Introduction
Saw palmetto (Serenoa repens [W.Bartram] Small [Arecaceae]; synonym: Sabal serrulata [Michx.] Schult.f.) is a small palm tree, native to southeastern North America, particularly
Florida [1]. Since the 1990s, saw palmetto has been one of the 10 top-selling herbal medicines
in the United States, with a worldwide turnover of about $700 million per year [2].
In Europe, numerous preparations containing saw palmetto extracts are marketed as
herbal medicinal products or food supplements and they are widely used to treat discomforts
related to prostatic hyperplasia [3], [4], [5]. Saw palmetto berry herbal drug and its extracts obtained by suitable extracting
procedures using hydroalcoholic mixtures, supercritical carbon dioxide, or n-hexane
are reported in United States and European pharmacopoeias in dedicated monographs
[6], [7].
Recently, the European Medicine Agency has indicated that preparations based on the
hexane soft extract (DER 7 – 11 : 1; 320 mg daily) can be designated as well-established
herbal medicinal products and can be used for the symptomatic treatment of benign
prostatic hyperplasia [4].
Moreover, the effectiveness of a special botanical product (Prostamev Plus) based
on saw palmetto (320 mg) plus pineapple stem extract (25 mg) and plus nettle root
extract (120 mg) has been recently compared to saw palmetto alone in reducing the
symptoms of inflammatory prostatitis (bacterial and nonbacterial ones). Prostamev
Plus represents a unique combination of botanicals because bromelain present in the
pineapple stem extract is particularly active in localized inflammation in the presence
of edema, while nettle extract is particularly effective in the symptomatic treatment
of micturition disorders [8]. Hence, the clinical study confirmed the advantage of using a cotreatment that associates
nettle and pineapple to saw palmetto because of the improvement of uroflowmetry values
and in the scores of the International Index of Erectile Function-5 and National Institute
of Health-Chronic Symptom Index questionnaires. The study reported no side effects
in addition to those provided for the antibiotic therapy alone. In conclusion, the
groups treated with Prostamev Plus showed an improvement of all the investigated parameters
compared to the groups treated with saw palmetto alone, with or without associated
antibiotic therapy [9].
Prostamev Plus is marketed in the form of soft gelatin capsules for oral administration
containing a coarse suspension due the mixture of lipid and water-soluble constituents,
namely saw palmetto CO2 extract plus nettle root extract and pineapple stem extract. For instance, the lipid
saw palmetto CO2 extract has a similar behavior of the low soluble drugs, which have high intra- and
inter-subject variability and lack of dose proportionality because the absorption
rate from the gastrointestinal lumen is controlled by the dissolution. Various approaches
can be used to improve the dissolution rate of these drugs and consequently to optimize
their bioavailability after oral administration. Among them are MEs, which are optically
isotropic and thermodynamically stable liquid preparations with an average structure
size below 300 nm. They consist of the oil phase, water, and surfactants and are especially
suitable for plant extracts and complex mixtures. Preconcentrates of MEs represent
alternative flexible formulations. These are homogeneous liquids, which contain oil,
surfactants, and the drug without aqueous phase. They are also known as SMEDDSs. When
a SMEDDS is in contact with water or gastrointestinal fluid, it spontaneously forms
a ME with gentle agitation. The digestive motility of the stomach and the intestine
can provide the agitation necessary for self-emulsification. SMEDDSs form transparent
MEs with a droplet size of less than 300 nm and are generally preferred over the regular
MEs for oral preparations because SMEDDSs can decrease the volume and it is easy to
fill in the soft gelatin capsules.
The aim of the present study is the development of innovative formulations based on
the complex mixture of lipophilic and hydrophilic constituents, namely from saw palmetto
CO2 extract plus nettle root extract and pineapple stem extract. Based on the solubility
studies of the different extracts, two MEs and two SMEDDSs were developed and fully
characterized by DLS, TEM, and HPLC-DAD in order to evaluate the size, the homogeneity,
the morphology, and the loading capacity and encapsulation efficiency. The chemical
and physical stabilities were also investigated including the stability in simulated
gastric fluid, followed by simulated intestinal fluid stability, avoiding precipitation
or increase in the globule size as this may affect the product performance in vivo. Therefore, the components used in the system should have high solubilization capacity
for the drug, ensuring the solubilization of the drug in the resultant dispersion.
The in vitro permeation studies were assessed by PAMPA to determine the suitability of the developed
nanoformulations and compare the permeation of the conventional coarse suspension
loaded in soft gelatin capsules.
Results and Discussion
The study started with the investigation of aqueous solubility of the extracts of
the CB. Both nettle (Urtica urens L. [Urticaceae]/Urtica dioica L. [Urticaceae]) dried extract (70% EtOH) root and pineapple (Ananas comosus L. [Bromeliaceae]) stem extract (1850 GDU/g bromelain) were highly soluble in water
according to the BCS classification. The doses of nettle (120 mg) and pineapple (25 mg)
extracts were soluble in less than 250 mL water. By contrast, saw palmetto CO2 extract (320 mg) resulted very poor soluble in 250 mL water [10].
The successive step was the selection of a representative marker among the constituents
of saw palmetto extract for the HPLC-DAD analysis, being a GC instrument not suitable
for the settled assays in order to develop and characterize the innovative drug delivery
systems. The saw palmetto extract was solubilized in a mixture of MeOH/dichloromethane
(1 : 1) and it was analyzed using different columns and solvent mixtures. The presence
of β-carotene was established on the basis of the characteristic UV spectrum and on the
value of the retention time by comparison with β-carotene standard. Accordingly, β-carotene was selected as a suitable marker of both saw palmetto commercial extract
and the CB because no interferences were found with the other constituents of the
preparation. An HPLC-DAD method previously developed and validated in our lab for
the for the analysis of goji berry and its extracts [11] was used for the quantification of β-carotene, which resulted ~ 0.18 mg/mL of extract. The chromatographic profile of
the commercial extract at 450 nm is reported in [Fig. 1]. Accordingly, the HPLC-DAD method was used to evaluate the solubility of the saw
palmetto extract in various oils and surfactants to select the appropriate components
of the MEs and SMEDDSs ([Table 1]). Soy oil was selected as the oily phase, while Tween 80 and Kolliphor EL were selected
as surfactants.
Fig. 1 Chromatographic profile of the Serenoa repens extract (top) and chromatogram of β-carotene standard reference (bottom) at 450 nm.
Table 1 Solubility of β-carotene from saw palmetto extract in various oils, surfactants, and solvents. Data
displayed as the mean ± SD (n = 3). Measurements were from three independent experiments.
|
Excipient/solvent
|
β-carotene solubility (mg/mL)
|
Percentage (%) of solubilised saw palmetto extract
|
|
Almond oil
|
0.095 ± 0.008
|
52.78
|
|
Soybean oil
|
0.116 ± 0.012
|
64.44
|
|
Vitamin E
|
0.101 ± 0.015
|
56.11
|
|
Sunflower oil
|
0.096 ± 0.010
|
56.33
|
|
Oleic acid
|
0.101 ± 0.014
|
56.11
|
|
C. sativa seed oil
|
0.111 ± 0.011
|
61.67
|
|
B. officinalis seed oil
|
0.123 ± 0.013
|
68.33
|
|
Labrafil
|
0.094 ± 0.007
|
52.22
|
|
Capryol 90
|
0.118 ± 0.012
|
65.56
|
|
Triacetin
|
0.020 ± 0.001
|
11.11
|
|
A. spinosa kerne oil
|
0.103 ± 0.011
|
57.22
|
|
Tween 80
|
0.121 ± 0.012
|
67.22
|
|
Tween 20
|
0.109 ± 0.010
|
60.56
|
|
Transcutol HP
|
0.087 ± 0.008
|
48.33
|
|
Kolliphor EL
|
0.122 ± 0.013
|
67.78
|
|
Dicloromethane : MeOH 1 : 1
|
0.180 ± 0.010
|
100.56
|
|
Water
|
Not detected
|
–
|
Accordingly, two MEs were obtained using a water titration method. Each sample was
visually checked after equilibrium and determined as being a clear ME, emulsion, or
gel. Their composition is reported in [Table 2]. The extracts of CB were added to the lipophilic phase and the corresponding loaded
MEs (CBM1 and CBM2) were obtained using deionized water added drop by drop, under
gentle agitation, to the oily mixture. CBM1 and CBM2 composition is reported in [Table 2]. CBM2 (Fig. 1S, Supporting Information) had a lower content of water when compared with CBM1 (30.2
vs. 17.3%). A pseudoternary diagram of CBM2 is available as Supporting Information
Table 2 Composition of the optimised MEs and SMEDDs.
|
Nanocarrier
|
Soy oil (g)
|
Kolliphor EL (g)
|
Tween 80 (g)
|
Water (g)
|
Saw palmetto extract (g)
|
Nettle extract (g)
|
Pineapple extract (g)
|
|
M1
|
0.2
|
–
|
1.8
|
4.9
|
–
|
–
|
–
|
|
M2
|
0.2
|
1.8
|
–
|
5.0
|
–
|
–
|
–
|
|
CBM1
|
0.055
|
–
|
0.50
|
2.25
|
3.20
|
1.20
|
0.25
|
|
CBM2
|
0.15
|
1.33
|
–
|
1.29
|
3.20
|
1.20
|
0.25
|
|
CBS1
|
0.28
|
–
|
2.52
|
–
|
3.20
|
1.20
|
0.25
|
|
CBS2
|
0.28
|
2.52
|
–
|
–
|
3.20
|
1.20
|
0.25
|
Two SMEDDSs loaded with the CB were also developed, namely CBS1 and CBS2. Their composition
is reported in [Table 2].
All developed preparations (M1, M2, CBM1, CBM2, CBS1, and CBS2) were characterized
by DLS in terms of size, polydispersity and ζ-potential. All the formulations were homogeneous systems, with a narrow size distribution
and low values of the PDI and mean diameter ([Table 3]). Among the loaded systems, CBM2 and CBS2 were the most appropriate. Analysis by
TEM of MEs confirmed the size of the internal phases. In [Fig. 2], TEM analysis of CBM2 is reported, confirming the presence of droplets with a size
of ~ 200 nm. TEM analysis of CBS1 and CBS2 were carried out using distilled water
after dilution 1 : 50 v/v. In [Fig. 3], TEM analysis of CBS2 after dilution is reported confirming the presence of droplets
with a size of not more than 200 nm.
Table 3 DLS characterization of MEs and SMEDDSs in term of size (nm), polydispersity, and
ζ-potential (mV). Data displayed as the mean ± SD (n = 3). Measurements were from three
independent experiments.
|
Nanocarrier
|
Size (nm)
|
Polydispersity
|
ζ-potential (mV)
|
|
M1
|
15.8 ± 2.1
|
0.22 ± 0.09
|
− 43.50 ± 2.33
|
|
M2
|
18.5 ± 4.0
|
0.18 ± 0.11
|
− 18.6 ± 3.12
|
|
CBM1
|
243.6 ± 11.4
|
0.31 ± 0.19
|
− 35.2 ± 6.31
|
|
CBM2
|
220.1 ± 9.6
|
0.30 ± 0.26
|
− 16.7 ± 4.11
|
|
CBS1
|
399.7 ± 18.4
|
0.41 ± 0.10
|
− 20.3 ± 1.55
|
|
CBS2
|
239.0 ± 13.4
|
0.28 ± 0.14
|
− 25.9 ± 2.56
|
Fig. 2 TEM image of nanoformulation CBM2.
Fig. 3 TEM image of nanoformulation CBS2 after dilution with water (1 : 50 v/v).
The optimized nanoformulations were stored away from light at 4 °C for 21 d in order
to assess their physical stability by monitoring the size of the dispersed phase by
DLS. CBS2 was quite stable: no phase separation occurred and the size of the droplets
remained nearly constant, as reported in [Fig. 4 A]. In the case of ME CBM2, no phase separation occurred during storage; however, the
size of the droplets decreased slightly at day 14 to return to the original values
at day 21. This behavior could be due to the fact that MEs are thermodynamically stable
and form spontaneously (or with very low energy input) under the right conditions,
but they are highly dynamic systems and, as such, undergo continuous and spontaneous
fluctuations that consist of phase inversion and changes in droplet size [12].
Fig. 4 Physical stability (A) and chemical stability (B) of CBM2 and CBS2 within 3 wk. Data are the mean ± SD (n = 3). Measurements were
from three independent experiments.
TEM analysis confirmed the data obtained by DLS analyses: sizes ranged between 200
and 250 nm for extract loaded nanocarriers. In addition, chemical stability was obtained
by quantifying the residual amount of β-carotene by using the HPLC-DAD method reported in the experimental part. As reported
in [Fig. 4 B], the concentration of β-carotene did not decrease significantly during the whole period of the test. After
3 wk the residual β-carotene was ~ 89% in the CBM2 and ~ 95% in the CBS2.
To further evaluate the appropriateness of the developed nanosystems for oral use,
intragastric stability was tested in SGM (pH 2) in the presence of pepsin for 2 h,
followed by treatment with SIM (pH 7) in the presence of the pancreatin-lipase-bile
extract mixture for 2 h. Samples were collected and analyzed by DLS analysis to check
their physical stability. The analyses confirmed the physical stability of the systems
in terms of size and homogeneity. CBM2 was stable and no aggregation or degradation
phenomena occurred. Sizes of the dispersed phase in SGM were 114.4 ± 1.5 nm, while
in SIM were 109.0 ± 2.5 nm at the end of the tests and were comparable with those
found before the tests. In addition, the CBS2 was stable after the tests in SGM (173 ± 3.5 nm)
and SIM (144.6 ± 2.7 nm).
Finally, in order to predict the oral permeability of saw palmetto extract, the PAMPA
test was utilized. Recently, PAMPA has gathered considerable interest in pharmaceutical
research as a helpful complement and in many cases an alternative test to the Caco-2
assay [13], [14], [15], [16]. The experiments were carried out measuring the ability of the saturated aqueous
solution of saw palmetto extract, of the saturated aqueous solution of the CB, of
CBM2 and CBS2, to diffuse from the donor to acceptor compartment, through a membrane,
in order to evaluate the influence of the formulation on the permeability of saw palmetto.
Both formulations displayed an appreciated permeation of β-carotene in comparison with the extract and the CB, used as control ([Table 4]). After 2 h of PAMPA test, permeated β-carotene was under the limit of detection (LOD = 0.87 µg) for all samples, with the
exception of CBS2, which displayed 2% of permeated β-carotene. After 4 h, the permeation increased for both the nanoformulations, while
it was detected a permeation for the extract and the CB. After 6 h the best formulation
for the oral delivery was the CBM2, with 17% of permeated β-carotene. This value is quite significant because according to the literature [17] for low soluble drugs a PAMPA flux in the range from 5 to 25% corresponds to the
3 – 70% passive oral human absorption in vivo. Finally, a required recovery more than 80% for an acceptable in vitro prediction was obtained for almost all the samples ([Table 4]).
Table 4 Quantity (%) of β-carotene permeated in the PAMPA test. Data are the mean ± SD (n = 3). Measurements
were from three independent experiments.
|
Sample
|
Incubation time
|
% permeated β-carotene
|
% of recovery
|
|
nd: not detected
|
|
CBM2
|
2 h
|
nd
|
–
|
|
4 h
|
2.0 ± 0.1%
|
99%
|
|
6 h
|
17.4 ± 2.1%
|
99%
|
|
CBS2
|
2 h
|
2.2 ± 0.3%
|
87%
|
|
4 h
|
4.2 ± 0.4%
|
83%
|
|
6 h
|
7.1 ± 0.3%
|
78%
|
|
CB
|
2 h
|
nd
|
–
|
|
4 h
|
nd
|
–
|
|
6 h
|
1.3 ± 0.2%
|
86.5%
|
|
Saw palmetto extract
|
2 h
|
nd
|
–
|
|
4 h
|
nd
|
–
|
|
6 h
|
3.1 ± 0.4%
|
98%
|
Accordingly, to the increase of passive diffusion, an enhancement of the oral bioavailability
is expected. ME and SMEDD manufacturing and scale-up is simple and do not require
specialized equipment and can represent a good alternative to the traditional formulations.
Additional imperative advantages of MEs over conventional formulations are almost
100% entrapment of drug, with a high stability over time and high versatility for
both polar and lipophilic constituents, and potentially excellent vectors for various
routes of administration.
Materials and Methods
Materials
The following products were supplied by Farmaceutica MEV: saw palmetto berry supercritical
CO2 extract (DER 8.0 – 14.3 : 1, containing not less than 70.0% and not more than 95.0%
of fatty acids and not less than 0.2% and not more than 0.5% of sterols, calculated
on an anhydrous basis, lot n. 15/01604); nettle (U. urens/U. dioica) root dried extract (70% v/v EtOH, DER 12 – 16 : 1, containing 0.82% β-sitosterol, lot n. 15/01509); pineapple (A. comousus) stem powder (bromelain was 1850 GDU/g, lot n. 15/00941). Authentic samples of these
extracts are maintained at the Department of Chemistry “Ugo Schiff” under the following
voucher specimens: 20 – 2016 (saw palmetto extract), 21 – 2016 (nettle extract), 22 – 2016
(pineapple extract).
β-carotene (purity ≥ 98%, UV) was from Extrasynthese. Soybean oil, almond oil, tween
20, tween 80, DL-α-tocopherol acetate (vitamin E, purity ≥ 96%, HPLC), kolliphor EL, and triacetin were
purchased from Sigma-Aldrich; oleic acid was from Farmitalia, Carlo Erba Spa; transcutol
HP, capryol 90, and labrafil were from Gattefossé; sunflower oil was from Coop; and
Cannabis sativa L. (Cannabaceae) seed oil, Borago officinalis L. (Boraginaceae) seed oil, and Argania spinosa (L.) Skeels (Sapotaceae) kernel oil were purchased from Galeno. EtOH and MeOH analytical
reagent, acetone, and MeOH HPLC grade and HCOOH (≥ 98%) were purchased from Sigma-Aldrich.
Water was purified by a Milli-Qplus system from Millipore. Phosphotungstic acid was from Electron Microscopy Sciences.
Cholesterol, lecithin, dichloromethane, DMSO, 1,7-pctadiene (≥ 98%), PBS bioperformance
certified, lipase from porcine pancreas, pepsin from porcine gastric mucose, bile
salts, HCl were purchased from Sigma-Aldrich.
Methods
HPLC-DAD assays
For qualitative and quantitative analysis, an HP 1100 L instrument with a diode array
detector and managed by a HP 9000 workstation (Agilent Technologies) was used. Data
were processed with HP ChemStation software (Agilent). Separation was performed at
24 °C on Luna RP C18 (250 × 4.6 mm), 5 µm particle size (Phenomenex). The mobile phase
consisted of an isocratic mixture of Acetone/MeOH 55 : 45 v/v. The flow rate was 1 mL/min
and the total run time was 20 min, with a post time of 3 min. The sample injected
volume was 10 µL. The UV spectra were recorded between 200 and 600 nm. Chromatographic
profiles were registered at 254, 350, 430, and 450 nm. The identification of β-carotene was performed by comparing the retention time and the UV spectra of the
peaks in the samples with those of authentic reference samples.
Calibration curve: Standard solutions were freshly prepared by a serial dilutions of stock solution
of β-carotene in acetone/MeOH 1 : 1 to obtain a range of concentration between 0.002 and
0.352 µg/mL.
Quantitative determination of β-carotene: External standard method was applied to quantify β-carotene using a regression curve and to increase confidence in our data. Samples
were analyzed in triplicate. Measurements were performed at 450 nm, the maximum absorbance
of β-carotene. Results were expressed as the mean ± standard deviation (SD) of three separate
experiments.
Preparation of samples for HPLC-DAD analysis: Commercial saw palmetto extract and the CB were solubilized in MeOH : dichloromethane
1 : 1, ultrasonicated for 5 min and centrifuged for 4 min at 14 000 rpm prior to injection
in HPLC. Aqueous solubility was determined by dissolving commercial extract in deionized
water at room temperature until saturation. The residue was eliminated by centrifugation
and the solution was analyzed by HPLC.
Solubility studies
To find out an appropriate excipient to develop MEs and SMEDDSs, the solubility of
saw palmetto extract and the CB in different oils and surfactant was determinate as
follows. An excess amount of SM extract was added to 5 mL of each selected excipient
(almond oil, soybean oil, tocopherol acetate, sunflower oil, C. sativa seed oil, B. officinalis seed oil, A. spinosa kernel oil, triacetin, oleic acid, labrafil, capryol 90, transcutol HP, kolliphor
EL, tween 20, tween 80, and water). Each mixture was shaken at 25 °C for 24 h and
then was centrifuged at 14 000 rpm for 10 min. The concentration of the components
of the extract was determined by HPLC-DAD after dilution with MeOH/dichloromethane
(1 : 1). The analyses were from three independent experiments.
Preparation of MEs, CBM1, and CBM2
Pseudo-ternary phase diagrams were constructed using Chemix School version 3.60 software
to obtain the concentration range of all components in which they form ME. The pseudo-ternary
phase diagrams were constructed using the water titration method. Each oil-surfactant
mixture was diluted at 50 °C under vigorous stirring dropwise with water. After equilibrium,
each sample was visually checked and the phase boundary was determined by observing
the changes in the sample appearance from turbid to transparent or from transparent
to turbid and by evaluating if ME, emulsion, or gel was present. Optimized preparations
are reported in [Table 2].
CBM1 and CBM2 were prepared by dissolving the CB into the oil-surfactant mixture,
by adding the required quantity of water, and stirring for 24 h to form a clear and
transparent dispersion ([Table 2]).
Preparation of SMEDDSs, CBS1, and CBS2
Empty SMEDDSs were prepared under magnetic stirring with weighted quantity of oil
and surfactant for 24 h at room temperature ([Table 2]). The mixture was diluted up 100 times with Milli-Q water and mixed by vortex until
a transparent preparation was obtained. CBS1 and CBS2 were prepared by dissolving
the CB in the mixture of oil and surfactant at room temperature for 24 h. After a
100-fold dilution with Milli-Q water, this mixture was mixed by vortex until a transparent
preparation was obtained. The resulting nanoformulations were stored at 4 °C.
Self-emulsification and precipitation assessment evaluation of SMEDDSs
Self-Emulsification time and precipitation assessment evaluation of the self-emulsifying
properties of SMEDDSs formulations were performed by visual assessment as previously
reported [17]. In brief, different compositions were categorized on speed of emulsification, clarity,
and apparent stability of the resultant emulsion. Visual assessment was performed
by dropwise addition of 1 mL of SMEDDS into 250 mL of distilled water. This was done
in a glass beaker at room temperature, and the contents were gently stirred magnetically
at ~ 100 rpm. Precipitation was evaluated by visual inspection of the resultant emulsion
after 24 h. The nanoformulations were then categorized as clear (transparent or transparent
with bluish tinge), nonclear (turbid), stable (no precipitation at the end of 24 h),
or unstable (showing precipitation within 24 h).
Robustness to dilution for SMEDDS
Robustness to dilution was studied by diluting SMEDDS to 50, 150, and 250 times with
distilled water and phosphate buffer with gastric and intestinal pH. The diluted SMEDDSs
were stored for 24 h and observed for any signs of phase separation, precipitation,
or coalescence. A confirmation of the robustness to dilution of CBS2 at acid pH was
obtained by TEM analysis.
Characterization of MEs and SMEDDSs in terms of particle size, polydispersity index,
and ζ- potential
Particle size of the developed formulations was measured by a DLS, Zetasizer Nano
series ZS90 (Malvern Instruments) equipped with a JDS Uniphase 22 mW He-Ne laser operating
at 632.8 nm, an optical fiber-based detector, a digital LV/LSE-5003 correlator, and
a temperature controller (Julabo water bath) set at 25 °C. Time correlation functions
were analyzed to obtain the hydrodynamic diameter of the particles and the particle
size distribution (PDI) using the ALV-60X0 software V.3.X provided by Malvern. Autocorrelation
functions were analyzed by distribution, fitting a multiple exponential to the correlation
function to obtain particle size distributions. In particular, polydispersity values
were calculated for each peak as peak width/mean diameter. Scattering was measured
in an optical quality 4 mL borosilicate cell at a 90-degree angle, diluting the samples
from 5 to 30-fold in Milli-Q water. ζ-potentials of the nanocarriers were measured using a Malvern Instruments Zetasizer
Nano series ZS90. For all samples, an average of three measurements at stationary
level was taken. The temperature was kept constant at 25 °C by a Haake temperature
controller. The ζ-potential was calculated from the electrophoretic mobility, µE, using the Henry correction to Smoluchowskiʼs equation. The analyses were from three
independent experiments.
Morphological analysis of MEs and SMEDDSs
Nanocarrierʼs dispersions were analyzed in terms of morphology and mean diameter by
transmission electron microscope (TEM, Jeol Jem 1010) and by scanning electron microscope
(SEM, Phenom G2 ProX, Phenom-World, Alfatest). Ten microliters of ME or SMEDDS dispersion
diluted 10 times was applied to a carbon film-covered copper grid. Most of the dispersion
was blotted from the grid with filter paper to form a thin film specimen, which was
stained with a phosphotungstic acid solution 1% w/v in sterile water. The samples
were dried for 3 min and then were examined under a JEOL 1010 electron microscope
at an accelerating voltage of 64 kV.
Stability studies of MEs and SMEDDSs
In order to evaluate the stability of the different formulations, the samples were
put into sealed glass vials and stored at 4 °C for 21 d. Their chemical and physical
stabilities were studied by monitoring the occurrence of phase separation, dispersed
phase size, and drug content at predetermined intervals (1 wk) by DLS and HPLC/DAD
analyses. Furthermore, to mimic physiological dilution process after oral administration,
the samples were diluted 10, 20, and 30-fold with Milli-Q water. The dilutions were
followed by gentle vortexing for 2 min at room temperature. DLS analyses confirmed
the physical stability of the systems in terms of size and homogeneity. The analyses
were from three independent experiments.
In vitro stability of MEs and SMEDDSs in the presence of SIM and SGM
The intragastric stability was tested in SGM as described previously [15]. Briefly, 5 mL of formulation were suspended in 5 mL SGM (0.32% w/v pepsin, 2 g
of sodium chloride, and 7 mL HCl dissolved in 1 L Milli-Q water and pH adjusted to
1.8 using 1 M HCl) and incubated in a water bath at 37 °C under shaking speed of 100
strokes/min. After 2 h, sample was collected to analyze the size and PDI. After digestion
in simulated stomach condition, the same sample was subjected to digestion under simulated
intestinal condition containing intestinal enzyme complex (lipase 0.4 mg/mL, bile
salts 0.7 mg/mL, and pancreatin 0.5 mg/mL) and calcium chloride solution 750 mM at
pH 7.0, 37 °C under shaking speed of 100 strokes/min. After 2 h digestion in SIM,
the sample was collected and its physical stability was checked by DLS analysis. The
analyses were from three independent experiments.
In vitro PAMPA
The PAMPA assay was used to predict passive absorption. The assay is carried out in
a 96-well, MultiScreen-IP PAMPA (Millipore Corporation) filter plate. The ability
of compounds to diffuse from a donor compartment, through a PVDF membrane filter pre-treated
with a lipid-containing organic solvent, into an acceptor compartment was evaluated.
Ten microliters of lecithin (10 g/L) and cholesterol (8 g/L) in 1,7-octadiene solution
were added to the filter of each well. Immediately after the application of the artificial
membrane, 150 µL of drug containing donor solutions (saw palmetto extract, blend of
extracts, CBS2 and CBM2 at the concentration) were added to each well of the donor
plate. Three hundred microliters of buffer (PBS, pH 2) was added to each well of the
acceptor plate. The acceptor plate was then placed into the donor plate, ensuring
that the underside of the membrane was in contact with buffer. The plate was covered
and incubated at room temperature under shaking for 6 h and permeation was evaluated
at 0.5, 2, 4, and 6 h.
At the end of the experiment, the samples from the donor and receptor plate were analyzed
for β-carotene concentration by HPLC-DAD. The experiment was performed in triplicate and
mean of three samples was used in the data analysis. Permeability of the compound
across the compartments and the recovery of the experiments were calculated using
the following formula:
