Bone morphogenetic protein (BMP) signaling pathway (increased expression of BMP5, BMPR2, SMAD5) [1]
p53 (phosphorylation at Ser15) [1]
Cdc42 signaling pathway [1]

In HEK001 keratinocytes, 3,4,5-Tricaffeoylquinic Acid (0-50 μM) decreases TNF-α-induced cytokine production [1]. IkBα phosphorylation and NF-κB activation produced by TNF-α are inhibited by 3,4,5-Tricaffeoylquinic acid (15 μM, 35 minutes) [1].

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TCQA (1-20 μM) increased cell viability of human neural stem cells (hNSCs) in a dose-dependent manner at 24, 48, 72, and 96 h compared to controls [1]
- TCQA (10 μM) significantly increased protein expression of β3-tubulin (neurons) at 24 h and 96 h, MBP (oligodendrocytes) at 48 h and 96 h, and showed an increasing tendency for GFAP (astrocytes) at 48 h and 96 h in hNSCs [1]
- TCQA (10 μM) increased neurite length in hNSCs compared to control [1]
- TCQA (10 μM) significantly increased the percentage of hNSCs in G0/G1 phase in both undifferentiated and differentiated cells (p=0.02), and significantly decreased S phase cells (p=0.007) [1]
- TCQA (10 μM) significantly increased phosphorylation of p53 at Ser15 at 24, 48, and 96 h in hNSCs [1]
- TCQA (10 μM) significantly increased intracellular Ca2+ levels in undifferentiated hNSCs after 1 min of treatment [1]
- TCQA (10 μM) significantly increased mitochondrial membrane potential (MMP) in hNSCs after 30 min of treatment [1]
- TCQA (10 μM) significantly decreased intracellular ROS levels in hNSCs after 180 min of treatment compared to differentiated controls [1]
- DNA microarray analysis (24 h treatment) showed that TCQA increased expression of BMP5, BMPR2, SMAD5, NEUROD1, MAPK14, MAP2K6, DYRK2 (1.32-fold), RIF1 (1.29-fold), and decreased expression of SETD8 (-1.31-fold), MCM4 (-1.26-fold), HUS1 (-1.30-fold), PLK4 (-1.24-fold), DSCC1 (-1.24-fold), ANAPC7 (-1.24-fold), TACC3 (-1.22-fold) in hNSCs [1]

Oral administration of TCQA (5 mg/kg) for 30 days to senescence-accelerated prone 8 (SAMP8) mice significantly decreased escape latency time from day 4 to day 7 in the Morris water maze (MWM) compared to water-treated SAMP8 mice [1]
- TCQA-treated SAMP8 mice spent significantly more time in the target quadrant and crossed the platform location more times than water-treated SAMP8 mice in MWM [1]
- TCQA-treated SAMP8 mice showed significantly higher numbers of BrdU+/GFAP+ cells (activated stem cells) and BrdU+/NeuN+ cells (newborn neurons) in the anterior dentate gyrus (DG) compared to untreated SAMP8 mice [1]
- TCQA increased the number of BrdU+/NeuN+ cells in the posterior DG of SAMP8 mice [1]
- TCQA significantly increased the total number of BrdU+ cells in the subventricular zone (SVZ) of SAMP8 mice compared to water-treated SAMR1 mice [1]

Cell Viability Assay [1]
Cell Types: Keratinocytes
Tested Concentrations: 15, 25 and 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Cell death rate ∼4-5% at 15 and 25 μM and ∼ 50 μM 9%.

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Western Blot Analysis[1]
Cell Types: Keratinocytes
Tested Concentrations: 15 μM
Incubation Duration: 20 min before addition of 10 ng/mL TNF-α, for 15 min
Experimental Results: TNF-α-induced IkBα phosphorylation and cytosolic NF-levels Reduce κb p65, cytosolic NF-κb p65.

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Cell viability was measured using MTT assay. hNSCs were treated with 5-30 μM TCQA for 72 h. After treatment, MTT was added and cells were incubated for another 12 h. MTT formazan was dissolved in SDS solution and absorbance at 570 nm was measured [1]
- For differentiation assay, hNSCs were seeded at 2.5×10⁴ cells/cm² and grown for 48 h. Growth medium was replaced with differentiation medium without growth factors. Cells were treated with or without 10 μM TCQA for 24-96 h. Immunocytochemistry was performed using antibodies against β3-tubulin, MBP, and GFAP. Nuclei were counterstained with DAPI [1]
- Western blotting: Protein samples (20 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Primary antibodies against β3-tubulin, MBP, GFAP, GAPDH, phospho-p53 (Ser15), and p53 were used. Secondary antibodies were IRDye-conjugated. Signal was detected using an imaging system [1]
- Cell cycle assay: hNSCs were treated with or without 10 μM TCQA in growth or differentiation medium for 24 h. Cells were fixed in 70% ethanol, stained with cell cycle reagent, and analyzed by flow cytometry [1]
- Intracellular Ca2+ measurement: hNSCs were pre-treated with Fluo-4 AM for 30 min, then treated with differentiation medium with or without 10 μM TCQA for 1-180 min. Fluorescence intensity (495/535 nm) was measured [1]
- Mitochondrial membrane potential measurement: hNSCs were treated with or without 10 μM TCQA for 30-180 min, then stained with rhodamine 123 (10 μg/ml) for 20 min. Fluorescence intensity (507/529 nm) was measured [1]
- Intracellular ROS measurement: hNSCs were pre-treated with DCFH-DA for 1 h, then treated with or without 10 μM TCQA for 15-180 min. Fluorescence intensity of DCF (485/528 nm) was measured [1]
- DNA microarray analysis: Total RNA was extracted from hNSCs treated with or without 10 μM TCQA for 6-24 h. Double-stranded cDNA was synthesized and biotin-labeled aRNA was hybridized to microarrays. Data analysis was performed using software [1]

Animal model: Senescence-accelerated prone 8 (SAMP8) mice and senescence-accelerated resistant 1 (SAMR1) control mice (age and sex not specified in methods) [1]
- TCQA administration: TCQA was administered orally at 5 mg/kg body weight for 30 days. Control mice received water [1]
- BrdU administration: Bromodeoxyuridine (BrdU) was administered (details of dose and route not specified in methods) followed by washout and sacrifice to detect stem cell activation and neurogenesis [1]
- Morris water maze: A circular pool (120 cm diameter, 45 cm height) was filled with water (23±2°C). A submerged platform (10 cm diameter, 1 cm below water surface) was placed in the northeast quadrant. Mice received 4 trial sessions daily for 7 days. On day 7, the platform was removed, and mice swam for 60 s. Time spent in target quadrant and number of platform crossings were recorded [1]
- Tissue processing: Brains were fixed in 4% paraformaldehyde for 24 h, cryoprotected in 30% sucrose for 48 h at 4°C. Coronal sections (30 μm) were obtained using a microtome. Sections were stored at -20°C in cryoprotectant solution [1]
- Immunohistochemistry: Sections were washed in PBS, blocked with glycine, and incubated in HCl (2N) at 37°C for 1 h for BrdU detection. Primary antibodies: anti-BrdU (1:400), anti-GFAP (1:500), anti-NeuN (1:400). Secondary antibodies were Alexa-conjugated (1:500) [1]

[1]. 3,4,5-Tricaffeoylquinic acid inhibits tumor necrosis factor-α-stimulated production of inflammatory mediators in keratinocytes via suppression of Akt- and NF-κB-pathways. Int Immunopharmacol. 2011;11(11):1715-1723.

[2]. 3,4,5-Tricaffeoylquinic acid induces adult neurogenesis and improves deficit of learning and memory in aging model senescence-accelerated prone 8 mice. Aging (Albany NY). 2019;11(2):401-422.

(3R,5R)-3,4,5-tris{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1-hydroxycyclohexane-1-carboxylic acid is an alkyl caffeic acid ester and quinic acid.

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TCQA is a natural polyphenol belonging to caffeoylquinic acid derivatives, found in coffee beans, sweet potatoes, and propolis [1]
- Among caffeoylquinic acid derivatives, TCQA has the highest ability to increase ATP production in human neuroblastoma SH-SY5Y cells [1]
- TCQA induced G0/G1 cell cycle arrest, actin cytoskeleton organization, chromatin remodeling, and neuronal differentiation in hNSCs [1]
- TCQA treatment rescued cognitive deficits in SAMP8 mice to levels similar to control SAMR1 mice [1]
- The study suggests therapeutic potential of TCQA for aging-associated diseases such as Alzheimer's disease [1]

C34H30O15

678.6

678.158

86632-03-3

6440783

Yellow to brown solid powder

1.6±0.1 g/cm3

916.0±65.0 °C at 760 mmHg

290.9±27.8 °C

0.0±0.3 mmHg at 25°C

1.737

2.68

8

15

13

49

1200

2

C1C(C[C@H](C([C@@H]1OC(=O)/C=C/C2=CC(=C(C=C2)O)O)OC(=O)/C=C/C3=CC(=C(C=C3)O)O)OC(=O)/C=C/C4=CC(=C(C=C4)O)O)(O)C(=O)O

OAFXTKGAKYAFSI-YOWOTECTSA-N

InChI=1S/C34H30O15/c35-21-7-1-18(13-24(21)38)4-10-29(41)47-27-16-34(46,33(44)45)17-28(48-30(42)11-5-19-2-8-22(36)25(39)14-19)32(27)49-31(43)12-6-20-3-9-23(37)26(40)15-20/h1-15,27-28,32,35-40,46H,16-17H2,(H,44,45)/b10-4+,11-5+,12-6+/t27-,28-,32-,34+/m1/s1

(1S,3R,4S,5R)-3,4,5-Tris[(E)-3-(3,4- dihydroxyphenyl)acryloyloxy]-1-hydroxy-cyclohexanecarboxylic Acid

3,4,5-Tri-CQA 3,4,5-TriCQA3,4,5-Tri CQA

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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