Green synthesis of silver nanoparticles using canthaxanthin from Dietzia maris AURCCBT01 and their cytotoxic properties against human keratinocyte cell line.

AIM: Nano-biotechnologically synthesizing silver nanoparticles via canthaxanthin pigment extracted from Dietzia maris AURCCBT01 and assessing their cytotoxic therapeutic potential against human keratinocyte cell line (HaCaT) were the key objectives of this study. METHODS AND RESULTS: The pigment extracted from D. maris AURCCBT01 was identified as canthaxanthin using UV-VIS spectroscopy, FTIR, NMR (1 H NMR and 13 C NMR) and MS. Canthaxanthin, treated with silver nitrate solution, produced canthaxanthin-mediated silver nanoparticles and they were characterized by UV-VIS spectroscopy, FTIR, XRD, FESEM-EDX and TEM-SAED techniques. UV-VIS spectroscopy pointed out an absorption band at 420 nm, relating to the surface plasmon resonance of silver nanoparticles. FTIR findings suggested that the diverse functional groups of canthaxanthin bio-molecules played a significant task in capping the silver nanoparticles. XRD analysis exhibited 40·20 nm for the crystal size of nanoparticles. FESEM and TEM exhibited that the biosynthesized silver nanoparticles were spherical in shape with crystalline nature and the particle size was 40-50 nm. Moreover, the cytotoxicity assessment of the synthesized nanoparticles in HaCaT revealed significant cytotoxicity in the cultured cells with an IC50 value of 43 µg ml-1 . CONCLUSION: Stable silver nanoparticles synthesized using canthaxanthin from D. maris AURCCBT01 were found effective for application in wound healing activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Biosynthesized silver nanoparticles via canthaxanthin bacterial pigment exhibited their cytotoxicity effect in HaCaT and testified their eventual therapeutic potential in the wound healing activity with no side effects in a cost effective and eco-friendly process.

Anti-Skin Cancer Activities of Apostichopus japonicus Extracts from Low-Temperature Ultrasonification Process.

OBJECTIVES: This work aimed to enhance anti-skin cancer activities of Apostichopus japonicus, spiky sea cucumber, through ultrasonification extraction process at low temperature. METHODS: Dried Apostichopus japonicus was extracted with an ultrasonification process at 50°C and 95 kHz for two hours (UE), and anti-skin cancer activities of the extract from the UE were also compared with those from conventional extraction processes using hot water (WE) or 70% ethanol at 80°C (EE) for 12 hours. RESULTS: The amount of canthaxanthin in the UE was higher than that in the WE or EE, and its cytotoxicity against human keratinocytes was less than the others. The extract from the UE showed 93.5% inhibition against human malignant cell growth, which was also higher than those from both WE and EE. The extract from the UE demonstrated the ability of inhibiting both cancer cell proliferation and metastasis by downregulating the skin tumor-promoting genes such as Bcl-2, STAT3, and MMP-9. CONCLUSIONS: The ultrasonification process was proved to be effective especially in extracting heat-sensitive marine biomass, A. japonicus having higher amounts of canthaxanthin and better anti-skin cancer activities, possibly due to less destruction and high elution of bioactive substances under low temperature extraction condition.

HPLC Quantification of astaxanthin and canthaxanthin in Salmonidae eggs.

Astaxanthin and canthaxanthin are naturally occurring antioxidants referred to as xanthophylls. They are used as food additives in fish farms to improve the organoleptic qualities of salmonid products and to prevent reproductive diseases. This study reports the development and single-laboratory validation of a rapid method for quantification of astaxanthin and canthaxanthin in eggs of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis М.). An advantage of the proposed method is the perfect combination of selective extraction of the xanthophylls and analysis of the extract by high-performance liquid chromatography and photodiode array detection. The method validation was carried out in terms of linearity, accuracy, precision, recovery and limits of detection and quantification. The method was applied for simultaneous quantification of the two xanthophylls in eggs of rainbow trout and brook trout after their selective extraction. The results show that astaxanthin accumulations in salmonid fish eggs are larger than those of canthaxanthin. As the levels of these two xanthophylls affect fish fertility, this method can be used to improve the nutritional quality and to minimize the occurrence of the M74 syndrome in fish populations.

Co-solvent selection for supercritical fluid extraction of astaxanthin and other carotenoids from Penaeus monodon waste.

In recent years, astaxanthin is claimed to have a 10 times higher antioxidant activity than that of other carotenoids such as lutein, zeaxanthin, canthaxanthin, and ß-carotene; the antioxidant activity of astaxanthin is 100 times higher than that of α-tocopherol. Penaeus monodon (tiger shrimp) is the largest commercially available shrimp species and its waste is a rich source of carotenoids such as astaxanthin and its esters. The efficient and environment-friendly recovery of astaxanthins was accomplished by using a supercritical fluid extraction (SFE) technique. The effects of different co-solvents and their concentrations on the yield and composition of the extract were investigated. The following co-solvents were studied prior to the optimization of the SFE technique: ethanol, water, methanol, 50% (v/v) ethanol in water, 50% (v/v) methanol in water, 70% (v/v) ethanol in water, and 70% (v/v) methanol in water. The ethanol extract produced the highest carotenoid yield (84.02 ± 0.8 µg/g) dry weight (DW) with 97.1% recovery. The ethanol extract also produced the highest amount of the extracted astaxanthin complex (58.03 ± 0.1 µg/g DW) and the free astaxanthin content (12.25 ± 0.9 µg/g DW) in the extract. Lutein and ß-carotene were the other carotenoids identified. Therefore, ethanol was chosen for further optimization studies.

Characterization of canthaxanthin isomers isolated from a new soil Dietzia sp. and their antioxidant activities.

Canthaxanthin (cx) is a potent antioxidant that is chemically synthesized at the industrial scale and has imperative applications in the cosmetic and feed industries. An orange pigmented mesophilic bacterium, designated as K44, was isolated from soil samples of Kargil, India. Biochemical tests, 16S rRNA gene sequencing, and FAME analysis of the bacterium indicated it to belong in the genus Dietzia and is distinct from human isolates. The strain showed 98% 16S rRNA gene sequence homology with Dietzia maris DSM 43102. High-performance liquid chromatography profile of the pigments isolated from K44 showed two major peaks absorbing at 465.3 and 475 nm. The liquid chromatography-mass spectrometry (LC-MS) analysis of both these peaks revealed their m/z to be 564. The molecular weights, LC-MS/MS fragmentation patterns, and lambdamax of these fractions corresponded to all-trans- (475 nm) and 9-cis-(465.3 nm) cx isomers. The antioxidant activities of cis- and trans-cx isomers isolated from this bacterium were found to differ, where the cis-isomer showed higher free radical, superoxide radical, and reactive oxygen species scavenging activities than the alltrans- isomer, suggesting that 9-cis-cx is more effective as an antioxidant than the all-trans-cx.

Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents.

Canthaxanthin has a substantial commercial market in aquaculture, poultry production, and cosmetic and nutraceutical industries. Commercial production is dominated by chemical synthesis; however, changing consumer demands fuel research into the development of biotechnology processes. Highly productive microbial systems to produce carotenoids can be limited by the efficiency of extraction methods. Extraction with hexane, acetone, methanol, 2-propanol, ethanol, 1-butanol, tetrahydrofuran and ethyl acetate was carried out with each solvent separately, and subsequently the most efficient solvents were tested in combination, both as mixtures and sequentially. Sequential application of methanol followed by acetone proved most efficient. Extraction efficiency remained stable over a solvent to biomass range of 100:1 to 55:1, but declined significantly at a ratio of 25:1. Application of this method to a canthaxanthin-producing Escherichia coli production system enabled efficient canthaxanthin extraction of up to 8.5 mg g(-1) dry biomass.

Extraction, purification and concentration of partially saturated canthaxanthin from Aspergillus carbonarius.

A mutant Aspergillus carbonarius produces partially saturated canthaxanthin (PSC; C(40)H(62)O(2)) during submerged fermentation. The pigment was extracted from dried biomass using various organic solvents and purified using nanofiltration (NF) and nonporous membranes. Particle size had a great influence; PSC extractability from fines fraction of biomass (75-105 microm) was 1.5-fold higher compared to the coarse fraction (850-920 microm) in ethanol. Among the four solvents, hexane exhibited the highest PSC extractability of 5.83 mg/g and purity of 32 mg/g. On a relative scale, the extraction performance of hexane, acetone, methanol and ethanol were in the order 100, 16.1, 7.5 and 5.4. An assessment based on enrichment factor and permeate flux revealed notable performance with NF-250 membrane in ethanol extract followed by NF-200 and NF-GKSS membranes in methanol extract. These results suggested the suitability of hexane for extraction followed by alcohol phase purification and concentration employing NF. Accordingly, a PSC purity of 206 mg/g was achieved.

[Simultaneous determination of canthaxanthin and astaxanthin in feedstuffs using solid phase extraction-reversed-phase high performance liquid chromatography].

A method was developed for the simultaneous determination of canthaxanthin and astaxanthin in feedstuffs using reversed-phase high performance liquid chromatography (RP-HPLC). The sample was extracted by acetonitrile, and cleaned up by an LC-NH2 column. An Agilent ZORBAX Eclipse XDB-C18 analytical column (150 mm x 4.6 mm, 5 microm) was used and kept at 25 degrees C. Acetonitrile-methanol (95 : 5, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The detection was performed by a diode array detector at 474 nm. The quantitive analysis of external standard calibration curves was used. The linear ranges of the method for canthaxanthin and astaxanthin were 1.0 - 30.0 mg/L (r = 0.999 0) and 1.0 - 20.0 mg/L (r = 0.999 1), respectively. The average recoveries were 90% - 101% with the relative standard deviations of 0.62% - 3.68%. The detection limits were 0.84 and 0.60 mg/L for canthaxanthin and astaxanthin, respectively. The method is simple, precise, sensitive and reproductive. It can be used to determine the contents of canthaxanthin and astaxanthin in feedstuffs.

Partially saturated canthaxanthin purified from Aspergillus carbonarius induces apoptosis in prostrate cancer cell line.

A mutant Aspergillus carbonarius selected for temperature tolerance after UV treatment, when grown in shake flasks, produced mycelia bearing yellow pigment. Since the mutant was affected in sterol biosynthetic pathway, the pigment was apparently produced to maintain membrane fluidity and rigidity for growth sustenance in low-pH culture broth. Nuclear magnetic resonance analyses characterizing the pigment as a partially saturated canthaxanthin, containing beta-ionone end rings, suggested its application as a retinoid. When tested for this property in retinoic acid receptor expressing prostate cancer cell line, LNCaP, the fungal partially saturated canthaxanthin induced apoptosis. Low apoptosis percentage in DU145 prostrate cancer cells that does not express functional retinoic acid receptor-beta (RAR-beta) suggested binding specificity of the partially saturated canthaxanthin for RAR-beta.

Isolation and purification of canthaxanthin from the microalga Chlorella zofingiensis by high-speed counter-current chromatography.

Certain microalgae are considered to be a potential source of canthaxanthin, which possesses strong antioxidant and anticancer activities. A high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of canthaxanthin from the microalga Chlorella zofingiensis. The crude canthaxanthin was obtained by extraction with organic solvents after the microalgal sample had been saponified. Preparative HSCCC, with a two-phase solvent system composed of n-hexane-ethanol-water (10:9:1 v/v), was successfully performed yielding canthaxanthin at 98.7% purity from 150 mg of the crude extract (2.1% canthaxanthin) in a one-step separation. The recovery of canthaxanthin was 92.3%. This was the first report that canthaxanthin was successfully separated and purified from microalgae.

Identification by HPLC-MS of carotenoids of the Thraustochytrium CHN-1 strain isolated from the Seto Inland Sea.

The orange-pigmented Thraustochytrium, CHN-1 strain was found to contain astaxanthin as the main carotenoid pigment. Echinenone, canthaxanthin, phoenicoxanthin and beta-carotene were also identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry. The total extractable carotenoid level was found to increase with culture age.

Production of canthaxanthin by Haloferax alexandrinus under non-aseptic conditions and a simple, rapid method for its extraction.

The production of carotenoids from Haloferax alexandrinus strain TM(T) was investigated at various concentrations of NaCl (10-25%) in culture media under non-aseptic conditions. PCR and dot blot hybridization assays were employed to monitor the growth of Hfx. alexandrinus in the culture under aseptic and non-aseptic conditions. The amplified PCR products of 16S rDNA from Hfx. alexandrinus grown under aseptic conditions were used as specific probes, which bound with amplified PCR products of 16S rDNA dots from both aseptic and non-aseptic conditions (20-25% NaCl). The results indicated that contamination of the culture was precluded at high NaCl concentrations (20-25%). Therefore, it is not necessary to perform asepsis during the biotechnological processes of carotenoid production by Hfx. alexandrinus. A 1-l-scale cultivation of the cells in flask cultures under non-aseptic conditions produced 3.12+/-0.5 g dry weight, 6.34+/-2.5 mg total carotenoids and 2,156.67+/-0.1 microg canthaxanthin. Further experiments in a batch fermenter, under non-aseptic conditions, also demonstrated increases in the biomass concentration and carotenoid production. When grown in a standard growth medium at 25% NaCl, the cells of Hfx. alexandrinus lysed spontaneously in fresh water and hence carotenoids could be extracted directly from the cells without any mechanical disintegration. These results demonstrate the feasibility and simplicity of commercial production of carotenoids using Hfx. alexandrinus.

Induction of gap junctional communication by 4-oxoretinoic acid generated from its precursor canthaxanthin.

The activity of 4-oxoretinoic acid as an inducer of gap junctional communication was investigated in C3H/10T1/2 murine fibroblasts. Two isomers of this retinoid, all-trans- and 13-cis-4-oxoretinoic acid, enhance gap junctional communication. This is accompanied by increased expression of connexin43 mRNA. Decomposition fractions of canthaxanthin were isolated by preparative high-performance liquid chromatography and shown to be active in the cell-cell communication assay. Two of the decomposition compounds were identified as all-trans- and 13-cis-4-oxoretinoic acid. Therefore, it is concluded that the biological activity of canthaxanthin regarding cell-cell communication is at least in part due to the formation of active decomposition products such as 4-oxoretinoic acid.