Natural flavone

Silybin is a flavonolignan extracted from Silybum marianum (milk thistle) with hepatoprotective, antioxidant, and anti-inflammatory activity. Several studies have shown that silybin is highly effective to prevent and treat different types of cancer and that its antitumor mechanisms involve the arrest of the cell cycle and/or apoptosis. An MTT assay was performed to study cell viability, lipid peroxidation, extracellular NO production, and scavenger enzyme activity were studied by Thiobarbituric Acid-Reactive Species (TBARS) assay, NO assay, and MnSOD assay, respectively. Cell cycle and apoptosis analysis were performed by FACS. miRNA profiling were evaluated by real time PCR. In this study, we demonstrated that Silybin induced growth inhibition blocking the Hepg2 cells in G1 phase of cell cycle and activating the process of programmed cell death. Moreover, the antiproliferative effects of silybin were paralleled by a strong increase of the number of ceramides involved in the modulation of miRNA secretion. In particular, after treatment with silybin, miR223-3p and miR16-5p were upregulated, while miR-92-3p was downregulated (p < 0.05). In conclusion, our results suggest that silybin-Induced apoptosis occurs in parallel to the increase of ceramides synthesis and miRNAs secretion in HepG2 cells. [1]

Silybin shows good effects against obesity and metabolic syndrome, but the systemic modulation effect of silybin has not been fully revealed. This study aims to investigate the metabolic regulation by silybin of nonalcoholic fatty liver disease (NAFLD). C57BL/6 J mice were fed a high-fat/high-cholesterol diet for 8 weeks and treated with silybin (50 or 100 mg/kg/day) and sodium tauroursodeoxycholate (TUDCA, 50 mg/kg/day) by gavage for the last 4 weeks. Blood biochemical indexes and hepatic lipid measurement as well as Oil red O staining of the liver were conducted to evaluate the model and the lipid-lowering effect of silybin and TUDCA. Furthermore, serum and liver samples were detected by a metabolomic platform based on gas chromatography-mass spectrometry (GC/MS). Multivariate/univariate data analysis and pathway analysis were used to investigate differential metabolites and metabolic pathways. The results showed that the mouse NAFLD model was established successfully and that silybin and TUDCA significantly lowered both serum and hepatic lipid accumulation. Metabolomic analysis of serum and liver showed that a high-fat/high-cholesterol diet caused abnormal metabolism of metabolites involved in lipid metabolism, polyol metabolism, amino acid metabolism, the urea cycle and the TCA cycle. Silybin and TUDCA treatment both reversed metabolic disorders caused by HFD feeding. In conclusion, a high-fat/high-cholesterol diet caused metabolic abnormalities in the serum and liver of mice, and silybin treatment improved hepatic lipid accumulation and modulated global metabolic pathways, which provided a possible explanation of its multiple target mechanism. [2]

Cell Proliferation Assay [1]
Cell viability tests were performed using MTT assays. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], is a vital dye, which is metabolized by the cell at the level of the mitochondria. Cells were plated at 2.5 × 103 in 96-well plates. After 24 h, cells were exposed to various concentrations of Silybin (0–200 μM) for 24, 48, and 72 h. After the time of incubation with the different treatments, the MTT was added to all the wells in a concentration of 10%. After a minimum incubation of 4 h, the medium/MTT solution was aspirated; later the formazan salts were solubilized with an isopropanol/HCL 0.1 N solution for 20 min in constant agitation. Finally, the absorbance measurement of the present solution was performed respectively in each well of the multiwell, with an ELISA reader Biorad 550. Absorbance readings were performed at 570 nm. Experiments were performed in triplicate.

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Western Blotting [1]
Cells were plated and treated with IC:50 silybin and sorafenib individually or in combination obtained from Calcusyn. After 72 h of treatment, cells were washed and collected in PBS 1X on ice. Pellets obtained after centrifugation a 14000× g at 4 °C, were lysed for an hour on ice in a buffer containing HEPES 50 mM, NaCl 150 mM, Glycerol 1%, TRITON 1%, MgCl2 1.5 mM. EGTA 5 mM, 20 nM Na pyrophosphate, 10 mM Na orthovanadate, 25 mM NaF, Aprotinin (5 µL/mL), and PMSF (Phenylmethylsulfonyl fluoridate) 0.5 mM. The samples were centrifuged at 4 °C for 10 min and the supernatant was used for the experiments. Total protein concentration was determined by Biorad assay. Protein lysates, before being loaded onto the SDS-PAGE polyacrylamide gel, were denatured for 5 min at 100 °C in Sample Buffer 1X (Sample Buffer 5X: Tris 10 mg/mL, SDS 30 mg/mL, β-mercaptoethanol 0.15 mL, glycerol 0.3 mL, bromophenol blue). After electrophoretic separation, the proteins were transferred by Western blotting on nitrocellulose filter. The filter was washed twice with 0.05% Tween20/TBS (200 mM Tris-HCl pH 7.5, 1.5 M NaCl) and incubated for 1 h at 37 °C in blocking buffer (T-TBS/Milk at 5%). After the incubation, the filter was exposed to a solution of T-TBS/Milk with the different antibodies properly diluted.

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

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

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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.)