Enhanced Stability and Oral Bioavailability of FA-DEX-CoQ10


Enhanced Stability and Oral Bioavailability of FA-DEX-CoQ10...

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Bioactive Constituents, Metabolites, and Functions

Enhanced Stability and Oral Bioavailability of FA-DEXCoQ10 Nanopreparation by High-Pressure Homogenization Meng Luo, Xuan Yang, Xin Ruan, Wenmiao Xing, Min Chen, and Fan-song Mu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02660 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018

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Journal of Agricultural and Food Chemistry

Enhanced Stability and Oral Bioavailability of

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FA-DEX-CoQ10 Nanopreparation by High-Pressure

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Homogenization

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Meng Luo†,‡,Xuan Yang†,Xin Ruan†,Wenmiao Xing†,Min Chen†,Fansong Mu*,†,‡

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Heilongjiang, Harbin 150040, China

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8

Heilongjiang, Harbin, 150040, China

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ABSTRACT:

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The

Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education,

Collaborative Innovation Center for Development and Utilization of Forest Resources,

preparation

of

folic-dextran-coenzyme

Q10

(FA-DEX-CoQ10)

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nanopreparation was optimized by high-pressure homogenization to improve the

12

dissolution and oral bioavailability of CoQ10. The preparation conditions of

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FA-DEX-CoQ10 nanopreparation were optimized by single factor and orthogonal

14

experimental design. The properties of CoQ10 raw materials, CoQ10 physical mixtures

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and FA-DEX-CoQ10 nanopreparation were characterized by SEM, XRD, IR-IR, and

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DSC. The concentration of CoQ10 in rat plasma was determined by HPLC, and the

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corresponding pharmacokinetic parameters were calculated. The optimal preparation

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method is as follows: mass ratio of CoQ10 to FA-DEX of 1:18, mass ratio of stabilizer

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to CoQ10 of 0.4:1, 6 homogenization cycles, and homogenization pressure of 800 bar.

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These conditions resulted in an MPS of 87.6 nm. SEM showed that the particles was

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spherical. DSC and XRD analyses showed that the crystallinity of FA-DEX-CoQ10

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nanopreparation decreased. FA-DEX-CoQ10 possesses long-term stability. By single

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factor and orthogonal experiments, the dissolution rate, Cmax and AUC of the

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optimized FA-DEX-CoQ10 nanopreparation were 3.95, 2.7 and 2.4 times as much as

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that of the raw materials. The results showed that FA-DEX-CoQ10 nanopreparation

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had better oral bioavailability.

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KEYWORDS: coenzyme Q10 nanopreparation, high pressure homogenization,

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dissolution, oral bioavailability

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INTRODUCTION

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Coenzyme Q10 (CoQ10), also known as ubiquinone or ubidecarenone, is a kind of

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natural lipid-soluble molecule.1 It acts as a redox component of the transmembrane

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electron transport system in the mitochondria respiratory chain2 as well as a stabilizer

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on the cell membrane.3 CoQ10 can also be used as an effective antioxidant and radical

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scavenger.4-6 At present, most studies on the antioxidant activities of CoQ10 focused

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on the relationship between CoQ10 and oxidative stress related diseases,7-10 such as

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cardiovascular disease,11-13 neurodegenerative diseases,14-16 aging and cancer.17-20

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CoQ10 has significant nutritional and medical benefits; however, it is a liposoluble

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substance with poor stability and low bioavailability after oral administration.

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Therefore, enhancing the bioavailability of CoQ10 via optimized preparation

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techniques has become an important goal of research and development in recent

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years.

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Xia prepared CoQ10 nanoliposomes by ethanol injection. The results showed that

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the encapsulation efficiency was greater than 95%, with a retention ratio higher than

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90%. The z-average diameter (Dz) was about 67 nm.21 Li prepared CoQ10

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nanoliposomes, using the same method. The t1/2 and AUC of CoQ10 nanoliposomes

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were 1.54 times that of CoQ10. The nanoliposomes can delay drug release and

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increase bioavailability and targeting effects.22 Olsen and co-workers used solid

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dispersion technology to dissolve CoQ10 with a certain proportion of a polymer

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solubilizer, such as polyvinyl pyrrolidone (PVP), and hydroxypropyl methyl cellulose

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phthalate (HPMC-P). Next, the organic solvent was removed to obtain a dry CoQ10

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solid dispersion powder.23 The release of modified and amorphous CoQ10 was

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significantly higher than that of crystalline CoQ10. Kommuru developed a CoQ10

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self-emulsifying drug delivery system (SEDDS), using polyethylene glycol glyceride

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(PGG) as an emulsifier. Compared to a powder formulation, the bioavailability of

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CoQ10 in the SEDDS preparation was two-fold greater.24 Balakrishnan prepared

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CoQ10 formulations using two oils (Labrafil M 1944 and Labrafil M 2125), surfactant

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(Labrasol) and cosurfactant (Lauroglycol FCC and Capryol 90). The minimum mean

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droplet size was approximately 240 nm, and the AUC was approximately 2-fold

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higher than CoQ10 powder.25 The above methods can reduce the particle size of CoQ10,

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and therefore increase its bioavailability.26-29

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In this study, CoQ10, folic acid (FA) and dextran (DEX) were used as raw

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materials for the preparation of FA-DEX-CoQ10 via high-pressure homogenization.

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DEX is commonly used as accessory in pharmaceutical research. Active substances

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released from DEX in vivo can react with drugs to produce drug carriers. FA

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possessed targeting. It is a FDA-approved nutritional supplement. High-pressure

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homogenization technology is used to prepare insoluble drugs as a multi-phase system

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of nanometer particles, thereby improving the stability and oral bioavailability and

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enhancing the pharmacological effects.

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MATERIALS AND METHODS

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Materials. CoQ10 was purchased from Shanxi Sciphar Natural Products Co. Ltd.

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FA and DEX, 4-dimethylaminopyridine (DMAP) and dimethyl sulfoxide (DMSO)

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were from Beijing Bailingwei Technology Co. Ltd. Dicyclohexylcarbodiimide (DCC)

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and lecithin were purchased from J&K Scientific GmbH Ltd. and Zhengzhou Siwei

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Biological Technology Co. Ltd., respectively. Ethylenediamine tetraacetic acid

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disodium salt (EDTA-2NA) was purchased from Tianjin Ruijin Chemicals Co. Ltd.

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Methanol and ethanol (HPLC grade) were purchased from Dikma Pure. N-hexane was

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from Tianjin Fuyu Fine Chemical Co. Ltd. Sodium dodecyl sulfate (SDS) was

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obtained from Tianjin Guangfu Fine Chemical Research Institute.

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Preparation of FA-DEX. Briefly, 0.4586 g of FA, 0.2144 g of DCC and 0.1271

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g of DMAP were dissolved in 25 mL DMSO and kept in the dark at 30°C, with

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stirring under nitrogen gas for 30 min. Upon the addition of 1.0012 g of DEX, the

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mixture was stirred for 20 h. The precipitate was filtered, and the filtrate was dialyzed

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with 10 mmol/L of phosphate buffered saline (PBS, pH 7.4, 0.15 mol/L NaCl, the

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relative molecular mass was 3.5 kDa) to remove FA, which did not participate in the

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reaction. The dialysis step was repeated with water. The precipitate was removed by

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centrifugation at 25 000 g for 30 min. Finally, the solution was freeze-dried to obtain

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a pure FA-DEX coupling carrier.30

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Preparation and Optimization of FA-DEX-CoQ10 nanopreparation by Single

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Factor Experiment. The mean particle size (MPS) of FA-DEX-CoQ10 prepared by

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high-pressure homogenization was measured by the single factor method. The

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analysis parameters include the mass ratio of the stabilizer to CoQ10 (A), cycle times

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(B), and homogenization pressure (C) the mass ratio of CoQ10 to FA-DEX (D). In

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order to determine the optimal conditions, a series of single factor experiments were

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carried out. The mass ratio of CoQ10 to FA-DEX was varied between 1:2 and 1:24.

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The mass ratio of stabilizer to CoQ10 was varied from 0.1:1 to 0.5:1. The number of

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cycles ranged between 3 and 15, and the homogenization pressure ranged from 200 to

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1000 bar.

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Preparation and Optimization of FA-DEX-CoQ10 Nanopreparation by

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Orthogonal Experiment Design. Based on the results of the single factor experiment,

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an orthogonal design L9(3)4, including 4 factors and 3 levels, was used to optimize the

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MPS parameters of the FA-DEX-CoQ10 nanopreparation. As shown in Table 1, the

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horizontal gradient of each factor was as follows: the mass ratio of CoQ10 to FA-DEX

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was 1:6, 1:12 and 1:18; the mass ratio of stabilizer and CoQ10 was 0.3:1, 0.4:1 and

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0.5:1; the number of cycles were 3, 6 and 9; and the homogenization pressure was 600,

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800 and 1000 bar.

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FA-DEX-CoQ10 Nanopreparation Characterization.

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Scanning electron microscopy analysis. The samples were attached to the

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observatory with double-sided conductive adhesive tape treated with gold, and the

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morphology of the samples was observed by SEM.

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X ray diffraction analysis. The crystal forms of the raw materials, physical

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mixtures and FA-DEX-CoQ10 nanopreparation samples were characterized by XRD.

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The diffraction angle range was 10–90°, the scanning rate was 5°/min, the scanning

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step was 0.05°, the voltage was 40 kV, and the current was 30 mA.

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Fourier-transform infrared spectroscopy analysis. The raw materials, physical

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mixtures and FA-DEX-CoQ10 nanopreparation samples were pressed into slices,

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respectively. The results were recorded by IRAffinity-1 FT-IR spectrum in the range

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of 4500 cm−1 to 500 cm−1.

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Differential scanning calorimetry analysis. Twenty milligram samples of the raw

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materials, physical mixtures and FA-DEX-CoQ10 nanopreparations were added,

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respectively. The temperature range was 0–90°C, the heating rate was 5°C /min, and

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the crystal properties were detected by DSC.

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Stability Experiment. The lyophilized powder of the FA-DEX-CoQ10

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nanopreparation was reconstituted in water, kept at room temperature (25°C), and

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sampled at 6 h, 12 h, 1 d, 7 d, 1 month, 3 months, 6 months and 12 months,

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respectively. The MPS, used as an index, was determined via a laser particle size

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analyzer.

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Dissolution Test. Two milliliters of the raw materials and FA-DEX-CoQ10

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nanopreparation (equivalent to 20 mg of CoQ10) were sampled at 10, 20, 30, 60, 90,

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120, 180, 240, and 360 min, respectively. The samples were dissolved in 0.5% SDS

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solution, the speed was 100 r/min, and the temperature was 37°C. Passing through a

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0.22-µm filter membrane, and 20 µL of the filtrate was injected into high HPLC

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(Agilent 1260, Santa Clara, CA) for analyzing the amount of dissolved CoQ10 in each

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sample, at different time points. A KromasilTM C18 column (5 µm, 250 × 4.6 mm) was

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used for the separation of CoQ10. The mobile phase consisted of ethanol and n-hexane

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(10:90, V/V) at a flow rate of 1 mL/min, and the detection wavelength was 275 nm.31

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Bioavailability Study. A total of 24 male Wistar rats (280–320 g body weight)

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were randomized into two groups of 12 each. They were fasted for 12 h before oral

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administration. Two groups of male rats (n = 12) were orally administered

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(Containing 60 mg/kg of CoQ10). Seventy milligrams of heparin was dissolved in 7

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mL deionized water and mixed by sonication. Blood was collected by orbital puncture

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at 0, 0.5, 1, 2, 4, 8, 12, 24 h post-CoQ10 administration, placed in heparin tubes, and

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centrifuged immediately at 25 000 g for 10 min. The plasma samples were stored at

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−20°C until analysis.

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Two hundred microliters of each plasma sample were centrifuged at 9 000 g and

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collected the supernatant fluid. To precipitate proteins, 500 µL of methanol were

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added to the plasma samples, and the samples were vortexed for 5 min with 1 mL of

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n-hexane. After centrifugation at 9 000 g for 10 min, the organic layer was transferred

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to a clean tube and dried by nitrogen stream. The residue was re-dissolved in 100 µL

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of methanol, and 20 µL of the extract was subjected to HPLC analysis (method of

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analysis was the same as dissolution test).

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RESULTS AND DISCUSSION

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Single Factor Experimental Results. The reaction process of FA-DEX-CoQ10

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nanopreparation is shown in Figure 1. The following mass ratios of stabilizer to

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CoQ10 were investigated: 0.1:1, 0.2:1, 0.3:1, 0.4:1 and 0.5:1. Figure 2A shows the

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effect of the mass ratio of stabilizer to CoQ10 on MPS. For the minimum MPS, the

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mass ratio of stabilizer to CoQ10 was 0.4:1. However, proper addition of the

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stabilizers can also reduce the MPS. Therefore, the optimum condition of the mass

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ratio of stabilizer to CoQ10 was 0.4:1.

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Cycle time optimization experiments were carried out at 3, 6, 9, 12 and 15 times.

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Other optimized conditions, such as the mass ratio of CoQ10 to FA-DEX, the mass

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ratio of stabilizer to CoQ10, and the homogenization pressure, were 1:2, 0.4:1 and 200

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bar, respectively. Figure 2B shows that the MPS decreased gradually with increasing

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number of cycles from 3 to 6, and 365.1 nm was the minimum MPS at 6 cycles. When

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the cycle number was greater than 6, MPS increased at first and then decreased. The

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reason may be that the number of cycles was more than a certain range, which led to

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the agglomeration of FA-DEX-CoQ10 nanopreparation. Therefore, the optimal number

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of cycles was 6.

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The homogenization pressure had a significant effect on MPS. The following

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homogenization pressures were investigated: 200, 400, 600, 800 and 1000 bar. The

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mass ratio of CoQ10 to FA-DEX, the mass ratio of stabilizer to CoQ10, and the number

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of cycles was fixed at 1:2, 0.4:1 and 6 cycles, respectively. Figure 2C shows that the

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MPS decreased from 200–800 bar, and the amplitude of the change was relatively

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large. In contrast, the MPS was stable from 800–1000 bar. Energy consumption of the

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process was also taken into account, and therefore the optimal homogenization

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pressure was 800 bar.

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The mass ratio of CoQ10 to FA-DEX had a significant effect on MPS. The

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following mass ratios of CoQ10 to FA-DEX were investigated: 1:2, 1:6, 1:12, 1:18 and

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1:24. Figure 2D shows that MPS was 218.5 nm when the mass ratio of CoQ10 to

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FA-DEX was 1:2. For mass ratios between 1:12 and 1:24, MPS remained stable at

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109 nm. Considering both optimal MPS and minimal consumption of materials, 1:12

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was selected as the optimal mass ratio for CoQ10 to FA-DEX.

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Orthogonal Design of Experiment Results. Basing on single factor experiment

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the orthogonal experimental design L9(3)4 was used to optimize the MPS parameters

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of the FA-DEX-CoQ10 nanopreparation. The four factors (A, B, C and D) used in the

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orthogonal experiment are listed in Table 1. The results of 9 groups of experiments

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under different conditions are shown in Table 2. Under orthogonal conditions, the

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range of MPS was from 89.3 nm to 200 nm. The four factors affected MPS in the

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following order: the number of cycles > homogenization pressure > the mass ratio of

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CoQ10 to FA-DEX > the mass ratio of stabilizer to CoQ10 (B > C > D > A). Based on

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the experimental data, the optimal condition was the combination of A2B2C2D3. The

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optimized conditions were as follows: the mass ratio of stabilizer to CoQ10 was 0.4:1,

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the number of cycles was 6, the homogenization pressure was 800 bar, and the mass

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ratio of CoQ10 to FA-DEX was 1:18. Under the optimal conditions, the MPS measured

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by laser particle size analyzer was 87.6 nm.

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FA-DEX-CoQ10 Nanopreparation Characterization Analysis.

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SEM Analysis. Figure 3A and 3B show the SEM images of the raw materials and

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FA-DEX-CoQ10 nanopreparation, respectively. Comparing the two pictures, the MPS

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of the raw materials was larger (3–20 µm), and they were scattered and irregular plate

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crystals. The MPS of FA-DEX-CoQ10 nanopreparation was smaller (100–500 nm),

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and the particles were mainly spherical. According to Figure 3C, the numbers ranging

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from 200–300 nm were the most, and under 100 nm were the least.

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XRD Analysis. In order to further confirm the physical state, XRD was used to

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analyze possible changes in the internal structure of the CoQ10 sample. As shown in

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Figure 4A, the diffraction angles of the characteristic peaks of the raw materials,

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physical mixtures and FA-DEX-CoQ10 nanopreparation were basically unchanged.

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The strongest characteristic peak greatly decreased after nanocrystallization. This

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indicated that CoQ10 changed the crystal structure and showed an amorphous state

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with the decrease of MPS. The size of the diffraction peak represented the size of

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crystallinity, which indicated that the crystallinity of FA-DEX-CoQ10 nanopreparation

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decreased. A decrease in the crystallinity of the drug powder can increase the

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bioavailability of the drug.

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FT-IR Analysis. As shown in Figure 4B, saturated CH stretching vibration peaks

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(-CH3) appeared at 2963, 2964 and 2964 cm-1 in the raw materials, physical mixtures

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and FA-DEX-CoQ10 nanopreparation, respectively. Saturated CH stretching vibration

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peak (-CH2) appeared at 2911, 2910 and 2913 cm-1, respectively. The

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keto-characteristic peak (C = C) appeared at 1652, 1647 and 1652 cm-1, respectively.

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They all showed a saturated CO stretching vibration peak (-OCH3) at 2852 cm-1 and

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the out-of-plane bending vibration peak of CH (H-C-H deformation) appears at 1448

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cm-1.The infrared absorption peaks of the three were basically the same, indicating

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that the functional groups or chemical structures were same basically. But it can be

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seen from this figure that the FA-DEX-CoQ10 nanopreparation had an absorption peak

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at 3375 cm-1. Since the MPS of FA-DEX-CoQ10 nanopreparation was reduced and

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made the van der Waals force increase, the O-H telescopic vibration absorption peak

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was produced.

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DSC Analysis. As shown in Figure 4C, the endothermic DSC curve shows

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endothermic peaks for the raw materials, physical mixture, and the FA-DEX-CoQ10

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nanopreparation at 52°C, 52°C, and 50°C, respectively, indicating that the melting

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point of the FA-DEX-CoQ10 nanopreparation was lower than the other two samples.

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The melting point of the stable crystallization was high and the melting point of the

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metastable crystallizing was lower. The results also show that the degree of

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crystallization for the FA-DEX-CoQ10 nanopreparation was less than that of the other

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two samples, and FA-DEX-CoQ10 exhibited a more amorphous structure than the

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other samples. Stability Analysis. As can be seen from Table 3, lyophilized powder of FA-DEX-CoQ10 possesses long-term stability.

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Dissolution Analysis. Figure 5 shows the dissolution curves of the raw materials

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and FA-DEX-CoQ10 nanopreparation. According to Figure 5, the dissolution rate of

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FA-DEX-CoQ10 nanopreparation was higher than the raw materials. At 120 min, the

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dissolution rate of FA-DEX-CoQ10 nanopreparation reached 83%, and the raw

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materials were only 21%. Nanopreparation was 3.95 times as much as raw materials

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at the dissolution rate.

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directly affect its dissolution, and these factors are related to particle size. Therefore,

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as the size of the FA-DEX-CoQ10 nanopreparation decreased, the surface area

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increased, making it more soluble.

The solubility and specific surface area of a compound

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Bioavailability Analysis CoQ10. In vivo experiments in rats showed that the

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nanopreparation method could significantly improve the oral bioavailability of CoQ10.

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Figure 6 shows the plasma concentration of FA-DEX-CoQ10 was higher than the raw

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materials. According to pharmacokinetic parameters (Table 4), after oral

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administration, the concentration of CoQ10 from raw materials in plasma was very low,

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and the Cmax and AUC values were 0.823 µg/mL and 10.885 µg h/mL, respectively.

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The drug absorbed slowly to reach Tmax after approximately 4 h, and the CoQ10 from

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the nanopreparation was absorbed after approximately 2 h. By measuring and

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comparing the Cmax and AUC of the raw materials and FA-DEX-CoQ10

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nanopreparation, it was found that the Cmax and AUC of the FA-DEX-CoQ10

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nanopreparation significantly increased and were 2.7 times and 2.4 times as much as

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that of the raw materials, respectively. This indicates that the decrease in MPS could

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significantly improve the oral bioavailability of CoQ10.

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In conclusion, FA-DEX-CoQ10 nanopreparation was successfully prepared by

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high-pressure homogenization, thus improving the dissolution in vitro and oral

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bioavailability in vivo of CoQ10. Single factor and orthogonal experiments showed

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that 87.6 nm was the smallest MPS achieved under the optimized conditions: mass

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ratio of CoQ10 to FA-DEX of 1:18, mass ratio of stabilizer to CoQ10 of 0.4:1, 6 cycles,

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and homogeneous pressure of 800 bar. SEM results showed that FA-DEX-CoQ10

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nanopreparation were spherical. FTIR, XRD and DSC data revealed that the degree of

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crystallization of FA-DEX-CoQ10 nanopreparation was lower than that of the raw

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materials. Stability experiments revealed the FA-DEX-CoQ10 nanopreparation

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possesses long-term stability. In addition, the dissolution rate of FA-DEX-CoQ10

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nanopreparation in vitro, Cmax and AUC in rats were 3.95, 2.7 and 2.4 times as much

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as that of the raw materials, respectively. This study provides a simple method for the

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preparation of FA-DEX-CoQ10 nanopreparation, effectively improving the oral

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bioavailability of CoQ10.

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AUTHOR INFORMATION

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Corresponding Author

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* Tel.: +86-451-8219-1517. Fax: +86-451-8219-1517. E-mail:

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[email protected]

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ORCID

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Fansong Mu: 0000-0002-7645-2017

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Funding

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The authors gratefully acknowledge the financial supports by Youth Fund of National

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Natural Science Foundation of China (No. 21403032).

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Notes

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The authors declare that they have no competing interest.

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REFERENCES (1) Overvad, K.; Diamant, B.; Holm, L.; Holmer, G.; Mortensen, S. A.; Stender, S. Coenzyme Q10 in health and disease. Eur. J. Clin. Nutr. 1999, 53 (10), 764-770. (2) Crane, F. L. Biochemical functions of coenzyme Q10. J. Am. Coll. Nutr. 2001, 20, 591. (3) Luo, M.; Yang, X.; Hu, J. Y.; Ruan, X.; Mu, F. S.; Fu, Y. J. The synthesis of coenzyme Q10. Curr. Org. Chem. 2017, 21, 489-502. (4) Rauscher, F. M.; Sanders, R. A.; Watkins, J. B. Effects of coenzyme Q10 treatment on antioxidant pathways in normal and streptozotocin-induced diabetic rats. J. Biochem. Mol. Toxicol. 2001, 15 (1), 41-46. (5) Liu, H. T.; Huang, Y. C.; Cheng, S. B.; Huang, Y. T.; Lin, P. T. Effects of coenzyme Q10 supplementation on antioxidant capacity and inflammation in hepatocellular carcinoma patients after surgery: a randomized, placebo-controlled trial. Nutr. J. 2015, 15 (1), 85. (6) Zaki, M. E.; El-Bassyouni, H. T.; Tosson, A. M.; Youness, E.; Hussein, J. Coenzyme Q10 and pro-inflammatory markers in children with down syndrome: clinical and biochemical aspects. J. Pediatr. 2017, 93 (1), 100-104. (7) Liu, H. T.; Cheng, S. B.; Huang, Y. C.; Huang, Y. T.; Lin, P. T. Coenzyme Q10 Liu, and oxidative stress: inflammation status in hepatocellular carcinoma patients after surgery. Nutrients 2017, 9 (1), 29. (8) De Blasio, M. J.; Huynh, K.; Qin, C.; Rosli, S.; Kiriazis, H.; Ayer, A.; Cemerlang, N.; Stocker, R.; Du, X.J.; McMullen, J.R.; Ritchie, R.H. Therapeutic targeting of oxidative stress with coenzyme Q10 counteracts exaggerated diabetic cardiomyopathy in a mouse model of diabetes with diminished PI3K (p110α) signaling. Free Radic. Biol. Med. 2015, 87 (6), 137-147. (9) Mirmalek, S. A.; Boushehrinejad, A. G.; Yavari, H.; Kardeh, B.; Parsa, Y.; Salimi-Tabatabaee, S. A.; Yadollah-Damavandi, S.; Parsa, T.; Shahverdi, E.; Jangholi, E. Antioxidant and anti-inflammatory effects of coenzyme Q10 on L-arginine-induced acute pancreatitis in Rat. Oxid. Med. Cell Longev. 2016, 2016 (1), 1-8. (10) Yeung, C. K.; Billings, I. V.; Claessens, A. J.; Baback, R.; Lori, L.; Sundell, M. B.; Ahmad, S.; Shao, B.; Shen, D. D.; Ikizler, T. A.; Himmelfarb, J. Coenzyme Q10 dose-escalation study in hemodialysis patients: safety, tolerability, and effect on oxidative stress. BMC Nephrol. 2015, 16, 183. (11) Rundek, T.; Naini, A.; Sacco, R.; Coates, K.; DiMauro, S. Atorvastatin decreases the coenzyme Q10 level in the blood of patients at risk for cardiovascular disease and stroke. Arch. Neurol. 2004, 61 (6), 889-892. (12) Kumar, A.; Kaur, H.; Devi, P.; Mohan, V. Role of coenzyme Q10 (CoQ10) in cardiac disease, hypertension and meniere-like syndrome. Pharmacol. Ther. 2009, 124 (3), 259-268. (13) Pepe, S.; Marasco, S. F.; Haas, S. J.; Sheeran, F. L.; Krum, H.; Rosenfeldt, F. L. Coenzyme Q10 in cardiovascular disease. Mitochondrion. 2007, 7Suppl (2), S154-167.

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(14) Mancuso, M.; Orsucci, D.; Volpi, L.; Calsolaro, V.; Siciliano, G. Coenzyme Q10 in neuromuscular and neurodegenerative disorders. Curr. Medicine. Targets 2010, 11 (1), 111-121. (15) De la Mata, M.; Cotán, D.; Oropesa-Ávila, M.; Garrido-Maraver, J.; Cordero, M. D.; Villanueva Paz, M.; Delgado Pavón, A.; Alcocer-Gómez, E.; de Lavera, I.; Ybot-González, P.; Paula Zaderenko, A.; Ortiz Mellet, C.; García Fernández, J. M.; Sánchez-Alcázar, J. A. Pharmacological chaperones and coenzyme Q10 treatment improves mutant β-glucocerebrosidase activity and mitochondrial function in neuronopathic forms of gaucher disease. Sci. Rep. 2015, 5, 10903. (16) Spindler, M.; Beal, M. F.; Henchcliffe, C. Coenzyme Q10 effects in neurodegenerative disease. Neuropsychiatr. Dis. Treat. 2009, 5 (5), 597-610. (17) Borek, C. Anti-aging effects of coenzyme Q10. Agro Food Ind. Hi Tec. 2004, 15 (3), 24-25. (18) Premkumar, V. G.; Yuvaraj, S.; Vijayasarathy, K.; Gangadaran, S. G.; Sachdanandam, P. Effect of coenzyme Q10, riboflavin and niacin on serum CEA and CA 15-3 levels in breast cancer patients undergoing tamoxifen therapy. Biol. Pharm. Bull. 2007, 30 (2), 367-370. (19) Chai, W.; Cooney, R. V.; Franke, A. A.; Shvetsov, Y. B.; Caberto, C. P.; Wilkens, L. R.; Le Marchand, L.; Henderson, B. E.; Kolonel, L. N.; Goodman, M. T. Plasma coenzyme Q10 levels and postmenopausal breast cancer risk: the multiethnic cohort study. Cancer Epidemiol. Biomarkers Prev. 2010, 19 (9), 2351-2356. (20) Cooney, R. V.; Dai, Q.; Gao, Y. T.; Chow, W. H.; Franke, A. A.; Shu, X. O.; Li, H.; Ji, B.; Cai, Q.; Chai, W.; Zheng, W. Low plasma coenzyme Q(10) levels and breast cancer risk in Chinese women. Cancer Epidemiol. Biomarkers Prev. 2011, 20 (6), 1124-1130. (21) Xia, S.; Xu, S.; Zhang, X. Optimization in the preparation of coenzyme Q10 nanoliposomes. J. Agric. Food Chem. 2006, 57 (17), 6358-6366. (22) Li, Z.; Deng, Y. J.; Yang, J. W.; Li, B. Q.; Zhang, X. Y.; Lei, G. F. Pharmacokinetics and tissue distribution of coenzyme Q10 liposomes in Mice. Chin. J. Pharm. 2006, 4 (4), 167-172. (23) Olsen, S.; Doney, J. A.; Shores, C. Benzoquinones of enhanced. Bioavaliability. U.S. Patent 20070026072, February 1, 2007. (24) Kommuru, T. R.; Gurley, B.; Khan, M. A.; Reddy, I. K. Self-emulsifying medicine delivery systems (SEDDS) of coenzyme Q10: formulation development and bioavailability assessment. Int. J. Pharm. 2001, 212 (2), 233-246. (25) Balakrishnan, P.; Lee, B. J.; Oh, D. H.; Kim, J. O.; Lee, Y. I.; Kim, D. D.; Jee, J. P.; Lee, Y. B.; Woo, J. S.; Yong, C. S.; Choi, H. G. Enhanced oral bioavailability of coenzyme Q10 by self-emulsifying medicine delivery systems. Int. J. Pharm. 2009, 374 (1-2), 66-72. (26) Ozaki, A.; Muromachi, A.; Sumi, M.; Sakai, Y.; Morishita, K.; Okamoto, T. Emulsification of coenzyme Q10 using gum arabic increases bioavailability in rats and human and improves food-processing suitability. J. Nutr. Sci. Vitaminol. 2010, 56 (1),

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41-47. (27) Sato, Y.; Mutoh, H.; Suzuki, M.; Takekuma, Y.; Iseki, K.; Sugawara, M. Emulsification using highly hydrophilic stabilizers improves the absorption of orally administered coenzyme Q10. Biol. Pharm. Bull. 2013, 36 (12), 2012-2017. (28) Cheuk, S. Y.; Shih, F. F.; Champagne, E. T.; Daigle, K. W.; Patindol, J. A.; Mattison, C. P.; Boue, S. M. Nano-encapsulation of coenzyme Q10 using octenyl succinic anhydride modified starch. Food Chem. 2015, 174, 585-590. (29) Zhao, Q.; Ho, C. T.; Huang, Q. Effect of ubiquinol-10 on citral stability and off-flavor formation in oil-in-water (O/W) nanoemulsions. J. Agric. Food Chem. 2013, 61 (31), 7462-7469. (30) Hao, H.; Ma, Q.; Huang, C.; He, F.; Yao, P. Preparation, characterization, and in vivo evaluation of doxorubicin loaded BSA nanoparticles with folic acid modified dextran surface. Int. J. Pharm. 2013, 444 (1-2), 77-84. (31) Sui, X. Y.; Liu, C.; Wang, J.; Yuan, C.; Ma, X. X.; Liu, T. T.; Han, C. Y. The preparation and properties of the coenzyme Q10-γ-cyclodextrin nanocrystal suspension. Chin. J. Pharm. 2015, 50, 1412-1418.

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392 393

Figure 1. The synthesis of FA-DEX-CoQ10 nanopreparation.

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Figure 2. The effect of each factor on the MPS of FA-DEX-CoQ10 nanopreparation: (A) mass ratio of stabilizer to CoQ10, (B) number of cycles, (C) pressure and (D) mass ratio of CoQ10 to FA-DEX. * P < 0.05, ** P < 0.01.

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402 403 A 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418

B 3.0 2.5 2.0

Number

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1.5 1.0 0.5 0.0

0

100

200

300

400

Particle size (nm)

Figure 3. SEM images: (A) raw materials, (B) FA-DEX-CoQ10 nanopreparation and (C) particle size distribution.

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500

600

Journal of Agricultural and Food Chemistry

Intensity

A

a

b

c 0

5

10

15

20

25

30

35

40

45

50

55

2θ (degrees)

B T (%)

a

b

Dissolve

a

c

a 5000

4000

3000

2000

1000

c

c

0

-1

Wavenumber(cm )

C DSC(mW)

419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444

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a

b

c 30

40

50 60 Temperatue(°C)

70

80

90

Figure 4. (A) XRD, (B) FT-IR and (C) DSC curves: (a) raw materials, (b) physical mixtures and (c) FA-DEX-CoQ10 nanopreparation.

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445

100

A

Dissolved(%)

80

60

40

B

20

0

50

100

150

200

250

300

350

400

Time(min)

446 447 448 449 450 451

Figure 5. Dissolution curves: (A) FA-DEX-CoQ10 nanopreparation and (B) raw materials.

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452

2.5

Plasma concentration(µg/ml)

2.0

1.5

1.0

A 0.5

B 0

5

10

15

20

25

Time(h)

453 454 455 456 457 458 459

Figure 6. Mean plasma concentration - time curves: (A) FA-DEX-CoQ10 nanopreparation and (B) raw materials. Table 1. Orthogonal test parameters and conditions. Parameter

Condition 1

Condition 2

Condition 3

0.3:1

0.4:1

0.5:1

3

6

9

600

800

1000

1:06

1:12

1:18

Mass ratio

A

of stabilizer and CoQ10

B C

Cycle times Pressure (bar) Mass ratio

D

of CoQ10 and FA-DEX

460 461

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Table 2. Orthogonal experiment design L9(3)4 and results Experiment number A

B

C

D

MPS (nm)

1

1

1

1

1

200

2

1

2

2

2

104.1

3

1

3

3

3

104.3

4

2

1

2

3

145

5

2

2

3

1

89.3

6

2

3

1

2

134

7

3

1

3

2

122.4

8

3

2

1

3

124.9

9

3

4

2

1

135.6

K1

408.4

467.4

459.1

424.9

K2

368.5

318.3

384.7

360.5

K3

382.9

373.9

316

374.2

1

k

136.1

155.8

153

141.6

k2

122.8

106.1

128.2

120.2

k3

127.6

124.6

105.3

124.7

R

13.3

49.7

47.7

21.4

O

A2

B2

C2

D3

Note: O represents the optimum condition (P < 0.05).

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Table 3. The stability of FA-DEX-CoQ10 nanopreparation at Ddifferent Times Time

6h

12 h

1d

7d

One month

Three

Six months

months MPS (nm)

87.5

88.3

89.5

90.1

94.7

469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505

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Twelve months

97.3

104.1

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Table 4. Pharmacokinetic Parameters of CoQ10 (60 mg/kg) by Oral Administration

Raw materials FA-DEX-CoQ10 nanopreparation

Tmax(h)

AUC(µg h/mL)

Cmax(µg/mL)

4

10.885

0.823

2

26.722

2.252

509 510

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