TLR4-MD-2 heterodimer (competes with LPS for binding; hydrogen bonding sites: Tyr296 in TLR4 and Ser120 in MD-2) [1]
Caspase-3, caspase-8, caspase-9 (suppresses activation) [2]

Fruits and juices include the polyphenolic flavonoid proanthocyanidin B1, which has anti-inflammatory properties and binds to the TLR4/MD-2 complex. When proanthocyanidin B1 concentrations exceed 100 μg/mL, cytotoxicity occurs. In THP1 cells, procyanidin B1 (100 μg/mL) suppresses the expression of MD-2, TRAF6, NF-κB mRNA, phosphorylated p38 MAPK, and NF-κB protein as well as the generation of TNF-α produced by LPS [1]. Proanthocyanidin B1 (50-100 µM) inhibits the neuronal mortality caused by Aβ oligomers. It has been observed that proanthocyanidin B1 efficiently inhibits caspase-3 at 100 µM, caspase-8 at 30, 50, and 100 µM, and caspase-9 at 10, 30, 50, and 100 µM. [2]. The expression of ACO and CPT1 was considerably and dose-dependently elevated by proanthocyanidin B1 (10, 20, 30 μM), but PPARα mRNA expression showed no discernible change [3].

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In THP1 cells (human monocytic cell line), procyanidin B1 at 100 μg/mL co-treated with LPS (1 μg/mL) for 18 h significantly inhibited LPS-induced TNF-α production (p<0.05) [1].
Procyanidin B1 (100 μg/mL, 18 h co-treatment with LPS) significantly suppressed levels of phosphorylated p38 MAPK and NF-κB protein in THP1 cells (p<0.05) [1].
Procyanidin B1 (100 μg/mL, 18 h co-treatment with LPS) significantly reduced LPS-induced mRNA expression of MD-2 (by 60-75%), TRAF-6, and NF-κB (all p<0.05) in THP1 cells, but did not significantly affect TLR4 mRNA [1].
In rat primary cultured cortical neurons, procyanidin B1 at 30 μM, 50 μM, and 100 μM significantly inhibited Aβ oligomer (3 μM)-induced neuronal death as measured by MTT assay (p<0.01 or p<0.001) [2].
Procyanidin B1 at 100 μM significantly suppressed Aβ oligomer-induced activation of caspase-3 (p<0.001) [2].
Procyanidin B1 at concentrations of 30, 50, and 100 μM significantly inhibited Aβ oligomer-induced activation of caspase-8 (p<0.001), and at 10, 30, 50, and 100 μM inhibited activation of caspase-9 (p<0.01 for 10 μM, p<0.001 for others) [2].
In HepG2 cells treated with 0.5 mM palmitic acid for 48 h, procyanidin B1 at 10, 20, and 30 μM significantly suppressed intracellular triacylglycerol accumulation in a dose-dependent manner; at 30 μM, triacylglycerol level was reduced to approximately 50% of the control group (p<0.05) [3].
In palmitic acid-treated HepG2 cells, procyanidin B1 (10, 20, 30 μM) significantly induced mRNA expression of ACO (acyl-CoA oxidase) and CPT1 (carnitine palmitoyltransferase) in a dose-dependent manner (p<0.01), but showed no significant effect on PPARα mRNA expression [3].

Molecular docking study: The X-ray crystallographic structure of the TLR4/MD-2 complex was obtained from the Protein Data Bank. Three-dimensional structures of procyanidin B1 and LPS were generated using software. The docking procedure involved removing ligand and water molecules, adding hydrogens, and correcting protein chemistry. The Dreiding force field with Gasteiger charges was used. The LigandFit module was used for molecular modeling. Hydrogen bonds were identified between procyanidin B1 and Tyr296 in TLR4 and Ser120 in MD-2. Consensus scores: ligScore1 6.31-6.37, ligScore2 7.32-7.40, DockScore 85.124-85.145 [1].
Caspase activity assay: Caspase-3/7, -8, and -9 activities were measured using a luminescent assay kit that contains proluminescent caspase substrates with tetrapeptide sequences (DEVD for caspase-3/7, IETD for caspase-8, LEHD for caspase-9). Upon cleavage by active caspases, aminoluciferin is released and reacts with luciferase to produce luminescence. After treatment with Aβ oligomers and test substances, the reagent was added to culture medium, incubated at room temperature for 1 hour, and chemiluminescence intensity was measured [2].

THP1 cell culture: Human acute monocytic leukemia THP1 cells were grown in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum and antibiotics at 37°C in 5% CO2. Cells were plated at 4×10^5 cells/well in 96-well plates and pre-incubated for 24 h before treatment. For cytotoxicity assay, cells were treated with procyanidin B1 at 50-200 μg/mL for 18 h, then CCK8 solution was added and incubated for 4 h, and OD at 450 nm measured [1].
TNF-α measurement: THP1 cells were treated with 1 μg/mL LPS with or without 100 μg/mL procyanidin B1 for 18 h. Culture supernatants were collected and TNF-α levels measured by ELISA, with OD at 450 nm [1].
Real-time PCR: Total RNA extracted from THP1 cells using RNAiso Plus kit, reverse transcribed to cDNA. Real-time PCR was performed with specific primers for MD-2, TLR4, TRAF6, NF-κB, and GAPDH. Relative mRNA expression was calculated using 2^-ΔΔCt method. Cells were treated with LPS (1 μg/mL) with or without procyanidin B1 (100 μg/mL) for 18 h [1].
Western blot: Proteins extracted from THP1 cells using lysis buffer. Protein samples (30 μg/lane) separated on 15% polyacrylamide gel, transferred to nitrocellulose membranes. Membranes blocked with 5% nonfat milk, incubated with primary antibodies (rabbit anti-NF-κB or anti-p38 MAPK, mouse anti-GAPDH) overnight at 4°C, then with HRP-conjugated secondary antibodies for 40 min. Detection with ECL reagent. Cells were treated with LPS (1 μg/mL) with or without procyanidin B1 (100 μg/mL) for 18 h [1].
Primary cortical neuron culture: Cortical neurons from 18-day-old rat embryos. Tissue dissected, digested with papain/DNase I, cells resuspended in Neurobasal medium with B27 supplement, seeded on poly-D-lysine-coated plates at 1×10^5 cells/cm^2. Cultures maintained at 37°C, 5% CO2. Medium replaced every 3 days. Experiments performed 14 days after seeding. Aβ oligomers prepared from Aβ1-42 peptides (200 μM in sterile water at 4°C for 30 min, then diluted to 100 μM in PBS and rotated at 37°C for 7 days). Cells treated with 3 μM Aβ oligomers plus procyanidin B1 (3-100 μM) for 48 h. MTT assay: MTT added at 1 mg/mL final, incubated 1 h, formazan dissolved in 10% SDS in 0.1 N HCl, absorbance at 570 nm measured [2].
Caspase activity in neurons: After treatment with Aβ oligomers (3 μM) and procyanidin B1 for 24 h (caspase-8, -9) or 48 h (caspase-3), Caspase-Glo reagent added, incubated 1 h, luminescence measured [2].
HepG2 cell culture: HepG2 cells maintained in DMEM (5.5 mM glucose) with 10% FBS, penicillin-streptomycin at 37°C in 5% CO2. Palmitic acid-BSA complex prepared by dissolving palmitic acid in 10% fatty acid-free BSA at 55°C overnight to make 5 mM stock. Sub-confluent cells treated for 48 h with 0.5 mM palmitic acid and 1.0% fatty acid-free BSA, with or without procyanidin B1 (10, 20, 30 μM). Triacylglycerol content measured by colorimetric assay after cell lysis, normalized to protein. mRNA expression of PPARα, ACO, CPT1, β-actin measured by real-time RT-PCR [3].

In THP1 cells, procyanidin B1 at concentrations >100 μg/mL induced cellular toxicity as assessed by CCK8 assay; therefore, 100 μg/mL was used as the safe concentration for subsequent experiments [1].
In rat primary cortical neurons, procyanidin B1 at 100 μM did not show toxicity; instead it showed neuroprotective effects. However, note that for another compound (glycycoumarin), 100 μM increased neurotoxicity [2].

[1]. Anti-inflammatory effect of procyanidin B1 on LPS-treated THP1 cells via interaction with the TLR4-MD-2 heterodimer and p38 MAPK and NF-κB signaling. Mol Cell Biochem. 2015 Sep;407(1-2):89-95.

[2]. Protective effects of glycycoumarin and procyanidin B1, active components of traditional Japanese medicine yokukansan, on amyloid β oligomer-induced neuronal death. J Ethnopharmacol. 2015 Jan 15;159:122-8.

[3]. Flavangenol (pine bark extract) and its major component procyanidin B1 enhance fatty acid oxidation in fat-loaded models. Eur J Pharmacol. 2012 Feb 29;677(1-3):147-53.

Proanthocyanidin B1 is a proanthocyanidin composed of (-)-epicatechin and (+)-catechin units linked by a β-configuration at positions 4 and 8', respectively. Proanthocyanidin B1 is found in Ceylon cinnamon (Cinnamomum verum, found in its pericarp, bark, or epidermis), Uncaria guianensis (found in its roots), grape (Vitis vinifera, found in its leaves), and peach. It is a metabolite and also an EC 3.4.21.5 (thrombin) inhibitor and anti-inflammatory agent. It is a hydroxyflavanol, proanthocyanidin, biflavonoid, and polyphenol compound. Functionally, it is associated with (-)-epicatechin and (+)-catechin. Proanthocyanidin B1 has been reported in tea (Camellia sinensis), rhododendron (Rhododendron dauricum), and other organisms with relevant data. See also: Pine (partial); Mangosteen pericarp (partial).

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Procyanidin B1 is a polyphenolic flavonoid (procyanidin dimer) found in fruits and fruit juices. It can compete with LPS for binding to the TLR4-MD-2 heterodimer and suppress downstream activation of p38 MAPK and NF-κB signaling pathways, thereby exerting anti-inflammatory effects [1].
Procyanidin B1 is a component of Uncaria Hook (a traditional Japanese medicine ingredient) and contributes to the neuroprotective effects of yokukansan against Aβ oligomer-induced apoptosis by suppressing caspase-3 activation, possibly via inhibition of upstream caspases-8 and -9 [2].
Procyanidin B1 is a major component of Flavangenol (pine bark extract, containing 5% procyanidin B1). It enhances fatty acid oxidation in hepatocytes by inducing expression of ACO and CPT1, thereby reducing fat accumulation [3].

C30H26O12

578.53

578.142

20315-25-7

Cyanidin Chloride;528-58-5;Procyanidin B2;29106-49-8;Procyanidin C1;37064-30-5;Procyanidin A2;41743-41-3;Procyanidin A1;103883-03-0;Procyanidin B3;23567-23-9

11250133

Light yellow to yellow solid powder

1.7±0.1 g/cm3

955.3±65.0 °C at 760 mmHg

231～232℃

531.6±34.3 °C

0.0±0.3 mmHg at 25°C

1.803

0.3

10

12

3

42

925

5

C1[C@@H]([C@H](OC2=C1C(=CC(=C2[C@@H]3[C@H]([C@H](OC4=CC(=CC(=C34)O)O)C5=CC(=C(C=C5)O)O)O)O)O)C6=CC(=C(C=C6)O)O)O

XFZJEEAOWLFHDH-UKWJTHFESA-N

InChI=1S/C30H26O12/c31-13-7-20(37)24-23(8-13)41-29(12-2-4-16(33)19(36)6-12)27(40)26(24)25-21(38)10-17(34)14-9-22(39)28(42-30(14)25)11-1-3-15(32)18(35)5-11/h1-8,10,22,26-29,31-40H,9H2/t22-,26+,27+,28+,29+/m0/s1

(2R,3S)-2-(3,4-Dihydroxyphenyl)-8-[(2R,3R,4R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H-chromene-3,5,7-triol

Epicatechin-(4beta->8)-ent-epicatechin Procyanidin B1Proanthocyanidin B1

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 : ~25 mg/mL (~43.21 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.32 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 25.0 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: ≥ 2.5 mg/mL (4.32 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)