Ethyl Caffeate primarily targets the transcription factor NF-κB by preventing its binding to DNA. At the molecular level, its effects are mediated through the inhibition of NF-κB-DNA complex formation. The compound does not affect upstream signaling components including IκBα phosphorylation/degradation, NF-κB (p65) translocation to the nucleus, or the activation of mitogen-activated protein kinases (SAPK/JNK, p38, p42/44). The IC₅₀ for inhibiting LPS-induced NO production in RAW 264.7 macrophages is 5.5 μg/mL. [1]
NF‑κB (by impairing NF‑κB‑DNA complex formation) [1]

Ethyl Caffeate significantly inhibits LPS-induced NO production in RAW 264.7 macrophages in a dose-dependent manner, with an IC₅₀ of 5.5 μg/mL. At 2 μg/mL, NO production was inhibited by 35% compared to LPS-only treated cells. [1]
The compound inhibits iNOS mRNA expression; at 1 μg/mL, a 56% reduction in iNOS mRNA was observed, and at 2 μg/mL, iNOS mRNA levels were similar to vehicle control. At the protein level, 0.5 μg/mL ethyl caffeate reduced iNOS protein to approximately 30% of that in LPS-only treated cells. [1]
Ethyl Caffeate suppresses LPS-induced COX-2 protein expression; at 5 μg/mL, COX-2 expression was reduced to 45% of LPS-only control. It also markedly suppresses PGE₂ production; at 1 μg/mL, PGE₂ production was significantly inhibited, and at 2-5 μg/mL, total inhibition of PGE₂ was observed. [1]
In TPA-treated MCF-7 cells, ethyl caffeate (10-100 μg/mL) downregulates cox-2 transcriptional activity in a dose-dependent manner, reducing full-length cox-2 promoter activity by 72% (at 10 μg/mL), 57% (at 50 μg/mL), and 48% (at 100 μg/mL). [1]
Ethyl Caffeate does not affect LPS-induced MAPK phosphorylation (SAPK/JNK, p38, p42/44) in macrophages at concentrations up to 10 μg/mL. It also does not affect IκBα phosphorylation and degradation, nor NF-κB p65 translocation to the nucleus. [1]
Using an in vitro EMSA binding assay, ethyl caffeate (10-20 μg/mL) dose-dependently inhibits NF-κB binding to DNA, with complete inhibition at 20 μg/mL. This inhibition is completely reversed by 50 μM DTT. [1]
Structure-activity relationship studies show that both the catechol moiety and the α,β-unsaturated ester group are essential for NF-κB DNA binding inhibition. Ethyl caffeate (containing both) is most effective; ethyl 3,4-dihydroxyhydrocinnamate (containing catechol only) shows weaker inhibition; ethyl cinnamate (containing α,β-unsaturated ester only) shows no inhibition. [1]

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Ethyl caffeine has an IC50 of 5.5 μg/ml and strongly suppresses the generation of nitric oxide (NO) caused by lipopolysaccharide (LPS)[1].

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- Ethyl caffeine markedly suppressed LPS‑induced NO production in RAW 264.7 macrophages with an IC₅₀ of 5.5 μg ml⁻¹; at 2 μg ml⁻¹, NO production was inhibited by 35% compared to LPS‑only control. [1]

- Ethyl caffeine inhibited iNOS mRNA expression: at 1 μg ml⁻¹, iNOS mRNA was reduced by approximately 56% (by densitometry) in LPS‑treated macrophages; at 2 μg ml⁻¹, iNOS mRNA level was similar to vehicle control. [1]

- Western blot analysis showed that Ethyl caffeine at 0.5 μg ml⁻¹ reduced iNOS protein level to about 30% of that in LPS‑only treated cells. [1]

- In transient transfection assays using human COX‑2 promoter constructs in MCF‑7 cells, Ethyl caffeine (10, 50, 100 μg ml⁻¹) decreased TPA‑induced full‑length (−1334/−1) COX‑2 promoter activity by 72%, 57%, and 48%, respectively. No effect was observed on TPA‑induced IL‑10 promoter activity, indicating specificity. Deletion analysis showed that the −646/−1 promoter region (containing NF‑κB, NF‑IL6 and CRE sites) was the minimal responsive element, and Ethyl caffeine (20 μg ml⁻¹) inhibited luciferase activity by 67% for the −1334/−1 construct and 80% for the −646/−1 construct. [1]

- Ethyl caffeine at 5 μg ml⁻¹ inhibited LPS‑induced COX‑2 protein expression in RAW 264.7 cells by 45% (densitometry). [1]

- Ethyl caffeine at 1 μg ml⁻¹ markedly suppressed PGE₂ production in LPS‑stimulated RAW 264.7 cells; at 2‑5 μg ml⁻¹, total inhibition of PGE₂ was observed. [1]

- Ethyl caffeine (1‑10 μg ml⁻¹) did not affect LPS‑induced phosphorylation of MAPKs (SAPK/JNK, p38, p42/44) in RAW 264.7 cells (by Western blot with phospho‑specific antibodies). [1]

- Ethyl caffeine (1‑20 μg ml⁻¹) did not affect IκBα phosphorylation, IκBα degradation, or nuclear translocation of NF‑κB p65 in LPS‑stimulated RAW 264.7 cells (by Western blot). [1]

- Ethyl caffeine inhibited NF‑κB binding to DNA in a dose‑dependent manner in an in vitro EMSA: at 10 μg ml⁻¹ significant inhibition, at 20 μg ml⁻¹ complete inhibition; this effect was completely reversed by 50 μM DTT. [1]

- In structure‑activity relationship studies using EMSA, Ethyl caffeine (50 μM) completely inhibited NF‑κB‑DNA binding; ethyl 3,4‑dihydroxyhydrocinnamate (100 μM) also completely inhibited, catechol (400 μM) totally blocked binding, while ethyl cinnamate (up to 400 μM) showed no inhibition. [1]

- Ethyl caffeine showed little or no cytotoxicity to RAW 264.7 cells at concentrations ≤10 μg ml⁻¹ (by MTT assay). At 5 μg ml⁻¹, >50% cells were viable; at 20 μg ml⁻¹, viability decreased but data not specified. [1]

In a mouse skin immunohistochemical study, topical application of Ethyl Caffeate significantly inhibits TPA-induced COX-2 expression in the epidermal layer in a dose-dependent manner. At 1 mg/200 μL/site (24 mM), ethyl caffeate showed comparable inhibition to 1 mg/200 μL/site celecoxib (13 mM). At 2 mg/200 μL/site (48 mM), ethyl caffeate was more effective than 10 mg/200 μL/site celecoxib (131 mM) in inhibiting TPA-induced COX-2 expression. [1]

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- In female ICR mouse skin, topical application of Ethyl caffeine (1 mg/200 μl/site, ~24 mM) significantly inhibited TPA (10 nmol)‑induced COX‑2 expression as shown by immunohistochemistry; the inhibitory effect was comparable to that of celecoxib at the same dose (1 mg/200 μl/site, ~13 mM). A higher dose of Ethyl caffeine (2 mg/200 μl/site, ~48 mM) was more effective than celecoxib at 10 mg/200 μl/site (~131 mM) in suppressing TPA‑induced COX‑2 expression. [1]

An electrophoretic mobility shift assay (EMSA) was performed to assess NF-κB binding to DNA. Nuclear extracts (4 μg) from LPS-stimulated RAW 264.7 cells were incubated with biotin end-labeled, 22-mer double-stranded NF-κB oligonucleotide in binding buffer containing 10% glycerol, 100 mM KCl, 1.5 mM MgCl₂, and 0.3 mM EDTA for 20 minutes at room temperature. For in vitro binding assays, nuclear extracts from LPS-stimulated cells were treated with different concentrations of test compounds (including Ethyl Caffeate at concentrations such as 10 and 20 μg/mL) at 37°C for 30 minutes before EMSA analysis. DNA-protein complexes were separated on a native 5-10% gradient polyacrylamide gel. Specificity was confirmed by supershift with anti-p65 antibody and competition with unlabeled NF-κB oligonucleotide. [1]

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- Electrophoretic mobility shift assay (EMSA) for NF‑κB‑DNA binding: Nuclear extracts were prepared from LPS‑stimulated RAW 264.7 cells (1 μg ml⁻¹ LPS for 30 min). The nuclear extracts (4 μg) were incubated with various concentrations of Ethyl caffeine at 37°C for 30 min. Then, a biotin‑end‑labeled double‑stranded 22‑mer NF‑κB oligonucleotide (5′‑ATGTGAGGGGACTTTCCCAGGC‑3′) was added and incubated for 20 min at room temperature in binding buffer containing 10% glycerol, 100 mM KCl, 1.5 mM MgCl₂ and 0.3 mM EDTA. The DNA‑protein complexes were separated from free oligonucleotides on a native 5‑10% gradient polyacrylamide gel. Specificity was confirmed by super‑shift with anti‑p65 antibody and competition with unlabeled NF‑κB oligonucleotide. DTT (50 μM) was used to test reversibility. [1]

RAW 264.7 murine macrophages were cultured in DMEM with 10% FBS. For NO production assays, cells (2×10⁵ cells/well) were pretreated with Ethyl Caffeate (0.1-20 μg/mL) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Nitrite accumulation was measured by Griess reaction (100 μL supernatant mixed with 100 μL Griess reagent, absorbance at 540 nm). Cell viability was determined by MTT assay to exclude cytotoxicity. [1]
For RT-PCR, cells (3×10⁶/well) were treated with ethyl caffeate (0.5-5 μg/mL) for 1 hour, then LPS (1 μg/mL) for 6 hours. Total RNA was isolated using TRIZOL, and iNOS mRNA was amplified with specific primers. GA3PDH was used as internal control. [1]
For Western blot, cells were treated with ethyl caffeate for 1 hour, then LPS (1 μg/mL) for 18 hours (iNOS, COX-2) or 30 minutes (MAPK) or 11 minutes (IκBα). Total, cytosolic, or nuclear proteins (20 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and detected with specific antibodies against iNOS, COX-2, phospho-MAPKs, IκBα, p65 NF-κB, PARP, and α-tubulin. [1]
For COX-2 promoter assay, MCF-7 cells (1.2×10⁵/well) were co-transfected with pCOX-2-Luc and pRL-TK-Luc using LipofectAMINE. After recovery, cells were treated with TPA (50 ng/mL) alone or with ethyl caffeate (10-100 μg/mL) for 6 hours. Firefly and Renilla luciferase activities were measured using dual luciferase assay. [1]
For PGE₂ measurement, RAW 264.7 cells were pretreated with 500 μM aspirin for 3 hours (to inactivate COX-1), washed, then treated with ethyl caffeate (0.1-20 μg/mL) for 1 hour, followed by LPS (1 μg/mL) for 16 hours. PGE₂ in culture media was measured by ACE competitive enzyme immunoassay. [1]

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- NO production measurement: RAW 264.7 cells (1×10⁴ cells/well in 96‑well plates) were pretreated with Ethyl caffeine (0.1‑20 μg ml⁻¹) for 1 h, then stimulated with LPS (1 μg ml⁻¹) for 24 h. Nitrite accumulation in culture supernatants was determined by Griess reaction: 100 μl supernatant mixed with 100 μl Griess reagent (1:1 mixture of 0.1% N‑(1‑naphthyl)ethylenediamine in H₂O and 1% sulfanilamide in 5% phosphoric acid), absorbance at 540 nm measured. [1]

- Cell viability assay (MTT): RAW 264.7 cells (1×10⁴ cells/well) were treated with Ethyl caffeine (0.1‑20 μg ml⁻¹) for 24 h, then MTT was added and formazan absorbance measured at 570 nm. Viability was calculated as (OD₅₇₀ treated / OD₅₇₀ vehicle control) × 100. [1]

- RT‑PCR analysis: RAW 264.7 cells (3×10⁶ cells/well in 6‑well plates) were pretreated with Ethyl caffeine (0.5‑5 μg ml⁻¹) for 1 h, then stimulated with LPS (1 μg ml⁻¹) for 6 h. Total RNA was extracted using TRIZOL reagent. First‑strand cDNA was synthesized from 2 μg total RNA using reverse transcriptase. iNOS primers: sense 5′‑CAGAAGCAGAATGTGACCATC‑3′, antisense 5′‑CTTCTGGTCGATGTCATGAGC‑3′; GA3PDH as internal control. PCR conditions: 94°C for 2 min (1 cycle); 94°C for 1 min, 55°C for 30 s, 72°C for 1 min for 30 cycles. Products resolved on 1% agarose gel, visualized with ethidium bromide. [1]

- Western blot analysis: Cells were lysed and total cellular, cytosolic or nuclear proteins (20 μg) separated on 5‑20% gradient SDS‑PAGE, transferred to PVDF membrane. Membranes were blocked with 3% skim milk, incubated with primary antibodies (anti‑iNOS, anti‑IκBα, anti‑phospho‑IκBα, anti‑NF‑κB p65, anti‑phospho‑MAPKs, anti‑PARP, anti‑α‑tubulin, anti‑GA3PDH) overnight at 4°C, then with HRP‑conjugated secondary antibodies, and visualized by enhanced chemiluminescence. [1]

- Transient transfection and luciferase assay: MCF‑7 cells (1.2×10⁵ cells/well in 24‑well plates) were co‑transfected with COX‑2 promoter‑luciferase constructs (full‑length or deletions) and pRL‑TK‑Luc using lipofectamine. After 4 h, medium replaced, cells recovered for 16 h. Then treated with vehicle (0.1% DMSO), TPA (50 ng ml⁻¹) alone, or TPA plus Ethyl caffeine (10‑100 μg ml⁻¹) for 6 h. Cell lysates were assayed for firefly and Renilla luciferase activities; promoter activity expressed as ratio of firefly/Renilla. [1]

- PGE₂ production assay: RAW 264.7 cells (1×10⁴ cells/well in 96‑well plates) were pretreated with 500 μM aspirin for 3 h to inactivate COX‑1, washed, then treated with Ethyl caffeine (0.1‑20 μg ml⁻¹) for 1 h, followed by LPS (1 μg ml⁻¹) for 16 h. Culture media were collected and PGE₂ measured by competitive enzyme immunoassay. [1]

Female ICR mice were used. Dorsal skin was shaven. Mice were treated topically with Ethyl Caffeate (1 mg/200 μL/site or 2 mg/200 μL/site) or celecoxib (1 mg/200 μL/site or 10 mg/200 μL/site) for 30 minutes, then treated with TPA (10 nmol/200 μL/site) for 4 hours. After treatment, mice were killed by cervical dislocation. Skin tissues were formalin-fixed, paraffin-embedded, sectioned (4 μm), and deparaffinized. For antigen retrieval, sections were heated in 10 mM citrate buffer (pH 6.0) for 10 minutes. Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 15 minutes. Sections were incubated with polyclonal COX-2 antibody (1:500 dilution) for 1-2 hours at room temperature, then developed using HPR EnVision system with DAB staining, and counterstained with Mayer's hematoxylin. [1]

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- Immunohistochemical study of COX‑2 expression in mouse skin: Female ICR mice were topically treated on shaved backs with acetone (vehicle control, 200 μl/site) or TPA (10 nmol in 200 μl/site) for 4 h. For compound treatment, mice were first treated with Ethyl caffeine (1 mg or 2 mg in 200 μl acetone/site) or celecoxib (1 mg or 10 mg in 200 μl acetone/site) for 30 min, then further treated with TPA for 4 h, then euthanized by cervical dislocation. Skin sections (4 μm) were deparaffinized, rehydrated, subjected to antigen retrieval in 10 mM citrate buffer (pH 6.0) by boiling for 10 min, then treated with 3% H₂O₂ in methanol to block endogenous peroxidase. Sections were blocked with 2% normal goat serum, incubated with polyclonal COX‑2 antibody (1:500 dilution) for 1‑2 h at room temperature, then developed using HRP EnVision system, stained with diaminobenzidine, and counterstained with Mayer‘s hematoxylin. [1]

Ethyl Caffeate was found to have little or no cytotoxicity to RAW 264.7 macrophages at concentrations of 10 μg/mL or below, as determined by MTT assay. At 5 μg/mL, >50% of cells remained viable. [1]
In MCF-7 cells, no cytotoxic effects were observed with ethyl caffeate treatment at concentrations of 10-100 μg/mL over 6 hours as determined by MTT assay. [1]

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[1]. Ethyl caffeate suppresses NF-kappaB activation and its downstream inflammatory mediators, iNOS, COX-2, and PGE2 in vitro or in mouse skin. Br J Pharmacol. 2005 Oct;146(3):352-63.

Ethyl trans-caffeic acid is an ethyl ester formed by the condensation of the carboxyl group of trans-caffeic acid with ethanol. It possesses anti-inflammatory and antitumor activities. It is an alkyl caffeic acid ester and ethyl ester, functionally related to trans-caffeic acid. Ethyl caffeic acid has been reported in perilla, Himalayan sasanqua, and several other organisms with relevant data.

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Ethyl Caffeate was isolated from the ethyl acetate fraction of Bidens pilosa using bioactivity-guided fractionation. Its structure was elucidated by IR, NMR, and EI-MS. Molecular weight: 208 [M]⁺. Melting point: 147-149°C. [1]
The proposed mechanism of action is an "oxidation-inhibition" mechanism: the catechol moiety is oxidized to α-benzoquinone, which then reacts with cysteine sulfhydryl groups of the p65 subunit of NF-κB via Michael-type addition, preventing DNA binding. The α,β-unsaturated ester group provides additional reaction centers for covalent interaction with p65. [1]
The compound does not affect IL-10 promoter activity, suggesting specificity for COX-2 regulation. [1]

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- Ethyl caffeine suppressed iNOS and COX‑2 expression partly through inhibition of NF‑κB‑DNA complex formation, without affecting IκB degradation or MAPK phosphorylation. The catechol moiety and α,β‑unsaturated ester group are essential structural features for preventing NF‑κB‑DNA binding. An “oxidation‑inhibition” mechanism is proposed: oxidation of the catechol moiety to α‑benzoquinone may allow Michael‑type addition to cysteine residues of the p65 subunit, thereby blocking DNA binding. [1]

- The anti‑inflammatory properties of Ethyl caffeine may contribute to antitumor promoting activity, as demonstrated by inhibition of TPA‑induced COX‑2 expression in mouse skin. [1]

- Ethyl caffeine is isolated from Bidens pilosa (whole plant) via bioactivity‑guided fractionation: ethanol extraction, ethyl acetate partition, silica gel and RP‑18 column chromatography. [1]

C11H12O4

208.2106

208.073

102-37-4

Ethyl trans-caffeate;66648-50-8

5317238

Light yellow to yellow solid powder

1.3±0.1 g/cm3

377.0±32.0 °C at 760 mmHg

148.4±18.6 °C

0.0±0.9 mmHg at 25°C

1.612

1.72

2

4

4

15

237

0

CCOC(=O)/C=C/C1=CC(=C(C=C1)O)O

WDKYDMULARNCIS-GQCTYLIASA-N

InChI=1S/C11H12O4/c1-2-15-11(14)6-4-8-3-5-9(12)10(13)7-8/h3-7,12-13H,2H2,1H3/b6-4+

ethyl (E)-3-(3,4-dihydroxyphenyl)prop-2-enoate

Ethyl caffeate; 102-37-4; ethyl trans-caffeate; Ethyl 3,4-dihydroxycinnamate; Caffeic acid ethyl ester;

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 : ~250 mg/mL (~1200.71 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (9.99 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (9.99 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 3: ≥ 2.08 mg/mL (9.99 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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