Bioavailability enhancement of astilbin in rat through zein-caseinate

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Bioactive Constituents, Metabolites, and Functions

Bioavailability enhancement of astilbin in rat through zein-caseinate nanoparticles Dan Zheng, and Qing-Feng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00018 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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Journal of Agricultural and Food Chemistry

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Bioavailability enhancement of astilbin in rat through zein-caseinate

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nanoparticles

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Dan Zheng, Qing-Feng Zhang*

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Jiangxi Key Laboratory of Natural Product and Functional Food, College

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of Food Science and Engineering, Jiangxi Agricultural University, Nanchang

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330045, China

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*Corresponding author, Tel & Fax: 86-791-3813863, E-mail: [email protected]

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(Q.F. Zhang).

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ACS Paragon Plus Environment


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ABSTRACT

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The astilbin encapsulated zein-caseinate nanoparticles were fabricated through

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antisolvent method. The encapsulation and loading efficiency of astilbin in the

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nanoparticles were determined by HPLC. The nanoparticles were characterized by

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particle size, zeta potential, redispersibility, SEM, XRD, FTIR and DSC. Under the

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optimal formulation of astilbin, zein and sodium caseinate with mass ratio of 1:1:2,

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the size and zeta potential of the nanoparticles were 152.9 nm and -40.43 mV, while

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the encapsulation and loading efficiency of astilbin were 80.1% and 21.8%,

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respectively. The nanoparticles had good redispersibility in water without particle size

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change after freeze-drying. The nanoparticles showed spherical shape with smooth

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surface. XRD and DSC analysis showed that astilbin existed in amorphous form in the

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nanoparticles. The interactions between astilbin and the protein were found, and

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astilbin was encapsulated in nanoparticles rather than adsorbed. The diffusion of

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astilbin from nanoparticles was significantly faster than that of astilbin suspensions in

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both simulated gastric and intestinal fluids. Astilbin was relative stable in simulated

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intestinal fluids, and the encapsulation in the nanoparticles showed slight stability

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improvement effect. Pharmacokinetic study showed that the absolute bioavailability

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of astilbin was improved from 0.32% to 4.40% in rat through nanoparticles

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fabrication.

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Keywords: Astilbin; Zein; Nanoparticles; Pharmacokinetics; Bioavailability

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INTRODUCTION

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Astilbin is the rhamnoside of dihydroquercetin. The flavonoid is presented in many

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plant and plant-based foods with variety bioactivities.1-4 Particularly, the unique

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selective immunosuppressive activity of astilbin has attracted increasing attention.5-9

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Pharmacological studies revealed that astilbin could significantly inhibit delayed

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hypersen-sitivity,5,6 collagen-induced arthritis,7,8 and immune liver injury.9 It has

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reported that astilbin could selectively inhibit excess cellular immunity without

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affecting humoral immunity. Its strong immunomodulatory activity shows no obvious

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side-effects. Other compounds with similar activity has unfound yet. Astilbin may be

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an attractive candidate for novel immunomodulator development.10 However, astilbin

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is a poorly soluble compound with solubility of 0.221 g/L in water at 298 K.11 It is

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also unstable in alkaline solution, and the isomerization and decomposition were

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found.12 Due to the low solubility and permeability, the oral bioavailability of astilbin

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is very poor. Wang found that the apparent permeability coefficient of astilbin was

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less than 1*10-6 cm/s in Caco-2 cell and its oral bioavailability was only 0.066% in

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rat.13 The study of Lei et al. showed that the oral bioavailability of astilbin was 2.01%

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in rat.14 Hence, improving the bioavailability of astilbin is critical for its practical

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application. By developing self micro-emulsifying drug delivery system, the

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bioavailability of astilbin was enhanced 5.56-fold in beagle dogs.15 He et al. prepared

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the solid dispersions of astilbin in PVP K30-Tween 80 combined carries.

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Pharmacokinetic study in beagle dogs showed that the oral bioavailability of astilbin

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solid dispersions was about 205% of that of astilbin. 16 3



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Zein is the main storage protein of corn with more than 50% of hydrophobic amino

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acids. Hence, the protein is insoluble in water but has good solubility in 50-95%

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ethanol-aqueous solution.17 With the decrease of ethanol concentration, zein will lost

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its solubility and is able to self-assemble into micro- or nanoparticles,17 fiber

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film.19 The specific structure is depended on the preparation method and conditions,

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such as pH, zein concentration, shearing rate, etc.17 The unique self-assembly ability

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of zein to form nanoparticle makes it a promising delivering carrier for nutraceuticals

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and drugs.20 The internal hydrophobic environment of zein nanoparticles is suitable

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for encapsulating of hydrophobic compounds. Furthermore, zein is an edible protein

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with natural biocompatibility and biodegradability, and is generally recognized as safe

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(GRAS) biomaterial.17 The encapsulation in zein nanoparticles could significantly

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improve the stability, dispersibility and bioavailability of guest compounds.17,21

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Penalva et al. fabricated the resveratrol and quercetin loaded zein nanoparticles

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through desolvation method. The size of nanoparticles was 307 nm with resveratrol

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loading of 80 μg/mg. The oral bioavailability of resveratrol was enhanced by

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19.2-fold in rat.22 Similar results were found in quercetin loaded zein nanoparticles.23

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Zou et al. designed TPGS 1000 emulsified zein nanoparticles to improve the oral

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bioavailability of daidzin. Pharmacokinetic study showed the Cmax of daidzein in mice

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plasma as well as the bioavailability were significantly increased.24 Sodium caseinate

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is the sodium salt of casein, which is the main protein of milk. Sodium caseinate has

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superb water solubility, and is widely used for emulsifying and thickening functions

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in food industry. It has reported that sodium caseinate can adsorb on the surface of 4


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and

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zein nanoparticles due to its amphiphilic and charged nature, which can prevent the

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aggregation of colloids.25 Thus, sodium caseinate can be used as a stabilizer for zein

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nanoparticles.

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In the present study, zein-caseinate nanoparticles were fabricated to improve the

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bioavailability of astilbin. The formulation was optimized through comparing the

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encapsulation and loading efficiency of astilbin. The nanoparticles were characterized

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by particle size, zeta potential, redispersibility, scanning electron microscopy (SEM),

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differential scanning calorimeter (DSC), X-ray diffractometer (XRD), and Fourier

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transform infrared spectroscopy (FTIR). The diffusion profile of astilbin from the

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nanoparticles in simulated gastric (SGF) and intestinal fluids (SIF) was also studied.

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Finally, pharmacokinetic study was carried out to confirm the bioavailability

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enhancement of astilbin in rat.

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MATERIALS AND METHODS

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Chemicals. Astilbin (>98%) were purified from Rhizoma Smilacis Glabrae in our

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laboratory, and was identified by UV, IR, MS, and NMR. HPLC grade acetonitrile

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and methanol were purchased from Anhui Tedia High Purity Solvents Co., Ltd

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(Anqin city, Anhui province,China). Zein was obtained from Sigma Co. Ltd (St.

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Louis, MO, USA). Sodium caseinate, pepsin and pancreatin were purchased from

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Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). Milli-Q water was

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used throughout the study. All other reagents used were analytical grade.

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Preparation of astilbin encapsulated zein-caseinate nanoparticles. The

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nanoparticles were prepared by antisolvent method described elsewhere.17 The mass 5



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ratio of zein to astilbin in the nanoparticles was varied from 1:1 to 6:1, while the mass

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ratio of zein to sodium caseinate was maintained at 1:2. Briefly, 100 mg of zein mixed

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with 100, 50, 25 or 16.7 mg of astilbin were dissolved in 10 ml of 70% ethanol

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solution, respectively. The mixture was dropped into 30 ml water contains 200 mg of

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sodium caseinate using an injection syringe under quick magnetic stirring. The

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dropping rate was about 2 mL/min. After stirring for another 60 min, the

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nanoparticles suspensions were concentrated to 25 ml by rotary evaporators at 50

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oC.The

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large particles and free astilbin. Then, a little part of the suspensions were used for

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encapsulated astilbin determination, particle size and zeta-potential analysis, and the

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rest were freeze-dried.

suspensions were subsequently centrifugated at 2000 g for 10 min to remove

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Particle size, Polydispersity index (PDI) and Zeta-potential analyses. The

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particle size and zeta-potential of nanoparticles were measured by dynamic light

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scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). The

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measurement was replicated three times for each sample.

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Encapsulation and loading efficiency. The encapsulated astilbin in nanoparticles

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was determined by HPLC method. The encapsulation efficiency (EE) and loading

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efficiency (LE) were calculated with the equations:

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EE=

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LE=

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Briefly, the nanoparticles were dissolved in 70% ethanol. After appropriate dilution, a

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10 μL aliquot of sample was injected in an Agilent 1260 HPLC system (Agilent

𝐴𝑠𝑡𝑖𝑙𝑏𝑖𝑛 𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑒𝑑 𝑖𝑛 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠(𝑚𝑔) 𝑡𝑜𝑡𝑎𝑙 𝑎𝑠𝑡𝑖𝑙𝑏𝑖𝑛 𝑢𝑠𝑒𝑑 (𝑚𝑔)

×100%

𝐴𝑠𝑡𝑖𝑙𝑏𝑖𝑛 𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑒𝑑 𝑖𝑛 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠(𝑚𝑔) 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑦 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠(𝑚𝑔)

×100%

6


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Technologies, Palo Alto, CA, USA). The HPLC conditions were the same as we

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described before.12

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Scanning electron microscopy (SEM) analysis. The freeze-dried nanoparticles

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(Ast-Zein1:1) and its redispersion solution were used for morphology characterization

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by SEM (FEI Quanta 250 FEG, FEI Inc., OR, USA). The redispersion solution was

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first dropped on a conductive glass and then dried naturally. Before imaging, the

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samples were coated with a thin layer of gold first.

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Differential scanning calorimeter (DSC) analysis. DSC analysis was carried on a

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NETZSCH DSC 214 polyma instrument (Netzsch, Germany). About 5 mg of

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freeze-dried nanoparticles (Ast-Zein1:1) was added to a standard aluminum pan and

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hermetically sealed. The pan was placed in glass desicator for 24 h to exclude the

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influence of moisture. An empty sealed aluminum pan was used as control. Samples

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were heated from 50 to 300 oC at rate of 12 oC/min with a constant purging of dry

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nitrogen at rate of 50 mL/min. The thermal characteristics of astilbin, physical

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mixture of zein and sodium caseinate (with mass ratio of 1:2) were also analyzed.

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X-ray diffractometer (XRD) analysis. XRD patterns of Ast-Zein1:1 nanoparticles

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and astilbin were performed on a Bruker D8 ADVANCE diffractometer (Bruker,

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Germany). The instrument was equipped with a copper anode. The working

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accelerating voltage and tube current were 40 kV, 40 mA, respectively. The pattern

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was recorded over an angular range from 5o to 60o in continuous mode with a step

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size of 0.02o and scanning speed of 2o/min.

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Fourier transform infrared spectroscopy (FTIR) analysis. FTIR spectroscopy of 7



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Ast-Zein1:1 nanoparticles, astilbin and physical mixture of zein and sodium caseinate

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(with mass ratio of 1:2) were recorded on a Nicolet 5700 FTIR spectrometer (Nicolet,

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USA) by the KBr method.

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In vitro diffusion of astilbin from the nanoparticle in simulated gastric (SGF)

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and intestinal fluids (SIF). SGF (pH 1.2) and SIF (pH 6.8) were prepared in

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accordance with USP XXII.19. Ast-Zein1:1 nanoparticles and astilbin were dispersed

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in 5 ml of SGF or SIF (with enzyme) with astilbin concentration of 3 mg/mL. The

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solution was put into a dialysis bag (molecular cutoff: 7 kDa) and then placed in

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beaker containing 500 ml of SGF or SIF (without enzyme) under magnetic stirring at

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37 oC. At different time intervals, astilbin concentration in the beaker was analyzed by

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HPLC.

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Stability of free astilbin and Ast-Zein1:1 nanoparticles in simulated intestinal

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fluids (SIF). Astilbin solution was prepared in 60% ethanol with concentration of 1

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mg/mL. Ast-Zein1:1 nanoparticles were dispersed in water with astilbin concentration

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of 1 mg/mL. A 1.0 mL aliquot of the two solutions were mixed with 9 mL of SIF

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(without enzyme), respectively. The mixture was incubated at 37 oC in waterbath. At

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different time intervals, the remaining astilbin was determined by HPLC.

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Pharmacokinetic studies in rats. The animal studies were complied with the

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guidelines of Jiangxi Agricultural University on animal care. Sprague-Dawley (SD)

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rats were obtained from Hunan slack Jing Da laboratory animal Co., Ltd (Changsha

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City, Hunan province, China). The rats were breed in a controlled environment

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conditions with 12/12 h light/dark cycle and temperature of 23-25 ℃ . Before 8


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pharmacokinetic study, rats were fasted overnight with access to water. Eighteen rats

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were randomly divided into three groups (n=6) with mean weight of 300±15 g. For

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nanoparticles colloidal solution, freeze-dried Ast-Zein1:1 nanoparticles were dispersed

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in warm water (~40 oC) with astilbin concentration of 6 mg/mL. Similarly, astilbin

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suspensions were prepared by suspending astilbin powder (pass through 100 mesh,

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with average size of about 150 μm) in warm water (~40 oC) directly with

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concentration of 6 mg/mL. Because the solubility of astilbin is only about 0.2 mg/mL,

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it will precipitate soon. Thus, before oral administration, the suspensions should be

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shocked intensely. Group I and II were orally administered 1 mL of nanoparticles

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colloidal solution and astilbin suspensions with dose of 20 mg astilbin/kg body weight,

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respectively. Group III received intravenous (iv) administration of 0.3 ml astilbin

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solution (2 mg/mL in 10% ethanol) through caudal vein with dose of 2 mg astilbin/kg

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body weight. The iv solution was prepared by 5 times dilution of astilbin dissolved in

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50% ethanol (10 mg/mL) with water. Blood samples (~200 μL) were obtained from

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tail veins into K2-EDTA pretreated tubes at 0.167, 0.5, 1, 2, 3, 4, 6 and 8 h after

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administration. After centrifugation at 2000 g for 10 min, the plasma samples were

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obtained and stored at −80 °C before analysis.

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A liquot of 50 μL plasma sample was mixed with 150 μL of methanol containing

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350 ng/mL rutin (used as internal standard, IS). Then, a 5 μL liquot of acetic acid was

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added. The mixture was vortexed for 2 min to precipitate endogenous proteins. After

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centrifugation at 10000 g for 10 min, the supernatant was transferred to a new 1.5 mL

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tube and concentrated to about 100 μL on 75 oC waterbath for 12 min. The solution 9



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was centrifuged at 10000 g for 10 min again, and the supernatant was used for astilbin

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determination.

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The astilbin concentration was determined on an Agilent 1260 HPLC system

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equipped with an autosampler and DAD detector. A symmetry C18 column (4.6 mm

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× 250 mm, 5.0 μm; Waters, USA) was used. The mobile phase consisted of 24%

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acetonitrile and 76% water (with 0.1% acetic acid). The flow rate was 1.0 mL/min

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with column temperature of 40 oC and detection wavelength of 291nm (for astilbin)

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and 355 nm (for rutin). The injection volume was 50 μL and the analysis duration was

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10 min.

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Calibration curve was obtained in triplicates by plotting the peak area ratio

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(astilbin/rutin) against the astilbin concentration (16, 32, 64, 128, 256, 515, 721, 1030,

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5150, 10300 ng/mL in plasma). The regression equation is Y=2.2604X with linear

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coefficient of 0.9995, where Y is the peak area ratio and X is the concentration of

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astilbin in plasma (μg/mL).

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The pharmacokinetic data were simulated by Drug and Statistics (DAS) software

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(version 2.0) using a non-compartmental statistical model to calculated the

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pharmacokinetic parameters, including the maximal serum concentration (Cmax), the

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time in which Cmax is reached (tmax), the area under the concentration-time curve

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(AUC), the mean residence time (MRT), the clearance (CL), and the half-life in the

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terminal phase (t1/2). Furthermore, the absolute bioavailability (Fr, %) of astilbin was

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calculated by the equation

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Fr(%) =

AUCoral AUCiv

× 100

10


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Statistical analysis. Data were expressed as mean ± standard deviation of triplicates.

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Data analysis and plotting were performed with software of Origin 7.0 (Origin Lab

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Co., Northampton, MA, USA). One-way ANOVA was used for statistical analysis.

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Differences were considered significant when P

Bioavailability enhancement of astilbin in rat through zein-caseinate

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