Development of Organogel-Derived Capsaicin Nanoemulsion with

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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

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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).

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ACS Paragon Plus Environment


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ABSTRACT

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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

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

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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-

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

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

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

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

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

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products.

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

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gastric mucosa irritation

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Introduction

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

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

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

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

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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

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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

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

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

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

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

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

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

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

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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.

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

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

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

3

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developed a microemulsion system


16

also


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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.

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

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

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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

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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

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

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

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

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

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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

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

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irritation to stomach.

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Materials and Methods

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

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

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

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

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

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

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

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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

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also purchased from Sigma-Aldrich Company.

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

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

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

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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.

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

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

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

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

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

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

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

5



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

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

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buffer solutions. Experiments were carried out in triplicate.

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

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

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

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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

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

92

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

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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.

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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,

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

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as mean ± standard deviation (n = 3).

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Lipolysis Experiment. Lipolysis is the biochemical pathway responsible for the

104

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

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

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

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

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

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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

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animal studies. 23

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

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, 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

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

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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

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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

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

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

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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.

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



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

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

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follows:

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% Bioaccessibility =

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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.

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

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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

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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

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

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

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

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

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

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

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triplicate.

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

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

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

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

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the meal. Experiments were carried out in triplicate.

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

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

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

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

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

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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,

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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

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

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measurement was carried out in triplicate.

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

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

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

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

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

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

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

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

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

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

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

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

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

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Results and Discussion

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

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

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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,

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

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

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

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


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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

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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

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

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

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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.

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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

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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



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

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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.

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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


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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



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


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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



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

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328

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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



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


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Page 23 of 30


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



Figure 1.

24


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Figure 2.

25



Figure 3.

26


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Figure 4.

27



Figure 5.

28


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Figure 6.

29



TOC

30


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Development of Organogel-Derived Capsaicin Nanoemulsion with

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