(±)-Silybin (Silibinin (mixture of Silybin A and Silybin B))   中文名称: 水飞蓟宾

Natural flavone

水飞蓟宾是从水飞蓟（水飞蓟）中提取的黄酮木脂素，具有保护肝脏、抗氧化和抗炎活性。几项研究表明，水飞蓟宾对预防和治疗不同类型的癌症非常有效，其抗肿瘤机制包括阻止细胞周期和/或细胞凋亡。采用MTT法研究细胞活力，分别采用硫代巴比妥酸活性物质（TBARS）法、NO法和MnSOD法研究脂质过氧化、细胞外NO生成和清除酶活性。流式细胞仪进行细胞周期和凋亡分析。实时PCR检测miRNA谱。在本研究中，我们证明了水飞蓟宾诱导的生长抑制在细胞周期的G1期阻断Hepg2细胞并激活细胞程序性死亡过程。此外，水飞蓟宾的抗增殖作用与参与miRNA分泌调节的神经酰胺数量的强烈增加相平行。特别是水飞蓟宾处理后，miR223-3p和miR16-5p上调，miR-92-3p下调（p < 0.05）。综上所述，我们的研究结果表明，水飞蓟宾诱导的HepG2细胞凋亡与神经酰胺合成和miRNAs分泌的增加并行发生。[１]

水飞蓟宾对肥胖和代谢综合征有良好的治疗效果，但水飞蓟宾的全身调节作用尚未完全揭示。本研究旨在探讨水飞蓟宾对非酒精性脂肪性肝病（NAFLD）代谢的调节作用。C57BL/6 J小鼠连续8周饲喂高脂高胆固醇饲粮，后4周灌胃水飞蓟素（50或100 mg/kg/d）和牛磺酸脱氧胆酸钠（50 mg/kg/d）。采用血液生化指标、肝脏脂质测定及肝脏油红O染色评价水飞蓟宾和TUDCA的模型及降脂效果。此外，血清和肝脏样品通过基于气相色谱-质谱（GC/MS）的代谢组学平台进行检测。多变量/单变量数据分析和途径分析用于研究差异代谢物和代谢途径。结果表明，小鼠NAFLD模型成功建立，水飞蓟宾和TUDCA均能显著降低血清和肝脏脂质积累。血清和肝脏代谢组学分析表明，高脂/高胆固醇饮食导致脂质代谢、多元醇代谢、氨基酸代谢、尿素循环和TCA循环等代谢产物代谢异常。水飞蓟宾和TUDCA处理均能逆转HFD喂养引起的代谢紊乱。综上所述，高脂肪/高胆固醇饮食导致小鼠血清和肝脏代谢异常，水飞蓟宾治疗改善了肝脏脂质积累，调节了整体代谢途径，这可能解释了其多靶点机制。［2］

细胞增殖试验[1]
采用MTT法进行细胞活力检测。MTT[3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑]是一种重要的染料，在线粒体水平上由细胞代谢。细胞在2.5 × 103孔板中接种。24 h后，将不同浓度的水飞蓟宾（0 ~ 200 μM）作用于细胞24、48、72 h。不同处理孵育后，在所有孔中加入浓度为10%的MTT。至少孵育4小时后，抽吸培养基/MTT溶液；然后用异丙醇/盐酸0.1 N溶液不断搅拌20分钟。最后，用酶联免疫吸附仪Biorad 550在多孔的每孔中分别测定本溶液的吸光度。在570 nm处进行吸光度读数。实验一式三次。

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Western Blotting [1]
细胞被单独或联合从Calcusyn获得的IC:50水飞蓟宾和索拉非尼处理。处理72 h后，将细胞洗涤并在PBS 1X中冰敷。颗粒离心后获得一个14000×g在4°C,细胞溶解一小时在冰缓冲区包含消息灵通的50 mM,氯化钠150毫米,甘油1%,特里同1%,MgCl2 1.5毫米,EGTA 5毫米,20 nM Na焦磷酸,10毫米原钒酸钠,氟化钠25毫米,抑肽酶(5µL /毫升),和PMSF (Phenylmethylsulfonyl加氟)0.5毫米。样本离心机在4°C 10分钟,上层清液用于实验。用Biorad法测定总蛋白浓度。蛋白裂解物在被装载到SDS- page聚丙烯酰胺凝胶上之前，在Sample Buffer 1X （Sample Buffer 5X: Tris 10 mg/mL, SDS 30 mg/mL， β-巯基乙醇0.15 mL，甘油0.3 mL，溴酚蓝）中在100°C下变性5分钟。电泳分离后，用Western blotting将蛋白转移到硝化纤维素过滤器上。过滤器用0.05% Tween20/TBS （200 mM Tris-HCl pH 7.5, 1.5 M NaCl）洗涤2次，在37℃阻断缓冲液（T-TBS/Milk浓度为5%）中孵育1 h。孵育后，过滤器暴露于适当稀释不同抗体的T-TBS/Milk溶液中。

Male C57BL/6J mice (6–8 weeks old) were acclimatized under 12 h/12 h dark-light cycles at a constant temperature (22 ± 2℃) and had free access to water and food. All mice were fed a normal diet for one week to acclimate to the environment. After that, they were divided into 5 groups (n = 6): vehicle group, HFD (high-fat/high-cholesterol diet) group, LS (low-dose silybin) group, HS (high-dose silybin) group and TUDCA group. The vehicle group was continuously fed a standard normal diet, and the other groups were fed a high-fat/high-cholesterol diet (10 % lard, 10 % yolk, 1 % cholesterol, 0.2 % cholate and 78.8 % standard diet; 60 % of kcal as fat was the prescription) for 8 weeks. Silybin (50 or 100 mg/kg/day) and TUDCA (50 mg/kg/day) were ground in 0.5 % carboxymethylcellulose sodium (CMC-Na) and were given intragastrically for the last 4 weeks. At the end of the experiment, blood was collected from the orbital sinus after fasting overnight, and the levels of serum total cholesterol (TC), triglyceride (TG) and nonesterified fatty acid (NEFA) were assayed with commercial kits purchased from Nanjing Jiancheng Bio-Engineering Institute Co., Ltd (Nanjing, China). Livers were frozen by dry ice in OCT® compound during tissue collection and then sectioned into 8-μm-thick slices by a cryostat and stained with Oil red O as previously described. Both serum and liver samples were stored at -80 °C until analysis. [2]

Metabolism / Metabolites
Silybin has known human metabolites that include O-demethylated-silybin.

[1]. Silybin-Induced Apoptosis Occurs in Parallel to the Increase of Ceramides Synthesis and miRNAs Secretion in Human Hepatocarcinoma Cells. Int J Mol Sci. 2019 May 3;20(9):2190.

[2]. Silybin ameliorates hepatic lipid accumulation and modulates global metabolism in an NAFLD mouse model. Biomed Pharmacother. 2020 Mar;123:109721.

Silibinin is a flavonolignan isolated from milk thistle, Silybum marianum, that has been shown to exhibit antioxidant and antineoplastic activities. It has a role as an antioxidant, an antineoplastic agent, a hepatoprotective agent and a plant metabolite. It is a flavonolignan, a polyphenol, an aromatic ether, a benzodioxine and a secondary alpha-hydroxy ketone.
Silibinin is the major active constituent of silymarin, a standardized extract of the milk thistle seeds, containing a mixture of flavonolignans consisting of silibinin, isosilibinin, silicristin, silidianin and others. Silibinin is presented as a mixture of two diastereomers, silybin A and silybin B, which are found in an approximately equimolar ratio. Both in vitro and animal research suggest that silibinin has hepatoprotective (antihepatotoxic) properties that protect liver cells against toxins. Silibinin has also demonstrated in vitro anti-cancer effects against human prostate adenocarcinoma cells, estrogen-dependent and -independent human breast carcinoma cells, human ectocervical carcinoma cells, human colon cancer cells, and both small and nonsmall human lung carcinoma cells.
Silibinin has been reported in Aspergillus iizukae, Silybum eburneum, and other organisms with data available.
Silymarin is a mixture of flavonolignans isolated from the milk thistle plant Silybum marianum. Silymarin may act as an antioxidant, protecting hepatic cells from chemotherapy-related free radical damage. This agent may also promote the growth of new hepatic cells. (NCI04)
The major active component of silymarin flavonoids extracted from seeds of the MILK THISTLE, Silybum marianum; it is used in the treatment of HEPATITIS; LIVER CIRRHOSIS; and CHEMICAL AND DRUG INDUCED LIVER INJURY, and has antineoplastic activity; silybins A and B are diastereomers.

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Drug Indication
Currently being tested as a treatment of severe intoxications with hepatotoxic substances, such as death cap (Amanita phalloides) poisoning.

C25H22O10

482.44

481.113

36804-17-8

31553

White to off-white solid

1.527g/cm3

793ºC at 760 mmHg

160ºC

274.4ºC

1.684

3.384

159.05

5

10

4

35

750

4

COC1=C(O)C=CC(C2OC3C=C([C@H]4OC5=CC(=CC(O)=C5C(=O)[C@@H]4O)O)C=CC=3OC2CO)=C1

SEBFKMXJBCUCAI-HKTJVKLFSA-N

InChI=1S/C25H22O10/c1-32-17-6-11(2-4-14(17)28)24-20(10-26)33-16-5-3-12(7-18(16)34-24)25-23(31)22(30)21-15(29)8-13(27)9-19(21)35-25/h2-9,20,23-29,31H,10H2,1H3/t20-,23+,24-,25-/m1/s1

(2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one

Silibinin (mixture of Silybin A and Silybin B); 36804-17-8; 678-483-8; 802918-57-6; Legalon; SILYMARIN;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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。