Prevention of aspirin-mediated secondary toxicity by combined treatment of carotenoids in macrophages.

Upon understanding the boosting role of carotenoids on the endogenous anti-inflammatory system, it is vital to explore their role in reducing the use of high doses of non-steroidal anti-inflammatory drug (NSAIDs), and their mediated secondary toxicity during the treatment of chronic diseases. The current study investigates the carotenoids potential on inhibition of secondary complications induced by NSAIDs, aspirin (ASA) against lipopolysaccharide (LPS) stimulated inflammation. Initially, this study evaluated a minimal cytotoxic dose of ASA and carotenoids (ß-carotene, BC/lutein, LUT/astaxanthin, AST/fucoxanthin FUCO) in Raw 264.7, U937, and peripheral blood mononuclear cells (PBMCs). In all three cells, carotenoids + ASA treatment reduced the LDH release, NO, and PGE2 efficiently than an equivalent dose of carotenoid or ASA treated alone. Based on cytotoxicity and sensitivity results, RAW 264.7 cells were selected for further cell-based assay. Among carotenoids, FUCO + ASA exhibited an efficient reduction of LDH release, NO, and PGE2 than the other carotenoids (BC + ASA, LUT + ASA, and AST + ASA) treatment. FUCO + ASA combination decreased LPS/ASA induced oxidative stress, pro-inflammatory mediators (iNOS, COX-2, and NF-κB), and cytokines (IL-6, TNF-α, and IL-1ß) efficiently. Further, apoptosis was inhibited by 69.2% in FUCO + ASA, and 46.7% in ASA than LPS treated cells. A drastic decrease in intracellular ROS generation with the increase in GSH was observed in FUCO + ASA compared to LPS/ASA groups. The results documented on the low dose of ASA with a relative physiological concentration of FUCO suggested greater importance for alleviating secondary complications and optimize prolonged chronic disease treatments with NSAID's associated side effects. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03632-w.

Zein-Alginate-Phosphatidylcholine Nanocomplex Efficiently Delivers Lycopene and Lutein over Dietary-Derived Carotenoid Mixed Micelles in Caco-2 Cells.

This study evaluated the potency of zein-alginate-phosphatidylcholine nanoparticles (NPs) on bioaccessibility/intestinal uptake of encapsulated lycopene (LY) and lutein (LT) versus dietary absorption using simulated digestion and human intestinal Caco-2 cells. LY-zein-alginate-PC (LYZAP) and LT-zein-alginate-PC (LTZAP) NPs yield desired properties, which exhibit sustained release and are suitable for oral administration. Interestingly, co-treatment of LYZAP + LTZAP showed better release of carotenoids instead of individual treatment at intestinal pH. Bioaccessibility, cellular uptake, and basolateral secretion of LY and LT from NPs were significantly enhanced than micellar carotenoids (dietary mode of absorption). The increased absorption of carotenoids from NPs correlated with triglyceride levels. The intestinal cell uptake of carotenoids by nanoencapsulation may be due to endocytosis, paracellular, and SRB-1 protein-mediated transport. Overall, LYZAP and LTZAP NPs possess superior properties to control the release and cellular uptake of unique or distinct carotenoids. The inclusion of alginate and phosphatidylcholine in zein-based nanoencapsulation could be a promising strategy to improve carotenoid bioavailability.

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 ß-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 ß-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.

Fabrication of chitosan nanoparticles with phosphatidylcholine for improved sustain release, basolateral secretion, and transport of lutein in Caco-2 cells.

Biopolymers-based nanoparticles delivery emerged alternatively to improve nutraceuticals and drug bioavailability. The intestinal physiology suggested a prerequisite of lipid moiety for carotenoid absorption. This study aimed to fabricate chitosan-based nanoparticles with phosphatidylcholine (PC) to enhance lutein bioavailability. Lutein encapsulated chitosan nanoparticles with PC (LCNPC) or without PC (LCN) were assessed for bioaccessibility, sustain release, cellular uptake/internalization, and basolateral secretion of lutein in Caco-2 cells. Standard lutein mixed micelles (LMM), and micelles derived through in vitro digestion of green leafy vegetables (GMM) treated as controls. The LCNPC showed reduced particle size, higher colloidal stability, homogeneous dispersion, and suitable for oral administration compared to LCN. The cellular uptake of lutein (20 h) in LCNPC was higher than LCN, LMM, and GMM, respectively. Interestingly, lutein uptake was maximum at 8 h in LMM and gradually decreased against sustain-release response in LCNPC and LCN, whereas considerably low lutein uptake from GMM at all time points. Further, LCNPC significantly increased basolateral secretion of triglyceride (TG) and positively correlated enhanced lutein uptake/internalization process than LCN and micelles. Also, LCNPC demonstrated the upregulation of endocytosis, paracellular, scavenger receptor class B type 1 (SRB-1), and peroxisome proliferator-activated receptor gamma (PPARγ) mediated lutein transport mechanism. These results suggested that fabrication of biopolymer-based nanoparticles with PC could provide greater insight to improve lutein bioavailability at enterocyte levels, to avoid age-related macular degeneration and other chronic diseases.

Curcumin and capsaicin modulates LPS induced expression of COX-2, IL-6 and TGF-ß in human peripheral blood mononuclear cells.

The mechanism of action of treatment of either curcumin or capsaicin or in combination on LPS (Lipopolysaccharide) induced inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) was investigated using RT-PCR and in silico docking methods. RT-PCR analysis has shown that the curcumin and capsaicin significantly reduced LPS induced over expression of COX-2, IL-6 and TGF-ß in PBMCs. Whereas combined molecules demonstrated synergistic response on the reduction of COX-2, IL-6 and TGF-ß over expression in LPS induced PBMCs as compared to individual molecules. Further, The docking of curcumin and capsaicin at the active pockets of COX-2, IL-6 and TGF-ß has shown - 3.90, - 4.49 and - 5.61 kcal/mol binding energy for curcumin and - 3.80, - 4.78 and - 5.76 kcal/mol binding energy for capsaicin, while multiple ligand simultaneous docking (MLSD) of both molecules has shown higher binding energy of - 4.24, - 5.35 and - 5.83 kcal/mol respectively. This has demonstrated the efficacy of combined curcumin and capsaicin against the LPS induced expression of pro-inflammatory cytokines in PBMCs. These results attributed the coordinated positive modulation on biochemical and molecular cellular process by combined curcumin and capsaicin as compared to individual molecules.

Fractionation and Characterization of Lycopene-Oxidation Products by LC-MS/MS (ESI)+: Elucidation of the Chemopreventative Potency of Oxidized Lycopene in Breast-Cancer Cell Lines.

Lycopene (LYC) has been correlated with the reduction of certain cancers and chronic diseases. However, the existence and biofunctionality of degraded, oxidized, and biotransformed LYC products in vivo have not been revealed. Therefore, this study aimed to screen and elucidate the potential bioactive lycopene-derived products in breast-cancer and non-cancerous cells. LYC-oxidation or -cleavage products were generated using KMnO4. These oxidation products were separated as fractions I-III by silica column chromatography using gradient solvent systems. Further, LC-MS/MS (ESI)+ was used to elucidate their possible fragmentation patterns and structures. Fraction II showed higher cytotoxicity (IC50 value of 64.5 µM), cellular uptake, and apoptosis-inducing activity in MCF-7 cells. This fraction consists of major peak m/ z 323, identified as apo-8,6'-carotendial. The cytotoxicity-inducing activity may be due to partial ROS generation with mitochondrial dysfunction. Further, the role of apo-8,6'-carotendial in the induction of apoptosis is demonstrated for the first time. These results illustrated that LYC-oxidation derivatives or metabolites are involved in growth inhibition of cancer cells. Exploration of specific oxidized-carotenoid products will give further insight into the field of nutritional biochemistry.

Low-dose doxorubicin with carotenoids selectively alters redox status and upregulates oxidative stress-mediated apoptosis in breast cancer cells.

The combination of carotenoids and doxorubicin (DOX) selectively alters oxidative stress-mediated apoptosis in breast cancer cells. Primarily, cytotoxic efficiency of carotenoids (ß-carotene, BC; lutein, LUT; astaxanthin, AST; or fucoxanthin, FUCO) either with or without a minimal cytotoxic dose of DOX was evaluated in MCF-7 (0.12 µM) and MDA-MB-231 cells (0.28 µM). The higher cell growth inhibition of BC and/or LUT with DOX was selected for testing in further cell-based assays. Low-dose DOX significantly enhanced cytotoxicity in carotenoid (<5 µM)-treated cells compared to high-dose DOX (>1 µM) or carotenoid (20 µM) treatment alone. Depleted glutathione, increased lipid peroxides and increased ROS levels in cells confirmed the cytotoxic effect. Furthermore, mitochondrial dysfunction, cell growth arrest at G0/G1 phase and caspase cascades as well as up- and down-regulated expression levels of related proteins (p21, p27, Bax, p53, Bcl-2, and cyclin D1) revealed the synergistic effect of carotenoid and DOX treatment on ROS-mediated apoptosis. These observations demonstrated increased apoptosis in BC + DOX/LUT + DOX-treated cells due to the pronounced pro-oxidant action. Interestingly, normal breast epithelial cells (MCF 10A) exposed to similar treatments resulted in non-significant cytotoxicity. These newly observed mechanistic differences of anticancer drugs on the mitigation of toxicity with carotenoids may provide insight into the targeting of cancer therapy.

Astaxanthin from shrimp efficiently modulates oxidative stress and allied cell death progression in MCF-7 cells treated synergistically with ß-carotene and lutein from greens.

This study investigated the synergistic efficacy of keto-carotenoid astaxanthin (AST, from shrimp) plus hydrocarbon (ß-carotene, BC) and hydroxyl (lutein, L) carotenoids (from greens) on molecular events in MCF-7 cells. MCF-7 cells were treated with either of carotenoid (20 µM, AST or BC or L) separately or the mixture of them (an equimolar concentration of carotenoids mixture, CM) or saponified carotenoid extract from shrimp (SSCE) for 48 h and analyzed cellular uptake, cytotoxicity, and apoptosis. The IC50 and combination-index values of AST co-treatment with a lower concentration of BC and L (5 µM) exhibited enhanced cytotoxicity and oxidative stress as compared with individual carotenoids or SSCE. Further, higher cellular uptake/accumulation of AST along with BC and L found to synergistically induce apoptosis through modulation of cyclin D1, p53, Bax and Bcl-2 expressions by arresting cell cycle at G0/G1 phase. Further, CM or SSCE treatments are unlikely to affect proliferation of normal breast epithelial cells (MCF-10A). The results of selective killing of MCF-7 cells demonstrated a greater insight on the synergistic effect of shrimp AST plus BC and L. It is concluded that consumption of shrimp along with green leafy vegetables helps in combating cancer chemoprevention.

Enhanced cytotoxic and apoptosis inducing activity of lycopene oxidation products in different cancer cell lines.

Currently, upon understanding the metabolomics of carotenoids, it is important to address the key role of carotenoid derived products. In this regard, aim of the study was to elucidate and explore the role of lycopene (LYC) oxidative products generated through autoxidation (AOL) or chemical (KMnO4) oxidation (COL) against proliferation of selected cancer cells. Preliminary, we investigated the effect of LYC on cell viability of various cancer cell lines (PC-3, MCF-7, A431, HepG2, HeLa and A549). Based on the results of LYC treatment on cell cytotoxicity levels, MCF-7, PC-3 and HeLa cell lines were further tested with AOL and COL products. The decreased cell viability with depleted GSH and increased MDA levels were observed when treated with COL products than control, LYC and AOL. In addition, COL products increased ROS levels and percent apoptosis. The typical morphological changes and nuclear condensations showed that COL products have anti-proliferation and apoptosis inducing activity. Based on results, we hypothesized that ROS generation by LYC oxidation products may be one of intermediate step involved in apoptosis. The redox status and therapeutic approach of COL products in modulating ROS and induction of apoptosis in cancer cells were reported for the first time, to our knowledge. To conclude, COL products involves in cancer growth inhibition efficiently than intact LYC and AOL. Hence, there is a great potential for synthesizing or producing such carotenoid oxidation products to augment cancer complication.

Role of different vehicles in carotenoids delivery and their influence on cell viability, cell cycle progression, and induction of apoptosis in HeLa cells.

The objective of the present study was to determine the role of different vehicles in carotenoids delivery and their influence on cell viability, cell cycle progression and induction of apoptosis in HeLa cells. Cells (5 × 10(3)) were treated with different concentrations (25-100 µM) of ß-carotene (BC) or lutein (L) or astaxanthin (AST) dissolved in 0.5% of tetrahydrofuran (THF), dimethylsulfoxide (DMSO), and fetal bovine serum (FBS), respectively. The effect of delivery vehicle on carotenoids uptake, cytotoxicity, oxidative status, cell cycle distribution, and apoptosis was examined after 48 h of incubation. The results shown that, cell viability reduced significantly in a dose- and time-dependent manner irrespective of carotenoid delivered in vehicles. Cellular uptake of BC delivered in THF was higher by 49.1, 29.7% and L delivered through THF was higher by 41.7 and 37.5% than DMSO and FBS, respectively. While, AST delivered through DMSO was higher by 36.1 and 43.7% than the THF and FBS, respectively. In case of cells treated either with BC or L delivered through THF and AST in DMSO decreased the glutathione and increased the malondialdehyde levels. The net increase in the G 2/M phase percentage of cell cycle progression was observed in carotenoid-treated cells. The % induction of apoptosis by BC or L delivered with THF and AST in DMSO was higher than other treated groups. In conclusion, choice of suitable vehicle for specific carotenoids delivery is essential that in turn may influence on cell proliferation and cell-based assays.

Structural elucidation of possible lutein oxidation products mediated through peroxyl radical inducer 2,2'-Azobis (2-methylpropionamidine) dihydrochloride: antioxidant and cytotoxic influence of oxidized lutein in HeLa cells.

Aim of this study was to elucidate lutein oxidation products mediated through peroxyl radical inducer 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) and to study antioxidant and cytotoxic effects of oxidized lutein using liposome and HeLa cells. Lutein (20 µmol) with AAPH (5 mM) in liposome's was incubated at 37 °C in dark for 3 h, oxidized lutein products were characterized by LC-MS (APCI(+)) and studied for their free radical scavenging activity and cytotoxic effects in terms of cell viability, cellular glutathione, and malondialdehyde levels. AAPH mediated lutein fragmented ions were identified as 551 (M(+)+H(+)-H(2)O), 391 (M(+)+H(+)+O(2)-C(22)H(32)O) and 276 (M(+)+H(+)+O(2)-C(12)H(20)O) and its isomers as 13-Z lutein, 13-Z zeaxanthin, 13'-Z zeaxanthin and all-E zeaxanthin. Free radical scavenging activity of oxidized lutein was higher by 32.7% (IC(50), 2.64 µg) than lutein (IC(50), 5.28 µg). Oxidized lutein lowered the lipid peroxidation (21%), HeLa cells viability (22%) and glutathione levels (32%) than lutein. To conclude, the oxidized lutein may be highly reactive, since oxidation by AAPH results in peroxyl radical ions, which can react with conjugated polyene chain of lutein that could lead to higher antioxidant and cytotoxic effects on HeLa cells.

Antioxidant and cytotoxic effect of oxidized lutein in human cervical carcinoma cells (HeLa).

This study investigated the antioxidant and cytotoxic effect of oxidized lutein using human cervical carcinoma cell lines (HeLa). Liposome contained phosphatidylcholine (20 micromol) in Tris-HCl buffer and lutein (200 micromol) was exposed to sunlight for 100 min. Photo-oxidized lutein products were characterized by LC-MS (APCI(+)) and studied for their antioxidant property and apoptosis in terms of cell viability, glutathione and malondialdehyde (MDA) levels. Photo-oxidized lutein fragmented ions were identified as 523 (M+ + H+-3CH3), 476 (M+ + H+-6CH3), 551 (M+ + H+-H2O) and its isomers as 13-Z lutein, 13'-Z lutein, 13-Z zeaxanthin, all-E zeaxanthin, 9-Z lutein, 9'-Z lutein. Free radical scavenging activity of oxidized lutein was higher by 45.9% (IC(50), 3.71 microg) than lutein (IC(50), 5.28 microg). Oxidized lutein lowered the lipid peroxidation by 20.7% than lutein. The viability of HeLa cells, glutathione and MDA levels were decreased by 64%, 40% and 18% than lutein. To conclude, oxidized lutein may be highly reactive, since oxidation results in radical ions, which can combine with similar reactive oxidative species that could lead to higher antioxidant effect. This may be true in this study that antioxidant property of oxidized lutein was higher than lutein that correlates with free radical scavenging activity and cytotoxic effects on HeLa cells.

Possible degradation/biotransformation of lutein in vitro and in vivo: isolation and structural elucidation of lutein metabolites by HPLC and LC-MS (atmospheric pressure chemical ionization).

Metabolites of lutein are highly concentrated in the human macula and are known to provide protection against age-related macular degeneration. The aim of this investigation was to characterize the in vitro oxidation products of lutein obtained through photo-oxidation and to compare them with biologically transformed dietary lutein in intestine, plasma, liver, and eyes of rats. In vivo studies involved feeding rats a diet devoid of lutein for 2 weeks to induce deficiency. Rats were divided into two equal groups (n=6/group) and received either micellar lutein by gavage for 10 days or diet supplemented with fenugreek leaves as a lutein source for 4 weeks. Lutein metabolites/oxidation products obtained from in vivo and in vitro studies were characterized by HPLC and LC-MS (APCI) techniques to elucidate their structure. The characteristic fragmented ions resulting from photo-oxidation of lutein were identified as 523 (M(+)+H(+)-3CH(3)), 476 (M(+)+H(+)-6CH(3)), and 551 (M(+)+H(+)-H(2)O). In the eyes, the fragmented molecules resulting from lutein were 13-Z lutein, 13'-Z lutein, 13-Z zeaxanthin, all-E zeaxanthin, 9-Z lutein, 9'-Z lutein, and 3'-oxolutein. Epoxycarotenoids were identified in liver and plasma, whereas anhydrolutein was identified in intestine. This study emphasizes the essentiality of dietary lutein to maintain its status in the retina.

Lutein and zeaxanthin in leafy greens and their bioavailability: olive oil influences the absorption of dietary lutein and its accumulation in adult rats.

This study determined the lutein level in various green leafy vegetables (GLVs) and the influence of olive and sunflower oils on the postprandial plasma and eye response of dietary lutein in adult rats, previously induced with lutein depletion (LD). Fresh GLVs (n = 35) were assessed for lutein (L) and its isomer zeaxanthin (Z) levels by high-performance liquid chromatography and liquid chromatography-mass spectrometry. Among GLVs analyzed, Commelina benghalensis L. contained a higher level of L + Z (183 mg/100 g dry wt) and was used as a lutein source for feeding studies. Rats with LD were fed a diet containing powdered C. benghalensis (2.69 mg lutein/kg diet) with either olive oil (OO group), sunflower oil (SFO group), or groundnut oil (GNO group) for 16 days. The L + Z levels of the OO group were markedly (p > 0.05) higher than those of SFO and GNO groups, in plasma (37.6 and 40.9%) and eyes (22.7 and 30.8%), respectively. These results suggest that oleic acid or OO can be used as a suitable fat source to modulate the absorption of dietary lutein to manage age-related macular degeneration.

Influence of phospholipids on beta-carotene absorption and conversion into vitamin A in rats.

This study determines the effect of lysophosphatidylcholine (lysoPC) and phosphatidylcholine (PC) in mixed micelles on beta-carotene and retinyl palmitate levels in rats in order to delineate the role of micellar phospholipids in the intestinal uptake of beta-carotene and its conversion into vitamin A. The rats were fed a single dose of beta-carotene solubilized in lysoPC (LPC group), PC (PC group) or no phospholipids (NoPL, control group) in micellar form. The level of beta-carotene and retinyl palmitate in plasma and beta-carotene in liver was analyzed by HPLC after 1, 2, 3, 6 and 9 h of feeding. The postprandial levels of beta-carotene in plasma (599.9 pmol/mL, Area Under Curve (AUC)) and in liver (1,161.3 pmol/g) were significantly (p<0.05) higher in the LPC group compared with its level in plasma (207.2 pmol/mL) and in liver (616.5 pmol/g) of the PC group and in plasma (119.1 pmol/mL) and in liver (626.2 pmol/g) of the NoPL group. No difference was seen between the PC and NoPL groups. The results demonstrate that beta-carotene absorption and its accumulation in plasma and liver were unaffected by PC compared with NoPL, while lysoPC not only enhanced its accumulation but also increased cleavage of intestinally absorbed beta-carotene into vitamin A as the AUC of plasma BC was higher and the AUC of retinyl palmitate in plasma of the lysoPC group was significantly higher than those of the other two groups. The results suggest that the luminal hydrolysis of PC to lysoPC is necessary for intestinal uptake of beta-carotene solubilized in mixed micelles.

Determination of major carotenoids in a few Indian leafy vegetables by high-performance liquid chromatography.

Leafy vegetables [Basella rubra L., Peucedanum sowa Roxb., Moringa oleifera Lam., Trigonella foenum-graecum L., Spinacia oleracea L., Sesbania grandiflora (L.) Poir., and Raphanus sativus L.] that are commonly used by the rural population in India were evaluated in terms of their main carotenoid pattern. The extracted carotenoids were purified by open column chromatography (OCC) on a neutral alumina column to verify their identity by their characteristic UV-visible absorption spectra. Reverse-phase high-performance liquid chromatography (HPLC) on a C18 column with UV-visible photodiode array detection under isocratic conditions was used for quantification of isolated carotenoids. Acetonitrile/methanol/dichloromethane (60:20:20 v/v/v) containing 0.1% ammonium acetate was used as a mobile phase. The major carotenoids identified by both methods were lutein, beta-carotene, violaxanthin, neoxanthin, and zeaxanthin. Among the carotenoids identified, lutein and beta-carotene levels were found to be higher in these leafy vegetables. Results show that P. sowa and S. oleracea are rich sources of lutein (77-92 mg/100 g of dry wt) and beta-carotene (36-44 mg/100 g of dry wt) compared with other leafy vegetables. The purity of carotenoids eluted by OCC was clarified by HPLC, and they were found to be 92% +/- 3% for neoxanthin, 94% +/- 2% for violaxanthin, 97% +/-2% for lutein and zeaxanthin, and 90% +/- 3% for beta-carotene. It could be recommended to use P. sowa and S. oleracea as rich sources of lutein and beta-carotene for health benefits. The OCC method proposed is relatively simple and provides purified carotenoids for feeding trials.