Studies on the antioxidant properties and bioavailability of meso-zeaxanthin

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Meso-Zeaxanthin (MZ), a vital carotenoid, is renowned for its ocular health maintenance properties. Methods To assess MZ's protective attributes against oxidative stress in human retinal pigment epithelial cells (ARPE-19), H 2 O 2 was employed to induce oxidative stress in ARPE-19 cells. Subsequently, various parameters including cell viability, oxidative stress-related enzyme activities, reactive oxygen species (ROS) generation, cellular senescence, and cell cycle progression were evaluated. Additionally, in the realm of MZ bioavailability, microencapsulated MZ was investigated for its effects on human colorectal adenocarcinoma cells (Caco-2) and SD rat models. Subsequent evaluations encompassed cellular uptake, chiral structure assessment and bioavailability determination. Results The findings underscore MZ's remarkable antioxidant prowess, characterized by enhanced cell viability, diminished malondialdehyde (MDA) levels, augmented activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX), coupled with attenuated ROS production in H 2 O 2 -treated ARPE-19 cells. Remarkably, MZ downregulates ERK/JNK/P38MAPK expression in the MAPK pathway post-H 2 O 2 exposure, thereby ameliorating oxidative stress-induced cellular damage. MZ Microcapsule Powder enhances Caco-2 uptake compared to the Raw MZ group, after confirming negligible configurational selectivity differences in MZ uptake by Caco-2 cells. Furthermore, bioavailability experiments conducted in SD rats indicate elevated plasma zeaxanthin levels in the MZ Microcapsule Powder group compared to the Raw MZ group. Conclusions Consequently, MZ has a strong antioxidant capacity and products formulated utilizing microencapsulation technology hold promise for enhancing in vivo bioavailability. ARPE-19 Antioxidant activity meso-Zeaxanthin microencapsulation Bioavailability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Oxidative stress is characterized by the unregulated generation of reactive oxygen species (ROS) triggered by either endogenous or exogenous factors. Within living cells, there exists a plethora of low-molecular-weight antioxidants (e.g., vitamins E and C, carotenoids, flavonoids, etc.) alongside higher molecular-weight antioxidant enzymes, which collectively serve to prevent or mitigate damage induced by free radicals. Key antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and the TRX system [ 1 ] . The retina, being one of the most oxygen-consuming tissues, is responsible for the transduction of light signals essential for vision formation. However, prolonged exposure to light can instigate photooxidative reactions, thereby augmenting ROS production [ 2 ] . In physiological contexts, ROS may function as secondary messengers critical for maintaining cellular homeostasis [ 3 , 4 ] . Nevertheless, exposure to exogenous oxidative stressors such as ultraviolet light, ionizing radiation, or cigarette smoke elicits an overproduction of ROS, resulting in dysregulation of the redox system and subsequent oxidative injury to cellular lipids, proteins, and nucleic acids. The retinal pigment epithelium (RPE) comprises a single layer of cells positioned on the Bruch's membrane, located between the neurosensory retina and the choroid. It is instrumental in preserving retinal function [ 5 ] . Additionally, a critical role of the RPE involves the phagocytosis of photoreceptor outer segments (POS) harboring photosensitive moieties, diverse oxidants, and unsaturated fatty acids. This mechanism results in heightened production of ROS within the RPE [ 6 ] . Lutein (Lut), zeaxanthin (Z), and meso-zeaxanthin (MZ) represent three distinct forms of macular pigment (MP), which are carotenoids distributed across various regions of the human macula. Lut and Z are acquired through dietary intake since humans lack the ability to synthesize them endogenously. Conversely, previous studies have shown that MZ is rarely found in dietary sources. Its abundance in the human macula is attributed to enzymatic metabolism and the conversion of dietary lutein (Lut) by RPE enzyme RPE65. [ 7 ] . The principal roles of MP in ocular health encompass the filtration of high-intensity blue light, known for its phototoxic effects, and serving as potent antioxidants to scavenge free radicals [ 8 ] . Collectively, these functions safeguard the retina against oxidative stress-induced damage, thereby forestalling or delaying the onset of age-related ocular pathologies [ 9 ] . Research has demonstrated that daily supplementation of MZ can mitigate RPE atrophy in a murine model of mitochondrial oxidative stress [ 10 ] . Furthermore, regular consumption of carotenoid-rich foods has been linked to reduced incidence of various chronic ailments, including photosensitivity disorders, cataracts, age-related macular degeneration (AMD), and cardiovascular diseases [ 11 , 12 ] . MZ is a very important carotenoid with a unique conjugated double bond, and this structure determines its instability is highly susceptible to oxidative degradation under the action of light and thermal oxygen. Therefore, it is crucial to develop novel formulations or delivery systems with higher stability and bioavailability. The preparation of stable, less oxidized products by microencapsulation technology can improve their stability and bioavailability, thus promoting efficacy. Previous study reported that solid lipid nanoentrapment significantly improved cellular uptake of β-carotene by using an in vitro Caco-2 cell model [ 13 ] . Another study validates the in vivo enhancement of lutein bioavailability by lutein ester nanoparticles (LE-NPs) through a rat model [ 14 ] . Research findings indicate that the concentration of lutein in the adult retina increases with distance from the fovea, while the concentration of meso-zeaxanthin (MZ) decreases with increasing distance from the fovea [ 15 ] . The ratio of lutein to zeaxanthin at the foveal center is approximately 1:2. This suggests that the content and role of MZ in the retina are particularly significant. Previous studies on the effects of macular pigment (MP) have primarily focused on lutein, with limited in-depth and systematic research on MZ. In this study, we investigate the antioxidant efficacy and potential mechanisms of MZ. Using microencapsulation technology, we encapsulate the unstable, lipid-soluble MZ to create a formulation with improved water solubility, enhanced stability and higher bioavailability. The findings unequivocally demonstrated that MZ Microcapsule Powder markedly augmented the bioavailability of MZ. Moreover, our research has shown that MZ possesses substantial antioxidant activity in ARPE-19 cells. 2. Materials and Methods Experimental material The human retinal pigment epithelia (ARPE-19) cell line is obtained from Hunan Haixing Biotechnology Co., Ltd. Caco-2 cells were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co.Ltd. ARPE-19 cells and Caco-2 cells are cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12; Gibco.) and Roswell Park Memorial Institute (RPMI1640; Gibco) respectively, containing 10% FBS (ExCell Bio, Lnc.) and 1% penicillin/streptomycin antibiotic (Seven Biotech) at 37°C with 5% CO 2 . Meso-zeaxanthin used in this paper were provided by INNOBIO® Co., Inc. Dalian, China. Model of MZ pretreatment of ARPE-19 cells against oxidative stress damage ARPE-19 cells are seeded at a density of 1.5×10 5 cells per well in 6-well culture plates and subjected to treatment with or without MZ (20 µmol/L) for 24 hours. Following treatment, the cells are rinsed with phosphate-buffered saline (PBS) and replenished with complete medium. Subsequently, the ARPE-19 cells are exposed to H 2 O 2 ) at a concentration of 0.3 mM for a duration of 4 hours. The Caco-2 cells uptake model Caco-2 cells were seeded at a density of 3×10 6 cells per dish in 100mm culture dishes and incubated for 48 hours. Raw MZ and MZ Microcapsule Powder were dissolved in DMSO, and the culture medium was subsequently diluted to a concentration of 20 µM prior to administration to the cells for 24 hours. Following cell collection, MZ content was assessed using a UV spectrophotometer, and the cell uptake rate was determined. High-performance liquid chromatography (HPLC) was employed to analyze the composition of MZ across various configurations. Cell counting kit-8 (CCK-8) assay Cell viability is evaluated utilizing the cell counting kit-8 (CCK-8) assay from Seven Biotech, in accordance with the manufacturer's instructions. Prior to conducting the CCK-8 assay, ARPE-19 cells are seeded in 96-well culture plates at a density of 8×10 3 cells per well then treated with or without MZ and H 2 O 2 . Subsequently following incubation with the CCK-8 solution at 37°C for 4 hours, cell viability is quantified by measuring absorbance at 450 nm using a microplate reader (Tecan, Infinite F50, Swiss). ROS Assay ARPE-19 cells are seeded in 6-well culture plates at a density of 1.5×10 5 cells per well and treated with or without MZ and H 2 O 2 . An in-situ loading probe, DCFH-DA, is prepared using serum-free medium with a final concentration of 10 µmol/L, 1 mL per dish. The dishes are then incubated in a cell incubator at 37°C for 20 minutes and washed three times with PBS. Subsequently, the cells are harvested and centrifuged at 1000g for 3 minutes. The supernatant is removed, 1 mL of cold PBS is added to suspend the cells, followed by another round of centrifugation. Flow cytometry is employed to detect fluorescence with excitation at 485 nm and emission at 525 nm. Determination of malondialdehyde (MDA) Reagents are prepared following the instructions provided by the MDA kit (NanJingJianCheng Bio Lnc.). The procedure involves a sequential addition of reagent application solution, anhydrous ethanol, cell homogenate, and 50% glacial acetic acid, with thorough mixing after each addition. The mixture is then incubated for 40 minutes in a 95°C water bath, followed by cooling with flowing water. Subsequently, samples are centrifuged at 3500 ~ 4000 rpm for 10 minutes, and the supernatant is collected. Absorbance is measured at 532 nm to determine the level of MDA, which is calculated using the provided formula. Glutathione peroxidase (GSH-PX) assay Reagent preparation follows the instructions provided by the GSH-PX kit (NanJingJianCheng Bio Lnc.). In the enzymatic reaction, reagent application solution is added sequentially to the cell homogenate and thoroughly mixed. The mixture is then centrifuged at 3500 ~ 4000 rpm for 10 minutes, and 1 mL of the supernatant is utilized for the color reaction. For the color development reaction, reagent application liquid and top cleaning liquid are added sequentially. After thorough mixing, the mixture is left at room temperature for 15 minutes. The supernatant is collected, and the absorbance is measured at 412 nm. The activity of GSH-PX is determined using the provided formula. Determination of total superoxide dismutase (T-SOD) The reagent preparation adheres to the instructions provided by the T-SOD kit (NanJingJianCheng Bio Lnc.). Cell homogenate is mixed thoroughly with reagents, followed by incubation at a constant temperature of 37°C for 40 minutes. Subsequently, coloring liquid solution is added, and the mixture is thoroughly mixed again before being allowed to stand at room temperature for 10 minutes. Absorbance is measured at 550 nm, and SOD activity is determined using the provided formula. Cellular senescence assay. The reagent preparation adheres to the instructions provided by the Cellular senescence kit (Shanghai Beibo Biotechnology Co., Ltd.). ARPE-19 cells are seeded in 6-well culture plates at a density of 1.5×10 5 cells per well and treated with or without MZ and H 2 O 2 . After the cells are rinsed twice with PBS, 1 mL of β-galactosidase fixing solution is added to each well, and the cells are fixed at room temperature for 15 minutes. Subsequently, the six-well plate is washed three times with PBS on a shaker, with each wash lasting 3 minutes. Following the washes, 1 mL of staining solution is added to each well, and the plate is sealed with a sealing film and incubated at 37°C overnight. The cells are then observed and photographed under a microscope. Cell cycle analysis The ARPE-19 cells are seeded in a 6-well culture plate at a density of 1.5× 10^5 cells and treated with or without MZ and H 2 O 2 . Subsequently, the cells are harvested and centrifuged at 1000g for 3 minutes. After discarding the supernatant, the cells are resuspended in 0.5 mL of cold PBS, and this process is repeated twice. For cell fixation, 75% ethanol precool is prepared in advance and slowly added to the centrifuge tube containing the cells from the previous step. The cells are then fixed overnight at 4℃. Staining is performed by incubating each sample in a 37℃ water bath with 20 µL of staining solution for 30 minutes. After centrifugation and discarding the supernatant, the cells are resuspended in 0.5 mL of PBS. Subsequently, 5 µL of 7-AAD dye is added to each sample, mixed well, and incubated for 1 hour at 4 ℃ in the dark. Finally, the samples are analyzed by flow cytometry (LSRFortessaTM; BD, Franklin Lakes, NJ, USA), with data collection from at least 30,000 cells. The results are then analyzed using FlowJo 7.6.2 software (Tree Star Inc., Ashland, OR, USA). Western Blot Cells are harvested and lysed using lysis buffer containing proteinase inhibitor to extract total protein. The protein concentration is determined using the BCA protein assay. Subsequently, the protein lysates are resolved by 4–15% SDS-PAGE and transferred to PVDF membranes with a pore size of 0.22 µM. Following this, the membranes are blocked using 5% skim milk and incubated overnight at 4℃ with primary antibodies (MAPK Family Antibody Sampler Kit #9926,Cell Signaling Tecnology,Inc.), including ERK, JNK, p-38, and GAPDH. Following primary antibody incubation, the membranes are incubated with secondary antibodies (Goat anti-rabbit, Cell Signaling Tecnology,Inc.) IgG/HRP for 1 hour at room temperature. Pharmacokinetics studies This protocol of the animal experiment is approved by the Experimental Animal Ethics Committee, Dalian Medical University (Ethic number: AEE23131). Rats in both groups (n = 6) were given Raw MZ and MZ Microcapsule Powder (calculated according to zeaxanthin) by intragastric administration at a dose of 30 mg/kg. After oral administration, blood was extracted from the jugular vein of the rats in at 1, 2, 4, 6, 8, 12, and 24 hours. The blood sample was added to a centrifuge tube with 1% heparin sodium and centrifuged at 3,000 rpm for 20 minutes. The supernatant was placed in a refrigerator at -20°C. Plasma samples were added to 200 µL of butylated hydroxytoluene (BHT) ethanol solution (0.1 g/L) and vortexed for 3 minutes to allow for thorough mixing. Sub-sequently, 600 µL of butylated hydroxytoluene (BHT) n-hexane solution (0.1 g/L) was added and vortexed for 5 minutes, and the suspension was centrifuged at 12,000 rpm for 10 minutes. Finally, the supernatant was dried at room temperature under a gentle stream of nitrogen. The dried residue was redissolved in 100 µL of mobile phase (methanol: water = 88:12 and methyl tert-butyl ether volume ratio of 1:1). After vortexed for 3 minutes, the suspension was centrifuged at 12,000 rpm for 5 minutes, and 100 µL of the supernatant was extracted and injected into the HPLC apparatus. 3. Results 3.1 Meso-zeaxanthin increased the cell viability of ARPE-19 cells Initially, we evaluated the potential cytotoxicity of MZ on ARPE-19 cells by exposing them to different concentrations of the compound. We determined that at a concentration of 20 µΜ, cellular viability was optimized. Consequently, a concentration of 20 µmol/L MZ was employed in subsequent experiments. 3.2 A cellular oxidative stress model was established using a 300 µM concentration of H 2 O 2 H 2 O 2 is commonly employed to induce oxidative stress in in vitro studies. To ascertain the optimal concentration of H 2 O 2 for this investigation, ARPE-19 cells were exposed to varying concentrations of H 2 O 2 (100–500µM/L) for 4 hours. Cell viability was decreased by H 2 O 2 treatment dose-dependently. At 300µM, cell viability was halved. Consequently, a concentration of 300µM/L H 2 O 2 was employed in subsequent experiments. 3.3 MZ conferred protection to ARPE-19 cells against oxidative stress damage induced by H 2 O 2 The influence of MZ on the viability of H 2 O 2 -damaged ARPE-19 cells was investigated by pre-treating the cells with MZ for 24 hours. Subsequently, except for the control group, the remaining two groups underwent H 2 O 2 treatment for 4 hours. Changes in cell viability among those pre-treated with MZ were assessed using the CCK-8 assay. The results revealed a significant decrease in cell viability in the H 2 O 2 treatment group compared to the control group, confirming the successful induction of oxidative damage by H 2 O 2 . Statistical analysis indicated a marked increase in cell viability among those pre-treated with MZ compared to the H 2 O 2 treatment group. 3.4 Meso-zeaxanthin pretreatment decreased cellular ROS levels by H 2 O 2 -induced injury The overproduction of reactive oxygen species (ROS) is recognized as a primary contributor to H 2 O 2 -induced oxidative damage and cell death. As illustrated in the figure, exposure to H 2 O 2 results in a significant elevation in cellular ROS levels. However, pre-treatment of ARPE-19 cells with MZ substantially suppresses the excessive ROS accumulation induced by H 2 O 2 . 3.5 Meso-zeaxanthin treatment group exhibited an enhancement in the activities of GSH-PX and T-SOD in ARPE-19 cells during the H 2 O 2 damage, in contrast to the other treatment groups and demonstrated a reduction in MDA levels To investigate the influence of MZ on oxidative stress in H 2 O 2 -damaged ARPE-19 cells, cells were exposed to MZ for 24 hours. Subsequently, except for the control group, the remaining two groups were pre-treated with H 2 O 2 for 4 hours. Changes in antioxidant stress among cells pre-treated with MZ were assessed using assay kits. We observed a significant decrease in GSH-PX and T-SOD levels in the H 2 O 2 group compared to the control group, along with a marked increase in MDA content, confirming successful induction of oxidative damage by H 2 O 2 . Statistical analysis revealed that co-treatment with MZ and H 2 O 2 significantly enhanced GSH-PX and T-SOD activities in cells compared to the H 2 O 2 group, while reducing MDA levels. 3.6 Meso-Meszeaxanthin alleviated Cell Senescence caused by H 2 O 2 We examined the effect of MZ pre-treatment on the prevalence of senescent cells in H 2 O 2 -damaged ARPE-19 cells. ARPE-19 cells were pretreated with MZ for 24 hours, followed by H 2 O 2 treatment for 4 hours to induce senescence. Senescent cells were visualized using β-galactosidase staining and observed under fluorescence microscopy. Our findings demonstrated a substantial increase in staining precipitates in the H 2 O 2 -treated group compared to the blank control group, confirming successful induction of senescence by H 2 O 2 . Conversely, the group pre-treated with MZ exhibited a marked reduction in staining precipitates compared to the H 2 O 2 -treated group. 3.7 Influence of Meso-zeaxanthin on cell cycle progression To delve deeper into the impact of MZ pre-treatment on the cell cycle of ARPE-19 cells during H 2 O 2 -induced damage, we employed flow cytometry. ARPE-19 cells were stained with 7-AAD dye to evaluate the cell cycle of both H 2 O 2 -treated cells and cells pre-treated with MZ. The findings revealed that the cell cycle of ARPE-19 cells induced by H 2 O 2 was arrested at the G2/M phase, accompanied by a decrease in the number of cells in the G1 phase. Conversely, in the MZ pre-treatment group, there was a reduction in the number of cells in the G2/M phase, highlighting the significant amelioration of G2/M phase arrest induced by H 2 O 2 damage with MZ pre-treatment. 3.8 Influence of Meso-zeaxanthin on Cell MAPK Signaling Pathway To explore the impact of MZ on MAPK signaling pathway proteins in H 2 O 2 -damaged ARPE-19 cells, the expression levels of p38, JNK, and ERK proteins were assessed through Western blot analysis. Statistical analysis revealed that the H 2 O 2 group displayed significantly elevated expression of p38, JNK, and ERK compared to the Ctrl group, whereas the MZ-treated group exhibited a notable reduction in the expression levels of these proteins compared to the Ctrl group. 3.9 Characterization of MZ uptake by Caco-2 cells To evaluate the absorption efficacy of microencapsulated products, the Caco-2 model was utilized for cell uptake experiments. Results indicated that MZ Microcapsule Powder highly improved the absorption capacity of Caco-2 cells for MZ compared to Raw MZ, demonstrating a cell uptake rate approximately 3.6 times higher than that of Raw MZ. Furthermore, the conformational composition of zeaxanthin in the cellular uptake samples of MZ Microcapsule Powder remained largely unchanged before and after Table 1 . Hence, there seems to be no significant configuration selectivity difference in the uptake of zeaxanthin by Caco-2 cells, suggesting their ability to uptake various zeaxanthin configurations, including MZ. Table 1 Conformational composition of cells before and after uptake of MZ Microcapsule Powder. Ingredients(%) Before uptake After uptake Meso-zeaxanthin 83.72 82.50 3R,3’R-zeaxanthin 6.86 10.21 3S,3’S-zeaxanthin 0.27 0.22 Lutein 6.00 5.17 3.10 Bioavailability analysis The concentration-time curves depicting orally administered Raw MZ and MZ Microcapsule Powder are presented in Fig. 10 . Pharmacokinetic parameters of MZ Microcapsule Powder and Raw MZ were compared (Table 2 ). Notably, the microcapsule powder exhibited superior absorption efficacy compared to Raw MZ, as evidenced by higher AUC and C max values. Additionally, the AUC (0–24) values for zeaxanthin in rats were 137.48 µg/Lh and 103.341 µg/Lh in the MZ Microcapsule Powder and Raw MZ groups, respectively, indicating a 1.33-fold increase in bioavailability of zeaxanthin in the MZ Microcapsule Powder group compared to the Raw MZ group. Both microcapsule powder and Raw MZ displayed a T max of 8 hours, suggesting similar absorption rates. However, the microcapsule powder demonstrated enhanced absorption efficacy compared to Raw MZ. Therefore, pharmacokinetic studies suggest that MZ Microcapsule Powder, prepared using microencapsulation technology, can enhance in vivo bioavailability, improve zeaxanthin absorption. Table 2 Pharmacokinetic parameters of the MZ Microcapsule Powder and Raw MZ Room parameters Unit Raw MZ MZ Microcapsule Powder AUC (0−24) µg/L*h 103.341 137.48 T max hours 8 8 C max µg/L 9.494 15.427 Bioavailability % 100.000 133.035 4. Discussion Meso-zeaxanthin (MZ), along with lutein (Lut) and zeaxanthin (Z), forms the macular pigment (MP), playing a crucial role in protecting cells and organs from oxidative damage induced by oxygen free radicals, thus preventing certain eye diseases [ 16 , 17 ] . While MZ is a metabolite of Lut, it can also be obtained from dietary sources [ 18 , 19 ] . The concentration of MZ in the macula surpasses that in serum and liver, underscoring specific absorption and storage mechanisms within the retina, highlighting its pivotal role in retinal function [ 20 ] . Initially believed to be undetectable in human liver or serum [ 15 ] , subsequent research identified MZ in serum, and supplementation studies demonstrated a notable increase in macular pigment levels following oral administration of MZ, indicating its oral bioavailability and transport to the macula [ 18 ] . Despite its conversion from Lut in the retina, supplementation with high doses of MZ leads to elevated MZ levels in both the macula and serum compared to Lut and Z supplementation alone [ 21 ] . Our results suggest that MZ is directly absorbed and utilized in addition to being converted from lutein, as verified by in vivo and ex vivo models. In the outer retina, macular pigment acts as a potent antioxidant, countering oxidative damage to photoreceptor outer segments containing chromophores that serve as photosensitizers susceptible to oxidation [ 2 ] . MZ exhibits superior quenching capabilities against oxygen free radicals compared to Lut [ 15 ] , particularly in regions of the retina with the highest cone density and heightened risk of oxidative damage [ 22 ] .However, its underlying mechanisms of action remain to be thoroughly investigated. In this study, MZ treatment of ARPE-19 cells mitigated the adverse effects of H 2 O 2 on cell viability (Fig. 3 ) and suppressed excessive ROS accumulation induced by H 2 O 2 (Fig. 4). Furthermore, MZ treatment significantly bolstered intracellular GSH-PX and T-SOD activities, while reducing MDA levels (Fig. 5 ), and effectively mitigated cellular senescence (Fig. 6 ). Additionally, pre-treatment with meso-zeaxanthin markedly ameliorated H 2 O 2 -induced G2/M phase arrest (Fig. 7) and attenuated H 2 O 2 -induced activation of the MAPK pathway, indicative of oxidative stress damage (Fig. 8 ). Meso-zeaxanthin is an important fat-soluble compound that is difficult to dissolve in water and its application is greatly limited. Because of its structure contains 11 conjugated double bonds, and the tail group with hydroxyl, this structure determines its nature is very active and easy to oxidize and isomerize. Therefore, in order to prevent its isomerization and microencapsulation of zeaxanthin is technically difficult. In this study, MZ Microcapsule Powder was prepared utilizing a distinctive microencapsulation technique, and absorption was assessed both ex and in vivo using Caco-2 cells and rat models. Experimental results demonstrated a substantial enhancement in the uptake capacity of Caco-2 cells for meso-zeaxanthin facilitated by microencapsulation technology, with the cellular uptake rate in the MZ Microcapsule Powder group approximately 3.6 times higher than that in the Raw MZ group ( Fig. 9 ). Additionally, following administration of a 30 mg/kg dosage of MZ product to rats, the Cmax and AUC (0−24) values of MZ Microcapsule Powder surpassed those of Raw MZ (Fig. 10 ), indicating superior absorption of MZ Microcapsule Powder compared to Raw MZ. Utilizing Raw MZ as a benchmark preparation, the relative bioavailability of MZ Microcapsule Powder in rats was determined to be 133.035%, implying a noteworthy enhancement in MZ bioavailability post-encapsulation into microcapsules. 5. Conclusions Initially, MZ demonstrated robust antioxidant efficacy at the cellular level. Furthermore, in rat studies, MZ Microcapsule Powder, formulated with microencapsulation technology, exhibited superior bioavailability relative to Raw MZ. These observations indicate that MZ is capable of direct absorption, and the utilization of microencapsulation technology markedly enhances its bioavailability. 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Supplementary Files GelsandBlotsimages.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Aug, 2025 Reviews received at journal 18 Jul, 2025 Reviewers agreed at journal 15 Jul, 2025 Reviewers agreed at journal 10 Jul, 2025 Reviews received at journal 02 Jul, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviewers invited by journal 24 Jun, 2025 Editor assigned by journal 24 Jun, 2025 Submission checks completed at journal 23 Jun, 2025 First submitted to journal 11 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6869584","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475918407,"identity":"edfd85c2-5a21-4953-8a43-c54824edeeeb","order_by":0,"name":"Shuang Liu","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Liu","suffix":""},{"id":475918408,"identity":"f9d38c70-78d2-4966-899a-5dc354051fe8","order_by":1,"name":"Xiao Han","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Han","suffix":""},{"id":475918409,"identity":"f0bccb7a-2f90-487b-83a2-b945faeb1930","order_by":2,"name":"Xiaotong Zhang","email":"","orcid":"","institution":"INNOBIO Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Xiaotong","middleName":"","lastName":"Zhang","suffix":""},{"id":475918410,"identity":"34795ec7-ba06-4317-b887-3dd075adda9c","order_by":3,"name":"Jinqiu Li","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinqiu","middleName":"","lastName":"Li","suffix":""},{"id":475918411,"identity":"15f6656e-4390-4dee-89bb-6439078bb1d9","order_by":4,"name":"Jinye Wang","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinye","middleName":"","lastName":"Wang","suffix":""},{"id":475918413,"identity":"df17801c-b849-41c3-a461-5ee9674a0b1b","order_by":5,"name":"Xiang Ren","email":"","orcid":"","institution":"INNOBIO Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Ren","suffix":""},{"id":475918415,"identity":"166a27c5-e929-4bb1-b8f1-6e776632a2f0","order_by":6,"name":"Wenzhong Wu","email":"","orcid":"","institution":"INNOBIO Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Wenzhong","middleName":"","lastName":"Wu","suffix":""},{"id":475918417,"identity":"c801ed2a-6ca6-4ac8-8ac1-4eeeabfcf993","order_by":7,"name":"Linhua Liu","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Linhua","middleName":"","lastName":"Liu","suffix":""},{"id":475918419,"identity":"7fe30e52-ce6d-4a91-8b61-a5a485246abf","order_by":8,"name":"Chao Fan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYBAC9gYg8cEASEgwNhCnhecAAwPjDJK1MPOAWBLEOoyH/ezBzzYFd/L4pZsbHzDU3CFCC09esnSOwbNiyTkHmw0Yjj0jrMWeIceMOcfgcOKGG4ltQO8cJsIW/jdmzBakaZEA2sJAopY3xpI9QC0zQX5JOEaUw3IMP/z4czixX7r94YMPNURoQQUJpGoYBaNgFIyCUYAdAABEnTkds4g5PwAAAABJRU5ErkJggg==","orcid":"","institution":"INNOBIO Co., Ltd.","correspondingAuthor":true,"prefix":"","firstName":"Chao","middleName":"","lastName":"Fan","suffix":""}],"badges":[],"createdAt":"2025-06-11 08:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6869584/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6869584/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85513465,"identity":"f65fcdd8-2d9b-4b47-9668-aa6be257867a","added_by":"auto","created_at":"2025-06-26 17:14:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78773,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in cell viability after treatment of ARPE-19 cells with different concentrations of MZ. Experimental data were analyzed and plotted using GraphPad Prism 9. Statistical analysis was performed based on three independent replicate experiments. ****P﹤0.0001，***P﹤0.0005，*P﹤0.05.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/d06374b8589ca860eddee710.png"},{"id":85513468,"identity":"13bbaf68-07dd-429b-94e8-f8d6e69949b9","added_by":"auto","created_at":"2025-06-26 17:14:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92955,"visible":true,"origin":"","legend":"\u003cp\u003eARPE-19 cell viability following exposure to varying concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Experimental data were analyzed and plotted using GraphPad Prism 9. Statistical analysis was performed based on three independent replicate experiments. ****P﹤0.0001，***P﹤0.0005，*P﹤0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/a9672b0abb3804e7a2add225.png"},{"id":85513474,"identity":"d8f29ee8-30b9-4844-af5a-da1d28a667c6","added_by":"auto","created_at":"2025-06-26 17:14:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35948,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of MZ treatment on cell viability in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Experimental data were analyzed and graphed using GraphPad Prism 6. Statistical analysis was performed based on three independent replicate experiments. ****P﹤0.0001，***P﹤0.0005，*P﹤0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/f9ea2cce07b349bc5555ee3f.png"},{"id":85513467,"identity":"19a55153-b84c-4cbf-a77d-1a51e0e30e45","added_by":"auto","created_at":"2025-06-26 17:14:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55343,"visible":true,"origin":"","legend":"\u003cp\u003eROS Levels in ARPE-19 Cells Induced by H2O2 following MZ Pre-treatment. ROS levels were evaluated via flow cytometry and the data were analyzed and visualized using FlowJo software.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/d46e34250f5060e3c8de5c62.png"},{"id":85513912,"identity":"4d195cd3-46d5-47a4-8769-3e563d6e0577","added_by":"auto","created_at":"2025-06-26 17:22:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":184934,"visible":true,"origin":"","legend":"\u003cp\u003eMDA Levels, GSH-PX, and T-SOD Activity in ARPE-19 Cells during H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress following MZ pre-treatment. (A) Assessment of cellular MDA levels utilizing the MDA assay kit. (B) Evaluation of cellular GSH-PX activity using the GSH-PX assay kit. (C) Measurement of cellular T-SOD activity employing the T-SOD assay kit. Experimental data were analyzed and graphed using GraphPad Prism 9. Statistical analysis was conducted based on three independent replicate experiments. ****P﹤0.0001，***P﹤0.0005，*P﹤0.05.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/62bae7df611bb4486103d23e.jpeg"},{"id":85513470,"identity":"336292e5-fa5d-4694-85bd-ff851337866e","added_by":"auto","created_at":"2025-06-26 17:14:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":196386,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e effects on cellular senescence after MZ Pre-treatment of ARPE-19 cells utilizing a cellular aenescence Assay Kit.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/98327278677f0680b32d9090.jpeg"},{"id":85514487,"identity":"fbafb444-9d4f-4991-b40a-8c1ca1607f1b","added_by":"auto","created_at":"2025-06-26 17:30:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":97114,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of H2O2 induction on ARPE-19 cell cycle changes after MZ pre-treatment. Cell cycle analysis was performed using flow cytometry and the results were analyzed and visualized using FlowJo software.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/7b39885cbbc219adf86f8844.png"},{"id":85513914,"identity":"7fd380ef-7291-4f8c-8b77-3f7521377605","added_by":"auto","created_at":"2025-06-26 17:22:38","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":254200,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis evaluating the influence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on the expression of ERK, JNK, and p38 proteins in the MAPK pathway in ARPE-19 cells following MZ pre-treatment. Graphs were generated using Image J software. Experimental results from Western blotting were analyzed using GraphPad Prism 6 and depicted graphically. The experiments were conducted in triplicate, and statistical analysis was conducted. *P \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/9108942bfa9cd5f7b20658b0.jpeg"},{"id":85514489,"identity":"582a2813-2c91-41c0-8825-d2f83697bc92","added_by":"auto","created_at":"2025-06-26 17:30:38","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":11533,"visible":true,"origin":"","legend":"\u003cp\u003eUptake of Caco-2 cells for Raw MZ and MZ Microcapsule Powder. Experimental results from cell uptake were analyzed using GraphPad Prism 6 and depicted graphically. The experiments were conducted in triplicate, and statistical analysis was conducted. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/0d4bf26debfe20d5647dd57e.png"},{"id":85513477,"identity":"d02a0037-e346-4845-9ce4-ea3b2903d800","added_by":"auto","created_at":"2025-06-26 17:14:38","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":13572,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration-time profile of zeaxanthin in the plasma upon oral administration of MZ Microcapsule Powder and Raw MZ. The acquired data were analyzed by the pharmacoki-netic software DAS2.0. Plotting presentation of results using GraphPad Prism 6\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/ec7663970c9384bc25e75b7e.png"},{"id":85515437,"identity":"014a9728-88ca-43e5-8164-2a3e83e47bf6","added_by":"auto","created_at":"2025-06-26 17:46:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1965859,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/dc80eace-ae1b-4d0f-8f76-61bdb4016523.pdf"},{"id":85514491,"identity":"53c69855-af0d-4473-b4b8-784c499e5b41","added_by":"auto","created_at":"2025-06-26 17:30:38","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2274938,"visible":true,"origin":"","legend":"","description":"","filename":"GelsandBlotsimages.docx","url":"https://assets-eu.researchsquare.com/files/rs-6869584/v1/af1b264ed9e32f128b6afc5e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Studies on the antioxidant properties and bioavailability of meso-zeaxanthin","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOxidative stress is characterized by the unregulated generation of reactive oxygen species (ROS) triggered by either endogenous or exogenous factors. Within living cells, there exists a plethora of low-molecular-weight antioxidants (e.g., vitamins E and C, carotenoids, flavonoids, etc.) alongside higher molecular-weight antioxidant enzymes, which collectively serve to prevent or mitigate damage induced by free radicals. Key antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and the TRX system\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The retina, being one of the most oxygen-consuming tissues, is responsible for the transduction of light signals essential for vision formation. However, prolonged exposure to light can instigate photooxidative reactions, thereby augmenting ROS production\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. In physiological contexts, ROS may function as secondary messengers critical for maintaining cellular homeostasis\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, exposure to exogenous oxidative stressors such as ultraviolet light, ionizing radiation, or cigarette smoke elicits an overproduction of ROS, resulting in dysregulation of the redox system and subsequent oxidative injury to cellular lipids, proteins, and nucleic acids.\u003c/p\u003e \u003cp\u003eThe retinal pigment epithelium (RPE) comprises a single layer of cells positioned on the Bruch's membrane, located between the neurosensory retina and the choroid. It is instrumental in preserving retinal function\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Additionally, a critical role of the RPE involves the phagocytosis of photoreceptor outer segments (POS) harboring photosensitive moieties, diverse oxidants, and unsaturated fatty acids. This mechanism results in heightened production of ROS within the RPE\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLutein (Lut), zeaxanthin (Z), and meso-zeaxanthin (MZ) represent three distinct forms of macular pigment (MP), which are carotenoids distributed across various regions of the human macula. Lut and Z are acquired through dietary intake since humans lack the ability to synthesize them endogenously. Conversely, previous studies have shown that MZ is rarely found in dietary sources. Its abundance in the human macula is attributed to enzymatic metabolism and the conversion of dietary lutein (Lut) by RPE enzyme RPE65.\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The principal roles of MP in ocular health encompass the filtration of high-intensity blue light, known for its phototoxic effects, and serving as potent antioxidants to scavenge free radicals\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Collectively, these functions safeguard the retina against oxidative stress-induced damage, thereby forestalling or delaying the onset of age-related ocular pathologies\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Research has demonstrated that daily supplementation of MZ can mitigate RPE atrophy in a murine model of mitochondrial oxidative stress\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Furthermore, regular consumption of carotenoid-rich foods has been linked to reduced incidence of various chronic ailments, including photosensitivity disorders, cataracts, age-related macular degeneration (AMD), and cardiovascular diseases\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. MZ is a very important carotenoid with a unique conjugated double bond, and this structure determines its instability is highly susceptible to oxidative degradation under the action of light and thermal oxygen. Therefore, it is crucial to develop novel formulations or delivery systems with higher stability and bioavailability. The preparation of stable, less oxidized products by microencapsulation technology can improve their stability and bioavailability, thus promoting efficacy. Previous study reported that solid lipid nanoentrapment significantly improved cellular uptake of β-carotene by using an in vitro Caco-2 cell model\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Another study validates the in vivo enhancement of lutein bioavailability by lutein ester nanoparticles (LE-NPs) through a rat model\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eResearch findings indicate that the concentration of lutein in the adult retina increases with distance from the fovea, while the concentration of meso-zeaxanthin (MZ) decreases with increasing distance from the fovea\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The ratio of lutein to zeaxanthin at the foveal center is approximately 1:2. This suggests that the content and role of MZ in the retina are particularly significant. Previous studies on the effects of macular pigment (MP) have primarily focused on lutein, with limited in-depth and systematic research on MZ. In this study, we investigate the antioxidant efficacy and potential mechanisms of MZ. Using microencapsulation technology, we encapsulate the unstable, lipid-soluble MZ to create a formulation with improved water solubility, enhanced stability and higher bioavailability. The findings unequivocally demonstrated that MZ Microcapsule Powder markedly augmented the bioavailability of MZ. Moreover, our research has shown that MZ possesses substantial antioxidant activity in ARPE-19 cells.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cb\u003eExperimental material\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe human retinal pigment epithelia (ARPE-19) cell line is obtained from Hunan Haixing Biotechnology Co., Ltd. Caco-2 cells were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co.Ltd. ARPE-19 cells and Caco-2 cells are cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/nutrient mixture F-12 (DMEM/F12; Gibco.) and Roswell Park Memorial Institute (RPMI1640; Gibco) respectively, containing 10% FBS (ExCell Bio, Lnc.) and 1% penicillin/streptomycin antibiotic (Seven Biotech) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Meso-zeaxanthin used in this paper were provided by INNOBIO\u0026reg; Co., Inc. Dalian, China.\u003c/p\u003e \u003cp\u003e \u003cb\u003eModel of MZ pretreatment of ARPE-19 cells against oxidative stress damage\u003c/b\u003e \u003c/p\u003e \u003cp\u003eARPE-19 cells are seeded at a density of 1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well in 6-well culture plates and subjected to treatment with or without MZ (20 \u0026micro;mol/L) for 24 hours. Following treatment, the cells are rinsed with phosphate-buffered saline (PBS) and replenished with complete medium. Subsequently, the ARPE-19 cells are exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) at a concentration of 0.3 mM for a duration of 4 hours.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Caco-2 cells uptake model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCaco-2 cells were seeded at a density of 3\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per dish in 100mm culture dishes and incubated for 48 hours. Raw MZ and MZ Microcapsule Powder were dissolved in DMSO, and the culture medium was subsequently diluted to a concentration of 20 \u0026micro;M prior to administration to the cells for 24 hours. Following cell collection, MZ content was assessed using a UV spectrophotometer, and the cell uptake rate was determined. High-performance liquid chromatography (HPLC) was employed to analyze the composition of MZ across various configurations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell counting kit-8 (CCK-8) assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCell viability is evaluated utilizing the cell counting kit-8 (CCK-8) assay from Seven Biotech, in accordance with the manufacturer's instructions. Prior to conducting the CCK-8 assay, ARPE-19 cells are seeded in 96-well culture plates at a density of 8\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well then treated with or without MZ and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Subsequently following incubation with the CCK-8 solution at 37\u0026deg;C for 4 hours, cell viability is quantified by measuring absorbance at 450 nm using a microplate reader (Tecan, Infinite F50, Swiss).\u003c/p\u003e \u003cp\u003e \u003cb\u003eROS Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eARPE-19 cells are seeded in 6-well culture plates at a density of 1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and treated with or without MZ and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. An in-situ loading probe, DCFH-DA, is prepared using serum-free medium with a final concentration of 10 \u0026micro;mol/L, 1 mL per dish. The dishes are then incubated in a cell incubator at 37\u0026deg;C for 20 minutes and washed three times with PBS. Subsequently, the cells are harvested and centrifuged at 1000g for 3 minutes. The supernatant is removed, 1 mL of cold PBS is added to suspend the cells, followed by another round of centrifugation. Flow cytometry is employed to detect fluorescence with excitation at 485 nm and emission at 525 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of malondialdehyde (MDA)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eReagents are prepared following the instructions provided by the MDA kit (NanJingJianCheng Bio Lnc.). The procedure involves a sequential addition of reagent application solution, anhydrous ethanol, cell homogenate, and 50% glacial acetic acid, with thorough mixing after each addition. The mixture is then incubated for 40 minutes in a 95\u0026deg;C water bath, followed by cooling with flowing water. Subsequently, samples are centrifuged at 3500\u0026thinsp;~\u0026thinsp;4000 rpm for 10 minutes, and the supernatant is collected. Absorbance is measured at 532 nm to determine the level of MDA, which is calculated using the provided formula.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGlutathione peroxidase (GSH-PX) assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eReagent preparation follows the instructions provided by the GSH-PX kit (NanJingJianCheng Bio Lnc.). In the enzymatic reaction, reagent application solution is added sequentially to the cell homogenate and thoroughly mixed. The mixture is then centrifuged at 3500\u0026thinsp;~\u0026thinsp;4000 rpm for 10 minutes, and 1 mL of the supernatant is utilized for the color reaction. For the color development reaction, reagent application liquid and top cleaning liquid are added sequentially. After thorough mixing, the mixture is left at room temperature for 15 minutes. The supernatant is collected, and the absorbance is measured at 412 nm. The activity of GSH-PX is determined using the provided formula.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of total superoxide dismutase (T-SOD)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe reagent preparation adheres to the instructions provided by the T-SOD kit (NanJingJianCheng Bio Lnc.). Cell homogenate is mixed thoroughly with reagents, followed by incubation at a constant temperature of 37\u0026deg;C for 40 minutes. Subsequently, coloring liquid solution is added, and the mixture is thoroughly mixed again before being allowed to stand at room temperature for 10 minutes. Absorbance is measured at 550 nm, and SOD activity is determined using the provided formula.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCellular senescence assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe reagent preparation adheres to the instructions provided by the Cellular senescence kit (Shanghai Beibo Biotechnology Co., Ltd.). ARPE-19 cells are seeded in 6-well culture plates at a density of 1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and treated with or without MZ and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. After the cells are rinsed twice with PBS, 1 mL of β-galactosidase fixing solution is added to each well, and the cells are fixed at room temperature for 15 minutes. Subsequently, the six-well plate is washed three times with PBS on a shaker, with each wash lasting 3 minutes. Following the washes, 1 mL of staining solution is added to each well, and the plate is sealed with a sealing film and incubated at 37\u0026deg;C overnight. The cells are then observed and photographed under a microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell cycle analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe ARPE-19 cells are seeded in a 6-well culture plate at a density of 1.5\u0026times; 10^5 cells and treated with or without MZ and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Subsequently, the cells are harvested and centrifuged at 1000g for 3 minutes. After discarding the supernatant, the cells are resuspended in 0.5 mL of cold PBS, and this process is repeated twice. For cell fixation, 75% ethanol precool is prepared in advance and slowly added to the centrifuge tube containing the cells from the previous step. The cells are then fixed overnight at 4℃. Staining is performed by incubating each sample in a 37℃ water bath with 20 \u0026micro;L of staining solution for 30 minutes. After centrifugation and discarding the supernatant, the cells are resuspended in 0.5 mL of PBS. Subsequently, 5 \u0026micro;L of 7-AAD dye is added to each sample, mixed well, and incubated for 1 hour at 4 ℃ in the dark. Finally, the samples are analyzed by flow cytometry (LSRFortessaTM; BD, Franklin Lakes, NJ, USA), with data collection from at least 30,000 cells. The results are then analyzed using FlowJo 7.6.2 software (Tree Star Inc., Ashland, OR, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern Blot\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCells are harvested and lysed using lysis buffer containing proteinase inhibitor to extract total protein. The protein concentration is determined using the BCA protein assay. Subsequently, the protein lysates are resolved by 4\u0026ndash;15% SDS-PAGE and transferred to PVDF membranes with a pore size of 0.22 \u0026micro;M. Following this, the membranes are blocked using 5% skim milk and incubated overnight at 4℃ with primary antibodies (MAPK Family Antibody Sampler Kit #9926,Cell Signaling Tecnology,Inc.), including ERK, JNK, p-38, and GAPDH. Following primary antibody incubation, the membranes are incubated with secondary antibodies (Goat anti-rabbit, Cell Signaling Tecnology,Inc.) IgG/HRP for 1 hour at room temperature.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePharmacokinetics studies\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis protocol of the animal experiment is approved by the Experimental Animal Ethics Committee, Dalian Medical University (Ethic number: AEE23131). Rats in both groups (n\u0026thinsp;=\u0026thinsp;6) were given Raw MZ and MZ Microcapsule Powder (calculated according to zeaxanthin) by intragastric administration at a dose of 30 mg/kg. After oral administration, blood was extracted from the jugular vein of the rats in at 1, 2, 4, 6, 8, 12, and 24 hours. The blood sample was added to a centrifuge tube with 1% heparin sodium and centrifuged at 3,000 rpm for 20 minutes. The supernatant was placed in a refrigerator at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003ePlasma samples were added to 200 \u0026micro;L of butylated hydroxytoluene (BHT) ethanol solution (0.1 g/L) and vortexed for 3 minutes to allow for thorough mixing. Sub-sequently, 600 \u0026micro;L of butylated hydroxytoluene (BHT) n-hexane solution (0.1 g/L) was added and vortexed for 5 minutes, and the suspension was centrifuged at 12,000 rpm for 10 minutes. Finally, the supernatant was dried at room temperature under a gentle stream of nitrogen. The dried residue was redissolved in 100 \u0026micro;L of mobile phase (methanol: water\u0026thinsp;=\u0026thinsp;88:12 and methyl tert-butyl ether volume ratio of 1:1). After vortexed for 3 minutes, the suspension was centrifuged at 12,000 rpm for 5 minutes, and 100 \u0026micro;L of the supernatant was extracted and injected into the HPLC apparatus.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Meso-zeaxanthin increased the cell viability of ARPE-19 cells\u003c/h2\u003e\n \u003cp\u003eInitially, we evaluated the potential cytotoxicity of MZ on ARPE-19 cells by exposing them to different concentrations of the compound. We determined that at a concentration of 20 \u0026micro;\u0026Mu;, cellular viability was optimized. Consequently, a concentration of 20 \u0026micro;mol/L MZ was employed in subsequent experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 A cellular oxidative stress model was established using a 300 \u0026micro;M concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is commonly employed to induce oxidative stress in \u003cem\u003ein vitro\u003c/em\u003e studies. To ascertain the optimal concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for this investigation, ARPE-19 cells were exposed to varying concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (100\u0026ndash;500\u0026micro;M/L) for 4 hours. Cell viability was decreased by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment dose-dependently. At 300\u0026micro;M, cell viability was halved. Consequently, a concentration of 300\u0026micro;M/L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was employed in subsequent experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 MZ conferred protection to ARPE-19 cells against oxidative stress damage induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\n \u003cp\u003eThe influence of MZ on the viability of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-damaged ARPE-19 cells was investigated by pre-treating the cells with MZ for 24 hours. Subsequently, except for the control group, the remaining two groups underwent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 4 hours. Changes in cell viability among those pre-treated with MZ were assessed using the CCK-8 assay. The results revealed a significant decrease in cell viability in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment group compared to the control group, confirming the successful induction of oxidative damage by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Statistical analysis indicated a marked increase in cell viability among those pre-treated with MZ compared to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Meso-zeaxanthin pretreatment decreased cellular ROS levels by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced injury\u003c/h2\u003e\n \u003cp\u003eThe overproduction of reactive oxygen species (ROS) is recognized as a primary contributor to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage and cell death. As illustrated in the figure, exposure to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e results in a significant elevation in cellular ROS levels. However, pre-treatment of ARPE-19 cells with MZ substantially suppresses the excessive ROS accumulation induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003e3.5 Meso-zeaxanthin treatment group exhibited an enhancement in the activities of GSH-PX and T-SOD in ARPE-19 cells during the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e damage, in contrast to the other treatment groups and demonstrated a reduction in MDA levels\u003c/p\u003e\n \u003cp\u003eTo investigate the influence of MZ on oxidative stress in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-damaged ARPE-19 cells, cells were exposed to MZ for 24 hours. Subsequently, except for the control group, the remaining two groups were pre-treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 hours. Changes in antioxidant stress among cells pre-treated with MZ were assessed using assay kits. We observed a significant decrease in GSH-PX and T-SOD levels in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group compared to the control group, along with a marked increase in MDA content, confirming successful induction of oxidative damage by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Statistical analysis revealed that co-treatment with MZ and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e significantly enhanced GSH-PX and T-SOD activities in cells compared to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, while reducing MDA levels.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Meso-Meszeaxanthin alleviated Cell Senescence caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\n \u003cp\u003eWe examined the effect of MZ pre-treatment on the prevalence of senescent cells in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-damaged ARPE-19 cells. ARPE-19 cells were pretreated with MZ for 24 hours, followed by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 4 hours to induce senescence. Senescent cells were visualized using \u0026beta;-galactosidase staining and observed under fluorescence microscopy. Our findings demonstrated a substantial increase in staining precipitates in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated group compared to the blank control group, confirming successful induction of senescence by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Conversely, the group pre-treated with MZ exhibited a marked reduction in staining precipitates compared to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Influence of Meso-zeaxanthin on cell cycle progression\u003c/h2\u003e\n \u003cp\u003eTo delve deeper into the impact of MZ pre-treatment on the cell cycle of ARPE-19 cells during H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced damage, we employed flow cytometry. ARPE-19 cells were stained with 7-AAD dye to evaluate the cell cycle of both H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated cells and cells pre-treated with MZ. The findings revealed that the cell cycle of ARPE-19 cells induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was arrested at the G2/M phase, accompanied by a decrease in the number of cells in the G1 phase. Conversely, in the MZ pre-treatment group, there was a reduction in the number of cells in the G2/M phase, highlighting the significant amelioration of G2/M phase arrest induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e damage with MZ pre-treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8 Influence of Meso-zeaxanthin on Cell MAPK Signaling Pathway\u003c/h2\u003e\n \u003cp\u003eTo explore the impact of MZ on MAPK signaling pathway proteins in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e -damaged ARPE-19 cells, the expression levels of p38, JNK, and ERK proteins were assessed through Western blot analysis. Statistical analysis revealed that the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group displayed significantly elevated expression of p38, JNK, and ERK compared to the Ctrl group, whereas the MZ-treated group exhibited a notable reduction in the expression levels of these proteins compared to the Ctrl group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.9 Characterization of MZ uptake by Caco-2 cells\u003c/h2\u003e\n \u003cp\u003eTo evaluate the absorption efficacy of microencapsulated products, the Caco-2 model was utilized for cell uptake experiments. Results indicated that MZ Microcapsule Powder highly improved the absorption capacity of Caco-2 cells for MZ compared to Raw MZ, demonstrating a cell uptake rate approximately 3.6 times higher than that of Raw MZ. Furthermore, the conformational composition of zeaxanthin in the cellular uptake samples of MZ Microcapsule Powder remained largely unchanged before and after Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Hence, there seems to be no significant configuration selectivity difference in the uptake of zeaxanthin by Caco-2 cells, suggesting their ability to uptake various zeaxanthin configurations, including MZ.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eConformational composition of cells before and after uptake of MZ Microcapsule Powder.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIngredients(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore uptake\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter uptake\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMeso-zeaxanthin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3R,3\u0026rsquo;R-zeaxanthin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3S,3\u0026rsquo;S-zeaxanthin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLutein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.10 Bioavailability analysis\u003c/h2\u003e\n \u003cp\u003eThe concentration-time curves depicting orally administered Raw MZ and MZ Microcapsule Powder are presented in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e. Pharmacokinetic parameters of MZ Microcapsule Powder and Raw MZ were compared (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, the microcapsule powder exhibited superior absorption efficacy compared to Raw MZ, as evidenced by higher AUC and C\u003csub\u003emax\u003c/sub\u003e values. Additionally, the AUC\u003csub\u003e(0\u0026ndash;24)\u003c/sub\u003e values for zeaxanthin in rats were 137.48 \u0026micro;g/Lh and 103.341 \u0026micro;g/Lh in the MZ Microcapsule Powder and Raw MZ groups, respectively, indicating a 1.33-fold increase in bioavailability of zeaxanthin in the MZ Microcapsule Powder group compared to the Raw MZ group. Both microcapsule powder and Raw MZ displayed a T\u003csub\u003emax\u003c/sub\u003e of 8 hours, suggesting similar absorption rates. However, the microcapsule powder demonstrated enhanced absorption efficacy compared to Raw MZ. Therefore, pharmacokinetic studies suggest that MZ Microcapsule Powder, prepared using microencapsulation technology, can enhance in vivo bioavailability, improve zeaxanthin absorption.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePharmacokinetic parameters of the MZ Microcapsule Powder and Raw MZ\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRoom parameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUnit\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRaw MZ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMZ Microcapsule Powder\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUC\u003csub\u003e(0\u0026minus;24)\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026micro;g/L*h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e137.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026micro;g/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.494\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.427\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBioavailability\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e133.035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMeso-zeaxanthin (MZ), along with lutein (Lut) and zeaxanthin (Z), forms the macular pigment (MP), playing a crucial role in protecting cells and organs from oxidative damage induced by oxygen free radicals, thus preventing certain eye diseases\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. While MZ is a metabolite of Lut, it can also be obtained from dietary sources\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The concentration of MZ in the macula surpasses that in serum and liver, underscoring specific absorption and storage mechanisms within the retina, highlighting its pivotal role in retinal function\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Initially believed to be undetectable in human liver or serum\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, subsequent research identified MZ in serum, and supplementation studies demonstrated a notable increase in macular pigment levels following oral administration of MZ, indicating its oral bioavailability and transport to the macula\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Despite its conversion from Lut in the retina, supplementation with high doses of MZ leads to elevated MZ levels in both the macula and serum compared to Lut and Z supplementation alone\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Our results suggest that MZ is directly absorbed and utilized in addition to being converted from lutein, as verified by \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e models.\u003c/p\u003e \u003cp\u003eIn the outer retina, macular pigment acts as a potent antioxidant, countering oxidative damage to photoreceptor outer segments containing chromophores that serve as photosensitizers susceptible to oxidation\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. MZ exhibits superior quenching capabilities against oxygen free radicals compared to Lut\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, particularly in regions of the retina with the highest cone density and heightened risk of oxidative damage\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.However, its underlying mechanisms of action remain to be thoroughly investigated.\u003c/p\u003e \u003cp\u003eIn this study, MZ treatment of ARPE-19 cells mitigated the adverse effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and suppressed excessive ROS accumulation induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;4). Furthermore, MZ treatment significantly bolstered intracellular GSH-PX and T-SOD activities, while reducing MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and effectively mitigated cellular senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Additionally, pre-treatment with meso-zeaxanthin markedly ameliorated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced G2/M phase arrest (Fig.\u0026nbsp;7) and attenuated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced activation of the MAPK pathway, indicative of oxidative stress damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMeso-zeaxanthin is an important fat-soluble compound that is difficult to dissolve in water and its application is greatly limited. Because of its structure contains 11 conjugated double bonds, and the tail group with hydroxyl, this structure determines its nature is very active and easy to oxidize and isomerize. Therefore, in order to prevent its isomerization and microencapsulation of zeaxanthin is technically difficult.\u003c/p\u003e \u003cp\u003eIn this study, MZ Microcapsule Powder was prepared utilizing a distinctive microencapsulation technique, and absorption was assessed both ex and in vivo using Caco-2 cells and rat models. Experimental results demonstrated a substantial enhancement in the uptake capacity of Caco-2 cells for meso-zeaxanthin facilitated by microencapsulation technology, with the cellular uptake rate in the MZ Microcapsule Powder group approximately 3.6 times higher than that in the Raw MZ group ( Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Additionally, following administration of a 30 mg/kg dosage of MZ product to rats, the Cmax and AUC\u003csub\u003e(0\u0026minus;24)\u003c/sub\u003e values of MZ Microcapsule Powder surpassed those of Raw MZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e), indicating superior absorption of MZ Microcapsule Powder compared to Raw MZ. Utilizing Raw MZ as a benchmark preparation, the relative bioavailability of MZ Microcapsule Powder in rats was determined to be 133.035%, implying a noteworthy enhancement in MZ bioavailability post-encapsulation into microcapsules.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eInitially, MZ demonstrated robust antioxidant efficacy at the cellular level. Furthermore, in rat studies, MZ Microcapsule Powder, formulated with microencapsulation technology, exhibited superior bioavailability relative to Raw MZ. These observations indicate that MZ is capable of direct absorption, and the utilization of microencapsulation technology markedly enhances its bioavailability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare that there is no conflict of interest regarding the publication of this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThese authors have contributed equally to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eZhang SM, Fan B, Li YL, et al. Oxidative Stress-Involved Mitophagy of Retinal Pigment Epithelium and Retinal Degenerative Diseases [J]. 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Nutritional and clinical relevance of lutein in human health [J]. Br J Nutr. 2003;90(3):487\u0026ndash;502. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1079/bjn2003927\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAkuffo KO, Nolan JM, Howard AN, et al. Sustained supplementation and monitored response with differing carotenoid formulations in early age-related macular degeneration [J]. Eye (Lond). 2015;29(7):902\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/eye.2015.64\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKim SR, Nakanishi K, Itagaki Y, et al. Photooxidation of A2-PE, a photoreceptor outer segment fluorophore, and protection by lutein and zeaxanthin [J]. Exp Eye Res. 2006;82(5):828\u0026ndash;39.\u0026nbsp;\u003c/span\u003e\u003cspan\u003e1016/j.exer.2005.10.004.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nutrire","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Nutrire](https://www.springer.com/journal/41110)","snPcode":"41110","submissionUrl":"https://submission.nature.com/new-submission/41110/3","title":"Nutrire","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ARPE-19, Antioxidant activity, meso-Zeaxanthin, microencapsulation, Bioavailability","lastPublishedDoi":"10.21203/rs.3.rs-6869584/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6869584/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOxidative stress plays a pivotal role in the etiology of retinal degeneration, with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) serving as a significant oxidant triggering oxidative stress within cellular environments. Meso-Zeaxanthin (MZ), a vital carotenoid, is renowned for its ocular health maintenance properties.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo assess MZ's protective attributes against oxidative stress in human retinal pigment epithelial cells (ARPE-19), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was employed to induce oxidative stress in ARPE-19 cells. Subsequently, various parameters including cell viability, oxidative stress-related enzyme activities, reactive oxygen species (ROS) generation, cellular senescence, and cell cycle progression were evaluated. Additionally, in the realm of MZ bioavailability, microencapsulated MZ was investigated for its effects on human colorectal adenocarcinoma cells (Caco-2) and SD rat models. Subsequent evaluations encompassed cellular uptake, chiral structure assessment and bioavailability determination.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe findings underscore MZ's remarkable antioxidant prowess, characterized by enhanced cell viability, diminished malondialdehyde (MDA) levels, augmented activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX), coupled with attenuated ROS production in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated ARPE-19 cells. Remarkably, MZ downregulates ERK/JNK/P38MAPK expression in the MAPK pathway post-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure, thereby ameliorating oxidative stress-induced cellular damage. MZ Microcapsule Powder enhances Caco-2 uptake compared to the Raw MZ group, after confirming negligible configurational selectivity differences in MZ uptake by Caco-2 cells. Furthermore, bioavailability experiments conducted in SD rats indicate elevated plasma zeaxanthin levels in the MZ Microcapsule Powder group compared to the Raw MZ group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eConsequently, MZ has a strong antioxidant capacity and products formulated utilizing microencapsulation technology hold promise for enhancing in vivo bioavailability.\u003c/p\u003e","manuscriptTitle":"Studies on the antioxidant properties and bioavailability of meso-zeaxanthin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 17:14:33","doi":"10.21203/rs.3.rs-6869584/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-02T15:08:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T15:24:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121112001059897932187723751015326524650","date":"2025-07-15T10:04:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237981044980393357330956122375099841838","date":"2025-07-10T04:30:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-02T18:42:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175381358430734950508598066591102269737","date":"2025-06-24T18:29:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-24T11:36:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-24T11:30:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-24T00:30:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Nutrire","date":"2025-06-11T08:29:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nutrire","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Nutrire](https://www.springer.com/journal/41110)","snPcode":"41110","submissionUrl":"https://submission.nature.com/new-submission/41110/3","title":"Nutrire","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bf1cc356-6d21-48d2-b58b-6db9f70608a2","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-09-12T19:38:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-26 17:14:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6869584","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6869584","identity":"rs-6869584","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}