Enhances ARE-driven luciferase activity in a concentration-dependent manner in HepG2 cells; significant increase observed from 10 μM [1].

- Prevents tert-butylhydroperoxide (t-BHP, 500 μM, 4 h)-induced cell death in HepG2 cells after 12 h pretreatment (concentration-dependent); 1 h pretreatment shows no cytoprotective effect [1].

- Reduces intracellular ROS production induced by t-BHP (500 μM, 30 min) in HepG2 cells [1].

- Induces nuclear accumulation of Nrf2 in HepG2 cells (observed from 6 h exposure, concentration-dependent) [1].

- Upregulates mRNA and protein levels of antioxidant enzymes: catalytic subunit of glutamate-cysteine ligase (GCLC), modifier subunit of glutamate-cysteine ligase (GCLM), and NAD(P)H:quinone oxidoreductase 1 (NQO1) in HepG2 cells [1].

- Increases intracellular glutathione (GSH) content (~5-fold after 12 h) in HepG2 cells [1].

- Increases ERK phosphorylation (from 10 min up to 6 h) in HepG2 cells; ERK inhibition by PD98059 (10 μM) completely abolishes ARE-luciferase activity, nuclear Nrf2 accumulation, GCLC/GCLM mRNA upregulation, and cytoprotective effect [1].

- Inhibits growth of CaCo-2 colon cancer cells with IC50 = 185.6 μM (24 h treatment followed by 24 h recovery, MTT assay) [2].

ARE-luciferase reporter assay: HepG2 cells stably transfected with pGL4.37 plasmid (containing four copies of an ARE) were seeded at 2×10⁵ cells/well in 12-well plates. After overnight serum starvation, cells were treated with pachypodol for 12 h. Cells were lysed with passive lysis buffer, and luciferase activity was measured using a luciferase assay system in a luminometer [1].

- Cell viability assay (MTT): HepG2 cells were seeded at 5×10⁴ cells/well in 48-well plates. After overnight serum starvation, cells were pretreated with pachypodol for 1 h or 12 h, then exposed to 500 μM t-BHP for 4 h. MTT (0.3 mg/mL) was added for the last 1 h, formazan crystals dissolved in DMSO, and absorbance measured at 570 nm [1].

- Intracellular ROS measurement: HepG2 cells pretreated with pachypodol for 12 h were exposed to 500 μM t-BHP for 30 min, then incubated with 10 μM CM-H₂DCFDA for 25 min. Cells were fixed with 4% paraformaldehyde and examined by fluorescence microscopy; fluorescence intensity was quantified using a microplate reader (excitation 485 nm, emission 520 nm) and normalized to protein concentration [1].

- Nuclear extraction and Western blot: HepG2 cells were plated at 5×10⁵ cells/well in 6-well plates. After treatment, cells were lysed with hypotonic buffer, and nuclear fractions were extracted. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% skim milk, incubated with primary antibodies (anti-Nrf2, anti-lamin A/C, anti-α-tubulin, anti-phospho-ERK, anti-ERK, etc.) overnight at 4°C, then with HRP-conjugated secondary antibodies, and developed using ECL chemiluminescence [1].

- Real-time PCR: Total RNA extracted from HepG2 cells using Trizol, reverse transcribed to cDNA using oligo-d(T)16 primers and AMV reverse transcriptase. Real-time PCR performed with SYBR Green I Master mix using primers for GCLC, GCLM, NQO1, and GAPDH (internal control) on a LightCycler 480 II instrument [1].

- Intracellular GSH measurement: HepG2 cell pellets were resuspended in ice-cold 5% metaphosphoric acid, sonicated, centrifuged, and supernatant used for colorimetric assay using a commercial GSH kit; absorbance measured at 400 nm [1].

- CaCo-2 cytotoxicity assay (MTT-based, Promega CellTiter 96 non-radioactive cell proliferation assay): CaCo-2 cells were seeded at 8000 cells/well in 96-well plates and incubated for 48 h. Medium replaced with fresh medium containing pachypodol at final concentrations 0, 0.5, 1, 2.5, 5, 10, 15, 20, 25, 30 μM and incubated for 24 h. Medium removed, cells washed with PBS, fresh medium added, and incubated for another 24 h. MTT dye solution (15 μL) added and incubated for 4 h, then solubilization/stop solution (100 μL) added and incubated overnight. Absorbance recorded at 570 nm (reference 650 nm) [2].

Brine shrimp lethality assay: Brine shrimp eggs were hatched. Test compound (pachypodol) was applied at various concentrations. Mortality was recorded after 24 h. LD50 value (435.8 μM) was determined by Probit analysis. Percentage mortalities were adjusted using Abbott’s formula: P = (Pi - C) / (1 - C), where P = observed nonzero mortality rate, C = control mortality rate [2].

In HepG2 cells, pachypodol showed no cytotoxicity at concentrations of 3–100 μM for 24 h (MTT assay) [1].

- Brine shrimp lethality assay: LD50 = 435.8 μM after 24 h exposure [2].

- CaCo-2 colon cancer cells: IC50 = 185.6 μM (24 h treatment followed by 24 h recovery) [2].

[1]. Pachypodol, a Methoxyflavonoid Isolated from Pogostemon cablin Bentham Exerts Antioxidant and Cytoprotective Effects in HepG2 Cells: Possible Role of ERK-Dependent Nrf2 Activation. Int J Mol Sci. 2019;20(17):4082. Published 2019 Aug 21.

[2]. Pachypodol, a flavonol from the leaves of Calycopteris floribunda, inhibits the growth of CaCo 2 colon cancer cell line in vitro. Phytother Res. 2008;22(12):1684-1687.

Pachypodol is a trimethoxyflavonoid, a derivative of quercetin, with its hydroxyl groups at the 3, 7, and 3' positions replaced by methoxy groups. It has been isolated from Combretum quadrangulare and Euodia elleryana. Pachypodol functions as a plant metabolite and an antiemetic. It is a dihydroxyflavonoid and a trimethoxyflavonoid, functionally related to quercetin. Pachypodol has been reported in Melicope triphylla, Melicope semecarpifolia, and other organisms with relevant data.

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This is the first study to evaluate the antioxidant and cytoprotective effects of pachypodol in HepG2 cells and the underlying molecular mechanism (ERK-dependent Nrf2 activation) [1].

- Pachypodol (5,4′-dihydroxy-3,7,3′-trimethoxyflavone) was isolated from Pogostemon cablin Bentham (methanol extract, n-hexane fraction, centrifugal partition chromatography) and from Calycopteris floribunda Lam. (dichloromethane-methanol extract, silica gel column chromatography) [1,2].

C18H16O7

344.31

344.089

33708-72-4

5281677

White to yellow solid powder

1.5±0.1 g/cm3

598.8±50.0 °C at 760 mmHg

220.7±23.6 °C

0.0±1.8 mmHg at 25°C

1.654

2.56

2

7

4

25

532

0

KQFUXLQBMQGNRT-UHFFFAOYSA-N

InChI=1S/C18H16O7/c1-22-10-7-12(20)15-14(8-10)25-17(18(24-3)16(15)21)9-4-5-11(19)13(6-9)23-2/h4-8,19-20H,1-3H3

5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one

Pachypodol Ro-09-0179 Ro09-0179Ro 09-0179

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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 (~290.43 mM)

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.)