Arch Pharm Res Vol 30, No 1, 82-89, 2007

http://apr.psk.or.kr

Formulation and Biopharmaceutical Evaluation of Silymarin

Using SMEDDS
Jong Soo Woo, Tae-Seo Kim1, Jae-Hyun Park1, and Sang-Cheol Chi1

Pharmaceutical Research Lab. Hanmi Pharm. Co., Hwaseong 445-913, Korea and 1College of Pharmacy,
Sungkyunkwan University, Suwon 440-746, Korea
(Received October 11, 2006)

Silymarin has been used to treat hepatobiliary diseases. However, it has a low bioavailability
after being administered orally on account of its low solubility in water. In order to improve the
dissolution rate, silymarin was formulated in the form of a self-microemulsifying drug delivery
system (SMEDDS). The optimum formulation of SMEDDS containing silymarin was obtained
based on the study of pseudo-ternary phase diagram. The SMEDDS consisted of 15% silymarin, 10% glyceryl monooleate as the oil phase, a mixture of polysorbate 20 and HCO-50
(1:1) as the surfactant, Transcutol as the cosurfactant with a surfactant/cosurfactant ratio of 1.
The mean droplet size of the oil phase in the microemulsion formed from the SMEDDS was 67
nm. The % release of silybin from the SMEDDS after 6 hours was 2.5 times higher than that
from the reference capsule. After its oral administration to rats, the bioavailability of the drug
from the SMEDDS was 3.6 times higher than the reference capsule.
Key words: Silymarin, SMEDDS, Phase diagram, Dissolution, Bioavailability

INTRODUCTION

, 1992; Arcari
, 1992) and the incorporation of
silymarin into a solid dispersion (Chen
, 2005). The
solid dispersion of silymarin produced an approximately 2fold increase in bioavailability compared with the
conventional dosage form (Koo, 2002).
The solubility of silymarin may be further improved
using a microemulsion. A microemulsion is defined as an
O/W or W/O emulsion producing a transparent product
with a droplet size < 0.15 m, and unlike conventional
emulsions, does not have a tendency to coalesce (Gasco,
1997). It is a mixture consisting of oils, surfactants,
cosurfactants and water. A microemulsion can be used to
increase the solubility and bioavailability of poorly watersoluble drugs through the incorporation of the drug into
the oil phase (Ni
, 2002; Sinko, 2006). However, the
volume of the microemulsion per dose is too large to
administer or carry. Therefore, it is usually formulated in
the form of a self-microemulsifying drug delivery system
(SMEDDS), which is also known as a microemulsion
preconcentrate. The SMEDDS is filled directly into soft or
hard gelatin capsules for convenient oral administration.
After dilution with an aqueous media or gastric fluid after
ingestion, the SMEDDS forms a microemulsion spontaneously. Several studies have reported that the SMEDDS
promotes drug solubilization, drug release at the absorption

et al.

et al.

Silymarin is a purified extract from the seeds and fruits

of the milk thistle plant, Carduus marianus (L.) Gaertn.
The extract is a mixture of four isomeric flavonolignans,
silybin, isosilybinin, silydianin and silychristin (Fig. 1)
(Kvasnicka
., 2003). Among them, silybin is the major
active component. Silymarin has been used to treat toxic
liver disease and for the supportive treatment of chronic
active hepatitis and hepatic cirrhosis (Dewick, 1997;
Fitnleman, 1991; Flora ., 1998). However, the bioavailability of silymarin is quite low owing to its low solubility in
water (0.4 mg/mL) (Morazzoni and Bombardelli, 1995;
Basaga ., 1997; kottov ., 2000). Pharmacokinetic
studies have shown that only 23~47% of silymarin is
absorbed from the gastrointestinal tract after being
administered orally (Lorenz
, 1984; Schandalik and
Perucca, 1994; Schulz
., 1995). In order to increase
its bioavailability, many methods were used including the
complexation of silymarin with phosphatidylcholine, lecithin
or cyclodextrin clathrate (Barzaghi
, 1990; Morazzoni
et al

et al

et al

et al.

et al

et al.

et al.

et al

et al.

Correspondence to: Sang-Cheol Chi, College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea
Tel: 82-31-290-7709, Fax: 82-31-290-7729
E-mail: scchi@skku.ac.kr

82

Silymarin SMEDDS

83

(PC, Sigma Chemical Co., U.S.A.), D-a-tocopherol (Tama

Biochemical Co., Japan), ethyl linoleate and polyoxyethylene-50-hydrogenated castor oil (HCO-50, Nikkol Co.,
Japan), fractionated coconut oil (Miglyol 812, Hulls,
Germany), fish oil (Lysi, Iceland), castor oil (Dongyang,
Korea), diethylene glycol monoethyl ether (Transcutol),
PEG-8 glycol caprylate (Labrasol) and PEG-6 glyceryl
monooleate (Labrafil M 1944CS, Gattefosse Co., France).
The reference product, Legalon capsule, was purchased
from Bukwang Pharmaceutical Co. (Korea). Water was
freshly purified using a reverse osmosis method. All other
chemicals were of analytical grade.

Determination of silymarin solubility

To find out an appropriate solvent which has a good

solubilizing capacity of silymarin and, thus, can be used
as the oil or (co)surfactant phase in SMEDDS, the
solubility of silymarin in various solvents was measured as
follows: An excess amount of silymarin was added to 5
mL of each selected solvent (Transcutol, PEG 200,
ethanol, PG, PC and polysorbate 20) and shaken using
an isothermal shaker (Personality-11, Taitec Co., Japan)
at 251oC for 48 h. After centrifuged at 1,500 rpm for 10
min, the concentration of silybin in each solvent was
determined using a validated HPLC method.

Construction of pseudo-ternary phase diagrams

1. Chemical structure of the isomers of silymarin; silybin (A),
isosilybin (B), silydianine (C), silychristine (D)
Fig.

sites, and ultimately improve the oral bioavailability of the

drug (Pouton, 1997; Humberstone and Charman, 1997;
Lawrence and Rees, 2000; Attwood and Florence, 1983).
Therefore, this system has been regarded as an appropriate
system for increasing the bioavailability of many poorly
water-soluble drugs.
The aim of this study was to formulate a SMEDDS
containing silymarin and to assess its bioavailability compared with a conventional dosage form using rats.

MATERIALS AND METHODS

Materials

The following materials were purchased and used

without further purification. Silymarin (the content of silybin:
43%, Galena Opava, Czech Republic), polyethylene glycol
200 (PEG 200) and polyethylene glycol 400 (PEG 400,
Junsei Chemical Co., Japan), propylene glycol (PG, Dow
Chemical Korea, Korea), polysorbate 20 and glyceryl
monooleate (GMO, ICI Co., England), propylene carbonate

To obtain an optimum formula of the silymarin SMEDDS,

which can form a microemulsion upon dilution with water,
pseudo-ternary phase diagrams were constructed using the
water titration method at ambient temperature (Gattefosse,
1994). The silymarin concentration was fixed to 15%.
Based on preliminary experiments, GMO was used as the
oil phase, a mixture of polysorbate 20 and HCO-50 (1:1)
was used as the surfactant, and Transcutol was used as
the cosurfactant. The oil content used was 5, 10, 20 and
30%. The surfactant/cosurfactant ratio (S/CoS) used was
0.25, 0.5 and 1. The SMEDDS could not be formed at an
oil content 40% or S/CoS 2, because the drug was not
completely soluble in these systems.
After silymarin was added to the mixture of oil, surfactant
and cosurfactant, water was added drop by drop to this
mixture. During the titration, the samples were agitated
gently in order to reach equilibrium quickly. The phase
boundary was determined by observing the changes in
the sample appearance from turbid to transparent or from
transparent to turbid. All the ratios in this study are
reported as weight-to-weight ratios (W/W).

Determination of droplet size in formed microemulsion

A laser particle size analyzer (SALD-2001, Shimadzu,

Japan) was used to determine the droplet size distribution

84

of the oil phase in the microemulsions. The samples were

diluted with water (1,000 times), the droplet size of the oil
phase in the microemulsions was measured.

Dissolution study

The SMEDDS was encapsulated in a 22-oblong shape

soft capsule in order to evaluate the release of silymarin
from the SMEDDS. Each capsule contains 140 mg
silymarin, equivalent to 60 mg silybin. Legalon hard
capsule, containing the same amount of silymarin, was
used as the reference. The release of silymarin from the
SMEDDS was determined using a USP dissolution
apparatus II method. A six-position dissolution apparatus
(DT-80, Erweka, Germany) was used. The paddle was
run at a speed of 100 rpm. The medium was 900 mL
water, and the temperature was kept at 37 0.5oC.
A dialysis bag (Spectrapor/Por 3 membrane, MWCO
12,000, Spectrum, U.S.A.) was placed into a dissolution
vessel. The samples were introduced into the dissolution
medium outside the dialysis bag using sinkers. At predetermined times, the samples were withdrawn from inside
the dialysis bag and replaced immediately with the same
volume of fresh medium at 37 0.5oC. The silybin concentration in the medium was determined using a validated
HPLC method. The release pattern of silymarin was
investigated in not only water, but also in simulated gastric
(pH 1.2) and intestinal fluids (pH 6.8) to evaluate the effect
of medium pH on the dissolution of the drug.

Pharmacokinetic study

The pharmacokinetic characteristics of the SMEDDS

containing silymarin were evaluated using male SpragueDawley rats weighing 300 30 g. For this experiment, the
SMEDDS used was prepared with a S/CoS ratio of 1.
Legalon hard capsule was used as the reference product.
The dose was 140 mg/kg as silymarin.
One day before administering the drug, the femoral vein
was cannulated with a 23-gauge polyethylene cannula
under anesthesia with diethyl ether. Immediately before
dosing, 400 mg of silymarin SMEDDS was dispersed into
1 mL of saline while 120 mg of the content of the reference
product was suspended in 1 mL of saline. The solution or
suspension was administered orally to the rats using an
oral bougie, which was followed by the administration of 1
mL of water. About 0.25 mL of blood samples were
collected into a heparinized tube at 0, 0.5, 1, 2, 3, 4, 6, 8,
12, 16 and 24 h after dosing. The collected blood samples
were centrifuged at 12,000 rpm for 10 min and the plasma
was stored at -20oC until analysis.

HPLC analysis of silybin in rat plasma

The silybin concentrations in the solvent, dissolution

medium and rat plasma were determined with a slight

J. S. Woo et al.

modification of a reported HPLC method (Rickling

,
1995). The HPLC system consisted of an isocratic pump
(L-6200, Hitachi, Japan), an injector (7725i, Rheodyne,
U.S.A.), a UV detector (L-7400, Hitachi, Japan) and an
integrator (L-7000, Hitachi, Japan). The column used was
Inertsil ODS-2 (5 m, 4.6250 mm, GL Sciences Inc.,
Japan). The mobile phase consisted of a mixture of
methanol and pH 3.0 phosphate buffer (0.02M) (48:62 (V/
V)). The flow rate was 1.0 ml/min. The detector wavelength
was 285 nm. While the samples from the solubility and
dissolution study were injected onto the column after the
appropriate dilution with the mobile phase, silybin was
extracted from the rat plasma as follows: 400 L of pH 5.0
acetate buffer solution, 10 L of internal standard working
solution (naringenin 2.0 g/mL of methanol) and 50 L of
purified enzyme solution (-glucuronidase 13.48 unit/
arylsulfatase 4.5 unit/0.5M acetate buffer (pH 5.0)) were
added to 100 L of the rat plasma. The plasma was then
warmed to 37oC for 4 h in order to cause the cleavage of
glucuronides and sulphates of silybin. After the solution
had been cooled to room temperature, 0.5 mL of carbonate
buffer solution and 2 mL of diethyl ether were added and
shaken vigorously for 15 min. After centrifuging at 2000
rpm for 10 min, the organic phase was transferred to a 10
mL test tube and evaporated at 30C under a gentle
stream of nitrogen. The residue was reconstituted with
100 L of the mobile phase, vortexed for 1 min, and centrifuged at 12,000 rpm for 5 min. 50 L of the supernatant
was then injected onto the column.
et al.

Pharmacokinetic data analysis

The area under the drug concentration-time curve from

zero to 24 h (AUC) was calculated using the trapezoidal
rule (Gibaldi and Perrier, 1982). The maximum plasma
concentration of the drug (Cmax) and the time to reach Cmax
(Tmax) were obtained directly from the plasma profiles. The
relative bioavailability (BA) of the SMEDDS to the
reference was calculated as follows:
AUC
Dose
AUCreference Dosetest

reference
------------ -------------------------------Relative BA (%) = -------------------test

Where, AUCtest and AUCreference are AUCs obtained after

the oral administration of the SMEDDS and the reference,
respectively. Dosetest and Dosereference are the doses of the
two products.

Statistics

The data obtained from solubility and dissolution studies

are expressed as mean SD, while the data obtained
from pharmacokinetic study are expressed as mean SE.
The students t-test was used to compare the pharmacokinetic parameters. P value < 0.05 was considered
significant.

Silymarin SMEDDS

85

RESULTS AND DISCUSSION

et al.

Solubility of silymarin

To develop a microemulsion system for oral delivery of

poorly water-soluble silymarin, suitable oils and surfactants
need to be selected (Morazzoni and Bombardelli, 1995).
Table I shows the measured solubility of silymarin in
various solvents. According to the table, silymarin is
soluble in hydrophilic solvents such as PEG 200, PG and
Transcutol. In particular, silymarin was more soluble in
Transcutol than in the other solvents tested. Transcutol is
a powerful solubilizing agent used in several dosage
forms on account of its ability to solubilize many drugs
(Torrado
, 1997; Kim and Park, 2004). Therefore, it was
selected as the cosurfactant for the silymarin SMEDDS.
Silymarin showed a low solubility in the various oils
tested. Among the oils tested, silymarin showed the highest
solubility in GMO and was selected as the oil phase for
the formulation of the SMEDDS. This suggests that silymarin
has no lipophilic properties even though its solubility in
water is low (Log 7.4 is about 2.7) (Gak
, 2004).
When only polysorbate 20 was used as the surfactant,
the microemulsion formed was easily broken into an
opalescence emulsion after adding water. The use of a
mixture of polysorbate 20 and HCO-50 improved the
formation of a stable microemulsion. However, the clear
SMEDDS became turbid when too much ( 60%) HCO50 was added to polysorbate 20, which is possibly due to
the decreased solubility of silymarin in this mixture. Based
on this experiment, a 1:1 mixture of HCO-50 and polysorbate 20 was selected as the surfactant.
et al.

et al.

Construction of pseudo-ternary phase diagrams

The selection of oils, surfactants, cosurfactants, and the

S/CoS ratios plays an important role in the formation of
SMEDDS. The formulation of silymarin SMEDDS was

Table I.

Solubility of silybin in various solvents at 25oC

Solvents

Transcutol
PEG 200
PEG 400/Ethanol (1:1)
Ethanol
PG
Polysorbate 20
Propylene carbonate
Glyceryl monooleate
Tocopherol
Castor oil
Ethyl linoleate
Miglyol 812
Fish oil
Water
Mean SD (n=3)

optimized by evaluating the range of O/W microemulsions

using pseudo-ternary phase diagrams (Kim
, 2000).
The SMEDDS exists as a microemulsion apparently
without the addition of water because Transcutol behaves
as an aqueous phase. Georgakopoulos
(1992, 1993)
reported that Transcutol could work not only as the
surfactant but also as the aqueous phase. Therefore, in
the case of silymarin SMEDDS, there was no distinct
conversion from a W/O to O/W microemulsion. When
adequate water was added, the O/W microemulsion
became a coarse O/W emulsion, and even a turbid
suspension as a result of drug precipitation.
Fig. 2 shows the pseudo-ternary phase diagrams with
the different S/CoS. The gray areas indicate the clear O/
W microemulsion in the system. As shown in the figure,
the existence range of O/W microemulsion increased with
increasing S/CoS.
As explained above, the O/W microemulsion, formed
with the addition of water, became turbid when excess
water was added. Fig. 3 shows the water contents to form
an O/W microemulsion from the SMEDDS containing
silymarin at different S/CoS. As the S/CoS increased, the
maximum volume of water to form an O/W microemulsion
increased regardless of the oil percentage. In particular, at
10% oil phase and a S/CoS of 1, the maximum water
content reached 95.4%.
Therefore, an optimized SMEDDS was prepared using
10 % GMO as the oil phase with a S/CoS of 1. The final
SMEDDS consisted of 15% silymarin, 10% GMO, 37.5%
of a mixture of polysorbate 20 and HCO-50 (1:1), and
37.5% Transcutol.

Solubilitya (mg/mL)
350.1 10.4
345.9 19.5
342.1 17.1
225.2 15.2
162.4 13.6
131.3 16.3
159.1 13.3
133.2 12.8
120.0 11.9
117.1 11.2
112.1 10.8
110.8 10.5
110.5 10.2
110.4 10.1

et al.

Droplet size in formed microemulsion from

SMEDDS

The droplet size distribution is the most important

characteristics of an emulsion, including a microemulsion,
in evaluating its stability and
fate (Attwood, 1994;
Mayer, 1988; Schulman
, 1959). Therefore, the droplet
size of the oil phase in the formed microemulsion was
determined after adding water to the SMEDDS containing
silymarin. The concentration of the oil phase of SMEDDS
was fixed to 10%, while the S/CoS ratio was varied (0.25,
0.5 and 1). The result is shown in Fig. 4. The droplet size
of the oil phase in the microemulsion decreased with
increasing S/CoS. As the S/CoS was increased from 0.25
to 1, the mean droplet size of the oil phase in the microemulsion decreased from 123 nm to 67 nm. Constantinides
and Scalart (1997) reported that a small droplet size of the
oil phase provides a thermodynamically stable microemulsion.
in vivo

et al.

Dissolution study

After oral administration, the SMEDDS forms an O/W

86

J. S. Woo et al.

Pseudo-ternary phase diagram for the SMEDDS containing silymarin at a S/CoS of 0.25 (A), 0.5 (B) and 1 (C) (the gray area represents O/
W microemulsion existence region)
Fig. 2.

The effect of oil content and S/CoS on existence range of O/W

microemulsion. Key; : S/CoS = 0.25:1, : S/CoS = 0.5:1, : S/
CoS = 1:1 .
Fig. 3.

Droplet size distribution of the SMEDDS containing silymarin.

Key; : S/CoS = 0.25:1, : S/CoS = 0.5:1, : S/CoS = 1:1.
Fig. 4.

Silymarin SMEDDS

87

microemulsion with aqueous media in the gastrointestinal

tract. The release of the drug from the formed microemulsion was measured using an
dissolution test. In
the case of the conventional dissolution test, a 0.45 m
membrane filter is usually used after sampling the
medium in order to remove the undissolved drug. In the
preliminary test using the membrane filter, the amount of
drug released into the dissolution medium reached
approximately 100% at the first sampling time, 15 min
(data are not shown). This was due to the droplet size of
the internal phase (oil phase) of the microemulsion formed.
The oil phase could pass through the 0.45 m membrane
filter because its size was too small (<150 nm). Therefore,
when the dissolution tests for microemulsion formulation
were carried out, a dialysis bag with a molecular weight
cut-off of 12,000 was used to separate the microemulsionassociated drug from the truly dissolved drug (Chi, 1999;
Kang
, 2004). In this study, the same dialysis method
was used to evaluate the release of silybin from the
formed microemulsion.
Fig. 5 shows the release profiles of silybin from the
prepared SMEDDS capsule and the reference capsule. A
significant increase in drug release was observed with
SMEDDS over the reference capsule. The % release of
silybin from the SMEDDS at 360 minutes was approximately
2.5 times higher than that from the capsule.
Several experiments have shown similar results. Chi
(1999) reported that the % release of biphenyl dimethyl
dicarboxylate from SMEDDS was >12 fold higher than
that from the tablet containing the drug. The % release of
simvastatin from the SMEDDS was 1.5~2 times higher
in vitro

et al.

The dissolution profiles of silybin from SMEDDS and reference

capsule in water. MeanS.D. (n=3). Key; : SMEDDS, : reference
capsule.
Fig. 5.

than the conventional tablet (Kang

, 2004). This
highlights the advantage of a SMEDDS in improving the
rate of drug release over conventional dosage forms. Tarr
and Yalkowsky (1989) reported that the bioavailability of
cyclosporine administered in an emulsion might be
increased by reducing its droplet size. In the study of
SMEDDS containing idebenone, the decrease in the
droplet size of the internal phase of the microemulsion
formed could increase the rate of drug release (Kim
,
2000). In this study, very small droplets were formed
instantaneously when the SMEDDS was added to water,
which increased the rate of silymarin release.
The obtained release profiles in the other media were
not significantly different from that in water (data are not
shown). This suggests that pH had little effect on the
dissolution of the drug from the SMEDDS.
et

al.

et al.

Pharmacokinetic study

The bioavailability of the SMEDDS containing silymarin

was evaluated using rats. Fig. 6 shows the plasma profiles
of silybin in rats after the oral administration of the reference
capsule and prepared SMEDDS capsule containing
silymarin at a dose of 140 mg/kg. SMEDDS resulted in
significantly higher improvement of drug absorption than
the reference capsule. The necessary pharmacokinetic
parameters of silymarin in SMEDDS such as the AUC,
Cmax and Tmax were calculated from the profiles, and are
presented in Table II. The AUC and Cmax after the oral

6. The plasma concentration-time profiles of silybin after oral

administration of silymarin SMEDDS and reference capsule to rats at a
dose of 140 mg/kg as silymarin. MeanS.E. (n=7). Key; : SMEDDS, :
reference capsule.
Fig.

88

J. S. Woo et al.

Pharmacokinetic parameters of silybin after oral

administration SMEDDS and the reference capsule containing
silymarin to rats at a dose of 140 mg/kg
Parameters
Reference capsule
SMEDDS
1.10 0.48a
0.50 0.00
Tmax, h
Cmax, g/mL
3.47 0.20
24.79 4.69
AUC, gh/mL
22.75 3.19
81.88 12.86
Table

II.

Mean SE (n=7)
Significantly different from the reference capsule.

administration of SMEDDS were 3.6- and 7.1 times higher

than those of the reference capsule, respectively. However,
the Tmax was shorter than that of the reference capsule.
The calculated relative bioavailability of the SMEDDS
compared to the reference capsule was 360%.
The pharmacokinetic evaluation of silymarin showed
that the plasma level of silybin was very low in the conventional capsule (Lorenz
, 1984). After the oral
administration of the Legalon capsule to volunteers at a
dose of 560 mg of silymarin, the Cmax was in the range of
0.18 to 0.62 g/mL. After a single oral dose of silymarin
(200 mg/kg as silybin) in rats, the AUC and Cmax values
were 77.1 gh/mL and 6.7 g/mL, respectively (Morazzoni
, 1993). Similar data was obtained in the present
study.
As discussed above, the SMEDDS appears to be an
alternative dosage form, which increases the bioavailability
of silymarin. As mentioned earlier, the increase in the
bioavailability of silybin using an O/W microemulsion might
be due to the effect of drug dissolution and the improved
release rate. Morever, the presence of a surfactant and
cosurfactant in the microemulsion system might have
caused changes in the membrane permeability (Chi, 1999),
and the small droplet size in microemulsion provides a
large interfacial surface area for the improved bioavailability of silybin (Shah
, 1994).
et

al.

et al.

et al.

CONCLUSION
Silymarin was formulated as a SMEDDS in an attempt
to increase its release rate and bioavailability. An optimized
formula containing silymarin was developed through the
construction of pseudo-ternary diagrams of SMEDDS
containing the drug. The release rate of the drug from the
SMEDDS was approximately 2.5 times higher than that
from the reference capsule. After oral administration to
rats, the SMEDDS containing silymarin had a 360% higher
bioavailability compared with the reference capsule.

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