- AMPK (AMP-activated protein kinase) [1]
- MAPK (mitogen-activated protein kinase, including ERK, JNK, p38) [1]
- ACC (acetyl-CoA carboxylase) – downstream target of AMPK [1]
- PPARγ, C/EBPα, C/EBPβ – transcription factors involved in adipogenesis [1]

Myrosin (100 μg/mL; 24 h) causes cells to be arrested in the G0/G1 phase of the cell cycle and raises p21 and p27 expression [1]. The accumulation of lipid droplets and the formation of fat were significantly inhibited by the expression of EBPα, PPARγ, leptin, and aP2. Early in adipocyte proliferation, Sinigrin can increase AMPK, MAPK, and coenzyme A phosphorylase (ACC) phosphorylation. This suggests that Sinigrin has an anti-adipogenic effect by activating AMPK, MAPK, and ACC [1]. Pro-inflammatory cytokines, such as TNF-α and IL-6, IL-1β, and IL-18, are inhibited by myrosin (1-100 μg/mL; 8 days).

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- Sinigrin (1, 10, 100 μg/mL) significantly and concentration-dependently reduced lipid accumulation during adipocyte differentiation of 3T3-L1 cells as measured by Oil Red O staining (p < 0.05). [1]
- Sinigrin (1, 10, 100 μg/mL) downregulated the protein and mRNA expression of adipocyte differentiation markers including PPARγ, C/EBPα, SREBP1, aP2, Fas, and leptin. Western blot and qRT-PCR analyses showed decreased expression compared to MDI-treated control. [1]
- Sinigrin (100 μg/mL) arrested 3T3-L1 cells at the G0/G1 phase of the cell cycle during mitotic clonal expansion, as determined by flow cytometry. [1]
- Sinigrin increased the expression of p21 and p27, and suppressed the expression of CDK2 and c/EBPβ in MDI-induced adipocytes (Western blot). [1]
- Sinigrin (1, 10, 100 μg/mL) increased the phosphorylation of AMPK and its downstream target ACC in the early stage of adipocyte differentiation (2 days) without changing total AMPK or ACC protein levels (Western blot). [1]
- Sinigrin (1, 10, 100 μg/mL) significantly suppressed MDI-induced phosphorylation of ERK, JNK, and p38 MAPK (Western blot). [1]
- Sinigrin (1, 10, 100 μg/mL) reduced the expression and production of pro-inflammatory cytokines TNF-α, IL-6, IL-1β, and IL-18 at both protein (Western blot) and mRNA (qRT-PCR) levels, as well as in culture medium (ELISA). [1]
- Knockdown of AMPK by siRNA reversed the effect of sinigrin on AMPK and ACC phosphorylation, and increased expression of adipogenesis-related genes including PPARγ. [1]

By boosting antioxidant enzymes and modifying the MAPK buffer inhibitory marker cascade, myrosin (15–30 mg/kg; vessel wall; for 12 days) has both preventive and therapeutic benefits in DSS sensor vasculitis [2].

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Cell cycle analysis [1]
Cell Types: 3T3-L1 adipocytes
Tested Concentrations: 100 μg/mL
Incubation Duration: 24 h
Experimental Results: Cell cycle G0/G1 phase arrest, p21 and p27 expression increased.

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Western Blot Analysis[1]
Cell Types: 3T3-L1 Adipocyte
Tested Concentrations: 1, 10 and 100 μg/mL
Incubation Duration: 8 days
Experimental Results: Dramatically inhibited lipid droplet accumulation and adipogenesis by downregulating the expression of CCAAT enhancer binding protein alpha (C/EBPα), PPARγ, leptin, and aP2.

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RT-PCR[1]
Cell Types: 3T3-L1 Adipocyte
Tested Concentrations: 1, 10 and 100 μg/mL
Incubation Duration: 8 days
Experimental Results: Inhibition of pro-inflammatory cytokine production, including TNF-α and IL-6, IL- 1β and IL-18.

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- Cell proliferation assay (MTT): 3T3-L1 cells were seeded at 1×10^5 cells/well in 96-well plates and treated with various concentrations (0–200 μg/mL) of sinigrin for 48 hours. After removing supernatants, cells were incubated with MTT solution for 2 hours. Formazan crystals formed by viable cells were dissolved in DMSO, and absorbance was measured at 550 nm using a microplate reader. Cell proliferation was expressed as a percentage of untreated control. Sinigrin showed no cytotoxicity up to 200 μg/mL. [1]
- Oil Red O staining for lipid accumulation: After 8 days of adipocyte differentiation in the presence or absence of sinigrin (1, 10, 100 μg/mL), cells were washed with PBS, fixed with 3.7% formaldehyde for 10 minutes at room temperature, then stained with filtered Oil Red O solution for 2 hours at room temperature. After staining, cells were washed four times with PBS, and images of lipid droplets were collected using an optical microscope. The retained dye was eluted with isopropanol and quantified by spectrophotometry at 490 nm. [1]
- Western blot analysis: 3T3-L1 cells were seeded at 2×10^5 cells/well in 6-well plates and incubated with MDI or insulin in the presence of sinigrin (1, 10, 100 μg/mL) for 8 days (or specified time points). Cells were lysed in a homogenization buffer containing SDS, sodium deoxycholate, NP-40, protease inhibitors, and NaCl in Tris buffer (pH 8.0). After centrifugation at 13,000×g for 10 minutes at 4°C, protein concentration was determined by Bradford assay. Whole cell lysates were separated by 6–15% SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk in TBST for 1 hour at room temperature, and probed with appropriate antibodies. Blots were developed using an ECL kit. Densitometric analysis was performed using Image Gauge Ver 4.0 software, and relative intensities were normalized to β-actin. [1]
- Quantitative real-time PCR (qRT-PCR): 3T3-L1 cells were plated at 2×10^5 cells/mL in 6-well plates for 4 days and incubated with MDI or insulin only in the presence of sinigrin (1, 10, 100 μg/mL) for 8 days. Total RNA was extracted using Trizol. cDNA was amplified in 20 μL PCR reaction (8 μL cDNA, 1 μL each forward and reverse primer, 10 μL SYBR Green Master Mix) in a real-time PCR system. Gene expression was normalized to GAPDH as a reference gene. Primer sequences for PPARγ, C/EBPα, leptin, aP2, TNF-α, IL-6, IL-1β, IL-18, and GAPDH were used. [1]
- Flow cytometry cell cycle analysis: Post-confluent 3T3-L1 cells were treated with MDI in the presence or absence of sinigrin for 24 hours. Cells were harvested and fixed in 70% ethanol overnight at 4°C. After removing ethanol, cells were stained with propidium iodide solution (50 μg/mL) containing RNase (10 μg/mL) for 30 minutes in the dark. Fluorescence-activated cell sorting (FACS) analysis was performed, and data were analyzed using flow cytometry software. [1]
- RNA interference (siRNA): 3T3-L1 cells were plated in 6-well plates and transiently transfected with AMPK and ACC short interfering RNA (siRNA) mixed with siRNA transfection reagent according to the manufacturer's instructions. To knock down endogenous AMPK and ACC, cells were transfected with siRNA for 24 hours at a concentration of 100 mM. [1]

Animal/Disease Models: Dextran sodium sulfate (DSS)-induced mouse model [2]
Doses: 15 mg/kg or 30 mg/kg
Route of Administration: po (po (oral gavage)) lasted for 12 days
Experimental Results: Dramatically diminished DSS-induced weight loss and diminished colon Length shrinkage, and improved disease index scores. successfully eliminated DSS-induced IL-17 levels and improved the colon barrier in colon tissue.

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- Sinigrin did not show any cytotoxicity in 3T3-L1 cells at concentrations up to 200 μg/mL as assessed by MTT assay. [1]

[1]. Inhibitory effect of sinigrin on adipocyte differentiation in 3T3-L1 cells: Involvement of AMPK and MAPK pathways. Biomed Pharmacother. 2018 Jun;102:670-680.

[2]. Sinigrin Attenuates the Dextran Sulfate Sodium-induced Colitis in Mice by Modulating the MAPK Pathway. Inflammation. 2023 Jun;46(3):787-807.

- Obesity is characterized by increased number and size of differentiated adipocytes. Sinigrin inhibits early-stage adipogenesis of 3T3-L1 adipocytes through AMPK and MAPK signaling pathways, and reduces pro-inflammatory cytokine production. [1]
- Sinigrin suppresses adipocyte differentiation by attenuating mitotic clonal expansion (MCE) during the early phase of adipogenesis, associated with cell cycle arrest at G0/G1 phase and altered expression of cell cycle regulators (p21, p27, CDK2, c/EBPβ). [1]
- The anti-adipogenic effect of sinigrin is mediated by downregulation of major adipogenic transcription factors PPARγ and C/EBPα, as well as adipocyte-specific genes SREBP1, aP2, Fas, and leptin. [1]
- Sinigrin treatment reduces the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-18) in adipocytes, which are elevated in obesity and associated with inflammation. [1]

C10H16KNO9S2

397.4636

415

3952-98-5

534-69-0 (Parent);64550-88-5 (monohydrate salt/solvate)

23684362

White to off-white solid

125 - 127 °C

4

11

7

23

509

5

[K+].S(/C(/C([H])([H])C([H])=C([H])[H])=N\OS(=O)(=O)[O-])[C@@]1([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])O[H])O[H]

QKFAFSGJTMHRRY-FVDOMRANSA-M

InChI=1S/C10H17NO9S2.K/c1-2-3-6(11-20-22(16,17)18)21-10-9(15)8(14)7(13)5(4-12)19-10;/h2,5,7-10,12-15H,1,3-4H2,(H,16,17,18);/q;+1/p-1/b11-6-;/t5-,7-,8+,9-,10+;/m1./s1

potassium;[(Z)-1-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]sulfanylbut-3-enylideneamino] sulfate

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 and light.

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

H2O : ~12.5 mg/mL (~31.45 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.)