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Chitosan nanoparticles loaded with 2,5-dihydroxybenzoic acid and protocatechuic acid: Properties and digestion
Journal of Food Engineering 174 (2016) 8e14
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Chitosan nanoparticles loaded with 2,5-dihydroxybenzoic acid and protocatechuic acid: Properties and digestion Ana Raquel Madureira 1, Adriana Pereira 1, Manuela Pintado* rio Associado, Escola Superior de Biotecnologia, Universidade Cato lica Portuguesa/Porto, CBQF e Centro de Biotecnologia e Química Fina e Laborato ~o Vital, Apartado 2511, 4202-401 Porto, Portugal Rua Arquiteto Loba
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 September 2015 Received in revised form 3 November 2015 Accepted 6 November 2015 Available online 18 November 2015
Research efforts on the production of chitosan nanoparticles (NP) as delivery systems of bioactive compounds such as polyphenols have been made along the last decade. Nevertheless, the effect of the phenolic compound structure in the production of these NP was never evaluated so far. Low and high molecular weights chitosan (LMWC and HMWC) NP loaded with the phenolic acids, protocatechuic (PA) and 2,5-dihydroxybenzoic acids (2,5-DHBA) were produced by ionic gelation. Antioxidant activities were determined by ORAC assay. Physical and thermal properties were evaluated by dynamic light scattering (DLS) and differential scanning calorimetry (DSC), respectively. Stability and release of phenolic acids during simulation of gastrointestinal tract (GIT) conditions were also assessed. Nanoparticles sizes ranged from 300 to 600 nm and maintained stable during storage at 4 C during 30 d. Antioxidant activities of the phenolic acids decreased when loaded in the NP. High molecular weight chitosan NP adsorbed higher energy and melted at lower temperatures than LMWC NP. Nanoparticles produced with HMWC released higher phenolic acids % at GIT simulated conditions and with slight increases in their sizes. The most proper systems for delivery of PA and 2,5-DHBA were found to be LMWC and HMWC NP, respectively. These NP could be used to as functional food ingredients or as models for production of phenolic acids-rich extracts NP for future incorporation in food matrices. © 2015 Published by Elsevier Ltd.
Keywords: Chitosan Nanoparticles Phenolic compounds Gastrointestinal tract
1. Introduction Nowadays, there is a rising attention in the application of nanotechnology in food industry, namely in the production of nanoparticles (NP) as new functional food ingredients and as a way to improve the bioactive compounds functionality with proved benefits to human health. These new structures can protect the bioactive compounds during food processing and storage, and during passage by the gastrointestinal tract (GIT) (Ha et al., 2013; Zou et al., 2012). The polymeric NP are colloidal carriers with sizes ranging from 10 to 1000 nm. Great part of the studies use chitosan as entrapment material for NP production and bioactive/ drug compounds controlled delivery, protection from degradation and oxidation, production of nanocomposites, among other applications (Agrawal et al., 2010; Dehnad et al., 2014; Huh and Kwon, 2011). Chitosan is an abundant natural biopolymer obtained from
* Corresponding author. E-mail address: [email protected] (M. Pintado). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.jfoodeng.2015.11.007 0260-8774/© 2015 Published by Elsevier Ltd.
chitin deacetylation, and acquired from crustaceans shells or as a natural component of specific fungi genus (Mucoraceae) (Agrawal et al., 2010; Dash et al., 2011). This polymer is polycationic with constant acidity (pKa ¼ 6.5) and insoluble at neutral pH (Agrawal et al., 2010). Chitosan is claimed as non-toxic, biologically compatible polymer, non-immunogenic and highly antimicrobial (Kean and Thanou, 2010; Mukhopadhyay et al., 2012). The difference in the relative proportions of N-acetyl-D-glucosamine and Dglucosamine residues, provide specific structural changes, which give rise to chitosans that are distinguished on the basis of their degree of deacetylation (DD) and molecular weight. The polymers have different molecular weights (50e2000 kDa), viscosity and DD (40e98%). Commercial chitosans are sold as high and low molecular weight chitosans, and characterized containing between 20 and 190 kDa with DD > 75% and between 190 and 375 kDa with DD > 75%, respectively. On the other hand, there is a huge interest in encapsulation of phenolic acids or their extracts, due to its high biological activity, namely on the antioxidant capacity found in this large group of compounds present in plants (Mohammadi et al., 2015a, 2015b).
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The encapsulation of these compounds, especially the ones with hydrophobic character is justified by their higher level of reactivity, chemical susceptibility to the harsh conditions of the GIT, low oral bioavailability and intestinal absorption (Tang et al., 2013). They must be enough time in the intestinal lumen to adhere to cell apical surface and then, be transcytosed by intestinal cells. The phenolic compounds used in this study were protocatechuic acid (PA) and the 2,5-dihydroxybenzoic acid (2,5-DHBA) (also known as gentisic acid). Protocatechuic acid is the major metabolite of antioxidant phenolic compounds found in green tea, and has been described to possess several bioactivities such as antimicrobial, anticancer, anti-ulcerogenic, anti-ageing (Kakkar and Bais, 2014). In addition, 2,5-DHBA has been characterized as antimutagenic, anti-inflammatory and antimicrobial (Boaventura et al., 2013). Is widely present in regular foods, including cereals such as wheat and rye, actinidia (e.g., kiwi) fruit, aloe vera, mushrooms as well as other sources (Juurlink et al., 2014). Besides their
EE% ¼
9
2.2. Determination of the physical properties Physical properties such as particle size (PS), polydispersity index (PI) and zeta potential (ZP) were evaluated using the NP solutions, by dynamic light scattering (DLS) technique with a ZetaPALS, Zeta Potential Analyzer (Holtsville, NY, USA). The parameters were measured at the initial production time of NP and also after 1 month of storage time at 4 C. All assays were performed in triplicate. 2.3. Determination of NP entrapment efficiencies The NP suspensions were filtrated by a centrifugal filter units with a cut-off of 3 K (Amicon® Ultra-4, Millipore), and then centrifugated at 37,732 g, 25 C, for 1 h. Samples supernatants were subject to analyses by HPLC method (all assays were done in triplicate). The entrapment efficiency (EE%) was determined as follows:
Total amount of polyphenol Total amount of polphenols in supernatant Total amount of polyphenols
variety in terms of natural sources, these two phenolic acids are different in terms of their chemical structure, in what concerns the number of eOH groups which are 3 for PA and 2 for 2,5-DHBA. Thus, the aim of the present study was the development of model carrier systems of two phenolic acids with different structures, PA and 2,5-DHBA. These were produced with LMWC and HMWC and characterized for their physical, antioxidant and thermal properties. Afterwards their antioxidant activity and stability during a simulated pH gastrointestinal tract were also evaluated. 2. Material and methods 2.1. Preparation of NP The concentrations of chitosan and phenolic compounds used in the production of the NP were the following: 0.2% (m/v) low molecular weight chitosan (LMWC), 0.4% (m/v) high molecular weight chitosan (HMWC), 0.3% (m/v) PA (LMWC_PA and HMWC_PA), and 0.3% (m/v) DHBA (LMWC_DHBA and HMWC_DHBA). All compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). Production followed the ionic gelation method described by da Silva et al. (2014), but with some modifications. Chitosan solutions were first dissolved in acetic acid (where the concentration of acetic acid was 1.75 times higher than chitosan concentration). Hence, acetic acid at 3.5 mg/mL was used for LMWC at 0.2% (m/v) and 7 mg/mL for 0.4% of HMWC. pH solution was adjusted to the physiologic value of 5.8 with NaOH 1 M. Afterwards, the phenolic compounds were dissolved in ultra pure water. Sodium tripolyphosphate (TPP) (Sigma-Aldrich) was prepared at different concentrations according the chitosan MW at a proportion [chitosan:TPP] of 7:1. Four mL of chitosan solution were placed under gentle stirring and 1 mL of phenolic compound was slowly added drop wise, followed by 2 mL of TPP also drop wise, at room temperature. Nanoparticles controls without phenolic compound were also produced by the same method. At the end, the pH of the final NP solutions loaded with phenolic compounds and controls without phenolic compounds were measured and then stored at 4 C.
2.4. Determination of antioxidant activity The antioxidant activity of loaded phenolic compounds NP was determined by oxygen radical absorbance capacity (ORAC), a fluorometric method that measures in vitro the antioxidant activity through fluorescein (Sigma, Steinheim, Germany) oxidation by the peroxilos radicals in situ by thermal decomposition from AAPH (Aldrich, Steinheim, Germany). This method measures the ability of oxygen radical absorption through the method developed by Ou valos et al. (2004) using a microet al. (2002) and adapted by Da plate fluorescence reader. Twenty microliters of solution (NP and phenolic compounds) or sodium phosphate buffer (0.1 M, pH 7) in case of control, were mixed with 120 mL of fluorescein and preincubated for 10 min at 40 C. After this step, 60 mL of AAPH was rapidly added in a polystyrene black microplate (Nunc, Denmark) and incubated at 40 C until a total of 137 min was completed (with the 10 min included). The measurement was performed in a fluorometer FluoSTAR OPTIMA (BMG LABTECH GmbH, Offenburg, Germany) using a wavelength of excitation at 485 nm and the emission one at 520 nm, with a Fluostar Control version 1.32 R2 software. All analyses were performed in triplicate. 2.5. Determination of thermal properties The NP thermal properties were evaluated by differential scanning calorimetry (DSC) using a DSC-60 (Shimadzu, Maryland, DC, USA). Nanoparticle suspensions were concentrated by centrifugation at 37,732 g, 25 C, 1 h and 3 mg of the pellet were placed in an aluminium pans hermetically sealed. The thermal behaviour was determined in the range 20e200 C, at a heating rate of 10 C/ min. Optimal melting temperatures and enthalpies were calculated by the equipment software (ta60 version 2.10, DSC software, Shimadzu). The mixtures of the phenolic compounds and TPP compound at the two different concentrations used (0.29 and 058 mg/ mL) were also tested, to evaluate the effect of these compounds in the thermal properties of the NP. All NP samples were tested as well as the controls (without phenolic compounds) and raw compounds used in the NP production. All assays were performed in quadruplicate.
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2.6. In vitro simulated gastrointestinal tract conditions A simulation of pH, temperature, movements and time of digestion of human stomach and duodenum was performed as described elsewhere (Madureira et al., 2011). Stomach conditions were simulated by suspending the concentrated NP in NaCl buffer solution 0.5% (w/v) (Panreac, Barcelona, Spain) at a proportion of 1:1. pH was adjusted to 1.5e2 with 9 M HCl (Merck, Darmstadt, Germany). Samples were put into a water bath at 37 C (Clifton Shaking Baths NE5 Series, Weston-super-Mare, United Kingdom), with homogenization at 150 rpm (to simulate the stomach peristaltic movements) for 60 min. To simulate intestine conditions, samples were taken from the bath and pH was increased until 7, with 1 M NaHCO3 (Pronalab®, Lisbon, Portugal) and left 2 h at 37 C at 90 rpm. Controls were made at the same time without simulated conditions of stomach and intestine. At times 0, 1 (stomach) and 3 (intestine) h, samples were taken to determine the physical properties and to quantify the concentration of phenolic compound that was released by the NP during the simulation. The % of phenolic compounds released by the NP during each step was calculated as follows:
% Release ¼
ðCP CPr Þ 100% CP
where CP is the concentration of phenolic compound loaded by the NP, and CPr is the concentration of phenolic compound quantified in each simulation stage. The CP is calculated as follows:
CP ¼
ðEE% x CPi Þ 100
where EE% is the entrapment efficiency percentage, and CPi is the initial concentration of phenolic compound used to produce the NP. CPr is the phenolic compound concentration released by the NP during the simulation steps, and is calculated as follows:
CPr ¼ Ctotal free polyphenols Cpolyphenol not entrapped 2.7. Quantification of phenolic compounds by HPLC Phenolic compounds were chromatographically separated through the mobile phase A e water, methanol (Panreac, Barcelona, Spain) and formic acid (Merck, Darmstadt, Germany) (92.5:5:2.5, v/ v/v) - and mobile phase B e methanol, water and formic acid (92.5:5:2.5, v/v/v). The elution conditions were: a gradient starting at 40% mobile phase B until 2 min at a continuous flow of 0.5 mL/ min, between 2 and 17 min with 50% of mobile phase B, lastly between 17 and 25 min the gradient change with 80% of mobile phase B. The results were detected with wavelengths ranging from 280 to 320 nm. The peaks were reached at 320 nm (phenolic acids), and were analysed by comparison of retention time and spectra with pure phenolic compounds. The calibration curve was established for all of the pure phenolic compounds using a concentration range from 0.04 to 24 mg/mL. 2.8. Statistical analyses The statistical analysis to assay the several parameters and the achieved results was obtained by ShapiroeWilk test, Levene test, tstudent test, through running one-way ANalysis Of VAriance (ANOVA) carried out with the support of SPSS (v. 19, Chicago, IL, USA). Differences between means of the physical properties of the different NP produced at the time of production and after 1 month of storage, as well the antioxidant activities were analysed. The
differences between means of the physical properties of the NP at the same stage of simulated GIT conditions, and between the means of the same physical property but at the three stages of the simulated GIT conditions were analysed. The confidence level used in these tests was 95%. 3. Results and discussion Six different sets of chitosan NP were produced using two types of chitosan e LMWC and HMWC combined with two phenolic compounds e PA, 2,5-DHBA and fully characterized in terms of bioactivity and physical and structural properties. Production of NP was done, including besides chitosan and phenolic compounds, a cross-linking agent TPP. This cross-linking agent had the function to create inter and intra molecular reactions with amino groups positively loaded with chitosan, so that the gelation process can occur (Calvo et al., 1997; da Silva et al., 2014). The TPP and phenolic compounds interact with chitosan amine groups and the entrapment of compounds occurs efficiently. At the same time a competitive interaction between the groups of phenolic compounds (eOH) and the groups of TPP, by the protonated amino groups of chitosan is observed, resulting in low levels of productive particle formation when compared to the control chitosan NP (without phenolic compound entrapped). This event can be intensified with higher phenolic compound concentrations (Dudhani and Kosaraju, 2010). 3.1. Physical properties and entrapment efficiencies In Table 1 are depicted the means values of the physical properties and pH of the control NP and NP loaded with phenolic compounds measured at 25 C at the time of production and after one month of storage time at 4 C. In general, the highest sizes were obtained for NP produced with HMWC, specifically for controls with no loaded phenolic compound (ca. 743 nm). These sizes could be due to the MW and higher concentrations of chitosan, and TPP used in the formulation (Calvo et al., 1997; Neves, 2013; Pool et al., 2012). The same trend was observed for control LMWC NP (ca. 598 nm). This can be explained by the higher PI values, since a cluster formation can occur resulting in particles with different sizes (Hanaor et al., 2012). The NP produced with HMWC loaded with PA presented the lowest sizes (ca. 511 nm) (P < 0.05). The sizes obtained are the desired ones, since predicting the use of these NP for oral administration is recommended a desirable size 300 nm, in order to not exceed the cell gut epithelium size, avoiding passage to the bloodstream and toxicity of NP by accumulation in the organs (Severino et al., 2011). In what concerns PI this parameter is indicative of the width from the peaks that corresponds to the particle size, and a high polydispersity shows the existence of particles families with different sizes, which may mean the occurrence of certain aggregation (Hanaor et al., 2012). In general, all samples showed PI values near 0.3, which indicates a monodisperse distribution of the NP sizes. Zeta potential is the degree of repulsion between adjacent, similarly charged particles in dispersion. When ZP is high (whether they are positive or negative values) indicates stability between the particles, whereas when the potential is low, particles tend to coagulate/flocculate as attraction exceeds repulsion and the dispersion (Hanaor et al., 2012). For all NP the ZP values were in the range 20e30 mV, which means a moderate stability, and a positive surface charge of the NP (P > 0.05). The positive charge results from the chitosan amino groups available at the surface, which is determined by the degree of neutralization of groups eNH3 by the polyanionic groups from TPP. The NP loaded with PA have a slight
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Table 1 Physical properties, particle size (PS), polidispersity index (PI) and zeta potential (ZP) (means ± SD) and pH of control NP without polyphenols and loaded with polyphenols, both measured at the time of production and after 30 d of storage at 4 C. Nanoparticles
Storage time (d) 0
30
PS (nm) Control LMWC LMWC_PA LMWC_DHBA Control HMWC HMWC_PA HMWC_DHBA
598.3 360.3 373.5 743.6 511.2 593.7
± ± ± ± ± ±
PI 27.1aA 29.7bA 29.8bA 39.5aA 38.4aA 37.2aA
0.33 0.23 0.23 0.27 0.15 0.15
ZP (mV) ± ± ± ± ± ±
0.03aA 0.04bA 0.02bA 0.06aA 0.04bA 0.03bA
22.9 30.0 29.6 25.4 31.1 25.0
± ± ± ± ± ±
2.3aA 1.7bA 2.3bA 2.3bA 3.7bA 2.9bA
pH 5.9 5.2 5.0 5.5 5.5 5.2
± ± ± ± ± ±
0.07aA 0.20aA 0.10aA 0.20aA 0.13aA 0.15aA
EE%
PS (nm)
NA 76.0 69.0 NA 50.0 55.0
494.1 232.6 250.0 950.3 848.0 603.7
± 0.89aA ± 0.24bA ± 0.21aA ± 0.98bA
± ± ± ± ± ±
PI 29.0aA 18.6bA 24.0bA 51.8aB 40.3aB 37.4aA
ZP (mV)
0.29 0.17 0.17 0.24 0.22 0.15
± ± ± ± ± ±
0.03aA 0.02bB 0.02bB 0.03aA 0.03aB 0.03bA
24.7 28.7 25.4 14.1 22.2 24.4
± ± ± ± ± ±
3.1aA 2.9aA 3.2aB 2.8bB 1.9aB 3.3aA
pH 5.9 5.4 5.2 5.8 5.7 5.3
± ± ± ± ± ±
0.12aA 0.19aA 0.13aA 0.06aA 0.16aA 0.07aA
a,b The same letters in the same column correspond to the values of the same parameter but from the different formulations, show that there are no significant differences between the mean values (P > 0.05). A,BThe same letters in the same line correspond to the same value in the initial time and after one month, show that there are no significant differences between the mean values (P > 0.05) (n ¼ 18). LMWC e low molecular weight chitosan; HMWC e high molecular weight chitosan; PA e protocatechuic acid; DHBA e 2,5-dihydroxybenzoic acid; NA e not applicable.
higher value when produced with both types of chitosan (P > 0.05). This can be explained by the stability created between the chitosan and this phenolic acid, which in turn results in a strong attraction by the anionic and cationic charges from the compounds (da Silva et al., 2014). The pH of the chitosan solutions were adjusted to 5.8, which decreased after phenolic acids solutions addition. After one month of storage at 4 C, LMWC NP sizes decreased in contrast with HMWC NP sizes that increased. The highest size increment was registered for HMWC NP without phenolic compound (ca. 950.3 nm) (P < 0.05). In terms of loaded NP, the highest decrease observed was for LMWC_PA (232.6 nm) (P > 0.05). For ZP, the HMWC without phenolic compound and with PA suffered a significant decrease (P < 0.05). A highest value of ca. 28.7 mV was found for NP LMWC_PA and the lowest for NP produced with HMWC without phenolic compound (ca. 14.1 mV). This decrease could be associated with the increase in the NP size, which in turn indicates the beginning of NP agglomeration, compromising the stability. pH does not affect directly the PS, but both properties are related as smaller PS have higher ZP values (Neves, 2013). Also, no significant changes on pH values during storage time were observed (P > 0.05). Hence, these slight variations could not be attributed to the pH, since ZP is dependent on pH of the dispersant. Finally, EE% values were in general high, and the highest value was found for LMWC NP.
3.2. Antioxidant activities Oxygen radical absorption capacity (ORAC) assay reflects the peroxyl radical scavenging activity of the phenolic compounds, and is more relevant to the biological antioxidant activity. In fact, it was clearly demonstrated that the hydrogen atom transfer reaction concurs with the electron transfer reaction and plays a dominant role in the biological redox reactions (Wright et al., 2001). ORAC principle is therefore closely related to the biological functions of
chain-breaking antioxidants. Nanoparticles solutions have been first subjected to a previous centrifugation to guarantee that the free phenolic compound that was not entrapped in the NP would not contribute to the values of antioxidant activities (AA) obtained. Table 2 shows the AA of the NP produced, and is possible to observe that AA of encapsulated phenolic compounds decreases when compared to free phenolic compounds. The results were similar for the two types of NP (LMWC and HMWC), with no statistical significant differences (P > 0.05) between them, except for NP with DHBA (P < 0.05). The highest AA of free phenolic compounds was obtained for DHBA (ca. 0.133 mmol/mL), in contrast with PA (ca. 0.107 mmol/mL). When encapsulated, a decrease in AA of PA was observed in both type of NP. In similar work (da Silva et al., 2014), the values obtained for NP produced with LMWC and rosmarinic acid also decreased 10 times that the ORAC value of the free extract. The encapsulated phenolic compound which almost maintained the original AA value was DHBA in both types of NP produced (LMWC and HMWC). ORAC assay results are originated by a competition between the substrate and the phenolic compound for fluorescein reaction. The values of AA obtained can be related with the EE%, or even with the number of eOH groups of each of the chemical structure of the phenolic compounds used. The NP loaded with PA and DHBA show high AA, indicating that they react easily and rapid with the fluorescein. Protochateutic acid has 3 eOH groups and 2,5-DHBA have 2 groups. The higher AA was obtained for NP with DHBA. When at NP production, the eOH groups react with the positive amino groups of chitosan, hence it is supposed that all the eOH groups of DHBA and PA totally react with the chitosan amino groups.
3.3. Thermal properties In Table 3 are depicted the thermal properties of all NP and controls, showing the enthalpy peaks (DH) and melting
Table 2 Antioxidant activities values (means ± SD) of the polyphenols encapsulated in the NP and of the free polyphenols obtained through the ORAC method and expressed as mmol equivalent of Trolox/mL of sample. Polyphenol (%, m/v)
Free compound or entrapped in nanoparticles
mmol equivalent of Trolox/mL from sample
Protocatechuic acid (0.3%)
Free LMWC HMWC Free LMWC HMWC
0.107 0.039 0.038 0.133 0.110 0.097
2,5-Dihydroxybenzoic acid (0.3%)
± ± ± ± ± ±
0.003a 0.002b 0.003b 0.005a 0.012a 0.005b
a,b The same letters, in the same line show that there are no significant differences between the mean values (P > 0.05) (n ¼ 9). LMWC e low molecular weight chitosan; HMWC e high molecular weight chitosan.
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Table 3 Enthalpy (DH) and melting points ( C) (means ± SD) of NP and for the physical mixtures of the free compounds used in the production of the NP with the two concentrations of tripolyphosphate (TPP). Samples
Properties
Nanoparticles
DH (J/g)
Control LMWC LMWC_PA LMWC_DHBA Control HMWC HMWC_PA HMWC_DHBA Compounds TPP C1 PA þ TPP C1 DHBA þ TPP C1 TPP C2 PA þ TPP C2 DHBA þ TPP C2
Melting points ( C) a
148 469 156 539 778 488
± ± ± ± ± ±
5.7 1.2b 5.8a 0.4bc 0.3c 2.6b
63 50 60 59 71 57
± ± ± ± ± ±
0.6a 0.8b 0.4a 0.8a 2.1c 2.4a
139 404 193 449 477 288
± ± ± ± ± ±
0.5a 7.1b 2.2a 0.6b 1.5b 0.2c
71 65 42 55 59 48
± ± ± ± ± ±
0.5a 0.4ab 0.4c 0.7b 0.4b 1.2c
LMWC e low molecular weight chitosan; HMWC e high molecular weight chitosan; PA e protocatechuic acid; DHBA e 2,5-dihydroxybenzoic acid; TPP e tripolyphosphate; C1 e concentration of 0.29 mg/mL; C2 e concentration of 0.58 mg/mL. a,b,c The same letters in the same column show that there are no significant differences between the mean values (P > 0.05) (n ¼ 9).
temperatures. All NP produced had an endothermic behaviour, i.e. they absorbed energy to melt. In general, LMWC NP melted at higher temperatures than the others and absorbed lower energy. In contrast, HMWC NP melted at lower temperatures and absorbed more energy. HMWC_PA NP were the ones that absorbed more energy (778.3 J/g) and at higher melting temperature (70.8 C) (P < 0.05). All samples had melting points ranging 49e76 C. In the physical mixtures, the two TPP concentrations used in production (0.29 and 0.58 mg/mL, for LMWC and HMWC, respectively) according the chitosan MW were tested. TPP at 0.58 mg/mL (C2) exibited higher values of energy than at 0.29 mg/mL (C1), which influenced the absorbed energy. In NP, the presence of phenolic compound and type of chitosan could had more influence on the thermal behaviour than TPP. Actually, chitosan MW showed the greatest impact, related with the absorbed water evaporation, the presence of acetic acid and concerning the phenolic compounds, to a loss of water internally linked to the molecule (Tagliari et al., 2012) and/or a possible chain relaxation (Polexe et al., 2013). The endothermic behaviour observed followed the found for other chitosan NP produced (Honary et al., 2011; Shinde et al., 2013), which was also justified by the occurrence of water loss. However, there is no consensus regarding the melting temperatures. Some authors assume that occur below 100 C, others endotherms peaks showed at temperatures between 125 and 150 C (Shinde et al., 2013). Nevertheless, in such research works, dried chitosan NP were used instead of wet as ocurred in the present work, which may origin higher melting temperatures of the of NP.
3.4. Effects of the simulated pH and time of digestion on particle sizes and phenolic compounds release Gastrointestinal tract is a selective mucosal barrier which represents a considerable surface area (ca. 200 m2) in the adult human. The pH variance within the gastrointestinal compartments can affect NP aggregation status and alter surface chemistry, particularly in NP where ZP is highly dependent on pH (e.g. chitosan). Limited work has been done in understanding parameters of dissolution in gastrointestinal fluids that may help predict uptake and blood concentrations (Loretz and Bernkop-Schnürch, 2007). Fig. 1 represents the evolution of PS and % of phenolic compound release at the different stages of digestion (stomach and intestine).
Fig. 1. Evolution of the NP particle sizes (PS) (y2 axis, round shapes) and polyphenol release (%) (y1 axis, lines) of the NP produced with a) LMWC and b) HMWC without polyphenol (B, ——), with PA (C, .....) and with DHBA ( , e e) when exposed to the simulated GIT conditions.
At a first look, for LMWC NP can be detected a lower % release of phenolic compounds and increase in the PS. The increment of PS is more than 6 times than HMWC NP. In addition, the most relevant changes in PS and release of phenolic compound occurs in the intestinal stage. The NP without phenolic compound are the ones that suffer higher increment in their sizes and in the % of release. Specifically, at stomach conditions for LMWC NP, no significant changes on PS values (P > 0.05) were observed. The PS of HMWC NP increased when at stomach conditions (Fig. 1b). All LMWC NP loaded with phenolic compounds released ca. 40% at stomach conditions. The same observation was detected for the HMWC NP. Nevertheless, at intestine, the NP produced with LMWC increased their sizes significantly (P < 0.05). The NP loaded with 2,5-DHBA showed the higher sizes (ca. 5500 nm) (Fig. 1a). All LMWC NP continues to release phenolic compounds (ca. 70e80%), with exception of the NP loaded with PA (Fig. 1a). The HMWC NP maintained or increase their sizes at intestine stage, with exception of the NP loaded with PA. In terms of release profiles, all HMWC NP continued to release phenolic compound at intestinal stage reaching ca. 80%. In Table 4 are represented the other measured physical parameters, the ZP of all NP decreased along digestion. Especially in intestine, a decrease in stability was observed, owing to the NP flocculation and agglomeration (P < 0.05). This decrease is higher for NP produced with LMWC than with HMWC. The instability of LMWC NP produced in intestine is confirmed when looking to the PI results, that are much higher than the ones obtained HMWC NP. This indicates that at intestine the LMWC NP underwent changes
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Table 4 Physical properties, polydispersity index (PI) and zeta potential (ZP) (mean values ± SD) of control NP without polyphenols and loaded with polyphenols, after exposition to the simulated stomach and small intestine (duodenum) conditions. Samples
Initial time
Stomach
PI Control LMWC LMWC_PA LMWC_DHBA Control HMWC HMWC_PA HMWC_DHBA
0.29 0.20 0.20 0.25 0.13 0.15
ZP (mV) ± ± ± ± ± ±
aA
0.01 0.09bA 0.03bA 0.07aA 0.01bA 0.03bA
24.7 32.3 30.2 26.3 34.1 26.0
± ± ± ± ± ±
End of digestion
PI aA
0.8 0.4bA 1.6bA 0.8aA 1.2bA 1.3aA
0.30 0.30 0.20 0.30 0.10 0.20
ZP (mV) ± ± ± ± ± ±
aAD
0.02 0.02aBD 0.03bAD 0.02aAD 0.01bAD 0.03bAD
21.4 22.4 24.5 22.3 23.9 20.2
± ± ± ± ± ±
PI aAC
0.4 0.8aBC 0.5aBC 0.6aAC 0.8aBC 0.9aBC
0.69 0.58 0.59 0.13 0.23 0.12
ZP (mV) ± ± ± ± ± ±
aBE
0.02 0.04aCE 0.02aBE 0.02bBE 0.01bBE 0.04bAE
15.6 16.9 16.8 19.4 18.0 17.5
± ± ± ± ± ±
2.8aBD 1.7aBD 0.6aBD 2.1aBC 0.9aBD 2.2aBC
a,b
The results represent the average and the standard deviation of samples in triplicate (n ¼ 3). The same letters in the same column show that there are no significant differences between the mean values (P > 0.05). A,B,C,D,EThe same letters, in the same line are related to the same parameter in the different stages, show that there are no significant differences between the values (P > 0.05) (n ¼ 16). LMWC e low molecular weight chitosan; HMWC e high molecular weight chitosan; PA e protocatechuic acid; DHBA e 2,5-dihydroxybenzoic acid; NA e not applicable.
that origin the formation of several families of different sizes, increasing the PI values. Nevertheless, these changes were showed that not affect the release of phenolic compound at the intestine, which is similar to the ones produced with HMWC, with exception of LMWC_PA. Also, the decrease in ZP can also be related with the pH increase at the simulation of intestinal conditions. The ZP is no fixed value, but dependent on a variety of parameters, e.g. solvent, electrolyte concentration, nature of electrolyte, pH of the solvent and temperature. The surface charge of the particle is directly influenced by the pH, due to protonation and deprotonation of the used particle matrix. Regarding the PS which is most suitable for NP oral administration, the produced chitosan NP had a medium size under 500 nm and when reaching the intestine this value increased. This can be a positive feature, as studies have demonstrated that such NP are capable of translocation all throughout the mucous barrier of intestinal tissue and collaborate with the underlying absorptive epithelial cells. The PS influences absorption, e.g. greater absorption of smaller (50 nm) polystyrene particles was showed when compared to larger (100 nm) particles. The largest particles in this study (300 nm) were not absorbed. Additionally, larger particles remained within the sub-mucosa or GALT of the intestine and colon, while smaller particles entered the bloodstream and accumulated in the liver and spleen (Jani et al., 1990). Additionally, this can be useful when high phenolic compound concentrations are at the sites of absorption, and when the ZP is positive can also play a role in adhesion of the NP to intestinal surfaces (Dube et al., 2010). Nanoparticles are sensible to each digestion stage pH. This could be due to chitosan pKa value and then the electrostatic interactions between the chains (Piai et al., 2009). When at lower pH than the pKa of chitosan (6.5) as in the case of stomach, a higher number of positive charges exists, since the amino groups from chitosan became protonated. For higher pH, as in case of intestine, there is a decrease of those groups. Thus, an inferior pH is favourable in maintaining the NP stable, and their PS, PI and ZP, as shown by the results. At the intestine the pH is near 7, and with high pH than the pka of chitosan there is a weakening and/or rupture from the connections leading to coagulation/flocculation, as shown by our results, which consequently lead to a higher release of phenolic compound (Piai et al., 2009). The epithelial cells in the various tissues including gastrointestinal tract, carry a negative charge on their surface due to the presence negatively charged residues of proteins in the cells outer membrane and the selective active ion pumps of the membrane. Therefore, all epithelia are selective to positively charged solutes (Rojanasakul et al., 1992). The positively charged colloidal drug carriers, such as the ones produced here, may increase the
permeability and potential uptake of slightly soluble drugs when compared with neutral or negatively charged ones, thus improving their bioavailability and reducing their side effects. The positive charge on the NP surface can enhance paracellular transport of drugs through interactions between the negatively-charged cell membrane and the positive charges of the polymer, or by complexing Ca2þ involved in the structure of tight junctions (Carino and Mathiowitz, 1999). 3.5. Conclusions The present study can be used to predict the functionality of NP loaded with natural extracts rich in phenolic acids. These NP could be used as functional ingredients to be further incorporated in food matrices in order to increase their nutritional content. The phenolic acids loaded in chitosan NP have several proved health benefits associated, such as antioxidant, anti-inflammatory, anti-carcinogenic or antimicrobial (Boaventura et al., 2013). In order to protect these compounds and to enhance their bioavailability they were entrapped in chitosan NP. Chitosan NP were produced with protocatechuic acid and 2,5-dihydroxybenzoic acid, with PS of ca. 300e600 nm, monodisperse distribution and a moderate stability. The highest EE% values were obtained for NP produced with LMWC. During storage at 4 C for 30 days, occurs the decrease of LMWC NP sizes and increase HMWC NP sizes. Decrease of zeta potential values were also registered depending on the phenolic compound loaded. Chitosan MW influences the thermal behaviour of the NP. Phenolic compounds loading do not interfere with the thermal stability of the LMWC NP but affects the thermal behaviour of the NP produced with HMWC. During digestion, at stomach, the LMWC NP releases a lower % of phenolic compounds than HMWC and with no changes on PS. At small intestine, occurs an increase of LMWC NP size and a high % of phenolic compounds release from HMWC NP. About 80% of 2,5DHBA is released independently of the type of NP, while for PA the LMWC NP released ca. 40%. With all these results, is possible to conclude that for 2,5-DHBA the best delivery systems are LMWC NP, while for PA the best ones are the produced with HMWC. The main property of the phenolic compounds, which is their antioxidant activity is shown to be preserved after the NP production. These are stable at refrigerated temperatures and the final pH is at the physiological state. If these NP are incorporated in a food matrix, the loaded compounds will be protected from the interactions with other matrix compounds and from the negative processing parameters. When ingested, NP will pass by stomach acidic conditions, preserving the phenolic acids integrity and when reaching the intestine will adhere to the intestinal epithelium and the phenolic acids will be released and adsorbed to blood stream.
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Acknowledgements ~o para a Cie ^ncia e TecnoAuthors acknowledge FCT e Fundaça logia for funding research work trough project NANODAIRY (PTDC/ AGR-ALI/117808/2010) and by National Funds from FCT through project PEst-OE/EQB/LA0016/2013. Author Ana Raquel Madureira acknowledges FCT for the post-doctoral scholarship SFRH/BPD/ 71391/2010. References Agrawal, P., Strijkers, G.J., Nicolay, K., 2010. Chitosan-based systems for molecular imaging. Adv. Drug Deliv. Rev. 62 (1), 42e58. ^ncio, E.S., Maraschin, M., Murakami, F.S., Boaventura, B.C.B., Murakami, A.N.N., Prude Amante, E.R., Amboni, R.D.d.M.C., 2013. Enhancement of bioactive compounds content and antioxidant activity of aqueous extract of mate (Ilex paraguariensis A. St. Hil.) through freeze concentration technology. Food Res. Int. 53 (2), 686e692. Calvo, P., Remunan-Lopez, C., Vila-Jato, J., Alonso, M., 1997. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J. Appl. Polym. Sci. 63 (1), 125e132. Carino, G.P., Mathiowitz, E., 1999. Oral insulin delivery1. Adv. Drug Deliv. Rev. 35 (2e3), 249e257. da Silva, S.B., Amorim, M., Fonte, P., Madureira, R., Ferreira, D., Pintado, M., Sarmento, B., 2014. Natural extracts into chitosan nanocarriers for rosmarinic acid drug delivery. Pharm. Biol. 0, 1e11. Dash, M., Chiellini, F., Ottenbrite, R., Chiellini, E., 2011. Chitosan - a versatile semisynthetic polymer in biomedical applications. Prog. Polym. Sci. 36 (8), 981e1014. mez-Cordove s, C., Bartolome , B., 2004. Extending applicability of the Davalos, A., Go oxygen radical absorbance capacity (ORAC-fluorescein) assay. J. Agric. Food Chem. 52 (1), 48e54. Dehnad, D., Emam-Djomeh, Z., Mirzaei, H., Jafari, S.-M., Dadashi, S., 2014. Optimization of physical and mechanical properties for chitosanenanocellulose biocomposites. Carbohydr. Polym. 105, 222e228. Dube, A., Nicolazzo, J.A., Larson, I., 2010. Chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (þ)-catechin and ()-epigallocatechin gallate. Eur. J. Pharm. Sci. 41 (2), 219e225. Dudhani, A.R., Kosaraju, S.L., 2010. Bioadhesive chitosan nanoparticles: preparation and characterization. Carbohydr. Polym. 81 (2), 243e251. Ha, H.-K., Kim, J.W., Lee, M.-R., Lee, W.-J., 2013. Formation and characterization of quercetin-loaded chitosan oligosaccharide b-lactoglobulin nanoparticle. Food Res. Int. 52 (1), 82e90. Hanaor, D., Michelazzi, M., Leonelli, C., Sorrell, C.C., 2012. The effects of carboxylic acids on the aqueous dispersion and electrophoretic deposition of ZrO2. J. Eur. Ceram. Soc. 32 (1), 235e244. Honary, S., Ghajar, K., Khazaeli, P., Shalchian, P., 2011. Preparation, characterization and antibacterial properties of silver-chitosan nanocomposites using different molecular weight grades of chitosan. Trop. J. Pharm. Res. 10 (1). Huh, A.J., Kwon, Y.J., 2011. “Nanoantibiotics”: a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era. J. Control. Release 156 (2), 128e145. Jani, P., Halbert, G.W., Langridge, J., Florence, A.T., 1990. Nanoparticle uptake by the rat gastrointestinal mucosa: quantitation and particle size dependency. J. Pharm. Pharmacol. 42 (12), 821e826. Juurlink, B.H., Azouz, H.J., Aldalati, A.M., AlTinawi, B.M., Ganguly, P., 2014.
Hydroxybenzoic acid isomers and the cardiovascular system. Nutr. J. 13 (1), 63. Kakkar, S., Bais, S., 2014. A review on protocatechuic acid and its pharmacological potential. ISRN Pharmacol. 2014. Kean, T., Thanou, M., 2010. Biodegradation, biodistribution and toxicity of chitosan. Adv. Drug Deliv. Rev. 62 (1), 3e11. Loretz, B., Bernkop-Schnürch, A., 2007. In vitro cytotoxicity testing of non-thiolated and thiolated chitosan nanoparticles for oral gene delivery. Nanotoxicology 1 (2), 139e148. Madureira, A.R., Amorim, M., Gomes, A.M., Pintado, M.E., Malcata, F.X., 2011. Protective effect of whey cheese matrix on probiotic strains exposed to simulated gastrointestinal conditions. Food Res. Int. 44 (1), 465e470. Mohammadi, A., Jafari, S.M., Assadpour, E., Esfanjani, A.F., 2015a. Nano-encapsulation of olive leaf phenolic compounds through WPC-pectin complexes and evaluating their release rate. Int. J. Biol. Macromol. (in press). Mohammadi, A., Jafari, S.M., Esfanjani, A.F., Akhavan, S., 2015b. Application of nanoencapsulated olive leaf extract in controlling the oxidative stability of soybean oil. Food Chem. (in press). Mukhopadhyay, P., Mishra, R., Rana, D., Kundu, P.P., 2012. Strategies for effective oral insulin delivery with modified chitosan nanoparticles: a review. Prog. Polym. Sci. 37 (11), 1457e1475. ~o e caracterizaç~ Neves, A.L.d.P., 2013. Preparaça ao de nanopartículas de quitosana incorporadas com zinco com potential atividade cicatrizante e antimicrobiana. Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A., Deemer, E.K., 2002. Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative study. J. Agric. Food Chem. 50 (11), 3122e3128. Piai, J.F., Rubira, A.F., Muniz, E.C., 2009. Self-assembly of a swollen chitosan/chondroitin sulfate hydrogel by outward diffusion of the chondroitin sulfate chains. Acta Biomater. 5 (7), 2601e2609. Polexe, R.C., Terrat, C., Verrier, B., Cuvillier, A., Champier, G., Delair, T., 2013. Elaboration of targeted nanodelivery systems based on colloidal polyelectrolyte complexes (PEC) of chitosan (CH)-dextran sulphate (DS). Eur. J. Nanomed. 5 (1), 39e49. Pool, H., Quintanar, D., de Dios Figueroa, J., Mano, C.M., Bechara, J.E.H., Godínez, L.A., Mendoza, S., 2012. Antioxidant effects of quercetin and catechin encapsulated into PLGA nanoparticles. J. Nanomater. 2012, 86. Rojanasakul, Y., Wang, L.-Y., Bhat, M., Glover, D.D., Malanga, C.J., Ma, J.K., 1992. The transport barrier of epithelia: a comparative study on membrane permeability and charge selectivity in the rabbit. Pharm. Res. 9 (8), 1029e1034. Severino, P., Andreani, T., Macedo, A.S., Fangueiro, J.F., Santana, M.H.A., Silva, A.M., Souto, E.B., 2011. Current state-of-art and new trends on lipid nanoparticles (SLN and NLC) for oral drug delivery. J. Drug Deliv. 2012. Shinde, R.N., Pandey, A., Acharya, R., Guin, R., Das, S., Rajurkar, N., Pujari, P., 2013. Chitosan-transition metal ions complexes for selective arsenic (V) preconcentration. Water Res. 47 (10), 3497e3506. Tagliari, M.P., Granada, A., Kuminek, G., Stulzer, H.K., Silva, M.A., 2012. Development and validation of analytical method for determination of glycyrrhizic acid, salicylic acid, and caffeine in chitosan-alginate nanoparticles. Quím. Nova 35 (6), 1228e1232. Tang, D.-W., Yu, S.-H., Ho, Y.-C., Huang, B.-Q., Tsai, G.-J., Hsieh, H.-Y., Sung, H.-W., Mi, F.-L., 2013. Characterization of tea catechins-loaded nanoparticles prepared from chitosan and an edible polypeptide. Food Hydrocoll. 30 (1), 33e41. Wright, J.S., Johnson, E.R., DiLabio, G.A., 2001. Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. J. Am. Chem. Soc. 123 (6), 1173e1183. Zou, T., Percival, S.S., Cheng, Q., Li, Z., Rowe, C.A., Gu, L., 2012. Preparation, characterization, and induction of cell apoptosis of cocoa procyanidinsegelatinechitosan nanoparticles. Eur. J. Pharm. Biopharm. 82 (1), 36e42.
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Chitosan nanoparticles loaded with 2,5-dihydroxybenzoic acid and protocatechuic acid: Properties and digestion Ana Raquel Madureira 1, Adriana Pereira 1, Manuela Pintado* rio Associado, Escola Superior de Biotecnologia, Universidade Cato lica Portuguesa/Porto, CBQF e Centro de Biotecnologia e Química Fina e Laborato ~o Vital, Apartado 2511, 4202-401 Porto, Portugal Rua Arquiteto Loba
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 September 2015 Received in revised form 3 November 2015 Accepted 6 November 2015 Available online 18 November 2015
Research efforts on the production of chitosan nanoparticles (NP) as delivery systems of bioactive compounds such as polyphenols have been made along the last decade. Nevertheless, the effect of the phenolic compound structure in the production of these NP was never evaluated so far. Low and high molecular weights chitosan (LMWC and HMWC) NP loaded with the phenolic acids, protocatechuic (PA) and 2,5-dihydroxybenzoic acids (2,5-DHBA) were produced by ionic gelation. Antioxidant activities were determined by ORAC assay. Physical and thermal properties were evaluated by dynamic light scattering (DLS) and differential scanning calorimetry (DSC), respectively. Stability and release of phenolic acids during simulation of gastrointestinal tract (GIT) conditions were also assessed. Nanoparticles sizes ranged from 300 to 600 nm and maintained stable during storage at 4 C during 30 d. Antioxidant activities of the phenolic acids decreased when loaded in the NP. High molecular weight chitosan NP adsorbed higher energy and melted at lower temperatures than LMWC NP. Nanoparticles produced with HMWC released higher phenolic acids % at GIT simulated conditions and with slight increases in their sizes. The most proper systems for delivery of PA and 2,5-DHBA were found to be LMWC and HMWC NP, respectively. These NP could be used to as functional food ingredients or as models for production of phenolic acids-rich extracts NP for future incorporation in food matrices. © 2015 Published by Elsevier Ltd.
Keywords: Chitosan Nanoparticles Phenolic compounds Gastrointestinal tract
1. Introduction Nowadays, there is a rising attention in the application of nanotechnology in food industry, namely in the production of nanoparticles (NP) as new functional food ingredients and as a way to improve the bioactive compounds functionality with proved benefits to human health. These new structures can protect the bioactive compounds during food processing and storage, and during passage by the gastrointestinal tract (GIT) (Ha et al., 2013; Zou et al., 2012). The polymeric NP are colloidal carriers with sizes ranging from 10 to 1000 nm. Great part of the studies use chitosan as entrapment material for NP production and bioactive/ drug compounds controlled delivery, protection from degradation and oxidation, production of nanocomposites, among other applications (Agrawal et al., 2010; Dehnad et al., 2014; Huh and Kwon, 2011). Chitosan is an abundant natural biopolymer obtained from
* Corresponding author. E-mail address: [email protected] (M. Pintado). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.jfoodeng.2015.11.007 0260-8774/© 2015 Published by Elsevier Ltd.
chitin deacetylation, and acquired from crustaceans shells or as a natural component of specific fungi genus (Mucoraceae) (Agrawal et al., 2010; Dash et al., 2011). This polymer is polycationic with constant acidity (pKa ¼ 6.5) and insoluble at neutral pH (Agrawal et al., 2010). Chitosan is claimed as non-toxic, biologically compatible polymer, non-immunogenic and highly antimicrobial (Kean and Thanou, 2010; Mukhopadhyay et al., 2012). The difference in the relative proportions of N-acetyl-D-glucosamine and Dglucosamine residues, provide specific structural changes, which give rise to chitosans that are distinguished on the basis of their degree of deacetylation (DD) and molecular weight. The polymers have different molecular weights (50e2000 kDa), viscosity and DD (40e98%). Commercial chitosans are sold as high and low molecular weight chitosans, and characterized containing between 20 and 190 kDa with DD > 75% and between 190 and 375 kDa with DD > 75%, respectively. On the other hand, there is a huge interest in encapsulation of phenolic acids or their extracts, due to its high biological activity, namely on the antioxidant capacity found in this large group of compounds present in plants (Mohammadi et al., 2015a, 2015b).
A.R. Madureira et al. / Journal of Food Engineering 174 (2016) 8e14
The encapsulation of these compounds, especially the ones with hydrophobic character is justified by their higher level of reactivity, chemical susceptibility to the harsh conditions of the GIT, low oral bioavailability and intestinal absorption (Tang et al., 2013). They must be enough time in the intestinal lumen to adhere to cell apical surface and then, be transcytosed by intestinal cells. The phenolic compounds used in this study were protocatechuic acid (PA) and the 2,5-dihydroxybenzoic acid (2,5-DHBA) (also known as gentisic acid). Protocatechuic acid is the major metabolite of antioxidant phenolic compounds found in green tea, and has been described to possess several bioactivities such as antimicrobial, anticancer, anti-ulcerogenic, anti-ageing (Kakkar and Bais, 2014). In addition, 2,5-DHBA has been characterized as antimutagenic, anti-inflammatory and antimicrobial (Boaventura et al., 2013). Is widely present in regular foods, including cereals such as wheat and rye, actinidia (e.g., kiwi) fruit, aloe vera, mushrooms as well as other sources (Juurlink et al., 2014). Besides their
EE% ¼
9
2.2. Determination of the physical properties Physical properties such as particle size (PS), polydispersity index (PI) and zeta potential (ZP) were evaluated using the NP solutions, by dynamic light scattering (DLS) technique with a ZetaPALS, Zeta Potential Analyzer (Holtsville, NY, USA). The parameters were measured at the initial production time of NP and also after 1 month of storage time at 4 C. All assays were performed in triplicate. 2.3. Determination of NP entrapment efficiencies The NP suspensions were filtrated by a centrifugal filter units with a cut-off of 3 K (Amicon® Ultra-4, Millipore), and then centrifugated at 37,732 g, 25 C, for 1 h. Samples supernatants were subject to analyses by HPLC method (all assays were done in triplicate). The entrapment efficiency (EE%) was determined as follows:
Total amount of polyphenol Total amount of polphenols in supernatant Total amount of polyphenols
variety in terms of natural sources, these two phenolic acids are different in terms of their chemical structure, in what concerns the number of eOH groups which are 3 for PA and 2 for 2,5-DHBA. Thus, the aim of the present study was the development of model carrier systems of two phenolic acids with different structures, PA and 2,5-DHBA. These were produced with LMWC and HMWC and characterized for their physical, antioxidant and thermal properties. Afterwards their antioxidant activity and stability during a simulated pH gastrointestinal tract were also evaluated. 2. Material and methods 2.1. Preparation of NP The concentrations of chitosan and phenolic compounds used in the production of the NP were the following: 0.2% (m/v) low molecular weight chitosan (LMWC), 0.4% (m/v) high molecular weight chitosan (HMWC), 0.3% (m/v) PA (LMWC_PA and HMWC_PA), and 0.3% (m/v) DHBA (LMWC_DHBA and HMWC_DHBA). All compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). Production followed the ionic gelation method described by da Silva et al. (2014), but with some modifications. Chitosan solutions were first dissolved in acetic acid (where the concentration of acetic acid was 1.75 times higher than chitosan concentration). Hence, acetic acid at 3.5 mg/mL was used for LMWC at 0.2% (m/v) and 7 mg/mL for 0.4% of HMWC. pH solution was adjusted to the physiologic value of 5.8 with NaOH 1 M. Afterwards, the phenolic compounds were dissolved in ultra pure water. Sodium tripolyphosphate (TPP) (Sigma-Aldrich) was prepared at different concentrations according the chitosan MW at a proportion [chitosan:TPP] of 7:1. Four mL of chitosan solution were placed under gentle stirring and 1 mL of phenolic compound was slowly added drop wise, followed by 2 mL of TPP also drop wise, at room temperature. Nanoparticles controls without phenolic compound were also produced by the same method. At the end, the pH of the final NP solutions loaded with phenolic compounds and controls without phenolic compounds were measured and then stored at 4 C.
2.4. Determination of antioxidant activity The antioxidant activity of loaded phenolic compounds NP was determined by oxygen radical absorbance capacity (ORAC), a fluorometric method that measures in vitro the antioxidant activity through fluorescein (Sigma, Steinheim, Germany) oxidation by the peroxilos radicals in situ by thermal decomposition from AAPH (Aldrich, Steinheim, Germany). This method measures the ability of oxygen radical absorption through the method developed by Ou valos et al. (2004) using a microet al. (2002) and adapted by Da plate fluorescence reader. Twenty microliters of solution (NP and phenolic compounds) or sodium phosphate buffer (0.1 M, pH 7) in case of control, were mixed with 120 mL of fluorescein and preincubated for 10 min at 40 C. After this step, 60 mL of AAPH was rapidly added in a polystyrene black microplate (Nunc, Denmark) and incubated at 40 C until a total of 137 min was completed (with the 10 min included). The measurement was performed in a fluorometer FluoSTAR OPTIMA (BMG LABTECH GmbH, Offenburg, Germany) using a wavelength of excitation at 485 nm and the emission one at 520 nm, with a Fluostar Control version 1.32 R2 software. All analyses were performed in triplicate. 2.5. Determination of thermal properties The NP thermal properties were evaluated by differential scanning calorimetry (DSC) using a DSC-60 (Shimadzu, Maryland, DC, USA). Nanoparticle suspensions were concentrated by centrifugation at 37,732 g, 25 C, 1 h and 3 mg of the pellet were placed in an aluminium pans hermetically sealed. The thermal behaviour was determined in the range 20e200 C, at a heating rate of 10 C/ min. Optimal melting temperatures and enthalpies were calculated by the equipment software (ta60 version 2.10, DSC software, Shimadzu). The mixtures of the phenolic compounds and TPP compound at the two different concentrations used (0.29 and 058 mg/ mL) were also tested, to evaluate the effect of these compounds in the thermal properties of the NP. All NP samples were tested as well as the controls (without phenolic compounds) and raw compounds used in the NP production. All assays were performed in quadruplicate.
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2.6. In vitro simulated gastrointestinal tract conditions A simulation of pH, temperature, movements and time of digestion of human stomach and duodenum was performed as described elsewhere (Madureira et al., 2011). Stomach conditions were simulated by suspending the concentrated NP in NaCl buffer solution 0.5% (w/v) (Panreac, Barcelona, Spain) at a proportion of 1:1. pH was adjusted to 1.5e2 with 9 M HCl (Merck, Darmstadt, Germany). Samples were put into a water bath at 37 C (Clifton Shaking Baths NE5 Series, Weston-super-Mare, United Kingdom), with homogenization at 150 rpm (to simulate the stomach peristaltic movements) for 60 min. To simulate intestine conditions, samples were taken from the bath and pH was increased until 7, with 1 M NaHCO3 (Pronalab®, Lisbon, Portugal) and left 2 h at 37 C at 90 rpm. Controls were made at the same time without simulated conditions of stomach and intestine. At times 0, 1 (stomach) and 3 (intestine) h, samples were taken to determine the physical properties and to quantify the concentration of phenolic compound that was released by the NP during the simulation. The % of phenolic compounds released by the NP during each step was calculated as follows:
% Release ¼
ðCP CPr Þ 100% CP
where CP is the concentration of phenolic compound loaded by the NP, and CPr is the concentration of phenolic compound quantified in each simulation stage. The CP is calculated as follows:
CP ¼
ðEE% x CPi Þ 100
where EE% is the entrapment efficiency percentage, and CPi is the initial concentration of phenolic compound used to produce the NP. CPr is the phenolic compound concentration released by the NP during the simulation steps, and is calculated as follows:
CPr ¼ Ctotal free polyphenols Cpolyphenol not entrapped 2.7. Quantification of phenolic compounds by HPLC Phenolic compounds were chromatographically separated through the mobile phase A e water, methanol (Panreac, Barcelona, Spain) and formic acid (Merck, Darmstadt, Germany) (92.5:5:2.5, v/ v/v) - and mobile phase B e methanol, water and formic acid (92.5:5:2.5, v/v/v). The elution conditions were: a gradient starting at 40% mobile phase B until 2 min at a continuous flow of 0.5 mL/ min, between 2 and 17 min with 50% of mobile phase B, lastly between 17 and 25 min the gradient change with 80% of mobile phase B. The results were detected with wavelengths ranging from 280 to 320 nm. The peaks were reached at 320 nm (phenolic acids), and were analysed by comparison of retention time and spectra with pure phenolic compounds. The calibration curve was established for all of the pure phenolic compounds using a concentration range from 0.04 to 24 mg/mL. 2.8. Statistical analyses The statistical analysis to assay the several parameters and the achieved results was obtained by ShapiroeWilk test, Levene test, tstudent test, through running one-way ANalysis Of VAriance (ANOVA) carried out with the support of SPSS (v. 19, Chicago, IL, USA). Differences between means of the physical properties of the different NP produced at the time of production and after 1 month of storage, as well the antioxidant activities were analysed. The
differences between means of the physical properties of the NP at the same stage of simulated GIT conditions, and between the means of the same physical property but at the three stages of the simulated GIT conditions were analysed. The confidence level used in these tests was 95%. 3. Results and discussion Six different sets of chitosan NP were produced using two types of chitosan e LMWC and HMWC combined with two phenolic compounds e PA, 2,5-DHBA and fully characterized in terms of bioactivity and physical and structural properties. Production of NP was done, including besides chitosan and phenolic compounds, a cross-linking agent TPP. This cross-linking agent had the function to create inter and intra molecular reactions with amino groups positively loaded with chitosan, so that the gelation process can occur (Calvo et al., 1997; da Silva et al., 2014). The TPP and phenolic compounds interact with chitosan amine groups and the entrapment of compounds occurs efficiently. At the same time a competitive interaction between the groups of phenolic compounds (eOH) and the groups of TPP, by the protonated amino groups of chitosan is observed, resulting in low levels of productive particle formation when compared to the control chitosan NP (without phenolic compound entrapped). This event can be intensified with higher phenolic compound concentrations (Dudhani and Kosaraju, 2010). 3.1. Physical properties and entrapment efficiencies In Table 1 are depicted the means values of the physical properties and pH of the control NP and NP loaded with phenolic compounds measured at 25 C at the time of production and after one month of storage time at 4 C. In general, the highest sizes were obtained for NP produced with HMWC, specifically for controls with no loaded phenolic compound (ca. 743 nm). These sizes could be due to the MW and higher concentrations of chitosan, and TPP used in the formulation (Calvo et al., 1997; Neves, 2013; Pool et al., 2012). The same trend was observed for control LMWC NP (ca. 598 nm). This can be explained by the higher PI values, since a cluster formation can occur resulting in particles with different sizes (Hanaor et al., 2012). The NP produced with HMWC loaded with PA presented the lowest sizes (ca. 511 nm) (P < 0.05). The sizes obtained are the desired ones, since predicting the use of these NP for oral administration is recommended a desirable size 300 nm, in order to not exceed the cell gut epithelium size, avoiding passage to the bloodstream and toxicity of NP by accumulation in the organs (Severino et al., 2011). In what concerns PI this parameter is indicative of the width from the peaks that corresponds to the particle size, and a high polydispersity shows the existence of particles families with different sizes, which may mean the occurrence of certain aggregation (Hanaor et al., 2012). In general, all samples showed PI values near 0.3, which indicates a monodisperse distribution of the NP sizes. Zeta potential is the degree of repulsion between adjacent, similarly charged particles in dispersion. When ZP is high (whether they are positive or negative values) indicates stability between the particles, whereas when the potential is low, particles tend to coagulate/flocculate as attraction exceeds repulsion and the dispersion (Hanaor et al., 2012). For all NP the ZP values were in the range 20e30 mV, which means a moderate stability, and a positive surface charge of the NP (P > 0.05). The positive charge results from the chitosan amino groups available at the surface, which is determined by the degree of neutralization of groups eNH3 by the polyanionic groups from TPP. The NP loaded with PA have a slight
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11
Table 1 Physical properties, particle size (PS), polidispersity index (PI) and zeta potential (ZP) (means ± SD) and pH of control NP without polyphenols and loaded with polyphenols, both measured at the time of production and after 30 d of storage at 4 C. Nanoparticles
Storage time (d) 0
30
PS (nm) Control LMWC LMWC_PA LMWC_DHBA Control HMWC HMWC_PA HMWC_DHBA
598.3 360.3 373.5 743.6 511.2 593.7
± ± ± ± ± ±
PI 27.1aA 29.7bA 29.8bA 39.5aA 38.4aA 37.2aA
0.33 0.23 0.23 0.27 0.15 0.15
ZP (mV) ± ± ± ± ± ±
0.03aA 0.04bA 0.02bA 0.06aA 0.04bA 0.03bA
22.9 30.0 29.6 25.4 31.1 25.0
± ± ± ± ± ±
2.3aA 1.7bA 2.3bA 2.3bA 3.7bA 2.9bA
pH 5.9 5.2 5.0 5.5 5.5 5.2
± ± ± ± ± ±
0.07aA 0.20aA 0.10aA 0.20aA 0.13aA 0.15aA
EE%
PS (nm)
NA 76.0 69.0 NA 50.0 55.0
494.1 232.6 250.0 950.3 848.0 603.7
± 0.89aA ± 0.24bA ± 0.21aA ± 0.98bA
± ± ± ± ± ±
PI 29.0aA 18.6bA 24.0bA 51.8aB 40.3aB 37.4aA
ZP (mV)
0.29 0.17 0.17 0.24 0.22 0.15
± ± ± ± ± ±
0.03aA 0.02bB 0.02bB 0.03aA 0.03aB 0.03bA
24.7 28.7 25.4 14.1 22.2 24.4
± ± ± ± ± ±
3.1aA 2.9aA 3.2aB 2.8bB 1.9aB 3.3aA
pH 5.9 5.4 5.2 5.8 5.7 5.3
± ± ± ± ± ±
0.12aA 0.19aA 0.13aA 0.06aA 0.16aA 0.07aA
a,b The same letters in the same column correspond to the values of the same parameter but from the different formulations, show that there are no significant differences between the mean values (P > 0.05). A,BThe same letters in the same line correspond to the same value in the initial time and after one month, show that there are no significant differences between the mean values (P > 0.05) (n ¼ 18). LMWC e low molecular weight chitosan; HMWC e high molecular weight chitosan; PA e protocatechuic acid; DHBA e 2,5-dihydroxybenzoic acid; NA e not applicable.
higher value when produced with both types of chitosan (P > 0.05). This can be explained by the stability created between the chitosan and this phenolic acid, which in turn results in a strong attraction by the anionic and cationic charges from the compounds (da Silva et al., 2014). The pH of the chitosan solutions were adjusted to 5.8, which decreased after phenolic acids solutions addition. After one month of storage at 4 C, LMWC NP sizes decreased in contrast with HMWC NP sizes that increased. The highest size increment was registered for HMWC NP without phenolic compound (ca. 950.3 nm) (P < 0.05). In terms of loaded NP, the highest decrease observed was for LMWC_PA (232.6 nm) (P > 0.05). For ZP, the HMWC without phenolic compound and with PA suffered a significant decrease (P < 0.05). A highest value of ca. 28.7 mV was found for NP LMWC_PA and the lowest for NP produced with HMWC without phenolic compound (ca. 14.1 mV). This decrease could be associated with the increase in the NP size, which in turn indicates the beginning of NP agglomeration, compromising the stability. pH does not affect directly the PS, but both properties are related as smaller PS have higher ZP values (Neves, 2013). Also, no significant changes on pH values during storage time were observed (P > 0.05). Hence, these slight variations could not be attributed to the pH, since ZP is dependent on pH of the dispersant. Finally, EE% values were in general high, and the highest value was found for LMWC NP.
3.2. Antioxidant activities Oxygen radical absorption capacity (ORAC) assay reflects the peroxyl radical scavenging activity of the phenolic compounds, and is more relevant to the biological antioxidant activity. In fact, it was clearly demonstrated that the hydrogen atom transfer reaction concurs with the electron transfer reaction and plays a dominant role in the biological redox reactions (Wright et al., 2001). ORAC principle is therefore closely related to the biological functions of
chain-breaking antioxidants. Nanoparticles solutions have been first subjected to a previous centrifugation to guarantee that the free phenolic compound that was not entrapped in the NP would not contribute to the values of antioxidant activities (AA) obtained. Table 2 shows the AA of the NP produced, and is possible to observe that AA of encapsulated phenolic compounds decreases when compared to free phenolic compounds. The results were similar for the two types of NP (LMWC and HMWC), with no statistical significant differences (P > 0.05) between them, except for NP with DHBA (P < 0.05). The highest AA of free phenolic compounds was obtained for DHBA (ca. 0.133 mmol/mL), in contrast with PA (ca. 0.107 mmol/mL). When encapsulated, a decrease in AA of PA was observed in both type of NP. In similar work (da Silva et al., 2014), the values obtained for NP produced with LMWC and rosmarinic acid also decreased 10 times that the ORAC value of the free extract. The encapsulated phenolic compound which almost maintained the original AA value was DHBA in both types of NP produced (LMWC and HMWC). ORAC assay results are originated by a competition between the substrate and the phenolic compound for fluorescein reaction. The values of AA obtained can be related with the EE%, or even with the number of eOH groups of each of the chemical structure of the phenolic compounds used. The NP loaded with PA and DHBA show high AA, indicating that they react easily and rapid with the fluorescein. Protochateutic acid has 3 eOH groups and 2,5-DHBA have 2 groups. The higher AA was obtained for NP with DHBA. When at NP production, the eOH groups react with the positive amino groups of chitosan, hence it is supposed that all the eOH groups of DHBA and PA totally react with the chitosan amino groups.
3.3. Thermal properties In Table 3 are depicted the thermal properties of all NP and controls, showing the enthalpy peaks (DH) and melting
Table 2 Antioxidant activities values (means ± SD) of the polyphenols encapsulated in the NP and of the free polyphenols obtained through the ORAC method and expressed as mmol equivalent of Trolox/mL of sample. Polyphenol (%, m/v)
Free compound or entrapped in nanoparticles
mmol equivalent of Trolox/mL from sample
Protocatechuic acid (0.3%)
Free LMWC HMWC Free LMWC HMWC
0.107 0.039 0.038 0.133 0.110 0.097
2,5-Dihydroxybenzoic acid (0.3%)
± ± ± ± ± ±
0.003a 0.002b 0.003b 0.005a 0.012a 0.005b
a,b The same letters, in the same line show that there are no significant differences between the mean values (P > 0.05) (n ¼ 9). LMWC e low molecular weight chitosan; HMWC e high molecular weight chitosan.
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Table 3 Enthalpy (DH) and melting points ( C) (means ± SD) of NP and for the physical mixtures of the free compounds used in the production of the NP with the two concentrations of tripolyphosphate (TPP). Samples
Properties
Nanoparticles
DH (J/g)
Control LMWC LMWC_PA LMWC_DHBA Control HMWC HMWC_PA HMWC_DHBA Compounds TPP C1 PA þ TPP C1 DHBA þ TPP C1 TPP C2 PA þ TPP C2 DHBA þ TPP C2
Melting points ( C) a
148 469 156 539 778 488
± ± ± ± ± ±
5.7 1.2b 5.8a 0.4bc 0.3c 2.6b
63 50 60 59 71 57
± ± ± ± ± ±
0.6a 0.8b 0.4a 0.8a 2.1c 2.4a
139 404 193 449 477 288
± ± ± ± ± ±
0.5a 7.1b 2.2a 0.6b 1.5b 0.2c
71 65 42 55 59 48
± ± ± ± ± ±
0.5a 0.4ab 0.4c 0.7b 0.4b 1.2c
LMWC e low molecular weight chitosan; HMWC e high molecular weight chitosan; PA e protocatechuic acid; DHBA e 2,5-dihydroxybenzoic acid; TPP e tripolyphosphate; C1 e concentration of 0.29 mg/mL; C2 e concentration of 0.58 mg/mL. a,b,c The same letters in the same column show that there are no significant differences between the mean values (P > 0.05) (n ¼ 9).
temperatures. All NP produced had an endothermic behaviour, i.e. they absorbed energy to melt. In general, LMWC NP melted at higher temperatures than the others and absorbed lower energy. In contrast, HMWC NP melted at lower temperatures and absorbed more energy. HMWC_PA NP were the ones that absorbed more energy (778.3 J/g) and at higher melting temperature (70.8 C) (P < 0.05). All samples had melting points ranging 49e76 C. In the physical mixtures, the two TPP concentrations used in production (0.29 and 0.58 mg/mL, for LMWC and HMWC, respectively) according the chitosan MW were tested. TPP at 0.58 mg/mL (C2) exibited higher values of energy than at 0.29 mg/mL (C1), which influenced the absorbed energy. In NP, the presence of phenolic compound and type of chitosan could had more influence on the thermal behaviour than TPP. Actually, chitosan MW showed the greatest impact, related with the absorbed water evaporation, the presence of acetic acid and concerning the phenolic compounds, to a loss of water internally linked to the molecule (Tagliari et al., 2012) and/or a possible chain relaxation (Polexe et al., 2013). The endothermic behaviour observed followed the found for other chitosan NP produced (Honary et al., 2011; Shinde et al., 2013), which was also justified by the occurrence of water loss. However, there is no consensus regarding the melting temperatures. Some authors assume that occur below 100 C, others endotherms peaks showed at temperatures between 125 and 150 C (Shinde et al., 2013). Nevertheless, in such research works, dried chitosan NP were used instead of wet as ocurred in the present work, which may origin higher melting temperatures of the of NP.
3.4. Effects of the simulated pH and time of digestion on particle sizes and phenolic compounds release Gastrointestinal tract is a selective mucosal barrier which represents a considerable surface area (ca. 200 m2) in the adult human. The pH variance within the gastrointestinal compartments can affect NP aggregation status and alter surface chemistry, particularly in NP where ZP is highly dependent on pH (e.g. chitosan). Limited work has been done in understanding parameters of dissolution in gastrointestinal fluids that may help predict uptake and blood concentrations (Loretz and Bernkop-Schnürch, 2007). Fig. 1 represents the evolution of PS and % of phenolic compound release at the different stages of digestion (stomach and intestine).
Fig. 1. Evolution of the NP particle sizes (PS) (y2 axis, round shapes) and polyphenol release (%) (y1 axis, lines) of the NP produced with a) LMWC and b) HMWC without polyphenol (B, ——), with PA (C, .....) and with DHBA ( , e e) when exposed to the simulated GIT conditions.
At a first look, for LMWC NP can be detected a lower % release of phenolic compounds and increase in the PS. The increment of PS is more than 6 times than HMWC NP. In addition, the most relevant changes in PS and release of phenolic compound occurs in the intestinal stage. The NP without phenolic compound are the ones that suffer higher increment in their sizes and in the % of release. Specifically, at stomach conditions for LMWC NP, no significant changes on PS values (P > 0.05) were observed. The PS of HMWC NP increased when at stomach conditions (Fig. 1b). All LMWC NP loaded with phenolic compounds released ca. 40% at stomach conditions. The same observation was detected for the HMWC NP. Nevertheless, at intestine, the NP produced with LMWC increased their sizes significantly (P < 0.05). The NP loaded with 2,5-DHBA showed the higher sizes (ca. 5500 nm) (Fig. 1a). All LMWC NP continues to release phenolic compounds (ca. 70e80%), with exception of the NP loaded with PA (Fig. 1a). The HMWC NP maintained or increase their sizes at intestine stage, with exception of the NP loaded with PA. In terms of release profiles, all HMWC NP continued to release phenolic compound at intestinal stage reaching ca. 80%. In Table 4 are represented the other measured physical parameters, the ZP of all NP decreased along digestion. Especially in intestine, a decrease in stability was observed, owing to the NP flocculation and agglomeration (P < 0.05). This decrease is higher for NP produced with LMWC than with HMWC. The instability of LMWC NP produced in intestine is confirmed when looking to the PI results, that are much higher than the ones obtained HMWC NP. This indicates that at intestine the LMWC NP underwent changes
A.R. Madureira et al. / Journal of Food Engineering 174 (2016) 8e14
13
Table 4 Physical properties, polydispersity index (PI) and zeta potential (ZP) (mean values ± SD) of control NP without polyphenols and loaded with polyphenols, after exposition to the simulated stomach and small intestine (duodenum) conditions. Samples
Initial time
Stomach
PI Control LMWC LMWC_PA LMWC_DHBA Control HMWC HMWC_PA HMWC_DHBA
0.29 0.20 0.20 0.25 0.13 0.15
ZP (mV) ± ± ± ± ± ±
aA
0.01 0.09bA 0.03bA 0.07aA 0.01bA 0.03bA
24.7 32.3 30.2 26.3 34.1 26.0
± ± ± ± ± ±
End of digestion
PI aA
0.8 0.4bA 1.6bA 0.8aA 1.2bA 1.3aA
0.30 0.30 0.20 0.30 0.10 0.20
ZP (mV) ± ± ± ± ± ±
aAD
0.02 0.02aBD 0.03bAD 0.02aAD 0.01bAD 0.03bAD
21.4 22.4 24.5 22.3 23.9 20.2
± ± ± ± ± ±
PI aAC
0.4 0.8aBC 0.5aBC 0.6aAC 0.8aBC 0.9aBC
0.69 0.58 0.59 0.13 0.23 0.12
ZP (mV) ± ± ± ± ± ±
aBE
0.02 0.04aCE 0.02aBE 0.02bBE 0.01bBE 0.04bAE
15.6 16.9 16.8 19.4 18.0 17.5
± ± ± ± ± ±
2.8aBD 1.7aBD 0.6aBD 2.1aBC 0.9aBD 2.2aBC
a,b
The results represent the average and the standard deviation of samples in triplicate (n ¼ 3). The same letters in the same column show that there are no significant differences between the mean values (P > 0.05). A,B,C,D,EThe same letters, in the same line are related to the same parameter in the different stages, show that there are no significant differences between the values (P > 0.05) (n ¼ 16). LMWC e low molecular weight chitosan; HMWC e high molecular weight chitosan; PA e protocatechuic acid; DHBA e 2,5-dihydroxybenzoic acid; NA e not applicable.
that origin the formation of several families of different sizes, increasing the PI values. Nevertheless, these changes were showed that not affect the release of phenolic compound at the intestine, which is similar to the ones produced with HMWC, with exception of LMWC_PA. Also, the decrease in ZP can also be related with the pH increase at the simulation of intestinal conditions. The ZP is no fixed value, but dependent on a variety of parameters, e.g. solvent, electrolyte concentration, nature of electrolyte, pH of the solvent and temperature. The surface charge of the particle is directly influenced by the pH, due to protonation and deprotonation of the used particle matrix. Regarding the PS which is most suitable for NP oral administration, the produced chitosan NP had a medium size under 500 nm and when reaching the intestine this value increased. This can be a positive feature, as studies have demonstrated that such NP are capable of translocation all throughout the mucous barrier of intestinal tissue and collaborate with the underlying absorptive epithelial cells. The PS influences absorption, e.g. greater absorption of smaller (50 nm) polystyrene particles was showed when compared to larger (100 nm) particles. The largest particles in this study (300 nm) were not absorbed. Additionally, larger particles remained within the sub-mucosa or GALT of the intestine and colon, while smaller particles entered the bloodstream and accumulated in the liver and spleen (Jani et al., 1990). Additionally, this can be useful when high phenolic compound concentrations are at the sites of absorption, and when the ZP is positive can also play a role in adhesion of the NP to intestinal surfaces (Dube et al., 2010). Nanoparticles are sensible to each digestion stage pH. This could be due to chitosan pKa value and then the electrostatic interactions between the chains (Piai et al., 2009). When at lower pH than the pKa of chitosan (6.5) as in the case of stomach, a higher number of positive charges exists, since the amino groups from chitosan became protonated. For higher pH, as in case of intestine, there is a decrease of those groups. Thus, an inferior pH is favourable in maintaining the NP stable, and their PS, PI and ZP, as shown by the results. At the intestine the pH is near 7, and with high pH than the pka of chitosan there is a weakening and/or rupture from the connections leading to coagulation/flocculation, as shown by our results, which consequently lead to a higher release of phenolic compound (Piai et al., 2009). The epithelial cells in the various tissues including gastrointestinal tract, carry a negative charge on their surface due to the presence negatively charged residues of proteins in the cells outer membrane and the selective active ion pumps of the membrane. Therefore, all epithelia are selective to positively charged solutes (Rojanasakul et al., 1992). The positively charged colloidal drug carriers, such as the ones produced here, may increase the
permeability and potential uptake of slightly soluble drugs when compared with neutral or negatively charged ones, thus improving their bioavailability and reducing their side effects. The positive charge on the NP surface can enhance paracellular transport of drugs through interactions between the negatively-charged cell membrane and the positive charges of the polymer, or by complexing Ca2þ involved in the structure of tight junctions (Carino and Mathiowitz, 1999). 3.5. Conclusions The present study can be used to predict the functionality of NP loaded with natural extracts rich in phenolic acids. These NP could be used as functional ingredients to be further incorporated in food matrices in order to increase their nutritional content. The phenolic acids loaded in chitosan NP have several proved health benefits associated, such as antioxidant, anti-inflammatory, anti-carcinogenic or antimicrobial (Boaventura et al., 2013). In order to protect these compounds and to enhance their bioavailability they were entrapped in chitosan NP. Chitosan NP were produced with protocatechuic acid and 2,5-dihydroxybenzoic acid, with PS of ca. 300e600 nm, monodisperse distribution and a moderate stability. The highest EE% values were obtained for NP produced with LMWC. During storage at 4 C for 30 days, occurs the decrease of LMWC NP sizes and increase HMWC NP sizes. Decrease of zeta potential values were also registered depending on the phenolic compound loaded. Chitosan MW influences the thermal behaviour of the NP. Phenolic compounds loading do not interfere with the thermal stability of the LMWC NP but affects the thermal behaviour of the NP produced with HMWC. During digestion, at stomach, the LMWC NP releases a lower % of phenolic compounds than HMWC and with no changes on PS. At small intestine, occurs an increase of LMWC NP size and a high % of phenolic compounds release from HMWC NP. About 80% of 2,5DHBA is released independently of the type of NP, while for PA the LMWC NP released ca. 40%. With all these results, is possible to conclude that for 2,5-DHBA the best delivery systems are LMWC NP, while for PA the best ones are the produced with HMWC. The main property of the phenolic compounds, which is their antioxidant activity is shown to be preserved after the NP production. These are stable at refrigerated temperatures and the final pH is at the physiological state. If these NP are incorporated in a food matrix, the loaded compounds will be protected from the interactions with other matrix compounds and from the negative processing parameters. When ingested, NP will pass by stomach acidic conditions, preserving the phenolic acids integrity and when reaching the intestine will adhere to the intestinal epithelium and the phenolic acids will be released and adsorbed to blood stream.
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