Supersaturable self-emulsifying drug delivery system of krill oil with

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Bioactive Constituents, Metabolites, and Functions

Supersaturable self-emulsifying drug delivery system of krill oil with improved oral absorption and hypotriglyceridemic function Yoshiki Seto, Chikara Morizane, Kodai Ueno, Hideyuki Sato, and Satomi Onoue J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00693 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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Journal of Agricultural and Food Chemistry

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Supersaturable self-emulsifying drug delivery system of krill oil with improved

2

oral absorption and hypotriglyceridemic function

3 4

Yoshiki Seto, Chikara Morizane, Kodai Ueno, Hideyuki Sato, and Satomi Onoue*

5 6

Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical

7

Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan

8 9 10 11 12 13 14 15 16 17 18 19

*Corresponding author, Department of Pharmacokinetics and Pharmacodynamics,

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School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku,

21

Shizuoka 422-8526, Japan.

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[email protected]

Tel.: +81-54-264-5630; Fax: +81-54-264-5636, E-mail:

23

1 ACS Paragon Plus Environment


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ABSTRACT

25

This study aimed to develop a supersaturable self-emulsifying drug delivery

26

system (S-SEDDS) of krill oil (KO), a rich source of docosahexaenoic acid and

27

eicosapentaenoic acid (EPA), to improve its hypotriglyceridemic function.

28

of KO (KO/S-SEDDS) was prepared by the addition of lysolecithin, glycerin, and

29

hydroxypropyl methylcellulose (HPMC).

30

HPMC

31

physicochemical and pharmacokinetic properties of KO samples were characterized,

32

and the hypotriglyceridemic function of KO/S-SEDDS was evaluated.

33

droplets in KO/SEDDS and KO/S-SEDDS with a mean diameter of ca. 270 nm could be

34

observed compared with KO and KO/HPMC.

35

tended to enhance the dissolution behavior of KO, and the S-SEDDS formulation

36

improved the dissolution behavior of KO due to micronized droplets and the addition of

37

HPMC.

38

basis of the pharmacokinetic profiling of EPA, and repeated oral administration of

39

KO/S-SEDDS (250 mg-KO/kg/day) for 7 days had a potent hypotriglyceridemic effect

40

on rats with corn oil-induced hypertriglyceridemia compared with orally administered

41

KO.

42

option to enhance the nutraceutical properties of KO.

(KO/HPMC)

were

also

S-SEDDS

SEDDS of KO (KO/SEDDS) and KO with

prepared

for

comparison

purposes.

The

Micronized

Both KO/HPMC and KO/S-SEDDS

KO/S-SEDDS (60 mg-EPA/kg) improved the oral absorption of KO on the

Based on these findings, the S-SEDDS approach might be an efficacious dosage

43 44

KEYWORDS: krill oil, oral absorption, supersaturable self-emulsifying drug delivery

45

system, hypotriglyceridemic function

46


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47

ABBREVIATIONS: AUC0–8h, area under the concentration versus time curve from 0

48

to 8 h; AUC0–24h, area under the concentration versus time curve from 0 to 24 h; Cmax,

49

maximum concentration; DHA, docosahexaenoic acid; DLS, dynamic light scattering;

50

EPA,

51

methylcellulose, KO, krill oil; PK, pharmacokinetic; SEDDS, self-emulsifying drug

52

delivery system; SIR, selected ion recording; S-SEDDS, supersaturable self-emulsifying

53

drug delivery system; TEM, transmission electron microscopy; TG, triglyceride; Tmax,

54

time to reach the maximum concentration; UPLC/ESI-MS, ultra-performance liquid

55

chromatography equipped with electrospray ionization mass spectrometry; VLDL, very

56

low-density lipoprotein

eicosapentaenoic

acid;

GI,

gastrointestinal;


HPMC,

hydroxypropyl


57

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1. Introduction

58

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), essential

59

omega-3 polyunsaturated fatty acids, have various biological effects, including

60

hypolipidemic and antithrombolic effects 1, 2, and the main sources of DHA and EPA are

61

marine oils 3.

62

their oral bioavailability is limited 5.

63

(Euphausia superba), is abundant in DHA and EPA 6.

64

phospholipids in KO contain DHA and EPA, and DHA and EPA in KO are mainly

65

present in the form of phospholipids (ca. 70% of total DHA; and ca. 70% of total EPA) 4,

66

6

67

because they are emulsified in water

68

addition, KO was reported to contain the free forms of DHA and EPA (ca. 10% of total

69

DHA; and ca. 10% of total EPA); however, the free and phospholipid forms of DHA

70

and EPA in fish oil had been negligible in the previous report 4.

71

considered as an attractive source of DHA and EPA with enhanced oral absorption.

72

Although KO was reported to have many health-promoting properties, such as

73

anti-inflammatory, hypoglycemic, and hypolipidemic effects

74

DHA and EPA levels after KO intake might be insufficient compared with fish oil 10, 11.

75

The intake of KO at 1–3 g/day has been needed to take full advantage of its biological

76

functions, and the similar or relatively higher daily doses of fish oil also provided its

77

attractive biological functions compared with KO

78

the delivery of KO is required to gain access to its attractive biological benefits at a low

79

intake.

80

.

Fish oil is rich in DHA and EPA as triglyceride (TG) forms 4; however, Krill oil (KO), extracted from Antarctic krill Both triacylglycerols and

The phospholipid forms have advantages for the oral absorption of DHA and EPA 7

and incorporated into cell membranes 5.

6, 10

.

In

Thus, KO is

6, 8-10

, increase in plasma

Thus, further improvement in

To improve oral absorption and biological functions of poorly-water soluble


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81

chemicals, several pharmaceutical technologies were used including solid dispersion,

82

nanoparticles, solid lipid nanoparticles, emulsion, and self-emulsifying drug delivery

83

system (SEDDS) 12-16.

84

had been applied lipid-based formulations

85

supersaturable SEDDS (S-SEDDS) have been developed as attractive lipid-based

86

formulations to enhance the solubility and oral bioavailability of drugs with a limited

87

aqueous solubility

88

co-surfactants, and substances, and it forms an emulsion in digestive fluid.

89

approach has been used to improve the bioavailability and functionality of hydrophobic

90

bioactive nutrients

91

self-emulsifying potential.

92

co-surfactant to KO led to the formation of a fine oil-in-water emulsion, and favorable

93

in vitro anti-inflammatory effects had been observed using KO-in water emulsion

94

S-SEDDS was developed by a water-soluble polymeric precipitation inhibitor to

95

stabilize substances in a temporary supersaturated state

96

bioavailability of some drugs delivered through S-SEDDS was observed compared with

97

that delivered through SEDDS

98

especially the S-SEDDS approach, may be effective for improving the nutraceutical

99

properties of KO; however, the feasibility of using S-SEDDS to improve the

100

Especially, omega-3 polyunsaturated fatty acids, fish oil or KO,

17

.

12, 14

.

In these technologies, SEDDS and

The SEDDS formulation consists of lipids, surfactants,

15, 18-20

.

21

Phospholipids are surfactants

SEDDS

; therefore, KO has a

In a previous report, the addition of glycerol as a

22-24

.

12

.

22

, and the enhanced oral

In this context, lipid-based formulations,

bioavailability of KO is still unclear.

101

In the present study, KO was incorporated into S-SEDDS by mixing KO,

102

lysolecithin, glycerin, and hydroxypropyl methylcellulose (HPMC) to enhance its oral

103

bioavailability and thus its potential health benefits.

104

of KO/S-SEDDS were characterized with focusing on the particle size and dissolution

The physicochemical properties



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105

behaviors.

An in vivo test was also employed to investigate the plasma EPA level after

106

oral administration of KO/S-SEDDS as an indicator of oral absorbability of KO.

107

hypotriglyceridemic effect after repeated oral administration of KO and KO/S-SEDDS

108

was evaluated by measuring the TG level in rat plasma after oral administration of corn

109

oil.

110


The

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111

2. Materials and Methods

112

2.1. Materials

113

KO was kindly supplied by ACRESS (Osaka, Japan).

The content rate of

114

phospholipids was 40% in KO, and the content rates of DHA and EPA as phospholipid

115

forms in KO were 6% and 13%, respectively.

116

mPa・s (2% solution at 20°C)] was kindly supplied by Shin-Etsu Chemical (Tokyo,

117

Japan).

118

non-hydrogenation] was obtained from Tsuji Seiyu (Mie, Japan).

119

tamoxifen were bought from Wako Pure Chemical industries (Osaka, Japan).

120

chemicals were purchased from commercial sources.

HPMC [TC-5○R , Grade: R, viscosity: 6

Lysolecithin [SLP-paste lyso, the content rate of phospholipids: 95–98%, Glycerin and All other

121 122 123

2.2. Preparation of KO formulations The selection of surfactant, co-surfactant, and polymer was with reference to 17, 22, 24, 25

124

the previous reports

125

KO to prepare KO/HPMC, and KO/HPMC was agitated with vortex mixer (VORTEX

126

Genius 3; IKA, Staufen, Germany) and mixed by inverting under room temperature.

127

To obtain KO/SEDDS, KO, lysolecithin, and glycerin were simultaneously mixed by

128

inverting, and the oily liquid state of KO/SEDDS was agitated using vortex mixer

129

under room temperature.

130

powder to prepared KO/SEDDS, and the oily liquid was agitated with a spatula and

131

mixed by inverting under room temperature.

132

are described in Table 1.

.

HPMC powder, a hydrophilic polymer, was added to

KO/S-SEDDS was prepared by the addition of HPMC

The compositions of KO formulations

133 134

2.3. Transmission electron microscopy (TEM)



135

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An aliquot of KO samples suspended in distilled water (40 mg-KO/mL) using

136

vortex mixer was placed on a Formvar 200 mesh Cu (Nisshin EM, Tokyo, Japan).

137

sample was allowed to stand for 1 min, and then any excess solution was removed by

138

blotting.

139

heptamolybdate tetrahydrate and allowed to dry.

140

HT7700 TEM System (Hitachi High-Technologies, Tokyo, Japan).

The

The samples were negatively stained with 1% (w/v) hexaammonium They were then visualized under an

141 142

2.4. Dynamic light scattering (DLS)

143

The particle size distributions of KO and its formulations after dispersion in

144

distilled water were measured by the DLS method using a Zetasizer Nano ZS

145

(MALVERN, Worcestershire, UK).

146

distilled water (ca. 0.13 mg-KO/mL), and the samples were mixed with vortex mixer

147

before measurement.

The KO and its formulations were suspended in

148 149

2.5. Dissolution tests

150

To evaluate initial self-emulsifying potentials of KO samples, dissolution

151

tests of KO samples (1 g-KO) were carried out in pH1.2 solution (100 mL) at 37°C

152

with constant stirring at 50 rpm using a magnetic stirrer (SST-66, Shimadzu, Kyoto,

153

Japan).

154

120 min) and centrifuged at 6,000×g for 5 min.

155

with the same volume of acetonitrile, and the absorbance at 241 nm, the maximum

156

absorption wavelength of diluted KO samples within 230–700 nm, was measured by

157

SAFIRE (TECAN, Männedolf, Switzerland) to determine the KO concentration.

Samples were collected at the indicated periods (0, 5, 15, 30, 45, 60, 90, and Obtained supernatants were diluted

158


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159

2.6. In vivo preparation

160

Male Sprague-Dawley rats at 6–10 weeks of age (ca. 286±110 g body weight)

161

were purchased from SLC Inc. (Hamamatsu, Japan), housed in the laboratory with

162

free access to food and water, and maintained on a 12-h dark/light cycle in a room

163

with controlled temperature (24±1°C) and humidity (55±5%).

164

used in the present study were conducted according to the guidelines approved by the

165

Institutional Animal Care and Ethical Committee of the University of Shizuoka.

All of the procedures

166 167

2.7. Pharmacokinetic study after oral administration of KO samples

168

As an indicator of the oral absorption of KO, plasma EPA levels in rats were

169

measured after oral administration of KO samples.

Each KO sample was dispersed

170

in distilled water and orally administered to rats (60 mg-EPA/kg; the dose represents

171

the amount of phospholipid forms of EPA).

172

mL/kg) was orally administered.

173

tail vein of unanesthetized rats at the indicated times (0, 0.5, 1, 2, 3, 6, 12, and 24 h)

174

after oral administration of KO samples.

175

centrifugation (4°C, 10,000×g, 10 min) and stored at -20°C until they were analyzed.

176

The plasma samples (50 µL) were deproteinized by the addition of acetonitrile (150 µL)

177

containing an internal standard (tamoxifen) and centrifuged at 10,000×g for 10 min at

178

4°C.

179

USA), the plasma concentration of EPA was analyzed by ultra-performance liquid

180

chromatography with an electron spray ionization mass spectrometry (UPLC-ESI/MS)

181

system.

182

the concentration versus time curve from 0 to 24 h (AUC0–24h) was calculated using

As a control group, distilled water (10

Blood samples (300 µL) were collected from the

Plasma samples were obtained by

After filtration (0.20-µm membrane filter, Millex-LG; Millipore, Billerica, MA,

On the basis of the obtained EPA concentration in the plasma, the area under



183

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GraphPad Prism 5 for Windows (version 5.04, GraphPad Software, La Jolla, CA, USA).

184 185

2.8. UPLC analysis

186

The plasma concentration of EPA was determined by UPLC-ESI/MS analysis.

187

The UPLC-ESI/MS system consisted of a Waters Acquity UPLC system (Waters,

188

Milford, MA, USA), which included a binary solvent manager, a sample manager, a

189

column compartment, and a micromass SQ detector connected with Waters Masslynx

190

v. 4.1.

191

Φ2.1×50 mm; Waters) was used, and the column temperature was maintained at 40°C.

192

The standards and samples were separated using a gradient mobile phase consisting of

193

Milli-Q containing 5 mM ammonium acetate (A) and acetonitrile (B).

194

conditions of the mobile phase were 0–0.5 min, 70% B; 0.5–3.5 min, 70–95% B (linear

195

gradient curve), and the flow rate was set at 0.25 mL/min.

196

using selected ion recording (SIR) for specific m/z 302.5 for EPA [M–H]- and 372 for

197

tamoxifen [M+H]+.

A Waters Acuity UPLC BEH C18 (particle size: 1.7 µm and column size:

The gradient

Analysis was carried out

198 199

2.9. Functionality test of KO samples

200

Corn oil was used as a TG supplier, and 50% (w/w) corn oil emulsion was

201

prepared by adding the same weight of distilled water with lysolecithin to a final

202

concentration of 1% (w/w) in corn oil emulsion.

203

mg-KO/kg/day, once a day) were orally administered to rats for 7 days, and corn oil

204

emulsion (10 mL/kg) was orally administered at 24 h after final administration of KO

205

samples.

206

for 7 days, and distilled water (10 mL/kg) or corn oil emulsion (10 mL/kg) was orally

KO and KO/S-SEDDS (250

Distilled water (10 mL/kg/day, once a day) was orally administered to rats


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207

administered at 24 h after final administration of distilled water as a control or vehicle

208

group, respectively.

209

unanesthetized rats at the indicated times (0, 2, 4, 6, and 8 h) after oral administration

210

of corn oil emulsion, and then plasma samples were obtained by centrifugation

211

(3,200×g, 10 min).

212

Briefly, plasma samples (2 µL) and coloring substance (300 µL) were mixed in the

213

wells of 96-well microplate (Asahi Glass, Tokyo, Japan), and the absorbance at 600 nm

214

was measured by SAFIRE after incubation (5 min, 37°C).

Blood samples (300 µL) were collected from the tail vein of

Plasma TG levels were determined using LabAssayTM Triglyceride.

215 216

2.10. Data analysis

217

For statistical comparisons, one-way ANOVA with pairwise comparison by

218

Fisher’s least significant difference procedure was used. A p-value of less than 0.05

219

was considered significant for all analyses.

220



221

3. Results and Discussion

222

3.1. Physicochemical characterization of KO samples

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The physicochemical characteristics of KO samples after dispersion in distilled

223 224

water were evaluated in terms of droplet sizes and dissolution behaviors.

225

samples could disperse when introduced into distilled water, and the morphology of KO

226

samples after dispersion was observed using TEM (Fig. 1).

227

samples were found to be spherical particles, and KO/SEDDS and KO/S-SEDDS

228

formed fine droplets compared with KO and KO/HPMC.

229

samples after dispersion in distilled water were measured by DLS (Table 2).

230

basis of DLS analysis, the mean diameter of KO was calculated to be 566 nm, and the

231

droplet size of KO/HPMC was not changed, as evidenced by a mean diameter of 565

232

nm.

233

and 266 nm, respectively, and the polydispersity indexes of KO/SEDDS and

234

KO/S-SEDDS were lower than those of KO and KO/HPMC, suggesting micronized

235

droplets with narrow size distributions in KO/SEDDS and KO/S-SEDDS.

236

zeta-potential of KO ranged from ca. –36 to –32 mV owing to the presence of

237

negatively charged phospholipids and free fatty acids in KO.

238

samples in DLS analysis were mostly consistent with the diameters in TEM observation.

239

In a previous investigation, a S-SEDDS formulation of ginger extract was prepared, and

240

the obtained data from particle size analysis were similar between SEDDS and

241

S-SEDDS formulations of ginger extract

242

lysolecithin and glycerin would contribute to the formation of fine and uniform droplets

243

in KO/SEDDS and KO/S-SEDDS, whereas the addition of HPMC would not affect the

244

particle size of KO droplets.

All KO

All droplets in all KO

The droplet sizes of KO On the

The mean diameters of KO/SEDDS and KO/S-SEDDS were calculated to be 269

25

.

The

The droplet sizes of KO

Based on these findings, the addition of


Page 13 of 32


245

SEDDS formulations can spontaneously form an emulsion when introduced 17

246

into aqueous media in the gastrointestinal (GI) tract

247

self-emulsifying performances, the dissolution/dispersion profiles of KO samples were

248

monitored in acidic solution (pH1.2) at 37°C up to 120 min (Fig. 2).

249

gradually dispersed with the amount of ca. 2.6% of the total at 120 min under the

250

present conditions.

251

almost the same as that of KO, the initial dissolution/dispersion of KO/HPMC and

252

KO/SEDDS was slightly improved.

253

reached a plateau at 30 min, and the amount of KO dispersed in KO/S-SEDDS at 30

254

min was 5.6-fold higher than that in KO.

255

behaviors of KO in KO/S-SEDDS could be observed, and the S-SEDDS approach may

256

facilitate the rapid and high-level oral absorption of active components in KO.

.

To evaluate the

KO was

Although the amounts of these formulations at 120 min were

In KO/S-SEDDS, the level of KO dispersed

Therefore, rapid dissolution and dispersion

257 258

3.2. Pharmacokinetic behaviors of EPA after oral administration of KO samples

259

In preliminary study, plasma DHA and EPA concentrations were also

260

monitored after oral administration of KO formulations; however, the variation in DHA

261

levels in the plasma were too wide to use as an indicator of oral KO absorption.

262

to clarify the enhancement in the oral absorption of KO by the S-SEDDS approach,

263

plasma EPA levels, employed as an indicator of the oral absorption of KO, were

264

monitored after oral administration of KO samples (60 mg-EPA/kg) (Fig. 3), and

265

AUC0–24h values of EPA were calculated.

266

absolute increases in oral absorption of EPA by KO.

267

could be detected after 24-h fasting, and the concentrations of EPA in the plasma were

268

at comparable levels.

Thus,

A control group was employed to clarify In all groups, EPA in the plasma

In the control group, a decrease in the plasma concentration of



Page 14 of 32

269

EPA was observed within 1 h, and the plasma EPA level was maintained at ca. 3 µg/mL

270

until at least 24 h.

271

maximum concentration (Cmax) (5.4±0.2 µg/mL) at 3 h, and then the concentration

272

decreased to the initial EPA level at 6 h.

273

KO group (95±1.3 µg・h/mL) was found to be 1.5-fold higher than that in the control

274

group (62±7.5 µg・h/mL), and enhancement of the oral absorption of EPA by KO might

275

still be limited.

276

oil was needed to exhibit its functionality 6, 10; therefore, the potential of KO to improve

277

the oral absorption of EPA and DHA might not be high enough to produce their

278

attractive biological effects.

279

be observed at around 2 to 3 h after oral administration of KO/S-SEDDS compared with

280

the other groups.

281

Cmax levels of EPA compared with the KO group, at 8.3±1.3 and 7.5±0.7 µg/mL,

282

respectively, and KO/HPMC shortened the time to reach the plasma Cmax (Tmax)

283

compared with the KO group.

284

(Fig. 3), increases in plasma EPA levels were similar between KO/HPMC and

285

KO/S-SEDDS groups up to 1 h.

286

behaviors and oral absorption of poorly water-soluble chemicals, and HPMC might

287

inhibit the precipitation of free forms of EPA in the GI tract

288

increase in the plasma EPA level after oral administration of KO/S-SEDDS may mainly

289

be attributed to the addition of HPMC.

290

KO/SEDDS groups were calculated to be 130±4.3 and 105±5.2 µg・h/mL, respectively,

291

and the oral absorption of KO in these groups tended to be higher than that in the KO

After oral administration of KO, the plasma EPA level reached its

The calculated AUC0–24h value of EPA in the

In previous reports, the intake of KO at a dose similar to that of fish

The highest Cmax value of EPA at 10.8±1.6 µg/mL could

Oral administration of KO/HPMC and KO/SEDDS led to higher

On the basis of the plasma concentration-time curves

HPMC had been employed to improve the dissolution

23

.

Thus, the rapid

The AUC0–24h values of EPA in KO/HPMC and


Page 15 of 32


292

group.

KO/S-SEDDS exhibited significantly higher AUC0–24h value of EPA compared

293

with the KO group, as evidenced by 161±23 µg・h/mL (P＜0.05; versus KO group), and

294

improvement of the oral absorption of EPA would be more potent compared with

295

KO/HPMC and KO/SEDDS.

296

about a higher oral absorption of lipophilic active ingredients compared with SEDDS

297

formulations

298

increase the oral absorption of EPA due to its inhibitory effect on precipitation.

299

contains other active ingredients, and, to clarify the actual improvement of oral

300

absorption of KO, monitoring plasma concentrations of these ingredients would be still

301

needed after the oral administration of KO/S-SEDDS.

302

absorption of other active ingredients in KO is still unclear, an S-SEDDS approach

303

would be useful for improving the oral absorption of KO according to the PK profiles of

304

EPA in the KO/S-SEDDS group, possibly leading to enhanced functionalities of KO.

In previous reports, S-SEDDS formulations had brought

22, 23, 26

. Thus, the addition of HPMC might be of marked benefit to KO

Although enhancement in oral

305 306 307

3.3. Hypotriglyceridemic function of KO/S-SEDDS KO has attractive functions for health, and reduction of the blood TG level by 6, 27, 28

308

orally administered KO or EPA had been reported in several clinical trials

309

According to the results of the present pharmacokinetic study, only KO/S-SEDDS

310

indicated a significant improvement of plasma EPA level compared with KO.

311

evaluate the hypotriglyceridemic effect of KO/S-SEDDS, plasma TG levels in rats

312

pretreated with KO and KO/S-SEDDS (250 mg-KO/kg/day, 7 days, p.o.) were

313

monitored after oral administration of corn oil, and the AUC from 0 to 8 h (AUC0–8h) of

314

plasma TG was calculated (Fig. 4).

315

KO theoretically contains ca. 32.5 mg of EPA.

.

Thus, to

According to the content rate of EPA, 250 mg of Pretreatment with the ethyl ester form



Page 16 of 32

316

of EPA (300 mg/kg/day, 7 days, p.o.) had significantly reduced the rise of TG in rat

317

plasma after oral administration of corn oil

318

difference in AUC0–24h values of EPA between the ethyl ester form of EPA (60

319

mg-EPA/kg) and control groups was calculated to be ca. 13 µg・h/mL.

320

hand, difference in AUC0–24h values of EPA between control and KO/S-SEDDS groups

321

was found to be ca. 100 µg・h/mL based on the present pharmacokinetic profiles.

322

potential of enhancement in plasma EPA level by KO/S-SEDDS may be ca. 10-fold

323

higher than that by the ethyl ester form of EPA.

324

samples was set at 250 mg-KO/kg/day in the present functionality investigation.

325

vehicle group, the plasma TG level increased to around 350 mg/dL within 4 h after oral

326

administration of corn oil, and the calculated AUC0–8h value in the vehicle group was

327

significantly higher than that in the control group.

328

TG levels were increased within 3 h after intake of breakfast containing 20 g of corn oil

329

30

330

effects of KO samples.

331

intake of corn oil was suppressed by pretreatment with KO and KO/S-SEDDS

332

compared with the vehicle group, and the Cmax level of TG in the KO/S-SEDDS group

333

was lower than that in the KO group.

334

KO/S-SEDDS groups gradually decreased to become comparable to that in the control

335

group at 8 and 6 h after oral administration of corn oil, respectively.

336

AUC0–8h values in both KO and KO/S-SEDDS groups were significantly lower than that

337

in the vehicle group, and the hypotriglyceridemic potential of KO/S-SEDDS was

338

1.7-fold higher than that of KO on the basis of the AUC0–8h values.

339

EPA are known to have lipid-lowering effects, the mechanisms of which have been

29

.

In preliminary experiments, the

On the other

The

In this context, the dose of KO In the

In the previous clinical trials, blood

; therefore, the present rat model would be acceptable to evaluate hypotriglyceridemic Elevation of plasma TG levels in rats induced by the oral

Then, the plasma TG levels in the KO and


The calculated

Both DHA and

Page 17 of 32


340

thought to involve the inhibition of TG production and increase of TG clearance 28, 31, 32.

341

Ethyl ester forms of DHA and EPA suppressed the activity of fatty acid and TG

342

synthesizing enzymes in the rat liver 31.

343

(VLDL) production by omega-3 fatty acids was also reported 28, and the intake of fish

344

oil reduced VLDL-TG secretion from the liver

345

trials, blood VLDL levels were significantly increased in patients with metabolic

346

syndrome compared with healthy subjects

347

with omega-3 fatty acids (4 g/day, 6 weeks) was decreased to become comparable level

348

of control subjects

349

weeks) indicated no significant impact on low-density and high-density lipoproteins

350

levels in blood

351

suppression of increases in plasma TG levels in KO and KO/S-SEDDS groups after oral

352

administration of corn oil.

353

in the acceleration of TG-rich lipoprotein clearance 28.

354

sample groups could be reduced to levels comparable with that in the control group.

355

In this experiment, the plasma levels of TG derived from corn oil was monitored;

356

however, the verified functionality of KO/S-SEDDS may be still limited.

357

further investigations for monitoring the changes of TG, cholesterols and lipoproteins

358

using several metabolic syndrome models would be helpful to prove the functionality of

359

KO/S-SEDDS.

360

elimination of plasma TG were observed in the KO/S-SEDDS group compared with the

361

KO group, and the S-SEDDS approach would be efficacious for enhancing the

362

hypotriglyceridemic function of KO.

35, 36

.

36

.

The reduction of very low-density lipoprotein

32

.

According to the previous clinical

33, 34

, and blood VLDL level after treatment

On the other hand, intake of omega-3 fatty acids (4 g/day, 6

In this context, these mechanisms would contribute to the

EPA and DHA increase lipoprotein lipase activity, resulting Thus, plasma TG levels in KO

Therefore,

Based on these findings, lower plasma TG levels and faster

363



364

Page 18 of 32

4. Conclusion

365

In the present study, KO/S-SEDDS was prepared with the use of the SEDDS

366

technique with the addition of HPMC for improving the oral bioavailability and thus the

367

hypotriglyceridemic activity of KO.

368

and improved dissolution behavior of KO.

369

significant enhancement in the oral absorption of KO compared with other KO samples,

370

and the favorable hypotriglyceridemic function of KO could be observed after oral

371

administration of KO/S-SEDDS to rats with hypertriglyceridemia.

372

findings, the S-SEDDS would be an efficacious carrier to enhance the nutraceutical

373

values of KO.

KO/S-SEDDS indicated micronized droplet size Orally administered KO/S-SEDDS led to a

374


From these

Page 19 of 32


375 376

Acknowledgement The authors are grateful to ACRESS (Osaka, Japan) for providing KO.

This

377

work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 17K15516;

378

Y. Seto and 16K18950; H. Sato) from the Ministry of Education, Culture, Sports,

379

Science and Technology of Japan, and a grant from the Takeda Science Foundation.

380 381

Conflicts of interest

382

The authors declare that there are no conflicts of interest.

383 384

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385

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supplementation in hyperlipidemia: a systematic review and meta-analysis. Int. J.

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(2) Breslow, J. L. n-3 fatty acids and cardiovascular disease. Am. J. Clin. Nutr. 2006, 83,

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Schebb, N. H. Lipid Class Specific Quantitative Analysis of n-3 Polyunsaturated Fatty

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(8) Vigerust, N. F.; Bjorndal, B.; Bohov, P.; Brattelid, T.; Svardal, A.; Berge, R. K. Krill

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oil versus fish oil in modulation of inflammation and lipid metabolism in mice

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transgenic for TNF-alpha. Eur. J. Nutr. 2013, 52, 1315-1325.

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(9) Sampalis, F.; Bunea, R.; Pelland, M. F.; Kowalski, O.; Duguet, N.; Dupuis, S.

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(10) Maki, K. C.; Reeves, M. S.; Farmer, M.; Griinari, M.; Berge, K.; Vik, H.; Hubacher,

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R.; Rains, T. M. Krill oil supplementation increases plasma concentrations of

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eicosapentaenoic and docosahexaenoic acids in overweight and obese men and women.

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hypercholesterolemia in high-fat-fed mice. J. Agric. Food Chem. 2009, 57, 9339-9345.

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Emulsion Protects against Lipopolysaccharide-Induced Proinflammatory Activation of

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Macrophages In Vitro. Mar. drugs 2017, 15, pii: E74, 1-12.

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krill

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(15) Onoue, S.; Uchida, A.; Kuriyama, K.; Nakamura, T.; Seto, Y.; Kato, M.; Hatanaka,

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system of coenzyme Q10 with improved photochemical and pharmacokinetic behaviors.

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improved oral delivery of lipophilic drugs. Biomed. Pharmacother. 2004, 58, 173-182.

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solid dispersion for enhancing oral bioavailability of Lycopene--in vivo evaluation

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Lai-Kuen, R.; Chaumeil, J. C.; Sfar, S.; Charrueau, C. Resveratrol self-emulsifying

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system increases the uptake by endothelial cells and improves protection against

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oxidative stress-mediated death. Eur. J. Pharm. Biopharm. 2014, 86, 418-426.

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(20) Onoue, S.; Uchida, A.; Nakamura, T.; Kuriyama, K.; Hatanaka, J.; Tanaka, T.;

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Miyoshi, H.; Seto, Y.; Yamada, S. Self-nanoemulsifying particles of coenzyme Q10 with

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M. S.; Hageman, M. J. Development of a supersaturable SEDDS (S-SEDDS)

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function of ginger extract-loaded supersaturable self-emulsifying drug delivery systems.

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472

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473

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Laferrere, B. Impact of medium and long chain triglycerides consumption on appetite

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K. Effects of dietary alpha-linolenic, eicosapentaenoic and docosahexaenoic acids on

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hepatic lipogenesis and beta-oxidation in rats. Biosci. Biotechnol. Biochem. 1998, 62,

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489

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491

Newman, J. W. Abnormal lipoprotein oxylipins in metabolic syndrome and partial

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494

fatty acids in chronic kidney disease: nervonic acid predicts mortality. J. Renal Nutr.



495

2012, 22, 277-283.


Page 24 of 32

Page 25 of 32


496

Table 1

The composition of KO formulations KO (wt%)

Lysolecithin (wt%)

Glycerin (wt%)

HPMC (wt%)

KO/HPMC

95

–

–

5

KO/SEDDS

60

30

10

–

KO/S-SEDDS

57

29

9

5

497



498

Table 2

Page 26 of 32

Mean diameters and polydispersity index of KO samples

KO samples

Mean diameter (nm)

Polydispersity index

Zeta potential (mV)

KO

566

0.56

–36.1

KO/HPMC

565

0.54

–32.6

KO/SEDDS

269

0.35

–31.8

KO/S-SEDDS

266

0.36

–33.8

499


Page 27 of 32


500

Figure Legends

501

Fig. 1

502

KO/SEDDS, and (D) KO/S-SEDDS dispersed in distilled water.

503

500 nm.

Transmission electron microscopic images of (A) KO, (B) KO/HPMC, (C) Each bar represents

504 505

Fig. 2

506

KO/HPMC; □, KO/SEDDS; and ●, KO/S-SEDDS.

507

experiments.

▽, KO; △,

Dissolution behaviors of KO samples in pH1.2 solution.

Data represent mean±SE of 3

508 509

Fig. 3

Pharmacokinetic behaviors of EPA in rat plasma after oral administration of KO

510

samples (60 mg-EPA/kg).

511

▽, KO; △, KO/HPMC; □, KO/SEDDS; and ●, KO/S-SEDDS.

512

mean±SE of 4–6 rats.

Plasma concentration-time profiles of EPA.

◇, Control;

Data represent

513 514

Fig. 4

Hypotriglyceridemic effects of KO samples.

515

plasma TG after oral administration of corn oil.

516

and ●, KO/S-SEDDS.

517

plasma TG.

518

to vehicle group.

(A) Concentration-time curves of ◇, Control; ◆, Vehicle; ▼, KO;

Data represent mean±SE of 6 rats.

Data represent mean±SE of 6 rats.

(B) AUC0–8h values of

*P＜0.05; ***P＜0.001 with respect



Figure 1 229x172mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 28 of 32

Page 29 of 32


Figure 2 166x188mm (300 x 300 DPI)



Figure 3 160x166mm (300 x 300 DPI)


Page 30 of 32

Page 31 of 32


Figure 4 152x251mm (300 x 300 DPI)



TOC graphic 85x43mm (300 x 300 DPI)


Page 32 of 32

Supersaturable self-emulsifying drug delivery system of krill oil with

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