordance with the Panizzon et
al. method [18].

2.10. Pharmacokinetic Studies

The State University of Maringá Animal Ethics Committee approved the following procedures
and protocols on 7 June 2011 under 043/2011 project identification code. Twenty-four male Wistar
rats (Rattus norvegicus) at the age of 90 days were divided into two groups and kept under controlled
conditions in a 12 h light/dark cycle at 22 ± 2 ◦ C. We have provided them with water and a standard
rodent chow (Nuvital® ) diet ad libitum. Seventy-two hours before the treatment, soy-free chow was
supplied and maintained ad libitum until 14 h before the treatment. Twenty-four hours before the
treatment, rats were anesthetized (ketamine:Rompun® 1:1, v/v, 1 mL/kg) and underwent surgery.
A silicone cannula was implanted into the left jugular vein and stabilized on the dorsal region of
the neck. Then, 10 mg of DZ and 0.73 g of F10 was suspended in 0.98 mL and 0.6 mL of olive oil,
respectively. The final concentration of drug was 10 mg/mL. A dose of 10 mg/kg was administrated
by gavage. Blood samples (300 µL each sample, total blood volume collection of 1.8 mL per animal)
were collected at 0.083, 0.17, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h (n = 6) through the cannula and
mixed with heparinized saline (50 µL). The volume of collected blood was replaced by sterile saline.
Plasma was obtained by centrifugation (2500× g, 7 min) and stored frozen at −80 ◦ C until analysis.
Pharmaceutics 2019, 11, 492 6 of 17

HPLC-MS/MS analyses were carried out with a Waters 1525µ coupled to a Micromass Quattro
micro™ API triple-quadrupole mass spectrometer (Waters) with an electrospray ionization interface
under the following conditions: capillary voltage of 2.5 kV (positive mode), cone voltage of 30 V,
extraction cone voltage of 2 eV, source temperature of 130 ◦ C, desolvation temperature of 450 ◦ C,
cone gas flow of 50 L/h, and desolvation gas flow of 750 L/h. The spectrometer was tuned in the
multiple reaction-monitoring mode to monitor mass transitions m/z 254.94→90.83 (DZ) and m/z
285.91→270.87 (IS) with collision energies of 35 eV and 25 eV, respectively. MassLynx™ software
(version 4.0, Waters) was used for data processing. The used column was Luna C18(2)-HST (2.5 µm;
50 × 2 mm, Phenomenex, Torrance, CA, USA), and the mobile phase consisted of water with 0.1% (v/v)
formic acid (solvent A) and acetonitrile with 0.1% (v/v) of formic acid (solvent B) delivered at a rate of
0.3 mL/min, as follows: 5% solvent B at 0–0.5 min, 75% solvent B at 4 min, and 5% solvent B at 5 min.
In order to determine total DZ (free and conjugated DZ), samples were enzymatically hydrolyzed
as Qiu et al. method [9] with modifications. The enzyme β-glucuronidase (100 µL, 9000 U/mL in
0.05 mol/L acetate buffer, pH 5.0) was added to plasma (100 µL) and incubated at 37 ◦ C for 14 h.
An internal standard (glycitein, 35 µL; 1000 ng/mL) and purified water (2 mL) were added and samples
were loaded onto a solid phase extraction cartridge C18 Amprep (500 mg, Amersham Pharmacia
Biotech). The cartridge was flushed with acetic acid 0.1% (3 mL, v/v) followed by 25% methanol (3 mL)
containing 0.1% (v/v) acetic acid. The analytes were eluted with methanol (2 × 3 mL) and evaporated to
dryness in a MiVac Quattro Concentrator (Genevac, Ipswich, UK). The residue was reconstituted with
80% methanol (100 µL, v/v), and 10 µL was injected. The method was validated using bio-analytical
method validation guidance (FDA, 2001) (Supplementary Materials). Pharmacokinetic analyses have
been carried out by using a non-compartmental model. The maximum plasma concentration (Cmax)
and time to reach the maximum plasma concentration (Tmax) were directly obtained from the mean
blood concentration-time curve. The pharmacokinetic parameters were calculated by using Excel 2013
software. The areas under the concentration vs. time curves (0–24 h [AUC0–24 h] and 0 to infinity
[AUC0→∞]) were calculated by using the trapezoidal method.

2.11. Statistical Analysis

The software Statistica® 8.0 (StatSoft, Inc. 1984–2007, Tulsa, OK, USA) was used for statistical
analysis. Data are presented as mean ± standard deviation (S.D.) using a unilateral analysis of
variance (one-way ANOVA). Significant differences were determined with the Tukey test, with p < 0.05
considered statistically significant.

3. Results

3.1. Extraction and Purification of DZ

The overall recovery of DZ throughout the entire purification process was 63–69%. The final
content, determined by HPLC, was 95.6% ± 1.4% DZ and 1.2% ± 4.8% genistein. Figure S1 shows the
characterization of the material that was performed by using HPLC-MS/MS operating in full scan
mode. The identity was confirmed in daughter scan mode by comparing the ion products (MS/MS)
with published data [19]. The SEM and XRPD results showed that purified DZ was in a crystalline
form, which was then used as a polyphenolic BCS Class IV drug model for DZ SD manufacturing.

3.2. Effect of Excipients and Solvents on DZ Solubility

Daidzein can be classified as practically insoluble in water (1.50 µg/mL). As expected for a weak
acid, DZ has high solubility in basic pH in contrast to neutral and acidic pH (Figure 2).
Pharmaceutics 2019, 11, 492 7 of 17
Pharmaceutics 2019, 11, x FOR PEER REVIEW 7 of 17

Figure
Figure 2. 2.Solubility
SolubilityofofDaidzein
Daidzein(DZ)
(DZ)inindifferent
different solvents:(A)
solvents: (A)pH
pH1.2–10
1.2–10buffered
buffered solutions,
solutions, (B)(B) water
water
and hydroethanolic solvents (EtOH 20–80%,
and hydroethanolic solvents (EtOH 20–80%, v/v). v/v).

This
Thisshows
showsthat
thatDZDZsolubility
solubilityisispH-dependent,
pH-dependent,and andalkalinizers
alkalinizerswould
wouldbebethe thebest
bestchoice
choicetoto
prepare
preparepHM
pHMSDs.SDs.Table
TableSISIshows
shows the
theDZ-solubilizing
DZ-solubilizingcapacitycapacityofof1010polymers,
polymers,sixsixsurfactants,
surfactants,and
and
four alkalinizers at different concentrations (0.5, 1, and 2%, w/v). Among the investigated
four alkalinizers at different concentrations (0.5, 1, and 2%, w/v). Among the investigated polymers, polymers,
PVP
PVPK90
K90was
wasthethe
most effective.
most BAK
effective. BAKis a is
surfactant
a surfactantwithwith
a higher drug-solubilizing
a higher capacity,
drug-solubilizing but SDS
capacity, but
was
SDSchosen due to due
was chosen its lower
to its toxicity [20]. Na
lower toxicity 2 CO
[20]. Na3 provided the highest
2CO3 provided amount
the highest of dissolved
amount DZ and
of dissolved DZ
was used as an alkalinizer to prepare SG-pHM and TG-pHM SDs. Furthermore,
and was used as an alkalinizer to prepare SG-pHM and TG-pHM SDs. Furthermore, water that water that contained
0.5% (w/v) Na
contained 2 CO(w/v)
0.5% 3 could
Nareplace 80%replace
2CO3 could EtOH (v/v) 80% as the solvent.
EtOH (v/v) as the solvent.

3.3.
3.3.Manufacturing
Manufacturingand
andCharacterization
CharacterizationofofSDs
SDs
Daidzein
Daidzein(2.5
(2.5g/L)
g/L)was
wasdissolved
dissolvedinin80%
80%EtOH
EtOH(v/v)(v/v)for
forthe
thepreparation
preparationofofSG SG(F1(F1and
andF2F2
formulations)
formulations) and
andTG TG(F3 and
(F3 andF4F4
formulations).
formulations).DZDZ was
wasdissolved
dissolved inin
water
watercontaining
containing 0.5%
0.5%NaNa
2 CO 3 3
2CO
(w/v) for the preparation of SG-pHM SDs (F5 and F6 formulations) and TG-pHM SDs
(w/v) for the preparation of SG-pHM SDs (F5 and F6 formulations) and TG-pHM SDs (F7, F8, F9, and(F7, F8, F9, and
F10
F10formulations).
formulations).Table
Table2 2shows
showsthetheDZDZcontent,
content,LE,
LE,mean
mean particle
particlesize, and
size, span.
and span.The
TheLELEwaswas≥≥
95.6%.
95.6%.The mean
The particle
mean size and
particle size span
and ranged from 2.48
span ranged to 7.09
from 2.48µm toand
7.09from
µm1.13
andtofrom
3.88, respectively.
1.13 to 3.88,
respectively.
Table 2. Daidzein content (DZC), loading efficiency (LE), mean particle size, and span of solid
dispersions (SD) F1–F10. The results are shown as mean ± S.D.
Table 2. Daidzein content (DZC), loading efficiency (LE), mean particle size, and span of solid
dispersions (SD)SGF1–F10. The results
Parameter TGare shown as mean ± S.D.
SG-pHM TG-pHM
SD F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
Parameter 32.4
DZC (mg/g) 22.8 SG 20.9 TG
18.8 19.1 SG-pHM
15.1 18.6 14.8 TG-pHM
18.9 13.7
LE (%) 97 ± 1.6 110 ± 0.7 110± 0.9 104 ± 1.1 96 ± 1.6 105.4 ± 2.0 98 ± 4.3 109 ± 0.5 103 ± 1.5 105 ± 2.3
Mean Particle
5.33 2.48 F23.43 F3 7.09 2.94 3.18 2.60 4.24 4.02 3.01F10
Size (µm) SD F1 F4 F5 F6 F7 F8 F9
Span 2.4 2.9 3.2 3.5 1.1 1.2 1.2 3.3 3.9 1.4

DZC (mg/g) 32.4 22.8 20.9 18.8 19.1 15.1 18.6 14.8 18.9 13.7

Micrographs of purified
97 ±
DZ
110 ±
and SDs
110±
are shown
104 ±
in96Figure
±
3. Drug 98
105.4 ±
crystals
±
showed103
109 ±
a prismatic
± 105 ±
LE (%)
shape with similar sizes.1.6However,
0.7 we 0.9could not1.1 note drug
1.6 crystals
2.0 in the
4.3 SDs. 0.5
The morphological
1.5 2.3
results of all of the SDs were similar in terms of shape and matrix distribution. SDs had a smooth
Mean Particle Size
surface and concave depressions
5.33 (collapsed
2.48 3.43 walls),
7.09 with2.94
no agglomerates
3.18 or pinholes.
2.60 4.24 4.02 3.01
(µm)

Span 2.4 2.9 3.2 3.5 1.1 1.2 1.2 3.3 3.9 1.4

Micrographs of purified DZ and SDs are shown in Figure 3. Drug crystals showed a prismatic
shape with similar sizes. However, we could not note drug crystals in the SDs. The morphological
results of all of the SDs were similar in terms of shape and matrix distribution. SDs had a smooth
surface and concave depressions (collapsed walls), with no agglomerates or pinholes.
 Pharmaceutics 2019, 11, x FOR PEER REVIEW 8 of 17

Pharmaceutics 2019, 11, 492 8 of 17

Pharmaceutics 2019, 11, x FOR PEER REVIEW 8 of 17

Figure 3. Scanning electron microscopy micrographs of Daidzein (DZ) and SDs (F1–F10). Scale bar in
DZ, F1–F4, F6, F8–F10 is 5 µm; scale bar in F5 is 2 µm; scale bar in F7 is 10 µm.
Figure
Figure 3. Scanning electron
3. Scanning electron microscopy
microscopy micrographs
micrographs ofof Daidzein
Daidzein (DZ)
(DZ) and
and SDs
SDs (F1–F10).
(F1–F10). Scale
Scale bar
bar in
in
3.4.DZ,
Saturation Solubility
F1–F4, F6, F8–F10 is 5 µm; scale bar in F5 is 2 µm; scale bar in F7 is 10 µm.
DZ, F1–F4, F6, F8–F10 is 5 µm; scale bar in F5 is 2 µm; scale bar in F7 is 10 µm.
The results
3.4. Saturation of the saturation solubility assay are shown in Figure 4. When the results were
Solubility
3.4. Saturationwith
compared Solubility
the PMs, only the SG was unable to significantly increase the water solubility of DZ
(p >The
The
results
0.05). The of
results TG,the
of the
saturation
SG-pHM, andsolubility
saturation TG-pHMassay
solubility
are shown
SDs resulted
assay
in Figure 4.
in significant
are shown
When the
in Figure increases
results
in the
4. When the
were of
solubility
results were
compared
DZ (p < with
0.05).the
ThePMs,
pHM only the
SDs SG was
showed unable
the to
highestsignificantly
capability increase
of the
solubilizingwater
high solubility
doses of of
DZ DZ
(>20
compared with the PMs, only the SG was unable to significantly increase the water solubility of DZ
> 0.05). The TG, SG-pHM, and TG-pHM SDs resulted in significant increases in the solubility of DZ
(p mg/mL).
(p > 0.05). The TG, SG-pHM, and TG-pHM SDs resulted in significant increases in the solubility of
(p < 0.05). The pHM SDs showed the highest capability of solubilizing high doses of DZ (>20 mg/mL).
DZ (p < 0.05). The pHM SDs showed the highest capability of solubilizing high doses of DZ (>20
mg/mL).

Figure
Figure Saturation
4. 4. solubility
Saturation of of
solubility Daidzein
Daidzein(DZ) in in
(DZ) physical mixtures
physical (PM)
mixtures and
(PM) solid
and dispersion
solid (SD):
dispersion (SD):
(A)(A)
F1–F3 and (B) F4–F10 (mean ± S.D, n =
F1–F3 and (B) F4–F10 (mean ± S.D, n = 3).3).

Figure 4. Saturation
3.5. Differential Scanningsolubility of Daidzein
Calorimetry Analyses(DZ) in physical mixtures (PM) and solid dispersion (SD):
3.5. Differential Scanning Calorimetry Analyses
(A) F1–F3 and (B) F4–F10 (mean ± S.D, n = 3).
The DSC thermograms of DZ, excipients, PMs, and spray-dried formulations (F1–F10) are shown
The DSC thermograms of DZ, excipients, PMs, and spray-dried formulations (F1–F10) are
in Figure
3.5. 5A,B.Scanning
Differential The DSCCalorimetry
results showed no phase transition of the drug until the melting point of
Analyses
shown in Figure 5A,B. The DSC results showed no phase transition of the drug until the melting point
genistein (319.3 ◦ C) and DZ (336 ◦ C), where two sharp endothermic peaks that are characteristic of the
The DSC thermograms of DZ, excipients, PMs, and spray-dried formulations (F1–F10) are
shown in Figure 5A,B. The DSC results showed no phase transition of the drug until the melting point
Pharmaceutics 2019, 11, x FOR PEER REVIEW 9 of 17
Pharmaceutics 2019, 11, 492 9 of 17

of genistein (319.3 °C) and DZ (336 °C), where two sharp endothermic peaks that are characteristic of
the crystalline
crystalline solidsolid
statestate
werewere found.
found. The The
DSCDSC
tracetrace of PVP
of PVP K90 K90 showed
showed endothermic
endothermic peaks
peaks at and
at 153 153
and◦168
168 °C. Conversely,
C. Conversely, therethere wasnoticeable
was no no noticeable
peakpeak at melting
at melting pointpoint
of DZofin
DZ in of
any any ofexcipients
the the excipients
and
and The
PM. PM. melting
The melting
peak peak of crystalline
of crystalline DZ observed
DZ were were observed in all
in all PM. PM.DSC
Thus, Thus, DSC could
results resultsbecould
used beto
used to solid
evaluate evaluate
statesolid
of DZ.state
Withofthe
DZ. With the
exception exception
of F1, of F1, the
the DZ melting DZdisappeared
peak melting peak disappeared
completely in all
completely
other in all other formulations.
formulations.

Figure 5. Differential scanning

5. Differential scanning calorimetry
calorimetry (DSC)
(DSC) thermograms
thermograms ofof (A) (A) daidzein
daidzein (DZ),
polyvinylpyrrolidone
polyvinylpyrrolidoneK90 K90(PVP),
(PVP),sodium
sodiumdodecyl
dodecylsulfate
sulfate(SDS),
(SDS),sodium
sodiumcarbonate
carbonate(Na (Na22CO
CO33), physical
mixtures
mixtures of
of DZ:PVP
DZ:PVP 1:1
1:1 (PM
(PM 1), DZ:PVP:SDS
DZ:PVP:SDS 1:1:1
1:1:1 (PM
(PM 2),
2), DZ:PVP:
DZ:PVP: Na
Na22CO33 1:1:1
1:1:1 (PM
(PM 3)
3) DZ:PVP:SDS:
DZ:PVP:SDS:
Na22CO33 1:1:1:1
1:1:1:1 (PM
(PM 4),
4), and
and (B)
(B) DSC
DSC solid
solid dispersions
dispersions (F1–F10).
(F1–F10).

3.6. X-Ray Powder

3.6. X-Ray Powder Diffraction
Diffraction Analyses
Analyses
Figure
Figure 6 shows the XRPD patterns
6 shows the XRPD patterns ofof purified
purifiedDZ,
DZ,formulations,
formulations,andandNaNa22CO
CO33.. The
The XRPD
XRPD analysis
analysis
showed ◦ ◦ ◦ 24.2◦ ,
showed that
that DZDZ was
was aa crystalline
crystalline powder
powder withwith sharp
sharp peaks
peaks atat 2θ
2Ɵ equal
equal to
to 10.4
10.4°,, 15.8
15.8°,, 16.8
16.8°,, 24.2°,
24.9 ◦ ◦ ◦
24.9°,, 26.1
26.1°,, and
and 28.2
28.2°.. Among
Among thethe excipients,
excipients, only
only Na CO33 was
Na22CO was crystalline
crystalline and
and responsible
responsible for
for the
the
diffraction
diffraction peaks shown in F5 to F10. The F1 diffraction pattern showed that DZ was still present its
peaks shown in F5 to F10. The F1 diffraction pattern showed that DZ was still present in in
crystalline state,
its crystalline probably
state, duedue
probably to the
to low concentration
the low of theofpolymer.
concentration F2, TG,
the polymer. F2,SG-pHM,
TG, SG-pHM, and TG-pHM
and TG-
had
pHMpatterns that were
had patterns thatanalogous to the carriers.
were analogous to the carriers.
Pharmaceutics 2019, 11, 492 10 of 17
Pharmaceutics 2019, 11, x FOR PEER REVIEW 10 of 17

Figure 6.
Figure 6. X-ray
X-ray powder
powder diffraction
diffraction patterns
patterns of
of DZ
DZ and
and spray-dried
spray-dried formulations
formulations (F1–F10).
(F1–F10).

3.7.
3.7. Evaluation
Evaluation of
of PVP
PVP Structural
Structural Modification
Modification by
by Gel
Gel Permeation
Permeation Chromatography
Chromatography (GPC)
(GPC)
The
The results
results showed
showed that
that polymer
polymer Mw
Mw had
had no
no changes
changes in
in SG
SG or
orTG (Mw== 1200
TG(Mw 1200 kDa).
kDa). However,
However,
the
the size
size changing
changing for
for SG-pHM
SG-pHM and
andTG-pHM (Mw== 2920
TG-pHM(Mw 2920 kDa)
kDa) was
was shown
shown by
by GPC.
GPC.

3.8.
3.8. Dissolution
Dissolution Profile
Profile
Daidzein
Daidzein precipitation
precipitation andand significant
significant variations
variations inin pH
pH (>0.1)
(>0.1) were
were not
not noted
noted in
in the
the collected
collected
samples.
samples. The dissolution profile of DZ showed a slow release rate, and F1 was unable to
The dissolution profile of DZ showed a slow release rate, and F1 was unable to improve
improve
solubility
solubility (Figure
(Figure 7A).
7A). Some
Some enhancement
enhancement in in in
in vitro
vitro release
release was
was reached
reached with
with aa higher
higher amount
amount of of
the polymer (F2). A higher release rate was observed only when 10% SDS (w/w;
the polymer (F2). A higher release rate was observed only when 10% SDS (w/w; F4) was added (FigureF4) was added
(Figure
7B), but7B), but still
it was it was still incomplete.
incomplete. The SG-pHM
The SG-pHM SD (Figure
SD (Figure 7C) had 7C)faster
had faster and higher
and higher releaserelease
rates
rates
compared with the previous SDs. TG-pHM SDs, with 1:2 and 1:4 DZ:polymer ratios, were also
compared with the previous SDs. TG-pHM SDs, with 1:2 and 1:4 DZ:polymer ratios, were also
prepared
prepared (Figure
(Figure 7D).
7D). The
The TG-pHM
TG-pHM SDsSDs effectively
effectively increased
increased the
the release
release rate,
rate, even
even with
with lower
lower PVP
PVP
content
content (1:2
(1:2 DZ:polymer
DZ:polymer ratio).
ratio).
Pharmaceutics 2019, 11, 492 11 of 17
Pharmaceutics 2019, 11, x FOR PEER REVIEW 11 of 17

Figure7.7.Cumulative
Figure Cumulativerelease (%)(%)
release of (A) Daidzein
of (A) (DZ),
Daidzein SG (F1
(DZ), SGand
(F1 F2),
and(B)
F2),TG
(B)(F3
TGand
(F3F4),
and (C) SG-pHM
F4), (C) SG-
(F5 and(F5
pHM F6)and
andF6)
(D)and
TG-pHM (F7, F8, (F7,
(D) TG-pHM F9 and
F8, F10) (mean
F9 and S.D, n±=S.D,
F10)±(mean 3). n = 3).

3.9.
3.9.Daidzein-Release
Daidzein-ReleaseMechanism
MechanismandandKinetics
Kinetics
The coefficients of determination
The coefficients of determination (R2) of power
(R2) of powerlawlaw
bestbest
fit the
fit in vitro
the release
in vitro data, data,
release showing that
showing
DZ
thatrelease was run
DZ release wasbyrun
anomalous transport
by anomalous (0.43 < n(0.43
transport < 0.85;
< n Table
< 0.85;SII). However,
Table the diffusion
SII). However, process
the diffusion
was predominant for F3 (n = 0.40) and super case II was predominant for F6 (n =
process was predominant for F3 (n = 0.40) and super case II was predominant for F6 (n = 0.85).0.85).

3.10. Pharmacokinetic Studies

3.10. Pharmacokinetic Studies
HPLC-MS/MS results of specificity (Figure S2), linearity, precision, accuracy, extraction recovery,
HPLC-MS/MS results of specificity (Figure S2), linearity, precision, accuracy, extraction
and matrix effect (Table SIII) are shown in the Supplementary Information. Figure 8 shows the mean
recovery, and matrix effect (Table SIII) are shown in the Supplementary Information. Figure 8 shows
plasma concentration–time profiles of DZ following administration of a 10 mg/kg dose of optimized
the mean plasma concentration–time profiles of DZ following administration of a 10 mg/kg dose of
SD and DZ in rats. Two peaks of DZ occurred in plasma. The first peak of purified DZ occurred at 4 h,
optimized SD and DZ in rats. Two peaks of DZ occurred in plasma. The first peak of purified DZ
and the second peak occurred at 8 h, with low concentrations as reported in other studies [9]. For F10,
occurred at 4 h, and the second peak occurred at 8 h, with low concentrations as reported in other
the first plasma peak occurred at 15 min and the second peak at 12 h.
studies [9]. For F10, the first plasma peak occurred at 15 min and the second peak at 12 h.
The Tmax of purified DZ and F10 was 6.5 ± 1.9 and 0.26 ± 0.1 h, respectively. Cmax of purified
The Tmax of purified DZ and F10 was 6.5 ± 1.9 and 0.26 ± 0.1 h, respectively. Cmax of purified
DZ and F10 was 707.2 ± 91.6 and 4050.50 ± 506.7 ng/mL, respectively. The AUC0–24 h and AUC0→∞
DZ and F10 was 707.2 ± 91.6 and 4050.50 ± 506.7 ng/mL, respectively. The AUC0–24 h and AUC0→∞
for purified DZ and F10 were 9143.3 ± 1577.8 and 21995.3 ± 3025.5 ngh/mL and 11442.2 ± 2201 and
for purified DZ and F10 were 9143.3 ± 1577.8 and 21995.3 ± 3025.5 ngh/mL and 11442.2 ± 2201 and
28305.4 ± 4691.9 ngh/mL, respectively.
28305.4 ± 4691.9 ngh/mL, respectively.
Pharmaceutics 2019, 11, 492 12 of 17
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Pharmaceutics2019, 11,
11,xxxFOR
2019,11, FOR PEER
FORPEER REVIEW
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REVIEW 12
12 ofof17
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17

Figure
Figure 8.8.Mean
Figure8. Mean plasma
Meanplasma concentration–time
plasmaconcentration–time curves
concentration–timecurves ofofDaidzein
curvesof Daidzein (DZ)
Daidzein(DZ) after
(DZ)after oral
afteroral administration
oraladministration ofofpure
administrationof pure
pure
DZ ((▬
(▬ ▇
▇ ▬)
▬) ) and
and F10
F10 (▬
(▬
( ▼
▼ ▬)
▬)) (mean
(mean ±± S.D,
S.D,
DZ (▬▄▬) and F10 (▬▼▬) (mean ± S.D, n = 6).
DZ ± S.D,nn
n =
==6).
6).
6).

4.
4. Discussion
4.4.Discussion
Discussion
Discussion
Several
Severalstudies
Several
Several studieshave
studies
studies have
have
have shown
shown
shown
shown thethe
the
the biological
biological
biological
biological effects
effects
effects
effectsof of
DZ,
of DZ,
ofDZ, making
DZ, making
making
making it ait
itpromising
itaaapromising
promising
promising compound
compound
compound
compound for the for
for
for
treatment
the
the treatment
thetreatment of
treatmentof several
of diseases
several
ofseveral
severaldiseases [21].
diseases
diseases[21]. Nevertheless,
[21]. Nevertheless,
[21].Nevertheless, DZ
Nevertheless,DZ is aDZ BCS
DZis class
isisaaaBCS
BCS IV polyphenol,
class
BCSclassclassIV IV with
polyphenol,
IVpolyphenol,
polyphenol,withlow aqueous
with
withlow low
low
solubility
aqueous
aqueous
aqueoussolubilityand low permeability,
solubility
solubility and
and
andlow low thus limiting
permeability,
lowpermeability,
permeability, thus
thus
thus itslimiting
oral bioavailability.
limiting
limiting its
its oral
itsoral Solid dispersions
bioavailability.
oralbioavailability.
bioavailability. Solid
Solid are useful are
dispersions
Soliddispersions
dispersions for
are
are
increasing
useful
useful for
for the aqueous
increasing
increasing the
the solubility
aqueous
aqueous and bioavailability
solubility
solubility and
and of drugs
bioavailability
bioavailability
useful for increasing the aqueous solubility and bioavailability of drugs under these characteristics. under
of
of these
drugs
drugs characteristics.
under
under these
these characteristics.
characteristics.
High-purity
High-purityDZ
High-purity
High-purity DZwas
DZ
DZ waswasisolated
was isolatedfrom
isolated
isolated fromsoybean
from
from soybeanextract
soybean
soybean extractusing
extract
extract usingaaaamodified
using
using modified
modifiedmethod
modified methoddescribed
method
method describedby
described
described by
by Zhang
Zhang
byZhang
Zhang
et
et al.
al. [17]. In order to manufacture SDs by spray drying, it was necessary to test drug solubilityin
etetal.
al. [17].
[17].
[17]. In
In
In order
order
order to
to
to manufacture
manufacture
manufacture SDs
SDs
SDs by
by
by spray
spray
spray drying,
drying,
drying, it
it
it was
was
was necessary
necessary
necessary to
to
to test
test
test drug
drug
drug solubility
solubility
solubility in
inin
solvents
solvents
solvents
solventsthat that
that are
thatareare able
able
areable to
to dissolve
dissolve
abletotodissolve
dissolveboth both
both excipients
excipients
bothexcipients
excipientsand and
and DZ.
andDZ.DZ. In
In
DZ.InInorderorder
order to
to manufacture
manufacture
ordertotomanufacture
manufactureSG SG
SG and
and
SGandandTG,TG,
TG, 80%
TG,80%80%
80%
EtOH
EtOH(v/v)
EtOH
EtOH (v/v)was
(v/v)
(v/v) wasused
was
was useddue
used
used dueto
due
due totoits
to itsvolatility,
its
its volatility,low
volatility,
volatility, lowtoxicity
low
low toxicity[12,22]
toxicity
toxicity [12,22]and
[12,22]
[12,22] andability
and
and abilityto
ability
ability todissolve
to
to dissolvehigh
dissolve
dissolve highamounts
high
high amountsof
amounts
amounts of
ofof
DZ
DZ
DZ (2629
(2629
(2629 µg/mL).
µg/mL).
µg/mL). However,
However,
However, Na
Na
Na CO
CO
CO had
had
had limited
limited
limited solubility
solubility
solubility
DZ (2629 µg/mL). However, Na2CO3 had limited solubility in 80% EtOH (v/v). Consequently, Na2CO33 3
2 2
2 333 in
in
in 80%
80%
80% EtOH
EtOH
EtOH (v/v).
(v/v).
(v/v). Consequently,
Consequently,
Consequently, Na
Na
Na 22CO
2 CO
CO 3
was
wasdissolved
was
was dissolvedin
dissolved
dissolved inwater
in
in water(0.5%
water
water (0.5%w/v)
(0.5%
(0.5% w/v)and
w/v)
w/v) andandthen
and thenused
then
then usedto
used
used to
totodissolve
dissolveDZ.
dissolve
dissolve DZ.This
DZ.
DZ. Thissolution
This
This solutionwas
solution
solution wasspray-dried
was
was spray-driedto
spray-dried
spray-dried to
toto
manufacture
manufacture
manufacture
manufacturethe the
the pHM
pHM
thepHMpHMSDs. SDs.
SDs.
SDs.
In
In
InInthethedevelopment
the
the developmentstage,
development
development stage,the
stage,
stage, theselection
the
the selectionof
selection
selection of
of ofexcipients
excipientshas
excipients
excipients hasaaaasignificant
has
has significant
significanteffect
significant effecton
effect
effect onthe
on
on thesolid
the
the solidstate
solid
solid stateand
state
state and
and
and
stability
stability
stability
stabilityof of
of the
the
ofthe amorphous
amorphous
theamorphous
amorphousstate state
stateunder
under
stateunderunderinin vitro
in and
vitro
invitro
vitroand in
and vivo
andin in conditions.
vivo
invivo conditions.
vivoconditions. Considering
Considering
conditions.Considering this,
Consideringthis, solubility
this, tests
solubility
this,solubility
solubility
were
tests
tests carried
were
testswerewerecarried out with
carried
carried outseveral
out
out with
with
withseveralpolymers,
several
several polymers,surfactants,
polymers,
polymers, and alkalizers.
surfactants,
surfactants,
surfactants, and
and
andalkalizers. The aim
alkalizers.
alkalizers. The
The
Thewas aim
aim
aimtowas study
was
was to
to and
study
tostudy
study select
and
and
and
the
select
select excipients
selectthethe
theexcipients by
excipients
excipientsbyusingby a
byusing simple
using a
usingaasimple and
simple
simpleand effective
and approach,
effective
andeffective approach,
effectiveapproach, which
approach,which is
which based
is based
whichisisbased on
basedon individual
on individual
onindividual capability
capability
individualcapability
capability of
dissolving
of
of dissolving
ofdissolving
dissolvingthethe drug
the drug
thedrug to
drugto obtain
to obtain
toobtain different
different
obtaindifferent generations
generations
differentgenerations
generationsof of SDs
of [13,21,23].
SDs
ofSDs [13,21,23].
SDs[13,21,23].
[13,21,23].
TheThe characterization of SDs showed that spray drying providedhigh
The
The characterization
characterization
characterization of
of
of SDs
SDs
SDs showed
showed
showed that
that
that spray
spray
spray drying
drying
drying provided
provided
provided highdrug
high
high drugLE.
drug
drug LE.This
LE.
LE. Thismay
This
This mayoccur
may
may occur
occur
occur
due
due
due
dueto to
to rapid
rapid
torapid solvent
solvent
rapidsolvent evaporation
evaporation
solventevaporation
evaporationthat that
that led
thatledled
ledto to
to a rapid
toaaarapid
rapid increase
increase
rapidincrease
increasein in
in viscosity,
viscosity,
inviscosity,
viscosity,thus thus
thus permitting
permitting
thuspermitting
permittingkinetickinetic
kinetic
kinetic
trapping
trappingof
trapping
trapping of
ofofthethedrug
the
the drugin
drug
drug in
ininthethematrix
the
the matrix[10].
matrix
matrix [10].SEM
[10].
[10]. SEManalysis
SEM
SEM analysisrevealed
analysis
analysis revealedneedle-shaped
revealed
revealed needle-shapedDZ
needle-shaped
needle-shaped DZcrystals.
DZ
DZ crystals.Crystal
crystals.
crystals. Crystal
Crystal
Crystal
habit
habit
habit ofof
of drugdrug
drug was was
wasnot found
not
not on
found
found SEM on
on micrographs
SEM
SEM micrographs
micrographs
habit of drug was not found on SEM micrographs after spray drying. The SDs had similar after spray after
after drying.
spray
spray The SDs
drying.
drying. hadThe
The similar
SDs
SDs morphology
had
had similar
similar
to PVP-containing
morphology
morphology
morphology to
to microparticles
PVP-containing
toPVP-containing
PVP-containing that have been
microparticles
microparticles
microparticles that
that
thatobtained
have
have
havebeenbeen byobtained
been spray
obtained
obtained drying
by
bybyspray in a drying
spray
spray previous
drying
dryingin ininastudy
aaprevious
previous[24].
previous
The
study
study low
study[24].[24]. viscosity
[24].TheThe
Thelowlow of the
viscosity
lowviscosity solution
viscosityof of the
ofthe was
thesolutioninsufficient
solution
solutionwas was to maintain
insufficient
wasinsufficient
insufficientto to the physical
maintain
tomaintain
maintainthe the stability
physical
thephysical of the
stability
physicalstability droplets
stabilityof of the
ofthe the
during
droplets
droplets solvent
during
during evaporation,
solvent
solvent which
evaporation,
evaporation, may have
which
which ledmay
may to this
have
have
droplets during solvent evaporation, which may have led to this characteristic shape [25]. After characteristic
led
led to
to this
this shape [25].
characteristic
characteristic After shape
shapemanufacturing
[25].
[25]. After
After
the formulations,
manufacturing
manufacturing
manufacturing the
the
thethe influence ofthe
formulations,
formulations,
formulations, theinfluence
the
the spray-drying
influence
influence of
of the
ofthe technique
the spray-drying
spray-drying
spray-drying on the DZ aqueous
technique
technique
technique on
on
onthe thesolubility
the DZ
DZ was
aqueous
DZaqueous
aqueous
studied
solubility
solubility
solubility bywas
wascomparing
was studied
studied
studiedby the
by amount
comparing
bycomparing
comparing oftheDZ
the that
amount
theamount
amount wasof ofdissolved
of DZ
DZ that
DZthat thatwas by formulations
was
was dissolved
dissolved
dissolvedby by with the amount
formulations
byformulations
formulations with
with
with that
the
the
the
was
amount
amount dissolved
amountthat that was
thatwas by the
wasdissolvedPMs
dissolved
dissolvedby in the
by the
bythe solubility
thePMsPMs
PMsin ininthesaturation
the solubility
thesolubility tests. The
saturation
solubilitysaturation results
saturationtests. tests.have
tests.TheThe shown
results
Theresults that
resultshave have spray
shown
haveshown drying
shownthat that
that
effectively
spray
spray drying
spraydrying dryingincreased
effectively
effectively
effectivelyDZ increased
solubility
increasedcompared
increased DZ
DZ solubility
DZsolubility
solubility with the PMs. with
compared
compared
compared with
withthe the PMs.
thePMs.
PMs.
DSCDSC and XRPD assessed DZ solid state. The DSC analyses revealedthe
DSC
DSC and
and
and XRPD
XRPD
XRPD assessed
assessed
assessed DZ
DZ
DZ solid
solid
solid state.
state.
state. The
The
The DSC
DSC
DSC analyses
analyses
analyses revealed
revealed
revealed theabsence
the
the absenceof
absence
absence ofendothermic
of
of endothermic
endothermic
endothermic
or exothermic peaks at DZ melting point of (336 ◦ C), showing that there was no interference caused
or
or exothermic
orexothermic
exothermicpeaks peaks
peaksat atatDZ
DZ melting
DZmelting
meltingpoint point
pointof of (336
of(336
(336°C),°C), showing
°C),showing
showingthat that there
thatthere
therewas was
wasno no interference
nointerference
interferencecaused caused
caused
by
by the
bythe excipients
theexcipients or
excipientsor their
ortheir mixture
theirmixture on
mixtureon DZ
onDZ solid-state
DZsolid-state characterization
solid-statecharacterization in
characterizationin the
inthe formulations.
theformulations. DZ
formulations.DZ melting
DZmelting
melting
peak
peak in PM showed that the solid state of drug had no alterations. The absence of a DZ melting
peak in PM showed that the solid state of drug had no alterations. The absence of a DZ meltingpeak
in PM showed that the solid state of drug had no alterations. The absence of a DZ melting peak
peak
Pharmaceutics 2019, 11, 492 13 of 17

by the excipients or their mixture on DZ solid-state characterization in the formulations. DZ melting

peak in PM showed that the solid state of drug had no alterations. The absence of a DZ melting peak
may indicate the transformation of crystalline DZ into an amorphous form during the spray drying
process, which would partially explain the enhancement in solubility [26]. The PVP used in this study
presented two endothermic peaks at 154 and 168 ◦ C. Although they are normal temperatures for Tg
of PVP K90, this unusual DSC trace may be associated to changes in molecular weight, crystallinity
degree, and more notably in purity of PVP obtained from different origins [27,28].
The XRPD analyses of the formulations are consistent with previous DSC data, showing that
DZ has an amorphous solid state. The amorphous state consists of molecules that are randomly
distributed with high free energy. Little or no energy is required to separate them, thus facilitating
their dissolution [29] and improving their bioavailability [30].
Various excipients such as alkalinizing and acidifying agents and processes have been used to
obtain amorphous SDs by the spray-drying technique [12,13,31]. However, little information about
their influence on the polymeric carrier is available. The GPC technique was used to study the influence
of process conditions and excipients on PVP Mw characteristics. PVP is a water-soluble polymer in a
wide range of pH (1–10). However, under conditions of alkalinity and high temperatures, cross-linking
may occur. Thus, PVP may become insoluble, depending on the extent of cross-linking, but it remains
swellable [32]. PVP may be used for sustained or controlled release. When there is an increase in Mw,
there is also a modification of the drug release rate [33]. This may be related to the slowest release
rate of the TG-pHM SD with a higher polymer ratio (F8 and F10). Thus, during the development and
manufacturing of pHM SDs, molecular changes in PVP should be considered to ensure that the drug
release kinetics are not impaired.
The dissolution phase was performed under sink conditions by using a USP apparatus IV
(flow-through cell). This apparatus is the most suitable pharmacopeical method for testing poorly
water-soluble drugs and distinguishing between different formulations, thus decreasing the number of
tests and animals in pharmacokinetic studies [34]. The results showed that the F1 release profile may
be correlated with the crystalline state that was observed in the XRPD and DSC analyses. The highest
polymer:drug ratio is known to have a beneficial influence on solubility and consequently drug
release [35]. For this reason, for all of TG systems, a 1:4 DZ:polymer ratio was maintained, and the
influence of SDS (5% and 10% w/w) was evaluated. Notably, surfactants might enhance drug wettability,
decrease surface tension, trigger drug release from SDs, and prevent nucleation and crystal growth [36].
Moreover, SDS will be above the critical micelle concentration (CMC) during the drying stage of the
spray drying. Above CMC concentration, the drug can be entrapped in different regions of the micelle
and generally result in enhanced solubility of poorly soluble drugs [37].
These effects could be the reason for the improved results when 10% SDS was used. In recent
studies, pHM SDs were successfully used to improve the aqueous solubility of poorly soluble ionizable
drugs [12,13]. The in vitro release profile showed that SG-pHM SD (Figure 7C) had faster and higher
dissolution rates. TG-pHM SD were assessed by adding a surfactant and with high drug loading (F7
and F9). Even under this condition, the formulations reached fast release, comparable to previous
SDs. F8 and F10 had the highest DZ:polymer ratio (1:4) and reached some sustained release, which can
be an advantage compared with SG-pHM SD. The in vitro drug release was not changed by a higher
amount of SDS. In addition to the aforementioned factors, the enhancement of in vitro release by SDs
may also be due to a decrease in particle size at the molecular level and decrease in agglomeration,
porosity, and amorphization of the crystalline state [11]. The most important factor that controls drug
release from SDs obtained with hydrophilic polymers is the establishment of a gel layer around the
system where water is adsorbed [12]. At this site, release is caused by water infiltration, the swelling of
polymers, drug dissolution and diffusion, and matrix erosion [34]. The drug is soluble and released
into the dissolution medium until a saturation solubility limit is reached. Henceforth, dissolution
occurs under non-sink conditions, leading to nucleation and crystal growth in the gel layer, the extent
of which depends on the carriers and drug concentration.
Pharmaceutics 2019, 11, 492 14 of 17

Therefore, despite the apparent advantage of SG and TG, the generation of a supersaturated gel
layer with a low threshold of saturation solubility can lead to insufficient oral bioavailability of the
drug, which is likely to occur due to precipitation during release into the gastrointestinal tract [38].
Daidzein has pH-dependent solubility, and Na2 CO3 allows the formation of a pH microenvironment
with high saturation solubility threshold of the drug in the gel layer of SG-pHM SD. Furthermore, pHM
SDs can release the drug regardless the pH of the environment [12,39]. The advantage of TG-pHM
SD is the possibility to further increase the saturation solubility threshold in the gel layer due to
presence of surfactant. TG-pHM SD carriers also play a crucial role in maintaining supersaturation
and precipitation inhibition in vivo, which is widely accepted as critical in enhancing solubility in the
gastrointestinal tract [40]. The release profiles generated by the flow-through cell shows that both
pHM-SD systems can enhance DZ bioavailability.
Further characterization of the release kinetic profile, which is based on the data from the in vitro
release tests, was performed by fitting the data to mathematical models, including zero and first
orders, Higuchi, Hixson-Crowell, and Korsmeyer-Peppas (power law). These mathematical models
can provide insights into the physical mechanisms and kinetics of drug release from SDs. Anomalous
transport is a combination of Fickian diffusion and matrix swelling. The diffusion process was
predominant for F3, which is likely to occur due to rapid elimination of the polymer that may be caused
by its low amount and higher dissolution aided by the surfactant and causes consequent precipitation
of the drug in the flow-through cell. In the super case II, the mechanism involves the relaxation of
polymer chains and corresponds to zero-order kinetics. The release kinetics of SDs are controlled by
the gel layer and depend on composition, stability, concentration, viscosity, and the chemical structure
of the polymer [41].
In order to improve the oral absorption of BCS class IV polyphenol, we manufactured different
types of SD systems and assessed DZ release. The characterization, saturation solubility, and
in vitro release data were considered when choosing an optimized SD for comparisons with the oral
bioavailability of purified DZ, thus requiring fewer animals. Formulations with high amount of PVP
could be advantageous due to its mucoadhesive properties that are likely to help to enhance drug
delivery [42]. The possibility to further increase the saturation solubility threshold in the gel layer, the
role of Na2 CO3 and SDS in maintaining supersaturation and precipitation inhibition in vivo [40] and
the lack of data available about TG-pHM, made this an advantageous and interesting formulation to
be evaluated in vivo. Based on this, we decided to performed pharmacokinetic study on F10.
In order to ensure the reliability of the results of the pharmacokinetic study, a HPLC-MS/MS
method was validated. The plasma concentration profiles of DZ showed two peaks. The second
peak is described in the literature and corresponds to the enterohepatic recirculation of DZ that was
excreted in the bile and reabsorbed [43,44]. The plasma concentration profiles of purified DZ and
F10 had different pharmacokinetics behavior. After oral administration of purified DZ, the plasma
concentration increased slowly, unlike formulation F10. For F10, the first drug peak occurred at 15 min,
representing a 20-fold increase at this time. Pharmacokinetic parameters (Cmax, Tmax, AUC0–24 h,
and AUC0→∞) were significantly different between purified DZ and F10 (p < 0.05). For example, the
Cmax and AUC0–24 h of F10 increased 5.7-fold and 2.4-fold, respectively, compared with purified DZ.
These results show that F10 can improve bioavailability of drug.
Many efforts have been made to manufacture solid dosage forms with high bioavailability
by changing the solid state [31]. The obtained F10 formulations by spray-drying technique also
provided a higher Cmax and lower Tmax compared with DZ-loaded phospholipid complex polymeric
nanoparticles and DZ-loaded cyclodextrin inclusion complex polymeric nanoparticles at the same
dose [21,22]. The in vivo pharmacokinetic data have shown that the accelerated initial DZ release that
was achieved by the spray drying technique and incorporation of excipients into SDs dramatically
improved the extent of oral DZ absorption.
Pharmaceutics 2019, 11, 492 15 of 17

5. Conclusions
In this study, SG, TG, SG-pHM, and TG-pHM SDs that contained DZ were successfully
manufactured by the spray-drying technique using PVP (K90), SDS, and Na2 CO3 as the most
appropriate excipients. All of the formulations had high DZ content, no detectable crystallinity,
enhanced relative solubility, and an enhanced dissolution rate, with the exception of F1 formulation.
PVP Mw may change based on the specific conditions. The results showed that a major obstacle when
using amorphous SG and TG is the limited DZ solubilizing capacity. Thus, the pH-dependent solubility
of DZ was reflected by the dissolution rate, and only the pHM SDs reached complete dissolution even
with high DZ loading. The potential of TG-pHM SDs to increase solubility in the gastrointestinal
tract and enhance absorption was reflected by enhanced oral bioavailability, which was shown by the
pharmacokinetic analyses (i.e., increases in AUC and Cmax and a decrease in Tmax). TG-pHM SD
formulation has the potential to enhance the therapeutics effects of DZ by increasing its absorption,
thus broadening the therapeutic applications of such drugs. These results also provide an experimental
basis for using spray-dried pHM SDs that contain poorly water-soluble ionizable polyphenols and
drugs as a feasible drug delivery system to provide immediate release. Overall, it is an attractive
alternative to enhance the therapeutic effects of polyphenolic drugs belonging to BCS class IV.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/11/10/492/s1,

HPLC-ESI-MS/MS method development and validation; Figure S1: Total ion chromatogram of electrospray
ionization mass spectrum from purified material and daidzein ion products. Figure S2: Representative MRM
chromatograms of blank plasma for daidzein (DZ) (A) and for IS (B); blank plasma spiked with the analyte at
LLOQ (C); IS (350 ng/mL) (D); plasma samples after oral administration of pure DZ (10 mg/kg) (E); and F10 (10
mg/kg) (F); Table SI: The effect of hydrophilic polymers, surfactants, and alkalizers with different proportions
(0.5, 1, and 2% w/v) on the solubility of daidzein (DZ) at 37 ◦ C (mean ± S.D, n = 3). Table SII: Fitting (r2 ) and
release constant (K) parameters of mathematical models for daidzein (DZ) release from the solid dispersion (SD)
formulations (F1–F10). Table SIII: Intra-day and inter-day precisions and accuracies, extraction recoveries, and the
matrix effect for the determination of daidzein (DZ) from the assay samples (mean, n = 5).
Author Contributions: All authors have participated in the research and/or preparation of the article: B.P.D.F.
was responsible for conception and design of the study; G.P.P. and F.G.B. performed the in vitro and in vivo
experiments and wrote the manuscript; C.V.N. and T.U.-N. contributed by advising on the in vivo experiments,
critical reading and feedback on the manuscript. All authors have revised and approved the final article.
Funding: The research was financially supported by the National Council for Scientific and Technological
Development (CNPq), the Federal Agency for Support and Evaluation of Graduate Education (CAPES), and the
Postgraduate Program in Pharmaceutical Sciences of the State University of Maringá (PCF-UEM).
Acknowledgments: The authors acknowledge Complexo de Centrais de Apoio à Pesquisa (COMCAP),
which is a multi-user laboratory of analytical equipment and facilities at State University of Maringá, for
providing infrastructure.
Conflicts of Interest: The authors declare no conflict of interest.

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