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.

Carminic acid supplementation protects against fructose-induced kidney injury mainly through suppressing inflammation and oxidative stress via improving Nrf-2 signaling.

Excessive fructose (Fru) intake has become an increased risk for chronic kidney disease progression. Despite extensive researches that have been performed to develop effective treatments against Fru-induced renal injury, the outcome has achieved limited success. In this study, we attempted to explore whether carminic acid (CA) could influence the progression of Fru-induced kidney injury, and the underlying molecular mechanism. At first, our in vitro results showed that CA significantly reduced inflammation in mouse tubular epithelial cells and human tubule epithelial cells stimulated by Fru. The anti-inflammatory effects of CA were associated with the blockage of nuclear factor-κB (NF-κB) signaling. In addition, Fru-exposed cells showed higher oxidative stress, which was effectively restrained by CA treatment through improving nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) nuclear translocation. Importantly, we found that Fru-induced inflammation and oxidative stress were accelerated in cells with Nrf-2 knockdown. What's more, in Fru-stimulated cells, CA-alleviated inflammatory response and reactive oxygen species (ROS) production were evidently abolished by Nrf-2 knockdown. The in vivo analysis demonstrated that Fru led to metabolic disorder, excessive albuminuria and histologic changes in renal tissues, which were effectively reversed by CA supplementation. We confirmed that CA significantly reduced inflammation and oxidative stress in the kidneys of mice through regulating NF-κB and Nrf-2 signaling pathways, eventually alleviating the progression of chronic kidney injury. Taken together, these results identified CA as a potential therapeutic strategy for metabolic stress-induced renal injury through restraining inflammation and oxidative stress via the improvement of Nrf-2 signaling.

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.

Laccase induction by synthetic dyes in Pycnoporus sanguineus and their possible use for sugar cane bagasse delignification.

The use of synthetic dyes for laccase induction in vivo has been scarcely explored. We characterized the effect of adding different synthetic dyes to liquid cultures of Pycnoporus sanguineus on laccase production. We found that carminic acid (CA) can induce 722 % and alizarin yellow 317 % more laccase than control does, and they promoted better fungal biomass development in liquid cultures. Aniline blue and crystal violet did not show such positive effect. CA and alizarin yellow were degraded up to 95 % during P. sanguineus culturing (12 days). With this basis, CA was selected as the best inducer and used to evaluate the induction of laccase on solid-state fermentation (SSF), using sugarcane bagasse (SCB) as substrate, in an attempt to reach selective delignification. We found that laccase induction occurred in SSF, and a slight inhibition of cellulase production was observed when CA was added to the substrate; also, a transformation of SCB under SSF was followed by the 13C cross polarization magic angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). Results showed that P. sanguineus can selectively delignify SCB, decreasing aromatic C compounds by 32.67 % in 16 days; O-alkyl C region (polysaccharides) was degraded less than 2 %; delignification values were not correlated with laccase activities. Cellulose-crystallinity index was increased by 27.24 % in absence of CA and 15.94 % when 0.01 mM of CA was added to SCB; this dye also inhibits the production of fungal biomass in SSF (measured as alkyl C gain). We conclude that CA is a good inducer of laccase in liquid media, and that P. sanguineus is a fungus with high potential for biomass delignification.

Properties of reformulated hot dog sausage without added nitrites during chilled storage.

The aim of this study was to assess the effect of a complete nitrite replacement strategy using celery, carmine, sodium lactate and orange dietary fibre combined with vitamins C and E, on the quality characteristics (technological, sensorial and safety properties) of hot dog sausages (five samples) during chilled storage (2 ± 1℃ 60 days). Nitrite replacers (combined with vitamins C and E) presented antioxidant activity, reducing lipid oxidation in reformulated samples. At the end of storage redness (a*) was similar in the control sample (with added nitrite) and in the sample without added nitrite. Sensory evaluation detected no significant difference between samples with and without added nitrite. All the reformulated samples were judged acceptable by the panellists. At the end of storage, the control sample contained more than four times as much residual nitrite as the reformulated samples. Growth of presumptive Clostridium perfringens was not observed in any of the samples. Samples without added nitrite had longer shelf-lives than control sausage. Samples containing 0.1% vitamin C registered the lowest microbiological levels. This strategy could be a good alternative to reduce and/or eliminate added nitrite in hot dog sausages.

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.

Improved human tenocyte proliferation and differentiation in vitro by optimized silk degumming.

Tendon disorders are common clinical conditions. Tendon tissue engineering provides a new approach for tendon repair by integrating engineered substitutes with their native counterparts. Silk is considered to be a promising candidate for tendon engineering because of its biological and mechanical properties. However, a major concern with using silk for biomedical applications is the immune responses generated by sericin, a glue-like protein that coats the silk fibres. This study improves the existing protocols for silk 'degumming' which removes sericin and enables preparation of silk that is suitable for tendon regeneration. Bombyx mori silks were treated by sequential treatments with different proteases. The efficiency of degumming was determined by measuring weight loss, picric acid and carmine staining and scanning electron microscopy. To evaluate the cellular responses after degumming, the growth and differentiation of human tenocytes on silks were examined. The results showed that sequential protease treatment effectively degummed raw silks. The sequentially degummed silks showed enhanced tenocyte proliferation and upregulated mRNA levels of tendon markers. Thick cell multilayers formed on the treated silks, with cells and collagen fibres penetrating into the spaces in individual silk filaments, resulting in a structure resembling human tendon.

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.

Subcellular localization and antiviral activity of carminic acid/poly r(A-U) combinations.

Carminic acid (CAR) enhances the antiviral activity of poly r(A-U) twelve-fold without increasing interferon induction, inactivating the vesicular stomatitis virus or inducing host cell cytotoxicity. Phase contrast photomicrographs of human foreskin fibroblasts (HSF) incubated with CAR alone, poly r(A-U) alone or with a CAR/poly r(A-U) combination illustrate that the CAR/poly r(A-U) combinations display altered subcellular distribution with the CAR being localized in the nucleoli and chromatin. Phase contrast and fluorescence photomicrographs of adriamycin (ADR)-treated and ADR/poly r(A-U)-treated HSF cells corroborate these findings. These results suggest that modulation of one or more nucleolar processes may be responsible for the enhanced antiviral activity.

Polyribonucleotide-anthraquinone interactions: in vitro antiviral activity studies.

Twelve anthraquinones (AQ) were evaluated for their ability to potentiate the antiviral activity of poly r(A-U) using a human foreskin fibroblast-vesicular stomatitis virus bioassay in which the AQ was combined with 0.2 mM poly r(A-U) to produce an AQ/ribonucleotide ratio of 1/4. Poly r(A-U) and the AQ alone were not effective antiviral agents. Five of the twelve AQs tested, mitoxantrone, adriamycin, ametantrone, carminic acid and daunomycin, enhanced the antiviral activity of poly r(A-U) 9- to 13-fold. The interferon-inducing activity of the five active AQ/poly r(A-U) combinations was equal to the sum of the interferon-inducing activities of their constituents. These five AQs appear to potentiate the antiviral activity of poly r(A-U) without superinduction of interferon.

Enhancement of the antiviral and interferon-inducing activities of poly r(A-U) by carminic acid.

Experiments have been designed to systematically examine the effects of carminic acid (CAR) on the antiviral/interferon-inducing activity of poly r(A-U), using the human foreskin fibroblast-vesicular stomatitis virus bioassay system. Modulation of the antiviral/interferon-inducing activity of poly r(A-U) by carminic acid was examined at fixed poly r(A-U) concentrations of 0.05 mM or 0.2 mM while varying the carminic acid concentrations to produce variable CAR/ribonucleotide ratios ranging from 1:16 to 2:1. Carminic acid and poly r(A-U) were tested individually at the concentrations employed in the CAR/poly r(A-U) combinations. Neither the carminic acid alone nor poly r(A-U) alone were effective antiviral agents/interferon inducers. The antiviral/interferon-inducing activity of poly r(A-U) was potentiated twelve-fold at CAR/ribonucleotide ratios in the region of 1/6 to 1/4. These results suggest a synergism between the poly r(A-U) and the carminic acid at the concentrations employed in this study.

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.

Carminic acid, a non-competitive inhibitor of kidney UDP-glucose:galactosylhydroxylysine-collagen glucosyltransferase.

1. UDP-glucose:galactosylhydroxylsine-collagen glucosyltransferase was purified 12-fold from rat kidney. 2. An assay using calf-skin gelatin as substrate showed time- and enzyme-dependent incorporation; KmS for UDP-glucose and gelatin were 16-7 microM and 4.5 mg/ml, respectively. 3. Column chromatography of the alkaline hydrolysate of reaction product on Dowex 50W-4X(H+) showed that 84% of the radioactivity was in the glycosylgalactosylhydroxylsine peak. 4. Carminic acid inhibited collagen glycosyltransferase; a dose-dependent study showed a two-stage inhibition and kinetic analysis by double-reciprocal plots at varying UDP-glucose concentrations revealed a non-competitive mode of inhibition.

Identification of G6PDH-active sinusoidal cells as Kupffer cells in the rat liver.

The aim of this study was to identify the G6PDH-active sinusoidal cells in the rat liver described by Rieder et al. (1978). Because of their number and distribution in the liver parenchyma, endothelial cells and pit cells could be excluded. Fat-storing cells were specifically marked by vital staining with vitamin A and identified by fluorescence microscopy. Kupffer cells could be detected after vital staining with carmine. Both staining methods allowed a subsequent incubation for the demonstration of G6PDH activity in the same unfixed cryostat section. Whereas more than 80% of the fluorescent particles were found outside the enzyme-positive cells, all G6PDH-active cells contained carmine particles. After counting the G6PDH-active cells, an estimation of 0.217 x 10(8) cells/g liver tissue was obtained. The results indicate that high G6PDH activity is common to all Kupffer cells, and is therefore a highly specific marker enzyme for this class of sinusoidal liver cells.

Effects of carmine and carminic acid on embryonic tissue cell cultures.

The biological reaction to carmine and carminic acid at cellular level on 'in vitro' cultures was tested and significant variables were controlled. Results suggested that proliferation and metabolism of these cultures were not affected by the 2 stains.