Preparation and Characterization of Mucoadhesive Buccal

May 24, 2016 - The adhesive interactions of CS/DS NPs with mucin were not significantly different from those of CS/sodium triphosphate pentabasic (TPP...
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Preparation and Characterization of Mucoadhesive Buccal Nanoparticles Using Chitosan and Dextran Sulfate Ji Woon Suh, Ji-Soo Lee, Sanghoon Ko, and Hyeon Gyu Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00849 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 26, 2016

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

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Journal of Agricultural and Food Chemistry

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Preparation and Characterization of Mucoadhesive

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Buccal Nanoparticles Using Chitosan and Dextran

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Sulfate

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Ji Woon Suh,† Ji-Soo Lee,† Sanghoon Ko,‡ and Hyeon Gyu Lee*,†

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Department of Food and Nutrition, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea

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Department of Food Science and Technology, Sejong University, 98 Gunjadong, Gwangjin-gu, Seoul, 143-747, Republic of Korea

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*Corresponding author.

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Tel: +82-2-2220-1202; Fax: +82-2-2281-8285;

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E-mail: [email protected] (H.G. Lee).

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ABSTRACT

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The aim of this study was to formulate buccal mucoadhesive nanoparticles

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(NPs) using the natural mucoadhesive polymers. The natural mucoadhesive

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polymers chitosan (CS) and dextran sulfate sodium salt (DS) were used to

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prepare mucoadhesive nanoparticles using ionic gelation method. As the

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molecular weight of DS decreased, the amount of mucin and the number of

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buccal cells adsorbed on DS increased. The CS/DS NPs ranged from 100-200

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nm in diameter. The adhesive interactions of CS/DS NPs with mucin were not

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significantly different from those of CS/sodium triphosphate pentabasic (TPP)

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NPs; however, CS/DS NPs exhibited 5 times greater mucoadhesive activity to

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buccal cells compared with control CS/TPP NPs in ex vivo adhesion tests.

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These results indicate that the buccal mucoadhesive properties of NPs can be

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improved by using natural mucoadhesive polymers.

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KEYWORDS: mucoadhesive; dextran sulfate; chitosan; nanoencapsulation

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INTRODUCTION

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Mucoadhesion is defined as the adhesive capacity of synthetic or natural

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polymers to the mucous gel layer covering mucosal membranes.1 It is generally

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believed that prolonging the residence time on the adsorbing membrane by

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mucoadhesion provides optimal conditions for effective delivery. Therefore,

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mucoadhesive systems have been studied as an approach to increase the

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residence time of the dosage form on the absorbing mucosal surface and to

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localize drugs or biological nutrients in a particular region.2 Mucoadhesion also

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allows the diffusion and penetration of mucoadhesive materials into the mucous

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layer, resulting in improved absorption and bioavailability.3

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Buccal delivery provides a number of unique advantages including

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excellent accessibility, bypass of the first pass metabolism, and avoidance of

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presystemic elimination within the gastrointestinal tract. However, the major

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limitations of absorption across the oral mucosa include exposure to salivary

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flow, shearing forces due to tongue movements and swallowing, and short

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residence time. These limitations could in principle be effectively alleviated by

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the use of a buccal mucoadhesive delivery system.

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Buccal mucoadhesive delivery systems have been fabricated by several

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different approaches. Most studies have focused on buccal mucoadhesive

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tablets, patches, films, gels, and ointments for drug delivery to treat oral disease;

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these patches are based on synthetic mucoadhesive polymers.4 Tiyaboonchai

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et al.5 used polyethylenimine–dextran sulfate nanoparticles for the buccal

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mucoadhesive delivery of Punica granatum peel extract. This strategy resulted

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in prolonged release and significant antibacterial activity against oral pathogens

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that cause plaque, cavities, and oral malodor. For a mucoadhesive delivery

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system to be used in food applications, an encapsulation technique is

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necessary to protect the cargo from potentially hostile characteristics of the

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external environment such as pH, temperature, and moisture. In addition,

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encapsulation can mask unwanted flavor and taste. Nanoparticles also have

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sensory advantages relevant to the food industry, because nano-sized particles

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cannot be felt by the mouth due to their reduced particle size; in contrast, micro-

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sized particles can be felt. 6, 7 However, only a few studies have used a buccal

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mucoadhesive nanoparticle system for biological nutrients such as curcumin

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and plant phenolic extracts and reported improved absorption and bioavailability.

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These studies were based on natural biopolymers for their application in the

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food industry.

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For optimal formulation of mucoadhesive delivery systems, appropriate

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mucoadhesive polymers are required. The binding capacity of polymers to the

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mucosal membrane has been reported to be influenced by ionic bonds,

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hydrophobic interactions, covalent bonds, and physical entanglement.2,

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Mucoadhesive properties have also been reported to be affected by a number

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of factors such as molecular weight, flexibility, hydrogen bonding capacity,

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cross-linking density, charge, concentration, and polymer swelling.4 Various

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polymers including chitosan (CS), alginate, dextran sulfate sodium salt (DS),

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and carrageenan have been reported as mucoadhesive biopolymers.10

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Particularly, mucoadhesive biopolymers, CS and DS can be also used for

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nanoencapsulation. The positive charge of CS binds to a negatively charged

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mucous surface due to ionic bonds. Müller et al.11 observed that CS-modified

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nanosuspensions exhibited increased retention time in the gastrointestinal tract.

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In addition, the sulfate functional groups of DS form strong hydrogen bonding

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interactions with mucin.12 Tiyaboonchai et al.5 demonstrated the ability of DS to

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adhere to the mucosal surface. Although nanoencapsulation using CS and DS

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could enhance cellular uptake of curcumin,13 the use of CS and DS to enhance

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the mucoadhesive activity of nanoparticles has not been fully explored.

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For a buccal mucoadhesive nanoparticle system for biological nutrient to

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be used in food industry, the natural mucoadhesive polymers is preferable to

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wall material of nanoparticle system with regard to safety. Hence, the aim of this

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study was to formulate buccal mucoadhesive nanoparticles using natural

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mucoadhesive polymers CS and DS. The physical properties of the

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nanoparticles, including particle size, polydispersity index (PDI), and derived

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count rate (DCR) were investigated. In addition, the effect of CS and DS

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concentration on the mucoadhesive activity of the nanoparticles was evaluated

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using in vitro and ex vivo adhesion tests.

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MATERIALS AND METHODS

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Materials. CS (water soluble, M.W. 1,000-3,000, 24 cps, 95% deacetylated)

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was obtained from Kittolife Co. (Seoul, Korea). DS (MW: 15,000, 40,000, and

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200,000 kDa), sodium triphosphate pentabasic (TPP), and magnesium chloride

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were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All

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other chemicals were of reagent grade and all solvents were of HPLC grade.

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Preparation of nanoparticles. Two types of nanoparticles, CS/DS and

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CS/TPP, were obtained by ionic gelation of CS with DS and TPP, respectively.14

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CS was dissolved in distilled water at a concentration of 1 - 2 mg/mL. The

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CS/DS nanoparticles were prepared by adding 3 mL of DS solution (0.2 – 0.6

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mg/mL) into CS solution for 10 min under magnetic stirring (1,000 rpm, WiseStir

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MS-MP8, Wise Laboratory Instruments, Wertheim, Germany). The CS/TPP

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nanoparticles (control group) were prepared by adding 1 mL of TPP solution

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(0.3 – 0.6 mg/mL) into CS solution for 10 min under magnetic stirring.

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Physical properties of the nanoparticles. The physical properties of

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the CS/DS and CS/TPP nanoparticles, including particle size, PDI, zeta

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potential, and DCR, were determined by dynamic light scattering with a Malvern

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Zetasizer

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Worcestershire, UK). All measurements were performed in triplicate at multiple

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narrow modes at 25 ± 1°C.

Nano

ZS

instrument

(Malvern

Instruments

Ltd.,

Malvern,

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Determination

of

in

vitro

mucoadhesive

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properties.

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mucoadhesive properties of the nanoparticles to mucin were investigated as

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described previously,15 but with minor modifications. Briefly, 0.6 mL of mucin

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solution (0.5 mg/mL) was mixed with 0.6 mL of nanosuspension and incubated

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at 37°C in a shaking water bath for 1 h. After centrifugation at 14,000xg for 40

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min, the supernatant was collected and the amount of free mucin was

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measured using the Bradford protein assay. The supernatant was incubated

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with Bradford reagent for 5 min, after which the absorbance was measured at

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595 nm with a spectrophotometer (Biomate 3S, Thermo Scientific, Waltham, MA,

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USA). The amount of mucin-adsorbed nanoparticles was calculated as the

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difference between the total amount of mucin added and the residual mucin in

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the supernatant. Mucin concentration was calculated from a standard curve of

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mucin in concentration from 0-5 mg/mL .16

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Determination of ex vivo mucoadhesive properties. The ex vivo

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mucoadhesive properties of the nanoparticles were determined by the methods

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of Kockkisch et al.17 and Patel et al.,18 with minor modifications. All procedures

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for collecting buccal cells were approved by the Hanyang University Institutional

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Review Board. Buccal cells were collected from 10 healthy male and female

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volunteers by gently scraping the oral cavity with a tongue depressor. Donors

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were prohibited from eating or drinking for 1 h prior to buccal cell collection.

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Cells were immediately suspended in 0.25 M aqueous sucrose solution and the

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cell suspension was then added to 0.5% (w/v) trypan blue solution. The cell

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concentration was determined using a hemocytometer and standardized to 48 x

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106 cells per test. All cells were stored at 4°C and used within 4 h of collecting.

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The cells were separated from supernatants by centrifugation at 700 x g

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for 5 min and then reacted with samples (polymers or nanoparticles) in

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phosphate buffer in a 30°C shaking incubator for 30 min. The 0.1% Alcian blue

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was added and incubation for 1 h at 30°C was carried out. The cells were then

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washed using 0.25 M sucrose solution. The complexed dye with polymer

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treated cells was eluted by immersion in 5 M magnesium chloride for 1 h at

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30°C. Cells were then spun by centrifugation at 700 x g for 15 min and the

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absorbance of the supernatant was measured at 605 nm using a

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spectrophotometer. A control was performed following the same procedure

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without a test sample. Results are expressed as a percentage reduction as

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compared with control.

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Statistical analysis. Statistical analyses were conducted using SPSS

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(Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL, USA).

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One-way analyses of variance (ANOVA) were performed to investigate the

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significance of differences between conditions (p