- Sedanolide modulates the following targets/pathways :
- PI3K-I/Akt/mTOR pathway (decreases protein levels of PI3K-I, Akt, and mTOR). [2]
- Beclin-1/PI3K-III/LC3-II pathway (increases protein levels of Beclin-1, PI3K-III, and LC3-II). [2]
- p53 signaling (increases nuclear p53 and DRAM; decreases cytosolic p53 and TIGAR). [2]
- NF-κB activation (increases phosphorylation of IκB, nuclear translocation of p65, and NF-κB-DNA binding activity). [2]

Sedanolide activates the PI3K, p53, and NF-κB signaling pathways in human hepatoma cells to cause autophagy [2].

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- In HepG2 and CaCo-2 cells, a 24-hour exposure to Sedanolide at concentrations ranging from 7 to 500 μM did not affect cell viability as measured by MTT assay. However, when cells were cultured in Sedanolide-free medium for a further 72 hours (two cell cycles), a decrease in viability was observed in HepG2 cells previously exposed to 500 μM Sedanolide (IC₅₀ value for HepG2 cells: 431.4 ± 37.0 μM; for CaCo-2 cells: >500 μM). [1]
- Pretreatment of HepG2 or CaCo-2 cells with 100 μM Sedanolide for 24 hours followed by exposure to H₂O₂ or tBOOH (7-500 μM, 30 min) did not result in a statistically significant difference in cell viability compared to controls. IC₅₀ values for H₂O₂ alone: CaCo-2 233.7 ± 60.3 μM, HepG2 300.8 ± 45.8 μM; for tBOOH alone: CaCo-2 46.6 ± 21.6 μM, HepG2 126.1 ± 10.9 μM; for 100 μM Sedanolide + H₂O₂: >500 μM for both cell lines; for 100 μM Sedanolide + tBOOH: CaCo-2 122.6 ± 77.8 μM, HepG2 221.5 ± 110.6 μM. [1]
- In the comet assay, a 24-hour incubation with 500 μM Sedanolide induced a significant increase (p < 0.05) in DNA strand breaks in HepG2 cells (arbitrary units increased), but not in CaCo-2 cells. Lower concentrations (10-250 μM) did not increase strand breaks. Viability prior to lysis exceeded 80% by FDA/EtBr assay. Sedanolide (100 μM, 24 h pretreatment) did not protect against H₂O₂- or tBOOH-induced DNA strand breaks in either cell line. [1]
- In human liver cancer J5 cells, Sedanolide (100, 250, or 500 μM for 24 h) suppressed cell viability as measured by MTT assay: 101±8%, 96±7%, and 71±7% respectively, with significant reduction at 500 μM (p < 0.05) compared to control (100%). Morphological changes were observed at 500 μM. [2]
- Autophagy induction was assessed by monodansylcadaverine (MDC) staining. After 24 h treatment with 100, 250, or 500 μM Sedanolide, MDC fluorescence (indicating autophagic vacuoles) significantly increased to 35±5%, 46±8%, and 83±9% respectively, compared to control (6±3%) (p < 0.05). [2]
- Immunoblot analysis in J5 cells treated with Sedanolide (250 or 500 μM, 24 h) showed significant decreases in PI3K-I protein levels (89±7% and 78±4% of control), Akt (86±5% and 62±3%), and mTOR (75±4%, 65±3%, and 53±4% for 100, 250, and 500 μM respectively) (p < 0.05). [2]
- In J5 cells, Sedanolide (250 or 500 μM, 24 h) significantly increased Beclin-1 (144±14% at 500 μM), PI3K-III (124±12% at 250 μM, 164±18% at 500 μM), and LC3-II (167±13% at 250 μM, 185±15% at 500 μM) protein levels compared to control (100%) (p < 0.05). [2]
- Sedanolide (500 μM, 24 h) significantly increased nuclear p53 (183±17%) and DRAM (168±14%) protein levels, while decreasing cytosolic p53 (45±13%) and TIGAR (56±13%) compared to control (100%) (p < 0.05). [2]
- Sedanolide (250 or 500 μM, 24 h) increased p-IκB protein levels (256% and 278% of control), decreased nuclear p65 (334% and 345%), and increased NF-κB-DNA binding activity (p < 0.05). [2]

In the liver, small intestine mucosa, and forestomach of A/J mice, sedum lactone can enhance glutathione-S-transferase (GST) activity [1].

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- MTT cell viability assay (first study): HepG2 and CaCo-2 cells were seeded in 96-well microtiter plates and allowed to adhere overnight. Cells were incubated with increasing concentrations of Sedanolide (7-500 μM) for 24 hours. After washing twice with phosphate-buffered saline (PBS), cells were allowed to grow for a further two cell cycles (approximately 72 hours) in complete media without Sedanolide. Cell survival was measured by monitoring the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at 540 nm. MTT (0.6 mg/mL) was added for 3 hours at 37°C, then the medium was removed, and the dye was solubilized in isopropanol. Absorbance was read using a 96-well microtiter plate reader. IC₅₀ values were calculated. [1]
- Fluorescein diacetate/ethidium bromide (FDA/EtBr) viability assay: Cells were mixed 1:1 (v/v) with a solution of FDA and EtBr, incubated at 37°C for 2-5 minutes, then layered onto a microscope slide. Live cells fluoresce green, dead cells red, and dying cells have a green cytoplasm and red nucleus. Samples were examined at 400× magnification using a fluorescence microscope with blue light (450-490 nm). Two hundred cells were scored per slide. This assay was used to monitor cell viability prior to the lysis step of the comet assay. [1]
- Comet assay (single-cell gel electrophoresis): HepG2 or CaCo-2 cells were plated in 3.5-cm tissue culture dishes and allowed to adhere overnight. To assess background DNA damage, cells were treated with Sedanolide (10-500 μM) for 24 hours. To assess protection, cells were pretreated with 100 μM Sedanolide for 24 hours then exposed to H₂O₂ or tBOOH (1-100 μM, 30 min). Cells were embedded in 1% low melting point agarose on glass slides, immersed in lysing solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100, 10% DMSO, pH 10) for 90 minutes at 4°C. Alkaline treatment (40 min, 4°C) allowed DNA unwinding. Electrophoresis was performed at 20 V for 25 min (300 mA). Slides were rinsed, neutralized, and stained with EtBr. Nuclei were scored visually using a fluorescence microscope, with 100 nuclei per sample scored from 0 (undamaged) to 4 (severely damaged). Arbitrary units were calculated. [1]
- MTT cell viability assay (second study): J5 human hepatocellular carcinoma cells were plated at 1×10⁶ cells per 3-cm plate for 24 hours, then treated with 100, 250, or 500 μM Sedanolide for 24 hours. MTT reagent (5 mg/mL) was added, and cell viability was determined by measuring optical density at 570 nm. Morphological changes were examined using a phase-contrast microscope. [2]
- Monodansylcadaverine (MDC) staining for autophagy: J5 cells grown on coverslips were treated with 100, 250, or 500 μM Sedanolide for 24 hours. Cells were then incubated with 0.1 mM MDC for 15 minutes. Micrographs were acquired at 400× magnification on an inverted fluorescence microscope. MDC-stained cells were measured fluorometrically at excitation 360 nm and emission 530 nm. [2]
- Immunoblot analysis of protein expression: J5 cells were treated with Sedanolide (100, 250, or 500 μM) for 24 hours. Total protein was collected, and nuclear extracts were obtained using an NE-PER extraction kit. Cytoplasmic and nuclear protein expression of PI3K-I, PI3K-III, Akt, mTOR, Beclin-1, LC3-II, p53, DRAM, TIGAR, p-IκB, IκB, and NF-κB (p65) were determined by SDS-PAGE and immunoblotting. Bands were visualized using hydrogen peroxide/tetrahydrochloride diaminobenzidine or an enhanced chemiluminescent detection kit and quantitated with an AlphaImager 2000. [2]
- NF-κB-DNA binding activity assay: Nuclear extracts from J5 cells treated with Sedanolide (100, 250, or 500 μM for 24 h) were obtained using an NE-PER extraction kit. NF-κB-DNA binding activity was determined using an NF-κB (p65) transcription factor activity assay kit according to the manufacturer's instructions. [2]

- In HepG2 and CaCo-2 cells, a 24-hour exposure to Sedanolide up to 500 μM was not toxic as measured by MTT assay (120±10% survival for HepG2, 110±20% for CaCo-2). However, after culturing in Sedanolide-free medium for 72 hours, decreased viability was observed in HepG2 cells with an IC₅₀ of 431.4 ± 37.0 μM; CaCo-2 cells had IC₅₀ >500 μM. [1]
- In the comet assay, 500 μM Sedanolide (24 h) induced a significant increase in DNA strand breaks in HepG2 cells (p < 0.05), but not in CaCo-2 cells. This increase was not accompanied by decreased cell viability as measured by MTT or FDA/EtBr assays. [1]
- In J5 human liver cancer cells, Sedanolide at 500 μM for 24 h significantly reduced cell viability to 71±7% (p < 0.05) as measured by MTT assay. [2]

[1]. Sedanolide, a Natural Phthalide From Celery Seed Oil: Effect on Hydrogen Peroxide and Tert-Butyl Hydroperoxide-Induced Toxicity in HepG2 and CaCo-2 Human Cell Lines. In Vitr Mol Toxicol. Fall 2001;14(3):233-40.

[2]. Sedanolide Induces Autophagy Through the PI3K, p53 and NF-κB Signaling Pathways in Human Liver Cancer Cells. Int J Oncol. 2015 Dec;47(6):2240-6.

Sedanolide belongs to the 2-benzofuran class of compounds. It has been reported to exist in Ligusticum chuanxiong, Angelica sinensis, and other organisms with relevant data. See also: Neo-Nonide (note moved to).

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- Sedanolide is a phthalide-like compound (3-butyl-3a,4,5,6-tetrahydro-1(3H)-isobenzofuranone) present in celery (Apium graveolens L.) seed oil at very low levels (1-3%) in the essential oil. It is responsible for the characteristic aroma and flavor of celery. [2]
- Previous studies (referenced but not original in these papers) have shown that Sedanolide exhibits prostaglandin H endoperoxide synthase (COX-I and COX-II) inhibitory activities and topoisomerase-I and -II enzyme inhibitory activities. It also increased GST activity in mouse liver, small intestinal mucosa, and forestomach, and inhibited benzopyrene-induced forestomach tumor formation in mice (57% reduction in tumor incidence and 83% reduction in tumor multiplicity). [1][2]
- In the second study, the authors propose a model where Sedanolide induces autophagy in J5 cells by suppressing PI3K-I/Akt/mTOR expression and enhancing Beclin-1/PI3K-III/LC3-II protein levels for pre-autophagosome formation, and by decreasing cytosolic p53/TIGAR while increasing nuclear p53/DRAM to promote autophagosome formation. [2]

C12H18O2

194.27012

194.131

C, 74.19; H, 9.34; O, 16.47

6415-59-4

5018391

White to off-white solid powder

1.03

342.0±11.0 °C(Predicted)

120 °C

1.742

2.828

0

2

3

14

255

0

CCCCC1C2CCCC=C2C(=O)O1

UPJFTVFLSIQQAV-UHFFFAOYSA-N

InChI=1S/C12H18O2/c1-2-3-8-11-9-6-4-5-7-10(9)12(13)14-11/h7,9,11H,2-6,8H2,1H3

3-butyl-3a,4,5,6-tetrahydro-3H-2-benzofuran-1-one

Neocnidilide; Sedanolide

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 : ~100 mg/mL (~514.75 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.)