IC50: PCSK9
In HepG2 cells, Pinostrobin targets the gene expression of Proprotein convertase subtilisin/kexin type 9 (PCSK9) through the modulation of Forkhead box O3a (FoxO3a) protein. It increases nuclear FoxO3a protein levels and enhances FoxO3a/PCSK9 promoter complexes formation. [1]
In RAW 264.7 and J774A.1 cells, Pinostrobin targets the production of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β). The IC50 for TNF-α inhibition was 17.28 ± 1.01 μM (RAW 264.7) and 19.63 ± 3.54 μM (J774A.1). The IC50 for IL-1β inhibition was 23.51 ± 1.06 μM (RAW 264.7) and 37.46 ± 3.42 μM (J774A.1). [3]
In B16F10 cells, Pinostrobin targets the melanogenesis pathway by upregulating the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1 (TRP-1). It acts via the cAMP/PKA signaling pathway (increasing phosphorylation of CREB, GSK-3β, and β-catenin) and the p38 MAPK signaling pathway. [5]
In acute leukemia NB4 and MOLT-4 cells, Pinostrobin targets the expression of miR-410-5p and subsequently upregulates secreted frizzled related protein 5 (SFRP5), a negative regulator of the Wnt/β-catenin signaling pathway. [6]
In HeLa cervical cancer cells, Pinostrobin targets multiple pathways involved in apoptosis, including the intrinsic (mitochondrial) and extrinsic (death receptor) pathways. It increases ROS production, decreases mitochondrial membrane potential, and modulates the expression of Bcl-2 family proteins (Bad, Bax) and caspases. [7]

In HepG2 cells, Pinostrobin (20 and 40 μM) significantly inhibited PCSK9 promoter activity from 1.00 ± 0.16 (fold) to 0.85 ± 0.06 and 0.54 ± 0.05, respectively. It also suppressed PCSK9 mRNA expression from 1.00 ± 0.11 (fold) to 0.81 ± 0.07 and 0.58 ± 0.07, respectively. Pinostrobin significantly reduced the mature form of the PCSK9 protein, inhibited the catalytic activity of PCSK9, increased the protein level of LDLR (by approximately 2.0- and 3.3-fold at 20 and 40 μM), and increased LDL uptake activity (to 213.5 ± 10.1% at 40 μM) in HepG2 cells. It markedly increased the level of nuclear FoxO3a protein (by approximately 1.4- and 2.1-fold at 20 and 40 μM) and enhanced FoxO3a/PCSK9 promoter complexes formation while attenuating the promoter binding capacity of nuclear HNF-1α. The knockdown of FoxO3a by siRNA abolished the Pinostrobin-mediated PCSK9 reduction. Pinostrobin also attenuated simvastatin-induced PCSK9 overexpression in HepG2 cells. [1]
In RAW 264.7 and J774A.1 cells, Pinostrobin was found to be most active in inhibiting TNF-α (IC50 = 17.28 ± 1.01 μM and 19.63 ± 3.54 μM, respectively) and IL-1β (IC50 = 23.51 ± 1.06 and 37.46 ± 3.42 μM, respectively) production. The cell viability was >80% at a concentration of 100 μM. [3]
In B16F10 cells, Pinostrobin (25-100 μM) did not exhibit any cytotoxicity. It significantly increased melanin content (more than 2.3 times at 100 μM) and tyrosinase activity. It stimulated the expression of tyrosinase, TRP-1, and MITF. Pinostrobin promoted the phosphorylation of CREB, GSK-3β (by >2 folds at 50 and 100 μM), and β-catenin. The PKA inhibitor (H89) markedly reduced the Pinostrobin-induced melanin synthesis, tyrosinase activity, and expression of tyrosinase, TRP-1, and MITF, as well as the phosphorylation of CREB, GSK-3β, and β-catenin. Pinostrobin also augmented the phosphorylation level of p38 while reducing ERK phosphorylation. The p38 inhibitor (SB203580) reduced the Pinostrobin-induced melanin content, tyrosinase activity, and expression of MITF and tyrosinase. [5]
In NB4 and MOLT-4 acute leukemia cells, treatment with Pinostrobin at IC50 (130 μM for NB4, 150 μM for MOLT-4) for 48 hours induced apoptosis (34.37 ± 5.84% and 49.40 ± 4.76%, respectively), increased caspase-3 expression, upregulated proapoptotic BAK, and downregulated antiapoptotic BCL-W and MCL-1. LC-MS/MS analysis identified SFRP5 as a target protein, and RT-qPCR showed Pinostrobin increased SFRP5 mRNA and decreased β-catenin mRNA and protein. Pinostrobin significantly suppressed the expression level of miR-410-5p. Transfection with a miR-410-5p mimic increased β-catenin and decreased apoptosis, while co-treatment with Pinostrobin reversed this effect. [6]
In HeLa cervical cancer cells, Pinostrobin (50 and 100 μM) reduced cell viability (CT50 of 50 μM at 48h), decreased GSH and nitrite levels, and induced condensed nuclei with fragmented chromatin. It increased ROS production (MFI increased to 32.27 ± 6.67 at 50 μM and 54.20 ± 6.73 at 100 μM) and markedly reduced mitochondrial membrane potential (20.67 ± 4.6% increase in JC-1 monomer at 100 μM). Pinostrobin treatment resulted in cell cycle arrest at G1/S phase (54.69% cell population at 100 μM vs 40.77% in control). Proteome profiler array showed increased expression of proteins involved in both extrinsic (TRAIL R1/D4, TRAIL R2/D5, Fas, FADD) and intrinsic (Bad, Bax, cytochrome C, cleaved caspase-3) apoptotic pathways. [7]

In Sprague Dawley (SD) rats, oral administration of Pinostrobin at a dose of 20 mg/kg significantly lowered LPS-induced TNF-α levels by 48.6% (to 834 pg/ml) and IL-1β levels by 53.1% (to 428 pg/ml) compared to the LPS-treated group (1623 pg/ml for TNF-α and 912 pg/ml for IL-1β). The positive control dexamethasone (5 mg/kg) decreased TNF-α by 75.8% and IL-1β by 63.0%. [3]
In female Kunming rats, a single intragastric administration of Pinostrobin at 0.5 mg/kg was used for a pharmacokinetic study. [8]

The catalytic activity of PCSK9 protein in HepG2 cell lysates was measured using a fluorogenic peptide (FAQSIPK) corresponding to the PCSK9 cleavage site, labeled with 2,4-dinitrophenyl and 7-amino-4-methyl coumarin. Cell protein lysates (80 μg) were added to a reaction buffer (50 mM Tris pH 7.4, 2.5 mM CaCl2, and 0.5% Triton X-100). The fluorogenic peptide (100 μM) and cell lysate samples were incubated at 30°C, and fluorescence intensity was measured at λexcitation = 340 nm, λemission = 460 nm. Pinostrobin (20 and 40 μM) decreased the fluorescence intensity by approximately 20% and 45% at t=540 min, indicating suppressed PCSK9 catalytic activity. [1]
A cell-free assay for topoisomerase I activity was performed using molecular docking, spectroscopic, and topoisomerase I activity studies, which suggested that Pinostrobin allosterically inhibits topoisomerase I. [7]
Tyrosinase activity in B16F10 cells was evaluated. Cells were stimulated with Pinostrobin for 3 days. The extracted protein (1 μg/μL) was added to a 96-well plate containing 10 mM L-DOPA and 0.1 M potassium phosphate buffer (pH 6.8). The reaction mix was incubated for 1 hour at 37°C and then measured at 490 nm. Pinostrobin increased tyrosinase activity. [5]

Cell viability was assessed by MTT assay. HepG2 cells were seeded in 24-well plates, treated with vehicle or Pinostrobin (10-40 μM) for 24 h, and then incubated with MTT reagent (1 mg/mL) at 37°C for 3 h. The formed formazan crystals were dissolved in DMSO, and absorbance was measured at 550 nm. Pinostrobin showed no cytotoxicity in HepG2 cells. [1]
For RT-qPCR analysis of PCSK9, LDLR, HMGCR, and Mylip/Idol, HepG2 cells were treated with vehicle or Pinostrobin (20 and 40 μM) for 24 h. Total RNA was extracted, reverse-transcribed, and quantitative real-time PCR was performed using human-specific primers and SYBR Green master mix. Pinostrobin decreased PCSK9 mRNA and did not significantly alter LDLR, HMGCR, or Mylip/Idol mRNA. [1]
For Western blot analysis, HepG2 cells were treated with Pinostrobin (20 and 40 μM) for 24 h. Total cellular protein or nuclear extracts were prepared, separated by 10% SDS-PAGE, and transferred to PVDF membranes. Membranes were incubated with primary antibodies (anti-PCSK9, anti-LDLR, anti-HNF1α, anti-FoxO3a, etc.) and then with HRP-conjugated secondary antibodies. Protein levels were measured using chemiluminescence. Pinostrobin decreased mature PCSK9 and increased LDLR and nuclear FoxO3a. [1]
For the apoptosis assay in leukemia cells, NB4 and MOLT-4 cells were treated with Pinostrobin at IC50 (130 μM and 150 μM, respectively) for 48 h. Cells were harvested and stained with Annexin V-FITC and PI, then analyzed by flow cytometry to measure the percentage of apoptotic cells. [6]
For the measurement of melanin content in B16F10 cells, cells were exposed to Pinostrobin for 3 days, washed, and pelleted. The pellet was incubated in 1N NaOH solution at 80°C for 2 h to dissolve melanin, and the melanin content was measured by an ELISA reader at 405 nm. [5]
For morphological assessment of apoptosis in HeLa cells, cells were grown on cover slips and treated with Pinostrobin (50 μM). After 48 h, cells were stained with Wright-Giemsa for 5 min, and morphological changes like cell shrinkage, condensed nuclei, and membrane blebbing were examined under a light microscope. [7]

For the pharmacokinetic study, female Kunming rats (weighing 220-250 g) received a single intragastric administration of Pinostrobin at a dose of 0.5 mg/kg. Blood samples were collected in heparinized tubes at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 hours post-dose. The blood was immediately centrifuged at 3000 × g for 15 min, and the plasma was stored at -70°C until analysis. [8]
For the in vivo anti-inflammatory study, female Sprague Dawley (SD) rats (200-250 g) were divided into groups (n=6). The test groups received Pinostrobin (10 and 20 mg/kg) orally at an interval of 12 h. Lipopolysaccharide (LPS, 5 mg/kg) was administered 2 h after the final dose. Blood samples were collected from the tail vein 2 h (for TNF-α) and 6 h (for IL-1β) after LPS administration. Plasma was separated by centrifugation at 3000 rpm for 10 min, and cytokine concentrations were determined by ELISA. [3]

After a single intragastric administration of Pinostrobin (0.5 mg/kg) in female Kunming rats, the maximal concentration (Cmax) was 615.35 ± 32.89 ng/mL (equivalent to approximately 2.28 μM). The time to reach maximum concentration (Tmax) was 4 ± 0.18 hours. The terminal half-life (t1/2) was 4.34 ± 0.24 minutes. The area under the plasma concentration-time curve (AUC0→∞) was 3817.80 ± 352.89 ng·min/mL. [8]
In silico pharmacokinetic prediction using SwissADME indicated that Pinostrobin is predicted to be absorbed in the gastrointestinal tract (93.8%) and to penetrate the skin (Log Kp = -2.8). It is predicted to be an inhibitor of CYP1A2 and CYP2C19. [5]
In rats, Pinostrobin is mainly distributed in the gastrointestinal tract after a single oral administration. The parent compound exhibited less than 1.6% elimination into urine, bile, and feces, suggesting that it is mostly metabolized in vivo. The compound is rapidly metabolized to glucuronides. [1, 5]
The calculated oral bioavailability of S-Pinostrobin and R-Pinostrobin in rats was 1.83 ± 1.43% and 13.8 ± 3.42%, respectively. The serum half-life was approximately 6 hours after intravenous administration (10 mg/kg) in male Sprague Dawley rats. [1]

In HepG2 cells, Pinostrobin at concentrations of 10-40 μM caused no cytotoxicity as determined by MTT assay. [1]
In B16F10 cells, Pinostrobin did not exhibit any cytotoxicity in the concentration range of 25 to 200 μM. [5]
In HEK293 non-cancerous cells, Pinostrobin showed negligible toxicity even at higher concentrations (4x CT50). [7]
In Wistar rats, Pinostrobin at doses between 1 and 100 mg/kg for 7 days showed no mutagenic effect. [5, 6]
Pinostrobin interacts with human serum albumin (HSA), which may affect its bioavailability, pharmacokinetics, pharmacodynamics, and half-life in the human body. [1]

[1]. Pinostrobin Inhibits Proprotein Convertase Subtilisin/Kexin-type 9 (PCSK9) Gene Expression through the Modulation of FoxO3a Protein in HepG2 Cells.J Agric Food Chem. 2018 Jun 20;66(24):6083-6093.

[2]. Activity investigation of pinostrobin towards herpes simplex virus-1 as determined by atomic force microscopy. Phytomedicine. 2011 Jan 15;18(2-3):110-8.

[3]. Pinostrobin and Cajanus lactone isolated from Cajanus cajan (L.) leaves inhibits TNF-α and IL-1β production: in vitro and in vivo experimentation. Phytomedicine. 2014 Jun 15;21(7):946-53.

[4]. Hepatoprotective effect of pinostrobin against thioacetamide-induced liver cirrhosis in rats. Saudi J Biol Sci. 2023 Jan;30(1):103506.

[5]. Discovery of Pinostrobin as a Melanogenic Agent in cAMP/PKA and p38 MAPK Signaling Pathway. Nutrients. 2022 Sep 9;14(18):3713.

[6]. Pinostrobin induces acute leukemia cell apoptosis via the regulation of miR-410-5p and SFRP5. Life Sci. 2023 Jul 15;325:121739.

[7]. Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action. PLoS One. 2018 Feb 8;13(2):e0191523.

[8]. Determination of pinostrobin in rat plasma by LC-MS/MS: application to pharmacokinetics. J Pharm Biomed Anal. 2011 Dec 5;56(4):841-5.

Pinostrobin has been reported to be detected in bees, turmeric, and other organisms with relevant data.
See also: Pinostrobin (note moved to).

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Pinostrobin is a PCSK9 inhibitor and down-regulates PCSK9 gene expression through the up-regulation of the FoxO3a level in hepatic cells. It may serve as a novel agent for cholesterol regulation and lipid management. The combination treatment of Pinostrobin and statins may counter the unwanted overexpression of PCSK9 induced by statins. [1]
Pinostrobin exerts a stimulatory effect on tyrosinase activity and melanin production without cytotoxicity by inducing expression of tyrosinase, TRP-1 and MITF. It could be used as a novel, potent and safe melanogenic agent for the prevention and/or treatment of vitiligo. [5]
Pinostrobin induces apoptosis in acute leukemia cells via inhibition of miR-410-5p and stimulation of SFRP5, which suppresses the Wnt/β-catenin signaling pathway. It has a synergistic effect with daunorubicin (DNR), enhancing DNR's cytotoxicity. [6]
Pinostrobin efficiently induces apoptosis through ROS-mediated extrinsic and intrinsic dependent signaling pathways, as well as ROS-mediated mitochondrial damage in HeLa cells. [7]
Pinostrobin is a chiral flavonoid. Only the (-)-isomer is produced by natural sources like honey, propolis, and finger roots, while chemical synthesis often results in a racemic mixture. [5]

C16H14O4

270.284

270.089

480-37-5

4101463

White to yellow solid

1.3±0.1 g/cm3

494.9±45.0 °C at 760 mmHg

100ºC

188.8±22.2 °C

0.0±1.3 mmHg at 25°C

1.612

4.11

1

4

2

20

350

0

O1C2=C([H])C(=C([H])C(=C2C(C([H])([H])C1([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)O[H])OC([H])([H])[H]

ORJDDOBAOGKRJV-AWEZNQCLSA-N

InChI=1S/C16H14O4/c1-19-11-7-12(17)16-13(18)9-14(20-15(16)8-11)10-5-3-2-4-6-10/h2-8,14,17H,9H2,1H3/t14-/m0/s1

(2S)-5-hydroxy-7-methoxy-2-phenyl-2,3-dihydrochromen-4-one

(±)-Pinostrobin; Pinostrobin

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 : ~100 mg/mL (~369.99 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.25 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.25 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 25.0 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.)