Luminescent Alendronic Acid-Conjugated Micellar Nanostructures for Potential Application in the Bone-Targeted Delivery of Cholecalciferol.

Vitamin D, an essential micronutrient crucial for skeletal integrity and various non-skeletal physiological functions, exhibits limited bioavailability and stability in vivo. This study is focused on the development of polyethylene glycol (PEG)-grafted phospholipid micellar nanostructures co-encapsulating vitamin D3 and conjugated with alendronic acid, aimed at active bone targeting. Furthermore, these nanostructures are rendered optically traceable in the UV-visible region of the electromagnetic spectrum via the simultaneous encapsulation of vitamin D3 with carbon dots, a newly emerging class of fluorescents, biocompatible nanoparticles characterized by their resistance to photobleaching and environmental friendliness, which hold promise for future in vitro bioimaging studies. A systematic investigation is conducted to optimize experimental parameters for the preparation of micellar nanostructures with an average hydrodynamic diameter below 200 nm, ensuring colloidal stability in physiological media while preserving the optical luminescent properties of the encapsulated carbon dots. Comprehensive chemical-physical characterization of these micellar nanostructures is performed employing optical and morphological techniques. Furthermore, their binding affinity for the principal inorganic constituent of bone tissue is assessed through a binding assay with hydroxyapatite nanoparticles, indicating significant potential for active bone-targeting. These formulated nanostructures hold promise for novel therapeutic interventions to address skeletal-related complications in cancer affected patients in the future.

Rational Design of Trident Aptamer Scaffold for Rapid and Accurate Monitoring of 25-Hydroxyvitamin D3 Metabolism in Living Cells.

The level of 25-hydroxyvitamin D3 [25(OH)VD3] in human blood is considered as the best indicator of vitamin D status, and its deficiency or excess can lead to various health problems. Current methods for monitoring 25(OH)VD3 metabolism in living cells have limitations in terms of sensitivity and specificity and are often expensive and time-consuming. To address these issues, an innovative trident scaffold-assisted aptasensor (TSA) system has been developed for the online quantitative monitoring of 25(OH)VD3 in complex biological environments. Through the computer-aided design, the TSA system includes an aptamer molecule recognition layer that is uniformly oriented, maximizing binding site availability, and enhancing sensitivity. The TSA system achieved the direct, highly sensitive, and selective detection of 25(OH)VD3 over a wide concentration range (17.4-12,800 nM), with a limit of detection of 17.4 nM. Moreover, we evaluated the efficacy of the system in monitoring the biotransformation of 25(OH)VD3 in human liver cancer cells (HepG2) and normal liver cells (L-02), demonstrating its potential as a platform for drug-drug interaction studies and candidate drug screening.

Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells.

Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.

Surface, rheopexy, digestive stability and toxicity of olive oil emulsions stabilized by chitin nanocrystals for vitamin D3 delivery.

Pickering emulsions are of interest in medicament transport systems. The properties of emulsions are influenced by the type of oil and the surface structure of nanoparticles-stabilizers. The process of formation of o/w emulsions of olive oil stabilized by chitin nanocrystals was investigated, their stability under the influence of physical factors, rheological characteristics, acute toxicity after oral administration, stability under the conditions of a model of the gastrointestinal tract, and their potential for oral transport of vitamin D3 were analyzed. Physically stable emulsions were obtained at a stabilizer concentration of 3.6 g/l. The addition of electrolyte leads to a substantial reduction in the average size of microdroplets. The resulting emulsions have rheopexy properties and the rheopexy index increases at 37 °C. Emulsions are classified as non-toxic when taken orally, physically stable in the upper digestive system, and capable of efficiently transporting vitamin D3 with a full release in the small intestine.

Affinity-based protein profiling identifies vitamin D3 as a heat shock protein 70 antagonist that regulates hedgehog transduction in murine basal cell carcinoma.

Vitamin D3 (VD3) is a seco-steroid that inhibits the Hedgehog (Hh) signaling pathway. Initial studies suggested its anti-Hh activity results from direct inhibition of Smoothened, a seven-transmembrane cell surface receptor that is a key regulator of the Hh signaling cascade. More recently, a role for the Vitamin D Receptor in mediating inhibition of Hh-signaling by seco-steroid has been suggested. Herein, an affinity-based protein profiling study was carried out to better understand the cellular proteins that govern VD3-mediated anti-Hh activity. We synthesized a novel biotinylated VD3 analogue (8) for use as a chemical probe to explore cellular binding targets of the seco-steroidal scaffold. Through a series of pull-down experiments and follow up mass spectrum analyses, heat shock protein 70 (Hsp70) was identified as a primary binding protein of VD3. Hsp70 was validated as a binding target of VD3 through a series of biochemical and cellular assays. VD3 bound with micromolar affinity to Hsp70. In addition, both selective knockdown of Hsp70 expression and pharmacological inhibition of its activity with known Hsp70 inhibitors suppressed Hh-signaling transduction in murine basal cell carcinoma cells, suggesting that Hsp70 regulates proper Hh-signaling. Additional cellular assays suggest that VD3 and its seco-steroidal metabolites inhibit Hh-signaling through different mechanisms.

Sulindac and vitamin D3 synergically inhibit proliferation of MCF-7 breast cancer cell through AMPK/Akt/ß-catenin axis in vitro.

Breast cancer is associated with a high rate of recurrence, resistance therapy and mortality worldwide. We aimed at investigating the inhibitory effects of Sulindac and vitamin D3 (VD) on MCF-7 human breast cancer cells. MCF-7 cells were cultured with different concentrations of Sulindac and VD over a period of 24, 48 and 72 hours for cell viability and IC50 experiments. Hochst staining was used to evaluate apoptosis, whereas quantitative PCR (qPCR) was performed to measure mRNA levels of BCL-2 and BAX genes. Immunofluorescence staining was used to monitor intracellular ß-catenin expression. The protein levels of AKT, AMPK and P65 were measured by western blotting. The result showed that cell viability decreased in treated cells dose/time dependently (P < .05). Hochst staining showed an increase in fragmented nuclei in treated cells. The expression of BCL-2 and BAX genes decreased and increased in treated cells, respectively (P < .05). Immunofluorescence staining indicated that the expression of ß-catenin significantly reduced in treated cells. The AKT-1/p-Akt-1 and AMPK/p-AMPK ratio increased in treated cells (P < .05), but the P65/p-P65 ratio did not change significantly (P > .05). Our results indicated that the combination of Sulindac and VD has a growth-inhibiting effect on MCF-7 cells through AMPK/Akt/ß-catenin axis.

Investigation of Vitamin D2 and Vitamin D3 Hydroxylation by Kutzneria albida.

The active vitamin D metabolites 25-OH-D and 1α,25-(OH)2 -D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D2 (VD2 ) and vitamin D3 (VD3 ) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25-OH-D2 and 25-OH-D3 was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100-fold higher product concentrations of 25-OH-D3 and tenfold higher concentrations of 25-OH-D2 after addition of 5 % (w/v) 2-hydroxypropyl ß-cyclodextrin (2-HPßCD) were reached. Besides the single-hydroxylation products, the human double-hydroxylation products 1,25-(OH)2 -D2 and 1,25-(OH)2 -D3 and various other potential single- and double-hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD2 and VD3 metabolites.

Novel osteoprotective nanocochleate formulation: A dual combination therapy-codelivery system against glucocorticoid induced osteoporosis.

Phosphatidylserine nanocochleates (Nanocochs) are novel delivery systems that may play a prominent osteoprotective role with their cargo, vitamin D3 (Vit-D3), against osteoporosis. Therefore, this study was conducted to characterize a Nanococh containing vitamin D3 (Nanococh-D3) and investigate its potential role in improving GIO in a rat model. Roll-shaped Nanococh-D3 particles were obtained in a size range of 320 nm with a sustained release performance. Oral Nanococh-D3 significantly increased the bioavailability of Vit-D3, enhanced bone mechanical strength, and improved osteogenic biomarkers including B-ALP, osteocalcin, Ca, and OPG in GIO rats. This formulation markedly suppressed gene expression of RANK and RANKL in treated rats. Histomorphometric analysis showed significant repairs in bone tissues and TRAP staining indicated a significant decrease in osteoclasts using Nanococh-D3 in osteoporotic rats. Nanococh alone similar to Nanococh-D3 acted better than AL as a standard anti-osteoporotic drug in the improvement of bone strength. In conclusion, our results established the potential role of Nanococh-D3 against osteoporosis in rats.

Synthesis of peptide conjugates with vitamins for induction of antigen-specific immunotolerance.

In this report, we designed conjugates of an antigen peptide with the immunosuppressive vitamins all-trans retinoic acid (ATRA) and vitamin D3 for efficient induction of antigen-specific immunotolerance. We established a synthetic scheme for the preparation of the peptide-vitamin conjugates, which the chemically unstable vitamins tolerated. Among the obtained conjugates, the ATRA conjugate successfully suppressed inflammatory effects in macrophages and dendritic cells and induced antigen presentation in dendritic cells. This synthetic method of conjugate is conceivably applicable to other antigen peptides for induction of antigen-specific immunotolerance.

Encapsulation of vitamin D3 in gum arabic to enhance bioavailability and stability for beverage applications.

Delivery of vitamin D3 (VD3 ) in foods should exhibit desirable physicochemical characteristics and improves absorption. In this study, gum arabic (GA) was investigated as a VD3 carrier to encapsulate VD3 . VD3 dissolved in 5 mL ethanol corresponding to 0.3 to 6.0% mass of GA, was blended in 5.0% w/v GA solution, followed by freeze drying. The encapsulation efficiency decreased while loading capacity increased with an increased amount of VD3 . At the highest VD3 level, the loading capacity (3.47%) was the highest, and the encapsulation efficiency (61.24%) was satisfactory, and the treatment was further studied. The magnitude of negative zeta-potential increased from 3.1 to 31.0 mV at pH 2.0 to 7.4. During the 100-day storage at 3 °C of capsules reconstituted at pH 2.0 to 7.4, the hydrodynamic diameter decreased at all pH conditions, most evident for reduction to 81.3 nm at pH 7.4, and no precipitation was observed, indicating the significance of steric repulsion on capsule stability. Bioaccessibility of VD3 in capsules (95.76%) was significantly higher than the nonencapsulated VD3 (68.98%). The in vivo pharmacokinetic study in Sprague-Dawley rats after a single-dose of 300 µg VD3 showed the area-under-curve of serum 25(OHD) level in 48 hr of the encapsulation treatment was 4.32-fold of the nonencapsulated VD3 and more than twice higher than the VD3 -GA physical mixture. During 2-week supplementation of 60 µg VD3 /d, rats receiving capsules or physical mixture had 25(OH)D levels of at least 81 ng/mL higher than that of the nonencapsulated VD3 group. The studied encapsulation system holds great potential as a value-added ingredient to supplement VD3 in beverages with a wide pH range. PRACTICAL APPLICATION: The findings of this study demonstrated the improved dispersion stability and absorption of vitamin D3 after encapsulation in gum arabic. The capsules exhibited good dispersion stability across a pH range between 2.0 and 7.4, showing potential application in beverages. Furthermore, the enhanced absorption of VD3 after encapsulation highlights the nutritional benefits of the studied encapsulation system.

Photoprotective Properties of Vitamin D and Lumisterol Hydroxyderivatives.

We have previously described new pathways of vitamin D3 activation by CYP11A1 to produce a variety of metabolites including 20(OH)D3 and 20,23(OH)2D3. These can be further hydroxylated by CYP27B1 to produce their C1α-hydroxyderivatives. CYP11A1 similarly initiates the metabolism of lumisterol (L3) through sequential hydroxylation of the side chain to produce 20(OH)L3, 22(OH)L3, 20,22(OH)2L3 and 24(OH)L3. CYP11A1 also acts on 7-dehydrocholesterol (7DHC) producing 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone (7DHP) which can be converted to the D3 and L3 configurations following exposure to UVB. These CYP11A1-derived compounds are produced in vivo and are biologically active displaying anti-proliferative, anti-inflammatory, anti-cancer and pro-differentiation properties. Since the protective role of the classical form of vitamin D3 (1,25(OH)2D3) against UVB-induced damage is recognized, we recently tested whether novel CYP11A1-derived D3- and L3-hydroxyderivatives protect against UVB-induced damage in epidermal human keratinocytes and melanocytes. We found that along with 1,25(OH)2D3, CYP11A1-derived D3-hydroxyderivatives and L3 and its hydroxyderivatives exert photoprotective effects. These included induction of intracellular free radical scavenging and attenuation and repair of DNA damage. The protection of human keratinocytes against DNA damage included the activation of the NRF2-regulated antioxidant response, p53-phosphorylation and its translocation to the nucleus, and DNA repair induction. These data indicate that novel derivatives of vitamin D3 and lumisterol are promising photoprotective agents. However, detailed mechanisms of action, and the involvement of specific nuclear receptors, other vitamin D binding proteins or mitochondria, remain to be established.

Spectral transmission of solar radiation by plastic and glass materials.

It is well known that excessive exposure to solar ultraviolet (UV) radiation can have serious adverse effects. Many everyday materials influence the UV radiation received by humans, for example, those used in construction and on the exterior of buildings such as plastics and glass can reduce the UV exposure of persons exposed to solar radiation. In this paper we analyse the spectral transmission of solar radiation of widely used materials using the transmittance parameter. The measurements were performed on clear days, at 8 h and 12 solar hours, in July 2018 (five days) and in January 2019 (three days). The spectral transmittances of these materials and the integrated transmittances in the UVB from 300 nm, UVA, visible (VIS) and near infrared ranges (NIR) were calculated. In summer in the UVB range from 300 nm methacrylate and smoked glass have the highest transmittance values (56%) and polycarbonate present the lowest (30%). In the VIS and NIR ranges methacrylate (95%) and smoked glass (80%) have the highest transmittances and polycarbonate the lowest (45%). In general the 8 h transmittances are higher than those at 12 h and are also higher in winter than summer. For two biological functions (erythemal and DNA-damage) and for the UVB range from 300 nm, the transmittance for most materials (except fibreglass) is in the range 6-14%. The exposure times obtained show that erythemal damage could occur after long exposure to solar radiation through the materials studied, information which should be made available to the general public.

Bioaccessibility study of calcium and vitamin D3 co-microencapsulated in water-in-oil-in-water double emulsions.

Calcium and vitamin D3 were co-encapsulated in three types of water-in-oil-in-water (W/O/W) double emulsions stabilized with biopolymers: gum arabic, sodium alginate (Alg) and chitosan (Ch). Three calcium salts with different solubility were used: calcium carbonate (CaC), tricalcium phosphate (CaP) and calcium gluconate (CaG). In order to study the bioavailability of calcium and vitamin D3, the W/O/W double emulsions were subjected to digestion in simulated conditions using in vitro gastrointestinal models. The size of the oil droplets of all double emulsions increased in oral phase and decreased in gastric and intestinal phases. In the intestinal phase, the average diameter of oil globules in the W/O/W(Alg) and W/O/W(Ch) was d23 = 6.56 ±â€¯0.09 and d23 = 5.33 ±â€¯0.01 and the electro-kinetic potential was: ζ ≈ -25 mV and ζ ≈ -17 mV, respectively. Presence of calcium ions in the intestinal fluid decreased the free fatty acids content and decreased the bioaccessibility of vitamin D3 due to the inhibition of micellization process.

Thermal Stability and Kinetic Study on Thermal Degradation of Vitamin D3 in Fortified Canola Oil.

Nowadays, fortified vegetable oils with vitamin D3 are widely available in different countries and are consumed daily. The reduction rate of added vitamin D3 in fortified canola oil during heating process, the changes in oxidative status, and the thermal kinetic degradation of vitamin D3 in the fortified oil were investigated. For this purpose, canola oil was fortified at two levels of vitamin D3 with 5.625 µg/mL (low concentration or LC) and 13.585 µg/mL (a high concentration or HC). Samples were heated isothermally at 100, 150, and 180 °C for 30 min. The vitamin D3 concentration was determined by the high-performance liquid chromatographic method. The retention of vitamin D3 in samples treated at 100 °C for 30 min showed no significant reduction. Samples treated at 150 and 180 °C depending on the initial concentration showed the retention of 67.5% to 72.97% and 33.16% to 40.35% of vitamin D3 , respectively. An inverse relationship was found between the increment of lipid oxidation products (peroxide and anisidine values) and the retention of vitamin D3 . Kinetic parameters such as rate constant, activation energy, decimal reduction time, and quotient indicator were also calculated. An Arrhenius relationship was used for the assessment of temperature dependence of vitamin D3 degradation. Activation energies for vitamin D3 in LC and HC between 100 and 180 °C were found to be 44.01 and 38.77 kJ/mol, respectively. PRACTICAL APPLICATION: The oil can be fortified with vitamin D3 at low cost and offers a good bioavailability. A high-temperature cooking method may not be appropriate for the fortified products containing high lipid content.

Protective effects of novel derivatives of vitamin D3 and lumisterol against UVB-induced damage in human keratinocytes involve activation of Nrf2 and p53 defense mechanisms.

We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50-200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.

Impact of an indigestible oil phase (mineral oil) on the bioaccessibility of vitamin D3 encapsulated in whey protein-stabilized nanoemulsions.

These is some interest in replacing digestible fats with indigestible ones to decrease the energy-density of foods. The utilization of indigestible oils, however, may have unforeseen nutritional consequences, such as the reduction of vitamin bioavailability. In this study, the impact of an indigestible oil on the bioaccessibility of emulsified vitamin D3 (VD) was examined. We prepared four kinds of nanoemulsions using different combinations of a digestible oil (DO) and an indigestible oil (IO): DO only; IO only; an oil mixture (OM) consisting of 1:1 DO:IO mixed before homogenization; and, an emulsion mixture (EM) consisting of 1:1 DO:IO nanoemulsions mixed after homogenization. A gastrointestinal tract (GIT) model was employed to elucidate the kinetics of VD bioaccessibility from the nanoemulsions. Both the lipid digestion rate and vitamin bioaccessibility decreased in the same order: DO > OM ≈ EM > IO. The change in vitamin bioaccessibility over time under simulated small intestine conditions was also measured. With the exception of the IO nanoemulsions, the vitamin bioaccessibility increased to a maximum value after around 30 min, but then decreased during the following 24 h. This effect was attributed to an initial solubilization of the vitamin within the mixed micelles, followed by their precipitation during prolonged incubation. Our results show that lipid digestion, micelle solubilization, and micelle aggregation impact the in vitro bioaccessibility of vitamin D. This knowledge may be helpful for designing more efficacious nanoemulsion-based delivery systems for fat-soluble vitamins.

Improved Stability of Polyglycerol Polyricinoleate-Substituted Nanostructured Lipid Carrier Cholecalciferol Emulsions with Different Carrier Oils.

Cholecalciferol, also known as vitamin D3 , is a recognized therapeutic agent for treatment of bone diseases and cancer. However, instability and poor bioavailability have been major challenges for delivering Vitamin D3 . The objective of this study was to formulate improved nanostructured lipid carrier (NLC) vitamin D3 emulsions. We tested the effect of different carrier oils and the use of a solid lipid nanoparticle emulsifier, polyglycerol polyricinoleate (PGPR) on the stability of the vitamin D3 emulsions. In contrast to the control that used glyceryl monostearate (GMS) the PGPR substitution resulted in relatively small particle sizes (0.30 to 0.43 µm), with high absolute value of zeta potentials (39.5 to 67.8 mV) and high encapsulation efficiency (85.2% to 90.4%). The stability of the NLC emulsions against environmental stresses was evaluated under varying conditions of ionic strength, pH, freeze-thaw cycles, and storage at different temperatures. Although NLC emulsions were stable at high ionic strengths, they were found to be unstable at low pH (<3), which led to aggregation and coalescence of emulsion droplets. In case of freeze-thaw stress, although relatively stable compared to control NLC, the PGPR substituted groups exhibited a slight increase in particle size and a decrease in zeta potential when the cycle was repeated five times. Additionally, we found that PGPR-substituted emulsions showed higher liquid dispersion stability than controls at 25 and 65 °C. Thus, we have formulated a modified NLC vitamin D3 emulsion that can be widely used in the food industry. PRACTICAL APPLICATION: Vitamin D3 , an essential micronutrient, is often added as supplements in food products and beverages for added health benefits. However, the stability of vitamin D3 emulsions that are used in the preparation of such products has been a major concern. We have developed a modified emulsion that has improved stability against environmental stresses. We believe, in future, this formulation can be efficiently used in the food industry.

Fabrication of plant-based vitamin D3-fortified nanoemulsions: influence of carrier oil type on vitamin bioaccessibility.

The influence of carrier oil type (corn, fish, or flaxseed oil) on the production, stability, and simulated gastrointestinal behavior of vitamin-fortified nanoemulsions was studied. The nanoemulsions were formulated using pea protein as an emulsifier since there is increasing interest in substituting artificial and animal-based food ingredients with natural plant-based alternatives. Lipid digestion and vitamin D3 bioaccessibility were measured when the nanoemulsions were subjected to a three-stage in vitro gastrointestinal tract: oral, gastric, and small intestinal. The majority of all three lipids were digested within the first few minutes in the simulated small intestine, with the corn oil nanoemulsions being digested faster than the fish or flaxseed oils. Moreover, a greater fraction of triglycerides were digested by the end of the small intestine for the corn oil than for the fish and flaxseed oils. For the different carrier oils, vitamin bioaccessibility was ranked: corn oil > flaxseed oil ≈ fish oil. These results suggest that monounsaturated-rich oils (such as corn oil) are better for encapsulating and delivering vitamin D3 than polyunsaturated-rich ones (such as flaxseed or fish oil). The insights gained here may aid in the formulation of more efficacious vitamin-fortified foods and beverages from plant-derived ingredients.

Effects of six compounds with different chemical structures on melanogenesis.

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D3, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 µmol·L-1in vivo and 100 µmol·L-1in vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.