- Nuclear factor (erythroid-derived 2)-like 2 (Nrf-2): Carminic acid improves Nrf-2 nuclear translocation and activation. [1]
- Nuclear factor-κB (NF-κB): Carminic acid blocks NF-κB activation by reducing the phosphorylation of IKKβ, IκBα, and NF-κB. [1]
- c-Jun N-terminal kinase (JNK): Carminic acid reduces Fru-induced JNK phosphorylation. [1]
- AMP-activated protein kinase α (AMPKα): Carminic acid improves AMPKα activation (increases p-AMPKα expression). [1]

- In mouse tubular epithelial cells (TCMK-1) and human tubule epithelial cells (HK2) stimulated by fructose (Fru, 5 mM for 24 h), Carminic acid (10 and 20 μM) significantly reduced the mRNA expression of inflammatory cytokines/chemokines including IL-1β, IL-6, IL-4, IL-18, TNF-α, TGF-β1, MCP-1, TIMP-1, MIP-1α, and CXCL1. [1]
- Carminic acid (10 and 20 μM) blocked NF-κB and JNK activation in Fru-treated TCMK-1 and HK2 cells, as evidenced by reduced phosphorylation of IKKβ, IκBα, NF-κB, and JNK, and reduced NF-κB nuclear translocation. [1]
- In Fru-stimulated TCMK-1 and HK2 cells, Carminic acid (10 and 20 μM) suppressed oxidative stress by reducing intracellular ROS production, MDA levels, and H₂O₂ contents, while rescuing SOD activity. It also reversed Fru-induced changes in antioxidant gene expression (increased HO-1, Nrf-2, SOD1, SOD2, GCLM, GCLC, NQO1; decreased Keap-1, iNOS, Gp91phox, p22phox, p47phox, XO). [1]
- Carminic acid (20 μM) improved Nrf-2 activation in Fru-treated cells by increasing nuclear Nrf-2 expression and decreasing cytoplasmic Nrf-2 and Keap-1 expression. [1]
- In TCMK-1 cells with Nrf-2 knockdown (siNrf-2), the anti-inflammatory and anti-oxidative effects of Carminic acid (20 μM) were largely abolished, evidenced by increased inflammatory factors, p-NF-κB, nuclear NF-κB, p-JNK, ROS, MDA, and decreased SOD and antioxidants. [1]
- Carminic acid (10 and 20 μM) reduced the mRNA expression of IL-1β, IL-6, IL-18, and TNF-α in TCMK-1 and HK2 cells stimulated with LPS (100 ng/mL for 24 h). [1]
- Carminic acid (10 and 20 μM) reduced the mRNA expression of α-SMA, collagen I, collagen III, and MMP-9 in TCMK-1 and HK2 cells stimulated with TGF-β (10 ng/mL for 24 h). [1]
- In a behavioral study using rat brain tissue, Carminic acid at doses of 500, 1500, and 3000 mg/kg was not detectable in hippocampus, brain, and brain stem tissues, indicating it does not cross the blood-brain barrier. However, it increased dose-dependently in peripheral tissues like liver, kidney, and blood. [3]
- In a heterologous production study, Carminic acid was successfully produced in Aspergillus nidulans engineered to express a semi-natural biosynthetic pathway including a plant type III PKS (OKS), bacterial cyclase (ZhuI), bacterial aromatase (ZhuJ), and the D. coccus C-glucosyltransferase DcUGT2. The strain expressing OKS, ZhuI, ZhuJ, and UGT2 produced Carminic acid and dcll. [4]

- In C57BL/6 mice fed with 30% fructose (Fru) in water for 16 weeks, supplementation with Carminic acid (0.5% or 1% in Fru solution, equivalent to ~0.75 and 1.5 g/kg/day) significantly ameliorated Fru-induced metabolic disorders, including reduced body weight gain, kidney weight, blood glucose, serum insulin, improved glucose intolerance and insulin resistance (OGTT and ITT), and reduced serum TG, TC, and LDL. [1]
- Carminic acid (0.5% or 1%) reduced Fru-induced renal dysfunction in mice, as evidenced by decreased serum creatinine, urinary albumin, and BUN levels. [1]
- Carminic acid (0.5% or 1%) attenuated Fru-induced pathological changes in mouse kidneys, including reduced glomerular hypertrophy, collagen accumulation (Sirius red and Masson's trichrome staining), and reduced mRNA levels of fibrotic genes (TGF-β1, α-SMA, collagen I, collagen III, MMP-9) and restored podocin expression. [1]
- Carminic acid (0.5% or 1%) inhibited Fru-induced inflammation and oxidative stress in mouse kidneys by reducing inflammatory factors, blocking NF-κB and JNK activation, reducing ROS, MDA, H₂O₂, 8-OHdG, and 4-HNE, increasing SOD activity, and improving Nrf-2 nuclear translocation. [1]
- Carminic acid (0.5% or 1%) improved AMPKα activation (increased p-AMPKα) in the kidneys of Fru-fed mice. [1]
- In rats, oral administration of Carminic acid at 1500 and 3000 mg/kg significantly increased locomotor activity (total distance and velocity) in the open field test and increased anxiety-like behavior (decreased time spent in open arm, increased time in closed arm) in the elevated plus maze test compared to control. The 500 mg/kg dose showed no statistically significant effect. [3]

- Cell Viability Assay (MTT): TCMK-1 and HK2 cells were incubated with Carminic acid at concentrations ranging from 1.25 to 100 μM for 24 hours. Cell viability was measured using an MTT assay to determine non-cytotoxic concentrations. [1]
- RNA Extraction and RT-qPCR: Total RNA was extracted from treated cells using Trizol reagent. After reverse transcription, PCR was conducted with SYBR Green to measure the mRNA expression levels of various inflammatory, oxidative stress, and fibrotic factors. Gene expression was quantified using the 2^(-ΔΔCT) method and normalized to GAPDH. [1]
- Western Blot Analysis: Treated cells were homogenized, and nuclear and cytoplasmic proteins were extracted. Protein concentrations were determined. Total protein (40-50 μg) was separated by SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies (e.g., against p-IKKβ, p-IκBα, p-NF-κB, Nrf-2, Keap-1, p-JNK, p-AMPKα). Signals were detected with an ECL system and quantified using Image Lab Software, normalized to GAPDH or Lamin B. [1]
- ROS Measurement (in vitro): Cellular ROS production was measured using DCFH-DA. After treatments, cells were incubated with 10 μM DCFH-DA for 30 minutes at 37°C. Fluorescence was observed under a fluorescence microscope, and relative ROS levels were quantified. [1]
- Biochemical Assays (in vitro): The contents of malondialdehyde (MDA), H₂O₂, and superoxide dismutase (SOD) activity in cell lysates were evaluated using commercial kits according to the manufacturers' instructions. [1]
- Gene Knockdown (siRNA): TCMK-1 cells were transfected with Nrf-2-specific siRNA (siNrf-2) or negative control using a transfection reagent for 24 hours. Transfection efficiency was measured by western blot analysis. After knockdown, cells were treated with Fru and/or Carminic acid for another 24 hours. [1]

- Fructose-Induced Kidney Injury Model: Male C57BL/6 mice (6-7 weeks old, 18-20 g) were randomly divided into 5 groups: Control (Con), Con + 1% CA (CAH), Fru (30% w/v fructose in water), Fru + 0.5% CA (Fru+CAL), and Fru + 1% CA (Fru+CAH). Carminic acid was dissolved in the 30% fructose solution. The precise daily doses were approximately 0.75 g/kg and 1.5 g/kg. Mice were treated for 16 weeks. Body weight was measured weekly. After 16 weeks, mice were sacrificed; blood was collected from the eyeball, and kidney samples were isolated. [1]
- Locomotor and Anxiety Behavior Study: 32 Wistar albino male rats (150-200 g) were divided into 4 groups: Control (1 mL distilled water by gavage), CA-500 (500 mg/kg/day CA in 1 mL water by gavage), CA-1500 (1500 mg/kg/day), and CA-3000 (3000 mg/kg/day). Carminic acid was dissolved in distilled water and administered by gavage daily. [3]
- Oral Glucose Tolerance Test (OGTT): After fasting for 8 hours, mice were orally administered glucose (2 g/kg body weight). Blood samples were collected from the tail vein at 0, 15, 30, 60, and 120 minutes post-administration, and blood glucose levels were measured. [1]
- Insulin Tolerance Test (ITT): Mice were fasted for 8 hours before intraperitoneal (i.p.) injection with insulin (1 U/kg body weight). Blood glucose levels were measured at indicated times post-injection. [1]
- Histological Analysis: Major organs (kidney, heart, liver, spleen, lung) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Renal sections were stained with H&E, PAS, Sirius Red, and Masson's trichrome to evaluate histological alterations. For immunohistochemistry, sections were incubated with primary antibodies against 8-OHdG and 4-HNE, followed by a secondary antibody, and developed with DAB. [1]
- ROS Measurement (in vivo): ROS levels in kidney samples were measured using dihydroethidium (DHE). Kidney sections were incubated with 2 μmol/L DHE dye at 37°C for 30 minutes, then analyzed and quantified using Image J software. [1]

- Carminic acid does not cross the blood-brain barrier. In rats, after oral administration of 500, 1500, and 3000 mg/kg, the compound was not detectable in hippocampus, brain, and brain stem tissues. [3]
- Carminic acid shows dose-dependent accumulation in peripheral tissues. In rats, levels in liver, kidney, and blood increased with the administered dose (500, 1500, 3000 mg/kg) (p < 0.05). [3]

- Carminic acid at concentrations ranging from 1.25 to 100 μM was non-cytotoxic to TCMK-1 and HK2 cells in vitro (MTT analysis). [1]
- In an in vivo toxicity study in mice, Carminic acid (1% CA, approx. 1.5 g/kg/day for 16 weeks) showed no significant histological alterations in heart, liver, lung, and spleen compared to control. Serum ALT, AST, and ALP levels (markers of hepatic function) were not markedly changed, indicating little toxicity at the used concentration. [1]
- The acute oral toxicity (LD50) of Carminic acid in mice is 6,250 mg/kg. [4]
- The "No-Observed-Adverse-Effect Level" (NOAEL) for carmine (a related colorant) is reported as 500 mg/kg/day (Acceptable Daily Intake, ADI) when a safety factor of 100 is considered. The European Food Safety Authority (EFSA) reports an acceptable daily intake of 2.5 mg CA/kg/day. [3][4]
- At high doses, Carminic acid (1500 and 3000 mg/kg) caused hyperactivity and anxiety-like behaviors in rats, as measured by open field and elevated plus maze tests. [3]
- Carminic acid is a known feeding deterrent to ants (Monomorium destructor). A 10⁻¹ M solution (approx. 5%) showed significant deterrence. [2]
- Carminic acid can cause an extreme allergic reaction in some people and is also associated with asthma and hyperactivity. [3]

[1]. Carminic acid supplementation protects against fructose-induced kidney injury mainly through suppressing inflammation and oxidative stress via improving Nrf-2 signaling. Aging (Albany NY). 2021 Apr 4;13(7):10326-10353.

[2]. Red cochineal dye (carminic Acid): its role in nature. Science. 1980 May 30;208(4447):1039-42.

[3]. Investigation of the dose-dependent effect of carminic acid on brain and peripheral tissues. 2023.

[4]. Heterologous production of the widely used natural food colorant carminic acid in Aspergillus nidulans. Sci Rep. 2018 Aug 27;8(1):12853.

Carmine acid appears as a dark purplish-brown mass or a bright red or dark red powder. Its color deepens at 248 °F (120 °C). It dissolves in water to form a deep red color. In acidic aqueous solutions, it is yellow to purple. (NTP, 1992)
Carmine acid is a tetrahydroxyanthraquinone with the structure 1,3,4,6-tetrahydroxy-9,10-anthraquinone, substituted with a methyl group at position 8, a carboxyl group at position 7, and a 1,5-dehydrated-D-glucol group linked to position 2 via a C-glycosidic bond. It is a natural dye isolated from various insects (e.g., Dactylopius coccus). It can be used as an animal metabolite and histological dye. It is a tetrahydroxyanthraquinone, monocarboxylic acid, and C-glycosidic compound. It is the conjugate acid of carmine (2-).
Carmine is a pigment extracted from the cochineal insect (Coccus cacti L.). It is used as a dye in food, pharmaceuticals, cosmetics, etc., and also as a microscope staining agent and biomarker.
See also: Cochineal (note moved to).

---

Mechanism of Action
The food coloring carmine acid generates free radicals through a redox cycle. These free radicals readily damage membrane lipids and degrade carbohydrate deoxyribose in the presence of trace amounts of iron salts. Damage to membrane lipids appears to primarily involve organic oxygen free radicals, such as alkoxy radicals and peroxy radicals, while damage to deoxyribose involves hydroxyl radicals formed in Fenton-type reactions. Antioxidants and iron chelators can prevent this damage.
The antitumor drug carmine acid does not bind to DNA but slowly cleaves DNA, with faster cleavage rates during in-situ reduction and even faster cleavage rates during pre-reduction of the quinone moiety.

---

- Carminic acid (CA) is a natural red food colorant (E120) produced from scale insects like Dactylopius coccus (cochineal). Current industrial production is estimated at 800 tons per year, mainly in Peru, Canary Islands, Chile, and Mexico. [4]
- In nature, Carminic acid acts as a potent feeding deterrent to ants (Monomorium destructor) and may have evolved in cochineals as a chemical weapon against predation. [2]
- The carnivorous caterpillar of a pyralid moth (Laetilia coccidivora) feeds on cochineals and is not deterred by Carminic acid. The larva regurgitates a fluid containing unaltered Carminic acid (2.2-3.3% of body weight) for its own defense against predators like ants. [2]
- A heterologous microbial production system for Carminic acid was developed in Aspergillus nidulans using a semi-natural biosynthetic pathway. The pathway includes a plant type III polyketide synthase (OKS) to form a non-reduced octaketide, bacterial cyclase (ZhuI) and aromatase (ZhuJ) to form flavokermesic acid, endogenous monooxygenases to form kermesic acid, and the Dactylopius coccus C-glucosyltransferase DcUGT2 to form Carminic acid. [4]
- Carminic acid has been used as a red colorant for at least 2800 years for textile dyeing, cosmetics, and food. [4]

C22H20O13

492.38600

492.09

1260-17-9

10255083

Brown to red solid

1.9±0.1 g/cm3

907.6±65.0 °C at 760 mmHg

136ºC

316.1±27.8 °C

0.0±0.3 mmHg at 25°C

1.792

4.8

9

13

3

35

864

5

CC1=C2C(=CC(=C1C(=O)O)O)C(=O)C3=C(C2=O)C(=C(C(=C3O)O)[C@H]4[C@@H]([C@H]([C@@H]([C@@H](CO)O4)O)O)O)O

DGQLVPJVXFOQEV-JNVSTXMASA-N

InChI=1S/C22H20O13/c1-4-8-5(2-6(24)9(4)22(33)34)13(25)10-11(15(8)27)16(28)12(18(30)17(10)29)21-20(32)19(31)14(26)7(3-23)35-21/h2,7,14,19-21,23-24,26,28-32H,3H2,1H3,(H,33,34)/t7-,14-,19+,20-,21+/m1/s1

3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-7-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]anthracene-2-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~253.86 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.22 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)