Carotenoid Transport Research Articles

The Absorption, Storage, and Transport of Ocular Carotenoids and Retinoids.

Carotenoids, yellow and red pigments found abundantly in nature, play essential roles in various aspects of human physiology. They serve as critical molecules in vision by functioning as antioxidants and as filters for blue light within the retina. Furthermore, carotenoids are the natural precursors of vitamin A, which is indispensable for the synthesis of retinaldehyde, the visual chromophore, and retinoic acid, a small molecule that regulates gene expression. Insufficient levels of carotenoids and retinoids have been linked to age-related macular degeneration and xerophthalmia, respectively. Nevertheless, the mechanisms by which the eye maintains carotenoid and retinoid homeostasis have remained a mystery. Recent breakthroughs identified the molecular players involved in this process and provided valuable biochemical insights into their functioning. Mutations in the corresponding genes disrupt the homeostasis of carotenoids and retinoids, leading to visual system pathologies. This review aims to consolidate our current understanding of these pathways, including their regulatory principles.

Imaging macular carotenoids and their related proteins in the human retina with confocal resonance Raman and fluorescence microscopy

Lutein and zeaxanthin are highly concentrated at the central region of the human retina, forming a distinct yellow spot known as the macula lutea. The delivery and retention of the macular pigment carotenoids in the macula lutea involves many proteins, but their exact roles remain incompletely understood. In our study, we examined the distribution of the twelve known macular carotenoid-related proteins within the human macula and the underlying retinal pigment epithelium (RPE) using both fluorescence and Raman modes on our confocal resonance Raman microscope. Additionally, we assessed protein and gene expression through Western blot analysis and a single-cell RNA sequencing database. Our findings revealed that GSTP1, BCO2, and Aster-B exhibited distribution patterns similar to the macular carotenoids, with higher expression levels within the macular region compared to the periphery, while SR-BI and ABCA1 did not exhibit specific distribution patterns within the macula or RPE. Interestingly, LIPC, SR-BI's partner, accumulated specifically in the sub-foveal RPE. All three of these carotenoid transport proteins were found to be highly expressed in the RPE. These results offer valuable insights into the roles these proteins play in the formation of the macula lutea.

Carotenoid basics: from food to skin

The aim of this review is to provide a comprehensive and simple graphical overview on carotenoids. The review describes in four detailed figures the course of carotenoids from food to skin. Differences in chemical structures of the six most prominent carotenoids, namely, α-carotene, β-carotene, lutein, zeaxanthin, lycopene and β-cryptoxanthin, and their seasonal variations in plasma concentrations resulting from different availability of fruits and vegetables are highlighted. Furthermore, factors affecting carotenoid status, sites of storage and how and where they can be assessed (dietary intake and in vivo) are discussed. The route of carotenoid transport after ingestion, transport across the enterocyte and receptors involved in carotenoid uptake in peripheral cells are shown followed by the routes of carotenoid delivery to the skin and reasons for variations in skin carotenoid status are visualized.

RNA-Seq analysis of the pyloric caecum, liver, and muscle reveals molecular mechanisms regulating fillet color in rainbow trout

BackgroundThe characteristic pink-reddish color in the salmonids fillet is an important, appealing quality trait for consumers and producers. The color results from diet supplementation with carotenoids, which accounts for up to 20–30% of the feed cost. Pigment retention in the muscle is a highly variable phenotype. In this study, we aimed to understand the molecular basis for the variation in fillet color when rainbow trout (Oncorhynchus mykiss) fish families were fed an Astaxanthin-supplemented diet. We used RNA-Seq to study the transcriptome profile in the pyloric caecum, liver, and muscle from fish families with pink-reddish fillet coloration (red) versus those with lighter pale coloration (white).ResultsMore DEGs were identified in the muscle (5,148) and liver (3,180) than in the pyloric caecum (272). Genes involved in lipid/carotenoid metabolism and transport, ribosomal activities, mitochondrial functions, and stress homeostasis were uniquely enriched in the muscle and liver. For instance, the two beta carotene genes (BCO1 and BCO2) were significantly under-represented in the muscle of the red fillet group favoring more carotenoid retention. Enriched genes in the pyloric caecum were involved in intestinal absorption and transport of carotenoids and lipids. In addition, the analysis revealed the modulation of several genes with immune functions in the pyloric caecum, liver, and muscle.ConclusionThe results from this study deepen our understanding of carotenoid dynamics in rainbow trout and can guide us on strategies to improve Astaxanthin retention in the rainbow trout fillet.

ASTER-B regulates mitochondrial carotenoid transport and homeostasis

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by β-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon β-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by β-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-β-cyclodextrin treatment enhanced zeaxanthin but not β-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.

Comparative Absorption and Transport of Five Common Carotenoids in Preruminant Calves

Preruminant calves, maintained in a monogastric state by feeding an all-liquid diet, were used to compare the serum appearance and lipoprotein transport of five different carotenoids over 144 h. Thirty newborn calves were fed milk replacer for 1 wk and then randomly assigned to six groups (n = 5), with each group receiving a single 20-mg oral dose of β-carotene in water-soluble beadlets, canthaxanthin in water-soluble beadlets, lutein in oil, lycopene in oil, crystalline α-carotene in oil or crystalline β-carotene in oil as part of a morning meal. Serial blood samples were taken by jugular puncture for up to 1 wk post-dosing. Lipoprotein separation and analysis were completed with selected animals. All carotenoids were absorbed, but in variable amounts. At peak serum carotenoids levels, HDL contained 70–90% of the carotenoids. Canthaxanthin and lutein peaked earlier in serum (8 and 12 h) than did the less polar lycopene, α-carotene, and β-carotene (16, 24 and 24 h). Canthaxanthin and lutein were also cleared more quickly from the serum. Serum concentrations of α-carotene and lycopene displayed slower disappearance rates than did β-carotene. The peak serum level (nmol/L ± SEM) of canthaxanthin (392 ± 136) was lower than that of β-carotene (1245 ± 425), and carotenoids levels of calves receiving these commercial beadlet sources were higher than the serum levels of calves receiving β-carotene (45 ± 17.5), α-carotene (42 ± 18.0), lutein (51 ± 9.5) and lycopene (18 ± 4.6), which were fed in oil. The results show differential uptake and clearance of various carotenoids and enhanced uptake of beadlet forms of two carotenoids. We suggest that the preruminant calf is an excellent model in which to evaluate uptake and transport of carotenoids, especially for those carotenoids that are not approved for human feeding studies.

GWAS resolves molecular mechanisms underlying natural variation for carotenoids in Cucurbita maxima Duchesne

Carotenoid levels and composition are essential nutraceutical traits in Cucurbita maxima influencing the fruit flesh and rind colors. In this study, Mr Fugly, Boston Marrow, Baby Green Hubbard, Chicago Warted Hubbard, Golden Hubbard, and Bush Buttercup of USA; Australian Butter (Australia); Marmellata and Marina di Chioggia of Italy contained the highest carotenoid content. EMMAX (Efficient Mixed-Model Association eXpedited) was employed for a Genome-wide association study (GWAS) to identify associated genomic regions underlying the regulation of carotenoids. Here, we profiled the contents of α-carotene, β-carotene, β-cryptoxanthin, lutein, and zeaxanthin from a diversity panel of C. maxima consisting of classical horticulture groups including buttercup, turban, banana, zapallito, hubbard, mammoth and australian. Lutein and β-carotene were the major carotenoids among C. maxima accessions. Furthermore, we used genotyping by sequencing to identify 12,996 single nucleotide polymorphisms (SNPs) that were used to resolve population structure with three primary clusters and their admixed genotypes. Our GWAS revealed several SNPs related to 289 genes involved in plastid biogenesis, protein ubiquitination, transcriptional regulation at the carotenoid biosynthesis level, fruit ripening, and regulation of carotenoid transport. Together, these results contribute to a better understanding of the molecular mechanisms of carotenoid biosynthesis in fruit flesh and would facilitate breeding for enhanced carotenoids in winter squashes.

Chromoplast differentiation: a central role for plastoglobule lipid droplets comes into focus.

This article is a Commentary on Morelli et al. (2023), 237: 1696–1710.

The Effect of Background Color on Skin Color Variation of Juvenile Plectropomus leopardus.

Fish skin color is usually strongly affected by the background color of their environment. The study investigated the effects of five different background colors on the skin color of leopard coral groupers (Plectropomus leopardus). More than 450 juveniles were reared in Blue, Red, Black, White, and Transparent background tanks for 56 days. The paraffin section showed that the skin melanin zone of fish in the White group was smaller, whereas the Black and Red groups (especially Black) were nearly the largest. The apparent skin color of P. leopardus was red on the white background, which darkened in response to the other color backgrounds. The Black group revealed the blackest skin color, followed by the transparent group. Moreover, the White group had the highest L*, a*, and b* values. The melanin content and tyrosinase activity in the dorsal and ventral skin of the Black group were significantly higher than those in the other groups (p < 0.05), and the serum α-MSH level was higher in the Black group as well. The carotenoid and lutein contents showed completely different trends among the experimental groups, as carotenoid content was higher in the Red and White groups, while lutein content was higher in the Transparent group. The expression level of scarb1 was highest in the Blue and White groups, followed by the Transparent group, and lowest in the Black group (p < 0.05). The expression trend of scarb1 was similar to the skin color in different backgrounds, indicating that the background color regulated scarb1 expression level through visual center, then influenced the uptake and transport of carotenoids, then influenced the skin color formation of P. leopardus. Moreover, lighter colors inhibited the formation of melanocytes and had a significant effect on carotenoid and lutein contents. Pigment-related genes were involved in the regulation of fish skin color, and they were affected by background color in P. leopardus. These results indicate that a white background is more conducive to maintaining red skin color in juvenile P. leopardus.

Microalgal lutein biosynthesis: Recent trends and challenges to enhance the lutein content in microalgal cell factories

Photosynthetic organisms such as eukaryotic microalgae and prokaryotic cyanobacteria synthesize a wide range of valuable chemicals. They are predicted to become efficient and renewable sources for valuable products in the future due to their high biomass synthesis using CO2 and solar energy. Microalgae are producers of several carotenoids including lutein, which is a xanthophyll carotenoid with several health advantages, including the prevention of age-related macular degeneration. Currently, it is extracted on commercial scale from marigold flower petals, however, production from plant sources is highly affected by seasonal variations, requires arable land, and has high production cost. Microalgae, on the other hand, are an ideal alternative for lutein synthesis due to their rapid growth and high biomass and lutein yield. It is, however, necessary to further improve lutein productivity, for a successful transition to commercial production. This article describes lutein biosynthesis in microalgae by using their native biochemical pathways, as well as possible target genes for genetic engineering to enhance lutein production. Understanding the processes behind lipid droplet synthesis in chloroplasts, as well as carotenoid transport across chloroplast membranes and carotenoid esterification, might lead to novel ways to boost lutein levels in microalgae.

Tissue Lycopene Accumulation in Transgenic Mice Lacking One or Both Carotenoid Cleaving Enzymes

ObjectivesTo evaluate the impact of gender and ablation of β-carotene oxygenase 1 (BCO1), β-carotene oxygenase 2(BCO2) or both on tissue accumulation of lycopene dosed transgenic mice. Previous work from our laboratories suggests that BCO2 appears to be the primary carotenoid cleavage enzyme for lycopene in vivo. MethodsThree-week old C57BL/6 male and female mice (wild type [WT], Bco1-/-, Bco2-/-, Bco1-/- X Bco2-/- double knock out [DKO]) were divided into groups based on genotype (n = 8 per group/per gender) and fed a powdered AIN 93G diet for 2 weeks. The mice were gavaged daily for 2 weeks with 1 mg of lycopene dissolved in cottonseed oil after which they were fasted and sacrificed. Liver, serum, and extra-hepatic tissues were harvested. Lycopene concentration was measured with high-performance liquid chromatography. Data analyses were performed using one-way ANOVA. ResultsOn a concentration basis, liver, duodenum and adrenal lycopene were higher than other tissues. Serum and tissues of DKO mice accumulated the highest lycopene. DKO mice had significantly higher lycopene than Bco1-/- mice in the liver (P < .002), heart (P < .004), adipose (P < .03), and the testes (P < .004). Compared to Bco2-/- mice, DKO mice had greater accumulation in the serum (P < .001), intestine (P < .04), heart (P < .0001), kidneys (P < .0001), adipose (P < .04), and testes (P < .0001). Liver (P < .007) and adrenal (ns) tissues in Bco2-/- mice had higher levels of lycopene than Bco1-/- mice whereas Bco1-/- mice had significantly higher levels in the kidneys (P < .001) and tended to have greater accumulation in other tissues. ConclusionsAccumulation of lycopene in tissues depended upon gender, genotype and tissue type. The abolition of both enzymes and mice of a female sex generated a higher accumulation. We are probing whether tissue-specific expression levels of BCO1, BCO2 or carotenoid transport proteins explain differential tissue accumulation across genotypes. Funding SourcesN/A.

Carotenoid Profile in Maternal/Cord Plasma and Changes in Breast Milk along Lactation and Its Association with Dietary Intake: A Longitudinal Study in a Coastal City in Southern China.

In this study, changes of carotenoids in breast milk were observed longitudinally for up to one year. Our study aimed to analyze the profile of carotenoids in breast milk and maternal/cord plasma and its correlation with dietary intake in Guangzhou. Plasma and breast milk samples of five stages during lactation (i.e., colostrum; transitional milk; and early, medium, and late mature milk) were collected from lactating mothers. The food frequency questionnaire (FFQ) was used for collecting data on dietary intake in the corresponding stages. Levels of lutein, zeaxanthin, β-cryptoxanthin, β-carotene, and lycopene were analyzed by high-performance liquid chromatography. We found that the total carotenoid level decreased gradually with the extension of lactation and eventually stabilized. Among them, the content of lutein increased from colostrum to transitional milk and decreased thereafter until it plateaued in the mature milk. Furthermore, lutein was reported as the dominant nutrient in maternal plasma, cord plasma, transitional milk, and mature milk at up to 400 days postpartum, while beta-carotene was predominant in colostrum. The content of β-carotenoid in middle and late mature breast milk was related to dietary intake (r = 1.690, p < 0.05). Carotenoid level in cord blood was lower than that in the mother’s plasma and was related to the carotenoid intake in the mother’s diet. Correlation of carotenoids between maternal and umbilical cord blood, breast milk, and maternal blood could well reflect the transport of carotenoids. These findings may help to guide mothers’ diets during breastfeeding.

Aster proteins mediate carotenoid transport in mammalian cells

Some mammalian tissues uniquely concentrate carotenoids, but the underlying biochemical mechanism for this accumulation has not been fully elucidated. For instance, the central retina of the primate eyes displays high levels of the carotenoids, lutein, and zeaxanthin, whereas the pigments are largely absent in rodent retinas. We previously identified the scavenger receptor class B type 1 and the enzyme β-carotene-oxygenase-2 (BCO2) as key components that determine carotenoid concentration in tissues. We now provide evidence that Aster (GRAM-domain-containing) proteins, recently recognized for their role in nonvesicular cholesterol transport, engage in carotenoid metabolism. Our analyses revealed that the StART-like lipid binding domain of Aster proteins can accommodate the bulky pigments and bind them with high affinity. We further showed that carotenoids and cholesterol compete for the same binding site. We established a bacterial test system to demonstrate that the StART-like domains of mouse and human Aster proteins can extract carotenoids from biological membranes. Mice deficient for the carotenoid catabolizing enzyme BCO2 concentrated carotenoids in Aster-B protein-expressing tissues such as the adrenal glands. Remarkably, Aster-B was expressed in the human but not in the mouse retina. Within the retina, Aster-B and BCO2 showed opposite expression patterns in central versus peripheral parts. Together, our study unravels the biochemical basis for intracellular carotenoid transport and implicates Aster-B in the pathway for macula pigment concentration in the human retina.

Transcriptomic analysis identifies candidate genes for shell colour formation in the Venus clamCyclina sinensis

ABSTRACTSome bivalves are important in fisheries and aquaculture, and provide significant economic benefits to humans. The diversity in shell colour among bivalves has received attention due to its potential in breeding and its relevance to the study of speciation and adaptation. In this study, the relationship between whole-genome-scale gene expression and shell coloration in the clam Cyclina sinensis was analysed using high-throughput Illumina sequencing technology. A total of 15,367 annotated genes and 4,425 novel genes were detected based on transcript assembly. The results of the transcriptome analysis show that 49 genes were differentially expressed in the outer mantle fold tissue of purple- vs white-shelled specimens. These genes include three lipoprotein receptor genes, low-density lipoprotein receptor gene (ldlr), low-density lipoprotein receptor-related protein 4 (lrp4) and scavenger receptor cysteine-rich protein gene (srcr). These genes may be involved in the transport of carotenoid pigments. Moreover, as shown by the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, several receptor activity terms and a glycosphingolipid biosynthesis pathway were detected, and these might be involved in shell coloration. Together with the analyses of differentially expressed genes, our results suggest a possible molecular mechanism governing the selective transport of carotenoids in the mantle of purple-shelled clams and shell colour formation. The results of this study may help to elucidate the colour formation mechanism in clam shells.

Transcriptome analysis based on dietary beta-carotene supplement reveals genes potentially involved in carotenoid metabolism in Crassostrea gigas.

Carotenoids are essential micronutrients for animals, and they can only be obtained from the diet for mollusk as well as other animals. In the body, carotenoids undergo processes including absorption, transport, deposition, and metabolic conversion; however, knowledge of the involved genes is still limited. To elucidate the molecular mechanisms of carotenoid processing and identify the related genes in Pacific oyster (Crassostrea gigas), we performed a comparative transcriptome analysis using digestive gland tissues of oysters on a beta-carotene supplemented diet or a normal diet. A total of 718 differentially expressed genes were obtained, including 505 upregulated and 213 downregulated genes in the beta-carotene supplemented group. Function Annotation and enrichment analyses revealed enrichment in genes possibly involved in carotenoid transport and storage (e.g., LOC105342035), carotenoid cleavage (e.g., LOC105341121), retinoid homeostasis (e.g., LOC105339597) and PPAR signaling pathway (e.g., LOC105323212). Notably, down-regulation of mRNA expressions of two apolipoprotein genes (LOC105342035 and LOC105342186) by RNA interference significantly decreased the carotenoid level in the digestive gland, supporting their role in carotenoid transport and storage. Based on these differentially expressed genes, we propose that there may be a negative feedback mechanism regulated by nuclear receptor transcription factors controlling carotenoid oxygenases. Our findings provide useful hints for elucidating the molecular basis of carotenoid metabolism and functions of carotenoid-related genes in the oyster.

Cloning, characterization and expression prolife analysis ofldlr: Insight into purple shell formation inCyclina sinensis

The clam Cyclina sinensis is an economically important marine bivalve, and the diversity of its shell colour has potential applications in breeding. In this study, the distribution of purple pigments in the clam shell was examined, and Raman spectroscopy was conducted. The levels of total carotenoids and four common carotenoids in various tissues were also determined and analysed. Furthermore, the full-length cDNA of a low-density lipoprotein receptor gene (ldlr) was cloned and characterized, and the tissue distribution of ldlr gene expression was analysed. The results showed that purple pigments existed mainly in the nacre layer of the shell from the purple-shell clam, in which strong Raman peaks for carotenoids were observed, suggesting that carotenoids might be the main components of purple pigments in the purple shell. The TCC and the levels of four common carotenoids were higher in the outer margin of the mantle and shell of purple-shell clams than in those of white-shell clams, and the ldlr mRNA was highly expressed in the outer margin of the mantle in purple-shell clams. Taken together, our results suggest a possible molecular mechanism governing the selective transport of carotenoids in the mantle of purple-shell clams and shell colour formation. The results of this study will help to elucidate the colour formation mechanism in clam shells.

Expression and biochemical analyses of proteins involved in the transport of carotenoids and retinoids.

Animals acquire carotenoids from the diet and convert them to retinoids. These lipids must be distributed in the body to support retinoid signaling in peripheral tissues and photoreceptor function in the eyes. However, the hydrophobicity of carotenoids and retinoids limit their diffusion in the aqueous environment of the body. Therefore, membrane proteins and cellular binding proteins transport these lipids between extra- and intracellular compartments and facilitate their metabolism. Mutations in genes encoding these transport proteins are associated with a wide spectrum of blinding disorders. Here, we describe approaches used by our laboratories that have proven successful in expressing these proteins and examining their biochemical properties in the test tube and in cell-based assays. These assays can be utilized for screening of small molecule modulators of their activities to correct pathologies associated with retinoid metabolism.

De novo assembly transcriptome analysis reveals the genes associated with body color formation in the freshwater ornamental shrimps Neocaridina denticulate sinensis.

The body color of Neocaridina denticulate sinensis is a compelling phenotypic trait, in which a cascade of carotenoid metabolic processes plays an important role. The study was conducted to compare the transcriptome of cephalothoraxes among three pigmentation phenotypes (red, blue, and chocolate) of N. denticulate sinensis. The purpose of this study was to explore the candidate genes associated with different colors of N. denticulate sinensis. Nine cDNA libraries in three groups were constructed from the cephalothoraxes of shrimps. After assembly, 75022 unigenes were obtained in total with an average length of 1026bp and N50 length of 1876bp. There were 45977, 25284, 23605, 21913 unigenes annotated in the Nr, Swissprot, KOG, and KEGG databases, respectively. Differential expression analysis revealed that there were 829, 554, and 3194 differentially expressed genes (DEGs) in RD vs BL, RD vs CH, and BL vs CH, respectively. These DEGs may play roles in the absorption, transport, and metabolism of carotenoids. We also emphasized that electron transfer across the inner mitochondrial membrane (IMM) was a key process in pigment metabolism. In addition, a total of 6328 simple sequence repeats (SSRs) were also detected in N. denticulate sinensis. The results laid a solid foundation for further research on the molecular mechanism of integument pigmentation in the crustacean and contributed to developing more attractive aquatic animals.

Identification of a key gene StAR-like-3 responsible for carotenoids accumulation in the noble scallop Chlamys nobilis

Carotenoids play important roles in living organisms. However, animals cannot synthesize carotenoids by themselves, and they must absorb and accumulate carotenoids from their diets in which some key genes are involved. In present study, a gene named StAR-like-3 was characterized in the noble scallop Chlamys nobilis, and its function was identified using golden scallops with higher carotenoids content and brown scallops with less carotenoids content by immunohistochemistry, carotenoid binding assay and RNAi. Results showed that the StAR-like-3 encodes a 54.7 kDa transmembrane protein (named as StAR3) of 481 amino acids containing a MENTAL domain and a START (Steroidogenic acute regulatory protein-related lipid transfer) domain, and its expression level in hemocytes and intestine of golden scallops were significantly higher than those of brown ones. Subsequently, the StAR3 protein was detected in the intestinal epithelial cells of golden scallops, and recombinant StAR3 could bind lutein conjugated to protein G and antibody to form a yellow complex, suggesting it is a carotenoid binding protein involving in carotenoids accumulation in golden scallops. Furthermore, total carotenoids content of hemolymph in golden scallops was significantly decreased when the expression of StAR-like-3 suppressed, suggesting this gene plays an important role in transport of carotenoids. Conclusion, the present results indicated that the StAR-like-3 is a key gene responsible for the carotenoids accumulation in the scallop.

Turmeric, red pepper, and black pepper affect carotenoids solubilized micelles properties and bioaccessibility: Capsaicin/piperine improves and curcumin inhibits carotenoids uptake and transport in Caco-2 cells.

This study aimed to evaluate the role of spices/spice active principles on physical, biochemical, and molecular targets of bioaccessibility/bioavailability. Carotenoids-rich micellar fraction obtained through simulated digestion of green leafy vegetables (GLV) with individual or two/three combinations were correlated to their influence on bioaccessibility, cellular uptake, and basolateral secretion of carotenoids in Caco-2 cells. Results suggest that carotenoids' bioaccessibility depends on micelles physicochemical properties, which is affected due to the presence of co-treated dietary spices and their composition. Increased bioaccessibility of β-carotene (BC) and lutein (LUT) is found in GLV (spinach) digested with turmeric (TM) than red pepper (RP) and black pepper (BP). In contrast, enhanced cellular uptake and secretion of BC and LUT-rich triglyceride-rich lipoprotein is observed in the presence of RP and BP compared to the control group. In contrast, TM inhibited absorption, while retinol levels significantly reduced in the presence of TM and RP than BP. Control cells have indicated higher cleavage of β-carotene to retinol than the spice-treated group. Besides, spice active principles modulate facilitated transport of carotenoids by scavenger receptor class B type 1 (SR-B1) protein. The effect of spices on carotenoids' bioavailability is validated with active spice principles. Overall, carotenoids' bioavailability (cellular uptake and basolateral secretion) was found in the following order of treatments; piperine>capsaicin>piperine + capsaicin>curcumin + capsaicin + piperine>control>turmeric. These findings suggested that the interaction of specific dietary factors, including spice ingredients at the enterocyte level, could provide greater insight into carotenoid absorption. PRACTICAL APPLICATION: Spices/spice active principles play a role in the digestion process by stimulating digestive enzymes and bile acids secretion. Since carotenoids are lipid soluble and have low bioavailability, spice ingredients' influence on intestinal absorption of carotenoids is considered crucial. Hence, understanding the interaction of co-consumed spices on the absorption process of carotenoids may help to develop functional foods/formulation of nutraceuticals to improve their health benefits.