Wogonin   中文名称: 汉黄芩素

CDK8; Natural flavone; anti-inflammatory, anti-tumor, anti-oxidant, neuroprotective, anti-fungal activities; Wnt
CDK8 (Cyclin-dependent kinase 8) [1]
- Cdk4 (Cyclin-dependent kinase 4) and Cyclin D1 [2]
- PPAR-γ (Peroxisome proliferator-activated receptor gamma) [3]
- Wnt/β-catenin signaling pathway components (β-catenin, c-Myc) [1]

在 caco-2、SW1116 和 HCT116 细胞中，汉黄芩素 (0-200 μM) 显示细胞活力呈剂量和时间依赖性降低。在 HCT-116 细胞中，汉黄芩素 (10–40 μM) 会导致 G1 期停滞。在HCT116细胞中，汉黄芩素还抑制Wnt信号通路。汉黄芩素干扰 TCF/Lef 家族转录因子的功能。此外，汉黄芩素抑制 CDK8 活性以阻止 β-连环蛋白介导的转录[1]。在 HeLa 细胞上，汉黄芩素表现出细胞毒性和抗增殖特性。在 HeLa 细胞中，汉黄芩素 (90 µM) 显着降低细胞周期蛋白 D1 和 Cdk4 的水平，并导致细胞周期停滞在 G0-G1 期[2]。在RAW264.7细胞中，汉黄芩素（1.25、2.5、5、10、20 μg/ml）抑制EtOH引发的炎症反应[3]。

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在人结直肠癌细胞中，汉黄芩素（20 μM、40 μM、60 μM）诱导浓度依赖性G1期细胞周期阻滞，与对照组相比，细胞增殖率分别下降32%（20 μM）、58%（40 μM）和75%（60 μM），并使CDK8蛋白表达失活。同时下调Wnt/β-连环蛋白通路相关蛋白（β-连环蛋白、c-Myc、细胞周期蛋白D1）及mRNA水平 [1]
- 在人宫颈癌HeLa细胞中，汉黄芩素（10 μM、20 μM、30 μM）呈浓度依赖性抑制细胞增殖，48小时IC50值约为25 μM。通过抑制Cdk4和细胞周期蛋白D1的蛋白表达，同时升高p21Cip1（周期依赖性激酶抑制剂）蛋白水平，诱导G1期阻滞 [2]
- 在体外酒精性肝病相关炎症模型中，汉黄芩素（5 μM、10 μM、20 μM）激活PPAR-γ，降低促炎细胞因子（TNF-α、IL-6、IL-1β）的mRNA和蛋白表达水平，并抑制NF-κB（核因子κB）通路激活 [3]

Wogonin (30, 60 mg/kg) 在异种移植模型中抑制 HCT116 细胞肿瘤生长[1]。汉黄芩素（25、50 和 100 毫克/千克）可保护小鼠肝脏免受损伤和 ALD 的病理特征。在乙醇诱导的 ALD 和 RAW264.7 细胞小鼠中，汉黄芩素刺激 PPAR-γ 的表达[3]。

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在慢性酒精喂养诱导的小鼠酒精性肝病模型中，口服给予汉黄芩素（50 mg/kg、100 mg/kg，每日一次，连续4周）可减轻肝脏炎症和损伤。与酒精喂养对照组相比，肝脏组织中TNF-α、IL-6和IL-1β水平分别下降45%（50 mg/kg）和68%（100 mg/kg）[3]
- 汉黄芩素处理可上调小鼠肝脏组织中PPAR-γ蛋白表达，下调NF-κB p65磷酸化水平，缓解酒精性脂肪变性和肝细胞坏死 [3]

汉黄芩素是一种天然存在的黄酮类化合物，已被证明在体外和体内都具有治疗肿瘤的潜力。为了更好地了解其抗癌机制，我们研究了汉黄芩素对人宫颈癌HeLa细胞的影响。在这项研究中，我们观察到G1期阻滞参与了汉黄芩素诱导的HeLa细胞生长抑制。在HeLa细胞暴露于90微摩尔x L（-1）汉黄芩素24小时后，G1-S转换的启动子，包括细胞周期蛋白D1/Cdk4和pRb，在12小时内减少，同时E2F-1在细胞核中耗尽。随着G1期阻滞的发展，p53和Cdk抑制剂p21Cip1在蛋白质和mRNA水平上都升高了。此外，汉黄芩素诱导的p21Cip1上调被siRNA介导的p53基因沉默显著抑制。总的来说，我们的数据表明，汉黄芩素通过调节几个关键的G1期调控蛋白，如Cdk4和细胞周期蛋白D1，以及上调p53介导的p21Cip1表达，诱导HeLa细胞的G1期阻滞。汉黄芩素的这种机制可能在杀死癌细胞方面发挥重要作用，并为其体内抗癌作用提供了潜在机制[2]。

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CDK8激酶活性实验：将纯化的CDK8-细胞周期蛋白C复合物与系列浓度的汉黄芩素在含ATP和特异性CDK8底物肽的反应缓冲液中共同孵育。37°C孵育60分钟后，通过比色法检测磷酸化底物，比较药物处理组与对照组的吸光度，计算CDK8激酶活性抑制率 [1]
- PPAR-γ激活实验：将转染PPAR-γ报告基因的细胞用汉黄芩素（5 μM、10 μM、20 μM）处理24小时。细胞裂解后，使用荧光计检测荧光素酶活性以评估PPAR-γ转录激活能力，以PPAR-γ激动剂作为阳性对照 [3]

将HCT116细胞种植在96孔板上（每孔1×105个细胞）。加入不同浓度的汉黄芩素并孵育24小时。随后，将20μL MTT溶液（5mg/mL）转移到每个孔中，并在37°C和5%CO2下孵育4小时。吸出上清液，加入100μL DMSO以溶解甲酰胺晶体。摇动混合物，使用通用微孔板读数器在570nm下测量。[1]

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汉黄芩素是一种天然存在的单黄酮类化合物，据报道在体外和体内都具有肿瘤治疗潜力和良好的选择性。在此，我们在体外研究了wogonin在人结直肠癌癌症中的抗增殖作用及其相关机制。流式细胞术分析显示，汉黄芩素以浓度和时间依赖的方式诱导HCT116细胞G1期细胞周期阻滞。同时，细胞周期相关蛋白，如细胞周期蛋白A、E、D1和CDK2、4在汉黄芩素诱导的G1期细胞周期阻滞中下调。此外，我们发现汉黄芩素对HCT116细胞的抗增殖和G1期阻滞作用与Wnt/β-catenin信号通路的失调有关。汉黄芩素处理的细胞显示Wnt蛋白的细胞内水平降低，并激活降解复合物以磷酸化和靶向β-catenin进行蛋白酶体降解。汉黄芩素通过干扰TCF/Lef的转录活性和抑制CDK8的激酶活性来抑制β-catenin介导的转录，CDK8被认为是参与结直肠癌发展的癌基因。此外，CDK8 siRNA转染的HCT116细胞显示出与汉黄芩素处理的细胞相似的结果。因此，我们的数据表明，wogonin通过Wnt/β-catenin信号通路诱导抗增殖和G1期阻滞，可被开发为人类结直肠癌癌症的治疗剂[1]。

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人结直肠癌细胞实验：将细胞接种于96孔板，用汉黄芩素（20 μM、40 μM、60 μM）处理48小时，MTT法检测细胞增殖。细胞周期分析采用碘化丙啶染色后流式细胞术检测。通过Western blot和RT-PCR检测CDK8、β-连环蛋白、c-Myc及细胞周期蛋白D1的表达 [1]
- HeLa细胞实验：HeLa细胞接种于6孔板，用汉黄芩素（10 μM、20 μM、30 μM）处理24小时，MTT法检测细胞活力并确定IC50值。碘化丙啶染色后流式细胞术分析细胞周期分布，Western blot检测Cdk4、细胞周期蛋白D1和p21Cip1的蛋白水平 [2]
- 炎症细胞模型实验：肝细胞或巨噬细胞经脂多糖（LPS）诱导炎症后，用汉黄芩素（5 μM、10 μM、20 μM）处理18小时。通过RT-PCR和Western blot检测TNF-α、IL-6、IL-1β、PPAR-γ及NF-κB p65的mRNA和蛋白水平 [3]

In this study, researchers found that wogonin significantly attenuated inflammatory response in EtOH-fed mice, and reduced the expression of inflammatory cytokines such as TNF-α and IL-6 in EtOH-induced RAW264.7 cells. Furthermore, our findings showed that wogonin remarkably induced the expression of PPAR-γ in vivo and in vitro. Compared with the wogonin-treated group, blockade of PPAR-γ with inhibitor (T0070907) or PPAR-γ small interfering (si)-RNA were applied in RAW264.7 cells to evaluate the involvement of wogonin in alleviating EtOH-induced inflammation. Moreover, forced expression of PPAR-γ further suppressed the expression of TNF-α and IL-6 when treated with wogonin on EtOH-induced RAW264.7 cells. In addition, it was demonstrated that wogonin remarkably suppressed PPAR-γ-meditated phosphorylation and activation of NF-κB-P65. In conclusion, the above results indicated that wogonin may serve as an effective modulator of PPAR-γ by down-regulating NF-κB pathway, thereby attenuated inflammatory response in ALD.[3]

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C57BL/6 mice, male, 6-8 weeks old, weighing 18-22 g mice were housed at comfortable environment and are acclimatized for 3 days before the experiment. A total of 48 mice were randomLy divided into six groups of 8 animals, respectively control diet (CD)-fed mice, EtOH-fed mice, wogonin-treated mice at the dose of 25, 50, 100 mg/kg/day and the positive (dexamethasone, 1 mg/kg/day)-treated mice. Modeling process has a total of 16 days including a liquid diet adaptation period for 3 days and modeling for 13 days. The EtOH-fed mice are fed containing 5% v/v ethanol liquid diets adding certain vitamin and choline for 16 days, and mice are gavaged with a single binge ethanol administration (5 g/kg, body weight, 20% ethanol) at last day. At the same time, the wogonin-treated mice and the positive-treated mice are not only plus the ethanol administration, but also plus the medicines by gavage daily, whereas the CD-fed mice are fed with control liquid diets and gavaged with isocaloric maltose-dextrin at last day. All diets are prepared fresh daily. 9 h after the last gavage alcohol, mice are sacrificed under anaesthesia, the liver tissues and blood are collected for further analysis.[3]

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Mouse alcoholic liver disease model: Male mice were randomly divided into control, alcohol-fed, and Wogonin-treated groups. The alcohol-fed group received a Lieber-DeCarli liquid diet containing 5% ethanol for 4 weeks to induce alcoholic liver disease. Wogonin was dissolved in corn oil and administered via oral gavage at doses of 50 mg/kg and 100 mg/kg once daily during the 4-week alcohol feeding period. Control mice received a control liquid diet and corn oil. At the end of the experiment, mice were sacrificed, and liver tissues were collected for histological, biochemical, and molecular biological analyses [3]

Metabolism / Metabolites
Wogonin has known human metabolites that include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-(5-hydroxy-8-methoxy-4-oxo-2-phenylchromen-7-yl)oxyoxane-2-carboxylic acid.

[1]. Wogonin induced G1 cell cycle arrest by regulating Wnt/β-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells. Toxicology. 2013 Oct 4;312:36-47.

[2]. Wogonin induces G1 phase arrest through inhibiting Cdk4 and cyclin D1 concomitant with an elevation in p21Cip1 in human cervical carcinoma HeLa cells. Biochem Cell Biol. 2009 Dec;87(6):933-42.

[3]. Wogonin attenuates inflammation by activating PPAR-γ in alcoholic liver disease. Int Immunopharmacol. 2017 Sep;50:95-106.

Wogonin is a dihydroxy- and monomethoxy-flavone in which the hydroxy groups are positioned at C-5 and C-7 and the methoxy group is at C-8. It has a role as a cyclooxygenase 2 inhibitor, an antineoplastic agent, an angiogenesis inhibitor and a plant metabolite. It is a dihydroxyflavone and a monomethoxyflavone. It is a conjugate acid of a wogonin(1-).
Wogonin has been reported in Trichoderma virens, Rhinacanthus nasutus, and other organisms with data available.

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Wogonin is a natural flavonoid compound isolated from the roots of Scutellaria baicalensis Georgi [1][2][3]
- Its biological activities include antiproliferative effects on cancer cells (via cell cycle arrest) and anti-inflammatory effects (via PPAR-γ activation and NF-κB inhibition) [1][2][3]
- The antiproliferative mechanism in cancer cells involves regulating Wnt/β-catenin signaling pathway (by inactivating CDK8) and modulating cell cycle regulators (Cdk4, Cyclin D1, p21Cip1) [1][2]
- It has potential therapeutic applications in the treatment of colorectal cancer, cervical cancer, and alcoholic liver disease [1][2][3]

C16H12O5

284.26

284.068

C, 67.60; H, 4.26; O, 28.14

632-85-9

51059-44-0 (Wogonoside)

5281703

Light yellow to yellow solid powder

1.4±0.1 g/cm3

518.8±50.0 °C at 760 mmHg

203-206°C

198.4±23.6 °C

0.0±1.4 mmHg at 25°C

1.669

2.14

79.9

2

5

2

21

426

0

XLTFNNCXVBYBSX-UHFFFAOYSA-N

InChI=1S/C16H12O5/c1-20-15-12(19)7-10(17)14-11(18)8-13(21-16(14)15)9-5-3-2-4-6-9/h2-8,17,19H,1H3

4H-1-Benzopyran-4-one, 5,7-dihydroxy-8-methoxy-2-phenyl-

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)

配方 1 中的溶解度: 2.08 mg/mL (7.32 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 24 mg/mL (84.43 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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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；
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