Nano Astaxanthin enhances cellular uptake, antioxidant and hypoglycemic activity in multiple cell lines

Astaxanthin is a colorant classified as a carotenoid, with high economic value. It can be found in a variety of aquatic organisms, including salmon, shrimp, crabs and even fish eggs. Astaxanthin can also be synthesized by some microorganisms, especially the freshwater microalga Haematococcus pluvialis which can accumulate astaxanthin up to 4-6% of dry biomass [1]. Astaxanthin is an antioxidant and its antioxidant activity can be 10 times higher than that of other carotenoids, specifically zeaxanthin, lutein, canthaxanthin and β-carotene [2].

Many studies have shown that astaxanthin acts as a protector against oxidative damage through various mechanisms such as scavenging free radicals to inhibiting chain reactions, Neutralizes singlet oxygen, protects cell membrane structure by inhibiting lipid peroxidation, strengthens the immune system and regulates genes. expression [3]. Choi et al. [4] reported that astaxanthin supplementation (5–20 mg/day) reduced oxidative stress biomarkers including malondialdehyde and isoprostane, increased superoxide dismutase and total antioxidant capacity in adults. become overweight and obese.

In an ex vivo study of 24 volunteers (mean age 28.2 years), consumption of 3.6 mg/day or more of astaxanthin reduced LDL oxidation [5]. In addition, astaxanthin has also been reported to inhibit hyperlipidemia and metabolic syndrome in in vitro and in vivo experiments as well as in human trials [[6], [7], [8 ], [9]]. In Apolipoprotein E knockout mice fed a high-fat and cholesterol diet, astaxanthin supplementation increased mRNA levels of LDL receptor (LDLR), carnitine palmitoyl transferase 1 (CPT-1), acetyl-CoA carboxylase β (ACC) and acyl-CoA oxidase (ACO), leading to improved cholesterol and triglyceride (TG) metabolism [6].

In a placebo-controlled astaxanthin study of 61 non-obese subjects with fasting serum TG of 120–200 mg/dl and free of diabetes and hypertension, astaxanthin supplementation 12 and 18 mg/day significantly reduced TG concentrations, and at 6 and 18 mg/day. 12 mg, the amount of HDL-cholesterol increased significantly. Serum adiponectin concentration also increased due to the effect of astaxanthin (12 and 18 mg/day), and the change of adiponectin was proportional to the change of TG and HDL-cholesterol [7]. Furthermore, other studies have also shown that astaxanthin plays a key role in the prevention and treatment of non-alcoholic fatty liver disease, cirrhosis, liver cancer, drug-induced liver injury and ischemia [8]. . Additionally, a particularly important feature is that astaxanthin shows no signs of acting as a provitamin A in humans or mammals; therefore, these organisms are not at risk of poisoning due to excessive accumulation of astaxanthin [10].

Experimental results on both male and female mice showed no harmful effects when using astaxanthin at 12 g/kg body weight/day for 14 days as well as 256, 513 and 1033 mg/kg body weight/day for 13 days [11, 12]. For humans, the safe dose for use can reach 14.4 mg of astaxanthin/day for 2 weeks. Therefore, in recent years astaxanthin has become increasingly popular as a nutritional and pharmaceutical ingredient for humans. Although astaxanthin offers various health benefits and is easily accessible, the potential for using astaxanthin in medicines and water-based food products is largely limited due to its practical insoluble in water leading to bioavailability. low availability (oral bioavailability less than 10%) [13] .

Some studies have shown that the Z isomers of astaxanthin have higher bioavailability and bioactivity than (all-E)-astaxanthin [14]. However, the stability of the Z-isomer is lower than that of the full-E isomer, which is a serious problem that affects their practical use [15]. In addition, astaxanthin in trans or cis form has the disadvantage of being poorly soluble in water (7.9 × 10-10 mg/l at 25°C) so bioavailability is not high [16]. Currently, nanotechnology is an effective solution in improving dispersion, absorption, enhancing pharmaceutical properties and enhancing the durability of active ingredients. Nanoparticle-based formulations are one of several approaches aimed at improving the bioavailability of lipophilic entities such as astaxanthin.

In this study, astaxanthin nanoparticles were prepared through emulsion solvent evaporation [17,18] and lyophilization to improve the solubility and biological activity of astaxanthin. The emulsification/solvent evaporation method is a common, conventional, simple, non-high energy consuming and feasible method for fabricating nanoparticles consisting of two organic/aqueous phases. In this study, astaxanthin was produced in nanoparticle form using a two-step process. In the first step, an aqueous solution of nano astaxanthin was prepared by the emulsification/solvent evaporation method, in which the success of the emulsification process depends on the choice of surfactant [19].

Surfactants stabilize nanoastaxanthin by self-localizing at the interface between nanoparticles and water with hydrophilic head groups protruding into water and hydrophobic tail groups in astaxanthin. Therefore, it reduces the surface tension between water and astaxanthin to form a stable colloid [20]. Most surfactants used in medicine are nonionic surfactants (e.g., cremophor RH40, Tween 80, spans, and fatty acids) [21]. In this study, we developed astaxanthin-containing surfactant-based nanocarriers using emulsification evaporation technique and freeze-drying method, and evaluated the biological activity of nano-astaxanthin such as cell uptake, cytotoxicity, antioxidant and lipid-lowering effects.

Astaxanthin (purity >95%) is extracted from the green microalga Haematococcus pluvialis. Poly(ethylene glycol) methyl ether-block-poly(D,L lactide) (PLA-PEG copolymer) was synthesized according to previous reports and has a molecular weight of 8400 corresponding to a PDI of 1.2 [ 17]. Cremophor RH40 and Tween 80 were purchased from Sigma-Aldrich and used without further purification. All solvents used in this study were of high performance liquid chromatography (HPLC) or analytical grade.

Using the emulsification/solvent evaporation method, astaxanthin nanoparticles with 5% astaxanthin content were prepared. The detailed synthesis process is as follows: the mixture of astaxanthin and surfactant (Table 1) was dissolved in 20 ml dichloromethane (DCM), then mixed thoroughly and sonicated for 10 min. The organic phase was added dropwise to 100 ml of an aqueous solution containing PLA-PEG copolymer with Ultra-Turrax homogenizer (T18 IKA, Germany) at 8400 rpm for 30 min. Then, the nanosuspension was freeze-dried to produce bright red astaxanthin nanopowder.

The particle size of nano astaxanthin was measured with a LitesizerTM 500 (from Anton Paar, Austria) using the dynamic light scattering (DLS) technique. All transmission electron microscopy (TEM) images were captured by a JEM 1400 (JEOL, Japan) with an accelerating voltage of ∼100 kV.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was performed according to the modified method of Xiao et al [18]. The absorbance of the reaction mixture was measured at 517 nm using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The DPPH inhibition removal efficiency in percentage (%) was calculated according to the equation:

Where A0 is the absorbance of DPPH solution in methanol and A1 is the absorbance of astaxanthin or nano astaxanthin. All experiments were performed in triplicate

HepG2 cells were cultured in Dulbecco’s Minimum Essential Medium (DMEM)/high glucose supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/ streptomycin. HT29 cells were cultured in DMEM/low glucose supplemented with 10 % (v/v) fetal bovine serum (FBS) and 1 % (v/v) penicillin/streptomycin. All cells were cultured in 5 % CO2 at 37 oC. After evaluating the antioxidant activity and gene expression levels of antioxidant enzymes, HepG2 cells were cultured for 24 hours in DMEM/high glucose medium in a 6-well culture plate at a density of 1 x 106 cells. /well. HepG2 cells were then incubated with nano astaxanthin at concentrations of 50 and 100 mg/mL for 24 hours. Distilled water was used as control. Each experiment was repeated at least 3 times.

After evaluating the lipid-lowering activity and expression levels of lipid metabolism-related genes, HepG2 cells were incubated with 800 µM oleic acid to induce hyperlipidemia within 4 hours. Then, cells were incubated with different concentrations of nanomolar astaxanthin (50 and 100 µg/mL) for 24 h. Distilled water was used as control. Each experiment was repeated at least 3 times.

The toxicity of nano astaxanthin at different concentrations (0, 10, 20, 50, 100 and 500 µg/mL) on HT29 and HepG2 cell lines was analyzed using 3-4,5-dimethylthiazol-2- reagent. yl-2,5-diphenyltetrazolium bromide (MTT) according to the instructions of Nga et al [19].

HT29 cells were seeded in 60 mm petri dishes at a density of 1 × 10^6 cells per dish in DMEM for 24 h. Then, the culture medium was replaced with medium containing free astaxanthin (5 µg/mL) or nano astaxanthin (with astaxanthin concentration of 5 µg/mL and polymer concentration of 100 µg/mL) and incubated at 37 °C for 24 hours. After equilibration, the experiment was stopped by washing the cells three times with cold phosphate-buffered saline (PBS). Then, cells were lysed, centrifuged at 2000 rpm for 3 minutes and stored at -20 oC until use. Astaxanthin from cells was extracted using the method described by Xiao et al [20]. Cellular Astaxanthin content was analyzed using high-performance liquid chromatography (Agilent 1260 Infinity Quanternary Pump VL series) and a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 µm; Agilent Co .). The mobile phase consisted of two main phases A (methanol solvent) and B (water and 0.1% formic acid) with a flow rate of 0.5 mL/min. UV wavelength ranges from 200 to 400 nm, UV-VIS wavelength ranges from 400 to 800 nm. The chromatography and data analysis processes are performed using Agilent’s specialized software. Chromatograms were recorded at 265 nm. Standard astaxanthin was supplied by Sigma.

The cell uptake efficiency was calculated by applying the following formula:

Where Ca is the astaxanthin content in the culture medium and Cb is the total astaxanthin content in the cells.

Protective effect against oxidative stress of nano astaxanthin against oxidative stress caused by H2O2 in HepG2 cells. Evaluation of the ability of nano astaxanthin to protect against oxidative stress by H2O2 on HepG2 cells was conducted using the method method of Wang and colleagues [21]. First, HepG2 cells were cultured for 24 hours in DMEM/high glucose medium in a 6-well culture plate at a density of 1 x 106 cells/well. Then, cells were incubated with nano astaxanthin (50 and 100 µg/mL) or ascobate as positive control for another 24 h, followed by incubation for 1 h with H2O2 solution (5 mM). The cryoprotective effect of nano astaxanthin against oxidative stress damage on HepG2 was indicated by the cell survival rate. Cell survival was determined using the MTT assay method.

HepG2 cells, after being cultured at different concentrations of nano-astaxanthin, were washed twice with phosphate-buffered saline (PBS, pH 7.4), lysed, extracted by ultrasound and centrifuged at high speed. 12000 rpm for 10 minutes at 4°C. The suspension was used to determine catalase (CAT) and glutathione peroxidase (GPx) enzyme activities as described by Weydert et al [22].

Lipid staining using ORO was performed according to the instructions of Hoang et al [23]. Cell images after staining were taken with a Canon IXY 70 digital camera (Canon, Tokyo, Japan). Stained intracellular lipids were quantified with Oil Red O in 100% isopropanol and measured spectrophotometrically at 500 nm using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA

HepG2 cells after incubation with nano astaxanthin or distilled water were wasted twice by PBS. Then, intracellular lipids were extracted with n-hexane:isopropanol (2:1, v:v) for 30 min as described by Hoang et al [23]. Intracellular cholesterol and triglyceride concentrations were determined enzymatically with an Olympus AU400 Clinical Chemistry Analyzer (Olympus Analyzers, Tokyo, Japan). Total protein was determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA) for normalization.

Total RNA was extracted from HepG2 using TRIzol™ reagent (Invitrogen, Singapore) according to the manufacturer’s instructions. Quantitative real-time PCR was performed in a MyGo Pro real-time PCR instrument (IT-IS Life Sciences Ltd., Dublin, Republic of Ireland) using the LunaUniversal One-Step RT-qPCR Kit (New England BioLabs Inc., United Kingdom). Primer sequences are shown in Table S1. Expression levels of target genes were normalized to β-actin levels using the expression normalization (CT) method according to the manufacturer’s instructions.

Data are expressed as mean ± standard error of the mean (SEM). Statistically significant differences were assessed using Student’s t test. Differences were considered statistically significant at P < 0.05.

Surfactants play an important role in the preparation of nano astaxanthin. That not only avoids the agglomeration of nanoparticles but also helps them disperse well in water. The effects of surfactants Tween 80 (A1) and cremophor RH40 (A2) in the preparation of astaxanthin nanoparticles were investigated (Figure 1).

Figure 1a shows the particle size distribution diagram of samples A1 and A2 in water indicating the presence of narrow polydisperse nanoparticles with average particle diameters of 96 and 90 nm, respectively. There are no significant differences in the morphology of the two samples. However, the toxicity of cremophor RH40 is lower than that of Tween 80. Therefore, cremophor RH40 was chosen to be used as a surfactant for astaxanthin nano preparations. Then, the effect of the weight ratio of astaxanthin/cremophor RH40 on the formation of nanoparticles was investigated. The average nanoparticle sizes of samples A2, A3, and A4 measured by DLS technique were 90, 115, and 85 nm, respectively (Fig. 1b).

On the other hand, the graph shows that the particle size is proportional to the astaxanthin/cremophor RH40 ratio. In particular, the average particle diameter decreased significantly from 115 nm to 90 nm, corresponding to the weight ratio between astaxanthin and cremophor RH40 decreasing from 1:1 to 1:2. Continuing to decrease the ratio from 1:2 to 1:3, the tendency for the nanoparticle size to decrease is not significant. According to some studies, the surfactant content present in nanoparticle formulations should be moderate to avoid final product side effects such as hyperlipidemia, abnormal lipoprotein patterns, erythrocyte aggregation and peripheral neuropathy [24,25]. In addition, the particle size distribution chart shows that sample A2 has a narrow distribution and the average particle size is less than 100 nm. This result shows the role of cremophor RH40 in protecting astaxanthin nanoparticles and the appropriate weight ratio of astaxanthin/cremophor RH40 is 1:2.

In addition, TEM images were used to observe the morphology of astaxanthin nanoparticles (Figure 1c). These nanoparticles have a clear spherical shape and especially do not have clumping. TEM image of sample A2 shows that the particles are small in size (about 90 nm) and most uniformly distributed. The sizes of these particles are in good agreement with those obtained from DLS.

Furthermore, the formation of astaxanthin nanoparticles is controlled by surfactants and encapsulants. The combination of surfactant (Cremophor RH40) and encapsulant (PLA-PEG amphiphilic copolymer) produces well-defined astaxanthin nanoparticles with high water dispersibility (Figure S1 in supplementary material). Furthermore, all compounds used in this study are biocompatible with food and pharmaceuticals.

Nano astxanthin improves DPPH free radical scavenging activity

One of the outstanding activities of astaxanthin is its antioxidant activity [26]. DDPH is a free radical used to evaluate the antioxidant level of research compounds. Therefore, the antioxidant activity of astaxanthin and nano astaxanthin (sample A2) using DPPH free radical scavenging assay was compared. Both astaxanthin and nano astaxanthin showed free radical scavenging activity in a dose-dependent manner (Fig. 2a). Consistent with other studies [21,27], nano astaxanthin had higher antioxidant activity than natural astaxanthin (Fig. 2a). At concentrations of 4, 20, 100 and 250 µg/mL, the rate of DPPH radical scavenging activity of nano astaxanthin was 13.2, 18.3, 43.3 and 92.1%, respectively, while that of astaxanthin was were 2.9, 7.9, 25.8 and 49.5 % (Figure 2a). ). This can be explained by the fact that astaxanthin is less soluble in its natural form while it is soluble in nanoparticle form [27].

Non-toxic effects of nano astaxanthin on HT9 and HepG2 cells

The effects of nano astaxanthin at different concentrations on the viability of HT29 and HepG2 cells were tested (Figures 2b and 2c). The results showed that HT29 and HepG2 cells could still survive with a survival rate of 98% when incubated with nano astaxanthin at concentrations of 10, 20, 50, 100 and 500 µg/mL for 24 hours (Figures 2b and 2 C). This shows that nano astaxanthin does not cause any cytotoxic effects on HT29 and HepG2 cells at the maximum concentration of 500 µg/mL. 3.4 Nano astaxanthin improves cellular uptake of HT29 The uptake of carotenoids, especially astaxanthin, in cells is often affected by their hydrophobicity, particle size distribution and bioaccessibility. Reducing the molecular size of the lipophilic component can improve the bioavailability of the active compounds [28]. Several studies have demonstrated that emulsifier- and colloid-based delivery systems enhance the solubility, dissolution rate, and bioavailability of compounds with poor water solubility [14, 29]. HT29 is a human colon adenocarcinoma cell line. In addition to being a xenograft tumor model for the study of colorectal cancer, it is also used as an in vitro model to study absorption, intestinal cell transport and secretion [30]. In this study, the intracellular uptake of nano astaxanthin and astaxanthin was compared on HT29 cells (Fig. 2d). It was found that the nanoparticle form of astaxanthin improved its solubility as well as increased the amount of astaxanthin delivered into HT29 cells (Fig. 2d). The absorption efficiency of astaxanthin increased from 2.5% to 12.6% in nanoparticle form. HPLC chromatography images (Figure S2 in the supplementary material) showed that nanoparticle astaxanthin increased uptake into HT29 cells by 5 times compared to conventional astaxanthin. Our results are consistent with those of Edelman et al [31].

Resistance to oxidative stress damage caused by H2O2 results from increased concentrations and gene expression of antioxidant enzymes. Oxidative stress is considered an important factor contributing to many diseases including cardiovascular diseases, chronic obstructive pulmonary diseases, neurodegenerative diseases [32]. Besides, studies have also shown that direct addition of H2O2 to the culture medium can lead to oxidative stress and cell death [27]. Therefore, in this study, we evaluated the cytoprotective effect of nano astaxanthin against oxidative damage caused by H2O2-induced oxidative stress in HepG2 cells (Fig. 3a). The results in Figure 3a show that treatment with 5mM H2O2 significantly reduced cell viability by 56.4% while this rate reached 100% when cells were cultured in medium without added H2O2 ( control). Incubating cells with nano astaxanthin before adding H2O2 significantly inhibited the damaging effect of H2O2 on cells, cell viability increased from 56.4% in cells treated with H2O2 to 64%. ,2% and 71.7% in cells pretreated with nano astaxanthin at concentrations of 50 and 100 µg/mL, respectively. Compared with the cell group pre-treated with nano astaxanthin, the ascorbic acid group also achieved a survival rate of 71.9%. Lu et al [33] showed a significant increase in cell viability from 63.9% to 73.9% when the cells were pretreated with 316 nM free astaxanthin before exposure to H2O2 . Wang et al. [21] and Oh et al. [27] did not find any cytoprotective effect of free astaxanthin at concentrations ranging from 10 to 40 µM on H2O2-induced oxidative stress in cells. Caco-2 and ARPE-19 cells.

However, in this study, we found that small amounts of astaxanthin (4.2 – 8.3 nM) in nanoparticle form can protect cells from H2O2-induced oxidative stress. The reduction in particle size and increase in solubility and cellular uptake of astaxanthin in nanoparticle form can be considered as an improvement in the antioxidant capacity of nano astaxanthin. CAT and GPx are important antioxidant enzymes that help maintain the dynamic balance of oxidative stress. Under oxidative stress, these enzymes act as endogenous antioxidants against free radicals and eliminate excessive free radicals generated in cells by direct catalysis during decomposition. H2O2 to O2 [34]. The results in Figures 3b and 3c show that Astaxanthin supplementation at doses of 50 and 100 µg/mL stimulated the activity of these enzymes. CAT activity increased by 62% and 105%, respectively (Fig. 3b), while GPx activity increased by 37% and 60%, respectively, in cells incubated with 50 and 100 µg/mL nanoastaxanthin ( Figure 3C). Antioxidant enzymes such as Keap1 (encoding Kelch-like Epichlorohydrin binding protein) and Nrf2 (encoding nuclear factor, erythroid-like 2) are used to maintain balance in the reduction-oxidation process. When the cell balances the oxidation-reduction process, Nrf2 combines with Keap1 in the cytoplasm and is inactive. Upon oxidative stress damage, Nrf2 dissociates itself from Keap1, translocates into the nucleus, and reacts with ARE, leading to upregulation of the antioxidant enzymes HO-1 (encoding heme oxygenase 1) and NQO-1 (encoding NAD(P)H quinone dehydrogenase), resulting in cells being protected against oxidative stress [35]. In this study, Keap1 mRNA levels decreased by 16% and 26% in cells incubated with nano astaxanthin when compared with the control group (Figure 3d). Gene expression of Nrf2 and its target genes HO-1 and NQO-1 increased significantly in astaxanthin nano-incubated cells. At a nanomolar astaxanthin concentration of 100 µg/mL, the cells improved the expression of Nrf2, HO-1, and NQO-1 by 119%, 89%, and 133%, respectively, compared with the control (Fig. 3d).

Based on the above results, we believe that low concentrations of astaxanthin when converted into nanoparticles can improve the activity against oxidative stress by protecting cell survival and regulating activity and expression. Gene expression of antioxidant enzymes.

One of the best known primary biological effects of astaxanthin is the reduction of lipid levels. Many studies on the hypoglycemic effect of astaxanthin have been reported in in vitro, in vivo and human trials [6,7,36,37]. Jia et al [36] showed that astaxanthin at a concentration of 10 µM could reduce cellular cholesterol and triglycerides by -14% and -20% in HepG2 cells. Hussein et al [37] reported that administration of astaxanthin increased good cholesterol (HDL-C) and decreased the content of unesterified fatty acids and triglycerides in animal models.

Furthermore, a diet supplemented with a rich astaxanthin extract from green algae for 4 weeks in mice with a mutated apoE gene significantly reduced plasma triglyceride concentrations [6]. Clinical results of Yoshida and colleagues conducted on 61 people with mild dyslipidemia treated with astaxanthin at 6 and 18 mg/day showed reduced plasma triglyceride and HDL content. -C increased with astaxanthin supplementation at 6 and 12 mg/day [7]. In this study, the lipid-reducing effect of nano astaxanthin was performed. Similar effects were achieved in cells treated with fenofibrate, intracellular triglyceride and cholesterol concentrations in cells treated with nano astaxanthin were significantly reduced in a dose-dependent manner compared to the control. Treatment with nano astaxanthin 100 µg/mL significantly reduced cholesterol and TG concentrations by 28% and 17%, respectively, compared to the control (Figures 4a and 4b). ORO staining results also showed similar results when analyzing intracellular lipid content (Figure 4c). Therefore, nano astaxanthin at concentrations ranging from 1 to 8.3 nM showed lipid-reducing ability at a similar or higher relative level to cells taking up astaxanthin above 100 nM as described in Jia et al [36 ]. Some studies indicate that cremophors compounds are often associated with severe anaphylactic shock, hyperlipidemia, abnormal lipoproteins [38]. To reduce this side effect, several methods have been applied, such as replacing Ccremophor with solutol HS 15 (and adding surfactants or solvents) [39]. In this study, astaxanthin-free surfactant (SWA)-based nanocarriers, at different concentrations (50 and 100 µg/mL) showed no change in cholesterol and intracellular triglycerides (Figures 4a and 4b). ORO analysis also showed similar results (Fig. 4c). Taken together, we believe that the nanofabrication method in this study limited the unwanted side effects caused by cremophors.

Too much cholesterol and TG can lead to atherosclerosis, so controlling plasma cholesterol and TG levels is important. The liver plays an important role in plasma cholesterol balance which is determined primarily by the rate of removal of low-density lipoprotein (LDL) from the circulation by the LDL receptor (LDLR). Once attached to LDLR on liver cells, LDL releases cholesterol and triglycerides. Significant amounts of cholesterol are stored or removed from the body by excretion into the bile as free cholesterol or after conversion to bile acids, a process regulated by the microsomal enzyme cholesterol 7α358 hydroxylase (CYP7A1). [40]. Fatty acid synthase (FAS) is a gene that quantifies de novo fatty acids synthesized in tissues. Many studies reported that down-regulation of FAS leads to decreased fatty acid synthesis [41]. Yang et al [6] reported that LDLR mRNA expression was significantly upregulated in the liver of ApoE knockout mice fed astaxanthin, leading to a decrease in plasma cholesterol.

In the same study, mRNA expression of the peroxisomal and mitochondrial fatty acid β-oxidation marker genes ACOX and CPT-1, respectively, was increased in the liver of given ApoE knockout mice. eat astaxanthin; suggests that increased fatty acid β-oxidation is responsible for the TG-lowering effect of astaxanthin. Additionally, Yao et al. [36] suggested that astaxanthin reduced hepatic lipid accumulation in high367 fat-fed rats via activation of peroxisome proliferator-activated receptor (PPAR) alpha, while CPT-1 was implicated as a factor transcription regulated by PPAR. Here, we found that low concentrations of astaxanthin (<10nM) in nanoparticle form were able to upregulate LDLR and CYP7A1 mRNA levels, while FAS mRNA levels decreased compared with the control. Nanoastaxanthin 100 µg/mL significantly increased the mRNA levels of LDLR and CYP7A1 by 190% and 148%, respectively. Treatment with nanoastaxanthin (50 and 100 µg/mL) reduced FAS mRNA levels by 23% and 41%, respectively (P < 0.1) compared with control. Peroxisome proliferator-activated receptor-alpha (PPARα), a nuclear hormone receptor, is expressed in many organisms such as liver, kidney, heart and muscle and plays an important role in regulating hepatic lipid metabolism. Natural or synthetic PPAR-α agonists, including fenofibrate, act as potential therapeutic agents in the treatment of hypertriglyceridemia. PPAR-α agonists reduce TG disorders through promoting genes involved in lipid metabolism including fatty acid uptake, fatty acid oxidation, thereby regulating fatty acid synthesis in the liver, leading to to reduce plasma triglycerides and cholesterol [42]. Here, we found that it stimulates the expression of genes encoding PPARα and its target genes. Specifically, at concentrations of 50 and 100 µg/mL, nano astaxanthin increased the mRNA content of PPARα by 37% and 73% compared to the control. CTP-1 and LXRα are PPARα target genes, in which CPT-1 participates in converting free fatty acids into acyl CoA, the initial substrate for β-oxidation in the mitochondrial membrane [42]. Here, cells incubated with nano-astaxanthin significantly increased the expression of CTP-1 and LXR in a dose-dependent manner. The results of our study are consistent with the results of Yao et al [36]. However, the concentration of active astaxanthin in nanoparticle form was 3-9nM, while that of free astaxanthin was 5-100 mM.

   By applying the emulsion solvent evaporation technique, we have produced nano astaxanthin with 5% astaxanthin content and 90 nm particle size. Nano astaxanthin can be easily dissolved in water, significantly improving the absorption of HT29 as well as its antioxidant activity compared to the natural form. Therefore, it can significantly improve the cytoprotective activity against oxidative stress and reduce intracellular lipids through increasing the activity and regulating the expression of antioxidant-related genes. metabolism and lipid metabolism without causing cell toxicity.