Natural flavonoid

Syringetin (3,5,7,4'-tetrahydroxy-3',5'dimethoxyflavone), a flavonoid derivative, is present in grape and wine. By means of alkaline phosphatase (ALP) activity, osteocalcin, and type I collagen ELISA, we have shown that syringetin exhibits a significant induction of differentiation in MC3T3-E1 mouse calvaria osteoblasts and human fetal osteoblastic 1.19 cell line human osteoblasts. ALP and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicate that syringetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by syringetin is associated with increased bone morphogenetic protein-2 (BMP-2) production. The BMP-2 antagonist noggin blocked syringetin-mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP-2 production is required in syringetin-mediated osteoblast maturation and differentiation. Induction of differentiation by syringetin is associated with increased activation of SMAD1/5/8 and extracellular signal-regulated kinase 1/2 (ERK1/2). Cotreatment of ERK1/2 inhibitor 2'-amino-3'-methoxyflavone inhibited syringetin-mediated ALP upregulation and osteocalcin production. In conclusion, syringetin increased BMP-2 synthesis, and subsequently activated SMAD1/5/8 and ERK1/2, and this effect may contribute to its action on the induction of osteoblast maturation and differentiation, followed by an increase of bone mass. [1]

Cell proliferation assay (XTT) [1]
Inhibition of cell proliferation by Syringetin was measured by XTT assay. Briefly, cells were plated in 96-well culture plates (8×103 cells/well). After 24 h incubation, the cells were treated with vehicle (0.05% DMSO) or syringetin (1, 5, 10, and 20 μM) for 48 and 72 h. In total, 50 μL of XTT test solution, which was prepared by mixing 5 mL of XTT-labeling reagent with 100 μL of electron coupling reagent, was then added to each well. After 4 h of incubation, absorbance was measured on an ELISA reader at a test wavelength of 492 nm and a reference wavelength of 690 nm.

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Assaying the levels of type I procollagen [1]
Cells were treated with various concentrations of Syringetin for 72 and 96 h. The type I procollagen assay, which measures the propeptide portion of the molecule and reflects the synthesis of the mature form of the protein, was carried out using Prolagen-C kit by following the manufacturer's protocol (Metra Biosystems, Mountainview, CA, USA). The type I procollagen levels obtained were normalized to total protein concentrations, as determined by BCA protein assay.

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Assaying the levels of osteocalcin and BMP-2 [1]
Osteocalcin and BMP-2 ELISA kits were used to detect osteocalcin and BMP-2 levels, respectively. Briefly, cells were treated with various concentrations of Syringetin for the indicated times. The culture medium was then collected and measured for osteocalcin and BMP-2. These samples were placed in 96-well microtiter plates coated with monoclonal detective antibodies and incubated for 2 h at room temperature. After removing unbound material with washing buffer (50 mM Tris, 200 mM NaCl, and 0.2% Tween 20), horseradish peroxidase-conjugated streptavidin was added to bind to the antibodies. Horseradish peroxidase catalyzed the conversion of a chromogenic substrate (tetramethylbenzidine) to a colored solution, with color intensity proportional to the amount of protein present in the sample. The absorbance of each well was measured at 450 nm. Results are presented as the percentage of change of the activity compared with the untreated control 34.

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Analysis for immunoblot [1]
Cells treated with Syringetin for the indicated times were lysed and the protein concentrations determined by Bio-Rad Protein Assay. For immunoblot, 50 μg of total cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The protein was transferred to polyvinylidene difluoride membranes using transfer buffer (50 mM Tris, 190 mM glycin, and 10% methanol) at 100 V for 2 h. The membranes were incubated with blocking buffer (50 mM Tris, 200 mM NaCl, 0.2% Tween 20, and 3% bovine serum albumin) overnight at 4°C. After washing three times with washing buffer (blocking buffer without 3% bovine serum albumin) for 10 min each, the blot was incubated with primary antibody (SMAD1/5/8, ERK1/2, phosphorylated ERK, and phospho-SMAD1/5/8) for 2–15 h, followed by horseradish peroxidase-labeled secondary antibody for 1 h. The membranes were washed again, and detection was performed using the enhanced chemiluminescence Western blotting detection system.

Metabolism / Metabolites
Eugenol's known human metabolites include (2S,3S,4S,5R)-6-[5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxetane-2-carboxylic acid and (2S,3S,4S,5R)-6-[3,5-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4-oxochromen-7-yl]oxy-3,4,5-trihydroxyoxetane-2-carboxylic acid.

[1]. Syringetin, a flavonoid derivative in grape and wine, induces human osteoblast differentiation through bone morphogenetic protein-2/extracellular signal-regulated kinase 1/2 pathway. Mol Nutr Food Res. 2009 Nov;53(11):1452-61.

Eucinol is a dimethoxyflavonoid, a derivative of myricetin, with its 3' and 5' hydroxyl groups replaced by methoxy groups. It has an inhibitory effect on platelet aggregation and is also a metabolite. It is a tetrahydroxyflavonoid, dimethoxyflavonoid, 7-hydroxyflavonol, 3'-methoxyflavonoid, and 3',5'-dimethoxyflavonoid compound. Functionally, it is related to myricetin. It is the conjugate acid of eugenol (1-). Eugenol has been reported to be present in pomegranate, pepper, and other organisms with relevant data. During in vitro differentiation, osteoblast phenotypic markers appear in the following order: collagen matrix accumulation, alkaline phosphatase (ALP) expression, osteocalcin secretion, and ultimately, bone nodules. Our results indicate that the presence of eugenol significantly increases alkaline phosphatase (ALP) activity, osteocalcin production, type I collagen synthesis, and mineralization. Since ALP activity is an early phenotypic marker of mature osteoblasts, our results suggest that the presence of styraxin may stimulate the early stages of osteoblast differentiation. Styraxin treatment increased the production of osteocalcin and type I collagen, both of which are phenotypic markers of the later stages of osteoblast differentiation. Furthermore, bone formation, as measured by mineralization, was also increased in MC3T3-E1 and hFOB cells treated with styraxin. In addition, the strong inhibitory effect of cyclohexylimide on the styraxin-induced increase in ALP activity and osteocalcin production strongly suggests that de novo protein synthesis is crucial for this response. In summary, these results indicate that styraxin-stimulated osteoblast maturation and differentiation may be involved at various stages of cell differentiation, from early to terminal.
Bone morphogenetic proteins (BMPs) play an important role in bone formation and remodeling.⁸ Extensive literature has documented that stimulation of osteoblast differentiation primarily manifests as increased expression of alkaline phosphatase (ALP), type I collagen, and osteocalcin. The role of bone marrow protein (BMPs) is mediated through heterotetrameric serine/threonine kinase receptors and downstream transcription factors SMAD1/5/8. Upon phosphorylation of serine residues, these transcription factors form a complex with the common mediator SMAD4, which translocates to the nucleus and activates the transcription of specific genes. Several natural or chemical compounds have been reported to induce osteoblast differentiation by inducing BMP and/or SMAD signaling pathways, such as daidzein, parsleyol, and leucopicrin. Our study showed increased BMP-2 production in eugenol-treated MC3T3-E1 and hFOB cells. Simultaneously, SMAD1/5/8 phosphorylation levels were also enhanced in eugenol-treated osteoblasts. In fact, the BMP antagonist noggin not only blocked eugenol-mediated SMAD1/5/8 activation, but also showed a similar inhibitory effect on eugenol-mediated cell differentiation (ALP upregulation and osteocalcin production). These results support the hypothesis that the BMP-2 signaling pathway plays an important role in eugenol-mediated osteoblast maturation and differentiation. ERK1/2 also plays an important role in osteoblast proliferation and differentiation. Multiple studies have shown that ERK is an important mediator of BMP-2-induced osteoblast differentiation, and inhibition of ERK1/2 leads to decreased expression of differentiation markers.^{18, 41} In this study, we observed that ERK1/2 activity increased after BMP-2 production and SMAD1/5/8 phosphorylation, and that co-treatment with noggin to inhibit the BMP-2 signaling pathway eliminated ERK1/2 activation in eugenol-treated cells. Furthermore, inhibiting ERK1/2 activity using the specific inhibitor PD98059 reduced the effect of eugenol on osteoblast maturation and differentiation. These data suggest that ERK1/2 activation plays an important role in eugenol-induced osteoblast differentiation.
In summary, this study clearly demonstrates that eugenol can stimulate osteoblast differentiation at different stages of MC3T3-E1 and hFOB cells. The effects of eugenol on cell maturation and differentiation are closely related to the BMP-2/SMAD1/5/8/ERK1/2 signaling pathway. Therefore, this suggests that eugenol may help stimulate osteoblast activity, thereby promoting bone formation. [1]

C17H14O8

346.29

346.069

C, 58.96; H, 4.08; O, 36.96

4423-37-4

5281953

Light yellow to yellow solid

1.591 g/cm3

622.4ºC at 760mmHg

287-289ºC

1.591ºC

1.707

2.299

4

8

3

25

533

0

COC1=CC(=CC(=C1O)OC)C2=C(C(=O)C3=C(C=C(C=C3O2)O)O)O

UZMAPBJVXOGOFT-UHFFFAOYSA-N

InChI=1S/C17H14O8/c1-23-11-3-7(4-12(24-2)14(11)20)17-16(22)15(21)13-9(19)5-8(18)6-10(13)25-17/h3-6,18-20,22H,1-2H3

3,5,7-trihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)chromen-4-one

3',5'-Dimethoxy-3,5,7,4'-tetrahydroxyflavone; Syringetin; 4423-37-4; 3',5'-Dimethoxy-3,5,7,4'-tetrahydroxyflavone; 3',5'-O-Dimethylmyricetin; CHEBI:18215; 3,5,7-trihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one; J68JG79B9W; 3,5,7-trihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)chromen-4-one; Myricetin-3',5'-dimethyl ether; 3',5'-O-Dimethylmyricetin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)