Activity depended on the assay system: 1 µM caffeic acid did not significantly reduce leukotriene-pathway products in stimulated human polymorphonuclear leukocytes, with or without exogenous arachidonic acid. [2]
In IL-1β-stimulated human CCD-18Co colon myofibroblasts, caffeic acid reduced PGE2 release and COX-2 expression at 10–50 µM after 24 h. IL-8 reduction was significant only at 50 µM; MCP-1 and IL-6 were unaffected. PGE2 reduction at 1 µM was not significant. [3]
Comparative inhibition in cell-derived enzyme preparations
Biochemical assay · Guinea-pig polymorphonuclear-leukocyte supernatant for 5-LO and PG synthase; human platelet supernatant for 12-LO. [1]
Caffeic acid inhibited 5-LO at a lower concentration than 12-LO or PG synthase in this assay panel. These are cell-derived enzyme assays, not intact-cell or animal-treatment experiments.
Literature-Reported Treatment Conditions
- Exposure duration
- 5-LO: 20 min; 12-LO and PG synthase: 30 min, all at 37 °C.
- Method
- 3.2 µM [1-14C]arachidonic acid substrate; inhibition relative to compound-free control.
- Vehicle
- Ethanol used to add inhibitors and removed before assay; ethanol substituted for compound solution in controls. 5-LO and PG synthase reaction mixtures contained 0.95% propylene glycol.
- Reported limits
- Table I does not report the parent concentration series, confidence intervals or number of independent preparations; each enzyme assay is described as done in duplicate.
Methods and source locations
Compound reported in the paper: Caffeic acid; Compound label in the paper: Parent caffeic acid
Parent-compound findings under the reported assay conditions; no Adooq lot validation, salt conversion or clinical inference.
p.1039 Materials; p.1041 Enzyme Preparation, Enzyme Assays, Addition of Inhibitors, Table I and associated Results
5-LO products (5-HETE and LTB4): Radiolabeled arachidonic acid conversion; TLC separation and scintillation counting. — p.1041, Table I caffeic acid row; Enzyme Assays
12-LO product (12-HETE): Radiolabeled arachidonic acid conversion; TLC/scintillation. — p.1041, Table I caffeic acid row; Enzyme Preparation and Enzyme Assays
PG synthase products (PGE2 and TXB2): Radiolabeled arachidonic acid conversion; TLC/scintillation. — p.1041, Table I caffeic acid row; Enzyme Assays
Limited leukotriene-pathway response in human leukocytes
Cell-based study · Isolated polymorphonuclear leukocytes from healthy human donors, stimulated with thapsigargin, with or without exogenous arachidonic acid. [2]
At 1 µM, parent caffeic acid did not significantly reduce 5-LO product formation in either stimulus condition shown in Figure 3A/B. Active CAPE and zileuton comparators demonstrate assay responsiveness.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 µM caffeic acid.
- Exposure duration
- 5 min preincubation followed by 15 min stimulation at 37 °C.
- Method
- 1 µM thapsigargin, in the presence or absence of 10 µM arachidonic acid.
- Vehicle
- Diluent control: 0.5% DMSO.
- Co-treatment
- Assay stimulation with thapsigargin ± arachidonic acid; CAPE and zileuton were separate comparator treatments.
- Reported limits
- No exact numerical caffeic-acid bar values or parent-specific n per condition given. Parent supplier/purity not specified in the acquired methods. No parent IC50 derived.
Methods and source locations
Compound reported in the paper: Caffeic acid; Compound label in the paper: Parent caffeic acid
Parent-compound findings under the reported assay conditions; no Adooq lot validation, salt conversion or clinical inference.
Figure 2 compound 5; Methods: PMNL isolation, Stimulation of PMNL for 5-LO products, Statistical analysis; Figure 3A/B and caption
Total 5-LO products: RP-HPLC; sum of LTB4, trans isomers, 20-COOH-/20-OH-LTB4 and 5-HETE. Figure caption: means ± SEM, 3–5 independent experiments, each in duplicate. — Results: The biosynthesis of 5-LO products in stimulated human PMNL; Figure 3A/B and caption
Inflammatory mediator responses in human colon myofibroblasts
Cell-based study · Human CCD-18Co colon myofibroblast-like cells (ATCC CRL-1459), IL-1β stimulation. [3]
Caffeic acid co-treatment reduced PGE2 release and COX-2 expression at 10 and 50 µM. IL-8 reduction reached significance only at 50 µM; MCP-1 and IL-6 were unaffected. The 1 µM PGE2 response was not significant.
Literature-Reported Treatment Conditions
- Treatment concentration
- PGE2: 1, 10 and 50 µM; COX-2 and cytokines: 10 and 50 µM; viability: 50 µM.
- Exposure duration
- 24 h co-treatment; PGE2 protocol includes 12 h serum deprivation before treatment.
- Method
- Co-treatment with 1 ng/mL IL-1β; vehicle-only controls with and without IL-1β. COX-2 examined after collection of medium for PGE2.
- Vehicle
- 0.5% (v/v) DMSO final concentration.
- Co-treatment
- IL-1β is the inflammatory stimulus; no second therapeutic compound.
- Reported limits
- No exact effect sizes digitized from plots; parent lot purity and stereochemical analysis not reported in acquired Materials. MTT is a viability surrogate, not comprehensive toxicity testing.
Methods and source locations
Compound reported in the paper: Caffeic acid; Compound label in the paper: Parent caffeic acid
Parent-compound findings under the reported assay conditions; no Adooq lot validation, salt conversion or clinical inference.
§§2.1–2.6, 2.13, 3.1–3.4; Figures 1–4 and captions; Supplementary Figure S1
PGE2 released into culture medium: ELISA; three independent experiments, each treatment in triplicate; average ± SD. — §§2.4, 3.2; Figure 2 and caption
COX-2 protein normalized to GAPDH: Western blot; three independent experiments, average ± SD. — §§2.5, 3.3; Figure 3A/B and caption
IL-8, MCP-1 and IL-6 in culture medium: ELISA; three independent experiments, each treatment in triplicate; average ± SD. — §§2.6, 3.4; Figure 4A–C and caption
Cell viability in the highest-concentration co-treatment: MTT after 24 h; three independent experiments, each replicate analyzed in triplicate; average ± SD in SI. — §§2.3, 3.1; Supplementary Figure S1
