- Xanthine oxidase (XO): IC₅₀ = 30 μM [1].
- DPPH radical: IC₅₀ = 7.5 μM [1].
- Superoxide anion (O₂•⁻): IC₅₀ = 10 μM [1].
- Peroxyl radical (RO₂•): IC₅₀ = 30 μM [1].
- Iron ions (Fe²⁺/Fe³⁺): metal chelation [1].
- NF‑κB, ERK, p38 (involved in apoptosis regulation) [2].

By causing cell opening, ending the sub-G1 phase, increasing the number of cells, DNA fragmentation, and oxygen activity content, thounningianin A efficiently suppresses the proliferation of HepG-2 cells [2].

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- Thonningianin A (Th A) (10 μM) inhibited NADPH‑induced lipid peroxidation in rat liver microsomes by 61% and Fe²⁺/ascorbate‑induced lipid peroxidation by 65% [1].
- At 10 μM, Th A inhibited NADPH‑dependent lipid peroxidation without affecting cytochrome P450 activity (aniline hydroxylase) [1].
- Th A dose‑dependently scavenged DPPH radical (IC₅₀ = 7.5 μM; complete scavenging at 25 μM), superoxide anion (IC₅₀ = 10 μM), and peroxyl radical (IC₅₀ = 30 μM) as measured by ESR [1].
- In the deoxyribose assay, Th A (10 μM) inhibited deoxyribose degradation by 15% in the presence of EDTA and by 77% in the absence of EDTA, indicating strong iron‑chelating ability and moderate hydroxyl radical scavenging [1].
- Th A inhibited xanthine oxidase activity dose‑dependently: 18% inhibition at 10 μM, IC₅₀ = 30 μM [1].
- Th A (25 and 100 μM) inhibited aniline hydroxylase (CYP) activity by 5% and 40%, respectively; no inhibition at 10 μM [1].
- Th A inhibited proliferation of HepG‑2 cells (IC₅₀ = 32.61 ± 0.78 μg/mL) and LO2 normal hepatocytes (IC₅₀ = 50.91 ± 1.03 μg/mL) after 24 h treatment [2].
- Th A (25–45 μg/mL, 24 h) induced apoptosis in HepG‑2 cells: apoptosis rate reached 82.6% at 45 μg/mL (Annexin V‑FITC/PI staining) [2].
- Th A induced sub‑G1 cell fraction dose‑dependently: 1.7% at 12.5 μg/mL, 3.0% at 25 μg/mL, 13.3% at 35 μg/mL (24 h); also increased G0/G1 phase percentage [2].
- Th A (45 μg/mL, 24 h) increased intracellular ROS levels by approximately 250% compared to control (DCFH‑DA probe) [2].
- Th A (25–45 μg/mL, 24 h) decreased mitochondrial membrane potential (Δψₘ): red fluorescence reduced from 99.4% to 69.3%, green fluorescence increased from 0.6% to 30.7% (JC‑1 staining) [2].
- Th A down‑regulated Bcl‑xL, cyclin D1, and CDK4 mRNA expression in HepG‑2 cells (qPCR) [2].
- Th A induced cleavage of caspase‑3 and caspase‑9 in a dose‑dependent manner (Western blot) [2].
- Th A decreased nuclear p65 protein level (NF‑κB), decreased phosphorylated ERK, and increased phosphorylated p38 (Western blot) [2].

- Xanthine oxidase activity assay: The assay mixture (total volume 535 μL) contained 50 mM potassium phosphate buffer (pH 7.5), 0.15 mM xanthine solution, and 1% methanol or various concentrations of Th A. The reaction was initiated by adding 35 μL of xanthine oxidase solution (final concentration 0.2 U/mL). Changes in absorbance were recorded at 295 nm for 3 min at room temperature. Allopurinol was used as a standard inhibitor [1].
- Aniline hydroxylase (CYP) activity assay: The activity was evaluated by estimating p‑aminophenol formation in washed rat liver microsomes. Microsomes were incubated with aniline and NADPH‑generating system, and p‑aminophenol was measured spectrophotometrically. The effect of Th A (1–100 μM) on this monooxygenase activity was tested [1].
- Deoxyribose assay for hydroxyl radical scavenging and iron chelation: The reaction mixture (1 mL final volume) contained 20 mM KH₂PO₄‑KOH buffer (pH 7.4), 2.8 mM deoxyribose, 100 μM FeCl₃, 104 μM EDTA (when added), 300 μM H₂O₂, and various concentrations of Th A (1–10 μM) or T. sanguinea extract. Ascorbic acid (100 μM) was added to start the reaction. After 1 h incubation at 37 °C, 1 mL of 1% thiobarbituric acid (in 0.05 M NaOH) and 1 mL of 2.8% trichloroacetic acid were added, followed by heating at 100 °C for 20 min. The absorbance of the malonaldehyde product was measured at 532 nm. Controls without deoxyribose were performed to exclude false positives [1].

- MTT cytotoxicity assay: HepG‑2 and LO2 cells (5 × 10⁴ cells/mL) were seeded in 96‑well plates and incubated for 24 h. After removing supernatant, fresh media containing different concentrations of Th A (20–100 μg/mL) were added and incubated for another 24 h. Then 100 μL of 0.1% MTT (0.5 mg/mL) was added to each well for 4 h at 37 °C. Formazan crystals were dissolved in dimethyl sulfoxide, and absorbance was measured at 570 nm. Cell proliferation inhibition rate was calculated as 1 – (CI_sample/CI_control) × 100% [2].
- Real‑time cell proliferation monitoring: HepG‑2 cells (4 × 10³ cells/well) were seeded in 96‑well E‑plates with integrated microelectronic sensors. After 30 min equilibration, the plate was placed in a Real‑Time Cell Electronic Sensing System. After 24 h, media were replaced with fresh media containing different concentrations of Th A, and cell index was recorded continuously for 24 h [2].
- Apoptosis analysis by flow cytometry: HepG‑2 cells treated with Th A (25, 35, 45 μg/mL) for 24 h were harvested, washed with PBS, resuspended in binding buffer (2 × 10⁶ cells/mL), and stained with 5 μL Annexin V‑FITC and 5 μL propidium iodide for 15 min on ice. Apoptotic rate was determined at 488 nm using flow cytometry (10,000 events per sample) [2].
- Cell cycle analysis: HepG‑2 cells treated with Th A (12.5, 25, 35 μg/mL) for 24 h were fixed in 75% ethanol at –20 °C overnight, then stained with 500 μL propidium iodide working solution for 4 h in the dark. Cell cycle distribution (including sub‑G1 peak) was analyzed by flow cytometry using MultiCycle software [2].
- ROS measurement: Treated HepG‑2 cells were harvested, washed with PBS, and suspended in PBS containing 10 μM DCFH‑DA. After 20 min incubation at 37 °C, fluorescence intensity was measured with excitation at 488 nm and emission at 525 nm. Relative DCF fluorescence was expressed as percentage of control [2].
- Mitochondrial membrane potential (Δψₘ) assay: HepG‑2 cells treated with Th A (25, 35, 45 μg/mL) for 24 h were washed with PBS and incubated with 2 μg/mL JC‑1 for 30 min at 37 °C in 5% CO₂. The red and green fluorescence intensities were measured by flow cytometry [2].
- Quantitative real‑time PCR: Total RNA was extracted using Trizol reagent and reverse transcribed. qPCR was performed using SYBR Green I with primers for Bcl‑xL, CDK4, cyclin D1, and β‑actin as internal control. Relative expression was calculated by 2⁻ΔΔCt method [2].
- Western blot analysis: Total cellular proteins were lysed in RIPA buffer (50 mM Tris‑HCl pH 7.4, 150 mM NaCl, 1% Nonidet P‑40, 0.1% SDS). Protein concentrations were determined by BCA assay. Equal amounts (20 μg) were separated by 12% SDS‑PAGE, transferred to PVDF membranes, blocked with 5% non‑fat milk, and incubated with primary antibodies (caspase‑3, caspase‑9, NF‑κB, phospho‑ERK, phospho‑p38, GAPDH) at 1:1000 dilution overnight at 4 °C, followed by HRP‑conjugated secondary antibodies (1:2000) for 1 h. Bands were visualized by ECL and densitometry [2].

- Th A showed moderate cytotoxicity in normal human hepatocyte LO2 cells with an IC₅₀ of 50.91 ± 1.03 μg/mL after 24 h treatment [2].
- At 10 μM, Th A did not inhibit rat liver microsomal aniline hydroxylase (CYP) activity, suggesting low acute enzyme inhibition at that concentration; higher concentrations (25 μM, 100 μM) caused 5% and 40% inhibition, respectively [1].

[1]. Antioxidant properties of Thonningianin A, isolated from the African medicinal herb, Thonningia sanguinea. Biochem Pharmacol. 2002 May 1;63(9):1725-37.

[2]. Effects of thonningianin A in natural foods on apoptosis and cell cycle arrest of HepG-2 human hepatocellular carcinoma cells. Food Funct. 2015 Aug;6(8):2588-97.

Thonningianin A is a tannin. It functions as a metabolite. There are reports that Thonningia sanguinea contains Thonningianin A, and relevant data are available for reference.

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- Thonningianin A is an ellagitannin isolated from the roots of Thonningia sanguinea Vahl (Balanophoraceae), a medicinal herb used prophylactically against bronchial asthma in Africa [1].
- The structure of Th A consists of gallic acid esters of glucose and dihydrochalcone; its molecular weight is approximately 875 Da [2].
- Th A exhibits strong antioxidant properties including radical scavenging (DPPH, superoxide, peroxyl), anti‑superoxide formation (via XO inhibition), and metal chelation (iron) [1].
- In HepG‑2 cells, Th A induces apoptosis through the intrinsic mitochondrial pathway: ROS generation → loss of Δψₘ → down‑regulation of Bcl‑xL → activation of caspase‑9 and caspase‑3. It also causes G0/G1 cell cycle arrest by down‑regulating cyclin D1 and CDK4, and inhibits the NF‑κB survival pathway while modulating MAPK (decreasing p‑ERK, increasing p‑p38) [2].
- The anti‑cancer activity of Th A was confirmed by DNA fragmentation and cell cycle arrest patterns [2].

C42H34O21

874.7068

872.179

271579-11-4

10328286

White to yellow solid

1.7±0.1 g/cm3

1365.2±65.0 °C at 760 mmHg

412.3±27.8 °C

0.0±0.3 mmHg at 25°C

1.733

6.13

12

21

9

63

1590

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]2([H])[C@@]1([H])C([H])([H])OC(C1=C([H])C(=C(C(=C1C1=C(C(=C(C([H])=C1C(=O)O2)O[H])O[H])O[H])O[H])O[H])O[H])=O)OC(C1C([H])=C(C(=C(C=1[H])O[H])O[H])O[H])=O)O[H])OC1C([H])=C(C(C(C([H])([H])C([H])([H])C2C([H])=C([H])C([H])=C([H])C=2[H])=O)=C(C=1[H])O[H])O[H]

XQVKQEFQGYTUAR-VHBRHXFYSA-N

InChI=1S/C42H34O21/c43-20(7-6-15-4-2-1-3-5-15)30-21(44)10-17(11-22(30)45)60-42-36(55)38(63-39(56)16-8-23(46)31(50)24(47)9-16)37-27(61-42)14-59-40(57)18-12-25(48)32(51)34(53)28(18)29-19(41(58)62-37)13-26(49)33(52)35(29)54/h1-5,8-13,27,36-38,42,44-55H,6-7,14H2/t27-,36-,37-,38-,42-/m1/s1

(10R,11R,12R,13S,15R)-13-[3,5-dihydroxy-4-(3-phenylpropanoyl)phenoxy]-3,4,5,12,21,22,23-heptahydroxy-8,18-dioxo-9,14,17-trioxatetracyclo[17.4.0.02,7.010,15]tricosa-1(23),2,4,6,19,21-hexaen-11-yl] 3,4,5-trihydroxybenzoate

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 (~114.32 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.86 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 (2.86 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.)