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Drug Dev Ind Pharm, Early Online: 1–8

! 2013 Informa UK Ltd. DOI: 10.3109/03639045.2012.749888

RESEARCH PAPER

Mixed lipid phase SMEDDS as an innovative approach to enhance

resveratrol solubility
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Katarina Bolko, Alenka Zvonar, and Mirjana Gašperlin

Faculty of Pharmacy, University of Ljubljana, Askerceva 7, Ljubljana, Slovenia

Abstract Keywords
Context: Despite its promising therapeutic activities, clinical use of resveratrol (RSV) is Castor oil, diglyceride, lipid-based delivery,
compromised with unfavorable biopharmaceutical properties, namely low water solubility. long-chain lipid, medium-chain lipid,
Objective: This work deals with improving RSV solubility and release rate through its incorporation microemulsion, phase diagram, self-
in innovative mixed lipid phase self-microemulsifying drug delivery systems (SMEDDS). microemulsifying
Methods: (Pseudo)ternary diagrams were constructed for different oils and surfactant mixtures.
Selected systems were further evaluated for RSV solubility, self-emulsification ability, accelerated History
stability, dynamic viscosity, compatibility with hard gelatin capsules and in vitro dissolution of RSV.
Results: Lipid phase composed of diverse lipid species, castor oil (long-chained triglyceride) and Received 31 July 2012
Capmul MCM (mixture of medium chain mono and diglycerides) allowed formulation of mixed Revised 30 October 2012
lipid SMEDDS with lower surfactants content (60% Cremophor EL/RH 40/RH 60). Mixed lipid Accepted 6 November 2012
For personal use only.

phase SMEDDS showed best self-emulsifying ability with regard to self-emulsifying time as well as Published online 10 January 2013
droplet size and monodispersity of microemulsions obtained upon SMEDDS dilution with
aqueous phase. Overall, incorporation of RSV in SMEDDS resulted in improved solubility (over 23-
fold) and dissolution rate compared to crystalline RSV. All SMEDDS formulations were adequately
viscous for filling into hard gelatin capsules (4150 mPas for empty SMEDDS;4400 mPas for RSV-
loaded SMEDDS) and no leaking was observed during three months of storage.
Conclusion: The presented work indicates the promising potential of mixed lipid SMEDDS
formulations for future development of SMEDDS with lower surfactant content and no added
cosolvents for incorporation of RSV and other poorly soluble drugs.

Introduction some of the parent molecule bioactivities and can serve as an

0 in vivo reservoir of RSV mobilizable by the intra-conversion10.
Resveratrol (RSV; 3,5,4 -trihydroxystilbene) is a natural
RSV exhibits sufficient permeability, with its apparent perme-
polyphenol, predominantly found in the skin of many
ability coefficient for apical to basolateral movement of
berries. Its reported benefits include antioxidant, anticancer,
2.0  105 m/s in Caco-2 cells11. Therefore, enhancement of its
anti-inflammatory and phytoestrogenic1,2. Additionally, it is held
water solubility was suggested as a viable option for improvement
primarily accountable for ‘‘French paradox’’, linking red wine
consumption with decreased risk of cardiovascular diseases2,3.
of poor RSV oral bioavailability, reportedly not exceeding
1%12,13. With the aim to improve RSV absorption by improving
13
20
However, its content in wine is very low (0.1–14.3 mg/L)4.
its solubility and dissolution rate, self-microemulsifying drug
Increased cardiovascular risk caused by alcohol consumption in
delivery systems (SMEDDS) were developed. To our knowledge,
large quantities presents an important drawback of using wine as a
no such systems with RSV have already been published.
source of RSV5. Supplementation of RSV therefore seems as a
SMEDDS are isotropic mixtures of lipids and surfactants that
logical solution. However, exploitation of RSV health promoting
form o/w microemulsions under dilution with intestinal fluids
effects is challenging due to its very limited bioavailability
following oral application14. The drug bioavailability is thereby
following oral application, caused by its poor biopharmaceutical
improved via enhanced solubilization and release rate, as shown
properties, namely low solubility and first-pass metabolism.
many times15–17. Furthermore, SMEDDS were also shown to
Unsurprisingly, the search for an appropriate RSV delivery
improve drug permeability and overcome adverse influence of
system has been of a great interest lately6–9.
P-glycoprotein18,19. Unfortunately, these dosage forms often
Following oral application, RSV undergoes extensive first-pass
contain large amounts of surfactants to ease microemulsion
intestinal and hepatic metabolism. The main metabolites identi-
formation. Therefore, biological tolerance of surfactants, which
fied are glucuronides and sulfates. They have been shown to exert
can disrupt biological membranes, must be considered. Chronic
application of surfactants in high doses could disturb some
Address for correspondence: Mirjana Gašperlin, Faculty of Pharmacy, physiological processes20. To lower the surfactants concentration,
University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia. cosolvents are often added to SMEDDS. Yet, many cosolvents can
Tel: þ386 1 4769634. Fax: þ386 1 4258031. E-mail: mirjana.gasperlin impact phase behavior, promote phase separation and drug
@ffa.uni-lj.si
 2 K. Bolko et al.
precipitation upon dilution of SMEDDS with water and have also
been reported as non-compatible with hard gelatin capsules,
which are often used to transform liquid SMEDDS to solid dosage
forms21–23.
Lipids used in SMEDDS can be of diverse chemistry.
However, the majority of SMEDDS described in the literature
are featuring single lipid components. Lately, there has been
emerging interest in more rational approach to selecting
SMEDDS excipients24. Our aim was therefore to improve
RSV solubility and dissolution profile by developing self-
microemulsifying formulation. Furthermore, we aimed to inves-
tigate whether heterogeneous oil phase composed of medium- and
long-chain mixed glycerides has a beneficial impact on SMEDDS
self-emulsifying properties in comparison to a single lipid phase.
Upon SMEDDS dilution with intestinal fluids, the drug remains
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entrapped in the oily droplets of microemulsions and is
advantageously delivered in its dissolved form throughout its
transit through the gastrointestinal tract. We propose that avoiding
of drug dissolution step will result in enhanced solubility of RSV.
Entrapment of RSV in the oily droplets could also benefit to
decrease cytotoxicity of RSV solution used at higher concentra-
tion. As reported previously by Caddeo et al.25 the incorporation
of RSV in liposomes eliminated its cytotoxicity at 100 mM
compared to the aqueous solution. In addition, we aimed to design
SMEDDS with the lowest surfactant content possible, while
maintaining high RSV incorporation capacity and its improved
dissolution profile.
Materials and methods
For personal use only.
Materials
RSV was purchased from Merck, Darmstadt, Germany. Capmul
MCM and Capmul PG-8 were obtained as a sample from Abitec,
Columbus, OH. Cremophor EL, Cremophor RH40 and
Cremophor RH60 were received as a sample from BASF,
Ludwigshafen, Germany. Miglyol 812 was obtained from Sasol,
Hamburg, Germany and Peceol from Gattefossé, Saint-Priest,
France. Castor oil was purchased from Lex, Koper, Slovenia, olive
oil from Henry Lamotte, Bremen, Germany and glycerol from
Fluka, Newport News, VA. All other chemicals used in this study
were of analytical grade.
Solubility studies
A total of 5 mL of selected vehicle with added excess of RSV was
placed in a beaker and magnetically stirred at 21.0  0.2  C for
48 h. Resulting homogeneous mixture was transferred to a vial and
ultra-centrifuged (Thermo Scientific, Kanagawa, Japan) at
40 000 rpm for 20 min. A sample of supernatant was adequately
diluted with 70% (v/v) ethanol, filtered through 0.45 mm membrane
filter and analyzed by high performance liquid chromatography
(HPLC). Each solubility assay was conducted in duplicate.
Construction of (pseudo)ternary phase diagrams
The water titration method was employed to develop (pseudo)-
ternary diagrams of lipid phase, surfactant and water. For each
phase diagram, lipid phase (Capmul MCM, castor oil or Capmul
MCM/castor oil 50/50 m/m) and surfactant (Cremophor EL,
Cremophor RH40 or Cremophor RH60) were mixed at the weight
ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1. When
constructing (pseudo)ternary diagrams by the so-called titration
method, these weight ratios are customarily used to cover the
whole diagram area, presenting a compromise between the
amount of components and time needed to perform experiments
and diagram coverage density. The mixtures were diluted
dropwise with water, under moderate agitation in water bath at
Drug Dev Ind Pharm, Early Online: 1–8

37.0  0.1 C. Upon each dilution step – addition of 5% water –
samples were visually assessed and classified as microemulsions
(dot on the diagram) when they appeared as clear liquids. The
presence of transparent gel phase was indicated with a square
marker.
Preparation of SMEDDS and determination of maximum
RSV loading in selected SMEDDS formulations
SMEDDS formulations were prepared by weighing the excipients
and blending them at room temperature until homogeneous
transparent mixture was formed. Samples of SMEDDS were
assayed for saturated RSV solubility as described previously
for vehicles (‘‘Solubility studies’’ section). RSV-loaded
SMEDDS were prepared with 80% of saturated concentration
of RSV.
Accelerated stability testing
To evaluate the stability of developed SMEDDS, empty and RSV-
loaded formulations were subjected to four freeze-thawing cycles.
Approximately 2 g of each formulation was placed in cap vial.
Each cycle consisted of freezing at 4  C for 24 h followed by
thawing at 40  C for 24 h. Afterward, samples were centrifuged at
4000 rpm for 5 min and visually observed for phase separation.
Following dilution with 50% (v/v) methanol, samples were also
evaluated for RSV content by means of HPLC as further
described in ‘‘HPLC analysis of RSV’’ section.
Viscosity of SMEDDS and its compatibility with hard
gelatin capsules
Developed SMEDDS and RSV-loaded SMEDDS were further
evaluated for dynamic viscosity using a Physica MCR 301
rheometer (Anton Paar, Graz, Austria) with a cone and plate
measuring system CP50-2 (cone radius 24.981 mm, cone angle
2.001 and sample volume 1.15 mL) at a constant temperature
37.0  0.1  C. Afterward, 0.5 g of each empty and RSV-loaded
formulation was encapsulated in hard gelatin capsules size 0 and
observed for leaking after three months of storage at
21.0  0.2  C.
Assessment of self-emulsification ability of SMEDDS and
precipitation
To evaluate the efficacy of emulsification of selected SMEDDS
formulations, time required for SMEDDS to form a homogeneous
mixture under gentle agitation following aqueous dilution of the
formulation was measured. Dispersion media used were either
250 mL purified water, HCl solution of pH 1.2 or pH 6.8
phosphate buffer solution. Visual assessment was conducted at
37  C following dropwise addition of 1 g of formulation to
250 mL magnetically stirred media (100 rpm). Droplet size of
resulting dispersions was determined by a photon correlation
spectroscopy using a Zetasizer nano series instrument (Malvern
Instruments Inc, Southborough, MA). Prepared dispersions of
SMEDDS were further analyzed by HPLC, as described in
‘‘HPLC analysis of RSV’’ section, to assess the amount of
precipitation of the drug from the formulations.
In vitro dissolution test
In vitro dissolution studies were conducted using USP Apparatus
2 (VK7000, VanKel, Cary, NC). A total of 0.5 g of selected RSV-
loaded SMEDDS was filled into hard gelatin capsules in
triplicates and placed in 900 mL of dissolution medium (either
pH 1.2 hydrochloric acid aqueous solution or pH 6.8 phosphate
buffer). Fifty milligrams of pure RSV filled in a hard gelatin
 DOI: 10.3109/03639045.2012.749888 Mixed lipid SMEDDS to enhance RSV solubility 3
capsule were used as a reference. All capsules were agitated at
50 rpm and 37.0  0.1  C. Five milliliters of samples were
withdrawn at predetermined time intervals and assayed by
HPLC. Cumulative percentages of dissolved RSV from formula-
tions were calculated and plotted versus time.
The dissolution profiles of tested formulations were evaluated
by fitting the experimental data to equations describing different
kinetic orders. Linear regression analyses were made for zero-
order (Mt/M0 ¼ k0t), first-order (ln(M1  Mt) ¼ k1t), Higuchi
(Mt/M1 ¼ (kHt)1/2) and Hixon–Crowell (Mt1/3  M11/3 ¼ kHCt)
kinetics, where Mt is the fraction of drug released at time
t, M1 the fraction of drug remaining to release and k0, k1, kH and
kHC the coefficients of the said equations.

HPLC analysis of RSV

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The concentration of RSV in samples was determined by means

of reverse-phase HPLC analysis on Agilent 1100 Series system.
The chromatographic column used was YMC-Pack ODS-AM,
4.6  250 mm2, 5 mm (YMC, Kyoto, Japan). Acetic acid (0.5%) in
a mixture of methanol and water (50:50% vol/vol) was used as a
mobile phase. The chromatographic conditions were as follows:
flow rate 1 mL/min, injection volume 20 mL, column temperature
25  C, detection wavelength 303 nm and retention time of RSV
6.6  0.5 min.
Results and discussion
Saturated solubility of RSV in selected excipients and
formulation of SMEDDS
For personal use only.
Figure 1. Saturated solubility of RSV in selected excipients at
21.0  0.2  C. The results are shown as average of two measurements
with the corresponding SD.
microemulsion region, regardless of the type of Cremophor
used as a surfactant. This is in lieu with Capmul MCM’s
cosurfactant properties28. When constructing (pseudo)ternary
diagrams with castor oil as mono-lipid phase, we often
encountered formation of gel phase, which can represent
transition between w/o and o/w (micro)emulsions. The gel area
was reduced with the addition of Capmul MCM to castor oil.
Similar effect was reported after addition of Capmul MCM to
medium chain di- or triglyceride29.
Among the surfactants tested, Cremophor EL gained the

For development of the most economical formulation with the

broadest microemulsion range, followed by RH40 and RH60.
highest RSV loading, potential excipients were screened for RSV
Based on each (pseudo)ternary diagram developed, the self-
solubility. Of the excipients tested, olive oil exhibited the lowest
microemulsifying formulations with the lowest surfactant-to-oil
RSV solubility (0.01 mg RSV/100 mg) and Cremophor RH60
ratio S1–S9 were chosen (Table 1), to ensure the lowest irritation
the highest (22 mg RSV/100 mg; Figure 1). On the basis of
of tissue22. Incorporation of RSV in SMEDDS resulted in
solubility studies, two lipids of diverse chemism, Capmul MCM
improved solubility of all tested formulations compared to water.
(mixture of medium chain mono and diglycerides) and castor oil
Following exposure of selected formulations (S1–S9, empty or
(long chain triglyceride) were selected as lipid phases to
RSV-loaded) to four freeze-thaw cycles, no phase separation and
formulate SMEDDS. On the same premise, Cremophor EL,
minimal loss of RSV content (less than 2%) was observed
Cremophor RH40 and Cremophor RH60 were chosen as potential
(Table 1). This was a clear indicator of formulation’s stability,
surfactants.
therefore all systems were submitted to further studies.
Constructed (pseudo)ternary phase diagrams and stabi-
SMEDDS viscosity and compatibility with hard gelatin
lity of developed SMEDDS
capsules
Employing five different vehicles with highest solubilization
Due to benefits of solid dosage forms, liquid SMEDS are often
capacity for RSV (‘‘Saturated solubility of RSV in selected
filled in hard gelatin capsules as an economical solution. To
excipients and formulation of SMEDDS’’ section), nine different
minimize the risk of leaking and to ensure efficient capsule filling
(pseudo)ternary diagrams were constructed (Figure 2). Lipid
process, SMEDDS exhibiting viscosities of at least 100 mPas are
phases used were Capmul MCM, castor oil or 50/50 (w/w)
preferred30. Dynamic viscosities of all empty and RSV-loaded
mixture of both. Added surfactant phase was varied among three
systems surpassed that value (Figure 3). In general, the systems
Cremophors of different hydrogenation degrees. Systems were
comprising of long-chain glycerides (castor oil) exhibited higher
evaluated regarding their capability of forming microemulsion
viscosity than systems with lipid phase containing medium chain
upon addition of water in broadest range possible.
glycerides (Capmul MCM or Capmul MCM/castor oil ¼ 1/1
According to the results presented in Figure 2, castor oil was
mixture). Incorporation of RSV considerably increased the
found to be less efficient at forming microemulsions with
viscosity of all formulations examined, in particular of those
Cremophors than Capmul MCM. This corresponds to easier
composed solely with castor oil as lipid phase.
self-microemulsification of Capmul MCM, a mixture of C8-C10
To further confirm compatibility of selected SMEDDS with
mono- and diglycerides, in comparison to castor oil, a fixed oil
hard gelatin capsules, formulations were filled in capsules and
comprising of long-chain triglycerides, as already reported26.
stored for three months. All hard gelatin capsules remained intact;
Capmul MCM, previously already established as an appropriate
no leaking of SMEDDS was observed.
lipid phase for formulating SMEDDS, was more likely to form
microemulsions particularly at lower water content27. This can be
Self-emulsifying properties
explained by Capmul MCM’s low required HLB value of 5 and
therefore its greater affinity to form water-in-oil microemulsions The performance of selected SMEDDS (Table 1) was further
in comparison to castor oil (HLB ¼ 14). Nevertheless, combina- evaluated with respect to the progress of self-emulsification after
tion of Capmul MCM and castor oil yielded the greatest addition of 250 mL of hydrophilic phase. Parameters studied with
 4 K. Bolko et al. Drug Dev Ind Pharm, Early Online: 1–8
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For personal use only.

Figure 2. (Pseudo)ternary diagrams for systems composed of Cremophors (EL, RH40 or RH60, respectively) as surfactants and Capmul MCM, castor
oil, or mixture of Capmul MCM/Castor oil ¼ 1/1 as lipid phase at 37  C. Black dots represent the presence of a microemulsion. Gel phase is indicated
using blank square marker.

Table 1. Components content in selected SMEDDS formulations, saturated solubility (at 21.0  0.2  C) and accelerated stability of RSV in
formulations (n ¼ 2), respectively.

Saturated solubility Accelerated stability

SMEDDS Composition of RSV (mg/100 g) of RSV (% remained)
S1 Capmul MCM 40%, Cremophor EL 60% 11.34  0.17 99.63  0.01
S2 Capmul MCM 30%, Cremophor RH40 70% 13.89  0.02 98.65  0.09
S3 Capmul MCM 30%, Cremophor RH60 70% 14.92  0.05 99.68  0.05
S4 Castor oil 30%, Cremophor EL 70% 13.57  0.00 99.94  0.02
S5 Castor oil 30%, Cremophor RH40 70% 15.44  0.21 99.44  0.12
S6 Castor oil 20%, Cremophor RH60 80% 17.51  0.22 99.61  0.07
S7 Capmul MCM 20%, Castor oil 20%, Cremophor EL 60% 11.41  0.31 98.01  0.03
S8 Capmul MCM 20%, Castor oil 20%, Cremophor RH40 60% 12.60  0.06 100.85  0.08
S9 Capmul MCM 20%, Castor oil 20%, Cremophor RH60 60% 12.64  0.22 100.37  0.12

the purpose of monitoring self-emulsifying ability of selected
SMEDDS included emulsification time measurement, droplet size
and drug precipitation analysis.
The time required for formation of microemulsion was
assessed upon diluting formulation in different media. To observe
the effect of pH and ionic strength on self-emulsifying properties,
purified water, hydrochloric acid solution with pH 1.2 and
phosphate buffer solution with pH 6.8 were used as media since
following oral administration formulations are exposed to gastric
fluids rather than purified water. As shown in Table 2, the fastest
formation of microemulsion was generally observed in acidic
media (pH 1.2), with exception of formulations S5 and S6, which
were dispersed most rapidly in purified water. SMEDDS
containing Capmul MCM (S1–S3) were expected to self-emulsify
 DOI: 10.3109/03639045.2012.749888 Mixed lipid SMEDDS to enhance RSV solubility 5
Figure 3. Dynamic viscosities of selected
SMEDDS formulations (S1–S9) with
(SMEDDS þ RSV) or without RSV
(SMEDDS); n ¼ 3.
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Table 2. Emulsification time of SMEDDS upon dilution with aqueous media (purified water, hydrochloric acid solution with pH
1.2 and phosphate buffer solution with pH 6.8).

SMEDDS Purified water pH 1.2 pH 6.8

For personal use only.

S1 39 min 2 s  1 s 20 min 40 s  14 s 21 min 12 s  8 s

S2 1 min 55 s  4 1 min 1 s  3 s 1 min 36 s  4 s
S3 1 min 49 s  6 s 1 min 52 s  2 s 2 min 4 s  0 s
S4 17 min 33 s  1 min 42 s 13 min 44 s  53 s 18 min 41 s  3 min 53 s
S5 12 min 31 s  2 min 42 s 17 min 11 s  3 min 28 s 12 min 35 s  5 min 59 s
S6 14 min 10 s  3 min 34 s 14 min 21 s  5 min 21 s 20 min 34 s  4 min 53 s
S7 1 min 43 s  3 s 1 min 39 s  4 s 2 min 37 s  6 s
S8 1 min 20 s  6 s 1 min 14 s  0 s 1 min 8 s  2 s
S9 3 min 6 s  6 s 2 min 2 s  2 s 2 min 36 s  3 s

faster than systems containing castor oil (S4–S6) owing to their
more polar nature. This was indeed confirmed for formulations S2
and S3 in comparison with S5 and S6. Nevertheless, S1 exhibited
the longest self-emulsification time among all formulations tested
in spite of the highest Capmul MCM content. Self-emulsification
time of formulations S7–S9, containing mixture of Capmul
MCM and castor oil as lipidic phase, was considerably shorter
when compared to S4–S6 (SMEDDS containing castor oil) and
slightly prolonged in comparison to S2 and S3
(SMEDDS containing Capmul MCM). On contrary to our
expectation, S7 exhibited considerably shorter emulsifying
time compared to S1, a formulation containing only Capmul
MCM as lipid phase. The latter could be attributed to the fact that
Capmul MCM and Cremophor EL mixture at weight ratio of
40:60 form gel phase upon dilution with water medium, as seen in
Figure 2. On the contrary, this is not the case for S7 formulation,
forming only low viscous microemulsions upon dilution.
The droplet sizes of microemulsions obtained from different
SMEDDS with corresponding polydispersity index (PDI) are
presented in Table 3. Upon dilution of empty SMEDDS (without
RSV) with aqueous phase homogeneous microemulsion with
droplet sizes below 50 nm (PDI50.2) were obtained for all
formulations tested, with the exception of S1. This allows us to
classify those systems as SMEDDS, a Class IIIB formulation
according to lipid formulation classification system (LFCS)31.
Once again, poorer dispersing ability was observed for S1 (droplet
size  200 nm) that exhibited also the longest self-emulsifying
time. Incorporation of RSV into selected SMEDDS resulted in,
however, considerably increased droplet size as well as hetero-
genicity of microemulsion formed. This was especially evident for
formulations S1–S3, S5 and S7. Following incorporation of RSV
into those systems, the droplet size of microemulsions increased
so strongly that aforementioned formulations can no longer be
classified as SMEDDS (Class IIIB) but can be considered as self-
emulsifying drug delivery systems (Class IIIA) according to
LFCS. Overall, loading SMEDDS with RSV resulted in
higher PDI; the reason could be its precipitation as discussed
further below.
Systems with higher Capmul MCM content were
expected to form microemulsions with the smallest droplets due
to its mono-/diglycerides associated amphiphilic nature and
properties resulting from medium-chain length glycerides.
In agreement with the literature data, very fine microemulsions
were indeed obtained upon dilution of empty formulations S2 and
S3. Yet, upon incorporation of RSV, the most pronounced effect
on droplet size increase was observed for those systems
(S2 and S3) precisely. This was in agreement with our
precipitation study data, yielding the most precipitated RSV in
the case of aforementioned formulations S2 and S3 (Table 3),
which consequently influenced the droplet size
measurement results.
Considering the parameters presented, SMEDDS containing
mixed glycerides as lipid phase showed best self-emulsifying
ability with regard to self-emulsifying time as well as droplet size
 6 K. Bolko et al. Drug Dev Ind Pharm, Early Online: 1–8

Table 3. Droplet size (d) with corresponding PDI of empty and RSV-loaded SMEDDS upon dilution and percentage of precipitated drug in diluted
samples.

Purified water pH 1.2 pH 6.8

Precipitated Precipitated Precipitated
SMEDDS d (nm) PDI RSV (%) d (nm) PDI RSV (%) d (nm) PDI RSV (%)
S1 Empty 183.10 0.487 – 186.80 0.480 – 193.10 0.465 –
RSV loaded 375.80 0.456 3.96 572.20 0.539 11.15 715.00 0.401 8.72
S2 Empty 14.82 0.082 – 17.86 0.181 – 15.25 0.025 –
RSV loaded 113.50 0.399 27.18 305.30 1.000 34.81 433.30 0.532 28.65
S3 Empty 15.33 0.110 – 15.95 0.065 – 15.73 0.031 –
RSV loaded 276.40 0.300 35.80 221.40 0.542 44.56 393.60 0.465 30.88
S4 Empty 26.46 0.083 – 31.09 0.067 – 29.07 0.054 –
RSV loaded 68.36 0.398 0 80.43 0.478 5.34 89.05 0.589 9.37
S5 Empty 29.69 0.068 – 37.37 0.141 – 35.76 0.058 –
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RSV loaded 174.40 0.216 17.2 162.10 0.257 26.56 55.56 0.561 19.44
S6 Empty 24.71 0.064 – 24.71 0.064 – 26.38 0.064 –
RSV loaded 24.93 0.234 29.61 35.64 0.463 33.00 35.07 0.423 18.85
S7 Empty 23.00 0.045 – 25.64 0.050 – 22.52 0.031 –
RSV loaded 152.90 0.284 0.00 105.50 0.133 16.55 300.90 0.342 0.00
S8 Empty 21.77 0.040 – 24.00 0.050 – 22.18 0.044 –
RSV loaded 27.64 0.039 14.14 29.76 0.149 18.68 30.51 0.184 12.50
S9 Empty 23.44 0.048 – 23.13 0.052 – 25.59 0.056 –
RSV loaded 36.69 0.376 13.51 39.52 0.373 25.78 46.29 0.454 19.90
For personal use only.

Figure 4. In vitro RSV release from selected SMEDDS formulations (S4, S5, S6, S8 and S9) compared to dissolution of pure RSV. All samples were
filled in hard gelatin capsules, containing approximately 50 mg RSV; n ¼ 3.

and homogeneity of microemulsions obtained upon SMEDDS
dilution with aqueous phase.
Solubilization capacity of SMEDDS
The greatest solubilization of RSV (18 mg/100 g SMEDDS) was
achieved by formulation S6, containing highest amount of
emulsifier Cremophor RH60 that exhibited also the best RSV
solubility among tested excipients. With increasing oil content in
SMEDDS, their solubilization capacity for RSV expectedly
decreases, being the lowest in systems S1 and S7
(11 mg/100 g SMEDDS), both containing Cremophor EL
as surfactant. After dilution of selected SMEDDS with water,
hydrochloric acid solution and phosphate buffer solution (1 g
SMEDDS/250 mL), the solubility of RSV in S6 as best
formulation (0.70 mg/mL) was still over 23-fold higher than its
solubility in water (0.03 mg/mL), although considerable precipita-
tion was observed. Overall, lower precipitation was observed in
 DOI: 10.3109/03639045.2012.749888
Table 4. Coefficient of determination values for fitting of the release data to the following kinetic models: the zero-order equation, the first-order
equation, the Higuchi square root equation and the Hixson–Crowell cube root law.
R2Zero order R2First order
Formulation pH 1.2 pH 6.8 pH 1.2 pH 6.8
S4 0.8012 0.8372 0.7097 0.6021
S5 0.7631 0.8490 0.7641 0.6664
S6 0.7043 0.7114 0.6187 0.5749
S8 0.7372 0.8157 0.7765 0.9471
S9 0.4921 0.6613 0.6549 0.6312
systems containing higher amount of lipids as reported pre-
viously32. Due to the presence of bile salts and other endogenic
surfactants expansion of solubilization capacity is expected
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in vivo, though33.
In vitro dissolution studies
According to results of droplet size analysis and solubilization
capacity, formulations S4, S5, S6, S8 and S9 were submitted to
dissolution studies. The effect of SMEDDS on in vitro RSV
release was examined in hydrochloric acid solution pH 1.2
(Figure 4, left) and phosphate buffer solution pH 6.8 (Figure 4,
right). Drug release profiles of RSV-loaded SMEDDS (filled in
hard gelatin capsules) were compared with dissolution profile of
pure RSV powder also filled in hard gelatin capsules. As already
in dissolved state, RSV was released from all SMEDDS
formulations faster and in greater extent compared to powdered
RSV that was used as a reference. More than 85% of RSV was
For personal use only.
released from formulation S9 in the first 15 min. Furthermore,
immediate RSV release (100% of RSV released within the first
30 min) was confirmed for both mixed lipid SMEDDS formula-
tions (S8, S9) in both dissolution media. To compare, only 50%
of powdered RSV was dissolved in the same time interval,
similarly as already reported34. All tested SMEDDS with lipid
phase composed solely of castor oil (S4, S5 and S6) exhibited
slower drug release (between 70% and 80% of RSV released in the
first 30 min in both media) in comparison to formulations
containing also Capmul MCM.
Moreover, reduced gel phase formation was observed upon
diluting SMEDDS with aqueous media in the presence of Capmul
MCM. As shown by Prajapati et al.29, formation of gel could
retard drug release from SMEDDS for as long as 60 min. This is
in agreement with our results, as formulations S4, S5 and S6
exhibited comparable drug release profile with other tested
formulations from 60 min onward. Subsequently, the release data
were analyzed by application of four kinetic equations – the zero-
order equation, the first-order equation, the Higuchi square root
equation and the Hixson–Crowell cube root law. The best fit
(highest coefficient of determination value) for all formulations in
both media was Hixson–Crowell cube root law (Table 4),
describing the release from system, where there is a change in
surface area and diameter of the drug carrier. As the RSV is
already dissolved in SMEDDS, its release is dependent on the
capsule degradation, microemulsion formation process and
release of RSV from formed oily droplets.
Conclusion
In the presented paper, we have focused on development of new
drug delivery system to improve solubility of polyphenol
antioxidant RSV with the aim to exploit its health-promoting
effects. In order to improve poor water solubility of RSV and its
dissolution profile, innovative SMEDDS formulations were
originated. To our knowledge, no such systems with RSV have
Mixed lipid SMEDDS to enhance RSV solubility 7
R2Hixson Crowell R2Higuchi
pH 1.2 pH 6.8 pH 1.2 pH 6.8
0.9737 0.9654 0.9543 0.9515
0.9574 0.9715 0.9509 0.9679
0.9883 0.9769 0.9293 0.9157
0.9584 0.9821 0.9344 0.9728
0.8259 0.9602 0.8032 0.9011
already been published. According to the literature data about
SMEDDS used for improvement of poor biopharmaceutical
properties of various drugs, researchers focused mostly on
surfactants used to achieve desirable self-emulsifying properties.
The lipid phase was considered merely a vehicle; consequently,
systems with single lipid components were predominantly
developed. Therefore, one of our objectives was to formulate
SMEDDS composed of mixed lipid phase (long-chain triglycer-
ides, castor oil, and medium-chain mono- and diglycerides
Capmul MCM) and compare its characteristics with correspond-
ing single lipid systems. According to the presented results, mixed
lipid SMEDDS were more likely to form microemulsions in
the presence of aqueous phase and exhibited superior self-
emulsifying properties and dissolution profile. Furthermore,
developed formulations were also found stable and compatible
with hard gelatin capsules.
The results of our study indicate the promising potential of
mixed lipid formulations for future development of SMEDDS
with lower surfactant content and no added cosolvents for
incorporation of other poorly soluble drugs.
Acknowledgements
The authors thank Assist. Romana Rošic, MPharm for her help with
viscosity measurements.
Declaration of interest
The authors report no conflict of interest. The authors alone are
responsible for the content and writing of this article.
This research was partially supported by Ministry of Higher
Education, Science and Technology (no. 314-392).
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