Syringetin   中文名称: 丁香亭

Natural flavonoid

丁香素（3,5,7,4'-四羟基-3',5'-二甲氧基黄酮）是一种存在于葡萄和葡萄酒中的黄酮衍生物。通过碱性磷酸酶（ALP）活性、骨钙素和I型胶原ELISA检测，我们发现丁香素能显著诱导MC3T3-E1小鼠颅骨成骨细胞和人胎儿成骨细胞1.19细胞系的成骨分化。ALP和骨钙素分别是早期分化成骨细胞和终末分化成骨细胞的表型标志物。研究结果表明，丁香素能在从成熟到终末分化的各个阶段促进成骨细胞分化。 丁香素诱导分化的作用与骨形态发生蛋白-2（BMP-2）产量增加相关。BMP-2拮抗剂noggin能阻断丁香素介导的ALP活性增强和骨钙素分泌增加，表明BMP-2的产生是丁香素介导成骨细胞成熟和分化所必需的。丁香素诱导分化的作用还涉及SMAD1/5/8和细胞外信号调节激酶1/2（ERK1/2）的激活。ERK1/2抑制剂2'-氨基-3'-甲氧基黄酮共处理可抑制丁香素介导的ALP上调和骨钙素产生。 综上所述，丁香素通过增加BMP-2合成，进而激活SMAD1/5/8和ERK1/2信号通路，这一机制可能有助于其诱导成骨细胞成熟和分化，最终增加骨量。[1]

细胞增殖检测（XTT法）[1]
采用XTT法检测丁香素对细胞增殖的抑制作用。简要步骤：将细胞接种于96孔培养板（8×10³个细胞/孔），孵育24小时后，分别用载体（0.05% DMSO）或丁香素（1、5、10、20 μM）处理48和72小时。每孔加入50 μL XTT测试液（由5 mL XTT标记试剂与100 μL电子耦合试剂混合配制），继续孵育4小时后，用酶标仪在492 nm检测波长和690 nm参考波长下测定吸光度。

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I型前胶原水平检测[1]
细胞经不同浓度丁香素处理72和96小时后，使用Prolagen-C试剂盒检测I型前胶原水平。该检测通过测量分子的前肽部分反映成熟蛋白的合成量，具体操作遵循制造商说明书。检测获得的I型前胶原水平通过BCA蛋白测定法进行总蛋白浓度标准化。

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骨钙素与BMP-2水平检测[1]
分别采用骨钙素和BMP-2 ELISA试剂盒进行检测。简要流程：细胞经指定浓度丁香素处理相应时间后，收集培养上清。样本加入包被单克隆检测抗体的96孔微孔板，室温孵育2小时。用洗涤缓冲液（50 mM Tris、200 mM NaCl、0.2% Tween 20）去除未结合物质后，加入辣根过氧化物酶标记链霉亲和素与抗体结合。辣根过氧化物酶催化显色底物（四甲基联苯胺）转化为有色溶液，其颜色强度与样本中蛋白含量成正比。在450 nm波长下测定各孔吸光度，结果以相对于未处理对照组活性变化的百分比表示。

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免疫印迹分析[1]
经丁香素处理的细胞裂解后，通过Bio-Rad蛋白测定法确定蛋白浓度。取50 μg总细胞裂解物进行SDS-聚丙烯酰胺凝胶电泳，使用转移缓冲液（50 mM Tris、190 mM甘氨酸、10%甲醇）在100 V条件下将蛋白转印至聚偏二氟乙烯膜2小时。膜片在封闭缓冲液（50 mM Tris、200 mM NaCl、0.2% Tween 20、3%牛血清白蛋白）中4℃封闭过夜。用洗涤缓冲液（不含3%牛血清白蛋白的封闭液）洗膜3次（每次10分钟）后，与一抗（SMAD1/5/8、ERK1/2、磷酸化ERK及磷酸化SMAD1/5/8）孵育2-15小时，再与辣根过氧化物酶标记二抗孵育1小时。洗膜后采用增强化学发光Western blot检测系统进行显影。

Metabolism / Metabolites
Syringetin has known human metabolites that include (2S,3S,4S,5R)-6-[5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxane-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-trihydroxyoxane-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.

Syringetin is a dimethoxyflavone that is myricetin in which the hydroxy groups at positions 3' and 5' have been replaced by methoxy groups. It has a role as a platelet aggregation inhibitor and a metabolite. It is a tetrahydroxyflavone, a dimethoxyflavone, a 7-hydroxyflavonol, a member of 3'-methoxyflavones and a 3',5'-dimethoxyflavone. It is functionally related to a myricetin. It is a conjugate acid of a syringetin(1-).
Syringetin has been reported in Punica granatum, Capsicum annuum, and other organisms with data available.

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During differentiation in vitro, osteoblast phenotypic markers appear in the following order: accumulation of collagenous matrix, expression of ALP, secretion of osteocalcin, and finally, mineralization of bone nodules. Our results indicate that the presence of Syringetin causes a significant increase in ALP activity, osteocalcin production, type I collagen synthesis, and mineralization. As the appearance of ALP activity is an early phenotypic marker for mature osteoblasts, our results suggest that the presence of syringetin stimulates an early stage of osteoblast differentiation. The production of osteocalcin and type I collagen, both phenotypic markers for the later stage of osteoblast differentiation, was increased by Syringetin treatment. In addition, bone formation, as measured by mineralization, was also increased in MC3T3-E1 and hFOB cells treated with syringetin. Furthermore, the inhibitory protein synthesis effect of cycloheximide on the syringetin-induced increase in ALP activity and octeocalcin production strongly suggests that de novo protein synthesis is essential for this response. In summary, these results indicate that syringetin-stimulated maturation and differentiation of osteoblasts could be affected at various levels, from early to terminal stages of the cell differentiation process.

BMPs play an important role in the process of bone formation and remodeling 8. It has been well documented that stimulation of osteoblast differentiation is characterized mainly by increased expression of ALP, type I collagen, and osteocalcin. The action of BMPs is mediated by heterotetrameric serine/threonine kinase receptors and the downstream transcription factors SMAD1/5/8. After these transcription factors are phosphorylated on serine residues, they form a complex with a common mediator, SMAD4, and the complex is translocated into the nucleus to activate the transcription of a specific gene. Several natural or chemical compounds have been reported to induce osteoblast differentiation by induction of BMP and/or SMAD signaling, such as daidzein, osthole, and fraxetin. Our study indicates that the production of BMP-2 increases in Syringetin-treated MC3T3-E1 and hFOB cells. Also, phosphorylations of SMAD1/5/8 are simultaneously enhanced in syringetin-treated osteoblasts. Indeed, BMPs antagonist noggin not only blocked syringetin-mediated SMAD1/5/8 activation, but also exhibited a similar effect against syringetin-mediated cell differentiation (ALP upregulation and osteocalcin production). These results support the hypothesis that the BMP-2 signaling system plays an important role in syringetin-mediated cell maturation and differentiation in osteoblasts.

ERK1/2 is also important in osteoblast cell proliferation and differentiation. A number of studies have reported that ERK is an important mediator of BMP-2-induced osteoblast differentiation, and that inhibition of ERK1/2 results in the suppression of differentiation markers 18, 41. Our study observed an increase in ERK1/2 activity after BMP-2 production and SMAD1/5/8 phosphorylation, and suppression of BMP-2 signaling by cotreating noggin abrogated ERK1/2 activation in syringetin-treated cells. In addition, inhibition of ERK1/2 activity by specific inhibitor PD98059 decreased the effects of Syringetin on osteoblastic maturation and differentiation. These data suggest that activation of ERK1/2 plays an important role on the cell differentiation of syringetin activity in osteoblasts.

In summary, our study has clearly demonstrated that Syringetin stimulates osteoblast differentiation at various stages in MC3T3-E1 and hFOB cells. Syringetin's effect on cell maturation and differentiation is strongly associated with BMP-2/SMAD1/5/8/ERK1/2 signaling pathway. This, therefore, suggests that syringetin may be beneficial in stimulating the osteoblastic activity resulting in 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

129.59

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

注意： 本产品在运输和储存过程中需避光。

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。