Dihydrocoumarin (DHC) inhibits yeast Sir2p and human SIRT1 and SIRT2 deacetylases.
SIRT1 inhibition IC₅₀ = 208 µM in an in vitro enzymatic assay [2].
SIRT2 inhibition shows similar dose dependency [2].
SIRT3 deacetylase activity was not affected by DHC [2].

Dihydrocoumarin inhibits SIRT1 in an in vitro enzymatic assay in a concentration-dependent manner (IC50 of 208 μM). Even at micromolar concentrations, there was a reduction in SIRT1 deacetylase activity (85±5.8% and 73±13.7% activities at 1.6 μM and 8 μM, respectively). Similar dose-dependent inhibition of microtubule SIRT2 deacetylase was observed (IC50 of 295 μM) [1]. After 24 hours of treatment, dihydrocoumarin (1–5 mM) boosted the TK6 cell line's cytotoxicity in a dose-dependent way. At the 6-hour mark, dihydrocoumarin (1–5 mM) enhanced apoptosis in the TK6 cell line in a dose-dependent way. In the TK6 cell line, dihydrocoumarin at a dosage of 5 mM promotes apoptosis at the 6-hour time point [1]. After a 24-hour exposure period, dihydrocoumarin (1–5 mM) enhances p53 lysine 373 and 382 acetylation in a dose-dependent manner in the TK6 cell line [1].

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Dihydrocoumarin (DHC) disrupts heterochromatic silencing in S. cerevisiae in a dose-dependent manner, similar to the established Sir2p inhibitor splitomicin [2].
DHC increases p53 acetylation at lysine 373 and 382 in a dose-dependent manner in TK6 lymphoblastoid cells following 24-hour exposure [2].
DHC increases cytotoxicity and apoptosis in TK6 cells in a dose-dependent manner, with apoptosis levels increasing more than 3-fold compared to controls [2].
DHC enhances cell killing by etoposide in TK6 and HEK293 cell lines [2].
DHC is not mutagenic, clastogenic, or aneugenic in previous in vivo and in vitro studies [2].

The histone deacetylation assay with recombinant SIRT1 and SIRT2 was performed using a ³H-acetylated histone H4 peptide to measure deacetylase activity. DHC was tested for its ability to inhibit these enzymes in a concentration-dependent manner [2].
A separate assay measured the inhibition of yeast Sir2p using a galactose-inducible SIR2 overexpression system in yeast. Growth on galactose medium containing DHC was compared to controls to assess reversal of Sir2p-induced lethality [2].

For immunoblot analysis, TK6 lymphoblastoid cells were exposed to various concentrations of DHC (1-5 mM) for 24 hours. Cell lysates were collected, and protein concentrations were determined. Equal amounts of protein were resolved by PAGE, transferred to nitrocellulose membranes, and probed with antibodies against acetylated p53 (lysine 373 and 382) [2].
Cellular cytotoxicity was assessed using the Trypan blue exclusion assay. TK6 cells exposed to DHC for 24 hours were counted, and the percentage of non-viable cells was calculated [2].
Apoptosis was measured by flow cytometry using annexin V-FITC and propidium iodide staining. TK6 cells were treated with DHC for 6 hours, stained, and analyzed to distinguish apoptotic from necrotic cells [2].

Interactions
The following drugs may enhance the response to coumarin or indanedione derivatives: alcohol (acute poisoning), allopurinol, aminosalicylic acid, amiodarone, anabolic steroids, chloral hydrate, chloramphenicol, cimetidine, clofibrate, trimethoprim-sulfamethoxazole, danazol, dexthylexin sodium, diazoxide, diflunisal, disulfiram, erythromycin, ethacrynic acid, fenoprofen calcium, glucagon, ibuprofen, indomethacin, influenza vaccine, isoniazid, meclofenamic acid, mefenamic acid, methylthiouracil, metronidazole, miconazole, nalidixic acid, neomycin (oral), pentoxifylline, phenylbutazone, propoxyphene, propylthiouracil, quinidine, quinine. Salicylates, streptokinase, sulfinpyrazone, sulfonamides, sulindac, tetracyclines, thiazide diuretics, thyroid medications, tricyclic antidepressants, urokinase, vitamin E. /Coumarins and Indanedione Derivatives/
The following drugs...may...reduce...the response to coumarin or indanedione derivatives: alcohol (chronic alcoholism), barbiturates, carbamazepine, corticosteroids, adrenocorticotropic hormone, ethylclofenac, glutamine, griseofulvin, mercaptopurine, methylquinone, estrogen-containing oral contraceptives, rifampin, spironolactone, vitamin K. /Coumarins and Indanedione Derivatives/

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Non-human toxicity values
Oral LD50 in rats: 1460 mg/kg
Intraperitoneal LD50 in mice: 200 mg/kg
Oral LD50 in guinea pigs: 1760 mg/kg

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Previous studies cited in the literature have shown that dihydrocoumarin (DHC) is not a mutagen, chromosome breakage agent, or aneuploidy agent [2].
In this study, exposure of TK6 cells to DHC resulted in a concentration-dependent increase in cytotoxicity and apoptosis[2].

[1]. The flavoring agent Dihydrocoumarin reverses epigenetic silencing and inhibits sirtuindeacetylases. PLoS Genet. 2005 Dec;1(6):e77.

3,4-Dihydrocoumarin is a white to pale yellow, transparent, oily liquid with a sweet taste. It solidifies at room temperature. (NTP, 1992)
3,4-Dihydrocoumarin is a chromone, a 3,4-dihydro derivative of coumarin. It is a plant metabolite functionally related to coumarin.
3,4-Dihydrocoumarin has been reported in organisms with available data, such as Lasiolaena morii and Daphnia pulex.
See also: 4-Cromone (note moved to).
Mechanism of Action 4-Hydroxycoumarin derivatives and indanediones (also known as oral anticoagulants) are both vitamin K antagonists. Their use as rodenticides works by inhibiting the vitamin K-dependent steps in the synthesis of various blood clotting factors. Vitamin K-dependent proteins in the coagulation cascade include procoagulant factors II (prothrombin), VII (prothrombin convertase), IX (Christmas factor), and X (Stuart-Proll factor), as well as coagulation inhibitory proteins C and S. All of these proteins are synthesized in the liver. Before being released into the bloodstream, these precursor proteins undergo numerous (intracellular) post-translational modifications. Vitamin K acts as a coenzyme in one of these modifications, specifically by carboxylating 10–12 glutamate residues to γ-carboxyglutamate (Gla) at a specific site. The presence of these Gla residues is crucial for the procoagulant activity of various coagulation factors. Vitamin K hydroquinone (KH2) is the active coenzyme, whose oxidation to vitamin K 2,3-epoxide (KO) provides the energy required for the carboxylation reaction. The epoxide is then recycled through a two-step reduction reaction catalyzed by KO reductase… KO reductase is the target of coumarin anticoagulants. Coumarin anticoagulants inhibit KO reductase activity, leading to rapid depletion of KH2 supply and effectively preventing the formation of Gla residues. This results in the accumulation of uncarboxylated clotting factor precursors in the liver. In some cases, these precursors are further processed without carboxylation and (depending on the species) may appear in the bloodstream. At this point, the uncarboxylated protein is called decarboxylated clotting factor. Normal clotting factors circulate as proenzymes, participating in the coagulation cascade only after activation through limited proteolytic degradation. Decarboxylated clotting factors lack procoagulant activity (i.e., cannot be activated) and cannot be converted to active proenzymes by the action of vitamin K. While high levels of circulating decarboxylated clotting factors can be detected in humans receiving anticoagulation therapy, their levels are negligible in rats and mice treated with warfarin. /Anticoagulant rodenticide/

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Dihydrocoumarin (DHC) is a natural compound found in sweet clover (Melilotus officinalis), and can also be synthesized artificially. It is used as a flavoring agent in beverages, chewing gum, gelatin, pudding, gummies, frozen dairy products and baked goods, as well as a flavoring agent in perfumes, cosmetics, lotions and soaps[2].
The concentration of DHC in some foods exceeds 100 ppm (about 670 µM)[2].
Studies have shown that DHC is an epigenetic toxin that inhibits sirtuin deacetylase, which is associated with aging, and may promote p53-mediated apoptosis and potential tissue senescence[2].

C9H8O2

148.1586

148.052

119-84-6

660

Colorless to light yellow <24°C powder,>25°C liquid

1.2±0.1 g/cm3

272.0±15.0 °C at 760 mmHg

24-25 °C(lit.)

108.4±17.8 °C

0.0±0.6 mmHg at 25°C

1.562

1.8

0

2

0

11

165

0

O1C(C([H])([H])C([H])([H])C2=C([H])C([H])=C([H])C([H])=C12)=O

VMUXSMXIQBNMGZ-UHFFFAOYSA-N

InChI=1S/C9H8O2/c10-9-6-5-7-3-1-2-4-8(7)11-9/h1-4H,5-6H2

3,4-dihydrochromen-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)

DMSO : ~100 mg/mL (~674.95 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (16.87 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 (16.87 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (16.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)