Determination of Flavonolignan Compositional Ratios in Silybum marianum (Milk Thistle) Extracts Using High-Performance Liquid Chromatography.

Milk thistle is one of the most popular ingredients in the liver protection products market. Silymarin is the main component of milk thistle and contains multiple isomers. There have been few studies focusing on the compositional ratios of silymarin isomers. In this study, we developed an HPLC method for the separation and quantification of silymarin isomers, thereby elucidating their compositional ratios. Through the analysis of more than 40 milk thistle extract products on the market, we found that the ratios, specifically Ratio 1 (the silybin B content to the silybin A content, SBNB/SBNA) and Ratio 2 (the sum of the contents of silybin B and isosilybin B to the sum of the contents of silybin A and isosilybin A, (SBNB + IBNB)/(SBNA + IBNA)), are highly consistent across milk thistle extracts, averaging approximately 1.58 and 1.28, respectively. Furthermore, such ratios were verified in milk thistle seed samples. This study introduces significant findings concerning the stable ratios among silymarin isomers in milk thistle extracts and seeds, thereby offering an innovative approach for quality assurance of milk thistle extracts.

Chemical Standardization of Milk Thistle (Silybum marianum L.) Extract Using UHPLC-MS/MS and the Method of Standard Addition.

Extracts prepared from the seeds of the medicinal plant milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)] are widely used as dietary supplements due to anti-inflammatory, antitumor, and hepatoprotective effects. Called silymarin, the main components of lipophilic extracts of milk thistle seeds are flavonoids and flavonolignans including silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, taxifolin, and 2,3-dehydrosilybins. The aim of this study was to develop a method based on UHPLC-MS/MS for the chemical authentication and standardization of milk thistle silymarin. Validation included the method of standard addition to account for the lack of a blank matrix. Potential matrix effects were investigated by analyzing silymarin standards dissolved only in the initial UHPLC mobile phase. Measurements of six flavonolignans and taxifolin in the milk thistle extract using UHPLC-MS/MS with standard addition or external standard calibration produced similar results for all analytes except silydianin and 2,3-dehydrosilybin B, which showed significant peak enhancement during negative ion electrospray due to botanical matrix effects. The UHPLC-MS/MS-based method of standard addition requires <10 min per injection and is suitable for the standardization of silymarin from milk thistle in support of preclinical and clinical studies of safety and efficacy.

Total Synthesis of (+)-Silybin A.

We report the first total synthesis of silybin A (1). Key synthetic steps include the construction of the 1,4-benzodioxane neolignan skeleton, a modified Julia-Kocienski olefination reaction between m-nitrophenyltetrazole sulfone (m-NPT sulfone) 10 and aldehyde 21, the formation of the flavanol lignan skeleton 28 via a quinomethide intermediate under acidic conditions, and stepwise oxidation of the benzylic position of flavanol 29.

Development, Optimization, and in vitro Evaluation of Silybin-loaded PLGA Nanoparticles and Decoration with 5TR1 Aptamer for Targeted Delivery to Colorectal Cancer Cells.

Chemotherapeutic agents often lack specificity, intratumoral accumulation, and face drug resistance. Targeted drug delivery systems based on nanoparticles (NPs) mitigate these issues. Poly (lactic-co-glycolic acid) (PLGA) is a well-studied polymer, commonly modified with aptamers (Apts) for cancer diagnosis and therapy. In this study, silybin (SBN), a natural agent with established anticancer properties, was encapsulated into PLGA NPs to control delivery and improve its poor solubility. The field-emission scanning electron microscopy (FE-SEM) showed spherical and uniform morphology of optimum SBN-PLGA NPs with 138.57±1.30nm diameter, 0.202±0.004 polydispersity index (PDI), -16.93±0.45mV zeta potential (ZP), and 70.19±1.63% entrapment efficiency (EE). The results of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) showed no chemical interaction between formulation components, and differential scanning calorimetry (DSC) thermograms confirmed efficient SBN entrapment in the carrier. Then, the optimum formulation was functionalized with 5TR1 Apt for active targeted delivery of SBN to colorectal cancer (CRC) cells in vitro. The SBN-PLGA-5TR1 nanocomplex released SBN at a sustained and constant rate (zero-order kinetic), favoring passive delivery to acidic CRC environments. The MTT assay demonstrated the highest cytotoxicity of the SBN-PLGA-5TR1 nanocomplex in C26 and HT29 cells and no significant cytotoxicity in normal cells. Apoptosis analysis supported these results, showing early apoptosis induction with SBN-PLGA-5TR1 nanocomplex which indicated this agent could cause programmed death more than necrosis. This study presents the first targeted delivery of SBN to cancer cells using Apts. The SBN-PLGA-5TR1 nanocomplex effectively targeted and suppressed CRC cell proliferation, providing valuable insights into CRC treatment without harmful effects on healthy tissues.

Formulation design and characterization of silymarin liposomes for enhanced antitumor activity.

Liposomes, a nanoscale carrier, plays an important role in the delivery of drug, affects the in vivo efficacy of drugs. In this paper, silymarin(SM)-loaded liposomes was optimized using the response surface method (RSM), with entrapment efficiency (EE%) as an index. The formulation was optimized as follow: lecithin (7.8mg/mL), SM/lecithin (1/26) and lecithin/cholesterol (10/1). The optimized SM liposomes had a high EE (96.58 ±3.06%), with a particle size of 290.3 ±10.5nm and a zeta potential of +22.98 ±1.73mV. In vitro release tests revealed that SM was released in a sustained-release manner, primarily via diffusion mechanism. In vitro cytotoxicity studies demonstrated that the prepared SM liposomes had stronger inhibitory effects than the model drug. Overall, these results indicate that this liposome system is suitable for intravenous delivery to enhance the antitumor effects of SM.

Tailored green synthesized silymarin-selenium nanoparticles: Topical nanocarrier of promising antileishmanial activity.

Poor drug penetration, emerging drug resistance, and systemic toxicity are among the major obstacles challenging the current treatment of cutaneous leishmaniasis. Hence, developing advanced strategies for effective and targeted delivery of antileishmanial agents is crucial. Several drug delivery carriers have been developed till current date for dermal/transdermal delivery, especially those which are fabricated using eco-friendly synthesis approaches, since they protect the environment from the harmful effects of chemical waste disposal. This work describes the preparation of selenium nanoparticles loaded with silymarin via one-pot green reduction technique, for treatment of cutaneous leishmaniasis. The selected silymarin loaded selenium nanoparticles (SSNs4-0.1) displayed good loading efficiency of 58.22 ± 0.56 %, zeta potential of -30.63 ± 0.40 mV, hydrodynamic diameter of 245.77 ± 11.12 nm, and polydispersity index of 0.19 ± 0.01. It exhibited good physical stability, as well as high ex vivo deposition % in the epidermis (46.98 ± 1.51 %) and dermis (35.23 ± 1.72 %), which was further proven using confocal laser microscopy. It also exhibited significant cytocompatibility and noticeable cellular internalization of 90.02 ± 3.81 % in human fibroblasts, as well as high trypanothione reductase inhibitory effect (97.10 ± 0.30 %). Results of this study confirmed the successful green synthesis of silymarin-loaded selenium nanoparticles; delineating them as one of the promising antileishmanial topical delivery systems.

Erythrocyte membrane-camouflaged mesoporous silica composite nanoparticles for loading and controlled release of the hepatoprotective agent silibinin: A-synthesis and physicochemical characterization.

OBJECTIVES: Milk thistle has long been used in the treatment of liver and biliary disorders. In the present study, to make a long-acting delivery system for silibinin (SBN, a major active constituent of milk thistle seeds with antioxidant and hepatoprotective function), mesoporous silica composite nanoparticles (NC) were synthesized and coated with RBC membrane. METHODS: A modified Stöber method was used for NC synthesis, which was then characterized using FE-SEM, DLS, TEM, FTIR, and EDAX techniques. A suitable lysis buffer was used to prepare RBC-ghost, and sonication was used to coat SBN-loaded NC (SBN-NC). The RBC-ghost coated SBN-NC (SBN-NC-RBCG) was evaluated by SDS-PAGE, Bradford, TEM, EDAX, and DLS methods. SBN release was then compared for the SBN-NC and SBN-NC-RBCG samples. KEY FINDINGS: the RBC membrane proteins were recovered from the coating of SBN-NC-RBCG, and SBN release was sustained over 24 h when compared with the SBN-NC. CONCLUSIONS: Overall, through prolonging circulation in the bloodstream and evading the immune system, the developed system can improve SBN bioavailability in liver inflammation and fibrosis conditions that need further research.

Silibinin solubilization: combined effect of co-solvency and inclusion complex formation.

OBJECTIVE: Belonging to the class II drugs according to the biopharmaceutics classification system, silibinin (SLB) benefits from high permeability but suffers poor solubility that negatively affects the development of any delivery system. This research aimed to improve SLB solubility by combined use of co-solvency and complexation phenomena. METHODS: Solubility studies were performed using the phase solubility analysis according to the shake-flask method in the presence of ethanol and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as a co-solvent and inclusion complexing agent, respectively. SLB release studies from chitosan nanoparticles were carried out in double-wall, diffusion cells using the optimized drug release medium. RESULTS: SLB solubility was mathematically optimized constraining to using the lowest concentrations of ethanol and HP-ß-CD. SLB solubility increased linearly with the increase of HP-ß-CD concentration. The solubility in PBS-ethanol mixtures followed a log-linear model. SLB solubility in the presence of the ethanol co-solvent and HP-ß-CD complexing agent was optimized by adopting a genetic algorithm suggesting the phosphate buffer saline solution supplemented by 6%v/v ethanol and 8 mM HP-ß-CD as an optimized medium. The optimized solution was examined to study SLB release from chitosan nanoparticles (4.5 ± 0.2% drug loading) at 37 °C under static conditions. The sigmoidal release profile of SLB from the particles indicated a combination of erosion and diffusion mechanisms governing drug release from the nanoparticles. CONCLUSION: SLB solubility in a buffered solution supplemented by ethanol co-solvent and HP-ß-CD complexing agent is a function of free drug present in the semi-aqueous media, the drug-ligand binary complex, and the drug/ligand/co-solvent ternary complex.

Synthesis, physiochemical characterization, molecular docking study, and anti-breast cancer activity of silymarin loaded zein nanoparticles.

Breast cancer is a major cause of death in women worldwide leading to requirement of new therapeutic strategies. Silymarin demonstrated the anti-cancer activity however, due to low bioavailability its use is restricted. This study aimed to improve the solubility of silymarin by developing a silymarin loaded zein nanoparticles (SLNPs) which was stabilized by beta cyclodextrin. Comprehensive physiochemical characterization studies based on DLS, FTIR, UV-Vis Spectroscopy, FE-SEM, TEM, XRD, DSC, NMR and TGA confirmed the successful synthesis of SLNPs via an anti-solvent precipitation method. FE-SEM and TEM images demonstrated the uniform size and spherical shape of nanoparticles with encapsulation and loading efficiencies of 84.32 ± 1.9 % and 15.25 ± 2.4 % respectively. The zein protein interaction with silymarin, and ß-cyclodextrin was shown to be beneficial via the use of molecular simulations and binding energy calculations. Cellular studies demonstrated dose and time dependent cytotoxicity of SLNPs on MCF-7 breast cancer cell. FACS, qRT-PCR and Western blotting showed Bax (pro-apoptotic) upregulation while Bcl-2 (anti-apoptotic) downregulation. Our findings suggest that these loaded nanoparticles are more efficient than pure drug, enhancing its bioavailability and paving the path for developing it as a promising nutraceutical to treat breast cancer.

Preparation and in vitro evaluation of protective effects of Silibinin-loaded polymeric micelles on human hair against UV-B radiation.

BACKGROUND: The purpose of this study was to investigate the protective effect of Silibinin-loaded polymeric micelles from human hair against UV-B radiation. METHODS: Eight formulations with different concentrations of Silibinin, Pluronic F-127, and Labrasol-Labrafil were made by a solvent evaporation method, and the selected formulation was chosen by examining their properties like particle size and loading efficiency. Six groups of human hair, including a group that received the selected formulation, were exposed to UV-B radiation and by calculating its factors such as peak-to-valley roughness, RMS roughness, FTIR, and the amount of protein loss, the protective effect of the selected formulation was judged. RESULTS: According to the results, the loading efficiency and particle size of the selected formulation were 45.34% and 43.19 nm. The Silibinin release profile had two parts, fast and slow, which were suitable for creating a drug depot on hair. Its zeta potential also confirmed the minimum electrostatic interference between the formulation and hair surface. The zeta potential of selected formulation was -5.9 mv. Examination of AFM images showed that the selected formulation was able to prevent the increase in peak-to-valley roughness and RMS roughness caused by UV-B radiation. RMS roughness after 600 h of UV radiation in Groups 5 and 6 was significantly lower than the negative control group and the amount of this factor did not differ significantly between 0 and 600, so it can be concluded that the selected formulation containing Silibinin and the positive control group was able to prevent the increase of RMS roughness and hair destruction. In other hands, the two positive control groups and the selected formulation containing Silibinin were able to effectively reduce hair protein loss. CONCLUSION: Silibinin-loaded polymeric micelles were able to effectively protect hair from structural and chemical changes caused by UV-B radiation.

Biochemical bases of the pharmacological action of the flavonoid silymarin and of its structural isomer silibinin

The flavonoid silymarin and one its structural components, silibinin, have been well characterized as hepato-protective substances. However, little is known about the biochemical mechanisms of action of these substances. This review deals with recent investigations to elucidate the molecular action of the flavonoid. Three levels of action have been proposed for silymarin in experimental animals: a) as an antioxidant, by scavenging prooxidant free radicals and by increasing the intracellular concentration of the tripeptide glutathione; b) regulatory action of the cellular membrane permeability and increase of its stability against xenobiotic injury; c) at the nuclear expression, by increasing the synthesis of ribosomal RNA by stimulating DNA polymerase I and by exerting a steroid-like regulatory action on DNA transcription. The specific hepatoprotective action of silibinin against the toxicity of ethanol, phenylhydrazine and acetaminophen is also discussed. It is suggested that the biochemical effects observed for the flavonoid in experimental models may settle the basis for understanding the pharmacological action of silymarin and silibinin