Silymarin in liposomes and ethosomes: pharmacokinetics and tissue distribution in free-moving rats by high-performance liquid chromatography-tandem mass spectrometry.

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Silymarin in liposome and ethosome on pharmacokinetics and tissue distribution in free-moving rats by HPLC-MS/MS Li-Wen Chang, Mei-Ling Hou, and Tung-Hu Tsai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504139g • Publication Date (Web): 06 Nov 2014 Downloaded from http://pubs.acs.org on November 8, 2014

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Journal of Agricultural and Food Chemistry

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Silymarin in liposome and ethosome on pharmacokinetics and tissue distribution in

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free-moving rats by HPLC-MS/MS

3 4

Li-Wen Chang1, Mei-Ling Hou1, Tung-Hu Tsai1,2,3,4*

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1

Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan

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2

Graduate Institute of Acupuncture Science, China Medical University, Taichung,

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Taiwan

9

3

School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung,

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Taiwan

11

4

Department of Education and Research, Taipei City Hospital, Taipei, Taiwan

12 13

*Author for correspondence:

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Tung-Hu Tsai, Ph.D.

15

Professor

16

Institute of Traditional Medicine, School of Medicine, National Yang-Ming University

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155, Li-Nong Street Section 2,

18

Taipei 112, Taiwan

19

Fax: (886-2) 2822 5044 ; Tel: (886-2) 2826 7115

20

E-mail: [email protected]

1

ACS Paragon Plus Environment


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Abstract

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The aim of this study was to prepare silymarin-formulations (silymarin entrapped in

23

liposomes and ethosomes, formulations referred to as LSM and ESM, respectively) to

24

improve oral bioavailability of silymarin and evaluate its tissue distribution by liquid

25

chromatography with tandem mass spectrometry (LC-MS/MS) in free-moving rats.

26

Silibinin is the major active constituent of silymarin, which is the main component to be

27

analyzed. A rapid, sensitive, and repeatable LC-MS/MS method was developed and

28

validated in terms of precision, accuracy, and extraction recovery. Furthermore, the

29

established method was applied to study the pharmacokinetics and tissue distribution of

30

silymarin in rats. The size, zeta potential and drug release of the formulations were

31

characterized. These results showed that the LSM and ESM encapsulated formulations

32

of silymarin may provide more efficient tissue distribution and increased oral

33

bioavailability, thus improving its therapeutic bioactive properties in the body.

34 35

Keywords:

silymarin;

ethosome;

liposome;

36

spectrometry; encapsulated formulation.

pharmacokinetics;

37 38 39

Introduction

2


tandem

mass

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40 41

Silybum marianum, commonly known as milk thistle in the family Asteraceae, has been

42

used for more than 2,000 years to treat a range of liver and gallbladder disorders,

43

including hepatitis and cirrhosis

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seeds of milk thistle, is composed primarily of silibinin with small amounts of other

45

stereoisomers, such as silydianin and silychristin 3. Pharmacology properties have

46

demonstrated that silymarin, a hepatoprotective agent, has a positive effect on the

47

function of liver cells

48

protein-restoring activities 7.

1, 2

. Silymarin, a flavonoid complex isolated from the

4, 5

, influencing their regenerative capacity by antioxidant-

6

and

49 50

Despite its powerful value in vitro, silymarin suffers from poor absorbance with a low

51

bioavailability and does not provide effective treatment 8. The present commercial

52

formulations of silymarin are in the forms of tablets and capsules; patients have to take

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the medicine three times a day (approximately 1200-1500 mg/day) to maintain an

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effective plasma concentration 9. Therefore, novel nanotechnology-based drug delivery

55

systems have been developed to improve the oral bioavailability of silymarin to achieve

56

high-efficacy, increase drug-release pattern time, and avoid drug administration

57

frequency.

58 3



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Liposomes are spherical vesicles, prepared from a variety of natural and synthetic

60

phospholipids that encapsulate hydrophilic molecules in its aqueous core and

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incorporate lipophilic molecules in its lipid bilayer

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permeability of drugs through biological membranes. Moreover, there have been many

63

studies about modifying liposomes to solve various problems for delivery of biological

64

components, including solubility, bioavailability, toxicity, and immunogenicity of active

65

compounds to improve therapeutic effects 11.

10

, which could increase the

66 12

.

67

Ethosomes have good improvement among these modified phospholipid vehicles

68

They are soft, malleable, tiny, bubble-like lipid vesicles composed mainly of

69

phospholipids, water and ethanol (commonly found in pharmaceutical preparations).

70

Previous studies have demonstrated that ethosomes were not toxic to cultured cells

71

and they have enhanced the oral bioavailability of a poorly water-soluble flavonoid

72

compound, curcumin 14.

13

,

73 74

Of the many causes of limited silymarin bioavailability, the major one is low aqueous

75

solubility 9. Based on morphological structure, ethosomes are very similar to liposomes;

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however, ethosomes provide high encapsulation efficiency for lipophilic compounds

77

because alcohol increases the solubility of fat-soluble compounds 11. Therefore, it could 4


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be expected that ethosomes would improve bioavailability of silymarin.

79 80

Many formulations have been studied further to increase solubility and enhance the

81

bioavailability of silymarin, such as silymarin-loaded lipid microspheres

82

proliposomes

83

nanocarriers

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However, formulation-based drug delivery systems of silymarin in these studies are

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restricted to either in vitro dissolution studies or optimizing formulations. The major

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disadvantage of previous reports on the pharmacokinetics of silymarin is that the

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experimental animals are either restrained or under anesthesia. The stress associated

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with restraint and conventional blood sampling from conscious rodents affects their

89

physiology, biochemistry, metabolism and protein expression 21, 22.

16

, self-microemulsifying

17

, nanostructured lipid carriers

19

15

, silymarin

18

, oil based

, and glyceryl monooleate/poloxamer 407 liquid crystalline matrices

20

.

90 91

In the present investigation, we designed liposome- and ethosome-encapsulated

92

silymarin carriers to evaluate the differences between liposomes and ethosomes on

93

pharmacokinetic profiles and tissue distributions of free-moving rats.

94 95

Therefore, it is necessary to develop and validate an analysis method to investigate the

96

pharmacokinetics and tissue distribution of liposome- and ethosome-encapsulated 5



97

silymarin. Silibinin is the major active constituent of silymarin, which is the main target

98

of analysis. A number of analytical methods for determining bioactive components of

99

silymarin extracts in biological fluids and tissues have been reported, including liquid

100

chromatography–ultraviolet (LC-UV), mass spectrometry (MS), and tandem mass

101

spectrometry (MS/MS) in human, dog (beagle dogs and mongrel dogs), rat, rabbit, cat

102

plasma or tissue 17, 18, 23-28.

103 104

Among these analytical methods, HPLC has been most frequently used for

105

pharmacokinetic studies of silibinin due to its simplicity, sensitivity and selectivity. The

106

objective of this study to develop and validate a sensitive and rapid LC-MS/MS method

107

to quantify silymarin active compounds (silibinin in rat plasma and organ tissues,

108

including liver, heart, spleen, lung, kidney) after oral administration of silymarin loaded

109

in liposomes and ethosomes. Furthermore, the pharmacokinetics and organ tissue

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distribution of silymarin in free form and formulations following oral administration

111

were evaluated and compared.

112 113

Materials and methods

114 115

Chemicals and reagents 6


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Cholesterol, heparin, naringin (internal standard; IS) and silymarin were obtained from

117

Sigma-Aldrich

118

phosphatidylcholine (SPC, Phospholipon® 80H) was supplied by American Lecithin

119

Company (Oxford, CT, USA). Distearoyl-phosphatidyl-ethanolamine-polyethylene

120

glycol with a mean molecular weight of 5000 (DSPE-PEG 5000) was purchased from

121

Nippon Oil (Tokyo, Japan). The cellulose membrane (Cellu-Sep® T2, with a molecular

122

weight cutoff of 6000 to 8000) was obtained from Membrane Filtration Products

123

(Seguin, TX, USA). Acetonitrile, chloroform, ethyl acetate, and sodium chloride (NaCl)

124

were purchased from Merck (Darmstadt, Germany). Milli-Q grade water (Millipore,

125

Bedford, MA, USA) was used for the preparation of solution and mobile phases.

Chemical

(St.

Louis,

MO,

USA).

Hydrogenated

soybean

126 127

Preparation of formulations

128

The components of ethosome were similar to liposome, which contained soybean

129

phosphatidylcholine (3.0 %, w/v of the final product), cholesterol (0.5 %), and

130

DSPE-PEG 5000 (0.5 %) and triply deionized water with 20% ethanol (liposome only

131

with triply deionized water that didn’t contain 20% ethanol). Formulations were

132

prepared as follows: soybean phosphatidylcholine (3.0 %, w/v of the final product),

133

cholesterol (0.5 %), and DSPE-PEG 5000 (0.5 %) were dispersed in a chloroform :

134

methanol (2：1) solution (5 mL). The organic solvent was evaporated in a rotary 7



135

evaporator at 50 °C, and solvent traces were removed by retaining the lipid film under

136

vacuum overnight. The films were hydrated with triply deionized water or triply

137

deionized water with 20% ethanol containing silymarin using a probe-type sonicator

138

(VCX600, Sonics and Materials, Newtown, CT, USA) at 35 W for 30 min. The total

139

volumes of the resulting products (liposome and ethosome-encapsulated silymarin

140

formulation, and the formulations, referred to as LSM and ESM, respectively) were set

141

at 5 mL.

142

143

Formulation characteristics

144 145

Determination of particle size and Zeta potential

146 147

The mean particle size and polydispersity index (PDI) of formulations were measured by

148

dynamic light scattering (90 Plus, BIC, Holtsville, NY, USA). The Zeta potential of

149

formulations was determined using a Zeta potential analyzer (90 Plus, BIC, Holtsville,

150

NY, USA).

151 152

Encapsulation of silymarin in liposomes and ethosomes

153 154

Entrapment capacity estimated the amount of LSM and ESM. These formulations were

155

centrifuged at 48,000 × g at 4 °C for 30 min in Beckman Optima MAX® ultracentrifuge

156

(Beckman Coulter, Stanwood, Washington, USA) to separate the incorporated drug from

8


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the free form. The amount of silibinin in the formulation was analyzed by the

158

HPLC-MS/MS system. The entrapment capacities of formulations were calculated with

159

the following equation: [(total amount of drug − amount of drug detected only in the

160

supernatant)/ total amount of drug] × 100 12.

161 162

Morphological observations by transmission electron microscopy (TEM)

163 164

These vesicles were examined by TEM to characterize their microstructure. A drop of a

165

formulation was applied to a 400-mesh carbon film-covered copper grid to form a thin

166

film specimen, which was stained with 1% phosphotungstic acid. After the stained

167

samples were allowed to air dry, TEM samples were obtained. Finally, the sample was

168

then examined and photographed by a TEM (JEM-2000EXII, JEOL, Tokyo, Japan).

169 170

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) system and

171

analytical method validation

172 173

LC-MS/MS system

174 175

An Agilent-1100 HPLC system (Agilent Technologies, Waldbronn, Germany) with an

176

Applied Biosystems/MDS Sciex API 3000 tandem quadrupole mass spectrometer 9



177

(Framingham, MA, USA) equipped with quaternary pump, injector, vacuum degasser,

178

and autosampler, was used. The Applied Biosystems/MDS Sciex API 3000 tandem

179

quadrupole mass spectrometer equipped with electrospray ionization (ESI) turbo ion

180

interface was used with the following parameters: Turbo Ion Spray Gas (GAS 2): 6

181

L/min, Nebulizer Gas (NEB): 10, Curtain Gas (CUR): 10, Collisionally Activated

182

Dissociation Gas (CAD): 8, IonSpray Voltage (IS): -4000 V, and Turbo IonSpray

183

Temperature (TEM): 500 °C. Nitrogen was used in all cases. Negative ion mode with

184

multiple reaction monitoring (MRM) was used for HPLC-MS/MS analysis. The

185

following precursors to product ion transitions were used: m/z 481.2→301.0 for silibinin

186

(DP -155 V, FP -380 V, EP -12 V, CE -22 V, CXP -12 V) and m/z 579.3→271.6 for

187

naringin as an internal standard (IS) (DP -86 V, FP -200 V, EP -11.5 V, CE -49 V, CXP

188

-15 V). A reverse-phase C18 column (50 x 4.6 mm, particle size 5 µm, Phenomenex

189

Luna) was used for HPLC separation. The mobile phase consisted of acetonitrile and

190

0.1% formic acid (67:33, v/v) at a flow rate of 0.3 mL/min. The sample injection volume

191

was 10 µL.

192 193

Analytical method validation

194 195

The stock solution was silibinin and naringin (IS) in 100% acetonitrile (1 mg/mL). A set 10


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of standard solutions was prepared by appropriate dilution of the stock solution with

197

mobile phase to make the calibration curve. These solutions were stored at -20 °C before

198

use.

199 200

These solutions were further diluted to give a serial of working standard solutions and

201

samples for calibration in blank plasma and organ tissues (including liver, heart, spleen,

202

lung, and kidney) were prepared by spiking aliquots of the stock solutions into

203

biosamples to obtain final concentrations in the range of 5-1000 ng/mL for plasma and

204

10-1000 ng/mL for organ tissues (including liver, heart, spleen, lung ,and kidney);

205

samples were processed as described in the sample preparation section.

206 207

Pharmacokinetic studies and tissue distribution for bioanalytical assays were based on

208

method validation practices according to FDA guidelines 29, including tests of selectivity,

209

sensitivity, linearity, precision, accuracy, recovery, and matrix effect.

210 211

Selectivity of analytes was determined by confirming the presence or absence of

212

interference as well as the lot-to-lot variation regarding interference in blank plasma and

213

organ tissues.

214 11



215

The limit of quantification (LOQ) is defined as the lowest concentration of the linear

216

range, and the limit of detection (LOD) is defined as the concentration of analyte that

217

gives a signal-to-noise ratio (S/N) of 3. Calibration curves were constructed by plotting

218

the peak area ratio of silibinin and naringin (IS) versus concentration.

219 220

Quantitation of silibinin in plasma and organ samples (including liver, heart, spleen,

221

lung, and kidney) was based on calibration curves, ranging from 5–1000 ng/mL and

222

10–1000 ng/mL, respectively. These calibration curves were required to have a

223

correlation coefficient (r2) greater than 0.995.

224 225

The precision and accuracy of the analytical method for different biological matrices

226

were assayed by testing the samples in five replicates on the same day (intra-day) and on

227

five successive days (inter-day). The accuracy was calculated from the nominal

228

concentration (Cnom) and the mean value of observed concentration (Cobs) as follows:

229 230

accuracy (bias, %) = [(Cnom − Cobs)/Cnom] × 100.

231 232

The precision (relative standard deviation, RSD) was calculated from the standard

233

deviation and observed concentration as follows: 12


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precision (RSD, %) = [standard deviation (SD)/Cobs] × 100.

236 237

The percentage of bias (% bias) and the percentage of RSD (% RSD) for the lowest

238

acceptable reproducibility concentration were defined as being within ±15% (± 20 % at

239

the low limit of detection).

240 241

The matrix effect and recovery of silibinin in various biological samples were assessed

242

at three different concentration levels (25 ng/mL, 250 ng/mL, and 1000 ng/mL). The

243

naringin (IS) concentration was 250 ng/mL in rat plasma and organ tissues.

244 245

Matrix effect (%) and recovery (%) were calculated using the following formulas:

246 247

Matrix effect = (the peak areas for standards spiked after extraction/the peak areas

248

obtained in neat solution standards) × 100

249 250

Recovery = (the peak areas for standards spiked before extraction/the peak areas for

251

standards spiked after extraction) × 100

252 13



253 254

Animal Experiments

255 256

Freely-moving rat model

257 258

Adult male Sprague-Dawley rats weighing 200 ± 30 g were obtained from the National

259

Yang-Ming University Animal Center, Taipei, Taiwan. The animals were specifically

260

pathogen-free and had free access to food (Laboratory Rodent Diet 5001, PMI Nutrition

261

International LLC, St. Louis, MO, USA) and water. The rats were housed with a 12-h

262

light and 12-h dark cycle. All experimental protocols involving animals were reviewed

263

and approved by the Institutional Animal Care and Use Committee (IACUC number:

264

1020613) of the National Yang-Ming University.

265 266

Experimental rats were initially anesthetized by pentobarbital (50 mg/kg, i.p.), and then

267

polyethylene tubes were implanted in the right jugular and right femoral veins for i.v.

268

administration. For the oral administration group, only the right jugular was catheterized

269

for blood sampling. The catheter crossed the subcutaneous tissue and was fixed in the

270

dorsal neck region. The patency tubing was maintained by flushing with heparinized

271

normal saline (0.9% NaCl, w/v, solution containing 20 IU/mL heparin sodium salt). 14


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After surgery, the rat was placed in an experimental cage and allowed to recover for one

273

day. During the recovery period, the rat was kept warm under a light.

274

The oral administration group was given silymarin (free form) dissolved in triple

275

distilled deionized water and present in suspension; for i.v. administration, silymarin

276

(free form) was completely dissolved in PEG 400 with 15 % ethanol.

277 278

Bio-distribution

279 280

Blood samples were collected by cardiac puncture at 30 min after silibinin

281

administration (free drug and formulations) and then perfused with normal saline

282

through the left ventricle. The organs (including liver, heart, spleen, lung, and kidney)

283

were collected, weighed and homogenized. Finally, the biological samples were stored at

284

-20 °C for sample analysis.

285 286

Sample preparation

287 288

Plasma samples

289 290

The real plasma sample (45 µL) was mixed with 5 µL naringin (IS) and vortexed for 30 s. 15



291

Next, 1 mL of ethyl acetate was added, and the mixture was vortexed for 5 min. The

292

mixture was centrifuged at 16,000×g for 10 min at 4 °C. The supernatant (1 mL) was

293

transferred to an Eppendorf vial and dried at 40 °C in a centrifugation evaporator. The

294

dried sample was reconstituted in 100 µL of mobile phase (acetonitrile: 0.1% formic

295

acid = 67:33, v/v) and filtered through a 0.22 µm filter. Finally, the filtrate (10 µL) was

296

applied to the HPLC-MS/MS system for analysis.

297 298

Organ samples

299 300

After homogenization of the organs with 100% aqueous acetonitrile solution (1:3, w/v),

301

the homogenate was centrifuged at 16,000 × g for 10 min at 4 °C and the supernatant

302

was collected and frozen at -20 °C until analysis. To determine silibinin concentration in

303

the organs, sample preparation was similar to plasma samples (described in section of

304

plasma samples). Additionally, if the concentration of silibinin was over the limit of the

305

linearity range (1000 ng/mL), these analytes were be diluted with blank biological

306

samples.

307 308

Pharmacokinetics and statistics

309 16


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All pharmacokinetic analysis was processed by WinNonlin Standard Edition Version 1.0

311

(Scientific Consulting Inc.: Apex, NC, USA). The Akaike Information Criterion (AIC)

312

value was used to select the compartment model used for data analysis.

313 314

Moreover, pharmacokinetic parameters are explained as follows: the area under the

315

concentration-time curve (AUC), which is defined as the plot of concentration of drug in

316

blood plasma against time; t1/2,α and t1/2,β are the distribution half-life and the elimination

317

half-life, respectively; the peak plasma concentration of a drug after administration and

318

the time to reach this peak plasma are Cmax and Tmax, respectively; the clearance (Cl), an

319

indicator of drug elimination from the body, is calculated by Cl = D (dose)/AUC; mean

320

residence time (MRT) is the apparent average period drug molecules staying in the body.

321

Bioavailability (%) = 100 x (AUCoral/Doral) / (AUCi.v./Di.v.).

322 323

Pharmacokinetic results are represented as the mean±SEM. Statistical significance was

324

determined by using a one-way ANOVA, followed by a Schieff post-test (SPSS version

325

10 SPSS, Chicago, IL) with p < 0.05 as the minimal level of significance.

326

17



327

Results and Discussion

328

Physicochemical characteristics of the formulations

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329 330

These

formulations

can

be

prepared

successfully

by

a

thin-film

331

dispersion/homogenization method. The physicochemical characteristics of LSM and

332

ESM were investigated by evaluating the formulations’ appearance, vesicle size, zeta

333

potential, and drug encapsulation percentage.

334

In this study, a formulation system contained SPC, DSPE-PEG 5000 and cholesterol,

335

which functioned as the emulsifiers, were located at the oil/water interface as a

336

phospholipid membrane surrounding the quercetin-containing core.

337 338

The pictures of LSM and ESM under TEM are shown in Figure.1. These vesicles were

339

both spherical, and their sizes and zeta potentials are shown in Table 1. Dynamic light

340

scattering determinted the sizes of LSM and ESM were approximately 272 nm and 321

341

nm, respectively. The polydispersity index (PDI) is a measure of the width of the particle

342

size distribution. The values of these formulations were smaller than 0.32, demonstrating

343

highly-homogeneous populations of particles for the formulations 30. Zeta potentials of

344

the LSM and ESM were -27.14 and -36.32 mV, respectively.

345 18


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From a morphological point of view, the structure of ethosomes is very similar to

347

liposomes; however, the results showed that ESM exhibited higher encapsulation

348

efficiency (69.13±3.25%) than LSM (60.36±1.78 %). This may be because ethosomes

349

have ethanol, which provides better solubility for lipophilic drugs 31.

350 351

In vitro release

352 353

To examine if silibinin could be readily released from silymarin solution (free form) and

354

formulation groups, the in vitro release tests were performed. As shown in Figure 2,

355

release of silibinin from the silymarin solution (free form) was rapid at 24 hr; the release

356

kinetics of silibinin from LSM and ESM exhibited slower, more-continuous release over

357

48 h. The drug release percentage of silibinin from the silymarin solution (free form) at

358

the end of the experiment (48 h) showed enhancements of 2.33-fold compared to the

359

formulation groups.

360 361

The sustained release profiles of silymarin from LSM and ESM were consistent with the

362

Higuchi diffusion equation (r2 = 0.97) 32.There were no significant differentces between

363

LSM and ESM for in vitro release. LSM and ESM showed suppressed and sustained

364

release of silymarin compared to the control groups. 19



365 366

LC-MS/MS system and Method Validation

367 368

To develop a sensitive and accurate LC-MS/MS method for the determination of

369

silymarin’s major active compound, silibinin, in rat plasma, liver, heart, spleen, lung,

370

and kidney, the triple quadrupole mass spectrometer equipped with an electrospray

371

ionization (ESI) source is currently one of the most useful tools available because of its

372

high sensitivity and selectivity.

373 374

Therefore, the process of analytical method development and validation could be

375

divided into several parts, including optimization of mass and LC conditions, assessed

376

matrix and recovery of sample preparation, selected calibration range, precision (R.S.D.

377

%) and accuracy.

378 379

In the study, we used an MRM system for qualification analysis, which consisted of

380

selecting the precursor ion (MS 1) and selecting a specific fragment of precursor ion

381

(MS 2); therefore, the analysis method was specific and sensitive. Negative ion

382

electrospray ionization was used to optimize the HPLC-MS/MS analysis, and the

383

MS/MS conditions of silibinin and naringin (IS) are shown in Table S1. The mass transit 20


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384

ion patterns m/z 481.2 → 301.0 and m/z 579.3 → 271.2 were selected to monitor

385

silibinin and naringin (IS), respectively. The mass spectra and chemical structures of

386

silibinin and naringin (IS) are shown in Figure 3.

387 388

In this method, the degree of interference was assessed by inspection of the MRM mode.

389

Separation and quantification of silibinin in plasma and organs samples without other

390

endogenous interfering peaks were performed in an optimal mobile phase containing

391

acetonitrile with 0.1% formic acid (67:33, v/v) with a reverse-phase C18 column (50 x

392

4.6 mm, particle size 5 µm).

393 394

To assess pharmacokinetic and tissue distribution, a suitable assay system for the plasma

395

and different tissue sample should be developed and optimized. In the process

396

optimization, we have examined many methods for sample preparation, including

397

protein precipitation and liquid–liquid extraction. However, we found that background

398

interference appeared in the blank matrix extract after protein precipitation, whether the

399

samples were treated with methanol or acetonitrile. However, the background

400

interference did not exist when the samples were treated by liquid-liquid extraction. The

401

results showed that liquid–liquid extraction using the liquid solvent- ethyl acetate

402

provide the most effective extraction of silibinin from various biological samples. 21



403 404

Figure S1 (A)–(F) show the chromatograms of blank plasma, liver, heart, spleen, lung,

405

and kidney, respectively. The chromatograms showed that no significant interfering

406

peaks from the plasma and tissues (liver, heart, spleen, lung, and kidney) after

407

liquid-liquid extraction were found at the retention times or the ion channel of silibinin

408

or naringin (IS). Figure S1 (G)-(L) shows chromatograms of the plasma and different

409

organ samples containing the naringin (IS) and silibinin collected from rats after i.v.

410

administration of silymarin (100 mg/kg). The retention times of the silibinin and the

411

naringin (IS) in the chromatograms were 1.4 and 1.9 min, respectively. The analysis time

412

is shorter than previous analysis methods 16, 27.

413 414

Analyte matrix effect and recovery were used to assess suitability of the sample

415

preparation method. The matrix effect and recovery percentages, were determined at

416

three different concentrations (low, middle and high concentrations) for all analytes. The

417

data demonstrated that the mean matrix effect of silibinin in rat plasma and organ

418

samples was 98.26±3.93 % and within the ranges from 86.69 to 100.45 %, respectively.

419

No significant matrix effects of silibinin were observed in rat plasma and organ samples,

420

which could be ignored in quantitative analysis (Table S2). The extraction recovery of

421

silibinin in rat plasma and organ samples was 86.64±3.40 % and within the range of 22


Page 22 of 49

Page 23 of 49


422

88.40 to 95.57 %, which was both considered acceptable (Table S2).

423 424

The average matrix effects and recoveries of naringin (IS) in rat plasma and organ

425

samples were showed in Table S2. Because the value of the matrix effect in the organ

426

samples had only a small standard deviation, the naringin (IS) could be used as a

427

suitable internal standard for the analysis. To evaluate the analytical linearity of the

428

developed method, the peak area ratio of silibinin to naringin (IS) versus concentration

429

was used.

430 431

The data demonstrated that the linearity was related to concentration in the range of

432

5-1000 ng/mL and 10-1000 ng/mL for plasma and biological samples, respectively. The

433

results showed a good correlation coefficient (r2 ≥ 0.995) for silibinin over the

434

concentration range from separately prepared analytical runs on different days. The

435

inter- and intra-day precision and accuracy values for plasma and organ samples are

436

presented in Table S3 and Table S4. Precision (R.S.D. %) and accuracy (Bias %) of

437

plasma ranged from 0.77 % to 17.62 % and -6.23 to 12.78%, respectively; for organ

438

samples, the percentages ranged from 0.48 % to 15.26% and 15.41 % to 10.64 %,

439

respectively. The LOQ of silibinin in plasma and organ samples was 5 ng/mL and 10

440

ng/mL, respectively. 23



441 442

These results showed that the analytical method was sensitive, repeatable, and reliable.

443

The system could be applied to quantification of silibinin in pharmacokinetic studies and

444

tissue distribution.

445 446

Pharmacokinetics and oral bioavailability

447 448

Pharmacokinetic and biodistribution assessment of nanoparticles is necessary. Many

449

types of nanoparticles could improve drug distribution profiles, which usually influences

450

drug delivery properties 33. The morphological structure of ethosomes is similar to that

451

of liposomes, but there have not been any studies about pharmacokinetic differences

452

between them.

453 454

To assess the pharmacokinetics and bioavailability of silymarin (free form), LSM, and

455

ESM, each was administered orally (100 mg/kg, p.o.) and intravenously (10 mg/kg, i.v.)

456

to rats.

457 458

Rats have also been widely used to test drug efficacy and safety because they are

459

mammals. Their systems could react to medicines, formulations, food products or 24


Page 24 of 49

Page 25 of 49


460

cosmetics in a similar manner as human systems; therefore, studying drug efficacy on

461

rats is useful to test if a drug is considered safe enough for human consumption or

462

exposure. Therefore, this animal model is suitable to investigate pharmacokinetics of

463

silymarin loaded in liposomes and ethosomes.

464 465

The plasma drug concentration–time profile after intravenous and oral administration of

466

silymarin, LSM, and ESM in rats is presented in Figures 4 and 5. Observed in Figure 4,

467

there were no differences between silymarin (free form) and silymarin loaded in

468

formulations. However, we found that silibinin could be detected in plasma up to 90 min

469

after oral administration (100 mg/kg, p.o.), while the time was prolonged to 120 min in

470

the group administered LSM and ESM (Figure 5).

471 472

The pharmacokinetic parameters were calculated using the software program WinNonlin

473

(version 1.1), and the results are summarized in Table 2. The pharmacokinetic models

474

(one-versus two-compartment) were selected according to the Akaike's information

475

criterion (AIC) 34, which was calculated by WinNonlin. The smaller AIC values can be

476

observed because the model is more suitable to fit the concentration–time curve.

477

25



478

In this study, the average AIC values from the one-compartment model after silymarin

479

(10 mg/kg, i.v.), LSM (10 mg/kg, i.v.), and ESM (10 mg/kg, i.v.) administration were

480

56.97, 65.24, and 66.39, respectively. Additionally, the mean AIC values from the

481

two-compartment model after silymarin (10 mg/kg, i.v.), LSM (10 mg/kg, i.v.), and

482

ESM (10 mg/kg, i.v.) administration were 37.56, 52.99, and 57.47, respectively. Based

483

on the above results, the two-compartment model was more suitable than the

484

one-compartment model for intravenous administration in rats (Figure 4 and Table 2).

485 486

The two-compartmental pharmacokinetic model follow the equation Cp = Ae-αt + Be-βt,

487

which indicated that there are two different processes in the body, including a

488

distribution phase and an elimination phase, presented as Ae-αt and Be-βt, respectively.

489

The parameters of the equation, A and B, are the concentration (C) intercepts for fast

490

and slow disposition phases, respectively; α and β are the disposition rate constants for

491

fast and slow disposition phases, respectively. Analysis of the data after silymarin (10

492

mg/kg, i.v.), LSM (10 mg/kg, i.v.), and ESM (10 mg/kg, i.v.) administration yielded the

493

pharmacokinetic equations: C = 1.63e−0.20t + 0.12e−0.03t, C = 0.82e−0.23t + 0.09e−0.03t, and

494

C = 1.49e−0.61t + 0.06e−0.02t, respectively.

495 496

The data showed that the pharmacokinetic parameters for t1/2,β (43.1±20.9, 32.54±7.81, 26


Page 26 of 49

Page 27 of 49


497

and 41.19±6.69 min), CL (1.01±0.34, 1.33±0.16, and 0.78±0.10 L/min/kg), and MRT

498

(25.1±9.9, 27.12±5.93, and 23.23±5.33 min) of silymarin, LSM and ESM were not

499

found to be significantly different between the three groups of each parameter.

500 501

Pharmacokinetic

parameters

for

oral

administration

were

calculated

by

502

non-compartmental analysis, also called model independent analysis. The AUC values

503

of silymarin (free form), LSM, and ESM were found to be 2.04±0.74, 15.17±6.51,

504

4.48±1.03 min*µg/mL, respectively; the results showed that the AUC of LSM was

505

significantly higher than silymarin.

506 507

Cmax is often assessed in an effort to show bioequivalence between a known and

508

innovative drug product 35. The value for LSM and ESM were both significantly higher

509

(p < 0.05) than silymarin, but it was insignificant when LSM was compared with ESM

510

(p > 0.05). The AUC and Cmax of LSM after oral administration were 7.7-fold and

511

5.5-fold higher than for silymarin, respectively. These values for ESM, were 2.19-fold

512

and 4-fold higher than silymarin, respectively.

513 514

The mean of Tmax is the time to maximum concentration of a drug in plasma, which can

515

be used as an indicator of the drug absorption rate. In our study, after oral administration, 27



Page 28 of 49

516

the Tmax values for silymarin (free form), LSM, and ESM were not significantly different

517

(approximately 10 min), which was shorter than results reported by Parveen R et al.

518

Anesthesia has an influence on the disposition of drugs in animals

519

discrepancy may be because the previous study used blood samples collected from

520

anesthesia animals, whereas our blood samples were collected from conscious rats.

19

36

. Therefore, this

521 522

Additionally, LSM was significantly improved as evidenced by 13.65-fold reduction in

523

CL from silymarin. ESM also decreased by 4.05-fold from silymarin, indicating that

524

these formulations could increase the circulation time of silymarin in rats instead of

525

rapid elimination. Previous in vitro study indicated that silymarin was metabolized by

526

CYP2C8 in the liver 9. Weingarten et al. (1985) supported that liposome could protect

527

the drugs from enzymatic metabolism via oral administration 37. Our data also observed

528

that LSM and ESM could increase AUC and reduce CL of silibinin after oral

529

administration.

530 531

Bioavailability is a term used to indicate the fractional extent to which a dose of drug

532

reaches its site of action or a biological fluid from which the drug has access to its site of

533

action. The formula for calculating bioavailability of a drug administered orally is given

534

as Bioavailability (%) = 100 x (AUCoral/Doral) / (AUCi.v./Di.v.) 28


Page 29 of 49


535 536

According to the definition of pharmacokinetics, the bioavailability of i.v. administration

537

is 100%. However, bioavailability of oral administered usually decreases due to

538

incomplete absorption and first-pass metabolism. In this study, we used the AUCi.v. value

539

of silymarin (free form) to calculate the bioavailabilities of silymarin (free form), LSM,

540

and ESM. According to the experimental data, the oral bioavailability of silymarin (free

541

form), LSM, and ESM were around bioavailability = 100 x (2.04/100) / (12.9/10) = 1.58

542

%, bioavailability = 100 x (15.2/100) / (12.9/10) = 11.78 %, and bioavailability = 100 x

543

(4.48/100) / (12.9/10) = 3.47 %, respectively. The bioavailability of a substance in the

544

body can be improved by increasing the absorption of the substance in the

545

gastrointestinal tract or reducing its metabolism in the body 38.

546 547

As discussed above, the higher AUC and shorter CL for LSM and ESM caused their

548

relative bioavailabilities to decrease by 7.45-fold and 2.2-fold compared to silymarin

549

(free form), respectively. As a result, ethosomes have higher encapsulation efficiency

550

than liposomes. In vitro release tests showed no differences between liposomes and

551

ethosomes, but liposomes have better pharmacokinetic properties than ethosomes when

552

liposomes are encapsulating silymarin.

553 554

Bio-distribution study

555 556

Bio-distribution, which could be used to predict the possible mechanisms of action of 29



557

nanoparticles, was demonstrated to be important for the application of nanoparticles 39.

558

According to above the results from pharmacokinetic profiles, we observed that silibinin

559

was around the intersection between the first and second compartment at 30 min after a

560

single dose of silymarin was administered intravenously (10 mg/kg) of silymarin. Due to

561

the possible high distribution of silibinin in various tissues at 30 min after intravenous

562

administration, we selected this time point for assessing bio-distribution of silymarin.

563 564

Concentrations of silibinin were determined in rat plasma and various tissues including

565

liver, heart, spleen, lung, and kidney, at 30 min after a single dose of silymarin, and

566

injectable formulation groups (LSM and ESM) were administered intravenously (10

567

mg/kg). Bio-distribution study results are shown in Figure 6.

568 569

It was shown that the concentrations of silymarin in various organs were lower than for

570

the formulation groups. Distribution patterns of LSM and ESM were in the following

571

order (from high to low): lung > liver > spleen > kidney> heart for LSM; lung > spleen

572

> liver > kidney> heart for ESM. These formulation groups, when administered

573

intravenously, are unable to leave general circulation and rapidly accumulate in

574

mononuclear phagocyte systems (MPS), which are abundant in special tissues and

575

organs, such as liver, lung and spleen 40, 41. Therefore, the main organs of distribution in 30


Page 30 of 49

Page 31 of 49


576

the formation groups were the spleen, lung and liver instead of kidney.

577 578

From the perspective of treatment, silymarin has therapeutic efficacy for liver disease 42,

579

43

580

Relatively low levels of silibinin accumulate in the liver following administration of

581

silymarin. The exposures of total silibinin levels in the liver following administration of

582

LSM and ESM were approximately 9-fold and 3-fold higher than that for silymarin;

583

these results are beneficial to therapy of liver disease. Additionally, according to the data

584

of bio-distribution, LSM and ESM appear low accumulation in the kidney, which may

585

reduce nephrotoxicity.

; therefore, the level of silymarin accumulation in the liver should be considered.

586 587

The tissue distribution patterns of LSM and ESM are similar except, the lung and spleen

588

accumulation of ESM was higher than LSM. It is well known that the bio-distribution of

589

vectors could be firstly affected by their particle size. The sizes of LSM and ESM were

590

not significantly different. Therefore, if distribution differences between LSM and ESM

591

occur, the size factor could be ignored. From a morphological point of view, ethosomes

592

are very similar to liposomes; however, ethosomes exhibit high encapsulation and soft

593

structure, which may cause the high accumulation of ESM in some tissues.

594 31



595

To our knowledge, this is the first description of bio-distribution of ethosome. The

596

results clarify the difference between ethosomes and liposomes in tissue distribution.

597

ESM had high accumulation in the lung, suggesting that ethosomes have great potential

598

as a lung-targeting drug carrier for the treatment of lung diseases.

599 600

In this study, the experiment was divided into four major directions, including

601

development and establishment of analytical methods, formulation of design,

602

pharmacokinetic property studies in awake rats, and bio-distribution determination.

603 604

The LC-MS/MS method was developed and validated to assess pharmacokinetic profiles

605

and bio-distribution of silymarin loaded liposomes and ethosomes in preclinical study

606

phases. The analysis method is rapid, specific, sensitive, accurate, precise and linear in

607

the analytical range, which has been successfully used in pharmacokinetic and

608

bio-distribution studies of silymarin in free-moving rats.

609 610

The pharmacokinetic results demonstrated liposome and ethosome could improve the

611

bioavailability of silymarin, and that liposomes are better than ethosomes. Furthermore,

612

LSM and ESM were taken up by the RES with good targeting to the liver. Therefore,

613

ethosome have potential as a therapeutic for lung diseases. Although these results 32


Page 32 of 49

Page 33 of 49


614

provide preliminary clinical feasibility, more detailed assessments should be required

615

before clinical applications.

616 617

Acknowledgment

618

This work was supported in part by research grants from the National Science Council

619

Taiwan (NSC102-2113-M-010-001-MY3) and by TCH 103-02 and TCH 10301-62-021

620

from Taipei City Hospital, Taiwan.

621 622 623

Conflicts of Interest

624 625

The authors declare that there is no conflict of interests regarding the publication of this

626

article.

627 628

Supporting Information

629 630

Method validation results and typical HPLC-MS/MS chromatograms.

631 632

References 33



633 634

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Mayer, K. E.; Myers, R. P.; Lee, S. S., Silymarin treatment of viral hepatitis: a

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systematic review. Journal of viral hepatitis 2005, 12, 559-67. 2. Hackett, E. S.; Twedt, D. C.; Gustafson, D. L., Milk thistle and its derivative

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compounds: a review of opportunities for treatment of liver disease. Journal of

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veterinary internal medicine 2013, 27, 10-6. 3. Singh, R. P.; Agarwal, R., Flavonoid antioxidant silymarin and skin cancer.

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experimental pharmacology to clinical medicine. The Indian journal of medical research

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pharmacokinetics and biopharmaceutics 1978, 6, 165-75. 35. Midha, K. K.; Rawson, M. J.; Hubbard, J. W., The bioequivalence of highly

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1985, 26, 251–255. 38. Fasinu, P.; Pillay, V.; Ndesendo, V. M.; du Toit, L. C.; Choonara, Y. E., Diverse approaches for the enhancement of oral drug bioavailability. Biopharmaceutics & drug

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and

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Bordim, A.; Almeida, J. C.; Santos-Oliveira, R., Biodistribution of nanoparticles: initial

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and primary liver cancer. Current pharmaceutical biotechnology 2012, 13, 210-7. 43. Saller, R.; Meier, R.; Brignoli, R., The use of silymarin in the treatment of liver

758 759

diseases. Drugs 2001, 61, 2035-63.

760

Figure captions

infected

761 762

Figure 1. Transmission electron microscopy micrographs of (A) LSM and (B) ESM

763

Original magnification 120000 X, scale bars is 100 nm.

764

LSM: liposome-encapsulated silymarin formulation

765

ESM: ethosome-encapsulated silymarin formulation

766 767

Figure 2. In vitro release-time profiles of silibinin from silymarin (free form), LSM and

768

ESM across a cellulose membrane added to the donor compartment of a Franz diffusion

769

cell. (Mean ± SD, n = 4).

770


771

ESM: ethosome-encapsulated silymarin formulation 37



772 773

Figure 3. Representative product ion mass spectra and chemical structures of (A)

774

silibinin and (B) naringin (IS).

775 776 777

Figure 4. Concentration curve of silibinin in rat plasma after i.v. administration of

778

silymarin (10 mg/kg, iv.), LSM (10 mg/kg, iv.), and ESM (10 mg/kg, iv.); mean ± S.E.M,

779

n=5

780 781

Figure 5. Concentration curve of silibinin in rat plasma after oral administration of

782

silymarin (100 mg/kg, p.o.), LSM (100 mg/kg, p.o.), and ESM (100 mg/kg, p.o.); mean

783

± S.E.M, n = 5

784


785


786 787

Figure 6. Silibinin concentration of rat liver, heart, spleen, lung, kidney, and plasma at

788

30 min following i.v. administration of silymarin (free form), LSM and ESM 10 mg/kg

789

(mean ± SEM, n = 6).

790

LSM: liposome-encapsulated silymarin formulation 38


Page 38 of 49

Page 39 of 49


791


792 793

39



794

Page 40 of 49

Table 1. Characterization of LSM and ESM by vesicle size, zeta potential, PDI and drug

Size (nm)

Zeta (mV)

PDI

Encapsulation(%)

LSM

272.5±5.11

-27.14±0.84

0.32±0.02

60.36±1.78

ESM

321.1±22.4

-36.32±4.65

0.30±0.01

69.13±3.25

795

encapsulation.

796

PDI: polydispersity index

797


798


799

40


Page 41 of 49

800


Table 2. Pharmacokinetic parameters of silibinin in rat plasma. Data are presented the mean ± S.E.M. LSM Pharmacokinetic

ESM

Silymarin

Silymarin

LSM

ESM

(10 mg/kg, i.v.) (10 mg/kg, i.v.) (10 mg/kg, i.v.)

(100 mg/kg, p.o.) (100 mg/kg, p.o.) (100 mg/kg, p.o.)

parameters AIC of one-compartment

57.0±4.5

65.24±3.60

66.39±8.17

AIC of two-compartment

37.6±7.2

52.99±8.31

57.47±11.52

A (µg/mL)

1.63±0.64

0.82±0.14

1.49±0.34

B (µg/mL)

0.12±0.05

0.09±0.02

0.06±0.03

Alpha (1/min)

0.20±0.04

0.23±0.04

0.61±0.04

Beta (1/min)

0.03±0.01

0.03±0.01

0.02±0.01

t 1/2,α (min)

3.92±0.70

3.54±0.71

6.05±3.14

41



Page 42 of 49

t 1/2,β (min)

43.1±20.9

32.54±7.81

41.19±6.69

80.2±33.6

232±108

48.1±2.5

Cmax (µg/mL)

1.75±0.69

0.92±0.13

1.55±0.34

0.02±0.01

0.11±0.01*

0.08±0.02*

10.0±2.9

13.0±2.0

20.0±10.3

Tmax (min) CL (L/min/kg)

1.01±0.34

1.33±0.16

0.78±0.10

130±87

9.54±2.10*

32.1±12.2

MRT (min)

25.1±9.9

27.12±5.93

23.23±5.33

119±45

308±153

70.2±9.1

AUC (min*µg/mL)

12.9±3.5

7.99±0.9

14.7±3.2

2.04±0.74

15.2±6.5*

4.48±1.03

1.58

11.78

3.47

Bioavailability (%) 801

Data are expressed the mean±S.E.M, n = 5.

802

*

803


804


P < 0.05, significantly different from silymarin

42


Page 43 of 49


805

(A)

(B)

806

Figure 1. Transmission electron microscopy micrographs of (A) LSM and (B) ESM

807

Original magnification 120000 X, scale bars is 100 nm.

808


809


810 811

43



Page 44 of 49

812

10

Silymarin LSM ESM

Release (%)

8

6

4

2

0 0

10

20

30

40

50

Time (hr)

813 814

Figure 2. In vitro release-time profiles of silibinin from silymarin (free form), LSM and

815

ESM across a cellulose membrane added to the donor compartment of a Franz diffusion

816

cell. (Mean ± SD, n = 4).

817


818


819 820

44


Page 45 of 49


821 822

Figure 3. Representative product ion mass spectra and chemical structures of (A)

823

silibinin and (B) naringin (IS).

824 825

45



Plasma concentration of silibinin (ng/ml)

826

10000

Silymarin (100 mg/kg, i.v.) LSM (100 mg/kg, i.v.) ESM (100 mg/kg, i.v.)

1000

100

10

1 0

20

40

60

80 100 120 140 160 180 200 Time (hr)

827 828

Figure 4. Concentration curve of silibinin in rat plasma after i.v. administration of

829

silymarin (10 mg/kg, iv.), LSM (10 mg/kg, iv.), and ESM (10 mg/kg, iv.); mean ±

830

S.E.M, n = 5

831

46


Page 46 of 49


Plasma concentration of silibinin (ng/ml)

Page 47 of 49

1000 Silymarin (100 mg/kg, p.o.) LSM (100 mg/kg, p.o.) ESM (100 mg/kg, p.o.) 100

10

1 0

20

40

60

80 100 120 140 160 180 200 Time (hr)

832 833

Figure 5. Concentration curve of silibinin in rat plasma after oral administration of

834

silymarin (100 mg/kg, p.o.), LSM (100 mg/kg, p.o.), and ESM (100 mg/kg, p.o.);

835

mean ± S.E.M, n = 5

836


837


838 839

47



Silibinin concentration (ng/g)

25000

Silymarin,10 mg/kg, i.v. LSM, 10 mg/kg, i.v. ESM, 10 mg/kg, i.v.

20000 15000 10000 5000 0 Liver Heart Spleen Lung KidneyPlasma

840 841

Figure 6. Silibinin concentration of rat liver, heart, spleen, lung, kidney, and plasma at

842

30 min following i.v. administration of silymarin (free form), LSM and ESM 10

843

mg/kg (mean ± SEM, n = 6).

844


845


846 847

48


Page 48 of 49

Page 49 of 49


848

TABLE OF CONTENTS GRAPHICS

849 850

49


Pharmacokinetics, tissue distribution and excretion of peimisine in rats assessed by liquid chromatography-tandem mass spectrometry.

Pharmacokinetics in rats and tissue distribution in mouse of magnoflorine by ultra performance liquid chromatography-tandem mass spectrometry.

MS).

A sensitive liquid chromatography-tandem mass spectrometry method for pharmacokinetics and tissue distribution of nuciferine in rats.

Pharmacokinetic and tissue distribution profile of curculigoside after oral and intravenously injection administration in rats by liquid chromatography-mass spectrometry.

Determination of xanthotoxin using a liquid chromatography-mass spectrometry and its application to pharmacokinetics and tissue distribution model in rat.

Tissue distribution and excretion study of neopanaxadiol in rats by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of sweroside in rats.

Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, bioavailability and tissue distribution of neohesperidin dihydrochalcone in rats.

Pharmacokinetics and excretion study of sophoricoside and its metabolite in rats by liquid chromatography tandem mass spectrometry.

Metabolite Profiling, Pharmacokinetics, and In Vitro Glucuronidation of Icaritin in Rats by Ultra-Performance Liquid Chromatography Coupled with Mass Spectrometry.

Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry.

Mass Spectrometry Analysis and Pharmacokinetic Assessment of Ponatinib in Sprague-Dawley Rats.

Investigation on pharmacokinetics, tissue distribution and excretion of a novel platinum anticancer agent in rats by inductively coupled plasma mass spectrometry (ICP-MS).

Plasma protein binding, pharmacokinetics, tissue distribution and CYP450 biotransformation studies of fidarestat by ultra high performance liquid chromatography-high resolution mass spectrometry.

Pharmacokinetics of Rac inhibitor EHop-016 in mice by ultra-performance liquid chromatography tandem mass spectrometry.

Quantification of homoegonol in rat plasma using liquid chromatography-tandem mass spectrometry and its pharmacokinetics application.

Quantification of nardosinone in rat plasma using liquid chromatography-tandem mass spectrometry and its pharmacokinetics application.

Preclinical pharmacokinetics, tissue distribution and excretion studies of a potential analgesics - corydaline using an ultra performance liquid chromatography-tandem mass spectrometry.

Multidetection of antibiotics in liver tissue by ultra-high-pressure-liquid-chromatography-tandem mass spectrometry.

Determination of erythromycin A in salmon tissue by liquid chromatography with ion-spray mass spectrometry.

Quantification of neurotransmitters in mouse brain tissue by using liquid chromatography coupled electrospray tandem mass spectrometry.

Pharmacokinetics and tissue distribution of clevidipine and its metabolite in dogs and rats.

The Pharmacokinetics and Tissue Distribution of Honokiol and its Metabolites in Rats.