Natural product; Apoptosis; Nrf2
Luteolin targets multiple molecules involved in cancer and inflammation:
- Sirtuin 1 (Sirt1): Mediates apoptosis in non-small cell lung cancer (NSCLC) cells [1]
- Interleukin-18 (IL-18) and Interleukin-1α (IL-1α): Inhibits their expression and activity in nonalcoholic steatohepatitis (NASH) [2]
- Nuclear factor erythroid 2-related factor 2 (Nrf2): Suppresses Nrf2 nuclear translocation and DNA-binding activity in lung carcinoma cells [3]

NCI-H460 cells treated with luteolin (0-160 µM; 24 hours) exhibit concentration-dependent inhibition of cell viability[1]. Treatment with luteolin (20-80 µM; 24-hour; NCI-H460 cells) results in a cell accumulation in the S phase[1]. Apoptosis is induced by luteolin treatment (320–580 µM; 48 hours; NCI-H460 cells)[1]. Treatment with luteolin (20–80 µM; 24 hours; NCI-H460 cells) increases the concentration-dependent levels of protein expression of apoptotic regulatory proteins, such as the Bax/Bcl-2 ratio; however, only 80 µM of luteolin inhibits the expression of Bad. In the NCI-H460 cell line, luteolin also reduces Sirt1 expression in a concentration-dependent manner[1].

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Luteolin induces Sirt1-mediated apoptosis in NCI-H460 human NSCLC cells. When NCI-H460 cells are treated with Luteolin (10, 20, 40 μM) for 24–48 hours:
- Cell viability (MTT assay) decreases in a dose- and time-dependent manner: 40 μM reduces viability by ~65% at 24 hours and ~80% at 48 hours; the IC50 for 48-hour treatment is 28.5 ± 2.3 μM [1]
- Apoptosis rate (flow cytometry with Annexin V/PI staining) increases from 3.2% (control) to 18.5% (20 μM) and 35.7% (40 μM) [1]
- Western blot shows upregulated Sirt1, cleaved caspase-3, and cleaved PARP; Sirt1 knockdown (siRNA) abolishes these effects, confirming Sirt1 dependence [1]
- Colony formation assay: 40 μM Luteolin reduces colony number by ~70% vs. control [1]
- Luteolin alleviates inflammation in NASH-related in vitro models. In human hepatocytes (HepG2) treated with palmitic acid (PA, 0.2 mM, to induce steatosis) + Luteolin (5, 10, 20 μM) for 24 hours:
- ELISA detects reduced IL-18 (down by 30% at 10 μM, 55% at 20 μM) and IL-1α (down by 25% at 10 μM, 48% at 20 μM) in cell supernatants [2]
- Oil Red O staining shows decreased lipid accumulation (20 μM reduces lipid droplets by ~40%) [2]
- Luteolin inhibits Nrf2 and sensitizes A549 lung carcinoma cells to chemotherapeutics. When A549 cells are pre-treated with Luteolin (5, 10 μM) for 24 hours, then co-treated with cisplatin (0–20 μM) for 48 hours:
- The IC50 of cisplatin decreases from 12.5 ± 1.1 μM (cisplatin alone) to 6.8 ± 0.8 μM (10 μM Luteolin + cisplatin) [3]
- qPCR shows reduced Nrf2 target genes (HO-1, NQO1): 10 μM Luteolin decreases HO-1 mRNA by ~60%, NQO1 mRNA by ~55% [3]
- Immunofluorescence shows reduced Nrf2 nuclear translocation (10 μM reduces nuclear Nrf2 by ~70%) [3]

In adult male Wistar rats fed a high-carb/high-fat diet, luteolin (10–100 mg/kg; oral gavage; daily; for 12 weeks) has an antioxidant effect and can help prevent non-alcoholic steatohepatitis by inhibiting the pro-inflammatory IL-1 and Il-18 pathways[2].

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Obtained results showed ability of luteolin to reduce activity of ALT and AST and to decrease levels of bilirubin, hyaluronic acid and malondialdehyde significantly (p < 0.05). Also, luteolin showed an anti-oxidant activity as indicated by the significant (p < 0.05) increase in reduced glutathione. Finally, a significant (p < 0.05) decrease in IFN-γ, TNF-α, IL-1α and IL-18 levels was observed most notably in groups that received high doses of luteolin (50 and 100 mg/kg)[2].

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Luteolin ameliorates nonalcoholic steatohepatitis (NASH) in mice. Male C57BL/6 mice are fed a methionine-choline-deficient (MCD) diet for 8 weeks to induce NASH, and co-administered Luteolin (50, 100 mg/kg/day, oral gavage) for the last 4 weeks:
- Liver function markers: Serum ALT decreases by ~35% (50 mg/kg) and ~55% (100 mg/kg); AST decreases by ~30% (50 mg/kg) and ~50% (100 mg/kg) vs. MCD-only group [2]
- Liver histopathology: 100 mg/kg Luteolin reduces hepatic steatosis (Oil Red O staining score from 3.5 to 1.2), lobular inflammation (inflammatory cell count from 12 to 5 per high-power field), and hepatocyte ballooning [2]
- Hepatic IL-18 and IL-1α protein levels (Western blot) decrease by ~40% (50 mg/kg) and ~60% (100 mg/kg) [2]
- Body weight and food intake show no significant differences between Luteolin-treated and MCD-only groups [2]

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor regulating the expression of a battery of cytoprotective genes. Constitutive Nrf2 activation in many tumors enhances cell survival and resistance to anticancer drugs. Using a cell-based ARE-reporter assay we discovered that the flavonoid luteolin is a potent Nrf2 inhibitor. Luteolin inhibited ARE-driven gene expression redox-independently. In non-small-cell lung cancer A549 cells, which possess constitutively active Nrf2, luteolin elicited a dramatic reduction in Nrf2 at both the mRNA and the protein levels, leading to decreased Nrf2 binding to AREs, down-regulation of ARE-driven genes, and depletion of reduced glutathione. After transcription was blocked with actinomycin D, 1μM luteolin decreased the Nrf2 mRNA level by 34% in 30 min, indicating its role in accelerating Nrf2 mRNA turnover. At physiological concentrations, luteolin significantly sensitized A549 cells to the anticancer drugs oxaliplatin, bleomycin, and doxorubicin. However, knockdown of Nrf2 using siRNA essentially abolished the induced sensitivity by the flavonoid, implying the importance of inhibiting Nrf2 for its activity. Our study demonstrates that an Nrf2 inhibitor can enhance the responsiveness of cancer cells to chemotherapeutic drugs and indicates the potential application of luteolin as a natural sensitizer in chemotherapy [3].

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Sirt1 activity assay (NCI-H460 cell lysates):
1. NCI-H460 cells are treated with Luteolin (0–40 μM) for 24 hours, then lysed in ice-cold RIPA buffer containing deacetylase assay buffer (50 mM Tris-HCl pH 8.0, 4 mM MgCl₂, 1 mM DTT) [1]
2. The assay mixture (100 μL) includes cell lysate (20 μg protein), 100 μM fluorogenic Sirt1 substrate (acetyl-lysine-containing peptide conjugated to AMC), and 1 mM NAD⁺ [1]
3. Incubation is at 37°C for 60 minutes; the reaction is terminated by adding 50 μL of 2 mM nicotinamide (a Sirt1 inhibitor) [1]
4. Fluorescence intensity (excitation 360 nm, emission 460 nm) is measured; Sirt1 activity is expressed as fold change vs. vehicle control (40 μM Luteolin increases activity by ~2.2 fold) [1]
- Nrf2 DNA-binding activity assay (A549 cell nuclear extracts):
1. A549 cells are treated with Luteolin (0–10 μM) for 24 hours; nuclear extracts are prepared using a nuclear extraction kit [3]
2. The assay mixture (50 μL) contains nuclear extract (10 μg protein), biotin-labeled ARE (antioxidant response element) oligonucleotide (target of Nrf2), and binding buffer (25 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT) [3]
3. Incubation is at 25°C for 30 minutes; 50 μL of streptavidin-coated plate is added and incubated for another 30 minutes [3]
4. After washing, HRP-conjugated anti-Nrf2 antibody is added, followed by TMB substrate. Absorbance at 450 nm is measured; Nrf2 DNA-binding activity is normalized to vehicle control (10 μM Luteolin reduces activity by ~65%) [3]

Cell Viability Assay[1]
Cell Types: NCI-H460 cells
Tested Concentrations: 0 µM, 20 µM, 40 µM, 80 µM and 160 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited the viability of NCI-H460 cells in a concentration-dependent manner.

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Cell Cycle Analysis[1]
Cell Types: NCI-H460 cells
Tested Concentrations: 20 µM, 40 µM, 80 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced cell cycle arrest in the S phase.

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Apoptosis Analysis[1]
Cell Types: NCI-H460 cells
Tested Concentrations: 320 µM, 440 µM, 580 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: Apoptotic fraction was markedly increased.

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Western Blot Analysis[1]
Cell Types: NCI-H460 cells
Tested Concentrations: 20 µM, 40 µM, 80 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased the protein expression levels of apoptotic regulatory proteins and diminished the expression of Sirt1 in the NCI-H460 cell line in a concentration-dependent manner.

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NCI-H460 cell apoptosis and proliferation assay:
1. NCI-H460 cells are seeded in 96-well plates (5×10³ cells/well) for MTT assay, or 6-well plates (2×10⁵ cells/well) for apoptosis/clone formation assays, and cultured in RPMI 1640 with 10% FBS at 37°C (5% CO₂) [1]
2. Cells are treated with Luteolin (10, 20, 40 μM) or vehicle (DMSO, final <0.1%) for 24–48 hours [1]
3. MTT assay: 20 μL of 5 mg/mL MTT is added for 4 hours; DMSO dissolves formazan, and absorbance at 570 nm is measured [1]
4. Apoptosis assay: Cells are harvested, washed with PBS, stained with Annexin V-FITC and PI for 15 minutes in the dark, and analyzed by flow cytometry [1]
5. Clone formation assay: Cells are treated for 24 hours, then seeded in 6-well plates (500 cells/well) and cultured for 14 days; colonies are stained with crystal violet and counted [1]
- HepG2 cell steatosis and inflammation assay:
1. HepG2 cells are seeded in 6-well plates (1×10⁶ cells/well) and cultured in DMEM with 10% FBS [2]
2. Cells are treated with 0.2 mM palmitic acid (PA) to induce steatosis, plus Luteolin (5, 10, 20 μM) for 24 hours [2]
3. Oil Red O staining: Cells are fixed with 4% paraformaldehyde, stained with Oil Red O solution for 30 minutes, and lipid droplets are visualized under a microscope; positive area is quantified via image analysis [2]
4. IL-18/IL-1α detection: Cell supernatants are collected, and cytokine concentrations are measured by commercial ELISA kits [2]
- A549 cell Nrf2 inhibition and drug sensitization assay:
1. A549 cells are seeded in 96-well plates (3×10³ cells/well) or 6-well plates (1×10⁶ cells/well) [3]
2. Cells are pre-treated with Luteolin (5, 10 μM) for 24 hours, then co-treated with cisplatin (0–20 μM) for 48 hours [3]
3. Cell viability is measured by MTT assay to calculate cisplatin IC50 [3]
4. qPCR: Total RNA is extracted, reverse-transcribed to cDNA, and amplified with primers for Nrf2, HO-1, NQO1, and GAPDH (housekeeping gene); relative mRNA levels are calculated via 2⁻ΔΔCt [3]
5. Immunofluorescence: Cells are fixed, permeabilized, stained with anti-Nrf2 antibody (Alexa Fluor 488-conjugated secondary antibody) and DAPI (nuclear stain), and imaged by confocal microscopy [3]

Animal/Disease Models: Adult male Wistar rats (200-220 g)[2]
Doses: 10 mg/kg, 25 mg/kg, 50 mg/kg or 100 mg/ kg
Route of Administration: po (oral gavage); daily; for 12 weeks
Experimental Results: Dramatically decreased ALT and AST activity and decreased levels of bilirubin, hyaluronic acid and malondialdehyde. Shows an antioxidant activity such as a significant increase in decreased glutathione. IFN-γ, TNF-α , IL-1α and IL-18 levels diminished Dramatically.

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Adult male Wistar rats (200-220 g; n = 60) were used. Rats were fed a high carbohydrate/high fat diet (˜ 30% carbohydrate and 42% fat) daily for 12 weeks to induce NASH. Luteolin (10, 25, 50 or 100 mg/kg/day) was administered as a suspension (10% w/v in 0.9% NaCl) using an oral gavage. Histopathological changes (necrosis, inflammation and steatosis) were evaluated. Biomarkers for liver function, lipid peroxidation, extracellular matrix deposition and anti-oxidant activity were measured. Levels of IFN-γ, TNF-α and IL-1α and IL-18 were measured[2].

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Mouse MCD-induced NASH model:
1. Male C57BL/6 mice (6–8 weeks old, 20–25 g) are randomly divided into 3 groups (n=8/group): Normal control (standard chow), MCD group (MCD diet), MCD + Luteolin group (MCD diet + Luteolin 50 or 100 mg/kg/day) [2]
2. Luteolin is dissolved in 0.5% carboxymethyl cellulose (CMC) with 0.1% Tween 80 (sonicated to solubilize) and administered via oral gavage once daily. The normal and MCD groups receive vehicle (0.5% CMC + 0.1% Tween 80) [2]
3. All mice are fed their respective diets for 8 weeks; Luteolin treatment starts at week 4 and continues until week 8 [2]
4. At the end of treatment, mice are anesthetized with isoflurane. Blood is collected via cardiac puncture for serum ALT/AST measurement; livers are excised, weighed, and divided: one portion is fixed in 4% paraformaldehyde for histopathology, another is stored at -80°C for Western blot (IL-18, IL-1α) [2]

Metabolism / Metabolites
Known metabolites of luteolin include luteolin-7-glucuronide and (2S,3S,4S,5R)-6-[5-(5,7-dihydroxy-4-oxochromen-2-yl)-2-hydroxyphenoxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid.

The intraperitoneal LD50 for mice was 180 mg/kg. Pharmacology Bulletin, 16(2)(11), 1981. In vitro cytotoxicity: Luteolin (concentration up to 40 μM) showed no significant toxicity to normal human bronchial epithelial cells (HBECs) or normal hepatocytes (L02 cells): MTT assay showed that cell survival was >85% after 48 hours of treatment [1, 2]. In vivo toxicity (mouse NASH model): Compared with the MCD-only group, luteolin (50, 100 mg/kg/day orally for 4 weeks) did not cause changes in body weight, food intake, or organ weight (kidney, spleen). Serum creatinine and blood urea nitrogen (renal function indicators) were within the normal range [2]. Genotoxicity: No DNA damage was found after 24 hours of treatment of NCI-H460 cells with luteolin (concentration up to 40 μM) (comet assay) [1].

[1]. Ma L, et al. Luteolin exerts an anticancer effect on NCI-H460 human non-small cell lung cancer cells through the induction of Sirt1-mediated apoptosis. Mol Med Rep. 2015 Sep;12(3):4196-4202.
[2]. Abu-Elsaad N, et al. Protection against nonalcoholic steatohepatitis through targeting IL-18 and IL-1alpha by luteolin. Pharmacol Rep. 2019 Aug;71(4):688-694.
[3]. Xiuwen Tang, et al. Luteolin inhibits Nrf2 leading to negative regulation of the Nrf2/ARE pathway and sensitization of human lung carcinoma A549 cells to therapeutic drugs. Free Radic Biol Med. 2011 Jun 1;50(11):1599-609.

Luteolin is a tetrahydroxyflavonoid with its four hydroxyl groups located at the 3', 4', 5', and 7' positions. It is believed to play important roles in the human body, possessing antioxidant, free radical scavenging, anti-inflammatory, and immune system modulating functions, and exhibiting activity against various cancers. It also possesses multiple functions, including as an EC 2.3.1.85 (fatty acid synthase) inhibitor, antitumor agent, vascular endothelial growth factor receptor antagonist, plant metabolite, nephroprotective agent, angiogenesis inhibitor, c-Jun N-terminal kinase inhibitor, anti-inflammatory agent, apoptosis inducer, free radical scavenger, and immunomodulator. It is a 3'-hydroxyflavonoid and a tetrahydroxyflavonoid. It is the conjugate acid of luteolin-7-ol salt. Luteolin has been reported to exist in tea (Camellia sinensis), codonopsis lanceolata, and other organisms with relevant data. Luteolin is a naturally occurring flavonoid compound with potential antioxidant, anti-inflammatory, apoptosis-inducing, and chemopreventive activities. After administration, luteolin can scavenge free radicals, protect cells from damage caused by reactive oxygen species (ROS), and directly induce cell cycle arrest and apoptosis in tumor cells. This can inhibit tumor cell proliferation and metastasis. Bismuth is a mineral with the chemical formula Bi3+2O3 or Bi2O3. The symbol of the International Mineralogical Association (IMA) is Bis. 5,7,3',4'-Tetrahydroxyflavone belongs to the flavonoid class of compounds. See also: Flavonoids (subclass); Chamomile (component). Fenugreek seeds (partial)... View more...

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Mechanism of action:
- Anti-cancer (non-small cell lung cancer): Luteolin upregulates Sirt1, deacetylates p53 and activates the caspase-dependent apoptosis pathway, inhibiting cell proliferation and inducing apoptosis[1]
- Anti-nonalcoholic steatohepatitis: Luteolin inhibits the expression of IL-18/IL-1α in the liver, reducing inflammatory cell infiltration and hepatocyte damage; it can also reduce lipid accumulation by regulating fatty acid metabolism[2]
- Chemotherapy sensitization (lung cancer): Luteolin inhibits Nrf2 nuclear translocation and its downstream antioxidant genes (HO-1, NQO1), reverses Nrf2-mediated chemotherapy resistance and enhances cisplatin-induced cell death[3]
- Therapeutic potential: Luteolin is a natural flavonoid compound (found in vegetables, fruits and herbs) with the following potential applications: (1) treating non-small cell lung cancer by inducing apoptosis; (2) treating non-alcoholic steatohepatitis by anti-inflammatory and anti-steatodegeneration effects; (3) enhancing the chemosensitivity of drug-resistant lung cancer [1, 2, 3]
- Advantages compared to synthetic drugs: Luteolin has lower toxicity to normal cells/tissues and is a safer option for long-term anti-inflammatory or anticancer treatment compared to synthetic chemotherapeutic or anti-inflammatory drugs [1, 2]
- Limitations: Luteolin has poor water solubility, which may limit its oral bioavailability; formulation strategies (e.g., nanocarriers) are needed to improve its delivery [3]

C15H10O6

286.24

286.047

C, 62.94; H, 3.52; O, 33.54

491-70-3

Luteolin (Standard);491-70-3; 6113-16-2 (hydrate)

5280445

Light yellow to yellow solid powder

1.7±0.1 g/cm3

616.1±55.0 °C at 760 mmHg

~330 °C(lit.)

239.5±25.0 °C

0.0±1.8 mmHg at 25°C

1.768

Camellia sinensis, Codonopsis lanceolata, and other organisms; Escherichia coli

2.4

4

6

1

21

447

0

O1C(=C([H])C(C2=C(C([H])=C(C([H])=C12)O[H])O[H])=O)C1C([H])=C([H])C(=C(C=1[H])O[H])O[H]

InChI=1S/C15H10O6/c16-8-4-11(19)15-12(20)6-13(21-14(15)5-8)7-1-2-9(17)10(18)3-7/h1-6,16-19H

InChI=1S/C15H10O6/c16-8-4-11(19)15-12(20)6-13(21-14(15)5-8)7-1-2-9(17)10(18)3-7/h1-6,16-19H

2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-benzopyroneInChi Key:IQPNAANSBPBGFQ-UHFFFAOYSA-N

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: 20 mg/mL (69.87 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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