COX-2
Asaraldehyde targets cyclooxygenase-2 (COX-2), an enzyme involved in the production of prostaglandins and other inflammatory mediators. It is a natural COX-2 inhibitor with an IC50 value of 100 µg/mL. It exhibits 17-fold selectivity for COX-2 over COX-1. By inhibiting COX-2, it reduces the production of pro-inflammatory prostaglandins. This mechanism underlies its anti-inflammatory activity.

Asarylaldehyde (2,4,5-TMBA) is a naturally occurring COX-2 inhibitor that was isolated from carrot (Daucus carota L.) seeds. At 100 μg/mL, it significantly inhibits the activity of cyclooxygenase II (COX-2) compared to three commercial nonsteroidal anti-inflammatory drugs (IC50 values: 180, 2.52, and 2.06 μg/mL, respectively). In 3T3-L1 adipocytes, 2,4,5-TMBA, a natural cyclooxygenase-2 inhibitor, inhibits adipogenesis and oromotes lipolysis. It has been shown that at a concentration of 100 μg/mL, 2,4,5-Trimethoxybenzaldehyde (2,4,5-TMBA), which is found in plant roots, seeds, and leaves, significantly inhibits the activity of cyclooxygenase-2 (COX-2). Given that COX-2 is linked to preadipocyte development, the murine 3T3-L1 cells are cultivated with 100 μg/mL of 2,4,5-TMBA both during and after the cells have fully differentiated to examine the impact of the compound on lipolysis and adipogenesis. Oil The formation of lipid droplets during differentiation was found to be inhibited by 2,4,5-TMBA, as demonstrated by Red O staining and triglyceride assay. Additionally, 2,4,5-TMBA down-regulates the protein levels of adipogenic signaling molecules and transcription factors, including MAP kinase kinase (MEK), extracellular signal-regulated kinase (ERK), CCAAT/enhancer binding protein (C/EBP)α, β, and δ, PPAR (peroxisome proliferator-activated receptor)γ, adipocyte determination and differentiation-dependent factor 1 (ADD1), and the rate-limiting enzyme for lipid synthesis, acetyl-CoA carboxylase (ACC). Treatment of completely developed adipocytes with 2,4,5-TMBA for 72 hours resulted in a considerable reduction in lipid accumulation via boosting triglyceride hydrolysis through the up-regulation of hormone-sensitive lipase (HSL) and the suppression of perilipin A (lipid droplet coating protein). Treatment of completely differentiated 3T3-L1 adipocytes with 100 μg/mL of 2,4,5-TMBA for 24, 48, or 72 hours results in a decrease in viability of 8.35, 15.54, and 27.26%, respectively. Premadiocyte cell viability is reduced by 26.46% when they are treated with 100 μg/mL of 2,4,5-TMBA for 24 hours prior to the addition of differentiation medium[1]. The most prevalent component is 2,4,5-trimethoxybenzaldehyde (TMBA), a COX-2 inhibitor that is completely absent from both its natural host, C. kanehirae wood, and its cultured broth. In fruiting bodies, 2,4,5-trimethoxybenzaldehyde (TMBA) is the main component[2].

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In the coculture study (differentiation period), 2,4,5-Trimethoxybenzaldehyde (Asaraldehyde) at 100 μg/mL significantly suppressed the expression of phosphorylated MEK and ERK1 (but not ERK2) in 3T3-L1 preadipocytes; JNK and p38MAPK were also inhibited but not statistically significant [1].
It down-regulated the protein levels of transcription factors C/EBPβ, C/EBPδ, C/EBPα, PPARγ1, PPARγ2 (effect on PPARγ2 not significant), and ADD1 during differentiation [1].
2,4,5-Trimethoxybenzaldehyde (Asaraldehyde) significantly inhibited triglyceride accumulation by 27.64% during differentiation (Oil Red O staining and triglyceride assay) [1].
It inhibited COX-2 expression during differentiation; slightly down-regulated perilipin A (not significant); did not significantly affect pyruvate dehydrogenase (PD), pyruvate carboxylase (PC), or citrate synthase (CS); but significantly suppressed acetyl-CoA carboxylase (ACC) expression [1].
In fully differentiated adipocytes, treatment with 100 μg/mL for 72 h decreased lipid accumulation by 26.47% and increased glycerol release by 22.91%, indicating enhanced lipolysis [1].
It significantly suppressed perilipin A expression and up-regulated hormone-sensitive lipase (HSL) in mature adipocytes [1].
Treatment decreased leptin secretion by 27.11% and also inhibited adiponectin secretion (not statistically significant) [1].
In vitro, Asaraldehyde significantly inhibits COX-2 activity with an IC50 value of 100 µg/mL. It exhibits 17-fold selectivity for COX-2 over COX-1. At 100 µg/mL, it mildly inhibits COX-1 (3.32%) and significantly inhibits COX-2 (52.69%). Compared to three commercial NSAIDs (IC50 values: 180, 2.52, and 2.06 µg/mL), it shows significant inhibitory activity. These in vitro studies confirm its mechanism as a selective COX-2 inhibitor.

In vivo, Asaraldehyde has been studied in 3T3-L1 adipocytes. As a natural COX-2 inhibitor, it has potential anti-inflammatory effects. However, specific in vivo study results are not extensively documented in standard summaries. Its anti-inflammatory activity has been demonstrated in vitro, suggesting potential for in vivo efficacy.

Non-cellular enzyme assays for Asaraldehyde typically involve measuring its inhibition of COX-2 activity. These assays use purified COX-2 enzyme and a spectrophotometric or radiometric substrate to measure the rate of enzymatic reaction in the presence of varying concentrations of Asaraldehyde. The compound's ability to inhibit COX-2 is assessed by determining the half-maximal inhibitory concentration (IC50). Such assays are essential for characterizing its potency and selectivity as a COX-2 inhibitor.

Cell viability was assessed by MTT assay. 3T3-L1 preadipocytes or fully differentiated adipocytes were treated with 2,4,5-Trimethoxybenzaldehyde (Asaraldehyde) at 100 μg/mL for indicated times (24‑72 h or the whole 8‑day differentiation period), then incubated with 0.5 mg/mL MTT for 4 h. Formazan was dissolved in DMSO and absorbance measured at 570 nm [1].
Lipid droplet formation was observed by Oil Red O staining. Cells were fixed with 10% formalin for 1 h, stained with Oil Red O solution (0.35% in isopropanol diluted with water) for 15 min at room temperature, washed, and photographed. For quantification, stained lipid droplets were dissolved in isopropanol and absorbance read at 520 nm [1].
Glycerol release into culture medium was measured using an adipolysis assay kit (colorimetric method) [1].
Western blotting: Cells were lysed in buffer containing 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris‑HCl pH 7.5. Lysates were centrifuged at 10,000×g for 30 min then 105,000×g for 60 min. Supernatant proteins were quantified, separated by gel electrophoresis, transferred to PVDF membranes, blocked with 5% nonfat dry milk in TBST, incubated with primary antibodies (anti‑PPARγ, anti‑HSL, anti‑C/EBPα/β/δ, anti‑aP2, anti‑MEK, anti‑JNK, anti‑p38MAPK, anti‑ERK, anti‑ACC, anti‑COX‑2, anti‑perilipin A, etc.) for 3 h, then with HRP‑conjugated secondary antibody for 1 h at room temperature. Signals were detected by enhanced chemiluminescence [1].
Leptin and adiponectin secretion were measured by ELISA kits specific for each adipokine, using collected culture medium [1].
In vitro cell-based assays for Asaraldehyde are conducted using various cell lines to assess its effects on prostaglandin production and inflammation. Cells are treated with the compound, and prostaglandin E2 levels are measured using ELISA. Its effects on cell viability and proliferation can also be assessed. These experiments are crucial for confirming its mechanism of action at the cellular level.

In vivo animal studies for Asaraldehyde are not extensively documented in standard summaries. As a natural product with COX-2 inhibitory activity, it has potential for anti-inflammatory applications. However, specific in vivo efficacy studies would be required to validate its therapeutic potential.

Asaraldehyde has a molecular weight of 196.20 g/mol. It is a small, lipophilic molecule. As a natural product, its pharmacokinetic properties are not extensively documented. It is available as a research compound for studying COX-2 inhibition.

MTT assay showed that treatment of fully differentiated 3T3‑L1 adipocytes with 100 μg/mL 2,4,5-Trimethoxybenzaldehyde (Asaraldehyde) for 24, 48, and 72 h decreased cell viability by 8.35%, 15.54%, and 27.26%, respectively. When preadipocytes were treated with 100 μg/mL for 24 h before differentiation, viability decreased by 26.46%. Coculture during the 8‑day differentiation period decreased viability by 25.82% at the end [1].
Comprehensive toxicological data for Asaraldehyde are limited, as it is a natural product and research compound. It is a selective COX-2 inhibitor with potential anti-inflammatory activity. As with all research chemicals, appropriate safety precautions should be taken when handling Asaraldehyde.

[1]. 2,4,5-TMBA, a natural inhibitor of cyclooxygenase-2, suppresses adipogenesis and promotes lipolysis in 3T3-L1 adipocytes. J Agric Food Chem. 2012 Jul 25;60(29):7262-9.

[2]. Production of a COX-2 inhibitor, 2,4,5-trimethoxybenzaldehyde, with submerged cultured Antrodia camphorata. Lett Appl Microbiol. 2007 Apr;44(4):387-92.

2,4,5-Trimethoxybenzaldehyde is a beige powder. (NTP, 1992)
2,4,5-Trimethoxybenzaldehyde is a carbonyl compound.
2,4,5-Trimethoxybenzaldehyde has been reported in Alpinia flabellata, Mosla scabra, and other organisms with relevant data.

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2,4,5-Trimethoxybenzaldehyde (Asaraldehyde) (also known as 2,4,5‑trimethoxybenzaldehyde, TMBA) is a natural cyclooxygenase‑2 (COX‑2) inhibitor. It was identified as the most abundant volatile constituent in the fruiting bodies of Antrodia camphorata (a medicinal mushroom), with a peak area of 21.07% by GC‑MS [2]. It was absent in the liquid cultured broth of A. camphorata and in the wood of Cinnamomum kanehirae. Feeding vanillin (1 g/L) or acid‑digested sawdust of C. kanehirae to the culture broth resulted in production of 2,4,5-Trimethoxybenzaldehyde (Asaraldehyde) (3.32 mg/L and 2.60 mg/L respectively after 4 weeks), suggesting that the compound is derived from lignin degradation products or vanillin as a precursor [2]. In the fruiting bodies, other related compounds such as 1,2,3,4‑tetramethoxybenzene and methyl 3,4,5‑trimethoxybenzoate were also detected [2]. The presence of this COX‑2 inhibitor in A. camphorata fruiting bodies is consistent with the traditional anti‑inflammatory and anti‑cancer uses of the mushroom [2].
Asaraldehyde (Asaronaldehyde, 2,4,5-trimethoxybenzaldehyde) is a natural COX-2 inhibitor isolated from carrot seeds. It significantly inhibits COX-2 activity with an IC50 value of 100 µg/mL and exhibits 17-fold selectivity for COX-2 over COX-1. Asaraldehyde is not an approved drug and is available as a research compound for studying COX-2 inhibition and inflammation.

C10H12O4

196.2

196.073

4460-86-0

20525

White to off-white solid powder

1.1±0.1 g/cm3

334.7±37.0 °C at 760 mmHg

112-114 °C(lit.)

149.0±26.5 °C

0.0±0.7 mmHg at 25°C

1.525

1.62

0

4

4

14

183

0

IAJBQAYHSQIQRE-UHFFFAOYSA-N

InChI=1S/C10H12O4/c1-12-8-5-10(14-3)9(13-2)4-7(8)6-11/h4-6H,1-3H3

2,4,5-trimethoxybenzaldehyde

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 3.75 mg/mL (19.11 mM) (saturation unknown) in 10% EtOH + 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 37.5 mg/mL clear EtOH 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: ≥ 3.75 mg/mL (19.11 mM) (saturation unknown) in 10% EtOH + 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 37.5 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (12.74 mM) 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 4: ≥ 2.5 mg/mL (12.74 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 corn oil and mix evenly.

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