COX-2

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].

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].

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].

[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].

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.)