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

82

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 bioavailabilityafter being administered orally on account of its low solubility in water. In order to improve thedissolution rate, silymarin was formulated in the form of a self-microemulsifying drug deliverysystem (SMEDDS). The optimum formulation of SMEDDS containing silymarin was obtainedbased on the study of pseudo-ternary phase diagram. The SMEDDS consisted of 15% sily-marin, 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 67nm. The % release of silybin from the SMEDDS after 6 hours was 2.5 times higher than thatfrom the reference capsule. After its oral administration to rats, the bioavailability of the drugfrom the SMEDDS was 3.6 times higher than the reference capsule.

Key words: Silymarin, SMEDDS, Phase diagram, Dissolution, Bioavailability

INTRODUCTION

Silymarin is a purified extract from the seeds and fruitsof 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 et al., 2003). Among them, silybin is the majoractive component. Silymarin has been used to treat toxicliver disease and for the supportive treatment of chronicactive hepatitis and hepatic cirrhosis (Dewick, 1997;Fitnleman, 1991; Flora et al., 1998). However, the bioavail-ability of silymarin is quite low owing to its low solubility inwater (0.4 mg/mL) (Morazzoni and Bombardelli, 1995;Basaga et al., 1997; Škottová et al., 2000). Pharmacokineticstudies have shown that only 23~47% of silymarin isabsorbed from the gastrointestinal tract after beingadministered orally (Lorenz et al., 1984; Schandalik andPerucca, 1994; Schulz et al., 1995). In order to increaseits bioavailability, many methods were used including thecomplexation of silymarin with phosphatidylcholine, lecithinor cyclodextrin clathrate (Barzaghi et al., 1990; Morazzoni

et al., 1992; Arcari et al., 1992) and the incorporation ofsilymarin into a solid dispersion (Chen et al., 2005). Thesolid dispersion of silymarin produced an approximately 2-fold increase in bioavailability compared with theconventional dosage form (Koo, 2002).

The solubility of silymarin may be further improvedusing a microemulsion. A microemulsion is defined as anO/W or W/O emulsion producing a transparent productwith a droplet size < 0.15 µm, and unlike conventionalemulsions, 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 toincrease the solubility and bioavailability of poorly water-soluble drugs through the incorporation of the drug intothe oil phase (Ni et al., 2002; Sinko, 2006). However, thevolume of the microemulsion per dose is too large toadminister or carry. Therefore, it is usually formulated inthe form of a self-microemulsifying drug delivery system(SMEDDS), which is also known as a microemulsionpreconcentrate. The SMEDDS is filled directly into soft orhard gelatin capsules for convenient oral administration.After dilution with an aqueous media or gastric fluid afteringestion, the SMEDDS forms a microemulsion spontane-ously. Several studies have reported that the SMEDDSpromotes drug solubilization, drug release at the absorption

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

Silymarin SMEDDS 83

sites, and ultimately improve the oral bioavailability of thedrug (Pouton, 1997; Humberstone and Charman, 1997;Lawrence and Rees, 2000; Attwood and Florence, 1983).Therefore, this system has been regarded as an appropriatesystem for increasing the bioavailability of many poorlywater-soluble drugs.

The aim of this study was to formulate a SMEDDScontaining silymarin and to assess its bioavailability com-pared with a conventional dosage form using rats.

MATERIALS AND METHODS

MaterialsThe following materials were purchased and used

without further purification. Silymarin (the content of silybin:43%, Galena Opava, Czech Republic), polyethylene glycol200 (PEG 200) and polyethylene glycol 400 (PEG 400,Junsei Chemical Co., Japan), propylene glycol (PG, DowChemical Korea, Korea), polysorbate 20 and glycerylmonooleate (GMO, ICI Co., England), propylene carbonate

(PC, Sigma Chemical Co., U.S.A.), D-a-tocopherol (TamaBiochemical Co., Japan), ethyl linoleate and polyoxyethy-lene-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 glycerylmonooleate (Labrafil M 1944CS®, Gattefosse Co., France).

The reference product, Legalon® capsule, was purchasedfrom Bukwang Pharmaceutical Co. (Korea). Water wasfreshly purified using a reverse osmosis method. All otherchemicals were of analytical grade.

Determination of silymarin solubilityTo find out an appropriate solvent which has a good

solubilizing capacity of silymarin and, thus, can be usedas the oil or (co)surfactant phase in SMEDDS, thesolubility of silymarin in various solvents was measured asfollows: An excess amount of silymarin was added to 5mL of each selected solvent (Transcutol, PEG 200,ethanol, PG, PC and polysorbate 20) and shaken usingan isothermal shaker (Personality-11, Taitec Co., Japan)at 25±1oC for 48 h. After centrifuged at 1,500 rpm for 10min, the concentration of silybin in each solvent wasdetermined using a validated HPLC method.

Construction of pseudo-ternary phase diagrams

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 thewater titration method at ambient temperature (Gattefosse,1994). The silymarin concentration was fixed to 15%.Based on preliminary experiments, GMO was used as theoil phase, a mixture of polysorbate 20 and HCO-50 (1:1)was used as the surfactant, and Transcutol was used asthe cosurfactant. The oil content used was 5, 10, 20 and30%. The surfactant/cosurfactant ratio (S/CoS) used was0.25, 0.5 and 1. The SMEDDS could not be formed at anoil content ≥ 40% or S/CoS ≥ 2, because the drug was notcompletely soluble in these systems.

After silymarin was added to the mixture of oil, surfactantand cosurfactant, water was added drop by drop to thismixture. During the titration, the samples were agitatedgently in order to reach equilibrium quickly. The phaseboundary was determined by observing the changes inthe sample appearance from turbid to transparent or fromtransparent to turbid. All the ratios in this study arereported as weight-to-weight ratios (W/W).

Determination of droplet size in formed micro-

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

Japan) was used to determine the droplet size distribution

Fig. 1. Chemical structure of the isomers of silymarin; silybin (A),

isosilybin (B), silydianine (C), silychristine (D)

84 J. S. Woo et al.

of the oil phase in the microemulsions. The samples werediluted with water (1,000 times), the droplet size of the oilphase in the microemulsions was measured.

Dissolution studyThe SMEDDS was encapsulated in a 22-oblong shape

soft capsule in order to evaluate the release of silymarinfrom the SMEDDS. Each capsule contains 140 mgsilymarin, equivalent to 60 mg silybin. Legalon® hardcapsule, containing the same amount of silymarin, wasused as the reference. The release of silymarin from theSMEDDS was determined using a USP dissolutionapparatus II method. A six-position dissolution apparatus(DT-80, Erweka, Germany) was used. The paddle wasrun at a speed of 100 rpm. The medium was 900 mLwater, and the temperature was kept at 37 ± 0.5oC.

A dialysis bag (Spectrapor/Por 3 membrane, MWCO12,000, Spectrum, U.S.A.) was placed into a dissolutionvessel. The samples were introduced into the dissolutionmedium outside the dialysis bag using sinkers. At prede-termined times, the samples were withdrawn from insidethe dialysis bag and replaced immediately with the samevolume of fresh medium at 37 ± 0.5oC. The silybin concen-tration in the medium was determined using a validatedHPLC method. The release pattern of silymarin wasinvestigated in not only water, but also in simulated gastric(pH 1.2) and intestinal fluids (pH 6.8) to evaluate the effectof medium pH on the dissolution of the drug.

Pharmacokinetic study

The pharmacokinetic characteristics of the SMEDDScontaining silymarin were evaluated using male Sprague-Dawley rats weighing 300 ± 30 g. For this experiment, theSMEDDS 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 veinwas cannulated with a 23-gauge polyethylene cannulaunder anesthesia with diethyl ether. Immediately beforedosing, 400 mg of silymarin SMEDDS was dispersed into1 mL of saline while 120 mg of the content of the referenceproduct was suspended in 1 mL of saline. The solution orsuspension was administered orally to the rats using anoral bougie, which was followed by the administration of 1mL of water. About 0.25 mL of blood samples werecollected into a heparinized tube at 0, 0.5, 1, 2, 3, 4, 6, 8,12, 16 and 24 h after dosing. The collected blood sampleswere centrifuged at 12,000 rpm for 10 min and the plasmawas stored at -20oC until analysis.

HPLC analysis of silybin in rat plasmaThe silybin concentrations in the solvent, dissolution

medium and rat plasma were determined with a slight

modification of a reported HPLC method (Rickling et al.,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 anintegrator (L-7000, Hitachi, Japan). The column used wasInertsil ODS-2 (5 µm, 4.6×250 mm, GL Sciences Inc.,Japan). The mobile phase consisted of a mixture ofmethanol and pH 3.0 phosphate buffer (0.02M) (48:62 (V/V)). The flow rate was 1.0 ml/min. The detector wavelengthwas 285 nm. While the samples from the solubility anddissolution study were injected onto the column after theappropriate dilution with the mobile phase, silybin wasextracted from the rat plasma as follows: 400 µL of pH 5.0acetate buffer solution, 10 µL of internal standard workingsolution (naringenin 2.0 µg/mL of methanol) and 50 µL ofpurified enzyme solution (β-glucuronidase 13.48 unit/arylsulfatase 4.5 unit/0.5M acetate buffer (pH 5.0)) wereadded to 100 µL of the rat plasma. The plasma was thenwarmed to 37oC for 4 h in order to cause the cleavage ofglucuronides and sulphates of silybin. After the solutionhad been cooled to room temperature, 0.5 mL of carbonatebuffer solution and 2 mL of diethyl ether were added andshaken vigorously for 15 min. After centrifuging at 2000rpm for 10 min, the organic phase was transferred to a 10mL test tube and evaporated at 30×C under a gentlestream of nitrogen. The residue was reconstituted with100 µL of the mobile phase, vortexed for 1 min, and cen-trifuged at 12,000 rpm for 5 min. 50 µL of the supernatantwas then injected onto the column.

Pharmacokinetic data analysisThe area under the drug concentration-time curve from

zero to 24 h (AUC) was calculated using the trapezoidalrule (Gibaldi and Perrier, 1982). The maximum plasmaconcentration of the drug (Cmax) and the time to reach Cmax

(Tmax) were obtained directly from the plasma profiles. Therelative bioavailability (BA) of the SMEDDS to thereference was calculated as follows:

Relative BA (%)

Where, AUCtest and AUCreference are AUCs obtained afterthe oral administration of the SMEDDS and the reference,respectively. Dosetest and Dosereference are the doses of thetwo products.

StatisticsThe data obtained from solubility and dissolution studies

are expressed as mean ± SD, while the data obtainedfrom pharmacokinetic study are expressed as mean ± SE.The student’s t-test was used to compare the pharma-cokinetic parameters. P value < 0.05 was consideredsignificant.

AUCtest

AUCreference

-------------------------------

Dosereference

Dosetest

---------------------------------×=

Silymarin SMEDDS 85

RESULTS AND DISCUSSION

Solubility of silymarinTo develop a microemulsion system for oral delivery of

poorly water-soluble silymarin, suitable oils and surfactantsneed to be selected (Morazzoni and Bombardelli, 1995).Table I shows the measured solubility of silymarin invarious solvents. According to the table, silymarin issoluble in hydrophilic solvents such as PEG 200, PG andTranscutol. In particular, silymarin was more soluble inTranscutol than in the other solvents tested. Transcutol isa powerful solubilizing agent used in several dosageforms on account of its ability to solubilize many drugs(Torrado et al., 1997; Kim and Park, 2004). Therefore, it wasselected as the cosurfactant for the silymarin SMEDDS.

Silymarin showed a low solubility in the various oilstested. Among the oils tested, silymarin showed the highestsolubility in GMO and was selected as the oil phase forthe formulation of the SMEDDS. This suggests that silymarinhas no lipophilic properties even though its solubility inwater is low (Log P7.4 is about 2.7) (Gažák et al., 2004).

When only polysorbate 20 was used as the surfactant,the microemulsion formed was easily broken into anopalescence emulsion after adding water. The use of amixture of polysorbate 20 and HCO-50 improved theformation of a stable microemulsion. However, the clearSMEDDS became turbid when too much (≥ 60%) HCO-50 was added to polysorbate 20, which is possibly due tothe decreased solubility of silymarin in this mixture. Basedon this experiment, a 1:1 mixture of HCO-50 and polysor-bate 20 was selected as the surfactant.

Construction of pseudo-ternary phase diagrams

The selection of oils, surfactants, cosurfactants, and theS/CoS ratios plays an important role in the formation ofSMEDDS. The formulation of silymarin SMEDDS was

optimized by evaluating the range of O/W microemulsionsusing pseudo-ternary phase diagrams (Kim et al., 2000).The SMEDDS exists as a microemulsion apparentlywithout the addition of water because Transcutol behavesas an aqueous phase. Georgakopoulos et al. (1992, 1993)reported that Transcutol could work not only as thesurfactant but also as the aqueous phase. Therefore, inthe case of silymarin SMEDDS, there was no distinctconversion from a W/O to O/W microemulsion. Whenadequate water was added, the O/W microemulsionbecame a coarse O/W emulsion, and even a turbidsuspension as a result of drug precipitation.

Fig. 2 shows the pseudo-ternary phase diagrams withthe 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 withincreasing S/CoS.

As explained above, the O/W microemulsion, formedwith the addition of water, became turbid when excesswater was added. Fig. 3 shows the water contents to forman O/W microemulsion from the SMEDDS containingsilymarin at different S/CoS. As the S/CoS increased, themaximum volume of water to form an O/W microemulsionincreased regardless of the oil percentage. In particular, at10% oil phase and a S/CoS of 1, the maximum watercontent reached 95.4%.

Therefore, an optimized SMEDDS was prepared using10 % GMO as the oil phase with a S/CoS of 1. The finalSMEDDS consisted of 15% silymarin, 10% GMO, 37.5%of a mixture of polysorbate 20 and HCO-50 (1:1), and37.5% Transcutol.

Droplet size in formed microemulsion from

SMEDDSThe droplet size distribution is the most important

characteristics of an emulsion, including a microemulsion,in evaluating its stability and in vivo fate (Attwood, 1994;Mayer, 1988; Schulman et al., 1959). Therefore, the dropletsize of the oil phase in the formed microemulsion wasdetermined after adding water to the SMEDDS containingsilymarin. The concentration of the oil phase of SMEDDSwas 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 sizeof the oil phase in the microemulsion decreased withincreasing S/CoS. As the S/CoS was increased from 0.25to 1, the mean droplet size of the oil phase in the microemul-sion decreased from 123 nm to 67 nm. Constantinidesand Scalart (1997) reported that a small droplet size of theoil phase provides a thermodynamically stable microemul-sion.

Dissolution studyAfter oral administration, the SMEDDS forms an O/W

Table I. Solubility of silybin in various solvents at 25oC

Solvents Solubilitya (mg/mL)

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

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

a Mean ± SD (n=3)

86 J. S. Woo et al.

Fig. 2. 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. 3. 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. 4. Droplet size distribution of the SMEDDS containing silymarin.

Key; △: S/CoS = 0.25:1, ○: S/CoS = 0.5:1, ●: S/CoS = 1:1.

Silymarin SMEDDS 87

microemulsion with aqueous media in the gastrointestinaltract. The release of the drug from the formed microemul-sion was measured using an in vitro dissolution test. Inthe case of the conventional dissolution test, a 0.45 µmmembrane filter is usually used after sampling themedium in order to remove the undissolved drug. In thepreliminary test using the membrane filter, the amount ofdrug released into the dissolution medium reachedapproximately 100% at the first sampling time, 15 min(data are not shown). This was due to the droplet size ofthe internal phase (oil phase) of the microemulsion formed.The oil phase could pass through the 0.45 µm membranefilter because its size was too small (<150 nm). Therefore,when the dissolution tests for microemulsion formulationwere carried out, a dialysis bag with a molecular weightcut-off of 12,000 was used to separate the microemulsion-associated drug from the truly dissolved drug (Chi, 1999;Kang et al., 2004). In this study, the same dialysis methodwas used to evaluate the release of silybin from theformed microemulsion.

Fig. 5 shows the release profiles of silybin from theprepared SMEDDS capsule and the reference capsule. Asignificant increase in drug release was observed withSMEDDS over the reference capsule. The % release ofsilybin from the SMEDDS at 360 minutes was approximately2.5 times higher than that from the capsule.

Several experiments have shown similar results. Chi(1999) reported that the % release of biphenyl dimethyldicarboxylate from SMEDDS was >12 fold higher thanthat from the tablet containing the drug. The % release ofsimvastatin from the SMEDDS was 1.5~2 times higher

than the conventional tablet (Kang et al., 2004). Thishighlights the advantage of a SMEDDS in improving therate of drug release over conventional dosage forms. Tarrand Yalkowsky (1989) reported that the bioavailability ofcyclosporine administered in an emulsion might beincreased by reducing its droplet size. In the study ofSMEDDS containing idebenone, the decrease in thedroplet size of the internal phase of the microemulsionformed could increase the rate of drug release (Kim et al.,2000). In this study, very small droplets were formedinstantaneously when the SMEDDS was added to water,which increased the rate of silymarin release.

The obtained release profiles in the other media werenot significantly different from that in water (data are notshown). This suggests that pH had little effect on thedissolution of the drug from the SMEDDS.

Pharmacokinetic studyThe bioavailability of the SMEDDS containing silymarin

was evaluated using rats. Fig. 6 shows the plasma profilesof silybin in rats after the oral administration of the referencecapsule and prepared SMEDDS capsule containingsilymarin at a dose of 140 mg/kg. SMEDDS resulted insignificantly higher improvement of drug absorption thanthe reference capsule. The necessary pharmacokineticparameters of silymarin in SMEDDS such as the AUC,Cmax and Tmax were calculated from the profiles, and arepresented in Table II. The AUC and Cmax after the oral

Fig. 5. The dissolution profiles of silybin from SMEDDS and reference

capsule in water. Mean±S.D. (n=3). Key; ○: SMEDDS, ●: reference

capsule.

Fig. 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. Mean±S.E. (n=7). Key; : SMEDDS, :

reference capsule.

88 J. S. Woo et al.

administration of SMEDDS were 3.6- and 7.1 times higherthan those of the reference capsule, respectively. However,the Tmax was shorter than that of the reference capsule.The calculated relative bioavailability of the SMEDDScompared to the reference capsule was 360%.

The pharmacokinetic evaluation of silymarin showedthat the plasma level of silybin was very low in the con-ventional capsule (Lorenz et al., 1984). After the oraladministration of the Legalon® capsule to volunteers at adose of 560 mg of silymarin, the Cmax was in the range of0.18 to 0.62 µg/mL. After a single oral dose of silymarin(200 mg/kg as silybin) in rats, the AUC and Cmax valueswere 77.1 µg·h/mL and 6.7 µg/mL, respectively (Morazzoniet al., 1993). Similar data was obtained in the presentstudy.

As discussed above, the SMEDDS appears to be analternative dosage form, which increases the bioavailabilityof silymarin. As mentioned earlier, the increase in thebioavailability of silybin using an O/W microemulsion mightbe due to the effect of drug dissolution and the improvedrelease rate. Morever, the presence of a surfactant andcosurfactant in the microemulsion system might havecaused changes in the membrane permeability (Chi, 1999),and the small droplet size in microemulsion provides alarge interfacial surface area for the improved bioavail-ability of silybin (Shah et al., 1994).

CONCLUSION

Silymarin was formulated as a SMEDDS in an attemptto increase its release rate and bioavailability. An optimizedformula containing silymarin was developed through theconstruction of pseudo-ternary diagrams of SMEDDScontaining the drug. The release rate of the drug from theSMEDDS was approximately 2.5 times higher than thatfrom the reference capsule. After oral administration torats, the SMEDDS containing silymarin had a 360% higherbioavailability compared with the reference capsule.

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Table II. Pharmacokinetic parameters of silybin after oraladministration SMEDDS and the reference capsule containingsilymarin to rats at a dose of 140 mg/kg

Parameters Reference capsule SMEDDS

Tmax, h 1.10 ± 0.48a 0.50 ± 0.00†

Cmax, µg/mL 3.47 ± 0.20 24.79 ± 4.69†

AUC, µg·h/mL 22.75 ± 3.19 81.88 ± 12.86†

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

Silymarin SMEDDS 89

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