Natural flavonolignan from milk thistle (Silybum marianum) seeds

Growth inhibition is time-and dose-dependent for silybin (0-200 mM; for 72 hours)[1]. Silybin (68 μM; for 72 hours) causes apoptosis and raises the percentage of cells in the G1 phase to about 22% [1]. AKT activity inhibition is induced by silybin (68 μM) for a duration of 72 hours[1].

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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].

For the last four weeks, silybin (50–100 mg/kg/day; administered intragastrically) has been shown to dramatically reduce hepatic and serum lipid accumulation[2].

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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 Viability Assay[1]
Cell Types: HepG2 cell growth
Tested Concentrations: 0-200 mM
Incubation Duration: For 72 hrs (hours)
Experimental Results: Had growth inhibition in a time- and dose-dependent manner with an IC50 of 68 μM.

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Apoptosis Analysis[1]
Cell Types: HepG2 cell growth
Tested Concentrations: 68 μM
Incubation Duration: For 72 hrs (hours)
Experimental Results: Induced apoptosis in a higher number of cells (60%) when compared to untreated cells.

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Cell Cycle Analysis[1]
Cell Types: HepG2 cell growth
Tested Concentrations: 68 μM
Incubation Duration: For 72 hrs (hours)
Experimental Results: Increased the cells in G1-phase of ~22% and diminished of 47% the cells in S-phase.

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Western Blot Analysis[1]
Cell Types: HepG2 cell growth
Tested Concentrations: 68 μM
Incubation Duration: For 72 hrs (hours)
Experimental Results: Induced AKT activity inhibition.

Animal/Disease Models: Male C57BL/6J mice (6-8 weeks old) with nonalcoholic fatty liver disease (NAFLD)[2]
Doses: 50, 100 mg/kg
Route of Administration: Given intragastrically (po); daily; for the last 4 weeks
Experimental Results: Dramatically lowered both serum and hepatic lipid accumulation.

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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. The standard normal diet and high-fat/high-cholesterol diet were both obtained from Nanjing Qinglongshan Experimental Animal Center. 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 xxx. 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
Silymarin's known metabolites include O-demethylsilymarin.

[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.

Silybin is a flavonoid lignan isolated from milk thistle (Silybum marianum) and has been shown to possess antioxidant and antitumor activities. It has multiple functions including antioxidant, antitumor, hepatoprotective, and phytometabolism-enhancing effects. Silybin is a flavonoid lignan, polyphenol, aromatic ether, benzodioxin, and secondary α-hydroxyketone. Silybin is the main active ingredient of silybinin, a standardized extract of milk thistle seeds containing various flavonoid lignans, including silybin, isosilybin, silybinine, and silybinine. Silybin exists as a mixture of two diastereomers—silybin A and silybin B—in approximately equal molar ratios. In vitro and animal studies have shown that silybin possesses hepatoprotective (anti-hepatotoxic) properties, protecting hepatocytes from toxin damage. Silybin has also been shown to have in vitro anticancer effects, inhibiting human prostate adenocarcinoma cells, estrogen-dependent and non-estrogen-dependent breast cancer cells, human cervical os cancer cells, human colon cancer cells, and small cell and non-small cell lung cancer cells. Silybin has been reported to be found in Aspergillus iizukae, Silybum eburneum, and other organisms with relevant data. Silybin is a mixture of flavonoid lignans isolated from Silybum marianum. Silybin may have antioxidant effects, protecting hepatocytes from chemotherapy-related free radical damage. This substance may also promote the growth of new hepatocytes. (NCI04) Silybin is the main active ingredient extracted from the seeds of Silybum marianum, belonging to the flavonoid class of compounds; it is used to treat hepatitis, cirrhosis, and chemical and drug-induced liver damage, and has antitumor activity; silybin A and B are diastereomers.

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Drug indications
Currently being tested as a drug for the treatment of severe hepatotoxic poisoning (e.g., poison amanita phalloides poisoning).

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In summary, these data suggest that silybin may activate programmed cell death processes by synthesizing ceramides, inducing the secretion of specific miRNAs that may target the PTEN/AKT pathway. These findings provide new avenues for the treatment of cancers that are insensitive to drug therapy (e.g., advanced hepatocellular carcinoma). This study aims to explore the molecular mechanisms of silybin’s antitumor effects in a non-invasive and faster manner, beyond all in vivo experiments. [1] In summary, at least in our model, non-alcoholic fatty liver disease (NAFLD) is characterized by significant accumulation of lipids in serum and liver, accompanied by overall metabolic abnormalities involving lipid metabolism, polyol metabolism, amino acid metabolism, urea cycle and tricarboxylic acid cycle. Tauroursodeoxycholic acid (TUDCA) significantly modulates these metabolic pathways. Silybin also has significant regulatory effects on these metabolic pathways, and the pathways it regulates are consistent with its multi-target mechanism. Although most metabolites involved in energy metabolism, such as carbohydrates, amino acids, and fatty acids, were detected by gas chromatography-mass spectrometry (GC-MS), the coverage of endogenous metabolites is still not comprehensive enough. In addition, further molecular biology studies are needed to investigate the mechanisms by which silymarin regulates these pathways. [2]

C25H22O10

482.44

482.121

802918-57-6

Silybin A;22888-70-6;Isosilybin;72581-71-6;Silybin B;142797-34-0

31553

White to off-white solid powder

152-153°C

2.362

5

10

4

35

750

4

COC1=C(C=CC(=C1)[C@@H]2[C@H](OC3=C(O2)C=C(C=C3)[C@@H]4[C@H](C(=O)C5=C(C=C(C=C5O4)O)O)O)CO)O

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

36804-17-8; 678-483-8; 802918-57-6; Legalon; SILYMARIN; Silybin (Standard); CHEMBL9509;

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)

DMSO : 25 mg/mL (51.82 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.18 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)