Osmundacetone (OAC) reduces the phosphorylation of c‑Jun NH2‑terminal kinase (JNK), extracellular signal‑regulated kinase (ERK), and p38 mitogen‑activated protein kinases (MAPKs). It also increases the expression of heat shock protein 70 (HSP70) and heme oxygenase‑1 (HO‑1). [1]

The phosphorylation of MAPKs, such as JNK, ERK, and p38 kinase, is strongly inhibited by lithoacetone [1].

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Osmundacetone (OAC) at 2 μM restored cell viability to 98.26% ± 1.03% against 5 mM glutamate‑induced toxicity in HT22 cells, while N‑acetylcysteine (2 mM) restored viability to 90.39% ± 1.18%. OAC prevented glutamate‑induced morphological changes. [1]
In the DPPH radical scavenging assay, OAC showed antioxidant activity with an IC₅₀ of 7.88 ± 0.02 μM. [1]
OAC significantly reduced glutamate‑induced intracellular reactive oxygen species (ROS) accumulation in a dose‑dependent manner (1 and 2 μM), as measured by H₂DCF‑DA staining and fluorescence microscopy. [1]
Western blot analysis showed that OAC induced the expression of HSP70 and HO‑1 in HT22 cells. [1]
Hoechst 33342 staining revealed that OAC (2 μM) prevented glutamate‑induced chromatin condensation. [1]
Fluo‑4 AM staining showed that OAC reduced the accumulation of intracellular calcium (Ca²⁺) induced by glutamate after 8 h. [1]
An image‑based apoptosis assay (annexin V‑Alexa Fluor 488 / propidium iodide) demonstrated that glutamate increased apoptotic cells to 58% ± 2.08%, while treatment with OAC at 1 μM and 2 μM decreased apoptosis to 47.33% ± 0.88% and 35.33% ± 0.88%, respectively. [1]
Western blot analysis indicated that OAC markedly reduced the phosphorylation of JNK, ERK, and p38 kinases induced by glutamate in HT22 cells. [1]

The antioxidant activity of osmundacetone (OAC) was evaluated using the DPPH (2,2‑diphenyl‑1‑picrylhydrazyl) radical scavenging assay. OAC was tested at six concentrations (0–16 μM, including control) by mixing with 60 μM DPPH at a 1:1 ratio in ethanol as the solvent. After 30 min at room temperature protected from light, the absorbance of the reaction mixture was measured at 550 nm using a microplate reader. The IC₅₀ value was calculated as the concentration giving half‑maximal DPPH scavenging activity. [1]

HT22 mouse hippocampal neuronal cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37 °C in a humidified 5% CO₂ atmosphere. [1]
For cell viability assay, cells were seeded in 96‑well plates at 1×10⁴ cells/well, then co‑treated with 5 mM glutamate and osmundacetone (OAC) (0.5–4.0 μM) for 24 h. After treatment, 10 μL of EZ‑Cytox assay reagent was added to each well, incubated for 30 min at 37 °C, and absorbance was measured at 450 nm. Cell viability was expressed as percentage relative to the control group (non‑treated). [1]
For ROS assay, cells were co‑treated with 5 mM glutamate and OAC for 8 h, then stained with 10 μM H₂DCF‑DA in culture medium for 30 min. After washing with DPBS, fluorescence intensity was measured at excitation 488 nm and emission 525 nm using a fluorescence microplate reader. The fold increase was calculated relative to the control group. [1]
For intracellular calcium staining, cells were co‑treated with 5 mM glutamate and OAC for 8 h, then stained with 2.5 μM Fluo‑4 AM for 30 min. After washing, fluorescence was measured at excitation 488 nm and emission 525 nm. [1]
For Hoechst 33342 staining, cells were treated with OAC in the presence or absence of 5 mM glutamate for 12 h, then stained with Hoechst 33342 for 10 min protected from light, washed with DPBS, and observed under fluorescence microscopy for chromatin condensation. [1]
For image‑based apoptosis detection, cells were co‑treated with 5 mM glutamate and OAC for 12 h, then collected, washed, and stained with annexin V‑Alexa Fluor 488 and propidium iodide. Apoptotic and dead cells were determined using an image‑based cytometer (excitation 458 nm for green channel, 530 nm for red channel). [1]
For Western blotting, cells were treated with OAC in the presence or absence of 5 mM glutamate for 6 h, then lysed in RIPA buffer containing protease inhibitor cocktail. Protein concentration was determined by BCA assay. Equal amounts of protein (8 μg/lane) were electrophoresed on 10% SDS‑polyacrylamide gels, transferred to PVDF membranes, and probed with primary antibodies against phospho‑JNK, JNK, phospho‑ERK, ERK, phospho‑p38, p38, HSP70, HO‑1, and GAPDH (loading control), followed by secondary antibodies. Bound antibodies were detected by chemiluminescence. [1]

The provided reference notes that osmundacetone (OAC) at 2 μM slightly stimulated both ROS and Ca²⁺ accumulation as well as increased p38 phosphorylation compared to the control group, indicating a weak induction of oxidative stress in HT22 cells. However, no quantitative toxicity data (e.g., LD₅₀, cytotoxicity threshold) or toxicokinetic parameters are reported. [1]

[1]. Isolation and determination of osmundacetone in Osmundae Rhizoma. Journal of Chinese Pharmaceutical Sciences 45(21):1612-1614.

[2]. Protective Effect of Osmundacetone against Neurological Cell Death Caused by Oxidative Glutamate Toxicity. Biomolecules. 2021 Feb; 11(2): 328.

(E)-3,4-dihydroxyphenylmethylene acetone has been reported in Peltigera dolichorriza, Phellinus linteus and Inonotus obliquus, and available data are available.

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Osmundacetone (OAC) exerts neuroprotection against oxidative glutamate toxicity in HT22 cells by reducing ROS accumulation, inducing HSP70 and HO‑1 expression (activating the cellular self‑defense mechanism), inhibiting chromatin condensation and intracellular Ca²⁺ overload, suppressing apoptosis, and downregulating the phosphorylation of MAPKs (JNK, ERK, p38). The protective mechanism is illustrated in Figure 5 of the reference. The study suggests that OAC is a potential antioxidant agent for further in vivo studies and clinical trials in neurological disorders. [1]

C10H10O3

178.062

37079-84-8

(E)-Osmundacetone;123694-03-1

9942292

White to light yellow solid powder

1.3±0.1 g/cm3

382.5±32.0 °C at 760 mmHg

199.3±21.6 °C

0.0±0.9 mmHg at 25°C

1.637

1.18

2

3

2

13

210

0

CC(=O)/C=C/C1=CC(=C(C=C1)O)O

YIFZKRGUGKLILR-NSCUHMNNSA-N

InChI=1S/C10H10O3/c1-7(11)2-3-8-4-5-9(12)10(13)6-8/h2-6,12-13H,1H3/b3-2+

(E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)