Carminic acid mitigates fructose-triggered hepatic steatosis by inhibition of oxidative stress and inflammatory reaction.

Excessive fructose (Fru) consumption has been reported to favor nonalcoholic fatty liver disease (NAFLD). However, the molecular mechanism is still elusive, lacking effective therapeutic strategies. Carminic acid (CA), a glucosylated anthraquinone found in scale insects like Dactylopius coccus, exerts anti-tumor and anti-oxidant activities. Nevertheless, its regulatory role in Fru-induced NAFLD is still obscure. Here, the effects of CA on NAFLD in Fru-challenged mice and the underlying molecular mechanisms were explored. We found that Fru intake significantly led to insulin resistance and dyslipidemia in liver of mice, which were considerably attenuated by CA treatment through repressing endoplasmic reticulum (ER) stress. Additionally, inflammatory response induced by Fru was also attenuated by CA via the blockage of nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs) and tumor necrosis factor α/TNF-α receptor (TNF-α/TNFRs) signaling pathways. Moreover, Fru-provoked oxidative stress in liver tissues was remarkably attenuated by CA mainly through improving the activation of nuclear factor erythroid 2-related factor 2 (Nrf-2). These anti-dyslipidemias, anti-inflammatory and anti-oxidant activities regulated by CA were confirmed in the isolated primary hepatocytes with Fru stimulation. Importantly, the in vitro experiments demonstrated that Fru-induced lipid accumulation was closely associated with inflammatory response and reactive oxygen species (ROS) production regulated by TNF-α and Nrf-2 signaling pathways, respectively. In conclusion, these results demonstrated that CA could be considered as a potential therapeutic strategy to attenuate metabolic disorder and NAFLD in Fru-challenged mice mainly through suppressing inflammatory response and oxidative stress.

Inhibition of amyloid fibrillation of γD-crystallin model peptide by the cochineal Carmine.

γD-crystallin is among the most abundant γ-crystallins in the human eye lens which are essential for preserving its transparency. Aging, and environmental changes, cause crystallins to lose their native soluble structure and aggregate, resulting in the formation of cataract. Current treatment of cataract is surgical removal which is costly. Pharmaceutical therapeutics of cataract is an unmet need. We report a screen for small molecules capable of inhibiting aggregation of human γD-crystallin. Using a highly amyloidogenic hexapeptide model 41GCWMLY46 derived from the full-length protein, we screened a library of 68 anthraquinone molecules using ThT fluorescence assay. A leading hit, the cochineal Carmine, effectively reduced aggregation of the model GDC6 peptide in dose dependent manner. Similar effect was observed toward aggregation of the full-length γD-crystallin. Transmission electron microscopy, intrinsic Tryptophan fluorescence and ANS fluorescence assays corroborated these results. Insights obtained from molecular docking suggested that Carmine interaction with monomeric GDC6 involved hydrogen bonding with Ace group, Cys, Met residues and hydrophobic contact with Trp residue. Carmine was non-toxic toward retinal cells in culture. It also reduced ex vivo the turbidity of human extracted cataract material. Collectively, our results indicate that Carmine could be used for developing new therapeutics to treat cataract.

Combining dietary phenolic antioxidants with polyvinylpyrrolidone: transparent biopolymer films based on p-coumaric acid for controlled release.

Polyvinylpyrrolidone (PVP) has probably been one of the most utilized pharmaceutical polymers with applications ranging from a blood plasma substitute to nanoparticle drug delivery, since its synthesis in 1939. It is a highly biocompatible, non-toxic and transparent film forming polymer. Although high solubility of PVP in aqueous environment is advantageous, it still poses several problems for some applications in which sustained targeting and release are needed or hydrophobic drug inclusion and delivery systems are to be designed. In this study, we demonstrate that a common dietary phenolic antioxidant, p-coumaric acid (PCA), can be combined with PVP covering a wide range of molar ratios by solution blending in ethanol, forming new transparent biomaterial films with antiseptic and antioxidant properties. PCA not only acts as an effective natural plasticizer but also establishes H-bonds with PVP increasing its resistance to water dissolution. PCA could be released in a sustained manner up to a period of 3 days depending on the PVP/PCA molar ratio. Sustained drug delivery potential of the films was studied using methylene blue and carminic acid as model drugs, indicating that the release can be controlled. Antioxidant and remodeling properties of the films were evaluated in vitro by free radical cation scavenging assay and in vivo on a murine model, respectively. Furthermore, the material resorption of films was slower as PCA concentration increased, as observed from the in vivo full-thickness excision model. Finally, the antibacterial activity of the films against common pathogens such as Escherichia coli and Staphylococcus aureus and the effective reduction of inflammatory agents such as matrix metallopeptidases were demonstrated. All these properties suggest that these new transparent PVP/PCA films can find a plethora of applications in pharmaceutical sciences including skin and wound care.

Acaricidal Activity and Synergistic Effect of Thyme Oil Constituents against Carmine Spider Mite (Tetranychus Cinnabarinus (Boisduval)).

Studies examining the use of essential oils as replacements for synthetic insecticides require an understanding of the contribution of each constituent present, interactions among these components, and how they relate to overall toxicity. In the present study, the chemical composition of commercial thyme oil was identified by gas chromatography-mass spectrometry. Thyme oil and blends of its major constituents were tested for their acaricidal activitities against carmine spider mites (Tetranychus cinnabarinus (Boisduval)) using a slide-dip bioassay. Natural thyme oil showed greater toxicity than any single constituent or blend of constituents. Thymol was the most abundant component (34.4%), and also possessed the strongest acaricidal activity compared with other single constituents. When tested individually, four constituents (linalool, terpinene, p-cymene and carvacrol) also had activity, while α-pinene, benzoic acid and ethyl gallate had almost no activity. The toxicity of blends of selected constituents indicated a synergistic effect among the putatively active and inactive constituents, with the presence of all constituents necessary to reach the highest toxicity. The results indicated that thyme oil and some of its major constituents have the potential to be developed into botanical acaricides.

Promoting effects of carminic acid-enriched cochineal extracts on capsular invasive thyroid carcinomas through targeting activation of angiogenesis in rats.

Cochineal extracts (CE) is a coccid-derived natural food colorant containing carminic acid (CA) as an active ingredient that potentiates inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In our previous study, dietary administered CE (CA: 28.5% in CE) has shown to promote the macroscopic development of capsular invasive carcinomas (CICs) associated with up-regulation of angiogenesis-related genes in an intracapsular invasion model of experimental thyroid cancers using rats. However, the promoting effect of CE could not be confirmed histopathologically. The purpose of the present study was to confirm the promoting effect of CE through direct injections to animals on the development of CICs using this cancer invasion model. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSlc rats were administered CA-enriched CE (CA: 52.6% in CE) by intraperitoneal injections every other day (10 mg/kg body weight) during the promotion with 0.15% sulfadimethoxine in the drinking water for 8 weeks. The multiplicities of macroscopical CICs and the mean area of early capsular invasive foci estimated by Tenascin (TN)-C-immunoreactivity in the thyroid significantly increased with CE-treatment, while the number of TN-C-positive foci did not change with CE. Transcript level of Phbp and downstream genes unchanged; however, transcript level of angiogenesis-related genes, i.e, Vegfb and its transcription factor gene, Hif1a, those being downstream of phosphatase and tensin homolog (PTEN)/Akt signaling, up-regulated in the thyroid tissue with CE-administration. These results suggest that CE potentiates promotion activity by facilitating angiogenesis through activation of PTEN/Akt signaling without accompanying modification of PHBP-related proteolysis.

Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds, purpurin and alizarin.

Recently we have shown that anthraquinone food pigments such as purpurin and alizarin suppress the genotoxic activities of several mutagens including heterocyclic amines and polycyclic aromatic hydrocarbons in the Drosophila DNA repair test and in the Ames test. To investigate the mechanism of this inhibition, we have now examined the effects of these anthraquinone pigments on enzymes that metabolize xenobiotics. The activities of eight human recombinant cytochrome P450 (CYP) isozymes were measured in the presence of purpurin, alizarin or carminic acid. Purpurin and alizarin strongly inhibited the activities of CYP1A1, CYP1A2 and CYP1B1, and weakly suppressed those of CYP2A6 and CYP2E1 in a dose-dependent manner, but did not inhibit those of CYP2C19, CYP3A4 and CYP3A5. Carminic acid did not affect the activities of any CYPs tested. CYP1B1 was the most strongly affected CYP molecule by purpurin and alizarin among CYPs examined in this study. From kinetic analysis, it was shown that the inhibition by purpurin on CYP1B1 was both competitive and non-competitive, and that by alizarin was competitive. The values of slopes obtained from Lineweaver-Burk plots are proportional to the square of purpurin concentration. This observation suggests that two molecules of purpurin are interacting with one molecule of CYP1B1. The K(m) value of CYP1B1 was 11 microM, and the K(i) value of purpurin and alizarin against CYP1B1 was 0.7 microM(2) and 0.5 microM, respectively. We also examined the effects of these pigments on the mutagenicities of MeIQx and B[a]P in the Ames test, using Salmonella typhimurium TA1538 co-expressing each form of human CYP and NADPH-cytochrome P450 reductase (OR). The mutagenicity of MeIQx in TA1538 1A2/OR or 1B1/OR was suppressed by purpurin and alizarin but not by carminic acid. Purpurin also reduced the mutagenicity of B[a]P in TA1538 1A1/OR or 1B1/OR. These results suggest that the antigenotoxic activities of purpurin and alizarin can be explained by inhibition of CYP activities responsible for activating the mutagens.

Preventive effects of anthraquinone food pigments on the DNA damage induced by carcinogens in Drosophila.

We have previously demonstrated the inhibitory effect of chlorophyllin, a green food additive, on the genotoxicities of various carcinogens in Drosophila. Recently, we reported that purpurin, a component of a red food additive produced from madder root (Rubia tinctorium), inhibits the bacterial mutagenicity of heterocyclic amines. In the present study, we examined antigenotoxic activities of various pigments that are either constituents of food or food additives, using Drosophila in vivo DNA repair assay. Third instar larvae of Drosophila were fed a mutagen with or without pigment. The resulting adult flies were monitored for their male (repair deficient)/female (repair proficient) ratios, which reflect the DNA damage. We tested a total of 20 pigments, which are mainly of plant origins, including flavonoids, carotenoids, anthocyanins, anthraquinones and beta-diketone (curcumin)-derivatives, against the genotoxicities of eight carcinogens; IQ, MeIQx, AFB1, NDMA, 2-AAF, DMBA, 4NQO, and MNU. Four anthraquinone pigments (alizarin, purpurin, lac color, and cochineal extract) showed significant antigenotoxic activities. Alizarin and purpurin suppressed the DNA damage induced by IQ, MeIQx, AFB1, NDMA, 2-AAF, DMBA, and MNU. Lac color and cochineal extract showed inhibition against IQ, MeIQx, AFB1, 2-AAF and DMBA. In these inhibitions, suppression of metabolic enzymes may be involved. Since purpurin and alizarin suppressed the activity of MNU, a direct alkylating agent, there may also be a mechanism distinct from enzyme inhibitions in these anthraquinone-mediated suppressions of DNA damage.

Protection against Trp-P-2 mutagenicity by purpurin: mechanism of in vitro antimutagenesis.

Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) is a natural pigment isolated from madder root (Rubia tinctorum) which inhibits the mutagenicity of a number of heterocyclic amines in the Ames mutagenicity test. Two effects were observed in the presence of purpurin. The rate of degradation of 3-hydroxyamino-1-methyl-5H-pyrido¿4,3-bindole ¿Trp-P-2(NHOH) at neutral pH was increased. The major product of this purpurin-dependent degradation was identified as the parent amine 3-amino-1-methyl-5H-pyrido¿4,3-bindole (Trp-P-2). Secondly, the rate of Trp-P-2 N-hydroxylation, the major route of bioactivation, by PCB-treated rat hepatic microsomes was markedly decreased. Cytochrome P450-dependent O-dealkylation of methoxy-, ethoxy- and pentoxyresorufin by these microsomes was also significantly inhibited by purpurin. The nature of this inhibition was competitive. Spectrophotometric investigations suggest no direct interaction between Trp-P-2 and purpurin. Furthermore, no evidence for Trp-P-2 binding was observed with carminic acid, a structural analog of purpurin, when it was immobilized on omega-aminohexyl agarose. Therefore, in vitro the proposed mechanism by which purpurin protects against heterocyclic amine-induced mutagenesis involves competitive inhibition of cytochrome P450-dependent bioactivation and accelerated degradation of the N-hydroxylamine to the parent amine.

Sialyl Lewis X mimetics attenuate E-selectin-mediated adhesion of leukocytes to irradiated human endothelial cells.

Ionizing radiation causes histological changes in normal tissues that resemble those resulting from the inflammatory response. Inflammation is a multistep process requiring expression of adhesion molecules on the surface of endothelial cells which results in leukocyte extravasation. E-selectin is an adhesion molecule that mediates leukocyte "rolling" on the endothelium and is required for the inflammatory response. We quantified E-selectin expression and selectin-dependent adhesion of leukocytes to human endothelial cells after X irradiation to determine whether E-selectin participates in the radiation-mediated inflammation-like response. Immunofluorescence staining of irradiated endothelial cells demonstrated expression of E-selectin on the cell surface similar to that elicited by treatment with interleukin-1 (IL-1). Radiation-mediated expression of E-selectin was dependent on dose and time and occurred at doses as low as 0.5 Gy. Furthermore, the increased adhesion of leukocytes to irradiated endothelial cells was prevented by an E-selectin-blocking antibody. Sialyl Lewis X is one of the molecules on the surface of leukocytes that adheres to E-selectin. The anti-inflammatory agents glycyrrhizin and carminic acid, which are structural analogues of sialyl Lewis X, attenuated adhesion of leukocytes to endothelial cells treated with X rays or IL-1. These data implicate a new class of anti-inflammatory agents in the prevention of adhesions of leukocytes to the irradiated vascular endothelium.

Red dye No. 2 and the red pigment carmine enhance aryl hydrocarbon hydroxylase and guanylate cyclase activities.

The food coloring amaranth (F, D and C red No. 2) and the red pigment carmine, but not erythrosine (F, D and C red No. 3), caused a twofold enhancement of two enzymes (aryl hydrocarbon hydroxylase and guanylate cyclase) that have been linked with chemical carcinogenesis. Dose response relationships revealed that carmine enhanced both enzymes to near maximal levels at concentrations as low as 1 to 10 nM while amaranth needed a concentration of 100 nM to cause a maximal enhancement. These data suggest that amaranth and carmine mimic the effects of some chemical carcinogens at the cellular level, but erythrosine does not mimic any of these effects.