Target: p38 MAPK (reduces UVB-induced phosphorylation)
JNK (reduces UVB-induced phosphorylation to a lower extent)[1]

DHCA exhibited high antioxidant capacity in cell-free assays: ABTS⁺ scavenging ability of 5.09 ± 0.76 μM TE/μM sample; DPPH⁺ IC50 of 7.65 ± 0.08 μM; superoxide anion (O₂⁻) scavenging IC50 of 0.96 ± 0.03 μM in XOD assay. [1]
At concentrations 7-35 μM, DHCA showed no cytotoxicity to L929 fibroblasts (98.3% viability at 35 μM). [1]
Pretreatment with 35 μM DHCA significantly reduced UVB-induced cell death (600 mJ/cm²), increasing cell viability from 51.8% (UVB only) to 63.4%. [1]
DHCA (35 μM) decreased UVB-induced intracellular ROS production by 39.1% and extracellular H₂O₂ production by 72.2% (almost to basal levels). [1]
DHCA partially restored UVB-depleted endogenous antioxidants: increased catalase (CAT) activity by 32.5%, superoxide dismutase (SOD) activity by 28.5%, and restored 28.4% of reduced glutathione (GSH) levels compared to UVB-irradiated control. [1]
DHCA inhibited UVB-induced lipid peroxidation by 38.3% (DPPP assay). [1]
DHCA prevented UVB-induced loss of mitochondrial membrane potential (Δψm): UVB caused 50.1% loss, while DHCA pretreatment reduced loss to 10.6%. [1]
DHCA significantly attenuated UVB-induced apoptosis: UVB increased apoptosis by 42.5% (annexin V), and DHCA inhibited apoptosis by 41.6%. [1]
DHCA reduced UVB-induced late apoptotic and necrotic cells (acridine orange/propidium iodide staining) and decreased the number of cells with condensed nuclei (Hoechst 33342). [1]
DHCA significantly attenuated UVB-induced MMP-1 expression (western blot), but did not show significant reduction in MMP-3 or MMP-9 expression. [1]
DHCA reduced UVB-induced phosphorylation of p38 MAPK (from 2.4-fold to 1.3-fold compared to non-irradiated control) and JNK (from 2.0-fold to 1.4-fold). [1]

The ABTS⁺ scavenging ability of DHCA was evaluated by mixing ABTS⁺ radical cation solution (diluted to absorbance 0.70 ± 0.05 at 734 nm) with 7 μL of DHCA (35 μM) or quercetin dissolved in ethanol. Absorbance was measured after 6 min at 25°C in the dark. Results were expressed as μM trolox equivalent per μM of sample. [1]
The DPPH⁺ scavenging ability was evaluated by mixing 100 μL of DPPH⁺ methanol solution (65 μM) with 100 μL of DHCA or quercetin at different concentrations (2.7 - 16.5 μM). Absorbance was measured at 517 nm after 30 min incubation at 25°C in the dark. The percentage of DPPH⁺ scavenging was calculated, and results were expressed as IC50 values. [1]
The superoxide anion (O₂⁻) scavenging capacity was analyzed using a luminol-dependent chemiluminescent assay with the xanthine/luminol/xanthine oxidase (XOD) system. A reagent solution containing glycine buffer (0.1 M, 1 mM EDTA, pH 9.4), xanthine (6 mM), luminol (0.6 mM), and DHCA or quercetin (0.05 - 5.1 μM) dissolved in 50% ethanol was mixed. The reaction was started by adding fresh cold XOD (20 mU/mL), and chemiluminescence was read after 1 min. Scavenging percentage was calculated and results expressed as IC50 values. [1]

L929 fibroblasts were cultured in DMEM supplemented with 2 mM L-glutamine, 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 μg/mL) at 37°C in 5% CO₂. [1]
Cytotoxicity of DHCA was assessed by neutral red assay. Cells (2.5×10⁴/well) were treated with DHCA (7, 14, 21, 28, 35 μM) or 0.6% DMSO for 24 h. After washing, neutral red solution (40 μg/mL) was added for 3 h, then cells were fixed with formaldehyde/calcium chloride, and absorbance measured at 540 nm. [1]
For UVB irradiation studies, cells were pretreated with DHCA (35 μM) or 1 mM NAC for 1 h, then irradiated with UVB (600 mJ/cm²) using a UVB lamp at 20 cm distance. After irradiation, cells were kept in serum-free DMEM for 24 h before analyses. [1]
Intracellular ROS was measured using H₂DCFDA probe. Cells were treated with DHCA or NAC for 1 h, incubated with 10 μM H₂DCFDA for 45 min at 37°C, then irradiated. Fluorescence was measured immediately (λ_ex 488 nm, λ_em 525 nm). Protein concentration was quantified by Bradford method. [1]
Extracellular H₂O₂ was measured using Amplex Red kit. After irradiation, cells were incubated with 15 μM Amplex Red and 0.15 U/mL horseradish peroxidase in Tris-HCl pH 7.5 for 30 min at 25°C, and fluorescence measured (λ_ex 563 nm, λ_em 590 nm). [1]
Catalase activity was measured by monitoring H₂O₂ consumption at 240 nm. Samples (50 μg/mL protein) were mixed with 30 mM H₂O₂ in potassium phosphate buffer (50 mM, pH 7.0) and decomposition rate assessed spectrophotometrically. [1]
SOD activity was determined by pyrogallol oxidation. Sample (50 μg/mL protein) was mixed with 15 mM pyrogallol in Tris-HCl buffer (200 mM, 2 mM EDTA, pH 8.2), incubated for 2 min, and absorbance measured at 420 nm. One unit of SOD activity was defined as amount inhibiting 50% of pyrogallol oxidation. [1]
GSH content was measured by mixing cellular supernatants (50 μg/mL protein) with 7.5 mM o-phthalaldehyde in sodium phosphate buffer (100 mM, 5 mM EDTA, pH 8.0) for 15 min, and fluorescence measured (λ_ex 350 nm, λ_em 420 nm). [1]
Lipid peroxidation was evaluated using DPPP probe. After 24 h post-irradiation, cells were incubated with 20 μM DPPP for 30 min at 37°C, and fluorescence measured (λ_ex 351 nm, λ_em 380 nm). Fluorescence microscopy was also performed. [1]
Mitochondrial membrane potential was measured using rhodamine 123. After 24 h post-irradiation, cells were incubated with 26.2 μM Rh 123 for 15 min at 37°C, and fluorescence measured (λ_ex 488 nm, λ_em 525 nm). [1]
Apoptosis was detected by annexin V-FITC binding. Cells were dissociated, washed, resuspended in binding buffer (140 mM NaCl, 5 mM CaCl₂, 10 mM HEPES-Na, pH 7.4), stained with 2 μL annexin V-FITC for 15 min, and analyzed by flow cytometry (10,000 events). [1]
Acridine orange and propidium iodide double staining: cells were stained with 1 μg/mL acridine orange and 1 μg/mL propidium iodide for 10 min, then visualized by fluorescence microscopy. Viable (green), late apoptotic (orange nuclei), and necrotic (red nuclei) cells were counted (100 cells per experiment). [1]
Nuclear condensation was evaluated by Hoechst 33342 staining. After 24 h post-irradiation, cells were stained with 8 μM Hoechst 33342 for 15 min at 37°C and visualized by fluorescence microscopy. Condensed nuclei were quantitated (100 cells per experiment). [1]
Western blot analysis: cells were lysed in buffer containing Tris-HCl pH 7.4, 2% SDS, 5% 2-mercaptoethanol, 30% glycerine, protease and phosphatase inhibitor cocktails. Proteins (equal amounts) were separated on 12% SDS-PAGE, transferred to nitrocellulose membrane, blocked with 5% BSA in TBST, incubated overnight at 4°C with primary antibodies against JNK, p-JNK, p38, p-p38, MMP-1, MMP-3, MMP-9, caspase 9 (1:500), and β-actin (1:10000), then with HRP-conjugated secondary antibody (1:10000) for 2 h. Protein bands were detected with chemiluminescence reagent. [1]

Metabolism / Metabolites
Known metabolites of 3,4-dihydroxyphenylpropionic acid include 3-(3-hydroxyphenyl)propionic acid and 4-hydroxy-4-hydroxyphenylpropionic acid.

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DHCA was not cytotoxic to L929 fibroblasts at concentrations 7-35 μM, with cell viability of 98.3% at the highest concentration (35 μM) as assessed by neutral red assay after 24 h treatment. [1]

[1]. Dihydrocaffeic Acid Prevents UVB-Induced Oxidative Stress Leading to the Inhibition of Apoptosis and MMP-1 Expression via p38 Signaling Pathway. Oxid Med Cell Longev. 2019 Jan 17;2019:2419096.

3-(3,4-dihydroxyphenyl)propionic acid is a monocarboxylic acid, formed by substituting hydroxyl groups at the 3 and 4 positions of 3-phenylpropionic acid. Also known as dihydrocaffeic acid, it is a metabolite of caffeic acid and possesses antioxidant activity. It is both an antioxidant and an exogenous metabolite in the human body. Its function is related to 3-phenylpropionic acid; it is the conjugate acid of 3-(3,4-dihydroxyphenyl)propionic acid ester. 3-(3,4-dihydroxyphenyl)propionic acid has been reported to exist in Pyrrosia petiolosa, Lindera glauca, and several other organisms with relevant data.

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Chronic UVB exposure promotes oxidative stress, leading to skin photoaging characterized by deep wrinkling, loss of elasticity, dehydration, telangiectasia, and pigmentation alterations. DHCA has a catechol structure and carboxyl group attributed to its antioxidant activity. The study demonstrates for the first time that DHCA inhibits UVB-induced lipid peroxidation, apoptosis, and MMP-1 expression in L929 fibroblasts, thereby attenuating skin photoaging. These effects are associated with oxidative stress inhibition via decreased ROS production and regeneration of endogenous antioxidant defenses, leading to suppression of the p38 MAPK signaling pathway. DHCA is a promising compound for incorporation into topical delivery systems to prevent UVB-induced skin damage. [1]

C9H10O4

182.1733

182.057

1078-61-1

348154

White to off-white solid powder

1.4±0.1 g/cm3

417.5±30.0 °C at 760 mmHg

136-139 °C(lit.)

220.4±21.1 °C

0.0±1.0 mmHg at 25°C

1.620

0.5

3

4

3

13

181

0

DZAUWHJDUNRCTF-UHFFFAOYSA-N

InChI=1S/C9H10O4/c10-7-3-1-6(5-8(7)11)2-4-9(12)13/h1,3,5,10-11H,2,4H2,(H,12,13)

3-(3,4-dihydroxyphenyl)propanoic acid

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)

DMSO : ~250 mg/mL (~1372.34 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (11.42 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 (11.42 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 (11.42 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.)