Lipophilic Carotenoids Research Articles

Advances in Metabolic Engineering for the Accumulation of Astaxanthin Biosynthesis.

Astaxanthin, a lipophilic carotenoid renowned for itsstrong antioxidant activity, holds significant commercial value across industries such as feed, food, and cosmetics. Although astaxanthin can be synthesized through chemical methods, it may contain toxic by-products in the synthesized astaxanthin, limiting its application inmedicine or functional food. Natural astaxanthin can be extracted from algae, however, the cultivation cycle of algae is relatively longercompared to microorganisms. With the advancement of synthetic biology and metabolic engineering, the method of microbial fermentation has emerged asa promising strategy for the large-scale production of astaxanthin. This article provides a comprehensive overview of the research progress in astaxanthin biosynthesis, highlighting the use ofthe natural host Xanthophyllomyces dendrorhous, andthe heterologous hosts Yarrowia lipolytica and Saccharomyces cerevisiae. Additionally, future research prospects are also discussed.

Multidimensional Optimization of Saccharomyces cerevisiae for Carotenoid Overproduction.

Microbial synthesis of carotenoids is a highly desirable alternative to plant extraction and chemical synthesis. In this study, we investigated multidimensional strategies to improve the carotenoid synthesis in the industrial workhorse of Saccharomyces cerevisiae. First, we rewired the yeast central metabolism by optimizing non-oxidative glycolysis pathway for an improved acetyl-CoA supply. Second, we restricted the consumption of farnesyl pyrophosphate (FPP) by the down-regulation of squalene synthase using the PEST degron. Third, we further explored the human lipid binding/transfer protein saposin B (hSapB)-mediated metabolic sink for an enhanced storage of lipophilic carotenoids. Last, the copper-induced GAL expression system was engineered to function in the yeast-peptone-dextrose medium for an increased biomass accumulation. By combining the abovementioned strategies, the final engineered yeast produced 166.79 ± 10.43 mg/l β-carotene in shake flasks, which was nearly 5-fold improvement of the parental carotenoid-producing strain. Together, we envision that multidimensional strategies reported here might be applicable to other hosts for the future industrial development of carotenoid synthesis from renewable feedstocks.

Compartmentalization of lutein in simple and double emulsions containing protein nanoparticles: Effects on stability and bioaccessibility

Delivery systems designed through protein stabilized emulsions are promising for incorporating carotenoids in different products. Nevertheless, the versatility in structures of such systems raises questions regarding the effect of the bioactive compound localization on their bio-efficacy, in particular for double emulsions. In this context, the aims of this study were to determine the impact of the localization of lutein in different water/oil/water double emulsions versus a single oil/water emulsion on the stability and in vitro bioaccessibility of lutein, a lipophilic carotenoid. The inner aqueous phase, which contained whey protein isolate (WPI) nanoparticles obtained by desolvation, was emulsified in sunflower oil stabilized by polyglycerol polyricinoleate (PGPR). The primary emulsion was then emulsified in a continuous aqueous phase containing whey protein isolate (WPI) and xanthan gum, the latter to increase the viscosity of the outer phase and delay creaming. Lutein was incorporated using different strategies: (1) lutein entrapped by WPI nanoparticles within the inner water phase of a double emulsion (W-L/O/W); (2) lutein incorporated into the oil phase of the double emulsion (W/O-L/W); (3) lutein incorporated in the oil phase of a single emulsion (O-L/W). All systems contained similar whey protein concentrations, as well as all other stabilizers. W-L/O/W sample showed the lowest lutein stability against light exposure during storage, and the highest lutein bioaccessibility after in vitro digestion, for freshly made samples. Furthermore, the in vitro bioaccessibility of lutein incorporated into the single emulsion was considerably lower than those observed for the double emulsions. The results reinforce the importance of designing appropriate structures for delivering improved stability and bioaccessibility of bioactive compounds.

Increasing β-carotene bioavailability and bioactivity in spinach demonstrated using excipient nanoemulsions—especially those of long-chain triglycerides

This study sought to improve the biological fate of β-carotene obtained from spinach, using in vitro digestion, in situ single-pass intestinal perfusion, and in vivo approaches, to investigate the effects of excipient emulsions with medium- (MCT) and long-chain triglyceride (LCT) as a vehicle for improved health benefits of β-carotene. Results showed that the bioavailability and bioactivity of β-carotene were both significantly higher in the excipient emulsions relative to those without the emulsions. This was especially true when LCT was used as the vehicle. These results were confirmed by bioaccessibility, duodenal absorption, and in vivo absorption and metabolism. Furthermore, animal feeding studies revealed that LCT may have the potential to promote triglyceride and apo-B48 reconstitution and secretion. This suggested that LCT may facilitate the entry of carotenoids into circulation via the lymphatic pathway. These results highlight the importance of the optimization of excipient foods to improve the efficacy of lipophilic carotenoid.

Enzymatic pre-treatment in cold pressing: Influence on flaxseed, apricot kernel and grape seed oils

A commercial enzyme preparation consisting of pectolytic, cellulotic and hemicellulotic enzymes was applied to the oil extraction by cold pressing from apricot kernel, flaxseed and grape seed. The effects of enzyme pre-treatment varied depending on the different oil seed used as raw material. Although the increase in free fatty acidity can be considered as a negative effect (from 0.37 to 0.52), the decrease in peroxide number and p-anisidine values, increase in oil yield (22.75%), higher levels of total carotenoids and tocopherols, as well as a remarkable increase in phenolic content (x1.68) and radical scavenging effect (including hydrophilic and lipophilic-induced and total antioxidant capacity) showed that the use of enzyme application in the cold pressing of apricot kernel oil would be beneficial. Many of these positive results could not be achieved in the pressing of flaxseed or grape seed oils under the same conditions. A high negative correlation (r=-92.2) was found between p-anisidine value and δ-tocopherol for grapeseed oil. Hydrophilic and lipophilic antioxidant capacity, total phenolics, and total carotenoids negatively correlated well (r values above 80) with peroxide values for apricot seed oil. Correlation results showed that carotenoids play an important role in the oxidative stability of the oils, where it was much more evident for apricot seed oil (r=-97.5).

Antioxidant edible film based on a carrot pectin‐enriched fraction as an active packaging of a vegan cashew ripened cheese

SummaryAn orange‐coloured carrot pectin‐enriched fraction (CPEF), with 50% uronic acids (42%‐methylated), carrying lipophilic carotenoids and α‐tocopherol, developed homogeneous calcium‐crosslinked films at 0%, 25%, 50%, 75% and 100% CPEF proportions regarding a commercial pectin. CPEF gave higher relative elongation at break (65% for 50% CPEF film) when compared to 0% CPEF film (26% elongation). FTIR spectra showed a decreased intensity in the C = O group of the esterified carboxylate band as the CPEF content increased. CPEF maintained the orange colour (a* ≈ +37) and partly the carotenes (k ≈ 8.62 × 10−3 d−1) in films, even under 25 °C light storage. Also, CPEF gave water resistance to dissolution (100% CPEF film: 50% solubility), maintaining film integrity after 24 h soaking. Surface wettability decreased (40° contact angle) when CPEF proportion increased. These characteristics made the 100% CPEF film an efficient antioxidant interface that preserved a vegan cashew ripened cheese of high water activity (0.952) for 60 days at 7 °C, as determined through the assay of thiobarbituric acid‐reactive substances (TBARS).

Astaxanthin-Shifted Gut Microbiota Is Associated with Inflammation and Metabolic Homeostasis in Mice

Astaxanthin is a red lipophilic carotenoid that is often undetectable in human plasma due to the limited supply in typical Western diets. Despite its presence at lower than detectable concentrations, previous clinical feeding studies have reported that astaxanthin exhibits potent antioxidant properties. We examined astaxanthin accumulation and its effects on gut microbiota, inflammation, and whole-body metabolic homeostasis in wild-type C57BL/6J (WT) and β-carotene oxygenase 2 (BCO2) knockout (KO) mice. Six-wk-old male and female BCO2 KO and WT mice were provided with either nonpurified AIN93M (e.g., control diet) or the control diet supplemented with 0.04% astaxanthin (wt/wt) ad libitum for 8 wk. Whole-body energy expenditure was measured by indirect calorimetry. Feces were collected from individual mice for short-chain fatty acid assessment. Hepatic astaxanthin concentrations and liver metabolic markers, cecal gut microbiota profiling, inflammation markers in colonic lamina propria, and plasma samples were assessed. Data were analyzed by 3-way ANOVA followed by Tukey's post hoc analysis. BCO2 KO but not WT mice fed astaxanthin had ∼10-fold more of this compound in liver than controls (P <0.05). In terms of the microbiota composition, deletion of BCO2 was associated with a significantly increased abundance of Mucispirillum schaedleri in mice regardless of gender. In addition to more liver astaxanthin in male KO compared with WT mice fed astaxanthin, the abundance of gut Akkermansia muciniphila was 385% greater, plasma glucagon-like peptide 1 was 27% greater, plasma glucagon and IL-1β were 53% and 30% lower, respectively, and colon NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation was 23% lower (all P <0.05) in male KO mice than the WT mice. Astaxanthin affects the gut microbiota composition in both genders, but the association with reductions in local and systemic inflammation, oxidative stress, and improvement of metabolic homeostasis only occurs in male mice.

Vitamin and non-vitamin antioxidants and their interaction in food

Aerobic life has been challenged by the changing conditions appearing through evolution resulting in appearance of both hydrophilic and lipophilic antioxidants as an indispensable protection against the ubiquitous reactive oxygen- and nitrogen species. Interaction between hydrophilic and lipophilic antioxidants at interfaces often results in synergistic effects important for food stability. Such concerted actions by antioxidants also seem of relevance for human nutrition, but need a more mechanistic description. Non-vitamin antioxidants like the hydrophilic polyphenols including their glycosides thus show synergism through enhancing the lipophilic tocopherols as chainbreaking antioxidants. While this tocopherol/polyphenol synergism seems to be controlled kinetically, the synergism between the lipophilic tocopherols and the lipophilic carotenoids is rather thermodynamically controlled through a regeneration of the carotenoids by the tocopherols. Antioxidant interaction between carotenoids and polyphenols is less investigated and more complex, but may explain the special role of the non-vitamin carotenoids as antioxidants. Carotenoids act as electron donors reducing lipid radicals in membranes subsequently being regenerated at interfaces by hydrophilic polyphenols. Taking distribution phenomena into account, a kinetic description may be possible. Carotenoid radical cations of the less reducing carotenoids like astaxanthin is regenerated faster to form the parent carotenoids showing the importance of the keto group for electron transfer in favour of kinetic control as is further confirmed by linear-free-energy relationships.

Improved Bioavailability of β-Carotene by Amorphous Solid Dispersion Technology in Rats.

β-Carotene (BC) is a natural lipophilic carotenoid mainly present in vegetables and fruits. Although it has various beneficial pharmacological activities, its bioavailability is low owing to its low water solubility. Recently, we reported that BC solid dispersion prepared using hot-melt technology with polyvinylpyrrolidone and sucrose fatty acid esters was in an amorphous state and showed the highest solubility. We hypothesized that the absorption of BC solid dispersion would be better because of its increased water solubility. To verify this, we conducted a pharmacokinetic analysis of BC for application in functional foods. Crystalline or amorphous BC was orally administered to rats. Blood was collected at various time points, and the BC concentration in the plasma was measured by HPLC. Oral administration of amorphous BC showed increased absorption in rats compared with that of BC crystals. Using blood samples from rats that were intravenously injected with the plasma of rats that had been orally administered BC, pharmacokinetic parameters could be calculated without using organic solvents or surfactants. It was possible to calculate various pharmacokinetic parameters under physiological conditions according to amorphous BC characteristics. Thus, we were able to determine the bioavailability of BC after oral administration. This simple technology to improve BC solubility without the use of organic solvents can be applied not only in the pharmaceutical industry but also in the food industry, and it therefore has high utility value.

Characterization of Carotenoids Content and Composition of Saffron from Different Localities

The most essential carotenoids for humans are found in plants that are normally yellow, orange, and red coloured pigments. They are typically and mostly lipophilic in nature, but some unique plant species may yield water-soluble carotenoids. Saffron or Crocus sativus contains hydrophilic carotenoids named crocin. Thus, this paper will describe the extraction and characterization of hydrophilic and lipophilic carotenoids (colour properties) obtained from saffrons of different geographical origins. They are specifically the Iranian, Turkish, and Kashmiri saffron respectively. Maceration techniques have been employed to extract the targeted compounds, whereas the characterization of the compounds has been analysed using HPLC. The extraction and characterization of carotenoids in saffron from different geographical origins found that the amount of crocin content was substantially higher in Iranian saffron, which was 11414.67 ± 516.34 µg/g DW followed by Turkish and Kashmiri saffron. Lipohilic carotenoids (i.e. crocetin, β-carotene, and zeaxanthin) were detectable in Iranian and Turkish saffron but absent in Kashmiri saffron. Similarly, the highest amount of crocetin content was found in Iranian saffron at 1054.73 ± 50.31 µg/g DW, while the highest amount of β-carotene and zeaxanthin was found in Turkish saffron at 512.92 ± 79.98 µg/g DW and 252.04 ± 60.34 µg/g DW, respectively. There was a marked difference in carotenoid composition sourced from different localities. Various environmental factors like climatic conditions, agricultural practices, stigma separation, and storing and drying processes may play an important role to explain such difference.

Preparation and characteristics of octenyl succinic anhydride-modified partial waxy rice starches and encapsulated paprika pigment powder

Physicochemical properties and emulsion stability of octenyl succinic anhydride (OSA)-modified partial waxy rice Baegjinju starches which were made with different OSA additions and the color stability of the encapsulated paprika pigment powder using the OSA starch were investigated. The DSs of the OSA starches ranged from 0.0152 to 0.0269. The band at 3303 cm−1 reduced as the OSA levels increased, but the A-type crystallinity was maintained. The oil absorption and emulsion stability, the short chain (DP6-12) amylopectin, the resistant starch levels, and the peak and final viscosities increased as the OSA levels increased. The encapsulated pigment powder with freeze-drying had higher color stability and better emulsion, and heating stabilities occurred with spray drying and the native pigment. From these results, it is suggested that the OSA-modified Baegjinju rice starch is a good wall material to protect lipophilic carotenoids. The encapsulated pigment powders could be used as stable natural colorants.

Watermelon Juice: a Novel Functional Food to Increase Circulating Lycopene in Older Adult Women.

Because of accruing oxidative stress with advancing age, older adults may benefit from increased dietary intake of lycopene, a lipophilic carotenoid with potent antioxidant properties. Yet, intake of dietary lycopene as well as circulating lycopene levels are known to decrease with aging. Watermelon is one of the few food sources of dietary lycopene. Because heat treatment increases lycopene bioavailability, ingestion of watermelon in pasteurized juice form may be an optimal delivery vehicle to increase lycopene levels in older adults. However, due to its lipophilic nature, there are concerns that co-ingestion of dietary fat may be necessary for efficient intestinal absorption of lycopene. Thus, this feasibility study aimed to examine the effects of a one-time dose of 100% pasteurized watermelon juice on circulating lycopene concentrations of postmenopausal women after a 10-h overnight fast. Blood was sampled from eight women before and 2 h after ingestion of 360ml of juice, and serum lycopene was measured by ultra-high performance liquid chromatography. Circulating lycopene levels increased by three-fold (p < 0.001) with increases observed for every participant. Results demonstrate that 100% watermelon juice is a palatable, effective means of increasing serum lycopene in older adult women, a group at risk for low carotenoid intake. Trial registration: Clinicaltrials.gov identifier: NCT03608254 .

Effects of Lipids on in Vitro Release and Cellular Uptake of β-Carotene in Nanoemulsion-Based Delivery Systems.

β-Carotene (BC) nanoemulsions were successfully prepared by microfluidization. BC micellarization was significantly affected by bile salts and pancreatin concentration. Positive and linear correlation was observed between BC release and bile salts concentration. Pancreatin facilitated BC's release in simulated digestion. Compared to the control (bulk oil) (4.6%), nanoemulsion delivery systems significantly improved the micellarization of BC (70.9%). The amount of BC partitioned into micelles was positively proportional to the length of carrier oils. Unsaturated fatty acid (UFA)-rich oils were better than saturated fatty acid (SFA)-rich oils in transferring BC (p < 0.05). No significant difference was observed between monounsaturated fatty acid (MUFA)-rich oils and polyunsaturated fatty acid (PUFA)-rich oils (p > 0.05). A positive and linear relationship between the degree of lipolysis and the release of BC in vitro digestion was observed. Bile salts showed cytotoxicity to Caco-2 cells below 20 times dilution. BC uptake by Caco-2 cells was not affected by fatty acid (FA) compositions in micelles, but BC uptake was proportional to its concentration in the diluted micelle fraction. The results obtained are beneficial to encapsulate and deliver BC or other bioactive lipophilic carotenoids in a wide range of commercial products.

Preparation of vesicles entrapped lycopene extract.

Lycopene, a lipophilic carotenoid, has been known as an effective antioxidant in supporting the cutaneous defensive system. However, it is unstable when exposed to light and water. In this study, lycopene was isolated from tomatoes and a vesicular delivery system was developed to entrap and stabilize the lycopene in the aqueous system. A simple process, maceration in ethyl acetate, was used to extract lycopene from the tomatoes. The extract was then chromatographed on the Sephadex LH20 column using acetone as a solvent system to yield 995 μg of lycopene per gram of dried tomato weight. The vesicular delivery system was prepared from a combination of ascorbic acid-6-palmitate (AP), cholesterol and dicetyl phosphate using a thin film hydration method. The formulation was composed of AP, cholesterol and dicetyl phosphate at a 44:44:12 molar ratio and with 2.12 μmol/ml of the isolated lycopene. Both blank vesicles and lycopene loaded vesicles were kept for a period of 3 months at 4±2°C and at the room temperature (28±2°C) to evaluate the effect of the encapsulation on the characteristic of the vesicles and on the antioxidant activity of the encapsulated lycopene. The result implied that lycopene could be stabilized in the vesicles and its scavenging activity against DPPH free radicals was superior to that of the free lycopene solution.

A novel lipid-based solid dispersion for enhancing oral bioavailability of Lycopene – In vivo evaluation using a pig model

Lycopene is a potent anti-oxidant, which has been widely reported for its potential benefits at reducing the risks of certain types of cancer e.g. prostate cancer. The oral bioavailability of this highly lipophilic carotenoid is low and highly influenced by the extent of intestinal lymphatic uptake. The aim of this study was to develop an optimised formulation, which allows for efficient absorption following oral administration. A self-emulsifying drug delivery system (SEDDS) and solid dispersion of Lycopene were developed initially. Subsequently, a novel lipid based solid dispersion (LBSD) was designed. Processing via a solid dispersion approach was found to alter the solid state characteristics of Lycopene, as determined by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The bioavailability of Lycopene was significantly increased after oral administration of LBSD to fasted pigs, relative to the commercial product (Lycovit(®)). A clear distinction in terms of Cmax and AUC was observed between Lycovit(®) and LBSD. In conclusion, a novel LBSD formulation was developed to enhance the oral bioavailability of the model lipophilic compound, Lycopene, by enhancing dissolution in the gastrointestinal tract and promoting intestinal lymphatic uptake utilising digestible lipid excipients.

Lycopene induces retinoic acid receptor transcriptional activation in mice

Lycopene is a lipophilic carotenoid and provides the red colour to tomatoes and tomato product. Various studies indicated that lycopene and tomatoes/tomato products are able to positively influence various diseases associated with a chronic inflammation. The mechanism of action of lycopene to elicit these effects is partly unknown. A possible mechanism is that biological metabolites of lycopene may activate nuclear hormone receptors in mammalian cells. The aim of this study was to investigate the potential of orally administered lycopene and all-trans retinoic acid (ATRA) for the induction of the retinoic acid receptor (RAR) in a transgenic retinoic acid response-element (RARE)-reporter mouse system. Orally administered lycopene (100 mg/kg bw in beadlets, n= 6) and ATRA as an endogenous RAR ligand (50 mg/kg bw, n= 6) for the induction of the retinoic acid receptor in male mice using a transgenic RARE-reporter mouse system. Lycopene treatments induced RARE-mediated cell signalling indicated by quantified bioimaging, increased luciferase activity and up-regulated the retinoid target genes in selected organs of the mice. We conclude that lycopene can induce RAR-transcriptional activation in mice and lycopene might be a precursor of still non-identified biologically active metabolites.

Carotenoids in Antioxidant Networks. Colorants or Radical Scavengers

Optical and electronic properties of carotenoids as also reflected in their colors have been fine-tuned through evolution, resulting in a structural diversity important for carotenoid properties as radical scavengers and as quenchers of electronically excited states. Carotenoids form antioxidant networks based on one-electron transfer with other carotenoids depending on the balance between ionization energy and electron affinity of the individual carotenoids as has been demonstrated by real-time kinetic studies and later confirmed by quantum mechanical calculations. The more hydrophilic xanthophylls serve as molecular wiring across membranes in these networks through anchoring in water/lipid interfaces resulting in synergism with more lipophilic carotenoids. Radical scavenging of such networks seems to be thermodynamically controlled according to a two-dimensional classification of potential antioxidants. Carotenoids in birds' plumage, as reflected by their color and color intensity, seem to be indicators of good antioxidant status and health of the bird, and such antioxidant networks appear to be in "equilibrium". Carotenoids are under other conditions involved in networks with other types of antioxidants as in egg yolk and in some fish. For the more hydrophilic (iso)flavonoids and their glycosides, antioxidant synergism through regeneration of the lipophilic carotenoids active as radical scavengers becomes kinetically controlled at interfaces. Carotenoids appear accordingly, and also in food, as antioxidants under two types of conditions: (i) in "equilibrium" with other antioxidants in thermodynamically controlled networks serving as color indicators of good antioxidant status and (ii) as antioxidants active through radical scavenging in networks with kinetically controlled regeneration.

Fatty acids, tocols, and carotenoids in pulp oil of three sea buckthorn species (Hippophae rhamnoides, H. salicifolia, and H. tibetana) grown in the Indian Himalayas

AbstractSea buckthorn berries from Hippophae rhamnoides, H. tibetana, and H. salicifolia were collected from the cold deserts of the Himalayas (Lahaul, Ladakh, and Spiti; India) and characterized in terms of the FA, carotenoid, tocopherol, and tocotrienol composition in their pulp oil. These varied from species to species. Total carotenoids ranged from 692 to 3420 mg/kg in pulp oils of fresh berries, and total tocols, from 666 to 1788 mg/kg. Hippophae salicifolia berries contained substantially lower amounts of pulp oil, with lower levels of carotenoids and tocopherols. There was little difference in the proportion of individual tocols in pulp among the three species. α‐Tocopherol alone constituted 40–60% of total pulp tocols in berries. Pulp oils had palmitoleic acid (32–53%) as the most abundant FA followed by palmitic (25–35%), oleic (8–26%), linoleic (5–16%), and linolenic (0.6–2.6%) acids, with the highest deviation observed in the proportion of palmitoleic acid in these berries. Hippophae rhamnoides and H. tibetana contained the highest amount of the lipophilic carotenoids and tocols. Hippophae salicifolia berries had higher amounts of lipophobic constituents such as vitamin C and flavonols.

Safety evaluation of phytosterol-esters. Part 9: Results of a European post-launch monitoring programme

Phytosterol-esters were developed by Unilever as a cholesterol lowering novel food ingredient for use initially in vegetable oil spreads. In addition to an extensive package of safety studies and clinical studies a programme of post-launch monitoring (PLM) was developed. PLM was used to address the following questions: (a) Is the product use as predicted/recommended? (b) Are the known effects as predicted? (c) Does the product cause unexpected health effects? The overall conclusions from the PLM programme were: the product is being bought by the target population but intakes are less than the original assumptions made in the risk assessment; long-term use of phytosterol-ester enriched spreads results in a reduction in the serum levels of the most lipophilic carotenoids but at current levels of intake this is unlikely to result in reductions in carotenoids that are of biological significance; evaluation of health related consumer complaints have not indicated any unexpected health effects associated with the use of the product in the marketplace. As part of the European approval under Regulation (EC) No. 258/97 on Novel Foods and Food Ingredients the results of the PLM programme had to be submitted to the European Commission (EC) and reviewed by the Scientific Committee on Food (SCF). They concluded that the study provided valuable information, which complemented the pre-market safety evaluation studies, and that the EC mandatory requirement had been met.

Direct superoxide anion scavenging by a highly water-dispersible carotenoid phospholipid evaluated by electron paramagnetic resonance (EPR) spectroscopy

Synthetic carotenoid analogs, with increased utility for biological applications, are sparingly reported in the literature. Synthetic modification, which may increase the water solubility and/or water dispersibility of lipophilic carotenoids, allows their use in aqueous environments as potent antioxidants against potentially deleterious reactive oxygen species (ROS) that can be generated in vivo. Superoxide anion, produced by activated human neutrophils, can be a source of additional harmful ROS and nonradical species such as singlet oxygen in vivo. In the current study, direct scavenging of superoxide anion by a well-characterized C30 carotenoid phospholipid mixture was evaluated in a standard in vitro isolated human neutrophil assay by electron paramagnetic resonance (EPR) spectroscopy, employing the spin-trap DEPMPO. The carotenoid phospholipid was tested in aqueous formulation (aqueous dispersibility >60 mg/mL), in which supramolecular assembly takes place, as well as in ethanolic formulation as a monomeric solution of the carotenoid phospholipids. The carotenoid phospholipid (a highly unsaturated zwitterionic surfactant) was compared with a previously characterized rigid, long-chain, highly unsaturated dianionic bolaamphiphile, which contains an additional three conjugated double bonds in its extended conjugated system. As previously reported, direct scavenging by the carotenoid phospholipid derivatives in monomeric ethanolic formulation was superior at each tested concentration to aqueous, aggregated formulations of the compounds. Additionally, the percent inhibition of superoxide signal was related to the apparent or effective length of the conjugated chromophore, consistent with previous reports of radical inhibition and singlet oxygen quenching by polyene carotenoids of differing length.