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Preparation optimization and protective effect on 60Co-γ radiation damage of Pinus koraiensis pinecone polyphenols microspheres
Accepted Manuscript Preparation optimization and protective effect on 60Co-γ radiation damage of Pinus koraiensis pinecone polyphenols microspheres
Juanjuan Yi, Cuilin Cheng, Shubin Li, Dong Wang, Lu Wang, Zhenyu Wang PII: DOI: Reference:
S0141-8130(17)33530-4 doi:10.1016/j.ijbiomac.2018.02.131 BIOMAC 9188
To appear in: Received date: Revised date: Accepted date:
16 September 2017 14 January 2018 20 February 2018
Please cite this article as: Juanjuan Yi, Cuilin Cheng, Shubin Li, Dong Wang, Lu Wang, Zhenyu Wang , Preparation optimization and protective effect on 60Co-γ radiation damage of Pinus koraiensis pinecone polyphenols microspheres. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.02.131
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PREPARATION OPTIMIZATION AND PROTECTIVE EFFECT ON 60
Co-γ RADIATION DAMAGE OF PINUS KORAIENSIS PINECONE POLYPHENOLS MICROSPHERES
Juanjuan Yia,b, Cuilin Cheng a, Shubin Lia, Dong Wanga, Lu Wang a*, Zhenyu Wang a* Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin 150090, PR China
b
School of Life Sciences, Zhengzhou University, Zhengzhou, Henan, China
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a
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*Corresponding author: Lu Wang and Zhenyu Wang.
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Postal address: School of Chemical Engineering and Chemistry, Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin, 150090, PR China.
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E-mail address: [email protected] (Lu Wang).
E-mail address: [email protected] (Zhenyu Wang).
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Tell numbers: +86-0451-86282909, Fax numbers: +86-0451-86282909.
Nonstandard Abbreviations: Pinus koraiensis: P. koraiensis; pinecones polyphenols microspheres:
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PPM; Response surface methodology: RSM; Box-Behnken design: BBD;.Glutaraldehyde: GA; Scanning electron microscopy: SEM; Superoxide dismutase: SOD; Malondialdehyde: MDA;
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Phagocytic index: PI.
ABSTRACT: Here, the chitosan and the glutaraldehyde (GA) were used to encapsulate pinecones
polyphenols of Pinus koraiensis (P. koraiensis) by emulsification cross-linking technology. First, the prepared parameters (crosslinking agent amount, stirring speed, crosslinking temperature and emulsifying time) of the pinecones polyphenols microspheres (PPMs) were optimized by the response
ACCEPTED MANUSCRIPT 2 surface methodology (RSM). When chitosan concentration and crosslinking time were 2 % and 80 min, respectively, the optimal conditions were 7.91 mL of crosslinking agent, stirring speed of 660.98 r/min, crosslinking temperature of 41.18 °C and emulsifying time of 198.65 min. The prepared PPMs embedding rate was 73.57 %. The optimized PPM possessed a distinct core-shell structure and uniform
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spherical distribution with a particle size value of 3.4 μm. In addition, they had the excellent
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sustained-release characteristics in vitro. We also evaluated the radioprotective effects of PPMs against 60
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Co-γ radiation in vivo. PPMs improved significantly the activity of the antioxidant enzyme SOD and
reduce MDA level in the plasma of irradiated mice. Accordingly, PPMs could also significantly
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enhance the immunomodulation activity by promoting the proliferation of splenocytes and monocyte
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phagocytosis of irradiated mice. These results suggested that PPMs exert effective protection against radiation-induced injury by improving the antioxidant and immunomodulation activities.
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Key Words: Pinecone polyphenols of Pinus koraiensis; Microspheres preparation; Optimization;
1. Introduction
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Sustained-release characteristics; Radiation protection.
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In recent years, more attention has been paid plant polyphenols due to their multiple biological and pharmacological activities [1-4]. P. koraiensis pinecone was a byproduct in the processing of P. koraiensis seeds. Our previous studies have been proved that P. koraiensis pinecone is a rich source of polyphenols [5] and possesses strong anti-tumor activities in vitro [6] and in vivo [7], immunoregulation activity in vivo [8], antioxidant activity in vitro [9], and radioprotective effect [10]. The polyhydroxy structural characteristics of polyphenols played an important role in their biological effects [11]. However, further research has also found that the polyhydroxy structures of plant polyphenols exposed to high temperature, oxygen, extreme pH and other harmful environment can be
ACCEPTED MANUSCRIPT 3 easily oxidized and degraded, which could lead to functional composition degradation, ineffective dose concentrations and decreased bioactivity [12]. To improve the stability and utilization of polyphenols, many research efforts are ongoing [13-15]. Drug delivery systems (DDS) based on macromolecule polymers have been shown to improve the stability and release-control of the drugs [16]. Microspheres, as multiple-unit DDS for oral drugs,
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possess multiple significant and competitive benefits, such as more uniform distribution, good
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absorption, enhancement of drug stability, and thermodynamic stability against denaturation and degradation of polyphenols [17]. The study also has reported that microsphere technique could resolve
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the stability and sustained drug delivery problems of naturally active substances [18].
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Chitosan [α(1,4)-2-amino-2-deoxy-β-D-glucan, CS] is a type of natural carrier material applied in DDS and the food industry, it not only offers good biodegradability and multiple biological activities but
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also plays a protective role and could sustain release of the core drug [19-20]. Many methods, such as spray drying, ionotropic gelation, coacervation, solvent evaporation and crosslinking, have been
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developed for preparation of chitosan microspheres. Preparation of chitosan microspheres
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(CS-microspheres) by the traditional spray drying method is easy to perform, but it is difficult to encapsulate the drug in the polymer matrix. In the ionotropic gelation and coacervation processes, the
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pH of the solution must be strictly controlled, and the morphology and size of the microspheres are generally nonuniform [21-22]. In the method of solvent evaporation, organic solvent evaporation
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processes may reduce the bioavailability of the drug and destroy the biocompatibility of the microspheres [23]. Compared to the methods mentioned above, the crosslinking method has strong potential in the development of polymer microspheres to overcome the above defects, while providing narrow particle size distributions and good morphology microsphere, which prolongs the retention time frame and controls the release behavior of the active agent [24]. CS-microspheres crosslinked with chemical crosslinkers, including glutaraldehyde, ethylene glycol, diglycidyl ether, tripolyphosphate and disulfide, have been reported to provide favorable stability and a controlled release ability for many drugs [25-26]. Regarding the controlled release ability, it has been reported that the release of the active
ACCEPTED MANUSCRIPT 4 agent from CS-microspheres generally occurs in three different ways: (a) release from the surface of microspheres due to its porous structure, (b) diffusion resulting from swelling, and (c) release due to the degradation of the polymer [24]. Therefore, release is susceptible to pH, polarity of the release medium, and the activity of enzymes. Currently, the CS-microsphere technique has been widely applied in the biological, food and
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medical fields [27-29]. Cho et al prepared the resveratrol microspheres using high and medium
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molecular weight chitosan to improve resveratrol bioavailability [30]. In another study, resveratrol was encapsulated into chitosan microspheres for improving the stability and evaluating the ability of
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controlled release [31]. Park et al. investigated and characterized chitosan microspheres for anti-cancer
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bioactivity [32].
There have been no studies on the anti-radiation activity of chitosan-loaded-polyphenol
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microspheres. Therefore, the main purposes of this study were to prepare and optimize a novel chitosan-supported P. koraiensis pinecone polyphenol microsphere (PPM) by RSM to improve the
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radiation protection effect of a core drug (pinecone polyphenols) in mice. In this study, chitosan is a type
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of drug carrier, pine polyphenols are embedded, and an anti-radiation drug system is prepared, all of which enhance the effectivity of the drug system. This is a new approach in this field and has a strong
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practical significance for the research of anti-radiation drugs.
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2. Materials and methods 2.1 Materials
The dried pinecones of P. koraiensis were provided by the Forestry Bureau (Yichun, China). The chitosan (average molecular weight [MW] of 100 kDa and deacetylation degree of 92 %) was provided from sigma. The superoxide dismutase (SOD), malondialdehyde (MDA) and protein quantization measurement kits were purchased form Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Roswell Park Memorial Institute 1640 (RPMI-1640) and Fetal Bovine Serum (FBS) were provided
ACCEPTED MANUSCRIPT 5 from Hyelone Chemical Co., USA. All other chemicals were of analytical grade and purchased from local suppliers.
2.2 Extraction and separation of PP
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2.2.1 Extraction procedure The dried pinecones of P. koraiensis were smashed and passed through the 30 mesh sieves. The
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pinecones powders were added in the 60 % ethanol (1:30 g/mL), and carried on ultrasonic treatment at
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50 ℃ and 400 W for 30 min, then repeated extraction above twice. The extracts were combined and separated by filtration, then centrifuged at 4000 rpm for 10 min. The supernatant was concentrated by
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rotary evaporator for crude pinecones polyphenols (PPs).
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2.2.2 Separation procedure
In order to obtain higher purity, the separation of crude PPs extracts was carried out in glass
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columns packed with pretreated X-5 macroporous resins. The eluent concentration (ethanol-water) was
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60 %. Operating conditions were as follows: the concentration of crude PP: 2.5 mg/ mL, flow rate, 4.0 mL/min; collected volume: 2 bed volumes. Finally, the collected component was concentrated by a
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rotary evaporator to remove the ethanol. The purity of collected polyphenols was 40.16 % according to
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the Folin-Ciocalteau method.
2.3 Preparation of PPM Pinecone polyphenol microspheres were prepared using an emulsification and crosslinking method and slightly modified by reference to Patel et al [33]. Chitosans (50 mg) and the pinecone polyphenols (5 mg) were homogeneously dissolved in 3 % acetic acid solution (5 mL) by magnetic stirring for 2 hours and ultrasound treatment for 30 min. Then, the obtained mixed solution (5 mL) was dispersed by stirring at different stirring speeds (500-1000 r/min) into a continuous oil phase (60 mL) consisting of
ACCEPTED MANUSCRIPT 6 liquid paraffin and 2 % span80 (V:V, 30:1) at different crosslinking temperature (20-70 °C) for different emulsifying times (40-240 min) to form a water-in-oil micro-emulsion. Then, different amounts (2-12 mL) of 25 % crosslinking agent glutaraldehyde (GA) was added into the beaker under stirring at 600 r/min. The crosslinking reaction was allowed to proceed for a total of 1 h. Hardened microspheres were
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filtered and washed, first with distilled water to remove the unreacted
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polyphenols and residual CS. Then, the product was washed repeatedly with petroleum ether and
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anhydrous ethanol to remove liquid paraffin and unreacted GA, dried under a vacuum at 40 °C overnight
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2.4 Calculation of the embeding rate
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and kept in a desiccator until further use.
PPMs were dialyzed against the distilled water, which were shaken and centrifuged at 15000 r/min
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for 30 min at 4 ℃. The contents of the free polyphenols of the supernatant were determined according to
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Folin-reagent method [5]. The embeding rate of PPMs was calculated as follows[33]:
(1)
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E D CV D 100
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Where E is the embeding rate (%); D is the amount of total polyphenols (mg); C is the . concentration of the polyphenols of PPMs solution (mg GAE/mL), and V is the volume of PPMs solution (mL).
2.5 Experimental design and statistical analysis The single-test was used to determine the preliminary range of prepared parameters. Then, BBD was employed to optimize four independent variables (crosslinking agent amount X1, stirring speed X2, crosslinking temperature X3 and emulsifying time X4) for the preparation of PPMs. It employed 29
ACCEPTED MANUSCRIPT 7 experiments to optimize the prepared parameters of PPMs. Table 1s showed the four levels of these independent variables. The embeding rate (Y) was taken as a response for the design experiment in Table 1. The behavior of the system was explained by Equation (2):
Y A0
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Ai X i
Aii X i2
i 1
2
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Aij X i X j i j i 1
(2)
1
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i 1
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Where Y is the dependent variable, A0 is constant, and Ai, Aii, and Aij are coefficients estimated by
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model. Xi and Xj are levels of the independent variables while A0, Ai, Aii, and Aij are the regression
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2.6 Scanning electron microscopy (SEM)
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coefficients for the intercept, linearity, square, and interaction, respectively.
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The shape and surface morphology of PPMs were investigated using SEM. PPMs were coated
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with gold palladium under an argon atmosphere for 150 s to achieve a 20 nm film (sputter coater,
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SCD004, BAL-TEC Balzers, Furstentum, Lienchestein). The coated samples were examined in a scanning electron microscope (Jeol JSM-1600, Tokyo, Japan).
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2.7 Measurement of particle size distribution
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The particle size of PPM was analyzed by laser scattering particle size analyzer. Briefly, the PPMs were diluted with the distilled water, placed in a 1cm absorption dish and determined at 25 °C.
2.8 In vitro stimulated gastrointestinal digestion of PPMs The digestion of PPMs was investigated according to the method reported by Thakkar et al. with some modifications [34]. The stimulated gastric fluid (50 mL, pH 1.2) and stimulated intestinal fluid was separately put into two 250 mL beakers. During the digestion process, the same mass of PPMs were installed into two dialysis bags. Then, two dialysis bags were separately incubated in 50 mL of different incubation mediums (the stimulated gastric fluid, the stimulated intestinal fluid) at 37 °C, and
ACCEPTED MANUSCRIPT 8 gently shaken at oscillation speeds of 80 r/min. At predetermined time intervals, 5 mL of the different incubation mediums were separately collected to calculate the released amounts of polyphenols from PPMs. Meanwhile, 5 mL of fresh medium was compensated.
2.9 In vivo protective effect on 60Co-γ radiation damage of PPMs
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2.9. 1 Animal
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Male KM mice of SPF-level (6 – 8 weeks old, 18 – 20 g each) were housed in a mouse room at
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temperature (22 ± 2 °C), light (12 h light/dark cycles) and humidity (50 ± 10 %) and were provided with rodent laboratory chow pellets and tap water for a week to adapt to the environment of mouse
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room. The experimental protocol was approved by Institutional Animal Ethical committee.
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2.9.2 Groups and irradiation
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Mice were divided into eight groups of 20 animals each. Pinecone polyphenols (PPs, 25, 50 and
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100 mg/kg bwt/d) and pinecone polyphenol microspheres (PPMs, 25, 50 and 100 mg/kg bwt/d the encapsulated polyphenols) were separately administered to the mice by an oral probe in their water
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suspension for 14 consecutive days prior to irradiation. The normal control group and radiation model
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group were given an equal volume of normal saline every day. Irradiation was performed at a dose rate of 1.0 Gy/min using a
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Co irradiator (Heilongjiang Academy of Agricultural Sciences, China). The
irradiation dose was 6 Gy. After 24 h, the mice were sacrificed by cervical dislocation, and their organs, including the thymus, heart, liver and spleen, were separately excised and weighed to calculate the organ indexes.
2.9. 3 Antioxidant status assessment The activity of SOD and the content of MDA in the plasma were determined using commercial kits
ACCEPTED MANUSCRIPT 9 (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The SOD activity was expressed in U/mL, the content of MDA was expressed in nmol/mL.
2.9. 4 Assay of splenocyte proliferation in vivo
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After the mice sacrificed, the spleens collected from mice under aseptic conditions were grinded into small pieces and passed through sterilized meshes to obtain a homogeneous cell suspension at the
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room temperature. The red blood cells were removed by red blood cell lysis solution for 5 min.
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Recovered splenocytes were washed twice, then re-suspended in RMPI-1640 complete medium
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containing 5 % FBS [35-36]. Cells were seeded in a 96-well plate with or without Con A (7.5 μg/mL). After incubation for 48 h at 37 °C in a humidified 5 % CO2 incubator, the number of cells was
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determined by MTT assay using a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA).
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2.9. 5 Phagocytosis of monocyte assay
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After 14 days of oral administration, 25 % (v/v) India ink according to 0.2 mL/kg body weight was injected by a tail intravenous injection. A total of 20 μL of blood was collected through eye orbit after 2
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min (t1) and 10 min (t2), and added to 2 mL 0.1 % Na2CO3. The absorbance at 600 nm of blood after 2
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min (A1) and 10 min (A2) were measured, and the absorbance of normal control group of blood was set as zero. The mice were sacrificed by decapitation, and then the liver and spleen were weighed. Clearance index (K) and phagocytic index (α) were calculated as follows [37]:
K=
lg A1 -lgA 2 t2 -t1
2.10 Statistical analysis
α= K1/3 × body weight/(liver weight + spleen weight)
(3)
ACCEPTED MANUSCRIPT 10 All statistical analyses employed SPSS for Windows, Version 18.0. Data were expressed as means ± standard deviation (SD) of three independent measurements. Differences at p < 0.05 and p < 0.01 were considered statistically significant by Duncan’s new multiple-range test.
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3. Results and discussion
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3.1.1 Effects of crosslinking agent (GA) amount
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3.1 Effects of different parameters on embedding rate
GA, a crosslinking agent for the crosslinking reaction, was necessary for the preparation of
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chitosan microspheres due to the viscosity [38]. The effects of the GA contents on the embedding rate
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and the surface morphology of PPMs are shown in Fig. 1A and Fig. S1, respectively. The embedding rate of PPM exhibited an upward trend with increased GA contents. When the volume of GA was 8 mL,
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the embedding rate almost reached a maximum (78.7 %), and the particle size was smaller. This is
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possibly due to increase in the volume of crosslinking agent, the formation of more rigid polymer network and the shrinkage of particles, which might have decreased the particle size[39]. Subsequently,
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as the volume of GA increased, the embedding rate exhibited a fast downward trend. This was due to
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higher degrees of crosslinking, microspheres that are denser and a decrease in the free volume space within the matrix, resulting in reduced embedding rates[40]. Therefore, 0.8 mL was selected as the optimal volume of GA in the present study.
3.1.2 Effects of stirring speed High-speed stirring diminishes the droplet sizes based on turbulent flow and hydrodynamic shear stress. The emulsified droplets were seriously ruptured by the powerful shear force produced by the high-speed homogenizer [41]. Fig. 1B shows the effect of stirring speed on the embedding rate of PPMs.
ACCEPTED MANUSCRIPT 11 With the increase in the stirring speed (500–1000 r/min), the embedding rate increased first and then decreased, peaking with 82.1 % of the embedding rate at 700 r/min. However, compared with 700 r/min, the sphere is relatively smaller and better at 800 r/min (Fig. S2). To consider the comprehensive impacts of the stirring speed on the PPMs, 700 r/min was chosen as the best stirring speed for the
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preparation of PPMs.
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3.1.3 Effects of crosslinking temperature
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The crosslinking temperature is a decisive factor for the optimization of the embedding rate of PPM.
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Upon increasing the crosslinking temperature from 20 to 40 °C, the embedding rates of PPM increased from 59.8 % to 78.5 % (Fig. 1C), which may be attributed to the increase in temperature resulting in
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decreasing the viscosity of the dispersion system to obtain smaller sized particles [42]. However, with increasing crosslinking temperatures from 40 to 70 °C, the embedding rate later decreased slowly. The
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optimal embedding rate and morphology of PPMs were observed (Fig. S3) at a crosslinking temperature of 40 °C.
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3.1.4 Effects of emulsifying time
In the present study, it was necessary to determine the required emulsifying time and its effects on
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microsphere characteristics, including the embedding rate and the morphology. The effects of the emulsifying time on the embedding rate of PPMs were shown in Fig. 1D. A higher embedding rate was obtained with stirring for 200 min compared to other times. However, it was clear that the lowest embedding rate was obtained on emulsifying for 240 min. This may be due to the long stirring responsible for some more polyphenols to be leached out in the oil phase during the emulsification process. Moreover, smooth and uniform spherical distributions of PPMs were found at 200 min reaction time (Fig. S4). Therefore, the emulsifying time at 200 min is sufficient for producing the
ACCEPTED MANUSCRIPT 12 required microspheres of the high embedding rate and stable morphology.
3.2 Optimization of preparation parameters for PPM 3.2.1 Statistical analysis and the model fitting
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There were a total of 29 runs used to optimize the four individual parameters (crosslinking agent
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amount, stirring speed, crosslinking temperature and emulsifying time) to analyze the impacts of the independent variables on the embedding rate (Y) in the current BBD, as shown in Table 2. The model for
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the response variable could be expressed by the following quadratic polynomial equation in the form of
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coded values:
Y 74.2 0.89 X 1 0.73 X 2 0.97 X 3 0.21X 4 0.97 X 1 X 2 1.25 X 1 X 3 0.045 X 1 X 4 0.39 X 2 X 3 0.34 X 2 X 4 1.83 X 3 X 4 2.63 X 1 3.09 X 2 1.41X 3 1.95 X 4 2
2
2
(4)
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2
Where Y is the embedding rate of PPMs, and X1, X2, X3 and X4 are the coded variables for
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crosslinking agent amount (mL), stirring speed (r/min), crosslinking temperature (°C) and emulsifying
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time (min), respectively.
The analysis of variance (ANOVA) was performed for testing the significance of the fitted
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quadratic polynomial, which was obtained by the experimental data, as demonstrated in Table 3. The determination coefficient R2 was 0.9708, and it was suggested that 97.08 % of the variations could be
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illustrated by the fit model. The correlation between the experimental and predicted values can be reflected by the value of Radj 2, which should be closer to R2 in a well statistical model [43]. As shown in Table 3, the value of Radj2 was 0.9416 and was close to R2, which revealed that the model was highly significant. At the same time, the coefficient of variation (CV =0.81 %) was a relatively small value, which indicated a better reliability of the experimental values. The non-significant lack-of-fit for all the variables revealed that the polynomial model was statistically accurate for the responses [44]. Moreover, the significance of each coefficient was also checked by the P-value. Table 4 showed that the
ACCEPTED MANUSCRIPT 13 cross-product coefficients (X1X2, X1X3 and X3X4) were very significant with small P-values (p < 0.01). These results suggested the model could be used to predict these responses.
3.2.2 Effects of independent variables on embedding rate The 3D surface plots, which provide a method to visualize the relationship between responses and experimental levels of each variable [45], are shown in Fig. 2. Fig. 2A shows the effects of crosslinking
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agent amount and stirring speed on the embedding rate. When the stirring speed was fixed, the
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embedding rate increased with the increase of crosslinking agent amount until reaching a maximum and
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then decreased. Similarly, the increase in stirring speed at a fixed crosslinking agent amount led to an increase of the embedding rate and nearly reached a peak at a certain extent and then decreased. Fig. 2B
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shows the plot of embedding rate as affected by crosslinking temperature and crosslinking agent amount, which demonstrated a marked increase in embedding rate with the crosslinking temperature at a
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fixed crosslinking agent amount and then decreased. Additionally, an increase in crosslinking agent amount at a fixed crosslinking temperature led to an increase in the embedding rate and then decreased.
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Similarly, the effects of emulsifying time and crosslinking agent amount on the embedding rate (Fig.
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2C), which further illustrated that the embedding rate was positively affected by the crosslinking agent amount. However, the increase of viscosity could also cause the coalescence of the droplets that finally
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resulted in the decreased embedding rate. Fig. 2D and Fig. 2E respectively show the interactive effects of stirring speed with crosslinking temperature and emulsifying time on the embedding rate of PPMs,
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which further illustrated that the embedding rate was affected by GA content. With the increasing of the stirring speed, the embedding rate increased due to the high shear stress, which could rupture the viscous droplets and facilitate the package process [33]. Up to a certain level, the embedding rate started to decrease because the excessively high speed with the powerful energy could lead to the breaking down of resultant microspheres. Fig. 2F also shows that the interaction of the crosslinking temperature and the emulsifying time were demonstrated significantly on the embedding rate.
3.2.3 Optimal preparation conditions for PPMs
ACCEPTED MANUSCRIPT 14 The optimal values of embeding rate of PPMs for the independent variables were respectively carried out by solving the regression equations using Design-Expert 7.0 software. Through the comprehensive consideration with maximum desirability, the predicted optimal conditions for the preparation of PPMs were as follows: crosslinking agent amount of 7.19 mL, stirring speed of 660.98 r/min, crosslinking temperature of 41.18 ℃ and emulsifying time of 198.65 min. Under the optimal
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conditions, the embeding rate of PPMs was 73.57 %. These results confirmed that the model can be used
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to optimize the preparation parameters of PPMs.
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3.3 Morphology observation and particle size distribution of PPMs
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In the present work, the crosslinked polyphenols microspheres were successfully prepared by the
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emulsification and crosslinking method in response surface methodology. The shape and surface morphology of the PPM was investigated using scanning electron microscopy. The particle size
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distribution of the optimal PPM was measured by a laser particle size analyzer. The effect of the
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formulation variables on the morphology change and particle size of the PPM is shown in Fig. 3 and Fig. 4. The optimal microspheres had a smoother surface and were found to be discreet and spherical
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in shape with a stable form and uniform size (Fig. 3B). Compared with Fig. 3A, the embedding rate of
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the polyphenols almost reached a maximum with a value of 73.57 %, and the particle size was smaller with a uniform microsphere volume (Fig. 3B). Fig. 4 shows that the average particle size distribution was approximately 3.4 μm, which indicated that the microspheres were successfully prepared.
3.4 In vitro stimulated gastrointestinal digestion of the PPMs The release of polyphenols from the optimal microspheres was monitored using a two-model system: the stimulated gastric fluid and stimulated intestinal fluid were used as release media. Fig. 5 shows the release profiles of pinecone polyphenols from microspheres in the two-model medium. In
ACCEPTED MANUSCRIPT 15 general, there was an initial rapid increase in the amount of polyphenols released, followed by a more gradual release until a constant level was reached. Overall, the amount of polyphenols released was higher and the release rate was faster in the stimulated intestinal fluid compared with the stimulated gastric fluid, which can be attributed to the relative stability of the microspheres in the acid
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environment. This tendency was verified by many studies [46-47]. These results suggested that PPM
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had excellent sustained-release characteristic in the simulated gastric fluid and fast release
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characteristic in the stimulated intestinal fluid.
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3.5 Effects of PPMs on organ indexes
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Ionizing radiation is an electromagnetic wave or particle capable of producing ions and attacking living tissues and organs, which could cause multiple organ dysfunction and injury of biological
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systems, including immunomodulatory, hematopoietic, or liver systems in the human body [48-49].
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As shown in Table 5, compared with the normal control group, radiation resulted in multiple organ index declines, which indicated that ionizing radiation damaged the normal organs and affected their
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functions. Some in vitro and in vivo studies have confirmed that phenolic compounds are
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radioprotectors and have potentially radio-protective effects without obvious side effects [50-52]. Similarly, in the present study, the pinecone polyphenols also showed promising potential in improving organs indexes and preventing organ radiation damage. However, the pinecone polyphenols exposed in the aerobic and acidic environment can be easily oxidized and degraded, which results in a decrease of bioactivity. The preparation of microspheres improved the absorption efficiency and utilization rate of polyphenols in vivo. Table 5 showed that the pretreatment of PPMs improved the organ indexes most effectively, which initially indicated that PPMs possessed radiation protection function.
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3.6 Effects of PPMs on antioxidant status Endogenous antioxidant enzymes are considered to be the first line of defense in maintaining a redox balance and normal biochemical processes [53]. Homeostatic cellular functions require tight control of the redox environment [54]. However, radiation could attenuate the endogenous antioxidant
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enzymes to break the redox balance [53]. Therefore, agents that can repair and adjust the
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oxidation-reduction system have received extensive attention. In the present study, as shown in Fig. 6
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and Fig. 7, compared with the normal control group, there was a significant reduction in antioxidant enzyme SOD content and prominent enhancement of MDA level that were clearly observed in the
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model control group (p < 0.01). However, after the administrations of PPs (50 and 100 mg/kg) and
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PPMs (25, 50 and 100 mg/kg), the SOD contents were significantly increased (p < 0.05). In contrast, the MDA levels in plasma were significantly reduced (p < 0.05 or p < 0.01). The results above showed
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that the administrations of PPs and PPMs could effectively increase the contents of antioxidant
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enzymes and decrease the level of MDA in the irradiated mice.
3.7 Effect of PPMs on splenocytes proliferation
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The spleen is the largest immune organ in the vertebrate body, capable of producing a large
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number of lymphocytes [55]. T-lymphocyte is an important immunological active cell and plays an important role in enhancing the immune function of organisms [56-57]. The data showed that PPMs (25, 50 and 100 mg/kg) could enhance specific immune response by exhibiting significant comitogenic activity in Con A-induced splenocytes proliferation, with the better effect than the PP groups (Fig.10). The splenocytes proliferation response was related to immunity improvement of T-lymphocytes or B-lymphocytes [58]. The results indicated that PPMs possessed a definite and clear synergistic action on splenocytes proliferation after combining with ConA.
3.8 Effect of PPMs on phagocytosis of monocyte
ACCEPTED MANUSCRIPT 17 Monocyte plays an important role in immune response. The phagocytosis of monocytes was reflected by the test of carbon clearance [59]. Fig.11 showed the effect of PPMs on the phagocytosis in irradiated mice. The phagocytic index (PI) of model group decreased significantly compared with normal group (p < 0.01). After administration of PPM (50 and 100 mg/kg), the phagocytic indexes
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were significantly increased (p < 0.05). The results above revealed that PPMs could significantly
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improve the phagocytosis of monocyte in irradiated mice.
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4. Conclusions
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In summary, a novel chitosan microsphere for encapsulating pinecone polyphenols from p. koraiensis was successfully prepared and optimized for the first time using an emulsification
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crosslinking method. The morphology and particle size of the optimal microspheres is uniform. Embedding rate of microspheres could be achieved at up to 73.57 %. In vitro release studies suggested
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that the optimized microspheres had excellent release characteristics, and their release rates were higher
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in the simulated intestinal fluid while being somewhat lower in the simulated gastric fluid. Moreover, in vivo study clearly suggested that the optimized microspheres strengthened the radiation protection of
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pinecone polyphenols by improving the antioxidant and immunomodulatory activities. These results
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demonstrated that the bioactivity of pinecone polyphenols could be improved by a microspheric encapsulation method. Future research will be focused on the radiation protection mechanism of the polyphenol microspheres to reveal their radiation protection pathway.
Conflict of Interest The authors have declared no conflict of interest.
Acknowledgments
ACCEPTED MANUSCRIPT 18 The authors gratefully acknowledge the financial support of the NSFC (Grant No.31170510); Thirteen five national key research and development projects (No.2016YFC0500305-02). The scientific research innovation fund of Harbin Institute of Technology (HIT.NSRIF.2017023).
Animal Ethics
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The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):The experimental protocols were approved by Heilongjiang University of Chinese
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Medicine (SCXK Hei 2008004). All efforts were made to minimize animal suffering.
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Figure captions Fig.1. Effects of the variables on the embeding rate of PPM. (A): Crosslinking agent amount, (B) Stirring speed, (C) Crosslinking temperature and (D) Emulsifying time.
ACCEPTED MANUSCRIPT 21 Fig.2. Response surface plots showing the effects of crosslinking agent amount (X1), stirring speed (X2), crosslinking temperature (X3) and extraction temperature (X4) on the embeding rate of the prepared PPM. Fig.3. SEM images of PPM. (A) the preliminary prepared PPM, and (B) the optimal prepared PPM
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Fig.4. The particle size distribution of the optimal prepared PPM
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Fig.5. The release situations of the optimal prepared PPM in the simulated gastric fluid and the
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stimulated intestinal fluid, separately.
Fig.6. Effects of different doses PP and PPM pretreatments on the levels of antioxidant enzyme SOD
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in mice plasma. The irradiation dose was 6 Gy. *p < 0.05 and ** p < 0.01 compared with the normal
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group; # p < 0.05 compared with the model group (mean ± SD, n = 6). Fig.7. Effects of different doses PP and PPM pretreatments on the contents of MDA in mice plasma.
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The irradiation dose was 6 Gy. *p < 0.05 and ** p < 0.01 compared with the normal group; # p < 0.05
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and ##p < 0.01 compared with the model group (mean ± SD, n = 6). Fig.8. Effects of different doses PP and PPM pretreatments on the splenic lymphocytes proliferation in
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mice. The irradiation dose was 6 Gy. *p < 0.05 and ** p < 0.01 compared with the normal group; # p <
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0.05 compared with the model group (mean ± SD, n = 6). Fig.9. Effects of different doses PP and PPM pretreatments on the phagocytosis of monocyte in mice. The irradiation dose was 6 Gy. *p < 0.05 compared with the normal group; # p < 0.05 compared with the model group (mean ± SD, n = 6).
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Juanjuan Yi, Cuilin Cheng, Shubin Li, Dong Wang, Lu Wang, Zhenyu Wang PII: DOI: Reference:
S0141-8130(17)33530-4 doi:10.1016/j.ijbiomac.2018.02.131 BIOMAC 9188
To appear in: Received date: Revised date: Accepted date:
16 September 2017 14 January 2018 20 February 2018
Please cite this article as: Juanjuan Yi, Cuilin Cheng, Shubin Li, Dong Wang, Lu Wang, Zhenyu Wang , Preparation optimization and protective effect on 60Co-γ radiation damage of Pinus koraiensis pinecone polyphenols microspheres. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.02.131
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ACCEPTED MANUSCRIPT
PREPARATION OPTIMIZATION AND PROTECTIVE EFFECT ON 60
Co-γ RADIATION DAMAGE OF PINUS KORAIENSIS PINECONE POLYPHENOLS MICROSPHERES
Juanjuan Yia,b, Cuilin Cheng a, Shubin Lia, Dong Wanga, Lu Wang a*, Zhenyu Wang a* Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin 150090, PR China
b
School of Life Sciences, Zhengzhou University, Zhengzhou, Henan, China
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a
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*Corresponding author: Lu Wang and Zhenyu Wang.
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Postal address: School of Chemical Engineering and Chemistry, Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin, 150090, PR China.
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E-mail address: [email protected] (Lu Wang).
E-mail address: [email protected] (Zhenyu Wang).
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Tell numbers: +86-0451-86282909, Fax numbers: +86-0451-86282909.
Nonstandard Abbreviations: Pinus koraiensis: P. koraiensis; pinecones polyphenols microspheres:
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PPM; Response surface methodology: RSM; Box-Behnken design: BBD;.Glutaraldehyde: GA; Scanning electron microscopy: SEM; Superoxide dismutase: SOD; Malondialdehyde: MDA;
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Phagocytic index: PI.
ABSTRACT: Here, the chitosan and the glutaraldehyde (GA) were used to encapsulate pinecones
polyphenols of Pinus koraiensis (P. koraiensis) by emulsification cross-linking technology. First, the prepared parameters (crosslinking agent amount, stirring speed, crosslinking temperature and emulsifying time) of the pinecones polyphenols microspheres (PPMs) were optimized by the response
ACCEPTED MANUSCRIPT 2 surface methodology (RSM). When chitosan concentration and crosslinking time were 2 % and 80 min, respectively, the optimal conditions were 7.91 mL of crosslinking agent, stirring speed of 660.98 r/min, crosslinking temperature of 41.18 °C and emulsifying time of 198.65 min. The prepared PPMs embedding rate was 73.57 %. The optimized PPM possessed a distinct core-shell structure and uniform
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spherical distribution with a particle size value of 3.4 μm. In addition, they had the excellent
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sustained-release characteristics in vitro. We also evaluated the radioprotective effects of PPMs against 60
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Co-γ radiation in vivo. PPMs improved significantly the activity of the antioxidant enzyme SOD and
reduce MDA level in the plasma of irradiated mice. Accordingly, PPMs could also significantly
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enhance the immunomodulation activity by promoting the proliferation of splenocytes and monocyte
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phagocytosis of irradiated mice. These results suggested that PPMs exert effective protection against radiation-induced injury by improving the antioxidant and immunomodulation activities.
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Key Words: Pinecone polyphenols of Pinus koraiensis; Microspheres preparation; Optimization;
1. Introduction
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Sustained-release characteristics; Radiation protection.
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In recent years, more attention has been paid plant polyphenols due to their multiple biological and pharmacological activities [1-4]. P. koraiensis pinecone was a byproduct in the processing of P. koraiensis seeds. Our previous studies have been proved that P. koraiensis pinecone is a rich source of polyphenols [5] and possesses strong anti-tumor activities in vitro [6] and in vivo [7], immunoregulation activity in vivo [8], antioxidant activity in vitro [9], and radioprotective effect [10]. The polyhydroxy structural characteristics of polyphenols played an important role in their biological effects [11]. However, further research has also found that the polyhydroxy structures of plant polyphenols exposed to high temperature, oxygen, extreme pH and other harmful environment can be
ACCEPTED MANUSCRIPT 3 easily oxidized and degraded, which could lead to functional composition degradation, ineffective dose concentrations and decreased bioactivity [12]. To improve the stability and utilization of polyphenols, many research efforts are ongoing [13-15]. Drug delivery systems (DDS) based on macromolecule polymers have been shown to improve the stability and release-control of the drugs [16]. Microspheres, as multiple-unit DDS for oral drugs,
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possess multiple significant and competitive benefits, such as more uniform distribution, good
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absorption, enhancement of drug stability, and thermodynamic stability against denaturation and degradation of polyphenols [17]. The study also has reported that microsphere technique could resolve
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the stability and sustained drug delivery problems of naturally active substances [18].
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Chitosan [α(1,4)-2-amino-2-deoxy-β-D-glucan, CS] is a type of natural carrier material applied in DDS and the food industry, it not only offers good biodegradability and multiple biological activities but
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also plays a protective role and could sustain release of the core drug [19-20]. Many methods, such as spray drying, ionotropic gelation, coacervation, solvent evaporation and crosslinking, have been
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developed for preparation of chitosan microspheres. Preparation of chitosan microspheres
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(CS-microspheres) by the traditional spray drying method is easy to perform, but it is difficult to encapsulate the drug in the polymer matrix. In the ionotropic gelation and coacervation processes, the
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pH of the solution must be strictly controlled, and the morphology and size of the microspheres are generally nonuniform [21-22]. In the method of solvent evaporation, organic solvent evaporation
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processes may reduce the bioavailability of the drug and destroy the biocompatibility of the microspheres [23]. Compared to the methods mentioned above, the crosslinking method has strong potential in the development of polymer microspheres to overcome the above defects, while providing narrow particle size distributions and good morphology microsphere, which prolongs the retention time frame and controls the release behavior of the active agent [24]. CS-microspheres crosslinked with chemical crosslinkers, including glutaraldehyde, ethylene glycol, diglycidyl ether, tripolyphosphate and disulfide, have been reported to provide favorable stability and a controlled release ability for many drugs [25-26]. Regarding the controlled release ability, it has been reported that the release of the active
ACCEPTED MANUSCRIPT 4 agent from CS-microspheres generally occurs in three different ways: (a) release from the surface of microspheres due to its porous structure, (b) diffusion resulting from swelling, and (c) release due to the degradation of the polymer [24]. Therefore, release is susceptible to pH, polarity of the release medium, and the activity of enzymes. Currently, the CS-microsphere technique has been widely applied in the biological, food and
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medical fields [27-29]. Cho et al prepared the resveratrol microspheres using high and medium
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molecular weight chitosan to improve resveratrol bioavailability [30]. In another study, resveratrol was encapsulated into chitosan microspheres for improving the stability and evaluating the ability of
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controlled release [31]. Park et al. investigated and characterized chitosan microspheres for anti-cancer
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bioactivity [32].
There have been no studies on the anti-radiation activity of chitosan-loaded-polyphenol
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microspheres. Therefore, the main purposes of this study were to prepare and optimize a novel chitosan-supported P. koraiensis pinecone polyphenol microsphere (PPM) by RSM to improve the
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radiation protection effect of a core drug (pinecone polyphenols) in mice. In this study, chitosan is a type
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of drug carrier, pine polyphenols are embedded, and an anti-radiation drug system is prepared, all of which enhance the effectivity of the drug system. This is a new approach in this field and has a strong
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practical significance for the research of anti-radiation drugs.
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2. Materials and methods 2.1 Materials
The dried pinecones of P. koraiensis were provided by the Forestry Bureau (Yichun, China). The chitosan (average molecular weight [MW] of 100 kDa and deacetylation degree of 92 %) was provided from sigma. The superoxide dismutase (SOD), malondialdehyde (MDA) and protein quantization measurement kits were purchased form Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Roswell Park Memorial Institute 1640 (RPMI-1640) and Fetal Bovine Serum (FBS) were provided
ACCEPTED MANUSCRIPT 5 from Hyelone Chemical Co., USA. All other chemicals were of analytical grade and purchased from local suppliers.
2.2 Extraction and separation of PP
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2.2.1 Extraction procedure The dried pinecones of P. koraiensis were smashed and passed through the 30 mesh sieves. The
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pinecones powders were added in the 60 % ethanol (1:30 g/mL), and carried on ultrasonic treatment at
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50 ℃ and 400 W for 30 min, then repeated extraction above twice. The extracts were combined and separated by filtration, then centrifuged at 4000 rpm for 10 min. The supernatant was concentrated by
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rotary evaporator for crude pinecones polyphenols (PPs).
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2.2.2 Separation procedure
In order to obtain higher purity, the separation of crude PPs extracts was carried out in glass
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columns packed with pretreated X-5 macroporous resins. The eluent concentration (ethanol-water) was
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60 %. Operating conditions were as follows: the concentration of crude PP: 2.5 mg/ mL, flow rate, 4.0 mL/min; collected volume: 2 bed volumes. Finally, the collected component was concentrated by a
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rotary evaporator to remove the ethanol. The purity of collected polyphenols was 40.16 % according to
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the Folin-Ciocalteau method.
2.3 Preparation of PPM Pinecone polyphenol microspheres were prepared using an emulsification and crosslinking method and slightly modified by reference to Patel et al [33]. Chitosans (50 mg) and the pinecone polyphenols (5 mg) were homogeneously dissolved in 3 % acetic acid solution (5 mL) by magnetic stirring for 2 hours and ultrasound treatment for 30 min. Then, the obtained mixed solution (5 mL) was dispersed by stirring at different stirring speeds (500-1000 r/min) into a continuous oil phase (60 mL) consisting of
ACCEPTED MANUSCRIPT 6 liquid paraffin and 2 % span80 (V:V, 30:1) at different crosslinking temperature (20-70 °C) for different emulsifying times (40-240 min) to form a water-in-oil micro-emulsion. Then, different amounts (2-12 mL) of 25 % crosslinking agent glutaraldehyde (GA) was added into the beaker under stirring at 600 r/min. The crosslinking reaction was allowed to proceed for a total of 1 h. Hardened microspheres were
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filtered and washed, first with distilled water to remove the unreacted
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polyphenols and residual CS. Then, the product was washed repeatedly with petroleum ether and
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anhydrous ethanol to remove liquid paraffin and unreacted GA, dried under a vacuum at 40 °C overnight
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2.4 Calculation of the embeding rate
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and kept in a desiccator until further use.
PPMs were dialyzed against the distilled water, which were shaken and centrifuged at 15000 r/min
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for 30 min at 4 ℃. The contents of the free polyphenols of the supernatant were determined according to
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Folin-reagent method [5]. The embeding rate of PPMs was calculated as follows[33]:
(1)
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E D CV D 100
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Where E is the embeding rate (%); D is the amount of total polyphenols (mg); C is the . concentration of the polyphenols of PPMs solution (mg GAE/mL), and V is the volume of PPMs solution (mL).
2.5 Experimental design and statistical analysis The single-test was used to determine the preliminary range of prepared parameters. Then, BBD was employed to optimize four independent variables (crosslinking agent amount X1, stirring speed X2, crosslinking temperature X3 and emulsifying time X4) for the preparation of PPMs. It employed 29
ACCEPTED MANUSCRIPT 7 experiments to optimize the prepared parameters of PPMs. Table 1s showed the four levels of these independent variables. The embeding rate (Y) was taken as a response for the design experiment in Table 1. The behavior of the system was explained by Equation (2):
Y A0
3
Ai X i
Aii X i2
i 1
2
3
Aij X i X j i j i 1
(2)
1
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i 1
3
Where Y is the dependent variable, A0 is constant, and Ai, Aii, and Aij are coefficients estimated by
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model. Xi and Xj are levels of the independent variables while A0, Ai, Aii, and Aij are the regression
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2.6 Scanning electron microscopy (SEM)
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coefficients for the intercept, linearity, square, and interaction, respectively.
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The shape and surface morphology of PPMs were investigated using SEM. PPMs were coated
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with gold palladium under an argon atmosphere for 150 s to achieve a 20 nm film (sputter coater,
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SCD004, BAL-TEC Balzers, Furstentum, Lienchestein). The coated samples were examined in a scanning electron microscope (Jeol JSM-1600, Tokyo, Japan).
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2.7 Measurement of particle size distribution
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The particle size of PPM was analyzed by laser scattering particle size analyzer. Briefly, the PPMs were diluted with the distilled water, placed in a 1cm absorption dish and determined at 25 °C.
2.8 In vitro stimulated gastrointestinal digestion of PPMs The digestion of PPMs was investigated according to the method reported by Thakkar et al. with some modifications [34]. The stimulated gastric fluid (50 mL, pH 1.2) and stimulated intestinal fluid was separately put into two 250 mL beakers. During the digestion process, the same mass of PPMs were installed into two dialysis bags. Then, two dialysis bags were separately incubated in 50 mL of different incubation mediums (the stimulated gastric fluid, the stimulated intestinal fluid) at 37 °C, and
ACCEPTED MANUSCRIPT 8 gently shaken at oscillation speeds of 80 r/min. At predetermined time intervals, 5 mL of the different incubation mediums were separately collected to calculate the released amounts of polyphenols from PPMs. Meanwhile, 5 mL of fresh medium was compensated.
2.9 In vivo protective effect on 60Co-γ radiation damage of PPMs
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2.9. 1 Animal
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Male KM mice of SPF-level (6 – 8 weeks old, 18 – 20 g each) were housed in a mouse room at
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temperature (22 ± 2 °C), light (12 h light/dark cycles) and humidity (50 ± 10 %) and were provided with rodent laboratory chow pellets and tap water for a week to adapt to the environment of mouse
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room. The experimental protocol was approved by Institutional Animal Ethical committee.
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2.9.2 Groups and irradiation
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Mice were divided into eight groups of 20 animals each. Pinecone polyphenols (PPs, 25, 50 and
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100 mg/kg bwt/d) and pinecone polyphenol microspheres (PPMs, 25, 50 and 100 mg/kg bwt/d the encapsulated polyphenols) were separately administered to the mice by an oral probe in their water
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suspension for 14 consecutive days prior to irradiation. The normal control group and radiation model
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group were given an equal volume of normal saline every day. Irradiation was performed at a dose rate of 1.0 Gy/min using a
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Co irradiator (Heilongjiang Academy of Agricultural Sciences, China). The
irradiation dose was 6 Gy. After 24 h, the mice were sacrificed by cervical dislocation, and their organs, including the thymus, heart, liver and spleen, were separately excised and weighed to calculate the organ indexes.
2.9. 3 Antioxidant status assessment The activity of SOD and the content of MDA in the plasma were determined using commercial kits
ACCEPTED MANUSCRIPT 9 (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The SOD activity was expressed in U/mL, the content of MDA was expressed in nmol/mL.
2.9. 4 Assay of splenocyte proliferation in vivo
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After the mice sacrificed, the spleens collected from mice under aseptic conditions were grinded into small pieces and passed through sterilized meshes to obtain a homogeneous cell suspension at the
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room temperature. The red blood cells were removed by red blood cell lysis solution for 5 min.
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Recovered splenocytes were washed twice, then re-suspended in RMPI-1640 complete medium
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containing 5 % FBS [35-36]. Cells were seeded in a 96-well plate with or without Con A (7.5 μg/mL). After incubation for 48 h at 37 °C in a humidified 5 % CO2 incubator, the number of cells was
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determined by MTT assay using a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA).
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2.9. 5 Phagocytosis of monocyte assay
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After 14 days of oral administration, 25 % (v/v) India ink according to 0.2 mL/kg body weight was injected by a tail intravenous injection. A total of 20 μL of blood was collected through eye orbit after 2
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min (t1) and 10 min (t2), and added to 2 mL 0.1 % Na2CO3. The absorbance at 600 nm of blood after 2
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min (A1) and 10 min (A2) were measured, and the absorbance of normal control group of blood was set as zero. The mice were sacrificed by decapitation, and then the liver and spleen were weighed. Clearance index (K) and phagocytic index (α) were calculated as follows [37]:
K=
lg A1 -lgA 2 t2 -t1
2.10 Statistical analysis
α= K1/3 × body weight/(liver weight + spleen weight)
(3)
ACCEPTED MANUSCRIPT 10 All statistical analyses employed SPSS for Windows, Version 18.0. Data were expressed as means ± standard deviation (SD) of three independent measurements. Differences at p < 0.05 and p < 0.01 were considered statistically significant by Duncan’s new multiple-range test.
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3. Results and discussion
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3.1.1 Effects of crosslinking agent (GA) amount
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3.1 Effects of different parameters on embedding rate
GA, a crosslinking agent for the crosslinking reaction, was necessary for the preparation of
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chitosan microspheres due to the viscosity [38]. The effects of the GA contents on the embedding rate
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and the surface morphology of PPMs are shown in Fig. 1A and Fig. S1, respectively. The embedding rate of PPM exhibited an upward trend with increased GA contents. When the volume of GA was 8 mL,
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the embedding rate almost reached a maximum (78.7 %), and the particle size was smaller. This is
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possibly due to increase in the volume of crosslinking agent, the formation of more rigid polymer network and the shrinkage of particles, which might have decreased the particle size[39]. Subsequently,
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as the volume of GA increased, the embedding rate exhibited a fast downward trend. This was due to
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higher degrees of crosslinking, microspheres that are denser and a decrease in the free volume space within the matrix, resulting in reduced embedding rates[40]. Therefore, 0.8 mL was selected as the optimal volume of GA in the present study.
3.1.2 Effects of stirring speed High-speed stirring diminishes the droplet sizes based on turbulent flow and hydrodynamic shear stress. The emulsified droplets were seriously ruptured by the powerful shear force produced by the high-speed homogenizer [41]. Fig. 1B shows the effect of stirring speed on the embedding rate of PPMs.
ACCEPTED MANUSCRIPT 11 With the increase in the stirring speed (500–1000 r/min), the embedding rate increased first and then decreased, peaking with 82.1 % of the embedding rate at 700 r/min. However, compared with 700 r/min, the sphere is relatively smaller and better at 800 r/min (Fig. S2). To consider the comprehensive impacts of the stirring speed on the PPMs, 700 r/min was chosen as the best stirring speed for the
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preparation of PPMs.
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3.1.3 Effects of crosslinking temperature
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The crosslinking temperature is a decisive factor for the optimization of the embedding rate of PPM.
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Upon increasing the crosslinking temperature from 20 to 40 °C, the embedding rates of PPM increased from 59.8 % to 78.5 % (Fig. 1C), which may be attributed to the increase in temperature resulting in
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decreasing the viscosity of the dispersion system to obtain smaller sized particles [42]. However, with increasing crosslinking temperatures from 40 to 70 °C, the embedding rate later decreased slowly. The
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optimal embedding rate and morphology of PPMs were observed (Fig. S3) at a crosslinking temperature of 40 °C.
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3.1.4 Effects of emulsifying time
In the present study, it was necessary to determine the required emulsifying time and its effects on
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microsphere characteristics, including the embedding rate and the morphology. The effects of the emulsifying time on the embedding rate of PPMs were shown in Fig. 1D. A higher embedding rate was obtained with stirring for 200 min compared to other times. However, it was clear that the lowest embedding rate was obtained on emulsifying for 240 min. This may be due to the long stirring responsible for some more polyphenols to be leached out in the oil phase during the emulsification process. Moreover, smooth and uniform spherical distributions of PPMs were found at 200 min reaction time (Fig. S4). Therefore, the emulsifying time at 200 min is sufficient for producing the
ACCEPTED MANUSCRIPT 12 required microspheres of the high embedding rate and stable morphology.
3.2 Optimization of preparation parameters for PPM 3.2.1 Statistical analysis and the model fitting
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There were a total of 29 runs used to optimize the four individual parameters (crosslinking agent
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amount, stirring speed, crosslinking temperature and emulsifying time) to analyze the impacts of the independent variables on the embedding rate (Y) in the current BBD, as shown in Table 2. The model for
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the response variable could be expressed by the following quadratic polynomial equation in the form of
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coded values:
Y 74.2 0.89 X 1 0.73 X 2 0.97 X 3 0.21X 4 0.97 X 1 X 2 1.25 X 1 X 3 0.045 X 1 X 4 0.39 X 2 X 3 0.34 X 2 X 4 1.83 X 3 X 4 2.63 X 1 3.09 X 2 1.41X 3 1.95 X 4 2
2
2
(4)
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2
Where Y is the embedding rate of PPMs, and X1, X2, X3 and X4 are the coded variables for
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crosslinking agent amount (mL), stirring speed (r/min), crosslinking temperature (°C) and emulsifying
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time (min), respectively.
The analysis of variance (ANOVA) was performed for testing the significance of the fitted
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quadratic polynomial, which was obtained by the experimental data, as demonstrated in Table 3. The determination coefficient R2 was 0.9708, and it was suggested that 97.08 % of the variations could be
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illustrated by the fit model. The correlation between the experimental and predicted values can be reflected by the value of Radj 2, which should be closer to R2 in a well statistical model [43]. As shown in Table 3, the value of Radj2 was 0.9416 and was close to R2, which revealed that the model was highly significant. At the same time, the coefficient of variation (CV =0.81 %) was a relatively small value, which indicated a better reliability of the experimental values. The non-significant lack-of-fit for all the variables revealed that the polynomial model was statistically accurate for the responses [44]. Moreover, the significance of each coefficient was also checked by the P-value. Table 4 showed that the
ACCEPTED MANUSCRIPT 13 cross-product coefficients (X1X2, X1X3 and X3X4) were very significant with small P-values (p < 0.01). These results suggested the model could be used to predict these responses.
3.2.2 Effects of independent variables on embedding rate The 3D surface plots, which provide a method to visualize the relationship between responses and experimental levels of each variable [45], are shown in Fig. 2. Fig. 2A shows the effects of crosslinking
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agent amount and stirring speed on the embedding rate. When the stirring speed was fixed, the
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embedding rate increased with the increase of crosslinking agent amount until reaching a maximum and
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then decreased. Similarly, the increase in stirring speed at a fixed crosslinking agent amount led to an increase of the embedding rate and nearly reached a peak at a certain extent and then decreased. Fig. 2B
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shows the plot of embedding rate as affected by crosslinking temperature and crosslinking agent amount, which demonstrated a marked increase in embedding rate with the crosslinking temperature at a
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fixed crosslinking agent amount and then decreased. Additionally, an increase in crosslinking agent amount at a fixed crosslinking temperature led to an increase in the embedding rate and then decreased.
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Similarly, the effects of emulsifying time and crosslinking agent amount on the embedding rate (Fig.
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2C), which further illustrated that the embedding rate was positively affected by the crosslinking agent amount. However, the increase of viscosity could also cause the coalescence of the droplets that finally
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resulted in the decreased embedding rate. Fig. 2D and Fig. 2E respectively show the interactive effects of stirring speed with crosslinking temperature and emulsifying time on the embedding rate of PPMs,
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which further illustrated that the embedding rate was affected by GA content. With the increasing of the stirring speed, the embedding rate increased due to the high shear stress, which could rupture the viscous droplets and facilitate the package process [33]. Up to a certain level, the embedding rate started to decrease because the excessively high speed with the powerful energy could lead to the breaking down of resultant microspheres. Fig. 2F also shows that the interaction of the crosslinking temperature and the emulsifying time were demonstrated significantly on the embedding rate.
3.2.3 Optimal preparation conditions for PPMs
ACCEPTED MANUSCRIPT 14 The optimal values of embeding rate of PPMs for the independent variables were respectively carried out by solving the regression equations using Design-Expert 7.0 software. Through the comprehensive consideration with maximum desirability, the predicted optimal conditions for the preparation of PPMs were as follows: crosslinking agent amount of 7.19 mL, stirring speed of 660.98 r/min, crosslinking temperature of 41.18 ℃ and emulsifying time of 198.65 min. Under the optimal
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conditions, the embeding rate of PPMs was 73.57 %. These results confirmed that the model can be used
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to optimize the preparation parameters of PPMs.
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3.3 Morphology observation and particle size distribution of PPMs
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In the present work, the crosslinked polyphenols microspheres were successfully prepared by the
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emulsification and crosslinking method in response surface methodology. The shape and surface morphology of the PPM was investigated using scanning electron microscopy. The particle size
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distribution of the optimal PPM was measured by a laser particle size analyzer. The effect of the
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formulation variables on the morphology change and particle size of the PPM is shown in Fig. 3 and Fig. 4. The optimal microspheres had a smoother surface and were found to be discreet and spherical
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in shape with a stable form and uniform size (Fig. 3B). Compared with Fig. 3A, the embedding rate of
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the polyphenols almost reached a maximum with a value of 73.57 %, and the particle size was smaller with a uniform microsphere volume (Fig. 3B). Fig. 4 shows that the average particle size distribution was approximately 3.4 μm, which indicated that the microspheres were successfully prepared.
3.4 In vitro stimulated gastrointestinal digestion of the PPMs The release of polyphenols from the optimal microspheres was monitored using a two-model system: the stimulated gastric fluid and stimulated intestinal fluid were used as release media. Fig. 5 shows the release profiles of pinecone polyphenols from microspheres in the two-model medium. In
ACCEPTED MANUSCRIPT 15 general, there was an initial rapid increase in the amount of polyphenols released, followed by a more gradual release until a constant level was reached. Overall, the amount of polyphenols released was higher and the release rate was faster in the stimulated intestinal fluid compared with the stimulated gastric fluid, which can be attributed to the relative stability of the microspheres in the acid
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environment. This tendency was verified by many studies [46-47]. These results suggested that PPM
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had excellent sustained-release characteristic in the simulated gastric fluid and fast release
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characteristic in the stimulated intestinal fluid.
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3.5 Effects of PPMs on organ indexes
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Ionizing radiation is an electromagnetic wave or particle capable of producing ions and attacking living tissues and organs, which could cause multiple organ dysfunction and injury of biological
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systems, including immunomodulatory, hematopoietic, or liver systems in the human body [48-49].
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As shown in Table 5, compared with the normal control group, radiation resulted in multiple organ index declines, which indicated that ionizing radiation damaged the normal organs and affected their
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functions. Some in vitro and in vivo studies have confirmed that phenolic compounds are
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radioprotectors and have potentially radio-protective effects without obvious side effects [50-52]. Similarly, in the present study, the pinecone polyphenols also showed promising potential in improving organs indexes and preventing organ radiation damage. However, the pinecone polyphenols exposed in the aerobic and acidic environment can be easily oxidized and degraded, which results in a decrease of bioactivity. The preparation of microspheres improved the absorption efficiency and utilization rate of polyphenols in vivo. Table 5 showed that the pretreatment of PPMs improved the organ indexes most effectively, which initially indicated that PPMs possessed radiation protection function.
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3.6 Effects of PPMs on antioxidant status Endogenous antioxidant enzymes are considered to be the first line of defense in maintaining a redox balance and normal biochemical processes [53]. Homeostatic cellular functions require tight control of the redox environment [54]. However, radiation could attenuate the endogenous antioxidant
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enzymes to break the redox balance [53]. Therefore, agents that can repair and adjust the
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oxidation-reduction system have received extensive attention. In the present study, as shown in Fig. 6
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and Fig. 7, compared with the normal control group, there was a significant reduction in antioxidant enzyme SOD content and prominent enhancement of MDA level that were clearly observed in the
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model control group (p < 0.01). However, after the administrations of PPs (50 and 100 mg/kg) and
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PPMs (25, 50 and 100 mg/kg), the SOD contents were significantly increased (p < 0.05). In contrast, the MDA levels in plasma were significantly reduced (p < 0.05 or p < 0.01). The results above showed
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that the administrations of PPs and PPMs could effectively increase the contents of antioxidant
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enzymes and decrease the level of MDA in the irradiated mice.
3.7 Effect of PPMs on splenocytes proliferation
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The spleen is the largest immune organ in the vertebrate body, capable of producing a large
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number of lymphocytes [55]. T-lymphocyte is an important immunological active cell and plays an important role in enhancing the immune function of organisms [56-57]. The data showed that PPMs (25, 50 and 100 mg/kg) could enhance specific immune response by exhibiting significant comitogenic activity in Con A-induced splenocytes proliferation, with the better effect than the PP groups (Fig.10). The splenocytes proliferation response was related to immunity improvement of T-lymphocytes or B-lymphocytes [58]. The results indicated that PPMs possessed a definite and clear synergistic action on splenocytes proliferation after combining with ConA.
3.8 Effect of PPMs on phagocytosis of monocyte
ACCEPTED MANUSCRIPT 17 Monocyte plays an important role in immune response. The phagocytosis of monocytes was reflected by the test of carbon clearance [59]. Fig.11 showed the effect of PPMs on the phagocytosis in irradiated mice. The phagocytic index (PI) of model group decreased significantly compared with normal group (p < 0.01). After administration of PPM (50 and 100 mg/kg), the phagocytic indexes
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were significantly increased (p < 0.05). The results above revealed that PPMs could significantly
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improve the phagocytosis of monocyte in irradiated mice.
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4. Conclusions
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In summary, a novel chitosan microsphere for encapsulating pinecone polyphenols from p. koraiensis was successfully prepared and optimized for the first time using an emulsification
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crosslinking method. The morphology and particle size of the optimal microspheres is uniform. Embedding rate of microspheres could be achieved at up to 73.57 %. In vitro release studies suggested
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that the optimized microspheres had excellent release characteristics, and their release rates were higher
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in the simulated intestinal fluid while being somewhat lower in the simulated gastric fluid. Moreover, in vivo study clearly suggested that the optimized microspheres strengthened the radiation protection of
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pinecone polyphenols by improving the antioxidant and immunomodulatory activities. These results
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demonstrated that the bioactivity of pinecone polyphenols could be improved by a microspheric encapsulation method. Future research will be focused on the radiation protection mechanism of the polyphenol microspheres to reveal their radiation protection pathway.
Conflict of Interest The authors have declared no conflict of interest.
Acknowledgments
ACCEPTED MANUSCRIPT 18 The authors gratefully acknowledge the financial support of the NSFC (Grant No.31170510); Thirteen five national key research and development projects (No.2016YFC0500305-02). The scientific research innovation fund of Harbin Institute of Technology (HIT.NSRIF.2017023).
Animal Ethics
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The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):The experimental protocols were approved by Heilongjiang University of Chinese
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Medicine (SCXK Hei 2008004). All efforts were made to minimize animal suffering.
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Figure captions Fig.1. Effects of the variables on the embeding rate of PPM. (A): Crosslinking agent amount, (B) Stirring speed, (C) Crosslinking temperature and (D) Emulsifying time.
ACCEPTED MANUSCRIPT 21 Fig.2. Response surface plots showing the effects of crosslinking agent amount (X1), stirring speed (X2), crosslinking temperature (X3) and extraction temperature (X4) on the embeding rate of the prepared PPM. Fig.3. SEM images of PPM. (A) the preliminary prepared PPM, and (B) the optimal prepared PPM
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Fig.4. The particle size distribution of the optimal prepared PPM
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Fig.5. The release situations of the optimal prepared PPM in the simulated gastric fluid and the
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stimulated intestinal fluid, separately.
Fig.6. Effects of different doses PP and PPM pretreatments on the levels of antioxidant enzyme SOD
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in mice plasma. The irradiation dose was 6 Gy. *p < 0.05 and ** p < 0.01 compared with the normal
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group; # p < 0.05 compared with the model group (mean ± SD, n = 6). Fig.7. Effects of different doses PP and PPM pretreatments on the contents of MDA in mice plasma.
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The irradiation dose was 6 Gy. *p < 0.05 and ** p < 0.01 compared with the normal group; # p < 0.05
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and ##p < 0.01 compared with the model group (mean ± SD, n = 6). Fig.8. Effects of different doses PP and PPM pretreatments on the splenic lymphocytes proliferation in
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mice. The irradiation dose was 6 Gy. *p < 0.05 and ** p < 0.01 compared with the normal group; # p <
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0.05 compared with the model group (mean ± SD, n = 6). Fig.9. Effects of different doses PP and PPM pretreatments on the phagocytosis of monocyte in mice. The irradiation dose was 6 Gy. *p < 0.05 compared with the normal group; # p < 0.05 compared with the model group (mean ± SD, n = 6).
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