One possible treatment for glutamate-induced oxidative damage is gartanin, which works by upregulating the Nrf-2-independent HO-1 and AMPK/SIRT1/PGC-1α signaling pathways [1]. Gartanin inhibits the migration of human glioma cells by triggering cell cycle arrest and autophagy through the PI3K/Akt/mTOR and MAPK signaling pathways [1].

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In HT22 mouse hippocampal neuronal cells, Gartanin (1‑10 µM) significantly attenuated glutamate (2 mM, 24 h)-induced oxidative toxicity and cell death. MTT assay showed an optimal concentration of 3 µM, while 10 µM slightly inhibited MTT reduction. [1]
Gartanin (3 µM, 30 min pretreatment) decreased glutamate (2 mM, 12 h)-induced apoptosis in HT22 cells. Annexin V‑FITC/PI flow cytometry showed early apoptotic cells increased from 1.03% to 16.03% with glutamate alone, but were reduced to 1.19% with Gartanin pretreatment. [1]
Gartanin (3 µM) increased Bcl‑2 protein expression in a time-dependent manner (0‑24 h) without affecting Bax, thereby increasing the Bcl‑2/Bax ratio in HT22 cells. Gartanin also maintained Bcl‑2 levels down‑regulated by glutamate (2 mM). [1]
Gartanin (3 µM, 30 min pretreatment) reduced glutamate (2 mM, 12 h)-induced intracellular ROS accumulation in HT22 cells, measured by H₂DCF‑DA and DHE fluorescence (microscopy and flow cytometry). [1]
Gartanin (3 µM, 30 min pretreatment) significantly reduced glutamate (2 mM, 12 h)-induced mitochondrial membrane depolarization in HT22 cells, measured by Rhodamine 123 flow cytometry. [1]
Gartanin promoted HO‑1 protein expression in HT22 cells in a time‑dependent (0‑24 h) and concentration‑dependent (0.3‑10 µM) manner, independent of Nrf‑2 (no change in Nrf‑2 protein or ARE‑luciferase activity). HO‑1 siRNA (sequence 1) partially blocked the neuroprotective effect of Gartanin, confirming HO‑1 involvement. [1]
Gartanin (3 µM) activated AMPK signaling in HT22 cells: increased p‑AMPKα (Thr172) at 3‑12 h, and elevated SIRT1 and PGC‑1α protein levels. [1]
In human HCC cell lines Hep3B, HepG2 and Huh7, Gartanin (10‑40 µmol/L, 24 h) inhibited cell proliferation in a dose‑dependent manner (MTT assay). [2]
In Hep3B cells, Gartanin (40 µmol/L, 24 h) increased sub‑G1 phase population and Annexin V positivity, activated caspase‑8, ‑9 and ‑3, and enhanced Bax expression. [2]
Gartanin induced autophagy in Hep3B, HepG2 and Huh7 cells: increased acidic vesicular organelle (AO staining), LC3‑I to LC3‑II conversion, Atg5 expression, and GFP‑LC3 punctate formation. Gartanin failed to induce autophagy in ATG5 knockout MEFs. [2]
Gartanin induced dose‑dependent loss of mitochondrial membrane potential in Hep3B cells (MitoTracker staining, flow cytometry). Transmission electron microscopy confirmed mitochondrial disruption, autophagosomes and autolysosomes. [2]
In Hep3B cells, autophagy induced by Gartanin was protective; co‑treatment with autophagy inhibitors (3‑MA or bafilomycin A1) or Atg5 silencing enhanced apoptosis (increased sub‑G1 and caspase‑3 cleavage). [2]
Gartanin activated JNK (within 0.5 h, sustained for ≥6 h) and induced JNK‑dependent phosphorylation of Bcl‑2 at Ser70 in Hep3B cells. JNK inhibitor SP600125 inhibited Gartanin‑induced autophagy (AVO formation) and promoted Gartanin‑induced caspase‑3 activation. [2]
Gartanin induced autophagy in Hep3B cells via an mTOR‑independent pathway, as it increased phosphorylation of mTOR (S2448, S2481) and p70S6K. [2]

- HT22 cell viability (MTT): Cells seeded in 96‑well plates (4×10³ cells/well) overnight. Treated with Gartanin or DMSO for 30 min, then with/without 2 mM glutamate for 24 h. Added 10 µL of 5 mg/mL MTT per well for 2 h at 37 °C. Replaced medium with 100 µL DMSO, shook for 15 min at 37 °C, measured absorbance at 570 nm. [1]
- Apoptosis by flow cytometry (HT22): Cells harvested, centrifuged, resuspended in 200 µL binding buffer. Incubated with 5 µL Annexin V‑FITC for 10 min, then 10 µL PI for 15 min at room temperature, immediately analyzed by flow cytometry. [1]
- Intracellular ROS measurement (HT22): Cells in 48‑well plates (2×10⁴ cells/well) pretreated with Gartanin or DMSO for 30 min, then 2 mM glutamate for 12 h. Washed twice with PBS, incubated with 10 µM H₂DCF‑DA or DHE in serum‑free medium for 30 min at 37 °C in the dark. Washed twice with PBS, photographed under fluorescence microscope or analyzed by flow cytometry. [1]
- ΔΨm measurement (HT22): After treatment, cells incubated with 5 µM Rhodamine 123 in PBS for 15 min at 37 °C in the dark. Washed with PBS, incubated at 37 °C, then measured by flow cytometry. [1]
- Western blot (HT22 and HCC cells): Cells lysed, protein quantified and denatured. 20 µg protein per well separated by 10% SDS‑PAGE, transferred to PVDF membranes. Blocked with 5% skim milk for 2 h, incubated with primary antibodies overnight at 4 °C. Washed with TBST, incubated with HRP‑linked secondary antibodies (1:1000) for 1 h at room temperature. Detected by ECL system. [1][2]
- ARE‑luciferase reporter assay (HT22): Cells in 48‑well plates (1×10⁵ cells/well) transfected with pARE‑luc or pGL6‑luc plus pRL‑TK renilla using 200 ng DNA, 0.3 µL Lipofectamine 3000 and 0.4 µL P3000/well. After 24 h, treated with Gartanin or t‑BHQ. Luciferase activity measured at 24 h by dual luciferase assay; Renilla normalized to firefly. [1]
- HO‑1 siRNA transfection (HT22): Cells at ~50% confluence transfected with 100 nM HO‑1 siRNA duplexes or non‑targeting control using Lipofectamine 3000. After 6 h, medium changed, incubated for another 48 h, then processed for MTT or western blot. [1]
- Acidic vesicular organelle (AVO) staining (HCC): Cells treated with Gartanin, stained with 1 µg/mL acridine orange for 15 min. Observed under fluorescence microscope or detached and analyzed by flow cytometry (FACScan, CellQuest software). [2]
- GFP‑LC3 translocation assay (Hep3B): Cells transfected with GFP‑LC3 plasmid, then treated with Gartanin. Translocation observed by fluorescence microscopy; fluorescence intensity assessed by flow cytometry. [2]
- Mitochondrial labelling (Hep3B): Treated cells stained with 1 µg/mL MitoTracker for 15 min, observed under fluorescence microscope and analyzed by flow cytometry. [2]
- Cell cycle/DNA content analysis (HCC): Cells fixed with 1 U/mL RNase A and 10 µg/mL propidium iodide overnight at room temperature in the dark, analyzed by flow cytometry for sub‑G1 population. Annexin V staining also performed. [2]

[1]. Gartanin Protects Neurons against Glutamate-Induced Cell Death in HT22 Cells: Independence of Nrf-2 but Involvement of HO-1 and AMPK. Neurochem Res. 2016 Sep;41(9):2267-77.

[2]. Gartanin induces autophagy through JNK activation which extenuates caspase-dependent apoptosis. Oncol Rep. 2015 Jul;34(1):139-46.

Gartanin belongs to the xanthone class of compounds, with the structure 9H-xanthone-9-one, substituted with hydroxyl groups at positions 1, 3, 5, and 8, and with isopentenyl groups at positions 2 and 4. It possesses antitumor activity and is also a plant metabolite. Gartanin is a polyphenol. It has been reported to exist in plants of the genus Garcinia anomala, Maclura tinctoria, and other organisms with relevant data.

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Oxidative stress is implicated in neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Gartanin was investigated for neuroprotection against glutamate‑induced oxidative stress in HT22 cells, a model of oxidative neurotoxicity (these cells lack ionotropic glutamate receptors). [1]
The neuroprotection study was supported by Guangdong Provincial International Cooperation Project of Science & Technology (No. 2013B051000038), National Natural Science Foundation for China (No. 31371070) and the Fundamental Research Funds for the Central Universities (No. 15ykjc08b). [1]
Hepatocellular carcinoma (HCC) is the third leading cause of cancer‑related death worldwide. In HCC, Gartanin induced both extrinsic and intrinsic apoptotic pathways and protective autophagy; inhibition of autophagy enhanced its apoptotic effect. The JNK‑Bcl‑2 pathway was identified as a critical regulator of Gartanin‑induced protective autophagy and a potential drug target for chemotherapeutic combination. [2]
The HCC study was supported in part by the Daegu University Research Grant, 2011. [2]

C23H24O6

396.4331

396.157

C, 69.68; H, 6.10; O, 24.21

33390-42-0

5281633

Light yellow to yellow solid

1.3±0.1 g/cm3

644.4±55.0 °C at 760 mmHg

167 °C

224.9±25.0 °C

0.0±2.0 mmHg at 25°C

1.656

4.51

4

6

4

29

662

0

O1C2=C(C([H])=C([H])C(=C2C(C2=C(C(C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H])=C(C(C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H])=C12)O[H])O[H])=O)O[H])O[H]

OJXQLGQIDIPMTE-UHFFFAOYSA-N

InChI=1S/C23H24O6/c1-11(2)5-7-13-19(26)14(8-6-12(3)4)22-18(20(13)27)21(28)17-15(24)9-10-16(25)23(17)29-22/h5-6,9-10,24-27H,7-8H2,1-4H3

1,3,5,8-tetrahydroxy-2,4-bis(3-methylbut-2-enyl)xanthen-9-one

NSC692946; NSC 692946; NSC-692946; Gartanin

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 : ~125 mg/mL (~315.31 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.)