Nano astaxanthin exhibits stronger antioxidant capacity, longer lifetime, and stronger free radical scavenging ability than astaxanthin
The present study aimed to investigate the effect of chitosan (CS)-tripolyphosphate (TPP) nanoparticles (NPs) on the stability, antioxidant activity, and bioavailability of astaxanthin (ASX). Nano astaxanthin – Chitosan – TPP (NP – ACT) loaded with ASX (ACT-NP) was prepared by ionic gelation method between CS (0.571 mg/mL) and TPP (0.571 mg/mL) for particle size, zeta potential, polydispersity index and nanoization efficiency were 505.2 ± 184.8 nm, 20.4 ± 1.2 mV, 0.348 ± 0.044 and 63.9 ± 3.0%, respectively. An in vitro release study confirmed that ASX release in simulated gastric fluid (pH 1.2) and intestinal fluid (pH 6.8) was prolonged in ACT-NP. The in vitro antioxidant activity of ACT-NP was significantly improved compared with free ASX (FA) ( p < 0.05). Furthermore, cellular and in vivo antioxidant analysis verified that Nano astaxanthin could enhance the cytoprotective effect on BHK-21 cell line and demonstrated sustained release properties, leading to prolonged survival in rat plasma. The results show that the stability, antioxidant properties and bioavailability of ASX can be effectively enhanced through nanoization of Nano astaxanthin.
1.introduce
Astaxanthin (3,3′-dihydroxy-β-β′-carotene-4-4′-dione, ASX), a natural carotenoid pigment commonly found in marine organisms including shrimp, crabs, snakeheads and fish anise, is an effective natural antioxidant [ 1 ]. The polar structure of ASX, due to the hydroxyl (OH) and carbonyl (C=O) groups of each ionic ring, has been reported to show higher antioxidant activity compared to β-carotene, tocopherol and ascorbic acid. ( Figure 1 ) [ 2 , 3 ]. Furthermore, various physiological activities of ASX such as anti-inflammatory, anti-cancer and immunological activities, manifested from high antioxidant effects, have been reported [ 4 ]. However, despite these various beneficial effects, poor water solubility, leading to low in vivo absorption rates, has been reported as a limitation for the application of ASX as a functional material potential in food [ 5 ]. Furthermore, a previous study reported that ASX easily decomposes under heat, light, and oxygen due to its 11 conjugated carbon-carbon double bonds [ 6 ]. Therefore, overcoming the insolubility and instability of ASX has been investigated as an essential task to be solved so that it can be used as an effective food or pharmaceutical ingredient [ 7, 8 ].
Nanoporation is a technology that traps a bioactive compound within a nanoscale structure made from other wall materials [ 9 ]. Due to the protective effect of the wall material and the increased surface area due to nanosized carriers, nanonization has been widely used for potential delivery systems to enhance solubility. solubility, stability and absorption capacity of bioactive materials [ 10 ]. Recently, several studies applying various types of nanochemical techniques to ASX have been reported, such as poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) [ 11 ] and Lipid-based nanocarriers include nanoliposomes [ 12 ], nanoemulsions [ 13 ], and nanostructured lipid carriers [ 14 ]. Although these studies showed results in enhancing the stability, antioxidant activity and absorption of ASX, the process and materials were considered unsuitable for food application, which requires high safety due to the use of synthetic polymers and surfactants [ 15 , 16 ]. Furthermore, liposomes have some limitations such as poor stability and low nanoization efficiency [ 17 ].
On the other hand, NP using natural polysaccharides such as chitosan (CS) is considered a suitable technique for food applications because the production process is non-toxic, biodegradable, biocompatible and economics [ 18 ]. CS, produced by deacetylation of chitin obtained from crab or shrimp shells, is a biodegradable cationic biopolymer. Due to its cationic charge property in acidic solutions, CS can form NPs through electrostatic interactions with oppositely charged anionic polymers. CS-NPs have been studied to nanosize various functional materials such as resveratrol, quercetin, and curcumin to improve their water solubility and stability against temperature and gastrointestinal environments [ 19 , 20 , 21 ]. In addition, CS-NPs have been widely studied as a delivery system due to their positively charged properties, which have a strong affinity for anionic cell membranes, leading to enhanced mucosal adhesion and bioavailability. bioavailability, both ex vivo and in vivo [ 22 ]. However, only a few studies have attempted to investigate the nanoization of ASX in CS-NPs to enhance its stability, antioxidant activity, and bioavailability [ 23 , 24 ].
Therefore, the aim of this study was to encapsulate ASX in CS-NP to study the effect of CS-NP on the biodistribution and antioxidant activity of ASX. ASX-loaded CS-NPs were prepared using sodium tripolyphosphate (TPP), one of the most effective non-toxic anionic polymers for ionic cross-linking with the cationic amino groups of CS [ 21 ]. Physicochemical characteristics, including those of ASX-loaded CS-TPP NPs (ACT-NPs) were investigated, including particle size, zeta potential (ZP), polydispersity index (PDI) , nanoencapsulation efficiency (EE) and in vitro release characteristics. Furthermore, the impact of ACT-NP on the antioxidant activity of ASX was investigated by lipid peroxidation using iron thiocyanate (FTC) and thiobarbituric acid (TBA), free radical scavenging assays. 1,1-diphenyl-2-picrylhydrazyl (DPPH), cytoprotective. characterization and in vivo plasma ferric reducing capacity (FRAP) assay.
2. Materials and methods
2.1. Materials
Chitosan (CS, 50–190 kDa, 24 cps, 95% reduction), sodium triphosphate (TPP), 2,2-Diphenyl-1-pikryl-hydrazyl (DPPH), linoleic acid, and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Astaxanthin (ASX) was obtained from Neo Cremar Co. (Sungnam, Korea). Baby hamster kidney cells (BHK)-21 were obtained from the Korea Cell Bank (Seoul, Korea). Dulbecco’s modified Eagle culture medium (DMEM) and fetal bovine serum were obtained from Gibco Invitrogen Co. (Grand Island, NY, USA). Penicillin-streptomycin and phosphate-buffered saline (PBS) were obtained from Lonza (Walkersville, MD, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Company (St. Louis, MO, USA). All other chemicals were reagent grade and all solvents were HPLC grade.
2.2. Prepare ACT-NP
ACT-NP was prepared using the ionic gelation method with some minor improvements from previous research [ 25 ]. CS, ASX, and TPP were suspended in 1% acetic acid ( v / v ) and deionized water (DW) at final concentrations, respectively ( Table 1 ). Then, 0.5 mL of ASX solution was combined with 2 mL of CS solution under magnetic stirring at 1000 rpm for 5 min, then 4.5 mL of TPP solution was added using the main flexible pump (Master flex 77200-60, Cole Parmer Inc. , Vernon Hills, IL, USA) at a flow rate of 1 mL/min.
Table 1. Fabrication conditions and characteristics of astaxanthin-containing chitosan nanoparticles fabricated with different TPP concentrations. a–d Different letters in the same column indicate significant differences ( p < 0.05).
2.3. Characterization of ACT-NP
2.3.1. Particle size, ZP and PDI of ACT-NP
The particle size, ZP and PDI of NPs were analyzed by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). The resulting ACT-NP suspension immediately after fabrication was transferred into a DTS 1060C disposable zeta cell for measurement. All measurements were performed in multiple narrow modes at 25 ± 1 °C.
2.3.2. Nanoization efficiency of ACT-NP
The ACT-NP suspension was ultracentrifuged for 30 min at 30,000× g and 4 °C (Optima TL, Beckman Instruments, Fullerton, CA, USA). After separating the supernatant, ACT-NP was collected and freeze-dried (FD8508; Ilshin Co., Seoul, Korea). Lyophilized ACT-NP (14 mg) was suspended in 2.5 mL of acetone for ASX extraction, followed by UV measurement at 478 nm (Biomate 3S; Thermo Scientific, Madison, WI, USA). The EE of ACT-NP was calculated according to the following equation:
2.4. In vitro release properties
Simulated gastric fluid (SGF, pH 1.5) contained 0.1 M hydrogen chloride (HCl) and 0.05 M sodium chloride and simulated intestinal fluid (SIF, pH 6.8) contained 0.1 M sodium hydroxide and 0.05 M sodium dihydrogen phosphate buffer were used to study the release of ASX from ACT-NP. Briefly, lyophilized Nano astaxanthin ACT-NP (20 mg) was suspended in 5 mL of SGF and SIF, respectively, and kept in a horizontal incubator at 37°C, stirring at 100 rpm. To analyze the release rate at predetermined time points, 2 mL of each sample was taken at 2, 6, and 12 h and ASX was extracted with chloroform. The concentration of released ASX was measured using a UV spectrophotometer as mentioned above. The ASX issuance rate is calculated using the following equation:
2.5. Antioxidant activity in vitro
2.5.1. Lipid peroxidation assay
A linoleic acid model system was used to evaluate the antioxidant activity of unencapsulated and nanosized ASX [ 26 ]. Measured amounts of 0.143 g tween 20, 10 mL potassium phosphate buffer (20 mM, pH 7.0), 0.08 mL linoleic acid, and 10 mL 30% ethanol were used to create a model system for lipid peroxidation inhibition. The volume was adjusted to 25 mL using DW. Accurately weighed amounts of free ASX (FA) and ACT-NP containing 2 mg of ASX were added to the linoleic acid model system as test sample. Linoleic acid model system without FA and ACT-NP were used as controls. All samples were incubated at 40 ± 1 °C in a dark room to accelerate oxidation and then withdrawn at regular intervals (2, 4, 6 days). To estimate the degree of oxidation, FTC and TBA values were determined.
The FTC method was performed according to previous research with some minor modifications [ 27 ]. In the FTC method, the amount of peroxide produced at the early stage of linoleic acid peroxidation is estimated. Fe 2+ is oxidized to Fe 3+ by peroxide, and a thiocyanate complex is produced when Fe 3+ reacts with thiocyanate [ 28 ]. The above linoleic reaction solution (20 μL) withdrawn at regular intervals was mixed with 20 μL of 20 mM ferric chloride solution, 20 μL of 30% ( w / v ) ammonium thiocyanate in 3.5 HCl % ( v / v ) and 1.14 mL of 75% ethanol ( v / v ). After incubation for three minutes at 40 °C, the absorbance of the mixture was analyzed at 500 nm using an ELISA microplate reader (ELx800UV, Bio-Tek Instrument Inc., Windoski, VT, USA). Increased absorption indicates increased lipid peroxidation.
TBA values were measured using the method described by Ohkawa (1979) [ 29 ]. The TBA value reflects the degree of lipid peroxidation associated with malonaldehyde formation, an essential biomarker for assessing the secondary phase of linoleic acid peroxidation [ 30 ]. Briefly, samples (200 μL) drawn at predetermined intervals were spiked with 0.2 mL of 8.1% SDS, 1.5 mL of 20% acetic acid ( v / v ) adjusted for potency. pH 3.5 and 1.5 mL 0.8% ( w / v). ) TBA. After adjusting the mixtures to 4 mL with DW, the mixtures were reacted for one hour at 4°C before being heated in the dark for one hour at 95°C. Finally, the absorbance of the mixture was analyzed at 532 nm using an ELISA microplate reader after cooling at room temperature. The lipid peroxidation inhibitory activity performed by the FTC and TBA methods was calculated according to the following equation:
Where Ac is the absorbance of the control sample and As is the absorbance of the sample.
2.5.2. DPPH thorough trash picking activities
DPPH radical scavenging capacity was measured according to a previous study with some modifications [ 31 ]. FA and ACT-NP during storage, they were incubated at 25°C for 30 days and then removed at predetermined intervals to measure their antioxidant activity. An amount of 0.7 mL of DPPH ethanol solution (0.1 mM) was mixed with 0.3 mL of sample, followed by incubation in a shaking water bath at 37 °C for one hour. Absorbance was measured at 517 nm immediately after centrifugation (Combi 408, Hanil Co., Seoul, Korea) at 10,000 rpm for 10 min. DPPH free radical scavenging activity was calculated according to the equation:
Where C is the absorbance of the control sample, CB the absorbance of the control blank, S the absorbance of the sample, and SB the absorbance of the blank.
2.6. Ex Vivo Antioxidant Activity of ACT-NP
MTT assay was conducted to analyze cell viability to determine whether Nano astaxanthin ACT-NP has any protective and cytotoxic effects [32 ]. An aliquot of 180 µL of the baby hamster kidney fibroblast cell line BHK-21 was seeded in a 96-well plate at a density of 1.0 × 10 4 cells/well and cultured at 37°C in a 5% humidified atmosphere. CO 2 . After BHK-21 cells reached 80% confluence, cells were treated with 20 μL of FA, blank NP (BNP), and ACT-NP suspension at concentrations ranging from 12.5 to 500.0 μg /mL, then incubated for 24 hours. Cells were washed with PBS, after removing the cell culture medium and applying 1 mM hydrogen peroxide (H 2 O 2 ) to induce oxidative stress in the cells for one hour.
Then, 20 µL of freshly prepared MTT reagent (5 mg/mL) was introduced into each well and reacted at 37°C for another 4 h to simulate the formation of purple formazan crystals produced by the Turns tetrazolium bromide yellow due to mitochondrial succinate. dehydrogenase in living cells. The supernatant was removed after centrifugation for 5 min at 400× g and dimethyl sulfoxide was added to completely solubilize the formazan. The UV absorbance of formazan was determined at 540 nm using an ELISA microplate reader to evaluate cell viability.
2.7. Vivo FRAP test
Sprague−Dawley (SD) rats (male, 6 weeks old) were obtained from Woojung Bio Co., Ltd. (Suwon, Korea). Mice were housed on a 12-h light-dark cycle at 55 ± 5% humidity and 22 ± 2 °C temperature. They were acclimatized with free access to standard food and tap water for 1 week . All animal experiments followed Hanyang University IACUC guidelines. Before the study, all mice were starved overnight and then they were randomly divided into three groups of six animals each. One group was treated with freshly prepared ACT-NP at a dose of 10 mL/kg body weight (BW). Another group was given BNP, prepared without ASX. The final group was treated with FA. At 2, 4, 6, 8, 10, and 12 hours after sample processing, blood aliquots from retrograde orbital puncture were collected in 1 mL EDTA tubes, then centrifuged for 10 minutes at 2000 × g . Until analysis, plasma samples were promptly frozen at −70°C.
Antioxidant properties in plasma samples were evaluated using the FRAP assay, slightly modified from Benzie and Strain (1996) [ 33 ]. FRAP solution, containing acetate buffer (pH 3.6, 300 mM), 2,4,6-tripyridyl-s-triazine (10 mM) in HCl (40 mM), and FeCl 3·6H 2 O (20 mM) according to 10:1:1 ratio ( v / v / v ), freshly produced and warmed at 37°C before testing. Plasma samples (30 µL) were reacted with FRAP (900 µL) and DW (90 µL) reagent mixture in the dark for 30 min at 37°C. Antioxidant activity in plasma samples was analyzed by reading the UV absorbance of the mixture at 595 nm using a Synergy HT multi-microplate reader.
2.8. Statistical analysis
All experiments were conducted in triplicate and all data are expressed as mean ± standard deviation (SD). To assess significant differences between all groups, one-way ANOVA followed by Duncan’s multiple range test (SPSS Version 21.0, SPSS Inc., Chicago, IL, USA) was used. Significant differences in DPPH free radical scavenging effect between the two groups were determined using Student’s t -test. p values below 0.05 were considered statistically significant.
3. RESULTS AND DISCUSSION
3.1. Characteristics of ACT-NP
3.1.1. Particle size and ZP of ACT-NP
ACT-NPs were formulated in different ratios of cationic CS and anionic TPP with identical ASX concentrations as shown in Table 1 . In this study, TPP higher than 0.571 mg/mL and lower than 0.429 mg/mL were considered unsuitable for preparing Nano astaxanthin ACT-NP due to their irregular NP formation as previously determined in previous studies. our preliminary experiments. At the same CS concentration of 0.571 mg/mL, the particle size of ACT-NP ranged from 483.9 ± 148.4 to 505.2 ± 184.8 nm with TPP concentration ranging from 0.571 to 0.468 mg/mL , then increased significantly to 653.8 ± 215.1 nm. with a decrease in TPP concentration to 0.429 mg/mL (p < 0.05). Although NPs are typically defined as particles with a diameter of 100 nm or less, the particle size range in the food industry has been expanded to 1000 nm as accepted food-grade finished materials are able to potential to disturb the uniformity and small size of NPs due to their relatively low purity and large molecular weight [ 34 ]. According to a previous study, the physicochemical properties of NPs, formed by ionic gelation between positively and negatively charged polymers, can be influenced by the relative proportion of charged groups [ 35 ] . Therefore, TPP concentrations above or below the appropriate level at consistent CS concentrations will disrupt the charge balance between CS and TPP and affect the physicochemical properties of NPs, such as particle size, ZP and tendency to agglomerate particles. A similar result was reported that decreasing the mass ratio of CS and TPP from 5:1 to 2:1 led to an increase in the particle size of CS-NPs [ 36 ]. This can be explained by the relative increase in the amount of TPP, thus excess TPP in suspension can produce larger NPs [ 37 ]. However, decreasing the TPP concentration to 0.429 mg/mL showed a significant increase in particle size ( p < 0.05). These results highlight that the particle size of CS-TPP NPs is found to vary with different compositions such as the concentration and ratio of CS and TPP.
The ZP value of ACT-NP significantly increased from 20.4 ± 1.2 to 30.6 ± 0.6 mV as the TPP concentration decreased ( p < 0.05). The ZP value of NP depends on the composition of charged groups as mentioned above. At higher TPP concentrations, the degree of neutralization of protonated amino groups increased, leading to lower ZP values of CS-TPP NPs [ 38 ]. Furthermore, since CS and TPP carry positive and negative charges, respectively, it is reasonable to conclude that increasing the TPP concentration leads to a decrease in the positive charge of NPs. Since ZP illustrates the surface charge of NPs, better dispersion stability is associated with higher ZP values due to the electrical repulsion between each NP [ 18 ]. Furthermore, it has been shown that NPs with ZP values greater than +20 mV or less than −20 mV are said to be stable [ 39 ]. The dispersion stability of NP has been confirmed to be around 0.3 PDI levels, which is the recommended level for delivery systems [ 40 ]. Therefore, all ACT-NP formulations presented in Table 1 can be considered stable NPs.
3.1.2. Nanoization efficiency of ACT-NP
The EE of ACT-NP increased significantly with increasing TPP concentration as shown in Table 1 ( p <0.05). This may be due to the number of cross-linked units at different TPP concentrations. An increase in TPP concentration up to a certain ratio is associated with better cross-linking between CS and TPP, causing strong affinity [ 38 ]. The strong relationship between CS and TPP would result in ASX being tightly bound to the CS-TPP structure, thus making it difficult for ASX to release quickly from NPs. On the other hand, with a further increase in TPP concentration, EE may decrease due to the more compact structure between CS and TPP, which may hinder the interaction between TPP and the core material for CS binding [ 38 ]. However, because the ACT-NP presented in this study was prepared with the optimal ratio of CS and TPP for NP formation, therefore, Nano astaxanthin ACT-NP has the highest TPP concentration (0.571 mg/mL). showed significantly higher EE (63.9%). ) (p < 0.05). According to the discussion above, a ratio of CS and TPP of 1:1 was chosen for further research because astaxanthin ACT-NP Nanos prepared according to this ratio yielded the highest EE with a particle size and distribution of Acceptable.
3.2. In vitro release properties
In SGF medium, the ASX release rate from ACT-NP gradually increased without mass release during the incubation period, and the release rate reached 88.1 ± 2.4% after 12 hours ( Figure 2 ). The release of core material is mainly affected by the degradation of the NP structure. The cross-linking between CS and TPP can be affected because the charge state of the wall material can be changed by the external pH environment where the NPs are dispersed [ 20 ]. Furthermore, NPs with high ZP values tend to have strongly charged ions on the surface, which indicates a higher resistance to degradation of NPs in the presence of acidic environments [ 41 ]. In other words, NPs with low ZP values are easily decomposed in acidic environments leading to a higher release of core material. As mentioned above, the ZP value of ACT-NP prepared with a 1:1 ratio of CS and TPP is 20.4 ± 1.2 mV, lower than other formulations. Therefore, this denotes that the high release of Nano astaxanthin ACT-NP in SGF can be explained by the reduction of electrostatic attraction between CS and TPP in acidic medium. Another possible explanation for the increased ASX release in SGF is the solubility of CS in acidic media. In general, since CS is soluble in the acidic pH range, the interaction between CS-TPP NPs prepared by ionic gelation tends to reduce SGF and accelerate the release of ASX [42 ].
Figure 2. Astaxanthin release rate from nano astaxanthin in simulated gastric fluid (SGF, pH 1.2) and intestinal fluid (SIF, pH 6.8).
On the other hand, the release of ASX from ACT-NP was not induced in SIF medium, and the release rate reached 11.4 ± 2.7% after 12 h ( Figure 2 ). This difference can be explained by two main interaction mechanisms between CS-NP and the SIF environment. One is the deprotonation of CS molecules at neutral pH, which can stabilize the NP structure due to the formation of more hydrogen bonds [ 43 ]. Another possibility is the insolubility of CS at neutral pH. The structure of ACT-NP can be maintained without CS dissolution because the solubility of CS under neutral conditions is significantly lower than that under acidic media. Similarly, previous research observed the behavior of CS-NPs in different pH environments (pH 1.2, 6.5, 7.2) to study the insulin release characteristics. They confirmed that CS-NP showed lower insulin release at pH 6.5 than at pH 1.2, due to the solubility of chitosan in different media [ 42 ]. Therefore, ACT-NPs prepared with CS and TPP appeared to have sustained degradation in SIF, leading to prolonged ASX release.
3.3. Antioxidant activity in vitro
The results of lipid peroxidation analysis using the FTC and TBA methods are shown in Figure 3 a,b. The absorbance of the control substance began to increase rapidly within 2 days. However, it gradually decreases when it reaches its maximum level. The absorption value of FA continuously increased and reached a maximum level within 4 days, but decreased slightly over time. However, the absorption value of ACT-NP increased slightly after 6 days and showed a significantly lower level than the other values during the test period ( p < 0.05). The TBA method gives similar results. While the absorbance of control and FA increased rapidly, reaching maximum levels at 2 and 4 days, respectively, and gradually decreased over time. On the other hand, the absorbance of Nano astaxanthin ACT-NP remained at a significantly lower level without gradually increasing during the test (p <0.05).
Figure 3. Antioxidant activity of free ASX and nano astaxanthin (ACT-NP) in linoleic acid peroxidation system by iron thiocyanate method (a ) and thiobarbituric acid method (b ). a–c Values with different letters are significantly different ( p < 0.05).
As shown in Figure 4 , the DPPH free radical scavenging efficiency of both FA and Nano astaxanthin ACT-NP were 13.77 and 14.80%, respectively, on day 0, showing no significant difference. However, the effect of FA decreased significantly to 0.21% at day 30, while ACT-NP showed unchanged DPPH radical scavenging effect during the incubation period (p < 0.05).
Figure 4. Antioxidant activity of free ASX and nano astaxanthin (ACT-NP) using DPPH radical scavenging method. Single asterisks indicate significant differences (p < 0.05) between two groups at each time point using Student’s t -test.
The results obtained from lipid peroxidation and DPPH assay indicated that ACT-NP effectively maintained the antioxidant activity of ASX compared to FA. Furthermore, ACT-NP showed strong inhibition of linoleic acid peroxidation by 92% within 2 days both by FTC and TBA methods, indicating that ACT-NP was effective in hindering the process. linoleic acid oxidation in both the primary and secondary stages. The prolonged antioxidant activity of ACT-NP can be explained by three different mechanisms: increased solubility, sustained release properties, and large surface area. The solubility of the core material is one of the main factors influencing antioxidant activity as it reacts with free radicals in the soluble state [ 44 ]. For example, a previous study confirmed that the antioxidant activity of kaempferol was significantly increased by nanostimulation in CS-NP as its water solubility increased [ 45 ]. Additionally, CS-NPs with sustained release properties can enhance antioxidant activity by maintaining the stability of the core material [ 46 ]. Furthermore, the enhanced antioxidant activity of Nano astaxanthin ACT-NP may be related to the large surface area of CS-TPP NPs that allows ASX molecules to interact with the reaction medium more effectively [ 47 ]. Therefore, this study confirmed that the antioxidant activity of ASX was improved by nanosizing in CS-TPP NPs.
3.4. Ex Vivo Antioxidant Activity of ACT-NP
BHK-21 cell viability was 40% lower due to H2O2 induced injury, and viability was not improved by treatment with less than 50 μg/L ASX or ACT-NP containing the same amount of ASX , as shown in Figure 5 . However, cell viability increased significantly at 500 μg/L FA (46.7 ± 4.3%) (p < 0.05) and ASX activity increased markedly upon nanoporation in ACT-NP (52.9 ± 4.7%). Clearly, ACT-NP at 500 μg/L ASX concentration was effective in protecting BHK-21 cells against H2O2 induced injury.
Figure 5. Cytoprotective effects of blank NPs, free ASX, and nano astaxanthin ACT-NPs after hydrogen peroxide-induced damage of the BHK-21 cell line. a–c Values with different letters are significantly different ( p < 0.05).
Since the antioxidant activity of ASX is activated after cellular uptake, these results can be clarified by the cell permeation characteristics of CS-NP. The interior of the cell membrane is negatively charged because the Na+-K+ pump consumes energy from hydrolysis to pump Na+ and K+ across their electrochemical gradient [ 48 ]. Since CS carries a positive charge because it has amino groups in its structure, the electrostatic interaction between cationic CS and anionic cell surface may be one of the main factors affecting the cell permeability properties of CS-NPs. [ 49 ]. Therefore, prolonged contact between Nano astaxanthin ACT-NP and the cell surface enhances the absorption of CS-NP and thus increases the antioxidant activity of ASX.
3.5. Vivo FRAP test
The change in FRAP values in SD rats after oral administration of BNP, ASX, and ACT-NP is illustrated in Figure 6 . FRAP values of mice treated with BNP and FA increased rapidly within two hours to the highest levels, 95.26 ± 4.00 and 106.68 ± 17.93 μmol/L, respectively, while FRAP values of mice fed ACT-NP only increased to 85.33 ± 22.45 μmol/L during the same time period. However, FRAP values reversed after 2 hours of oral administration. In summary, the FRAP value of rats fed ASX decreased to 94.07 ± 20.85 μmol/L after 4 hours, continuously decreased over time and returned to the initial value after 8 hours, demonstrating that ASX has low stability under alkaline conditions [ 50 ]. On the other hand, the FRAP value of ACT-NP continuously increased and exceeded the FRAP value of FA after 4 h. Although the FRAP value of ACT-NP began to decrease 4 hours after administration, the antioxidant activity of ACT-NP was always superior to that of FA.
Figure 6. Changes in FRAP values in rat plasma after a single dose of blank NP, free ASX and nano astaxanthin ACT-NP.
A previous study demonstrated that the bioavailability of orally administered ASX was only 12% of ASX delivered by intraperitoneal injection, suggesting that ASX has low oral bioavailability due to its lipophilic properties. its [ 51 ]. This limitation of ASX can be overcome through nanoization in CS-TPP NPs. This may be due to the sustained release of ASX from CS-TPP NPs, as mentioned in the in vitro release studies, which indicated that CS-TPP NPs were effective in protecting ASX from degraded during absorption in the gastrointestinal tract. Furthermore, since nanoization in CS-NP has been reported to be an effective way to improve the solubility of core materials, Nano astaxanthin ACT-NP can enhance the solubility of ASX, thus promoting its oral bioavailability [45 ]. Therefore, nanoization in CS-TPP NPs may have the potential to be used as an oral delivery system for ASX to maintain its antioxidant capacity after oral administration.
4. CONCLUSION
In the present study, we encapsulated ASX in ACT-NP with CS and TPP using the ionic gelation method. Nano astaxanthin showed acceptable physicochemical properties such as particle size, ZP, PDI and EE. The physicochemical properties showed that the interaction between CS and TPP for NP formation was significantly influenced by the ratio between CS and TPP. CS-TPP NPs with a 1:1 ratio of CS and TPP were found to enhance the release properties, in vitro and in vivo antioxidant activities and cytoprotective effects of ASX. Nano astaxanthin ACT-NPs showed sustained release of ASX (11.4 ± 2.7%) in SIF medium, suggesting that CS-TPP NPs have the ability to increase the stability of ASX. This trend is consistent with the results of in vitro, ex vivo and in vivo antioxidant activities, suggesting that CS-TPP NPs can successfully influence the antioxidant activity and oral bioavailability of ASX vs. FA. The results show that CS-TPP NPs with desirable NP properties can be used as a potential oral delivery system to improve the stability, antioxidant activity, and bioavailability of ASX .
Reference source: Chitosan-Tripolyphosphate Nanoparticles Prepared by Ionic Gelation Improve the Antioxidant Activities of Astaxanthin in the In Vitro and In Vivo Model
by Eun Suh Kim 1,Youjin Baek 1,Hyun-Jae Yoo 1,Ji-Soo Lee 2,* and Hyeon Gyu Lee 1,*