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Preparation and Characterization of Microemulsions of Myricetin for Improving its Antiproliferative, Antioxidative Activity and Oral Bioavailability Ruixue Guo, Xiong Fu, Jian Chen, Lin Zhou, and Gu Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02184 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on July 26, 2016

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Journal of Agricultural and Food Chemistry

Preparation and Characterization of Microemulsions of Myricetin for Improving its Antiproliferative, Antioxidative Activity and Oral Bioavailability Rui Xue Guo†, Xiong Fu†, Jian Chen†, Lin Zhou†‡, Gu Chen*, †

† School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China ‡ School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, China Corresponding Author *Gu Chen, Tel: 86-13660887090. Fax: 86-20-87113849. E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT:

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To improve the bioactivity and oral bioavailability of myricetin, a microemulsion

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formulation was successfully developed, which consisted of Cremophor RH40 (12%),

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Tween 80 (6%), Transcutol HP (9%), WL 1349 (18%) and distilled water (55%).

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With lower content of surfactants and higher stability after dilution and storage for 6

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months, the optimized myricetin microemulsion (MYR-ME) could dramatically

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enhance the solubility of myricetin 1225 times of that in water. MYR-ME

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significantly increased antiproliferative activity against human cancer cell HepG2

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without influence on normal cell LO2. It also notably improved the cellular

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antioxidative activity of myricetin. Furthermore, the oral bioavailability of myricetin

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was remarkably enhanced by MYR-ME in Sprague-Dawley rats oral administration,

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which was 14.43-fold of that with myricetin suspension. Therefore, the MYR-ME

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developed here could be used as a potential carrier for myricetin with substantially

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enhanced bioactivities and bioavailability, and might promote myricetin future

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utilization in functional foods and cosmetics.

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KEYWORDS: myricetin, microemulsion, antiproliferation, cellular antioxidative

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activity, oral bioavailability

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INTRODUCTION

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Myricetin is a natural antioxidant flavonoid with hydroxyl substitutions at the 3,

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5, 7, 3', 4', and 5' positions.1 It is mainly isolated from the bark and roots of Myrica

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rubra or vine tea and is commonly consumed in our diet from natural foods such as

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teas, berries, grapes, vegetables, beans and red wine.2 As a functional nutraceutical,

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myricetin had many biological activities, such as antidiabetic, anti-oxidative,

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anticarcinogen, anti-bacterial, neuroprotective, and hepatoprotective activities.3-7 Also,

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myricetin was reported to be capable of quenching photoaging free radicals within

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skin through inhibition of ultraviolet-B-induced intracellular hydrogen peroxide

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production.8,9 In spite of various health benefits, myricetin still suffered from weak

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absorption efficacy and low bioavailability following oral administration due to its

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poor aqueous solubility.10,11

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Recently, some solubilization approaches, such as cyclodextrin inclusion

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complex, liposome and microemulsions, have been developed to overcome these

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drawbacks.12-16 Although these solubilization approaches could strengthen the

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bioavailability and bioactivity of myricetin to a certain extent, the thermodynamically

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stable, homogeneous and optically isotropic microemulsion encapsulated system

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especially gained more attention. Microemulsion encapsulated systems have

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characteristics of easy preparation, low viscosity, rapid absorption, strong

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solubilization effect and optical transparency, which make them very attractive in

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pharmaceutical and nutraceutical applications as vehicles.17,18 Microemulsions could

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form spontaneously by surfactant, co-surfactant, oil and aqueous solution with a

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particle size of less than 100 nm.19 In virtue of the negative interfacial tensions

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between oil and aqueous interface, drugs with poor aqueous solubility can be easily

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loaded and transported in microemulsions.20 In the field of nutraceuticals and food 3



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additives, microemulsions have many applications and have demonstrated excellent

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characteristics in improving solubility, oral absorption as well as the antioxidative and

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antiproliferative activity.21-26 Recently, the microemulsion for myricetin was reported

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to be constructed of surfactants (Tween 80: 20%, Tween 20: 10%, w/w), co-surfactant

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(ethanol: 15%, w/w), oil (oleic acid, 5%, w/w) and water (50%, w/w).15 This

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formulation could enhance the oral absorption efficacy of myricetin with relevant

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bioavailability up to 133.6%; however, the surfactant level was relatively high in this

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formula and its influence in bioactivities and cell cytotoxicity was not investigated yet.

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It is well known that the dosage of surfactant in food is only permissible at low levels,

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so we aimed to develop and evaluate a lower surfactants microemulsion of myricetin

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with higher bioactivity and oral bioavailability.

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Therefore, the purpose of present work was to (1) optimize microemulsion

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formations for myricetin with reduced amount of surfactants and better physical

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stability; (2) evaluate the antiproliferative effect and cytotoxicity of myricetin loaded

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in microemulsions (MYR-ME) on human hepatocellular cancer cells HepG2 and

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normal cells LO2, as well as the cellular antioxidative activities (CAA); (3)

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characterize the in vivo absorption of MYR-ME in rats to acquire the oral

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bioavailability of myricetin, which was compared with that in sodium carboxymethyl

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cellulose (CMC-Na) suspension and other related reports.

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MATERIALS AND METHODS

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Materials.

Myricetin,

quercetin,

2,

2’-azobis-amidinopropane

(ABAP),

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dichlorofluorescin diacetate (DCFH-DA), insulin, L- Glutamine, and hydrocortisone

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were purchased from Sigma-Aldrich Inc. (St. Louis, USA). Tween 80, Cremophor

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EL35, Isopropyl myristate (IPM), ethyl oleate (EO), soybean oil, glycerol, CMC-Na

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and propylene glycol were obtained from Aladdin (Shanghai, China). Labrasol, 4


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Capryol 90, Labrafil® M1944 CS (M1944 CS), LabrafacTM lipophile WL 1349 (WL

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1349) and Transcutol HP were obtained from Gattefosse (Shanghai, China).

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Cremophor RH40 was obtained from BASF Co., Ltd. (Germany). Both human liver

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cancer cells HepG2 and human hepatic cell LO2 were purchased from Sun Yat-Sen

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University. Williams’ medium E (WME), DMEM medium, fetal bovine serum,

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trypsin, PBS and other cell culture reagents were purchased from Gibico U.S.

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Biotechnology Co. Water was purified using a Millipore System (Millipore, Bedford,

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USA). All the other chemicals and solvents were analytical reagent grade.

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Preparation of Myricetin Microemulsions. Solubility Studies. The solubility

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of myricetin in different medium was evaluated by adding excess myricetin powder

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into medium. The centrifugal tubes containing the mixture were kept in a shaking

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incubator at ambient temperature for 24 h. The solubilized myricetin in various

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mediums was determined directly by HPLC as described below. Since the solubilized

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myricetin concentration in water was too low to be detected by HPLC accurately, the

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solubility was determined indirectly. The solubilized myricetin was calculated as the

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original weight minus the weight of undissolved myricetin, which was collected by

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centrifugation at 13000 rpm for 10 min, dissolved in methanol and quantified by

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HPLC.

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Construction of Pseudo-ternary Phase Diagrams. The pseudo-ternary phase

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diagrams, including surfactant, co-surfactant, oil, and aqueous phase, were

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constructed using water titration method at room temperature to optimize

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microemulsion formulations regarding the formative area of microemulsion region.

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The concentration range of components was obtained by the existence region of

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microemulsion. The surfactant and co-surfactant employed were mixed well in fixed

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mass ratios (abbreviated as “Km”) to produce a homogeneous liquid. For each phase 5



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diagram at a specific Km, aliquots of each surfactant–co-surfactant mixture (S-Cos)

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were mixed with oils at ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1. Then,

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distilled water was added dropwise into each blend under gentle magnetic stirring or

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vortexing at ambient temperature.3 Phase transparency and translucence were assessed

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visually as microemulsions after equilibrium. Therefore, the amount of water, at

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which the system turned at turbidity-to-transparency and transparency-to-turbidity,

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was acquired to establish the boundaries of microemulsion regions. The physical

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states were represented on the pseudo-ternary phase diagram with one axis

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representing the water phase, one representing oils, and the other one representing the

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S-Cos.27

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Screening of MYR-ME. On the basis of pilot studies of blank microemulsions

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from above, MYR-ME was further optimized by working out the best ratio of oils.

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The 10 mg oils were mixed with the fraction of surfactants and co-surfactant mixtures

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(Smix) at different K values (K: Smix/Oil, varied as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2

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and 9:1) with constant Km. An appropriate amount of water was added to the blend

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drop by drop until a critical clear point was reached. The mixture was then stirred for

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24 h at ambient temperature under gently stirring to allow equilibrium.25 For the

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myricetin loaded microemulsion preparation, excess myricetin powder was dispersed

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into the co-surfactant in advance and then mixed with the other fractions. The

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undissolved myricetin was removed by centrifugation at 13000 rpm for 10 min, and

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then the MYR-MEs were achieved. The particle size and loading capacity of different

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MYR-MEs were detected to select the best formula for further study.

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Determination of the Loading Capacity of Myricetin. After diluted with

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methanol, the total content of myricetin in microemulsions was determined by HPLC

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consisting of 1525 Binary pump, 717 plus auto sampler and Ultraviolet dual 6


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wavelength detector (Waters, US) using a C18 reverse-phase column (5 µm, 150 × 4.6

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mm, Waters, US) at 370 nm, 37 oC. Mixed distilled water and methanol (30:70, v/v)

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were used as mobile phase at a flow rate of 1.0 mL/min.28

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Physiochemical Characterization of MYR-ME. Type of Microemulsions. The

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type of microemulsion was recognized by the following method: the blank

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microemulsions were divided into two parts, into which the coloring agent Sudan red

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and methylene blue were added simultaneously. Then the diffusion speed of red and

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blue were observed. If the color of blue diffused faster than the red color, the type of

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the microemulsion was oil-in-water (o/w). Otherwise, it was the type of water-in-oil

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(w/o).25

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Droplet Size and Zeta Potential of MYR-ME: The mean droplet size and

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distribution (polydispersity index, PDI) of the MYR-ME were carried out by dynamic

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light scattering instrument (DLS, Nano-ZS type, Malvern, UK). The Zeta potential

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fall of MYR-ME was determined by automatic potentiometric titration instrument

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(MPT-2, UK, Malvern Co. Ltd). All measurements were performed at 25 oC and

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presented as average ± standard deviation (SD).29

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Morphological Evaluation: The morphology of microemulsions was observed

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by transmission electron microscopy (TEM) (JEM-100CXII, Hitachi Co. Ltd, Japan).

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A droplet of MYR-ME, diluted with water and stained for 3 min by a drop of 2wt%

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phosphotungstic acid solution, was placed on a copper grid with carbon film, followed

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by removal of the excess fluid with filter paper, and dried for 10 min before

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examination on TEM at an acceleration voltage of 200 kV.19

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Viscosity of MYR-ME: The viscosity of MYR-ME was analyzed by a rotary viscometer (DV-S, Brookfield) at 25 oC and expressed as m Pa/s.26

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Stability Studies: Experimental process was as follows: the optimized MYR-ME

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was stored at room temperature (25 ~ 30 oC) for 6 months. The visual inspection

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(phase separation), micromorphology, particle size and loading capacity were taken to

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estimate the shelf life of the stored system. In order to evaluate the metastable systems,

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the optimized formulation was also diluted at 100 and 200 times at room temperature

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and observed for any change in homogeneity of microemulsion.3,30

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Cell Culture. Human cancer cells HepG2 was cultured in WME medium

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supplemented with 5% CO2, 10 mM Hepes, 2 mM L-glutamine, 5 µg/mL insulin, 0.05

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µg/mL hydrocortisone, 50 units/mL penicillin, 50 µg/mL streptomycin and 100 µg/mL

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gentamycin and maintained at 37 oC in a humidified atmosphere of 5% CO2. Human

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hepatocytes LO2 was growth in DMEM medium supplemented with 10% fetal bovine

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serum, 50 units/mL penicillin, 50 µg/mL streptomycin, and 100 µg/mL gentamycin in

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5% CO2 at 37 oC.31

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Cell Proliferation Inhibiting Test of MYR-ME. The antiproliferative effects

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of myricetin loaded in microemulsion were evaluated in human cancer cells HepG2

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and human normal cells LO2 by the methylene blue colorimetric method reported

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before.32 The 100 µL of cell suspension was seeded in the 96-well microplate at a

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density of 2.5×104 per well and incubated for 4 h to allow sufficient attachment.

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Myricetin dissolved in DMSO or microemulsion beforehand was diluted by culture

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medium, and added into wells at various concentrations to replace the growth medium.

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Then, cells were continuously cultured for 72 h. After staining by methylene blue

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solution for 1 h, colored wells were eluted and read at 570 nm using a micro-plate

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reader. The antiproliferative activity was calculated by cell proliferation: cell

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proliferation (%) = (AS/AC) ×100, where AS is the absorbance of the well treated by

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samples, and AC appears as the absorbance of well without myricetin treatment but 8


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containing corresponding DMSO or blank microemulsion concentration in culture

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medium (0.5%, v/v) as the control group. The median effective dose (EC50) of

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myricetin was used to assess the antiproliferative effect.33

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Cell Cytotoxicity Test of MYR-ME. HepG2 or LO2 cells were cultivated at

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densities of 4.0×104 per well for 24 h, then samples at various concentrations were

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added and cultured for 24 h at 37 oC. After that, wells were treated as described in

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“antiproliferation test”. The cytotoxicity (%) was expressed as “100 minus cell

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proliferation”. Myricetin at certain concentration that decrease the absorbance by >

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10% is considered to possess cytotoxicity in comparison to the control.24,33

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Cellular Antioxidantive Activities (CAA) of MYR-ME. The CAA protocol

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was conducted as described previously.31 Briefly, HepG2 cancer cells were seeded at

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a density of 6×104 cells/well in a 96-well micro-plate and incubated for 24 h. Then the

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growth medium was removed and washed with PBS, to which 100 µL of treating

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medium, composed of tested samples and 50 µM DCFH-DA was added afterwards.

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After cultured for another 1 h, cells were treated as follows: some cells were washed

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with PBS (PBS wash protocol), while the other were not washed (No PBS wash

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protocol). Then, ABAP was supplied to treat each well except the blank well. At last,

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the micro-plate was placed into the Fluoroskan Ascent FL (Thermo Scientific, USA)

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plate-reader immediately to monitor the fluorescence subtraction. The area under the

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curve of fluorescence versus time was calculated as CAA value. With quercetin as the

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standard, the cellular antioxidant value for samples was expressed as micromoles of

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quercetin equivalents (QE) per 100 µM of myricetin.

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Oral Bioavailability Evaluation of MYR-ME in rats. The study was approved

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by the Ethical Committee of Guangdong Pharmaceutical University. Twelve male

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Sprague-Dawley rats of body weight 220-250 g were divided randomly into two 9



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groups. After fasted for 12 h and only supplied with water, they were fed orally with

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myricetin microemulsion or myricetin suspension (0.5% CMC-Na) at a dose of 25 mg

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of myricetin/kg of body weight, separately. Blood samples were taken at 0, 0.5, 1, 2, 3,

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4, 6, 8, 10, 14 and 24 h from the eye orbit veins after oral administration. Blood

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samples were put into tubes containing heparin and centrifuged at 4000 rpm for 15

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min to obtain the plasma. Then, 200 µL plasma samples were purified by adding 1mL

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ethyl acetate to remove the proteins. After centrifuged again, the supernatant was

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dried by nitrogen at 45 oC. At last, the residue was dissolved in 200 µL of methanol to

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obtain the test samples.14 Then samples were injected into HPLC to determine the

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myricetin concentration. Based on the concentration at various interval times, the

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pharmacokinetic parameters were calculated by the DAS 2.0 software program. The

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relative bioavailability (F) was expressed as: F (%) = (AUCS/AUCC) × 100, where

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AUCS is the area under the concentration-time curve of MYR-ME, and AUCC is the

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area under the curve of myricetin suspension.21,25

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Statistics Analysis. Results were the mean values and standard deviation (SD)

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from at least three different experiments. All measurements were expressed as mean ±

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SD and statistically analyzed by SPSS software, and p-value < 0.05 was considered

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significant in comparison between two experimental groups.

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RESULTS AND DISCUSSIONS

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Preparation and Optimization of Myricetin Microemulsion

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Screen Components for Myricetin Microemulsions

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Firstly, the solubility of myricetin in various oils, surfactants and co-surfactants

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was analyzed as shown in Table 1 to screen components for microemulsions. Among

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the five oral permissible oils tested (Soybean oil: complex fatty acid triglyceride, WL

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1349: caprylic/capric triglyceride, M1944 CS: oleoyl polyoxyl-6 glycerides, IPM: 10


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isopropyl myristate, EO: ethyl oleate), the myricetin solubility was similar in soybean

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oil, WL 1349 and M1944 CS, and higher than in IPM and EO. Thus it was suggested

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that triglyceride or branch-chain glyceride were better solvents of myricetin than

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short-chain alcohol ester of fatty acid. Therefore, soybean oil, WL 1349 and M1944

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CS were chosen as alternative oil phase for further studies in the phase diagram to

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measure their formulation ability. Among the five surfactants studied (Cremophor

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RH40, Tween 80, Span 80, Labrasol, Cremophor EL35), higher myricetin solubility

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was found in Tween 80 and Cremophor RH40. Given that these two surfactants have

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been applied generally in drugs as well as in foods and nutraceuticals to form

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microemulsions for oral absorption, and hemolysis might take place when Tween 80

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was employed in large quantities,34 both of them were chosen as the complex

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surfactants to reduce the dosage of Tween 80. When dispersed in five different co-

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surfactants (Transcutol HP, ethanol, propylene glycol, Transcutol P, glycerol),

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myricetin had the highest solubility in Transcutol HP. Thus, Transcutol HP was

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chosen as co-surfactant to study the pseudo-ternary phase diagram for microemulsions.

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Secondly, the Pseudo-ternary phase diagram of the blank microemulsions

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without myricetin was used to determine the concentration range of components. The

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translucent microemulsion region was presented in phase diagrams as “ME” while the

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rest of the region represented the turbid and conventional emulsions. Components

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were screened based on two principles: one is the percentage of the microemulsion

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region, and the other is that the less surfactant-co-surfactant employed the better. To

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find out the best proportion between the mixed surfactants, the microemulsion areas

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were calculated at different ratios of Tween 80 and Cremophor RH40 as 1:2, 1:1 and

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2:1, with Transcutol HP as the co-surfactant and WL 1349 as oil (Figure 1A). As the

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dosage of Cremophor RH40 increased, the microemulsion area was enlarged. So, the 11



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ratio of Tween 80 to Cremophor RH40 at 1:2 was chosen for further experiments.

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Then, the optimal oil was screened on the premise that Transcutol HP was used as co-

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surfactant phase along with the optimized ratio of complex surfactants. As shown in

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Figure 1B, the three oils, soybean oil, M1944 CS and WL 1349 could form

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microemulsion. But the area, made up by the mid-chain fatty acid-glycerides

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octanoic/decanoic acid, WL 1349, was much larger than the long-chain Oleoyl

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macrogol glyceride M1944 CS and the mixed fatty acid ester soybean oil. It was

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consistent with the previous report that macromolecular oil was difficult to embed

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into the surfactant to form the interface membrane.35 Thus, WL 1349 was chosen as

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oil phase for myricetin microemulsion preparation. The effect of surfactant and co-

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surfactant ratio (Km value) on pseudo-ternary phase diagrams was further

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investigated (Figure 1C). As Transcutol HP displayed the best solubility of myricetin,

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it was chosen as co-surfactant to examine the Km Value. According to the Km (Km =

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1:2, 1:1, 2:1) studies, it was found that the microemulsions region was increased

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sharply with the Km value increase from 1:2 to 2:1, suggesting that the

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microemulsions areas were closely related to the content of surfactants. Although

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more surfactants could formulate microemulsions with smaller droplet, it was not the

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more the better. Higher amount of surfactants, accordingly with more oils employed,

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might result in decreased solubilization of water phase, which leading to the interface

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disequilibrium and a smaller area as well as oral insecurity.36 Therefore, the ratio of

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2:1 was selected as the desirable surfactant and co-surfactant ratio.

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Optimization of Myricetin Microemulsions (MYR-ME) Preparation.

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After the components and ratio of surfactants to co-surfactants (Km) were

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optimized through pseudo-ternary phase diagram, the ratio of surfactants/co-

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surfactants mixture to oils (K value) should be optimized by investigating the criteria,

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such as loading capacity, droplet size, oral safety and stability.25,37,38

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Nine blank formulations were first prepared with different K value (from K1 1:9

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to K9 9:1) and optimized synergetic surfactants ratio (Tween 80: Cremophor RH40 =

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1:2) and Km (Km=2:1) (Supplemental Table 1). Phase separation was observed in K1

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(1:9) and K2 (2:8). K3 (3:7) contained 58.1% oil and 16.8% water, so that appeared as

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water in oil microemulsion and was unstable against dilution by water. K7 (7:3), K8

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(8:2) and K9 (9:1) had more than 36% surfactant/co-surfactant, which might be

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harmful to the gastrointestinal mucosa.39 Thus K4 (4:6), K5 (5:5) and K6 (6:4) were

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chosen to prepare myricetin microemulsions (MYR-ME).

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After myricetin was loaded in microemulsion, the myricetin loading capacity,

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droplet size and stability were evaluated and compared in K4, K5 and K6 formulation.

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The weight ratios of surfactants (Tween80: Cremophor RH40 = 1: 2), co-surfactant

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(Transcutol HP), oil (WL 1349) and water were as follows: 12: 6: 27: 55 (K4), 20: 10:

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30: 40 (K5) and 18: 9: 18: 55 (K6). The myricetin loading capacity were comparable

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in K5 (20.62 ± 1.15 mg/mL) and K6 (17.76 ± 1.34 mg/mL), and they were higher

284

than those in K4 (8.55 ± 0.98 mg/mL). Their droplet sizes ranged from 23.23 nm

285

(K5), 65.11 nm (K4) to 67.28 nm (K6), all within the reasonable size of

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microemulsion. Stability was assessed by centrifuging the MYR-MEs at 13000 rpm

287

for 10 min and diluting 100 times with water. At first, there was no precipitation and

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floccule out of the three formulations. However, after equilibrating for 24 h,

289

precipitation of myricetin occurred partly in K4, while no precipitation or

290

delamination occurred in K5 and K6. Though K5 and K6 had similar myricetin

291

loading capacity and stability, considering the principle that the less surfactant

292

employed in drug delivery systems, the safer oral administration, K6 formulation was 13



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selected for further studies. Therefore, the optimized formulation was 6% Tween 80,

294

12% Cremophor RH40, 9% Transcutol HP, 18% WL 1349 and 55% distilled water

295

with high loading capacity of myricetin as 17.76 mg/mL.

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The solubility of myricetin was significantly improved by the selected

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microemulsion formula. The loading capacity was about 1225 times of the solubility

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of myricetin in water as 14.50 µg/mL (14.50 µg/g). When the loading capacity was

299

converted to weight/weight ratio as about 20.19 mg/g, it was comparable to the

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previously reported 2% (w/w) in 45% surfactants and co-surfactant microemulsions.16

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But the surfactant ratio was reduced to 26.9% in our experiment. Maximal drug

302

loading with minimal surfactant had been the objective of many research efforts,

303

which might reduce intestinal mucosal damage.39-43

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Physiochemical Characterization of Myricetin Microemulsion

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Firstly, the MYR-ME was recognized as a type of oil in water (o/w) formulation

306

because the water-soluble methylene blue spread obviously quicker than oil-soluble

307

Sudan Red. This result suggested that the system could theoretically be diluted by

308

water. Under TEM, the microstructure of microemulsion appeared a homogeneous

309

distribution with nearly regular shape (Figure 2A).

310

Then, the physicochemical parameters of myricetin-loaded microemulsions were

311

measured (Table 2). The average droplet size of MYR-ME was around 67.28 nm, at a

312

polydispersity index (PDI) of 0.23. Such low PDI suggested that the formulation was

313

relatively uniform because lower PDI value meant higher homogeneous distribution.

314

The Zeta potential of MYR-ME was -5.50 mv, which was not very negatively charged.

315

Dynamic viscosity of microemulsions were 25.20 mPa·s-1, which was consistent with

316

the observation of oil in water system.19

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The formulation stability was another crucial parameter for microemulsion and it

318

was assessed in MYR-ME. In the accelerated destruction experiment by centrifuging

319

the microemulsions at a high speed of 13000 rpm for 10 min, there was no

320

precipitation and turbid phenomenon, as well as no stratification appearances at

321

ambient temperature (25 ~ 30 oC). To further assess the MYR-ME stability, they were

322

diluted with water by 100 and 200 times. As shown in Table 2, a slight decrease in

323

droplet size was found in 100 times dilution, from 67.28 nm to 60.65 nm; while the

324

droplet sizes were comparable between 200 times dilution and the original MYR-ME.

325

After dilution, the PDI of MYR-ME increased to 0.33, but it was still in an acceptable

326

range to keep the relevant uniform distribution of microemulsions. After 100 and 200

327

times dilution, the drug content of myricetin decreased around 104 and 209 times

328

respectively, which suggested the dilution stability of MYR-ME.

329

After stored for six months at 25 ~ 30 oC, the MYR-ME was transparent without

330

any precipitation or phase separation (Supplemental Figure 1). The Zeta potential,

331

viscosity and particle size of MYR-ME were similar with those of the intial MYR-ME

332

(Table 2). Also, there was no significant change in the myricetin loading capacity

333

after six months storage. More advantageously, the PDI value decreased from 0.23 to

334

0.078, indicating a more uniform distribution compared with the intial MYR-ME,

335

which was confirmed by the TEM images (Figure. 2). In comparison with the image

336

of newly prepared microemulsions (Figure. 2A), the system was even more regular in

337

spherical form and uniform distribution (Figure. 2B). Together, these data indicated

338

that the optimized MYR-ME was stable for up to 6 months. Long term stability of

339

microemulsion was also reported in literatures such as 2 months, 6 months and even

340

12 months.44-46 The high thermodynamic stability of MYR-ME might be due to its

341

lower free energy level compared with the phase separated status. 15



342

Evaluation of the Bioactivity of Myricetin Microemulsions

343

Antiproliferative Activity and Cytotoxicity of MYR-ME against HepG2 and

344

LO2. The inhibition of cell proliferation and cytotoxicity of MYR-ME were compared

345

with free myricetin (MYR) against human cancer cell HepG2 and human normal liver

346

cell LO2 (Figure. 3). After treated for 72h, both MYR-ME and MYR had

347

antiproliferative activity on HepG2 cells and followed a concentration-dependent

348

manner (Figure. 3). But MYR-ME showed a remarkable lower cell proliferation than

349

MYR, and had a significant lower EC50 as 32.66 µΜ compared with MYR as 103.87

350

µM. Thus it was indicated that the MYR-ME had stronger antiproliferative effect than

351

MYR. Since the same amount of myricetin was loaded in both systems, the difference

352

in antiproliferative activity might be due to the higher negotiability, homogeneity and

353

nano-scale particles of MYR-ME, which contributed to the infiltration and

354

transportation capacity of drugs through cytomembrane and/or endocytosis and

355

pinocytosis and resulted in the promotion of cells uptake.25,39,47,48 As a control, MYR-

356

ME showed little inhibitory effect in the proliferation of LO2 cells. Meanwhile, the

357

cytotoxicity to both cell lines was obtained. The cytotoxicity of MYR-ME towards

358

HepG2 was slightly higher than MYR, but both of which were below 10% at

359

myricetin concentration up to 200 µΜ, suggesting low cytotoxicity to cells growth.

360

The cytotoxicity of MYR-ME against LO2 was low as well. To assess whether

361

components in microemulsion affect the results, the blank microemulsion without

362

myricetin was tested for antiproliferative activity and cytotoxicity. There was no

363

apparent antiproliferative activity and cytotoxicity under the concentration used for

364

MYR-ME experiments (Supplemental Figure 2). Therefore, data here indicated that

365

the MYR-ME might be safe for drug delivery application and could substantially

16


Page 16 of 39

Page 17 of 39


366

improve the antiproliferative activity of myricetin against cancer cells, while MYR-

367

ME had little effect on normal cell growth.

368

Cellular Antioxidative Activity (CAA) Assay. The antioxidant activity of free

369

myricetin (MYR) and myricetin microemulsions (MYR-ME) was evaluated by the

370

CAA assay using two protocols, PBS wash protocol and no PBS wash protocol. Using

371

quercetin (QE) as standard, the CAA value was calculated and shown in Figure 4. The

372

CAA value of MYR-ME (118.13 ± 11.12 µM QE/100 µM) was significantly higher

373

than that of MYR (60.67 ± 2.67 µM QE/100 µM) in no PBS wash protocol (p < 0.05 ).

374

When taken the PBS wash protocol, MYR-ME also appeared much higher CAA value

375

(100.63 ± 5.62 µM QE/100 µM) than free myricetin (48.56 ± 5.72 µM QE/100 µM)

376

with a significant difference (p < 0.05), which was consist with the no PBS wash

377

protocol.

378

What’s more, the CAA values of MYR-ME under PBS wash and no PBS wash

379

protocol were not significantly different, while a significant difference was found in

380

CAA values of MYR under PBS wash and no PBS wash protocol. It implied that

381

most MYR-ME might enter cell before PBS washing. Given that CAA assay involved

382

the cellular absorption, transportation and metabolism of antioxidants, it was

383

relatively accurate to evaluate the ex vivo antioxidative activities.31,49 According to the

384

CAA value, MYR-ME could remarkably improve the cellular antioxidative activity of

385

myricetin. It was inferred that MYR-ME got an easier access to cells membrane than

386

MYR, leading to the topo-concentration enrichment, which was of great benefit to the

387

component absorption and diffusion.17,50

388

Evaluation of the Oral Bioavailability of Myricetin Microemulsion in Rats

389

The oral bioavailability of MYR-ME was further evaluated in Sprague-Dawley

390

rats and compared with myricetin CMC-Na suspension. The acute toxicity studies 17



391

suggested the oral tolerance and safety of MYR-ME below 500 mg/kg (Supplemental

392

Table 2) and dose of 25 mg/kg was chosen for bioavailability experiments to ensure

393

the oral safety of MYR-ME. After oral administration, myricetin would be

394

metabolized to other forms, such as conjugation with glycoside, glucuronidation and

395

sulfation, but the original form could also be found after 6 hours metabolism.14,51

396

Therefore, we detected the plasma concentration of original myricetin. The plasma

397

concentration-time curve of MYR-ME and myricetin CMC-Na suspension was

398

presented in Figure 5 and pharmacokinetic parameters were shown in Table 3. The

399

peak plasma concentration (Cmax) of MYR-ME (8.11 µg/mL) was significantly

400

higher than that of CMC-Na suspension (0.48 µg/mL) (p < 0.05), showing that the

401

highest content of MYR in plasma was 16.90-fold enhanced by the microemulsions.

402

The drug elimination half time (t/1/2) of MYR-ME (5.20 h) was longer than that in

403

suspension (3.53 h), which elucidated MYR-ME possessed a controlled release effect.

404

The area under the curve (AUC) of MYR-ME and MYR-suspension were 53.83 µg

405

h/mL and 3.73 µg h/mL, respectively. Compared with CMC-Na suspension, the

406

relative bioavailability of MYR-ME was as high as 1443.16% after oral

407

administration.

408

To our knowledge, the MYR-ME reported here had higher solubility and oral

409

bioavailability than any other previous reports. It was reported that the solubility and

410

oral bioavailability of myricetin was increased 6.93 times and 940.11% when

411

encapsulated in hydroxypropyl-beta-cyclodextrin inclusion as compared with CMC-

412

Na suspension,14 both of which were much lower than our MYR-ME. From the

413

literature,15 myricetin loaded in a microemulsion formulation composed with higher

414

surfactants (20% of Tween 80, 10% of Tween 20, 15% of ethanol, 5% of oleic acid

18


Page 18 of 39

Page 19 of 39


415

and 50% of water) had relevant bioavailability as 133.6%, which was lower than our

416

data as 1443.6%.

417

The remarkably improved myricetin loading capacity and bioavailability of our

418

formulation might attribute to the specific components and formula used in our study.

419

The synergetic surfactants of Tween 80 and Cremophor RH40 had higher emulsifying

420

capacity than Tween 80 and Tween 20 combination in the previous report. The novel

421

but oral permitted co-surfactant, Transcutol HP, which was equipped with hydrophilic

422

diethylene glycol and lipophilic ethyl ether structure, assisted the surfactants to form

423

larger and more stable microemulsion area than volatile ethanol used in previous

424

study16 and contribute to the solubilization of myricetin. In addition to this, the

425

reduced amount of short-chain co-surfactants (from 15% to 9%) facilitated to

426

decrease the interfacial tension of the membrane between the oil phase and the

427

aqueous phase, which was benefit to the system stability and might result in more

428

drugs embed into the interfacial film.52 Moreover, the mid-chain fatty acid-glyceride

429

WL 1349 was not so easy to be oxidized as free fatty acids oleic acid employed in the

430

previous study.16 This mid-chain triglycerides WL 1349 could form oil in water

431

microemulsions more easily and showed higher solubilizing capacity compared with

432

di-glyceride, mono-glyceride and free fatty acid,53 which contributed to the solubility

433

enhancement of myricetin.

434

The greatly enhanced bioavailability of our MYR-ME was speculated to the

435

following aspects. Firstly, the microemulsions significantly promoted the myricetin

436

solubility up to 17.76 mg/mL compared to aqueous solution at 14.50 µg/mL, which

437

contributed to the permeation rate of myricetin to the gastrointestinal tract. Secondly,

438

the medium-chain triglyceride WL1349 used in our experiment could increase the

439

drug absorption efficiently; since the poor water soluble myricetin could be well 19



440

embedded in oil drops at nano-scale size in the o/w microemulsions coupled with

441

ultralow interfacial tension, and was easier to pass through the lipid membrane.21,54

442

Thirdly, the drug elimination half-life was extended in MYR-ME during oral

443

administration, which was mainly attributed to that the viscous MYR-ME might

444

easily adhere to the intestinal mucosa and the control release of myricetin from

445

microemulsion gradually. Last but not least, compared with the previous reported

446

microemulsion, the enhancement of MYR-ME bioavailability was attributed to the

447

specific excipients composition and amount used here. The surfactants, Tween 80 and

448

Cremophor RH40 employed in our experiment were proposed to be potent P-

449

glycoprotein inhibitors, and their synergetic inhibitory effect was better than Tween

450

80 and Tween 20, thus their combination was beneficial to improve the permeability

451

and absorption efficacy of flavonoids into intestinal epithelial cells.55 The co-

452

surfactant, Transcutol HP with a longer alcohol molecule, was capable to increase the

453

transport rate of myricetin compared with ethanol used in the literature, since the

454

speed of transmembrane transport increased with the increasing length of alcohols

455

when the alkanol chain length was below nine.52 The oil, medium-chain triglyceride

456

WL 1349, was supposed to greatly enhance the permeability rate of myricetin through

457

the intestinal wall. According to the literatures, medium-chain triglycerides caused

458

higher permeability of hydrophobic drugs than di-glyceride, mono-glyceride and

459

unsaturated fatty acid oleic oil.53,56 As a consequence, myricetin incorporated in the

460

WL 1349 oil core could pass across the phospholipid membrane quickly, thus reduced

461

myricetin glucuronidation and sulfation in the liver and gastrointestinal tract.

462

Therefore, the in vivo absorption was enhanced by these microemulsion formulations,

463

leading to a better oral bioavailability.

20


Page 20 of 39

Page 21 of 39


464

In conclusion, a novel myricetin microemulsion formulation was designed and

465

optimized here to improve its poor aqueous solubility and low bioavailability. An oil-

466

in-water microemulsion was developed with oral permitted component of Cremophor

467

RH40 (12%, w/w, USP/NF), Tween 80 (6%, w/w, USP/EP/BP), Transcutol HP (9%,

468

w/w, EP/NF), WL1349 (18%, w/w USP/EP/NF) and distilled water (55%). The total

469

content of surfactants and co-surfactant (Cremophor RH40, Tween 80 and Transcutol

470

HP) was lower than any previous reports. The optimized MYR-ME could solubilize

471

myricetin up to 17.76 mg/mL, 1225–fold increase compared with the myricetin

472

solubility in water. The physicochemical properties demonstrated that this MYR-ME

473

was stable after dilution and storage for 6 months. The MYR-ME could significantly

474

increase antiproliferative activity against human cancer cell HepG2 while it had little

475

influence on the human normal hepatic cell LO2. The cellular antioxidative activity of

476

myricetin was also notably improved by MYR-ME. Furthermore, compared with

477

myricetin CMC-Na suspension, the oral bioavailability of myricetin was remarkably

478

enhanced by MYR-ME in Sprague-Dawley rats. Therefore, the MYR-ME developed

479

here could be used as a potential carrier for myricetin to promote its future utilization

480

in functional foods and cosmetic as well as pharmaceutical fields.

481

Supporting Information

482

Supplemental tables and figures are available via the Internet at http:// pubs.acs.org.

483

Supplemental Table 1. Optimization of ratios of surfactant/co-surfactant mixture to

484

oils (K value); Supplemental Table 2. Effects of MYR-ME on body weight of rats

485

after single-dosing oral administration; Supplemental Figure 1. The visual

486

appearances of MYR-MEs after storage; Supplemental Figure 2 Antiproliferative

487

activities and cytotoxicity of blank microemulsion against HepG2 and LO2.

488

Notes The authors declare no competing financial interest. 21



489

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Iyomasa, M. M.; Bentley, M. V.; Fonseca, M. J., Quercetin in w/o microemulsion: in

643

vitro and in vivo skin penetration and efficacy against UVB-induced skin damages

644

evaluated in vivo. Eur. J. Pharm. Biopharm. 2008, 69, 948-957.

645

(51) Griffiths, L. A.; Smith, G. E., Metabolism of myricetin and related compounds

646

in the rat. Metabolite formation in vivo and by the intestinal microflora in vitro.

647

Biochem. J. 1972, 130, 141-151.

648

(52) Plucinski, P.; Reitmeir, J., The influence of cosurfactants on the solubilization of

649

phenylalanine in water-in-oil microemulsion. Colloid Surface A 1995, 97, 157-167.

650

(53) Parris, N.; Joubran, R. F.; Lu, D. P., Triglyceride Microemulsions: Effect of

651

Nonionic Surfactants and the Nature of the Oil. J. Agric. Food Chem. 1994, 42, 1295-

652

1299.

653

(54) Kamm, W.; Jonczyk, A.; Jung, T.; Luckenbach, G.; Raddatz, P.; Kissel, T.,

654

Evaluation of absorption enhancement for a potent cyclopeptidic αν β3-antagonist in a

655

human intestinal cell line (Caco-2). Eur. J. Pharm. Sci. 2000, 10, 205-214.

656

(55) Yamagata, T.; Kusuhara, H.; Morishita, M.; Takayama, K.; Benameur, H.;

657

Sugiyama, Y., Effect of excipients on breast cancer resistance protein substrate uptake

658

activity. J. Control. Release 2007, 124, 1-5.

28


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Page 29 of 39


659

(56) Muranushi, N.; Takagi, N.; Muranishi, S.; Sezaki, H., Effect of fatty acids and

660

monoglycerides on permeability of lipid bilayer. Chem. Phys. Lipids 1981, 28, 269-

661

279.

662 663

Notes: This work was supported by Guangdong Science and Technology Program

664

(No.2013B090700008) and Guangdong Science and Technology Program (No.

665

2012B050500003).

666

29



667

Figure Captions:

668

Figure 1. Effects of various mixed surfactants, oils and Km values on pseudo-ternary

669

phase diagrams. A: screening of ratios of mixed surfactant: formulations containing

670

different ratios of Tween 80 to Cremophor RH40 with Transcutol HP as co-surfactant

671

and WL 1349 as oil phase. B: screening of oils: formulations containing various oils

672

with the S+Cos (Tween 80-Cremophor RH40-Transcutol HP) at fixed ratios. C:

673

screening of Km values: formulations containing different Km values with mixed

674

surfactants at ratio of 1:2 and WL 1349 as oil.

675

Figure 2. TEM Images of MYR-MEs (×10000). Where A represents the TEM images

676

of newly-prepared MYR-ME, while B is the MYR-ME 6 months later.

677

Figure 3. Antiproliferation effect and cytotoxicity of MYR-ME and free myricetin

678

(MYR) on HepG2 and LO2. MYR refers to myricetin dissolved in DMSO, and MYR-

679

ME represents myricetin loaded in microemulsion, and the content percentage of both

680

DMSO and ME are 0.5%.

681

Figure 4. CAA values of MYR-ME compared with free myricetin (MYR).

682

Figure 5. Plasma myricetin concentration-time curve after oral administration of

683

MYR-ME and myricetin suspension at doses of myricetin at 25 mg/kg of body weight.

30


Page 30 of 39

Page 31 of 39


Table 1. Solubility of Myricetin in Various Mediums at Room Temperature (Mean ± SD; n = 3)

Phase

Medium

Solubility of myricetin (mg/g)

Oil

Soybean oil

0.83 ± 0.11

WL 1349

0.78 ± 0.13

M1944 CS

0.76 ± 0.17

IPM

0.46 ± 0.09

EO

0.42 ± 0.06

Cremophor RH40

9.96 ± 0.72

Tween 80

9.05 ± 0.58

Span 80

7.99 ± 0.03

Labrasol

5.03 ± 0.69

Cremophor EL35

3.96 ± 0.08

Transcutol HP

21.37 ± 2.00

Ethanol

13.87 ± 0.37

Propylene glycol

12.98 ± 0.68

Transcutol P

12.30 ± 1.44

Glycerol

1.707 ± 0.06

Deionized water

0.015 ± 0.002

Surfactant

Co-surfactant

Water

31



Page 32 of 39

Table 2. Physicochemical Parameters of MYR-ME Value Parameters

Initial

Zeta potential (mv)

100 times

200 times

6 months

-5.50 ± 0.02

—

—

-5.07 ± 0.02

Viscosity (mPa•s )

25.20 ± 0.09

—

—

24.11 ± 0.12

Particle size (nm)

67.28 ± 0.90

60.65 ± 1.19

64.47 ± 5.88

69.56 ± 0.42

PDI

0.23 ± 0.01

0.33 ± 0.04

0.30 ± 0.02

0.078 ± 0.005

Drug content (mg/mL)

17.76 ± 1.34

0.17 ± 0.007

0.085 ± 0.003

17.59 ± 2.01

-1

Newly prepared MYR-ME was compared with MYR-ME diluted for 100 and 200 Times, and MYRME stored for 6 months at room temperature (25~30 oC) (Mean±SD; n=3).

32


Page 33 of 39


Table 3. Pharmacokinetic Parameters of MYR-suspension (25 mg/kg) and MYRME (25 mg/kg) after Oral Administration of Rats (Mean ± SD, n = 6) Parameters

Formulation MYR-suspension

MYR-ME

Cmax (µg/mL)

0.48 ± 0.08

8.11 ± 0.69

Tmax (h)

4.16 ± 0.28

2.25 ± 0.21

t1/2(h)

3.53 ± 0.30

5.20 ± 0.58

AUC(µg h/mL)

3.73 ± 0.31

53.83 ± 3.38

Relative bioavailability (%)

100

1443.16

33



Figure 1

A

0.00

A1:Tween 80:RH40=2:1 A2:Tween 80:RH40=1:1 A3:Tween 80:RH40=1:2

l Oi

C

0.25

0.50

1.00

0.75

Wa ter

0.75

ME

1.00 0.50

ME

A3

0.25

A2 ME 0.00 0.00

B

0.25

0.50

A1

0.75

1.00

0.00

B1: Soybean oil B2: M1944 CS B3: WL1349

0.25

l Oi

0.50

1.00

0.75

0.75

ME

Wa

ter

S+Cos

1.00

0.50

B3

ME 0.25

B2

ME 0.00 0.00

0.25

0.50

0.75

1.00

S+Cos

B1

C

0.00

C1: Km=1:2 C2: Km=1:1 C3: Km=2:1

0.25

0.50

l Oi

0.75

Wa ter

1.00

ME

1.00

0.75

C3 ME

0.50

C2

0.25

ME 0.00 0.00

0.25

0.50

C1

0.75

1.00

S+Cos

34


Page 34 of 39

Page 35 of 39


Figure 2

35



Page 36 of 39

Figure 3

100

100

Cell proliferation (%)

60

80

60

40

40

20

20

0

0 0

40

80

120

160

Concentration of myricetin (µM)

36


200

Cytotoxicity (%)

antiproliferation of MYR-ME on LO2 antiproliferation of MYR on HepG2 antintiproliferation of MYR-ME on HepG2 cytotoxicity of MYR-ME on HepG2 cytotoxicity of MYR on HepG2 cytotoxicity of MYR-ME on LO2

80

Page 37 of 39


Figure 4

CAA Value (µ M QE /100 µ M Myricetin)

a

MYR MYR-ME

120

a

80

b c 40

0 No PBS Wash

PBS Wash

37



Page 38 of 39

Figure 5

Myricetin concentration (µg/mL)

8

MYR-ME MYR-suspension

6

4

2

0 0

4

8

12 Time (h)

16

38


20

24

Page 39 of 39


TOC Graphic

39


Preparation and Characterization of Microemulsions of Myricetin for

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