Development of Organogel-Derived Capsaicin Nanoemulsion with

May 12, 2016 - ABSTRACT: Capsaicin (CAP) is the major active component in chili peppers with health-promoting benefits. However, the low bioavailabili...
1 downloads 9 Views 1MB Size
Subscriber access provided by University of Sussex Library

Article

Organogel-derived capsaicin nanoemulsion has improved bioaccessibility and reduced gastric mucosa irritation Muwen Lu, Yong Cao, Chi-Tang Ho, and Qingrong Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01095 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 12, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

Development of organogel-derived capsaicin nanoemulsion with improved bioaccessibility and reduced gastric mucosa irritation

Muwen Lu†, Yong Cao‡, Chi-Tang Ho†,*and Qingrong Huang†,*



Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New

Jersey 08901, USA; ‡ College of Food Science, South China Agricultural University, Guangzhou 510642, P. R. China.

* To whom correspondence should be addressed. Tel: 848-932-5514 (QH); 848-932-5553 (CH). Fax: 732-932-6776. Email: [email protected] (QH); [email protected] (CH).

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1

ABSTRACT

2

Capsaicin (CAP) is the major active component in chili peppers with health-promoting

3

benefits. However, the low bioavailability and irritating quality of CAP greatly limit its

4

applications in functional foods. The objective of this study was to develop a food-grade

5

nanoemulsion to increase the dissolution and bioaccessibility of CAP, and to alleviate its

6

irritating effects. To achieve this goal, CAP was first dissolved in medium-chain triacylglycerol

7

(MCT), followed by the addition of sucrose stearate S-370 as organo-gelator to develop CAP-

8

loaded organogel. The oil-in-water (O/W) emulsion was formed using organogel as the oil phase

9

and Tween 80 as the emulsifier. After ultrasonication treatment, droplet sizes of emulsion were

10

decreased to 168 nm with enhanced dissolution rate and bioaccessibility. In vivo study further

11

confirmed the reduced rat gastric mucosa irritation caused by CAP. The organogel-derived

12

nanoemulsion was proved to be an effective delivery system for CAP-based functional food

13

products.

14

KEYWORDS: Capsaicin; organogel-derived nanoemulsion; dissolution; bioaccessibility;

15

gastric mucosa irritation

16

2

ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

17

Journal of Agricultural and Food Chemistry

Introduction

18

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) (CAP) (structure shown in Figure 1), a

19

naturally occurring alkaloid that exists in placental tissue, the internal membranes as well as

20

other parts of fruits of capsicum plants,1 is responsible for its hot and spicy taste.2 Researches on

21

CAP’s beneficial effects have demonstrated its anti-inflammation,3 anti-cancer,4-7 analgesic,8

22

cardioprotective,9 anti-oxidation,10 and anti-obesity activities,11 which function through

23

activating the transient receptor potential vanilloid (TRPV) superfamily of cation-channel

24

receptors.8-12

25

However, as a lipophilic substance, CAP’s low aqueous solubility greatly hinders its oral

26

bioavailability,13 and the intensely pungent flavor further results in a burning sensation for

27

mammals, including humans. Choi et al.14 demonstrated that repeated oral exposure to CAP

28

significantly increased anxiety-like behaviors with a prolonged stress-response in rats.

29

To enhance the bioavailability of CAP and reduce its irritation in stomach, various lipid-

30

based formulations for oral delivery of capsaicin, such as oil-in-water (O/W) microemulsions,15

31

self-nanoemulsifying systems,16 liposomes,17 mixed polymeric micelles18 and colloidal

32

nanocapsules,19 have been recently developed. Zhu, et al.

33

by dissolving CAP into medium-chain triacylglycerol (MCT, oil phase), Cremophor EL, absolute

34

ethanol (co-surfactant) and water, which had an enhanced stability, increased oral bioavailability

35

and reduced gastric mucosa irritation compared to free unformulated CAP. Choi et al.

36

developed a CAP-loaded nanoemulsion system stabilized with alginate and chitosan through

37

self-assembly and emulsification, which showed an enhanced stability compared with other food

38

delivery systems. In summary, O/W nanoemulsion system has a great potential in improving the

3

15

developed a microemulsion system

ACS Paragon Plus Environment

16

also

Journal of Agricultural and Food Chemistry

39

40

Page 4 of 30

efficiency of delivery of CAP compounds in functional foods to improve human health. Organogel-based nanoemulsion systems, which were first developed by Huang et al.

20

41

are relatively novel for oral delivery of nutraceuticals. They involved the formation of a thermo-

42

reversible semi-solid organogel to immobilize some highly crystalline nutraceuticals, and the

43

emulsification of the organogel was achieved through ultrasonication or high-pressure

44

homogenization. Organogel systems have the advantages of high stability and high encapsulation

45

efficiency

46

demonstrated that organogel-based nanoemulsion systems had an improved dispersibilty,

47

enhanced waster solubility and oral bioavailability of hydrophobic compound. Similar approach

48

was later extended to the formation of D-limonene organogel-based nanoemulsion with good

49

storage stability.21

for

water-insoluble

nutraceuticals.

Physiochemical

characterization

studies

50

The objective of the current study was to prepare a capsaicin organogel-derived

51

nanoemulsion system by ultrasonication, and to investigate the effect of the types of oil, gelators

52

and emulsifiers on the formation of the nanoemulsions. The in vitro dissolution rate and

53

bioaccessibility of CAP were examined and compared with unformulated compound. The in vivo

54

gastric mucosa irritation was evaluated using experimental animals (i.e., rats) to observe the

55

inflammatory response caused by CAP. The present work may provide a promising oral delivery

56

system for CAP and its analogues with high loading capacity, oral bioavailability and reduced

57

irritation to stomach.

58

Materials and Methods

59

Materials. Capsaicin (purity ~99%) was purchased from Ji’an Shengda Fragrance Oils Company

60

(Ji’an, Jiangxi, China). Analytical standards for capsaicin (> 99%) were purchased from Sigma

4

ACS Paragon Plus Environment

Page 5 of 30

Journal of Agricultural and Food Chemistry

61

(St. Louis, MO, USA). Medium chain triacylglycerol (MCT) (Neobee 1053) was provided by

62

Stepan Company (Northfield, IL, USA). Canola oil and corn oil were purchased from a local

63

store. Tween 20 (polyoxyethylenesorbitan monolaurate), Tween 40, Tween 60 and Tween 80

64

were obtained from Sigma-Aldrich. Sugar ester (i.e., sucrose stearate S-370) was provided by

65

Mitsubishi-Kagaku Foods Company (White Plains, NY, USA). Pancreatin with 8X USP

66

specification, tris maleate and sodium taurodeoxycholate (NaTDC) were obtained from Sigma-

67

Aldrich Company (St. Louis, MO, USA). PC75 rapeseed lecithin containing 75%

68

phosphatidylcholine was provided by American Lecithin Co. (Oxford, CT, USA). HPLC-grade

69

water was from Alfa Aesar (Lawrence, KS, USA). HPLC grade-methanol and acetonitrile were

70

also purchased from Sigma-Aldrich Company.

71

Preparation of the Capsaicin (CAP)-Loaded Organogels and Organogel-Derived

72

Nanoemulsions. The CAP-loaded oil phase was first prepared by adding 2.3g CAP into 5.7g

73

MCT (w/w). The oil mixture was then heated to 70 °C under magnetic stirring to achieve

74

complete dissolution. Subsequently, 2.0 g sucrose stearate S-370 was added as gelator into the

75

hot oil mixtures, followed by cooling to room temperature to form the CAP-loaded organogel.

76

The loading for CAP in organogel was 23% (w/w).

77

The organogel-derived nanoemulsion was formed by mixing CAP-loaded organogel as

78

the oil phase, Tween 80 as the emulsifier and water with the ratio of 35%: 15%: 50% (w/w/w).

79

The loading of CAP in final emulsion formulation is 80.4 mg/mL. Then ultrasonication

80

technique at an output power of 250W was used to treat the emulsion mixture for 5 min, which

81

was enough to reduce the size of emulsion droplet to below 200 nm.

82

Selection of Oil Phase for Organogel. Three different oil samples (medium-chain

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

83

triacylglycerol MCT, Canola Oil and Corn Oil) containing 5% CAP were tested based on their

84

bioaccessibility and extent of lipolysis through the lipolysis tests in both fasted state and fed state

85

buffer solutions. Experiments were carried out in triplicate.

86

Selection of the Gelling Agent. In this study, food-grade sugar esters were used as

87

gelators for the formation of organogels. Sucrose stearates with different HLB values (between

88

1 to 9) were added to the preheated CAP-loaded MCT oil under stirring. The contents of sucrose

89

stearates in oil ranged from 15% to 25%. To examine the formation of organogels, vials were put

90

upside down on the table. If no gravitational flow was observed at ambient temperature, the

91

organogel system was considered to be formed. Experiments were carried out in triplicate.

92

Selection of Emulsifiers. To develop organogel-derived capsaicin nanoemulsions, four

93

common emulsifiers, Tween 20, Tween 40, Tween 60 and Tween 80 were compared in terms of

94

the particle size and polydispersity index (PDI) of different emulsions before and after

95

ultrasonicatoin. Experiments were carried out in triplicate.

96

Particle Size Measurements. Emulsion samples were diluted 1000 times with deionized

97

water and mixed well. The average particle sizes (hydrodynamic diameters) of the lipid droplets

98

were determined with a dynamic light scattering apparatus using a BIC 90Plus particle size

99

analyzer (Brookhaven Instrument, New York, NY) at a fixed scattering angle of 90° at ambient

100

temperature. The scattering signals were detected by a high-sensitivity avalanche photodiode,

101

and the autocorrelation functions were fitted by Cumulant method.28 The results were presented

102

as mean ± standard deviation (n = 3).

103

Lipolysis Experiment. Lipolysis is the biochemical pathway responsible for the

104

catabolism of triacylglycerol (TAG), which includes the hydrolysis of ester bonds between long

6

ACS Paragon Plus Environment

Page 6 of 30

Page 7 of 30

Journal of Agricultural and Food Chemistry

105

chain fatty acids and the glycerol backbone in TAG, as well as the release of free fatty acids.22

106

Through this process, the capsaicin initially entrapped into the liquid oils was redistributed into

107

micelles or mixed micelles. Therefore, lipophilic bioactive compounds with poor water solubility

108

become bioaccessible in the gastrointestinal tract. In this work, a dynamic in vitro lipolysis

109

model, which provides a good simulation of the in vivo lipid digestion process, was used to test

110

the bioaccessibility and the extent of lipolysis of the formulation before its further application in

111

animal studies. 23

112

According to the in vitro lipolysis experiment by Yu, et al.

24

, two types of lipolysis

113

buffer solutions were prepared to mimic different chemical environments in the fasted and fed

114

states, due to the fact that the concentrations of bile salts and phospholipids in the small intestine

115

lumen are higher in the fed state than in the fasted state during lipid digestion process. The

116

compositions of those buffer solutions were shown in Table 1. At the beginning of the test, 250

117

mg oil samples were added into 9 mL buffer solutions. Then by adding 1 mL pancreatin into the

118

mixtures, the lipolysis process was initiated. During the digestion process, one part

119

triacylglycerol molecule went through hydrolysis and released two parts of free fatty acids,

120

causing a pH drop. To maintain the pH of the samples at 7.5 ± 0.2, small amount of 0.25 M

121

sodium hydroxide solutions (NaOH) were added into the oil mixtures. The volume of NaOH was

122

recorded by a computer program that can be used to analyze the extent of lipolysis. The lipid oil

123

suspensions were constantly stirred during this 2h test at a fixed temperature of 37 °C. After the

124

lipolysis test, samples were ultra-centrifuged at 50,000 rpm (Type 60 Ti rotor, Beckman Coulter)

125

for 40 min. The supernatants were filtered through 220 nm filters and mixed with acetonitrile for

126

HPLC analysis. Experiments were carried out in triplicate.

127

Determination of Bioaccessibility and the Extent of Lipolysis for Different Oil 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 30

128

Samples. The bioaccessibility, which refers to the fraction of lipophilic compound (i.e., CAP)

129

released into the fluids of gastrointestinal tract to become bioaccessible, can be calculated as

130

follows:     

131

% Bioaccessibility =

132

The mass of CAP was calculated according to the concentration of capsaicin, the density

133

      !"  #

× 100%

(1)

of samples and mass of the oil or emulsion system.

134

The extent of lipolysis was determined as the percentage of triacylglycerols digested in

135

the system, which could be computed using the amount of NaOH added to neutralize the fatty

136

acids released from the complete digestion of triacylglycerols. Theoretically, complete digestion

137

of 1 mole of triglyceride releases 2 moles of fatty acids, which needs 2 moles of NaOH to

138

neutralize. The calculation method could be found as follows:    ,-. 

139

Extent of lipolysis = /!0 

140

Before each test, a control lipolysis test was conducted without the addition of lipid oil

141

samples. The amount of NaOH added in this test was subtracted when calculating the extent of

142

lipolysis.

  ,-. 

× 100%

(2)

143

In Vitro Dissolution Test. The dissolution rates of unformulated CAP, CAP-loaded

144

organogels and organogel-derived nanoemulsion were measured using flow-through cell USP

145

apparatus 4 (SOTAX Corporation, Westborough, MA, USA). Ten milligrams of samples

146

(referring to the content of CAP) were introduced into the dissolution medium containing 100

147

mL of phosphate-buffered medium (PBS) at 37.0 ± 0.5 °C at a speed of 10 mL/min. To better

8

ACS Paragon Plus Environment

Page 9 of 30

Journal of Agricultural and Food Chemistry

148

mimic the dissolution environment of GI tract, pH was set at 1.2 during the first one hour (pH in

149

stomach) and 7.5 during 1-3 hours (pH in small intestine). Samples were collected every 5 min

150

from the reservoir during 0-1 hour, every 10 min during 1-2 hours, and every 30 min during 2-3

151

hours. Collected samples were analyzed using HPLC at 280 nm. All studies were carried out in

152

triplicate.

153

In Vitro Lipid Digestion Test. The bioaccessibility of unformulated CAP, CAP-loaded

154

MCT oils, CAP-loaded organogels and organogel-derived nanoemulsion were studied through

155

the lipolysis experiment. Each test was conducted using fed-state and fasted-state buffer

156

solutions to better mimic the lipid digestion process when meal was taken or several hours after

157

the meal. Experiments were carried out in triplicate.

158

High-Performance Liquid Chromatography (HPLC). Capsaicin was quantified by an

159

automated high-performance liquid chromatography system (Dionex, Sunnyvale, CA, USA).

160

The system consisted of a quaternary solvent delivery system, an UV–vis diode array detector

161

and an automated injection system. The column used in this study is Dionex C18 column (150 x

162

4.6 mm, 3.5 µm) (Bellefonte, PA, USA). The mobile phase consists of (A) acetonitrile and (B)

163

water (HPLC grade). Ten µL samples were injected each time and then eluted under gradient

164

conditions: 0-2 min, 45% A and 55% B; 2-13 min, linear gradient from 45 to 90% A; 13-16 min,

165

held at 90% A; 17-18 min, A went back to 45% linearly; 19-20 min, held at 45% to balance the

166

column. The flow rate was set at 1.0 mL/min and the eluent was detected with UV wavelength at

167

280 nm. Different standard concentrations (5, 10, 20, 40, 60, 80 and 100 µg/mL) of CAP were

168

dissolved in methanol and analyzed using HPLC to generate the calibration curve. Each

169

measurement was carried out in triplicate.

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 30

170

Gastric Mucosa Irritation Test. This animal study protocol was reviewed and approved

171

by the South China Agriculture University Animal Ethics Committee. Male SPF Wistar rats

172

(250g-300g) were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou,

173

Guangdong, China). Rats were housed three per cage and maintained in a temperature/humidity-

174

controlled room with a 12 h light/dark cycle. After 5 days, rats were randomly divided into the

175

following three groups (n = 3), which were fed with physiological saline (negative control group),

176

free CAP suspended in physiological saline (positive control group) and CAP-loaded

177

nanoemulsions (test group), respectively, at a dose of 90 mg/kg of body weight (referring to only

178

capsaicin content). Rats were fasted but allowed free access to water throughout the experiment.

179

Two hours after oral gavage, they were sacrificed for autopsy, and the intact stomachs were

180

removed and fixed in 10% formaldehyde solution. All samples were dehydrated and embedded

181

in paraffin. The pathological section of the stomach, stained with hematoxylin and eosin, were

182

observed under light microscope equipped with a computer-controlled digital camera.

183

Results and Discussion

184

Selection of the Oil Phase for the CAP-Loaded Organogel. To decide which lipid oil

185

to use, three oil samples (MCT, canola oil and corn oil) containing 5% CAP were tested through

186

the in vitro dynamic lipolysis model. According to Figure 2A, capsaicin dissolved in MCT had

187

the highest level of bioaccessibility among all oils in both fasted state (78.0 ± 2.7%) and fed state

188

(76.2 ± 1.9 %), followed by canola oil and corn oil. The same trend can be observed in Figure 2B,

189

where the extent of lipolysis of MCT was much more complete than that of corn oil and canola

190

oil in both fasted state (92.8 ± 2.2 %) and fed state (76.9 ± 2.6 %). Therefore, MCT was most

191

effective in providing a high bioaccessibility and extent of lipolysis among all lipid oils, thus was

192

chosen as the oil for capsaicin formulation. This result agreed with previous researches 10

ACS Paragon Plus Environment

Page 11 of 30

Journal of Agricultural and Food Chemistry

193

concerning the digestion, absorption and metabolism of MCT as edible oils, which demonstrated

194

that MCT could be hydrolyzed into fatty acids and monoglycerides in the small intestine with a

195

rapid digestion rate.25

196

Selection of the Gelling Agent. Previous researches showed that phytochemicals

197

dissolved in MCT were metastable,24 so an organogel system was developed to increase its

198

stability and dissolution rate as well as to prevent precipitation during storage. In order to form

199

an organogel, the gelling agent is the key contributing factor to the gel network. Researches

200

revealed that sugar-derived compounds present organo-gelation properties through the formation

201

of certain intermolecular hydrogen bonds.26-27 Therefore, sugar derivatives, such as sugar esters,

202

were chosen in our experiment as the gelator to help create the organogel.

203

Food-grade sucrose stearates with different HLB values (between 1 to 9) were added to

204

the preheated CAP-loaded MCT oil under stirring. The content of sucrose stearates in oil ranged

205

from 15% to 25%. The result of the gel formation was shown in Table 2. According to the result,

206

gels could be formed by adding 15 – 20% S-270 and 15 – 25% S-370 to the system. However,

207

only the gel formed by 20% S370 was most stable without phase separation or precipitation

208

during one-month storage. Therefore, the CAP-loaded organogel was successfully formed with

209

20% surcrose stearate S-370 working as the gelling agent. The final loading of CAP in the

210

organogel was 23%.

211

Selection of Emulsifier. A series of organogel-derived emulsions was formulated using

212

non-ionic, oil-in-water emulsifiers (Tween 20, 40, 60 and 80). Figure 3 showed the particle sizes

213

and polydispersity indexes (PDI) of organogel-derived emulsions prepared at 15% (w/w) with

214

different Tween surfactants. PDI was used to measure the polydispersity of particle sizes in a

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

215

mixture.28 It is close to zero for monodisperse samples, but has a larger value for a sample with

216

broader distribution. Among four different emulsifiers tested, Tween 80 produced the smallest

217

mean droplet size (687.2 ± 63.7 nm, Figure 3A) with the lowest PDI (0.22, Figure 3B), meaning

218

that the formation of emulsion with Tween 80 is the most uniform and stable. The difference in

219

the oil droplet sizes may be owing to the hydrophilic - lipophilic balance (HLB) of emulsifiers,

220

which can influence the stability and formation of emulsions. HLB values for Tween 20 and 40

221

are 16.7 and 15.6, respectively, which are higher than Tween 60 (14.9) and Tween 80 (15.0).

222

Furthermore, even though Tween 60 and 80 have similar HLB values and molecular structures,

223

the hydrophobic chain of Tween 80 has a double bond yet the hydrophobic chain of Tween 60 is

224

saturated. Previous researches have reported that the presence of a double bond in the chain

225

favors the formation of emulsions with smaller droplet sizes.21,29 Therefore, Tween 80 as the

226

emulsifier can help produce the emulsion with the smallest particle sizes and the highest storage

227

stability among all tested emulsifiers.

Page 12 of 30

228

In the following experiments, after ultrasonication at 250 Watt for 5 minutes, the

229

organogel-derived nanoemulsion using Tween 80 as the emulsifier was formed with mean

230

droplet sizes of 167.9 ± 0.3 nm as determined by dynamic light scattering.

231

In Vitro Dissolution Test. To compare the in vitro dissolution rate of CAP-loaded

232

nanoemulsion, CAP-loaded organogel and free unformulated CAP after oral administration, the

233

dissolution test was carried out in two consecutive pH environments. During the first 60 min, the

234

pH was set at 1.2 to mimic the acidic environment in the stomach. And Between 60 to 180 min,

235

the pH was changed to 7.5 using the phosphate buffer solution (PBS) to simulate the pH

236

evolution in the small intestine. As shown in Figure 4, at pH=1.2, only 3.2 ± 0.4 % of the free

237

CAP was dissolved during the dissolution process after one hour. The dissolution rates of 12

ACS Paragon Plus Environment

Page 13 of 30

Journal of Agricultural and Food Chemistry

238

organogel and nanoemulsion reached plateau at around 10.1 ± 2.6% and 64.5 ± 2.9%, which

239

were more than 3 folds and 20 folds higher than the unformulated ones, respectively.

240

After one hour, the dissolution process continued as pH changed from 1.2 to 7.5. By the

241

end of the 3-hour dissolution test, free CAP, formulated organogel and nanoemulsion had stable

242

dissolution rates of 4.0 ± 0.5, 16.5 ± 3.0 and 75.7 ± 4.1 %, respectively. Therefore, the organogel

243

system had increased the in vitro dissolution rate of CAP by 4 times compared with coarse

244

samples, which might be attributed to the formation of soluble CAP-loaded micelles through

245

agitation during the dissolution process. However, by the end of dissolution test, a large portion

246

of CAP in the gel still remained undissolved, which might be attributed to the compact structure

247

and hydrophobic nature of the formed gel, requiring CAP to find its way out through the pores in

248

the gel matrix before it was dissolved in the solution. Furthermore, the CAP-loaded

249

nanoemulsion had the highest dissolution rate among all tested samples, which was more than 10

250

times higher than free CAP and 4 times higher than the original organogel. This was mainly due

251

to the reduced emulsion droplet sizes and enhanced surface area of the nanoemulsion, enabling

252

more CAP-loaded nanoscale oil droplets to get access to the PBS buffer solutions. In addition, by

253

comparing the dissolution result between CAP-loaded organogel and gel-derived nanoemulsion,

254

it is obvious that the transformation from the gel system into nanoemulsion system is necessary

255

in order to achieve a high dissolution rate of CAP compound.

256

In Vitro Bioaccessibility. To evaluate the in vitro bioaccessibility of CAP from different

257

formulations, the lipolysis experiment was carried out among CAP-loaded nanoemulsion, CAP-

258

loaded gel, CAP-loaded MCT and free unformulated CAP in both fasted and fed state buffer

259

solutions. The result was shown in Figure 5. The bioaccessibility of CAP-loaded gel was 56.5 ±

260

4.2% in fasted state and 63.9 ± 1.5% in fed state, which increased by approximately 10 times 13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

261

compared with the free CAP (5.7 ± 1.8% in fasted state and 6.9 ± 0.2% in fed state). The major

262

increase in the biaccessibility was due to the rapid digestion ability and high dissolution rate of

263

MCT. In addition, no major difference was observed for the bioaccessibility between CAP-

264

loaded organogel and CAP-loaded MCT oil in both situations, suggesting that the formation of

265

gel by adding gelling agent S-370 would not significantly reduce the in vitro bioaccessibility of

266

CAP. Moreover, after forming the nanoemulsion, the bioaccessibility of CAP was further

267

increased to 71.9 ± 1.3% in fasted state and 79.5 ± 0.6% in fed state. Also, by comparing the

268

fed-state lipolysis titration profiles shown in Figure 5C, the initial lipid digestion rate for

269

nanoemulsion was higher than that of the organogel. After 25 min, the extent of digestion for the

270

gel started to become stable and eventually reached 79.4 ± 3.2%, while the nanoemulsion

271

continued to be digested and reached a final digestion extent of 99.5 ± 1.3%. Therefore, the

272

nanoemulsion system had a much more complete lipid digestion and more sustained lipolysis

273

rate compared with the orgaongel system, which can be attributed to the formation of emulsion

274

droplets with smaller droplet sizes and larger surface area after lipase-catalyzed lipid hydrolysis.

275

Additionally, it was evident that the bioaccessibility and the extent of lipolysis in fed state were

276

higher than those in fasted state for all samples, which may suggest that the consumption of

277

developed CAP nanoemulsion and organogel should be taken with meal in order to guarantee a

278

high bioaccessibility.

279

Morphological and Histological Evaluation of Gastric Mucosal Irritation. In this

280

experiment, Male SPF Wistar rats (250g-300g) were used to study whether the gastric mucosal

281

irritation response caused by CAP could be relieved after CAP was encapsulated into the

282

nanoemulsion system. Previous research indicated that the oral LD50 (lethal dose which causes

283

the death of 50% of a group of test animals) value of capsaicin for male rats is 161.2 mg/kg.30

14

ACS Paragon Plus Environment

Page 14 of 30

Page 15 of 30

Journal of Agricultural and Food Chemistry

284

Saline group (A) was used as the negative control, while CAP suspension group (B) and CAP

285

nanoemulsion group (C) with the same dosage of capsaicin content (90 mg/kg of body weight)

286

were used as positive control and test group, respectively. Similar dose of 90 mg/kg of body

287

weight was also adopted in a previous paper,17 which is about 44% below the oral LD50 value. It

288

should be pointed out that the LD50 value for humans has been estimated at 0.5 to 5 g/kg body

289

weight. Therefore, it can be estimated that when consuming CAP with meal (including rice or

290

starch-containing foods), the dose of 90 mg/kg may not produce ulcers in humans.

291

Oral administration of CAP suspension for 6 h induced marked hemorrhagic and

292

ulcerative lesions on the gastric mucosal surface in rats according to Figure 6B; while in other

293

groups, hardly any hyperemia was observed (Figures 6A and 6C). The protective effect of the

294

organogel-derived nanoemulsion system was further confirmed by histological examination. For

295

the positive control group fed with free CAP suspension at dose of 90 mg/kg, the infiltration of

296

inflammatory cells was clearly observed in the gastric mucosa of rats as showed in Figure 6 B1

297

and B2. Rupture of membranes could also be seen in Figure 6 B3. However, for the negative

298

control group A and test group C, which were given physiological saline and CAP-loaded

299

nanoemulsion respectively, there was little evidence of gastric mucosal irritation (Figure 6 A

300

and C). Also, no inflammatory cell infiltration was seen in the histological sections of gastric

301

mucosa for rats fed with CAP nanoemulsion.

302

The above results suggested that the organogel-derived nanoemulsion possessed a

303

reducing effect against irritation in rat stomach tissues induced by CAP. This could be explained

304

by the encapsulation of CAP compound in the nanoemulsion system, reducing the direct contact

305

between CAP and surface of gastric mucosa. In addition, the nano-scale lipid droplet might be

306

able to biologically adhere to gastric tract, prolonging the retention time and avoiding the sudden 15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 30

307

release of CAP into the stomach. Furthermore, after the lipid hydrolysis in the gastric fluid, the

308

CAP nanoemulsion could be digested into micelles with even tiny droplet sizes (13.4 ± 0.4 nm,

309

according to the fasted state lipolysis model), thereby preventing the irritation caused by CAP

310

crystals getting in contact with gastric mucosa.

311

In summary, the organogel-derived CAP nanoemuslion with high in vitro dissolution rate

312

and lipid digestibility was developed and evaluated. The MCT was chosen as the lipid oil not

313

only because of its high bioaccessibility and extent of lipolysis, but also for its ability to increase

314

fat oxidation, energy expenditure and weigh loss as edible oil, which could work synergistically

315

with CAP as the anti-obesity agent. Then with the addition of sucrose stearate S-370, which was

316

working as the gelling agent to increase the stability of CAP-loaded MCT, the organogel was

317

formed. To better enhance the bioaccessibility of CAP, Tween 80 was added into organogel and

318

water with the ratio of 15 %: 35 %: 50 %, which was functioning as emulsifier to produce a

319

formulation with small droplet sizes and high stability. After ultrasonication process, the

320

nanoemulsion was developed with mean droplet sizes of 167.9 ± 0.3 nm and CAP loading of 8 %

321

(w/w). Animal studies using SPF rats showed that the gastric mucosa irritation caused by CAP

322

was alleviated effectively. These results indicated that the organogel-derived nanoemulsions

323

could be used as promising carriers for poorly water-soluble nutraceuticals with reduced

324

irritation in the stomach. This CAP-loaded nanoemulsions could also be further developed into

325

multi-functional beverages with weight-loss and cardio-protection effects.

326 327

16

ACS Paragon Plus Environment

Page 17 of 30

Journal of Agricultural and Food Chemistry

328

References

329

1.

330

applications. Curr. Anaesth. Crit. Care 2008, 19, 338-343.

331

2.

332

Chemical and pharmacological aspects of capsaicin. Molecules 2011, 16, 1253-1270.

333

3.

334

H.; Kim, T. Y., Capsiate inhibits ultraviolet B-induced skin inflammation by inhibiting Src

335

family kinases and epidermal growth factor receptor signaling. Free Radic. Biol. Med. 2010, 48,

336

1133-1143.

337

4.

338

J. G., Antitumor activity of capsaicin on human colon cancer cells in vitro and colo 205 tumor

339

xenografts in vivo. J. Agric. Food Chem. 2010, 58, 12999-13005.

340

5.

341

Vanilloid-mediated apoptosis in prostate cancer cells through a TRPV-1 dependent and a TRPV-

342

1-independent mechanism. Acta Biomed. 2009, 80, 13-20.

343

6.

344

Karlan, B.; Mehta, R.; Koeffler, H. P., Capsaicin causes cell-cycle arrest and apoptosis in ER-

345

positive and -negative breast cancer cells by modulating the EGFR/HER-2 pathway. Oncogene

346

2010, 29, 285-296.

347

7.

348

cisplatin-resistant stomach cancer cells by causing degradation of cisplatin-inducible Aurora-A

349

protein. Nutr. Cancer 2011, 63, 1095-1103.

Hayman, M.; Kam, P. C. A., Capsaicin: A review of its pharmacology and clinical

Reyes-Escogido, M. D.; Gonzalez-Mondragon, E. G.; Vazquez-Tzompantzi, E.,

Lee, E. J.; Jeon, M. S.; Kim, B. D.; Kim, J. H.; Kwon, Y. G.; Lee, H.; Lee, Y. S.; Yang, J.

Lu, H. F.; Chen, Y. L.; Yang, J. S.; Yang, Y. Y.; Liu, J. Y.; Hsu, S. C.; Lai, K. C.; Chung,

Ziglioli, F.; Frattini, A.; Maestroni, U.; Dinale, F.; Ciuffreda, M.; Cortellini, P.,

Thoennissen, N. H.; O'Kelly, J.; Lu, D.; Iwanski, G. B.; La, D. T.; Abbassi, S.; Leiter, A.;

Huh, H. C.; Lee, S. Y.; Lee, S. K.; Park, N. H.; Han, I. S., Capsaicin induces apoptosis of

17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 30

350

8.

351

Unravelling the mystery of capsaicin: a tool to understand and treat pain. Pharmacol. Rev. 2012,

352

64, 939-971.

353

9.

354

capsinoids. Eur. J. Pharmacol. 2011, 650, 1-7.

355

10.

356

(Capsicum annuum L.) stalk extracts: Comparison of pericarp and placenta extracts. J. Funct.

357

Foods 2013, 5, 1724-1731.

358

11.

359

J.; Tanaka, H.; Kiyonaga, A., Maximum tolerable dose of red pepper decreases fat intake

360

independently of spicy sensation in the mouth. Br. J. Nutr. 2004, 91, 991-995.

361

12.

362

A.; Ball, J. G.; Valentovic, M. A.; Dasgupta, P., Bioavailability of capsaicin and its implications

363

for drug delivery. J. Control. Release. 2014, 196, 96-105.

364

13.

365

transdermal delivery of capsaicin through rat skin. Int. J. Pharm. 2008, 358, 151-158.

366

14.

367

of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats. J. Biosci.

368

2013, 38, 561-571.

369

15.

370

Tong, S.; Xu, X.; Yu, J., In vitro and in vivo evaluation of capsaicin-loaded microemulsion for

371

enhanced oral bioavailability. J. Sci. Food Agric. 2015, 95, 2678-2685.

O'Neill, J.; Brock, C.; Olesen, A. E.; Andresen, T.; Nilsson, M.; Dickenson, A. H.,

Luo, X. J.; Peng, J.; Li, Y. J., Recent advances in the study on capsaicinoids and

Chen, L.; Kang, Y. H., Anti-inflammatory and antioxidant activities of red pepper

Yoshioka, M.; Imanaga, M.; Ueyama, H.; Yamane, M.; Kubo, Y.; Boivin, A.; St-Amand,

Rollyson, W. D.; Stover, C. A.; Brown, K. C.; Perry, H. E.; Stevenson, C. D.; McNees, C.

Zi, P.; Yang, X.; Kuang, H.; Yang, Y.; Yu, L., Effect of HPbetaCD on solubility and

Choi, Y. J.; Kim, J. Y.; Yoo, S. B.; Lee, J. H.; Jahng, J. W., Repeated oral administration

Zhu, Y.; Zhang, J.; Zheng, Q.; Wang, M.; Deng, W.; Li, Q.; Firempong, C. K.; Wang, S.;

18

ACS Paragon Plus Environment

Page 19 of 30

Journal of Agricultural and Food Chemistry

372

16.

373

Capsaicin-Loaded Nanoemulsions Stabilized with Alginate and Chitosan by Self-assembly. Food

374

Bioprocess Tech. 2011, 4, 1119-1126.

375

17.

376

S.; Shi, F.; Yu, J.; Xu, X.; Zhang, W., Improved oral bioavailability of capsaicin via liposomal

377

nanoformulation: preparation, in vitro drug release and pharmacokinetics in rats. Arch. Pharm.

378

Res. 2015, 38, 512-521.

379

18.

380

J., Enhanced oral bioavailability of capsaicin in mixed polymeric micelles: Preparation, in vitro

381

and in vivo evaluation. J. Funct. Foods 2014, 8, 358-366.

382

19.

383

Santander-Ortega, M. J.; Alonso, M. J., Chitosan-based nanocapsules: physical characterization,

384

stability in biological media and capsaicin encapsulation. Colloid. Polym. Sci 2012, 290, 1423-

385

1434.

386

20.

387

organogel-based nanoemulsions. J. Agric. Food. Chem. 2012, 60, 5373-5379.

388

21.

389

organogel-based nanoemulsion prepared by a high-pressure homogenizer. J. Agric. Food Chem.

390

2014, 62, 12563-12569.

391

22.

392

enzyme complex mediates the catabolism of cellular fat stores. Prog. Lipid Res. 2011, 50, 14-27.

Choi, A. J.; Kim, C. J.; Cho, Y. J.; Hwang, J. K.; Kim, C. T., Characterization of

Zhu, Y.; Wang, M.; Zhang, J.; Peng, W.; Firempong, C. K.; Deng, W.; Wang, Q.; Wang,

Zhu, Y.; Peng, W.; Zhang, J.; Wang, M.; Firempong, C. K.; Feng, C.; Liu, H.; Xu, X.; Yu,

Goycoolea, F. M.; Valle-Gallego, A.; Stefani, R.; Menchicchi, B.; David, L.; Rochas, C.;

Yu, H. L.; Huang, Q., Improving the oral bioavailability of curcumin using novel

Zahi, M. R.; Wan, P. Y.; Liang, H.; Yuan, Q. P., Formation and stability of D-limonene

Lass, A.; Zimmermann, R.; Oberer, M.; Zechner, R., Lipolysis - a highly regulated multi-

19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 30

393

23.

394

systems by an in vitro dynamic lipolysis model for improved oral bioavailability of poorly water

395

soluble drugs. J. Control. Release. 2008, 129, 1-10.

396

24.

397

high bioaccessibility and loading of curcuminoids. Food Chem. 2012, 131, 48-54.

398

25.

399

acids: edible oil with a suppressing effect on body fat accumulation. Asia Pac. J. Clin. Nutr.

400

2008, 17, 320-323.

401

26.

402

Reinhoudt, D. N., Sugar-integrated gelators of organic solvents - Their remarkable diversity in

403

gelation ability and aggregate structure. Chem. Eur. J. 1999, 5, 2722-2729.

404

27.

405

Insights into low molecular mass organic gelators: a focus on drug delivery and tissue

406

engineering applications. Soft Matter 2014, 10, 237-256.

407

28.

408

Enhancing anti-inflammation activity of curcumin through O/W nanoemulsions. Food Chem.

409

2008, 108, 419-424.

410

29.

411

self-nanoemulsifying drug delivery system. J. Colloid Interface Sci. 2009, 330, 443-448.

412

30.

413

Sci., 1996, 21, 195-200.

414 415

Note: Financial support from Chinese Scholarship Council for ML is acknowledged.

Dahan, A.; Hoffman, A., Rationalizing the selection of oral lipid based drug delivery

Yu, H. L.; Shi, K.; Liu, D.; Huang, Q. R., Development of a food-grade organogel with

Takeuchi, H.; Sekine, S.; Kojima, K.; Aoyama, T., The application of medium-chain fatty

Yoza, K.; Amanokura, N.; Ono, Y.; Shinkai, S.; Akao, T.; Shinmori, H.; Takeuchi, M.;

Skilling, K. J.; Citossi, F.; Bradshaw, T. D.; Ashford, M.; Kellam, B.; Marlow, M.,

Wang, X. Y.; Jiang, Y.; Wang, Y. W.; Huang, M. T.; Ho, C. -T.; Huang, Q. R.,

Wang, L. J.; Dong, J. F.; Chen, J.; Eastoe, J.; Li, X. F., Design and optimization of a new

Saito, A.; Yamamoto, M., Acute oral toxicity of capsaicin in mice and rats. J. Toxicol.

20

ACS Paragon Plus Environment

Page 21 of 30

Journal of Agricultural and Food Chemistry

Figure Captions

Figure 1. Chemical Structure of Capsaicin (CAP). Figure 2. Comparison of the lipolysis of CAP in three oils. (A) The percent bioaccessibility of CAP after lipolysis of three oils; (B) the extent of lipolysis of three oils. Data from fasted- and fed-state lipolysis are combined. Error bars represent standard deviation (n = 3). Figure 3. Effect of the type of emulsifiers on (A) the particle size before ultrasonication, (B) the particle size after ultrasonication, (C) the polydispersity (PDI) before ultrasonication and (D) the polydispersity (PDI) after ultrasonication of organogel-derived CAP-loaded emulsion. Emulsifiers were Tween 20, 40, 60 and 80, from the left to the right (data shown are the mean ± SD). Figure 4. Dissolution profiles of CAP-loaded organogel and free CAP. Between 0 to 60 min, pH was 1.2; between 60 to 180 min, pH was 7.5 (data shown are the mean ± SD). Figure 5. Comparison of the in vitro lipolysis result of free CAP, CAP-loaded MCT, CAPloaded organogel and organogel-derived nanoemulsion, in the aspect of (A) the lipolysis profile and (B) the extent of lipolysis (C) the lipolysis profile of organogel and nanoemulsion in fed state (data shown in (A) and (B) are the mean ± SD). Figure 6. Stomach and gastric mucosa. (A) Stomach of rats treated with physiological saline; (A1) - (A3) Histological tissues of gastric mucosa in group A without damage. (B) Stomach of rats treated with pure capsaicin suspension; (B1) - (B3) Histological tissues of gastric mucosa in group B with damage; (C) Stomach of rats treated with capsaicin-loaded nanoemulsion; (C1) (C3) Histological tissues of gastric mucosa in group C without damage. Only group B showed gastric ulcer and irritation response in gastric mucosa.

21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Table 1. Recipe of Lipolysis Buffer (1000 mL) for Fasted and Fed States. Components

Fasted-State Buffer

Fed State Buffer

Tris Maleate

11.8600 g

11.8600 g

NaCl

8.7664 g

8.7664 g

CaCl2.2 H2O

0.7351 g

0.7351 g

NaTDC

2.6084 g

10.4336 g

Phosphatidylcholine

0.9501 g

3.8004 g

22

ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30

Journal of Agricultural and Food Chemistry

Table 2. Formation of the Organogel System Using Different Sucrose Stearates of Different Weight Ratios. (√ Stands for Successful Formation of Gels; × Stands for Failure in Formation of Gels.)

Sucrose

Weight Ratio 15%

20%

25%

S170

×

×

×

S270





×

S370







S570

×

×

×

S770

×

×

×

S970

×

×

×

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 1.

24

ACS Paragon Plus Environment

Page 24 of 30

Page 25 of 30

Journal of Agricultural and Food Chemistry

Figure 2.

25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 3.

26

ACS Paragon Plus Environment

Page 26 of 30

Page 27 of 30

Journal of Agricultural and Food Chemistry

Figure 4.

27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 5.

28

ACS Paragon Plus Environment

Page 28 of 30

Page 29 of 30

Journal of Agricultural and Food Chemistry

Figure 6.

29

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

TOC

30

ACS Paragon Plus Environment

Page 30 of 30