Natural flavonolignan from Silybum marianum; anti-tumor; Silybin B is a typical multi-target natural product with multiple identified direct or indirect molecular targets. Regarding transporters, silybin B inhibits organic anion transporting polypeptides OATP2B1 (IC₅₀=800 nM), OATP1B3 (IC₅₀=5 μM), and OATP1B1 (IC₅₀=8.5 μM). This compound also acts as a direct modulator of P-glycoprotein (P-gp), inhibiting P-gp ATPase activity and downregulating the expression of multidrug resistance genes including ABCB1, ABCC1, and ABCG2. Furthermore, silybin inhibits xanthine oxidase (XO, 70% inhibition) and aldehyde oxidase (AO, 70% inhibition) activities

Silybins A and B (4 and 5) are the major components in the active extract of milk thistle (silymarin) and have been linked previously to skin cancer prevention effects via anti-inflammatory, antioxidant, and immunomodulatory mechanisms (Katiyar, Citation2005). Our study results now reveal that all seven major compounds in silymarin show good EBV-EA inhibition activities. In our testing model, silychristins A and B (1 and 2) and isosilybins A and B (6 and 7) had better inhibition effects compared with β.-carotene, and silybins A and B (4 and 5) showed lower activity compared with the other flavonolignans in silymarin. Further in vivo. testing on mouse skin papillomas is ongoing. In conclusion, seven pure compounds silychristin A (1), silychristin B (2), silydianin (3), silybin A (4), Silybin B (5), isosilybin A (6), and isosilybin B (7) were isolated from an extract of milk thistle. Evaluation with an in vitro. EBV-EA activation assay showed that silychristin B (2) was the most active compound with 94.9% EBV-EA inhibition at 1000 mol ratio/TPA. As it also showed low cytotoxicity, 2 could be valuable as an antitumor promoter or as a lead compound for new cancer preventive drug development. [1]

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Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS), and computational (all-atom molecular dynamics) techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, Silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aβ aggregation. Despite TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only Silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that silybin B interacts mainly with the C-terminal hydrophobic segment 35MVGGVV40 of Aβ40. Consequently to silybin B binding, the peptide conformation remains predominantly unstructured along all the simulations. By contrast, silybin A interacts preferentially with the segments 17LVFF20 and 27NKGAII32 of Aβ40 which shows a high tendency to form bend, turn, and β-sheet conformation in and around these two domains. Both 2,3-dehydrosilybin enantiomers bind preferentially the segment 17LVFF20 but lead to the formation of different small-sized, ThT-positive Aβ aggregates. [2]

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In vitro studies demonstrate that silybin B exhibits multiple biological activities. Regarding antioxidant activity, silybin B shows strong free radical scavenging capacity in oxygen radical absorbance capacity (ORAC) assays. For anti-inflammatory effects, this compound dose-dependently reduces the release of inflammatory cytokines including TNF-α and IL-6 in LPS-stimulated cells. Regarding reversal of drug resistance, silybin B is the only compound that directly acts upon P-glycoprotein and downregulates ABC transporter expression (ABCB1, ABCC1, ABCG2), and sensitizes drug-resistant ovarian carcinoma cells to doxorubicin. Regarding metabolic enzymes, 100 µM silybin inhibits rat hepatic xanthine oxidase and aldehyde oxidase activities by 70% and 70%, respectively.

Finally, in vivo studies in a transgenic Caenorhabditis elegans strain expressing human Aβ indicated that Silybin B is the most effective of the four compounds in counteracting Aβ proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of silybins and points to Silybin B as a promising lead compound for further development in anti-AD therapeutics [2].

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In vivo studies confirm the anti-tumor activity of silybin B in animal models. Silybin B has demonstrated cancer-suppressive effects in prostate cancer animal models. Regarding pharmacokinetic-pharmacodynamic relationships, the absorption and metabolism of this compound in vivo differ significantly from its diastereoisomer silybin A, suggesting isomer-dependent in vivo activity. Silymarin and silibinin have been shown to possess significant cancer chemopreventive and anticancer activities in various animal models of human epithelial malignancies.

In vitro enzyme inhibition assays for silybin B have been reported. A representative protocol for xanthine oxidase inhibition is as follows: Enzyme Source Preparation: Partially purify xanthine oxidase from rat liver homogenates. Substrate Preparation: Use xanthine as the substrate for enzyme activity detection. Inhibitor Incubation: Pre-incubate varying concentrations of silybin (10, 50, 100 µM) with xanthine oxidase in reaction buffer. Reaction Initiation and Detection: Initiate the reaction by adding substrate and detect product formation rate by spectrophotometry. Data Analysis: Calculate enzyme activity inhibition rates. Results show that 100 µM silybin inhibits rat hepatic XO activity by 70±1%.

Cell Culture: Culture drug-resistant cancer cell lines (e.g., doxorubicin-resistant ovarian carcinoma cells) in medium containing 10% fetal bovine serum at 37°C in a 5% CO₂ incubator. Drug Treatment: Dissolve silybin B in DMSO to prepare stock solution, dilute to working concentrations (e.g., 30 µM) with culture medium, and treat cells with or without chemotherapeutic agents (e.g., doxorubicin). Viability Assay: Assess cell viability using MTT or CCK-8 assays to evaluate silybin B cytotoxicity and its sensitizing effects. Drug Resistance Protein Expression Detection: Detect expression changes of drug resistance-related proteins including ABCB1, ABCC1, and ABCG2 by Western blot. P-gp ATPase Activity Assay: Assess the direct effect of the compound on P-gp activity using a P-gp ATPase assay kit. Inflammatory Cytokine Detection: Measure levels of inflammatory cytokines such as TNF-α and IL-6 in cell supernatants by ELISA. Data Analysis: Compare cell viability, protein expression, and inflammatory cytokine levels between treatment and control groups.

Animal Models: Use rats for pharmacokinetic studies or nude mice bearing human tumor xenografts for anti-tumor activity evaluation. Dosing Regimen: Administer silybin B by intragastric gavage at a typical dose of 200 mg/kg body weight (rats), dissolved in an appropriate vehicle (e.g., corn oil or CMC-Na). Pharmacokinetic Sampling: Collect plasma samples at specified time points (0.5, 1, 2, 3, 4, 6 hours) after administration and detect drug concentrations by HPLC-PDA or LC-MS/MS. Metabolite Identification: Identify metabolites in plasma using solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS). The major metabolite is silybin B-7-O-β-glucuronide. Anti-tumor Efficacy Assessment: In xenograft models, measure tumor volume and body weight periodically to calculate tumor growth inhibition rate. Data Analysis: Calculate pharmacokinetic parameters (Cmax, Tmax, T1/2, AUC) and compare tumor growth differences between treatment and control groups.

Silybin B exhibits superior pharmacokinetic properties compared to its diastereoisomer silybin A in rats. Following intragastric administration of 200 mg/kg in rats, total silybin B shows a Cmax of 14.50 µg/mL, Tmax of 2.6 hours, T1/2 of 2.9 hours, with AUC approximately 20-fold higher than that of silybin A. The estimated oral bioavailability is approximately 0.3%. Silybin B is extensively metabolized in vivo, with glucuronidation and sulfation as the major metabolic pathways; silybin B-7-O-β-glucuronide is the predominant plasma metabolite. In humans, silybin B has a Tmax of approximately 2-3 hours and a half-life of approximately 1.3-2.0 hours. Relative bioavailability varies significantly depending on formulation. Storage conditions: powder is stable for up to 3 years at 2-8°C protected from light.

Oral LD50 in mice >1600 mg/kg. (German patent document #2423725)
Silybin B, as a natural flavonolignan, exhibits a favorable safety profile. The predicted hepatotoxicity probability is relatively high (0.986), suggesting potential liver-related adverse effects under certain conditions. Diseases associated with this compound include cataract, peripheral neuropathy, muscle weakness, male infertility, osteoporosis, type 2 diabetes, and Alzheimer's disease. These associations are primarily based on systems toxicology predictions and literature reports, requiring further validation for causal relationships.

[1]. Cancer Preventive Agents. 7. Antitumor-Promoting Effects of Seven Active Flavonolignans from Milk Thistle (Silybum marianum.) on Epstein-Barr Virus Activation, Pharmaceutical Biology, 45:10, 735-738.

[2]. Inhibition of Aβ Amyloid Growth and Toxicity by Silybins: The Crucial Role of Stereochemistry. ACS Chem Neurosci. 2017 Aug 16;8(8):1767-1778.

Silybin B is a flavonoid lignan. Silybin B has been reported to exist in Aspergillus iizukae, Anastatica hierochuntica, and other organisms with relevant data. Silybin is a mixture of flavonoid lignans isolated from Silybum marianum. Silybin may have antioxidant properties, 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 and is used to treat hepatitis, cirrhosis, and chemical and drug-induced liver injury, and has antitumor activity; silybin A and B are diastereomers. See also: Silybum marianum (partial); Silybum marianum (note moved to).

C25H22O10

482.43618

482.121

C, 62.24; H, 4.60; O, 33.16

142797-34-0

Silybin A;22888-70-6;Isosilybin;72581-71-6;Silybin;802918-57-6;Silymarin;65666-07-1

1548994

White to off-white solid powder

1.5±0.1 g/cm3

793.0±60.0 °C at 760 mmHg

158-160℃ (methanol water )

274.5±26.4 °C

0.0±2.9 mmHg at 25°C

1.684

2.59

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-WAABAYLZSA-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+/m0/s1

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

Silibinin B; 142797-34-0; UNII-853OHH1429; DTXSID30858697; 4H-1-BENZOPYRAN-4-ONE, 2-((2S,3S)-2,3-DIHYDRO-3-(4-HYDROXY-3-METHOXYPHENYL)-2-(HYDROXYMETHYL)-1,4-BENZODIOXIN-6-YL)-2,3-DIHYDRO-3,5,7-TRIHYDROXY-, (2R,3R)-; 4H-1-Benzopyran-4-one, 2-[(2S,3S)-2,3-dihydro-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-1,4-benzodioxin-6-yl]-2,3-dihydro-3,5,7-trihydroxy-, (2R,3R)-; DTXCID703580; SILYBIN B (CONSTITUENT OF MILK THISTLE);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~518.20 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.31 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 20.8 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.)